Inna P. Gladysheva

Interests

Research

Molecular mechanisms of dilated cardiomyopathy, atrial cardiomyopathy and heart failure, and concurrent complications.Potential diagnostic and therapeutic targets for cardiovascular diseases, in particular in modulating progression of cardiomyopathy and heart failure. Specifically, we are interested in proteases and hormones as potential diagnostic and therapeutic targets for cardiovascular diseases, in modulating progression of cardiomyopathy and heart failure, and concurrent complications. Major long-term goal of the lab. translational research is to delay or prevent transition from cardiomyopathy to symptomatic heart failure characterized by fluid retention (edema) and progressive muscle wasting (cachexia/sarcopenia). Still, little is known about the predictors (biomarkers) and effectors (bio-targets) for this transition. The difficulty of objectively detecting and reproducible quantify edema and muscle mass is one of the major challenges in heart failure management. Toward this major goal we are interested in: • Biomarkers/bio-targets defining transition from cardiomyopathy to symptomatic heart failure• Mechanisms responsible for alteration of biological pathways associated with transition from cardiomyopathy to symptomatic heart failure• Preventive potential of dietary, pharmacologic & genetic interventions and impact of lifestyle • Sex-related alteration associated with transition from cardiomyopathy to symptomatic heart failure• Noninvasive objective technologies for edema and cachexia/sarcopenia monitoring • Precise consideration of the individual’s biomarker profile and sex.Research combines animal, biochemical and cell culture approaches, and clinical research.Area of expertise:Molecular Mechanisms of Cardiomyopathy and Heart Failure, Systolic Function, Cardiac Thrombus, Plasminogen-Plasmin System, Experimental Animal Models, Serine Proteases and their Inhibitors for biomedical applications, Drug Targeting and Drug Delivery.The research experience combines animal, biochemical, molecular biology, immunology and cell culture approaches and clinical research.

Courses

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Scholarly Contributions

Books

• Gladysheva, I. P., & Sullivan, R. D. (2022). Diagnosis and Management of Heart Failure. MDPI.

Journals/Publications

• Gladysheva, I. P., Sullivan, R. D., Saleem, S., Castellino, F. J., Ploplis, V. A., & Reed, G. L. (2024). Coagulation factor XII contributes to renin activation, heart failure progression, and mortality. bioRxiv : the preprint server for biology.
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  Symptomatic heart failure (sHF) with cardiac dysfunction, edema, and mortality are driven by overactivation of the renin-angiotensin-aldosterone system (RAAS). Renin is widely recognized as a key initiator of RAAS function, yet the mechanisms that activate renin remain a mystery. We discovered that activated coagulation factor XII generates active renin in the circulation and is directly linked to pathological activation of the systemic RAAS, development of sHF, and increased mortality. These findings suggest a new paradigm for therapeutically modulating the RAAS in sHF and other pathological conditions.
• Gladysheva, I. P., Wang, D., & Reed, G. L. (2024). Corin and Left Atrial Cardiomyopathy, Hypertension, Arrhythmia, and Fibrosis. The New England journal of medicine, 390(16), 1538-1539. doi:10.1056/NEJMc2313870
• Gladysheva, I. P., Sullivan, R. D., & Pellicori, P. (2023). Editorial: Edema in heart failure with reduced ejection fraction. Frontiers in cardiovascular medicine, 10, 1141937.
• Gladysheva, I. P., Sullivan, R. D., & Reed, G. L. (2023). Falling corin and ANP activity levels accelerate development of heart failure and cardiac fibrosis. Frontiers in cardiovascular medicine, 10, 1120487.
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  Available experimental and clinical evidence suggests that in DCM, dysregulation of the biological effects of ANP, at least in part by insufficient corin expression and/or activity, promotes cardiac fibrosis associated with relative cGMP deficiency and contributes to the progression of systolic dysfunction and symptomatic HFrEF. These insights may suggest a new therapeutic paradigm to prevent DCM from becoming a relentless, progressive and fatal form of HFrEF. Preserving or boosting the biological activity of the corin-ANP-cGMP axis by corin targeted interventions may offer potential therapeutic strategies for preventing or blocking progressive cardiac fibrosis in DCM-HFrEF.
• Gladysheva, I. P., Reed, G. L., Hernandez, M., McCune, M. E., & Sullivan, R. D. (2022). Suppression of Cardiogenic Edema with Sodium–Glucose Cotransporter-2 Inhibitors in Heart Failure with Reduced Ejection Fraction: Mechanisms and Insights from Pre-Clinical Studies. Biomedicines. doi:10.3390/biomedicines10082016
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  In heart failure with reduced ejection fraction (HFrEF), cardiogenic edema develops from impaired cardiac function, pathological remodeling, chronic inflammation, endothelial dysfunction, neurohormonal activation, and altered nitric oxide-related pathways. Pre-clinical HFrEF studies have shown that treatment with sodium-glucose cotransporter-2 inhibitors (SGLT-2i) stimulates natriuretic and osmotic/diuretic effects, improves overall cardiac function, attenuates maladaptive cardiac remodeling, and reduces chronic inflammation, oxidative stress, and endothelial dysfunction. Here, we review the mechanisms and effects of SGLT-2i therapy on cardiogenic edema in various models of HFrEF. Overall, the data presented suggest a high translational importance of these studies, and pre-clinical studies show that SGLT-2i therapy has a marked effect on suppressing the progression of HFrEF through multiple mechanisms, including those that affect the development of cardiogenic edema.
• Gladysheva, I. P., Reed, G. L., Ramanathan, K., & Sullivan, R. D. (2022). Soluble (Pro)Renin Receptor Levels Are Regulated by Plasma Renin Activity and Correlated with Edema in Mice and Humans with HFrEF. Biomedicines. doi:10.3390/biomedicines10081874
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  Symptomatic heart failure with reduced ejection fraction (HFrEF) is characterized by edema and chronic pathological activation of the classical renin–angiotensin–aldosterone system (RAAS). The soluble (pro)renin receptor (s(P)RR) is released into circulation by proteolytic cleavage of tissue expressed (P)RR and is a candidate biomarker of RAAS activation. However, previous studies linked elevated levels of s(P)RR in patients with HFrEF to renal dysfunction. Utilizing prospectively enrolled patients with comparable rEF, we show that increased plasma levels of s(P)RR are associated with symptomatic HF (characterized by edema), independent of chronic renal dysfunction. We also found that s(P)RR levels were positively correlated with patient plasma renin activity (PRA). Normotensive mice with dilated cardiomyopathy (DCM) and HFrEF, without renal dysfunction, showed plasma s(P)RR and PRA patterns similar to human HFrEF patients. Plasma s(P)RR levels positively correlated with PRA and systemic edema, but not with EF, resembling findings in patients with HFrEF without chronic kidney dysfunction. In female DCM mice with elevated PRA levels and plasma s(P)RR levels, a randomized, blinded trial comparing the direct renin inhibitor, aliskiren vs. vehicle control, showed that direct renin inhibition normalized PRA, lowered s(P)RR, and prevented symptomatic HFrEF. Considered in light of previous findings, these data suggest that, in HFrEF, in the absence of renal dysfunction, elevation of plasma s(P)RR levels is caused by increased PRA and associated with the development of systemic edema.
• Gladysheva, I. P., Sullivan, R. D., Ramanathan, K., & Reed, G. L. (2022). Soluble (Pro)Renin Receptor Levels Are Regulated by Plasma Renin Activity and Correlated with Edema in Mice and Humans with HFrEF. Biomedicines, 10(8).
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  Symptomatic heart failure with reduced ejection fraction (HFrEF) is characterized by edema and chronic pathological activation of the classical renin-angiotensin-aldosterone system (RAAS). The soluble (pro)renin receptor (s(P)RR) is released into circulation by proteolytic cleavage of tissue expressed (P)RR and is a candidate biomarker of RAAS activation. However, previous studies linked elevated levels of s(P)RR in patients with HFrEF to renal dysfunction. Utilizing prospectively enrolled patients with comparable rEF, we show that increased plasma levels of s(P)RR are associated with symptomatic HF (characterized by edema), independent of chronic renal dysfunction. We also found that s(P)RR levels were positively correlated with patient plasma renin activity (PRA). Normotensive mice with dilated cardiomyopathy (DCM) and HFrEF, without renal dysfunction, showed plasma s(P)RR and PRA patterns similar to human HFrEF patients. Plasma s(P)RR levels positively correlated with PRA and systemic edema, but not with EF, resembling findings in patients with HFrEF without chronic kidney dysfunction. In female DCM mice with elevated PRA levels and plasma s(P)RR levels, a randomized, blinded trial comparing the direct renin inhibitor, aliskiren vs. vehicle control, showed that direct renin inhibition normalized PRA, lowered s(P)RR, and prevented symptomatic HFrEF. Considered in light of previous findings, these data suggest that, in HFrEF, in the absence of renal dysfunction, elevation of plasma s(P)RR levels is caused by increased PRA and associated with the development of systemic edema.
• Hernandez, M., Sullivan, R. D., McCune, M. E., Reed, G. L., & Gladysheva, I. P. (2022). Sodium-Glucose Cotransporter-2 Inhibitors Improve Heart Failure with Reduced Ejection Fraction Outcomes by Reducing Edema and Congestion. Diagnostics (Basel, Switzerland), 12(4).
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  Pathological sodium-water retention or edema/congestion is a primary cause of heart failure (HF) decompensation, clinical symptoms, hospitalization, reduced quality of life, and premature mortality. Sodium-glucose cotransporter-2 inhibitors (SGLT-2i) based therapies reduce hospitalization due to HF, improve functional status, quality, and duration of life in patients with HF with reduced ejection fraction (HFrEF) independently of their glycemic status. The pathophysiologic mechanisms and molecular pathways responsible for the benefits of SGLT-2i in HFrEF remain inconclusive, but SGLT-2i may help HFrEF by normalizing salt-water homeostasis to prevent clinical edema/congestion. In HFrEF, edema and congestion are related to compromised cardiac function. Edema and congestion are further aggravated by renal and pulmonary abnormalities. Treatment of HFrEF patients with SGLT-2i enhances natriuresis/diuresis, improves cardiac function, and reduces natriuretic peptide plasma levels. In this review, we summarize current clinical research studies related to outcomes of SGLT-2i treatment in HFrEF with a specific focus on their contribution to relieving or preventing edema and congestion, slowing HF progression, and decreasing the rate of rehospitalization and cardiovascular mortality.
• Sullivan, R. D., & Gladysheva, I. P. (2022). Advances and Challenges in Diagnosis and Management of Heart Failure. Diagnostics (Basel, Switzerland), 12(5).
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  The prevalence of heart failure (HF) with reduced (r) and preserved (p) ejection fraction (EF) continues to rise globally despite current advances in diagnostics and improvements to medical management [...].
• Sullivan, R. D., McCune, M. E., Hernandez, M., Reed, G. L., & Gladysheva, I. P. (2022). Suppression of Cardiogenic Edema with Sodium-Glucose Cotransporter-2 Inhibitors in Heart Failure with Reduced Ejection Fraction: Mechanisms and Insights from Pre-Clinical Studies. Biomedicines, 10(8).
  More info

  In heart failure with reduced ejection fraction (HFrEF), cardiogenic edema develops from impaired cardiac function, pathological remodeling, chronic inflammation, endothelial dysfunction, neurohormonal activation, and altered nitric oxide-related pathways. Pre-clinical HFrEF studies have shown that treatment with sodium-glucose cotransporter-2 inhibitors (SGLT-2i) stimulates natriuretic and osmotic/diuretic effects, improves overall cardiac function, attenuates maladaptive cardiac remodeling, and reduces chronic inflammation, oxidative stress, and endothelial dysfunction. Here, we review the mechanisms and effects of SGLT-2i therapy on cardiogenic edema in various models of HFrEF. Overall, the data presented suggest a high translational importance of these studies, and pre-clinical studies show that SGLT-2i therapy has a marked effect on suppressing the progression of HFrEF through multiple mechanisms, including those that affect the development of cardiogenic edema.
• Gladysheva, I. P., Sullivan, R. D., & Reed, G. L. (2021). Neprilysin and Corin in HF: Does Combining 2 Biomarkers Double Our Insights?. JACC. Heart failure, 9(5), 406. doi:10.1016/j.jchf.2021.01.016
• Tripathi, R., Sullivan, R. D., Fan, T. M., Mehta, R. M., Gladysheva, I. P., & Reed, G. L. (2021). A Low-Sodium Diet Boosts Ang (1-7) Production and NO-cGMP Bioavailability to Reduce Edema and Enhance Survival in Experimental Heart Failure. International journal of molecular sciences, 22(8). doi:10.3390/ijms22084035
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  Sodium restriction is often recommended in heart failure (HF) to block symptomatic edema, despite limited evidence for benefit. However, a low-sodium diet (LSD) activates the classical renin-angiotensin-aldosterone system (RAAS), which may adversely affect HF progression and mortality in patients with dilated cardiomyopathy (DCM). We performed a randomized, blinded pre-clinical trial to compare the effects of a normal (human-equivalent) sodium diet and a LSD on HF progression in a normotensive model of DCM in mice that has translational relevance to human HF. The LSD reduced HF progression by suppressing the development of pleural effusions ( < 0.01), blocking pathological increases in systemic extracellular water ( < 0.001) and prolonging median survival (15%, < 0.01). The LSD activated the classical RAAS by increasing plasma renin activity, angiotensin II and aldosterone levels. However, the LSD also significantly up-elevated the counter-regulatory RAAS by boosting plasma angiotensin converting enzyme 2 (ACE2) and angiotensin (1-7) levels, promoting nitric oxide bioavailability and stimulating 3'-5'-cyclic guanosine monophosphate (cGMP) production. Plasma HF biomarkers associated with poor outcomes, such as B-type natriuretic peptide and neprilysin were decreased by a LSD. Cardiac systolic function, blood pressure and renal function were not affected. Although a LSD activates the classical RAAS system, we conclude that the LSD delayed HF progression and mortality in experimental DCM, in part through protective stimulation of the counter-regulatory RAAS to increase plasma ACE2 and angiotensin (1-7) levels, nitric oxide bioavailability and cGMP production.
• Sullivan, R. D., Houng, A. K., Gladysheva, I. P., Fan, T. M., Tripathi, R., Reed, G. L., & Wang, D. (2020). Corin Overexpression Reduces Myocardial Infarct Size and Modulates Cardiomyocyte Apoptotic Cell Death. International journal of molecular sciences, 21(10). doi:10.3390/ijms21103456
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  Altered expression of corin, a cardiac transmembrane serine protease, has been linked to dilated and ischemic cardiomyopathy. However, the potential role of corin in myocardial infarction (MI) is lacking. This study examined the outcomes of MI in wild-type vs. cardiac-specific overexpressed corin transgenic (Corin-Tg) mice during pre-MI, early phase (3, 24, 72 h), and late phase (1, 4 weeks) post-MI. Corin overexpression significantly reduced cardiac cell apoptosis ( < 0.001), infarct size ( < 0.001), and inhibited cleavage of procaspases 3, 9, and 8 ( < 0.05 to < 0.01), as well as altered the expression of Bcl2 family proteins, Bcl-xl, Bcl2 and Bak ( < 0.05 to < 0.001) at 24 h post-MI. Overexpressed cardiac corin also significantly modulated heart function (ejection fraction, < 0.0001), lung congestion (lung weight to body weight ratio, < 0.0001), and systemic extracellular water (edema, < 0.05) during late phase post-MI. Overall, cardiac corin overexpression significantly reduced apoptosis, infarct size, and modulated cardiac expression of key members of the apoptotic pathway in early phase post-MI; and led to significant improvement in heart function and reduced congestion in late phase post-MI. These findings suggest that corin may be a useful target to protect the heart from ischemic injury and subsequent post-infarction remodeling.
• Tripathi, R., Sullivan, R. D., Fan, T. M., Houng, A. K., Mehta, R. M., Reed, G. L., & Gladysheva, I. P. (2020). Cardiac-Specific Overexpression of Catalytically Inactive Corin Reduces Edema, Contractile Dysfunction, and Death in Mice with Dilated Cardiomyopathy. International journal of molecular sciences, 21(1), 203. doi:doi.org/10.3390/ijms21010203
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  Humans with dilated cardiomyopathy (DCM) and heart failure (HF) develop low levels of corin, a multi-domain, cardiac-selective serine protease involved in natriuretic peptide cleavage and sodium and water regulation. However, experimental restoration of corin levels markedly attenuates HF progression. To determine whether the beneficial effects of corin in HF require catalytic activity, we engineered cardiac overexpression of an enzymatically inactive corin transgene (corin-Tg(i)). On a wild-type (WT) background, corin-Tg(i) had no evident phenotypic effects. However, in a well-established genetic model of DCM, corin-Tg(i)/DCM mice had increased survival ( < 0.01 to 0.001) vs. littermate corin-WT/DCM controls. Pleural effusion ( < 0.01), lung edema ( < 0.05), systemic extracellular free water ( < 0.01), and heart weight were decreased ( < 0.01) in corin-Tg(i)/DCM vs. corin-WT/DCM mice. Cardiac ejection fraction and fractional shortening improved ( < 0.01), while ventricular dilation decreased ( < 0.0001) in corin-Tg(i)/DCM mice. Plasma atrial natriuretic peptide, cyclic guanosine monophosphate, and neprilysin were significantly decreased. Cardiac phosphorylated glycogen synthase kinase-3β (pSer9-GSK3β) levels were increased in corin(i)-Tg/DCM mice ( < 0.01). In summary, catalytically inactive corin-Tg(i) decreased fluid retention, improved contractile function, decreased HF biomarkers, and diminished cardiac GSK3β activity. Thus, the protective effects of cardiac corin on HF progression and survival in experimental DCM do not require the serine protease activity of the molecule.
• Tripathi, R., Sullivan, R. D., Fan, T. M., Mehta, R. M., Gladysheva, I. P., & Reed, G. L. (2020). In Experimental Dilated Cardiomyopathy Heart Failure and Survival Are Adversely Affected by a Lack of Sexual Interactions. International journal of molecular sciences, 21(15). doi:10.3390/ijms21155450
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  Nearly one in three people in the U.S. will develop heart failure (HF), characterized by fluid retention (edema) in the lungs and elsewhere. This leads to difficult breathing, deterioration of physical capacity, restriction of normal activities and death. There is little data about the safety and effects of sexual interactions in patients with HF. We tested whether a lack of sexual interactions affected pathophysiological outcomes in a pre-clinical mouse model of dilated cardiomyopathy that recapitulates the progressive stages of human HF. Male mice were randomly given access to, or deprived from, sexual interactions with female mice, which were confirmed by videography and generation of offspring. Cohousing with access to sexual interactions markedly prolonged survival, while cohousing without access to sexual activity did not. Sexual interactions improved systolic function, reduced HF-associated edema, altered transcription of heart contractile protein genes and decreased plasma testosterone levels. To determine whether testosterone levels contributed to survival, testosterone levels were experimentally reduced. Reduction of testosterone levels significantly prolonged survival. Taken together, in mice with dilated cardiomyopathy, sexual activity altered cardiac contractile gene transcription, improved systolic function, reduced edema and prolonged survival which may be in part due to lower testosterone levels.
• Sullivan, R. D., Mehta, R. M., Tripathi, R., Gladysheva, I. P., & Reed, G. L. (2019). Normalizing Plasma Renin Activity in Experimental Dilated Cardiomyopathy: Effects on Edema, Cachexia, and Survival. International journal of molecular sciences, 20(16). doi:10.3390/ijms20163886
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  Heart failure (HF) patients frequently have elevated plasma renin activity. We examined the significance of elevated plasma renin activity in a translationally-relevant model of dilated cardiomyopathy (DCM), which replicates the progressive stages (A-D) of human HF. Female mice with DCM and elevated plasma renin activity concentrations were treated with a direct renin inhibitor (aliskiren) in a randomized, blinded fashion beginning at Stage B HF. By comparison to controls, aliskiren treatment normalized pathologically elevated plasma renin activity ( < 0.001) and neprilysin levels ( < 0.001), but did not significantly alter pathological changes in plasma aldosterone, angiotensin II, atrial natriuretic peptide, or corin levels. Aliskiren improved cardiac systolic function (ejection fraction, < 0.05; cardiac output, < 0.01) and significantly reduced the longitudinal development of edema (extracellular water, < 0.0001), retarding the transition from Stage B to Stage C HF. The normalization of elevated plasma renin activity reduced the loss of body fat and lean mass (cachexia/sarcopenia), < 0.001) and prolonged survival ( < 0.05). In summary, the normalization of plasma renin activity retards the progression of experimental HF by improving cardiac systolic function, reducing the development of systemic edema, cachexia/sarcopenia, and mortality. These data suggest that targeting pathologically elevated plasma renin activity may be beneficial in appropriately selected HF patients.
• Sullivan, R. D., Mehta, R. M., Tripathi, R., Reed, G. L., & Gladysheva, I. P. (2019). Renin Activity in Heart Failure with Reduced Systolic Function-New Insights. International journal of molecular sciences, 20(13). doi:10.3390/ijms20133182
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  Regardless of the cause, symptomatic heart failure (HF) with reduced ejection fraction (rEF) is characterized by pathological activation of the renin-angiotensin-aldosterone system (RAAS) with sodium retention and extracellular fluid expansion (edema). Here, we review the role of active renin, a crucial, upstream enzymatic regulator of the RAAS, as a prognostic and diagnostic plasma biomarker of heart failure with reduced ejection fraction (HFrEF) progression; we also discuss its potential as a pharmacological bio-target in HF therapy. Clinical and experimental studies indicate that plasma renin activity is elevated with symptomatic HFrEF with edema in patients, as well as in companion animals and experimental models of HF. Plasma renin activity levels are also reported to be elevated in patients and animals with rEF before the development of symptomatic HF. Modulation of renin activity in experimental HF significantly reduces edema formation and the progression of systolic dysfunction and improves survival. Thus, specific assessment and targeting of elevated renin activity may enhance diagnostic and therapeutic precision to improve outcomes in appropriate patients with HFrEF.
• Gladysheva, I. P., Zaidi, S. S., Yu, X., Ward, R. D., Reed, G. L., Ramanathan, K., & Gladysheva, I. P. (2018). Possible Enzymatic Downregulation of the Natriuretic Peptide System in Patients with Reduced Systolic Function and Heart Failure: A Pilot Study.. BioMed research international, 2018, 7279036. doi:10.1155/2018/7279036
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  In patients with reduced systolic function, the natriuretic peptide system affects heart failure (HF) progression, but the expression of key activating (corin) and degrading enzymes (neprilysin) is not well understood..This pilot study (n=48) compared plasma levels of corin, neprilysin, ANP, BNP, and cGMP in control patients with normal ejection fractions (mean EF 63 ± 3%) versus patients with systolic dysfunction, with (EF 24 ± 8%) and without (EF 27 ± 7%) decompensated HF (dHF), as defined by Framingham and BNP criteria. Mean ages, use of beta blockers, and ACE-inhibitors-angiotensin receptor blockers were similar between the groups. Corin levels were depressed in systolic dysfunction patients (797 ± 346 pg/ml) versus controls (1188 ± 549, p
• Wang, D., Gladysheva, I. P., Sullivan, R. D., Fan, T. M., Mehta, R. M., Tripathi, R., Sun, Y., & Reed, G. L. (2018). Increases in plasma corin levels following experimental myocardial infarction reflect the severity of ischemic injury. PloS one, 13(9), e0202571. doi:10.1371/journal.pone.0202571
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  Following acute myocardial infarction, clinical studies show alterations in the blood levels of corin, a cardiac-selective activator of the natriuretic peptides pro-atrial natriuretic peptide (pro-ANP) and pro-B-type natriuretic peptide (pro-BNP). However, the temporal changes in circulating and cardiac corin levels and their relationships to the severity of myocardial infarction have not been studied. The main objective of this study was to examine the relationship between cardiac and circulating corin levels and their association with cardiac systolic function and infarct size during the early phase of acute myocardial infarction (
• Zaidi, S. S., Ward, R. D., Ramanathan, K., Yu, X., Gladysheva, I. P., & Reed, G. L. (2018). Possible Enzymatic Downregulation of the Natriuretic Peptide System in Patients with Reduced Systolic Function and Heart Failure: A Pilot Study. BioMed research international, 2018, Article ID 7279036. doi:10.1155/2018/7279036
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  In patients with reduced systolic function, the natriuretic peptide system affects heart failure (HF) progression, but the expression of key activating (corin) and degrading enzymes (neprilysin) is not well understood.
• Tripathi, R., Sullivan, R., Fan, T. M., Wang, D., Sun, Y., Reed, G. L., & Gladysheva, I. P. (2017). Enhanced heart failure, mortality and renin activation in female mice with experimental dilated cardiomyopathy. PloS one, 12(12), e0189315. doi:10.1371/journal.pone.0189315
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  Dilated cardiomyopathy (DCM) is the major cause of heart failure affecting both women and men. Limited clinical studies show conflicting data in sex-related differences in the progression of dilated cardiomyopathy and heart failure (HF) outcomes. We examined the comparative sex-related progression of cardiomyopathy and the development of HF (at 4, 7, 13 weeks of age) in a well-established, transgenic mouse model of DCM that recapitulates the progressive stages of human HF. By 13 weeks of age, female mice with DCM had more severe left ventricular systolic dysfunction, left ventricular dilation and wall thinning (P
• Wang, D., Gladysheva, I. P., Wang, D., Tripathi, R., Sun, Y., Sullivan, R. D., Reed, G. L., Mehta, R. M., Gladysheva, I. P., & Fan, T. M. (2017). Abstract P349: Plasma Corin Levels Reflect Dynamic Changes in Cardiac Expression Induced by Experimental Acute Myocardial Infarction. Hypertension, 70, AP349.
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  Introduction: Corin is a cardiac membrane protease that activates pro-ANP and pro-BNP. Changes in circulating corin levels have been linked to poor clinical outcomes following acute myocardial infa...
• Tripathi, R., Sullivan, R., Fan, M., Houng, N., Gladysheva, I. P., & Reed, G. L. (2016). Sodium Restriction Activates the Renin-angiotensin-aldosterone System but Fails to Improve Heart Failure or Survival in Experimental Dilated Cardiomyopathy. CIRCULATION, 134.
• Tripathi, R., Wang, D., Sullivan, R., Fan, T. H., Gladysheva, I. P., & Reed, G. L. (2016). Depressed Corin Levels Indicate Early Systolic Dysfunction Before Increases of Atrial Natriuretic Peptide/B-Type Natriuretic Peptide and Heart Failure Development. Hypertension (Dallas, Tex. : 1979), 67(2), 362-7. doi:10.1161/HYPERTENSIONAHA.115.06300
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  Dilated cardiomyopathy is a major cause of heart failure (HF) that affects millions. Corin cleaves and biologically activates pro-atrial natriuretic peptide (pro-ANP) and pro-B-type natriuretic peptide (pro-BNP). High corin levels reduce the development of systolic dysfunction and HF in experimental dilated cardiomyopathy. Yet, patients with significant HF unexpectedly show low corin levels with high plasma ANP/BNP levels. Therefore, we examined the relationship between cardiac corin expression, ANP/BNP levels, and the stages of HF. We used a well-established, dilated cardiomyopathy model to evaluate gene and protein expression as mice longitudinally developed Stages A-D HF. Cardiac systolic function (ejection fraction) continuously declined over time (P
• Wang, D., Sullivan, R. D., Gladysheva, I. P., Houng, A., Tripathi, R., Fan, T. M., & Reed, G. L. (2015). Corin Reduces Myocardial Infarction and Protects Against Cardiomyocyte Apoptosis. CIRCULATION, 132.
• Wang, D., Gladysheva, I. P., Fan, T. H., Sullivan, R., Houng, A. K., & Reed, G. L. (2014). Atrial natriuretic peptide affects cardiac remodeling, function, heart failure, and survival in a mouse model of dilated cardiomyopathy. Hypertension (Dallas, Tex. : 1979), 63(3), 514-9.
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  Dilated cardiomyopathy is a frequent cause of heart failure and death. Atrial natriuretic peptide (ANP) is a biomarker of dilated cardiomyopathy, but there is controversy whether ANP modulates the development of heart failure. Therefore, we examined whether ANP affects heart failure, cardiac remodeling, function, and survival in a well-characterized, transgenic model of dilated cardiomyopathy. Mice with dilated cardiomyopathy with normal ANP levels survived longer than mice with partial ANP (P
• Gladysheva, I. P., Wang, D., McNamee, R. A., Houng, A. K., Mohamad, A. A., Fan, T. M., & Reed, G. L. (2013). Corin overexpression improves cardiac function, heart failure, and survival in mice with dilated cardiomyopathy. (Subject of a featured editorial). Hypertension (Dallas, Tex. : 1979), 61(2), 327-32.
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  Heart failure, caused by dilated cardiomyopathy and other cardiac disorders such as hypertension, is a major public health problem with high morbidity and mortality. Corin, a cardiac enzyme that cleaves natriuretic peptides, is a promising biomarker of cardiomyopathy and heart failure, but its functional role in these processes is not understood. We evaluated the potential effects of corin in mice with a well-characterized model of dilated cardiomyopathy. Mice with dilated cardiomyopathy developed heart failure, reduced contractile function, cardiac fibrosis, and accelerated mortality in the setting of low corin expression. In wild-type mice, transgenic, cardiac-targeted, overexpression of corin enhanced cyclic guanosine monophosphate and blood pressure responses to pro-atrial natriuretic peptide, but did not affect heart size, contractility, body weights, survival, and blood pressure. In mice with dilated cardiomyopathy, corin overexpression significantly reduced the development of myocardial fibrosis (P
• Wang, D., Gladysheva, I. P., Fan, T. M., David, S. R., & Reed, G. L. (2013). More Than a Biomarker_ANP Modulates Heart Failure, Cardiac Function and Survival in Dilated Cardiomyopathy. CIRCULATION, 128(22).
• Ward, R. D., Zaidi, S. S., Ramanathan, K., Yu, X., Gladysheva, I. P., & Reed, G. L. (2013). Corin Levels Are Linked to Systolic Function and Serum Sodium. JOURNAL OF CARDIAC FAILURE, 19(8), S12-S12.
• Sazonova, I., Ibebuogu, U., Gladysheva, I., Kadle, N., Sharma, G., Figueroa, R., & Robinson, V. (2012). The role of plasma D-dimer testing in diagnosis of patients suspected to atrial source thrombi. THROMBOSIS RESEARCH, 130, S112-S112.
• Zhang, Y., Gladysheva, I. P., Houng, A. K., & Reed, G. L. (2012). Streptococcus uberis plasminogen activator (SUPA) activates human plasminogen through novel species-specific and fibrin-targeted mechanisms. The Journal of biological chemistry, 287(23), 19171-6.
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  Bacterial plasminogen (Pg) activators generate plasmin to degrade fibrin blood clots and other proteins that modulate the pathogenesis of infection, yet despite strong homology between mammalian Pgs, the activity of bacterial Pg activators is thought to be restricted to the Pg of their host mammalian species. Thus, we found that Streptococcus uberis Pg activator (SUPA), isolated from a Streptococcus species that infects cows but not humans, robustly activated bovine but not human Pg in purified systems and in plasma. Consistent with this, SUPA formed a higher avidity complex (118-fold) with bovine Pg than with human Pg and non-proteolytically activated bovine but not human Pg. Surprisingly, however, the presence of human fibrin overrides the species-restricted action of SUPA. First, human fibrin enhanced the binding avidity of SUPA for human Pg by 4-8-fold in the presence and absence of chloride ion (a negative regulator). Second, although SUPA did not protect plasmin from inactivation by α(2)-antiplasmin, fibrin did protect human plasmin, which formed a 31-fold higher avidity complex with SUPA than Pg. Third, fibrin significantly enhanced Pg activation by reducing the K(m) (4-fold) and improving the catalytic efficiency of the SUPA complex (6-fold). Taken together, these data suggest that indirect molecular interactions may override the species-restricted activity of bacterial Pg activators; this may affect the pathogenesis of infections or may be exploited to facilitate the design of new blood clot-dissolving drugs.
• Ibebuogu, U. N., Gladysheva, I. P., Houng, A. K., & Reed, G. L. (2011). Decompensated heart failure is associated with reduced corin levels and decreased cleavage of pro-atrial natriuretic peptide. (Subject of a featured editorial). Circulation. Heart failure, 4(2), 114-20. doi:10.1161/CIRCHEARTFAILURE.109.895581
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  By promoting salt and water excretion, the corin and the atrial natriuretic peptide (ANP) system should help to maintain fluid balance in heart failure. Yet, the development of fluid retention despite high levels of ANP-related peptides suggests that this compensatory system is limited.
• Ibebuogu, U. N., Gladysheva, I. P., & Reed, G. L. (2009). Is Heart Failure Due to Impaired Cleavage and Activation of Atrial Natriuretic Peptide?. JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, 53(10), A467-A468.
• Zamolodchikova, T. S., Popykina, N. A., Gladysheva, I. P., & Larionova, N. I. (2009). Effect of reactive center loop structure of antichymotrypsin on inhibition of duodenase activity. Biochemistry. Biokhimiia, 74(8), 824-33.
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  Interaction between duodenase (a granase family member) from bovine duodenal mucosa and recombinant antichymotrypsin (rACT) and its P1 variants has been studied. Association rate constants (k(a)) were 11, 6.8, and 17 mM(-1).sec(-1) for rACT, ACT L358M, and ACT L358R, respectively. Natural antitrypsin (AT) compared to ACT was a 20 times more effective duodenase inhibitor (in terms of k(a)). Duodenase interacted with P1 variants of ACT via a suicide mechanism with stoichiometry of the process SI = 1.2. The nature of the P1 residue of the inhibitor did not influence the interaction if other residues did not meet conformational requirements of the duodenase substrate-binding pocket. Also, interaction of duodenase with ACT variants containing residues from AT reaction center loop (rACT P2-P3', rACT P3-P4', rACT P4-P3', and rACT P6-P4') was studied. The inhibition type ([E](0) = 1.10(-7) M, 25 degrees C) was revealed to be reversible-like, and efficacy of inhibition decreased with increase in the substituted part of the reactive center loop. Constants of inhibition (K(i)) were measured. Efficacy of interaction between the enzyme (duodenase) and inhibitor depends on topochemical correspondence between a substrate-binding pocket of the enzyme and substrate structure.
• Gladysheva, I. P., King, S. M., & Houng, A. K. (2008). N-glycosylation modulates the cell-surface expression and catalytic activity of corin. Biochemical and biophysical research communications, 373(1), 130-5.
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  N-glycosylation may influence the subcellular localization and biological activity of the pro-ANP convertase, corin. In HEK293-corin cells, the inhibition of N-glycosylation, with tunicamycin, reduced the cell-surface expression of murine corin, but did not alter the total expression. Therefore, tunicamycin treatment likely caused the intracellular accumulation of non-glycosylated corin. Tunicamycin treatment also significantly reduced corin activity (pro-ANP cleavage) in these cells. We developed an assay to measure the effect of N-glycosylation on corin activity, independent of its effect on corin localization. We determined that the reduction in corin activity was due to a direct effect of N-glycosylation, and was not secondary to the effect of N-glycosylation on corin cell-surface expression. Our data provide evidence that N-glycosylation is essential for the cell-surface expression of murine corin and modulates its functional activity. N-Glycosylation represents a possible mechanism for the regulation of native corin on the surface of cardiomyocytes.
• Gladysheva, I. P., Robinson, B. R., Houng, A. K., Kováts, T., & King, S. M. (2008). Corin is co-expressed with pro-ANP and localized on the cardiomyocyte surface in both zymogen and catalytically active forms. Journal of molecular and cellular cardiology, 44(1), 131-42.
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  The multi-domain transmembrane serine protease corin cleaves pro-atrial natriuretic peptide (pro-ANP) in vitro to generate an active hormone, ANP. Corin may also contribute to the regulation of the natriuretic peptide system in vivo, and might be an attractive target for treatment of cardiovascular diseases. In order for corin to cleave its substrate pro-ANP, it should be catalytically active and located proximally. However, because knowledge of native corin is limited, we examined the expression, cardiac localization and molecular forms of the native corin protein. Immunofluorescence studies using a series of anti-corin antibodies directed against the stem and protease domains reveal that corin is present on the cell-surface of rat neonatal cardiomyocytes and murine HL-1 cardiomyocyte-like cells. Furthermore, we immunolocalized native corin in pro-ANP expressing cardiomyocytes. Immunoprecipitation of the membrane fraction of mouse heart extract showed that native corin had a relative mass of 205-210 kDa. Under reducing conditions native corin migrates as several different molecular weight forms corresponding to zymogen (uncleaved) and active (cleaved) forms. Studies using a FITC-tagged chloromethyl ketone that mimics the corin cleavage sequence in pro-ANP, suggest that an enzymatically active form of corin is localized to the cell surface of myocardial cells in vivo. Additionally, we showed that the 205-210 kDa form of corin is a glycosylated protein. Treatment of HL-1 cells with tunicamycin reduced the relative mass of expressed corin. We conclude that native corin is a glycosylated protease that is localized on the cell surface of pro-ANP-expressing cardiomyocytes in both zymogen and catalytically active forms.
• Gladysheva, I. P., Sazonova, I. Y., Houng, A., Hedstrom, L., & Reed, G. L. (2007). Regulation of nonproteolytic active site formation in plasminogen. Biochemistry, 46(30), 8879-87.
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  Streptokinase may be less effective at saving lives in patients with heart attacks because it explosively generates plasmin in the bloodstream at sites distant from fibrin clots. We hypothesized that this rapid plasmin generation is due to SK's singular capacity to nonproteolytically generate the active protease SK x Pg*, and we examined whether the kringle domains regulate this process. An SK mutant lacking Ile-1 (deltaIle1-SK) does not form SK x Pg*, although it will form complexes with plasmin that can activate plasminogen. When compared to SK, deltaIle1-SK diminished the generation of plasmin in plasma by more than 30-fold, demonstrating that the formation of SK x Pg* plays an important role in SK activity in the blood. The rate of SK x Pg* formation (measured by an active site titrant) was much slower in Glu-Pg, which contains five kringle domains, than in Pg forms containing one kringle (mini-Pg) or no kringles (micro-Pg). In a similar manner, Streptococcus uberis Pg activator (SUPA), an SK-like molecule, generated SUPA x Pg* much slower with bovine Pg than bovine micro-Pg. The velocity of SK x Pg* formation was regulated by agents that influence the conformation of Pg through interactions with the kringle domains. Chloride ions, which maintain the compact Pg conformation, hindered SK x Pg* formation. In contrast, epsilon-aminocaproic acid, fibrin, and fibrinogen, which induce an extended Pg conformation, accelerated the formation of SK x Pg*. In summary, the explosive generation of plasmin in blood or plasma, which diminishes SK's therapeutic effects, is attributable to the formation of SK x Pg*, and this process is governed by kringle domains.
• Sazonova, I. Y., Thomas, B. M., Gladysheva, I. P., Houng, A. K., & Reed, G. L. (2007). Fibrinolysis is amplified by converting alpha-antiplasmin from a plasmin inhibitor to a substrate. Journal of thrombosis and haemostasis : JTH, 5(10), 2087-94.
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  alpha(2)-Antiplasmin (alpha(2)-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against alpha(2)-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of alpha(2)-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to alpha(2)-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured alpha(2)-AP. Binding studies with chimeric alpha(2)-APs revealed that none of the mAbs bound to sites in alpha(2)-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of alpha(2)-AP, or the reactive center loop in the serpin domain of alpha(2)-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of alpha(2)-AP (L(13)GNQEPGGQTALKSPPGVCS(32)) near the site at which alpha(2)-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of alpha(2)-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by alpha(2)-AP (from 1.1 +/- 0.1 to 51 +/- 4 and 67 +/- 7) indicating that they convert alpha(2)-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of alpha(2)-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known alpha(2)-AP-plasmin contacts to enhance fibrinolysis by converting alpha(2)-AP from an inhibitor to a plasmin substrate.
• Sazonova, I. Y., Robinson, B. R., Gladysheva, I. P., Castellino, F. J., & Reed, G. L. (2004). alpha Domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator. The Journal of biological chemistry, 279(24), 24994-5001.
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  The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK alpha domain and converts SKDelta59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK alpha domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SKDelta59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SKDelta59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SKDelta59 does not protect plasmin from inhibition by alpha(2)-antiplasmin. At the same time, SKDelta59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SKDelta59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl(-)-deficient buffers. Taken together, these studies indicate that an intact alpha domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact alpha domain makes SKDelta59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by alpha(2)-antiplasmin.
• Gladysheva, I. P., Turner, R. B., Sazonova, I. Y., Liu, L., & Reed, G. L. (2003). Coevolutionary patterns in plasminogen activation. Proceedings of the National Academy of Sciences of the United States of America, 100(16), 9168-72.
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  The generation of plasmin by plasminogen (Pg) activators (PAs) is a physiologic process in animals that dissolves blood clots and promotes wound healing, blood vessel growth, and the migration of normal and cancerous cells. Pathogenic bacteria have evolved PAs [e.g., streptokinase (SK) and staphylokinase] that exploit the Pg system to infect animals. Animal PAs have a conserved ability to cleave a wide spectrum of animal Pgs, but the ability of bacterial PAs to cleave different animal Pgs is surprisingly restricted. We show that the spectrum of activity of an archetypal bacterial PA (SK) with animal Pgs can be profoundly altered by mutations that affect intermolecular complementarity at sites that participate in complex formation or substrate binding. Comparative sequence analysis of animal plasmins vs. close structural homologues (trypsin and chymotrypsin) that are not molecular targets for invading bacteria indicates that the sites in plasmin that interact with SK are preferentially targeted for mutation. Conversely, intermolecular contact sites in SKs that activate human Pg are more highly conserved than other loci in the molecule or than the same sites in other SKs that activate non-human Pgs. We propose that active modulation of intermolecular complementarity at sites of contact between SK and Pg may represent a competitive evolutionary strategy in a survival battle, whereby animals seek to evade bacterial invasion, and bacteria endeavor to invade their animal hosts.
• Popykina, N. A., Gladysheva, I. P., Zamolodchikova, T. S., & Larionova, N. I. (2003). [Interaction of duodenase with human blood serpins].. Bioorganicheskaia khimiia, 29(6), 605-10. doi:10.1023/b:rubi.0000008895.66892.9a
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  The interaction of duodenase, a new serine protease from a small group of Janus-faced proteases, with serpins, alpha 1-protease inhibitor (alpha 1-PI) and antichymotrypsin (ACT) from human blood serum, was studied. The stoichiometry of the inhibition process was found to be 1.2 and 1.3 mol/mol for alpha 1-PI and ACT, respectively. The presence of a stable enzyme-inhibitory complex duodenase-alpha 1-PI was confirmed by SDS-PAGE. No formation of the duodenase-ACT complex was demonstrated; instead, the band of the cleaved inhibitor was indicated upon the ACT hydrolysis. The suicide mechanism of the duodenase interaction with the human blood serpins was proved. The association rate constants (Ka, M-1 s-1) were 2.4 +/- 0.3 x 10(5) for alpha 1-PI and 3.0 +/- 0.4 x 10(5) for ACT. These results indicate the possibility of the regulation of duodenase activity by endogenous serpins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.
• Gladysheva, I. P., Popykina, N. A., Zamolodchikova, T. S., & Larionova, N. I. (2002). Study on interaction between duodenase protease with dual specificity and inhibitors of bowman-birk family. Protein and peptide letters, 9(2), 139-44.
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  The interaction between duodenase and inhibitors of Bowman-Birk type from soybeans (BBI) and lima beans (LBI) was investigated. Duodenase was shown to interact only with antichymotrypsin site of these inhibitors. The inhibition constants of duodenase by BBI, LBI, BBI-trypsin and LBI trypsin complexes were 4, 23, 400, 600 (n)M respectively.
• Gladysheva, I. P., Sazonova, I. Y., Chowdhry, S. A., Liu, L., Turner, R. B., & Reed, G. L. (2002). Chimerism reveals a role for the streptokinase Beta -domain in nonproteolytic active site formation, substrate, and inhibitor interactions. The Journal of biological chemistry, 277(30), 26846-51.
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  Streptokinase (SK) and staphylokinase form cofactor-enzyme complexes that promote the degradation of fibrin thrombi by activating human plasminogen. The unique abilities of streptokinase to nonproteolytically activate plasminogen or to alter the interactions of plasmin with substrates and inhibitors may be the result of high affinity binding mediated by the streptokinase beta-domain. To examine this hypothesis, a chimeric streptokinase, SKbetaswap, was created by swapping the SK beta-domain with the homologous beta-domain of Streptococcus uberis Pg activator (SUPA or PauA, SK uberis), a streptokinase that cannot activate human plasminogen. SKbetaswap formed a tight complex with microplasminogen with an affinity comparable with streptokinase. The SKbetaswap-plasmin complex also activated human plasminogen with catalytic efficiencies (k(cat)/K(m) = 16.8 versus 15.2 microm(-1) min(-1)) comparable with streptokinase. However, SKbetaswap was incapable of nonproteolytic active site generation and activated plasminogen by a staphylokinase mechanism. When compared with streptokinase complexes, SKbetaswap-plasmin and SKbetaswap-microplasmin complexes had altered affinities for low molecular weight substrates. The SKbetaswap-plasmin complex also was less resistant than the streptokinase-plasmin complex to inhibition by alpha(2)-antiplasmin and was readily inhibited by soybean trypsin inhibitor. Thus, in addition to mediating high affinity binding to plasmin(ogen), the streptokinase beta-domain is required for nonproteolytic active site generation and specifically modulates the interactions of the complex with substrates and inhibitors.
• Gladysheva, I. P., Moroz, N. A., Karmakova, T. A., Nemtsova, E. R., Yakubovskaya, R. I., & Larionova, N. I. (2001). Immunoconjugates of soybean Bowman-Birk protease inhibitor as targeted antitumor polymeric agents. Journal of drug targeting, 9(5), 303-16.
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  To enhance the antitumor potential of soybean Bowman-Birk inhibitor (BBI), the conjugate of BBI with an antibody via a macromolecular carrier was prepared. Clinical dextran (D) was used as a biocompatible biodegradable carrier for co-immobilization of BBI and antibody. A model immunoglobulin isolated from sheep serum (sIgG), raised against human IgM was utilized to develop the procedure of immunoconjugate synthesis. The molar ratio of the ingredients in the conjugate was the following BBI:D:sIgG=9:1:1. Comparison of the dose response curves for the native sIgG and the BBI-D-sIgG conjugate indicated that sIgG completely retained its specific activity (>90%) after modification with dextran. The determination of the Ki values for chymotrypsin interaction with the native BBI and the BBI-D-sIgG conjugate indicated high anti-chymotrypsin activity. In the next step, the monoclonal antibody (ICO 25 MAb) against the mucin-like human epithelial membrane antigen was used for conjugation as it is the most universal vector for targeting different agents to human tumors of epithelial origin. The influence of conjugation on the specificity of the Mab reaction with its antigen was studied. The conjugated MAb reacted with tumor cells of different epithelial genesis (breast, lung, gastric, ovarian and uterus tumors), but did not react with tumor cells of non-epithelial origin. It was shown that BBI-D-ICO 25 MAb conjugate has almost the same immunohistochemical activity as non-conjugated MAb. These results demonstrated the feasibility of exploiting the activities of covalently bound BBI and ICO 25 MAb for anticarcinogenic agent targeting.
• Gladysheva, I. P., Moroz, N. A., Papisova, A. I., & Larionova, N. I. (2001). Soybean bowman--birk inhibitor conjugates with clinical dextran. synthesis and antiproteolytic activity. Biochemistry. Biokhimiia, 66(4), 384-9.
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  Conjugates of the classical soybean Bowman-Birk inhibitor (BBI) with clinical dextran were synthesized. Clinical dextran was preliminarily oxidized with periodate to dialdehydedextran (DAD). The effect of the degree of oxidation of DAD on coupling of the inhibitor was evaluated. The binding of the protein was shown to increase with increasing degree of DAD oxidation (5, 10, 20%). Total coupling of the inhibitor occurred when the degree of oxidation of the dextran was 20%. The BBI-DAD (20%) conjugate contained 13% protein with BBI/DAD molar ratio 1 : 1. The conjugates retained the ability to inhibit trypsin (Ki = 0.2-0.3 nM) and alpha-chymotrypsin (Ki = 15-30 nM). Thus, the coupling of BBI with the polymeric carrier caused practically no decrease in the antiproteolytic activity of the inhibitor.
• Gladysheva, I. P., Polekhina, O. V., Karmakova, T. A., Nemtsova, E. R., Yakubovskaya, R. I., Shen, W. C., Kennedy, A. R., & Larionova, N. I. (2001). Potential of block copolymer- and immuno-conjugates for tumor-targeted delivery of Bowman-Birk soybean proteinase inhibitor. Journal of controlled release : official journal of the Controlled Release Society, 74(1-3), 303-8.
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  The present work reports the effect of conjugation of the anticarcinogenic and antitumor soybean Bowman-Birk protease inhibitor (BBI) with amphiphilic block copolymer of ethylene oxide and propylene oxide (PEO-PPO) as well as with monoclonal antibody via clinical dextran (D) on tumor-targeted delivery of BBI.
• Gladysheva, I. P., Popykina, N. A., Zamolodchikova, T. S., & Larionova, N. I. (2001). Interaction between duodenase and alpha1-proteinase inhibitor. Biochemistry. Biokhimiia, 66(6), 682-7.
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  The interaction between duodenase, a newly recognized serine proteinase belonging to the small group of Janus-faced proteinases, and alpha1-proteinase inhibitor (alpha1-PI) from human serum was investigated. The stoichiometry of the inhibition was 1.2 mol/mol. The presence of a stable enzyme-inhibitor complex was shown by SDS-PAGE. The mechanism of interaction between duodenase and alpha1-PI was shown to be of the suicide type. The equilibrium and inhibition constants are 13 +/- 3 nM and (1.9 +/- 0.3).105 M-1.sec-1, respectively. Based on the association rate constant of the enzyme-inhibitor complex and localization of duodenase and alpha1-PI in identical compartments, alpha1-PI is suggested to be a duodenase inhibitor in vivo.
• Gladysheva, I. P., Balabushevich, N. G., Moroz, N. A., & Larionova, N. I. (2000). Isolation and characterization of soybean Bowman-Birk inhibitor from different sources. Biochemistry. Biokhimiia, 65(2), 198-203.
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  A chromatographic procedure for isolation of different isoforms of Bowman--Birk soybean trypsin inhibitors was developed. The number of isoforms was shown to depend on soybean cultivar. The amount of the classical Bowman--Birk inhibitor (BBI) in different soybean cultivars, commercial flour, and processing products was analyzed. BBI reaches its highest concentration in freshly milled seeds. Storage conditions optimum for preservation of maximum inhibitory activity in soybean raw material were developed. The use of indirect enzyme immunoassay for BBI detection during its isolation from different sources was demonstrated.
• Gladysheva, I. P., Gladyshev, D. P., Dunaevsky, Y. E., Belozersky, M. A., & Larionova, N. I. (1999). Kinetics of interaction of trypsin with an anionic inhibitor of trypsin BWI-1a from buckwheat seeds. Biochemistry. Biokhimiia, 64(10), 1104-7.
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  The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k(ass)) was 2.2 x 10(6) M-1 x sec-1 and the dissociation rate constant (k(off)) of the enzyme--inhibitor complex was 3.5 x 10(-3) sec-1; the inhibition constant Ki was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.
• Gladysheva, I. P., Zamolodchikova, T. S., Sokolova, E. A., & Larionova, N. I. (1999). Interaction between duodenase, a proteinase with dual specificity, and soybean inhibitors of Bowman-Birk and Kunitz type. Biochemistry. Biokhimiia, 64(11), 1244-9.
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  The interaction between duodenase, which belongs to a group of Janus-faced proteinases, and classical Bowman--Birk (BBI) and Kunitz (STI) type inhibitors from soybean was investigated. Duodenase was shown to interact only with the antichymotrypsin site (Leu-Ser) of BBI, whereas the antitrypsin site (Lys-Ser) of the inhibitor appeared to be vacant and capable of interaction with trypsin. The inhibition constants of duodenase by BBI, the BBI--trypsin complex, and STI were 4, 400, and 40 nM, respectively.
• Popykina, N. A., Gladysheva, I. P., Zamolodchikova, T. S., & Larionova, N. I. (1999). [Interaction of duodenase with human blood serpins]. Bioorganicheskaia khimiia, 29(6), 605-10.
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  The interaction of duodenase, a new serine protease from a small group of Janus-faced proteases, with serpins, alpha 1-protease inhibitor (alpha 1-PI) and antichymotrypsin (ACT) from human blood serum, was studied. The stoichiometry of the inhibition process was found to be 1.2 and 1.3 mol/mol for alpha 1-PI and ACT, respectively. The presence of a stable enzyme-inhibitory complex duodenase-alpha 1-PI was confirmed by SDS-PAGE. No formation of the duodenase-ACT complex was demonstrated; instead, the band of the cleaved inhibitor was indicated upon the ACT hydrolysis. The suicide mechanism of the duodenase interaction with the human blood serpins was proved. The association rate constants (Ka, M-1 s-1) were 2.4 +/- 0.3 x 10(5) for alpha 1-PI and 3.0 +/- 0.4 x 10(5) for ACT. These results indicate the possibility of the regulation of duodenase activity by endogenous serpins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.
• Dunaevsky, Y. E., Gladysheva, I. P., Pavlukova, E. B., Beliakova, G. A., Gladyshev, D. P., Papisova, A. I., Larionova, N. I., & Belozersky, M. A. (1997). The anionic protease inhibitor BWI-1 from buckwheat seeds. Kinetic properties and possible biological role. Physiologia plantarum, 101(3), 483-488.
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  Kinetic characteristics and effects on the growth of filamentous fungi of one of the main anionic protease inhibitors, BWI-1, isolated from buckwheat seeds, have been studied. The inhibition constants of bovine trypsin, chymotrypsin and cathepsin G from human granulocytes with BWI-1 were found to be 1.1, 67 and 200 nM, respectively. Analysis of the amino acid sequence of BWI-1 in the vicinity of the reactive site revealed its homology to the potato proteinase inhibitor I family. It is suggested that the inability of BWI-1 to bind elastase of human granulocytes is due to the basic nature of the amino acid residue (Arg) at the P position in its reactive site. It was demonstrated that BWI-1 was able to suppress the germination of the spores and the growth of the mycelium of two filamentous fungi.
• Larionova, N. I., Gladysheva, I. P., & Gladyshev, D. P. (1997). Human leukocyte elastase inhibition by Bowman-Birk soybean inhibitor. Discrimination of the inhibition mechanisms. FEBS letters, 404(2-3), 245-8.
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  The reaction between human leukocyte elastase and soybean Bowman-Birk inhibitor has been studied. The inhibition was found to be due to slow tight binding of the inhibitor. The interaction of BBI with HLE was shown to involve two steps: the rapid formation of an initial EI complex, with a Ki of 28 nM, followed by a slow equilibrium conversion to a tighter-binding EI* complex with a final Ki* of 2.3 nM. At pH 7.5 and 25 degrees C, k(on) was 3.5 x 10(4) M(-1) s(-1) and k(off) was 1.0 x 10(-4) s(-1).
• Larionova, N. I., Gladysheva, I. P., Polekhina, O. V., Kurochkina, L. P., & Gorbatova, E. N. (1997). Synthesis and biodistribution of Bowman-Birk soybean protease inhibitor conjugate with amphiphilic polyester. Applied biochemistry and biotechnology, 61(1-2), 139-48.
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  The modification of Bowman-Birk soybean protease inhibitor (BBI) with the monoaldehyde derivative of block copolymer of ethylene oxide and propylene oxide (PE), M(r) 2,000 is described. The conjugate contains five covalently bound polymer chains per protein molecule, and retains the ability to inhibit trypsin and chymotrypsin-like proteinases. The distribution of native BBI and the BBI-PE conjugate was examined in mice. After i.v. injection of [125I]BBI and [125I]BBI-PE, both inhibitors distributed very rapidly to the liver, kidney, and lungs, and more slowly to the brain. At the same time-points (up to 24 h), radioactivity in the blood and organs of mice injected with modified inhibitor was higher than that of the native inhibitor. The blood concentration time profile following i.v. administration of two BBI preparations at a dose 3 mg/kg was reasonable well described by a two-compartment open model with first-order elimination kinetics. The total clearance of BBI-PE decreased by a factor of 8, body mean residence time increased by a factor of 5 in comparison with BBI. A physiological pharmacokinetic model was developed to describe the tissue-to-blood distribution of two inhibitors. One-compartment physiological organ model (flow limited) was used to describe of time-course profiles of BBI concentration in organs. A two-compartment physiological organ model (membrane limited) was used to predict tissue-to-blood distribution of conjugated BBI in some organs of mice (liver, lungs). The predicted concentration curves of BBI and BBI-PE in blood and organs in mice (with the exception of kidney) showed good agreement with the observed values.
• Gladysheva, I. P., Dunaevskiĭ, I. E., Belozerskiĭ, M. A., Gladyshev, D. P., Papisova, A. I., & Larionova, N. I. (1995). [Inhibition of exogenous serine proteinases by a trypsin inhibitor from the buckwheat IT-1 seeds]. Biokhimiia (Moscow, Russia), 60(9), 1530-5.
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  The possibility of inhibition of exogenous trypsin- and chymotrypsin-like proteinases by a proteinase inhibitor from buckwheat (IT-1) seeds has been studied. The inhibition constants for bovine trypsin and alpha-chymotrypsin and human granulocyte cathepsin G by IT-1 are equal to 1.1, 67 and 200 nm, respectively. The specificity of IT-1 with regard to its primary sequence adjacent to the active center and to its homology with inhibitors pertaining to the potato inhibitor I family has been carried out. It is concluded that by virtue of the basic nature of the P1 (Arg) residue in the active center IT-1 is not capable to bind human granulocyte elastase.
• Gladysheva, I. P., Polekhina, O. V., Shen, W. C., Shevchenko, A. A., Kazanskaia, N. F., & Larionova, N. I. (1995). [Structure and biological properties of a conjugate of Bowman-Birk type soy proteinase inhibitor with a block copolymer of ethylene oxide and propylene oxide]. Biokhimiia (Moscow, Russia), 60(4), 523-32.
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  The structure of the conjugate of Bowman-Birk soybean proteinase inhibitor (BBI) with the block copolymer of ethylene oxide and propylene oxide (proxanol) containing five moles of proxanol per mole of protein, has been studied. Data from reverse phase hydrophobic HPLC suggest that the conjugate is less hydrophobic compared to native BBI. A shift of the second derivative UV absorption spectrum for the conjugate towards the shortwave region indicates a greater accessibility of the Tyr-59 residue localized in the interdomain region of the BBI molecule for the solvent. It has been assumed that the conjugate-induced increase in Ki for chymotrypsin may be due to both disturbances in the intact structure of the interdomain region of BBI and screening of the anti-chymotrypsin reactive center as a result of hydrophobic interactions of propylene oxide blocks of proxanol with exposed hydrophobic groups around the reactive center. Supporting evidence in favour of BBI molecule hydrophilization as a result of modification by proxanol can be derived from decreased conjugate penetration into intestinal epithelial cells as well as from the slow elimination of the conjugate from mouse blood stream.
• Larionova, N. I., Vartanov, S. S., Sorokina, N. V., Gladysheva, I. P., & Varfolomeyev, S. D. (1997). Conjugation of the Bowman-Birk soybean proteinase inhibitor with hydroxyethylstarch. Applied biochemistry and biotechnology, 62(2-3), 175-82.
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  The classical Bowman-Birk soybean proteinase inhibitor was modified by hydroxyethylstarch. The modified inhibitor retained the capacity for simultaneous binding of trypsin and human leukocyte elastase. The inhibition constants, Ki, of bovine trypsin, alpha-chymotrypsin and human leukocyte elastase (HLE) increased not more than 10-, 1.5-, and 20-fold, respectively, on modification of the inhibitor. The less effective inhibition is presumably due to the steric hindrance brought about by the conjugation with polysaccharide molecules. The results obtained indicate the pronounced structure differences of the binding regions for trypsin and chymotrypsin/leukocyte elastase in the modified preparation.
• Tikhonova, T. V., Gladysheva, I. P., & Larionova, N. I. (1995). Retardation by the soybean Bowman-Birk inhibitor of elastin hydrolysis catalyzed by leukocyte proteinases. FEBS letters, 362(2), 225-8.
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  The classical Bowman-Birk inhibitor from soya retards strongly the hydrolysis of elastin catalyzed by leukocyte elastase, cathepsin G and a mixture of both. The inhibitory effect is practically unaffected by both the adsorption of the enzymes on elastin and prolongation of the enzymatic reaction.
• Gladysheva, I. P., Larionova, N. I., Gladyshev, D. P., Tikhonova, T. V., & Kazanskaia, N. F. (1994). [The classical Bowman-Birk soy inhibitor is an effective inhibitor of human granulocyte alpha-chymotrypsin and cathepsin G]. Biokhimiia (Moscow, Russia), 59(4), 513-8.
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  A kinetic study of the interaction of the classical Bowman-Birk type soybean inhibitor (BBI 2-IV) with human granulocyte alpha-chymotrypsin and cathepsin G has been carried out. The K(a) values for the inhibitor-proteinase systems--alpha-chymotrypsin and cathepsin G (2.0 x 10(5) and 6.4 x 10(6) M-1 s-1, respectively) have been established.
• Gladysheva, I. P., Larionova, N. I., Gladyshev, D. P., Tikhonova, T. V., & Kazanskaya, N. F. (1994). A CLASSICAL BOWMAN-BIRK SOYBEAN INHIBITOR ACTS AS AN EFFECTIVE INHIBITOR OF HUMAN GRANULOCYTE ALPHA -CHYMOTRYPSIN AND CATHEPSIN G. Biochemistry (Moscow), 59(4), 371-374.
• Gladysheva, I. P., Sharafutdinov, T. Z., & Larionova, N. I. (1994). [High molecular weight soy isoinhibitors of the Bowman-Birk type. Isolation, characteristics, and kinetics of interaction with proteinases]. Bioorganicheskaia khimiia, 20(3), 281-9.
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  Multiple forms of Bowman-Birk soybean inhibitor have for the first time been isolated from commercial soya flour and purified to homogeneity. Amino acid compositions and isoelectric points of the inhibitors were determined. The isolated inhibitors are shown to be related to classic (M 8000 Da, 2-II) and high molecular mass glycine-rich (M 17 000 Da, 3-II, 5-II) Bowman-Birk inhibitors. The inhibitor (2-II) was found to have two reactive sites and bind trypsin at one centre and alpha-chymotrypsin, cathepsin G and human leukocyte elastase at the other. Rate constants of the complex formation (ka) and complex dissociation (kd) were determined by following the kinetics of approaching to the steady state in a system including the enzyme, the substrate and various concentrations of the inhibitor.
• Larionova, N. I., Balabushevich, N. G., Gladysheva, I. P., Moroz, N., Nf, K., Polekhina, O. V., & Donetskii, I. A. (1994). Natural proteinase inhibitors as a basis for creating new drugs. Vopr Med Khim., 25-31.
• Tikhonova, T. V., Gladysheva, I. P., Kazanskaia, N. F., & Larionova, N. I. (1994). [Inhibition of elastin hydrolysis, catalyzed by human leukocyte elastase and cathepsin G, by the Bowman-Birk type soy inhibitor]. Biokhimiia (Moscow, Russia), 59(11), 1739-45.
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  Cathepsin G stimulates the hydrolysis of elastin from bovine neck ligament catalyzed by human leukocyte elastase. Stimulation factor depends on the ratio of the enzyme concentrations and ionic strength and equals 1.0-2.0. The classical Bowman-Birk inhibitor from soya retards strongly the hydrolysis of elastin catalyzed by leukocyte elastase, cathepsin G and the mixture of both. The inhibitory effect is practically unaffected by the adsorption of the enzymes on elastin, prolongation of the enzymatic reaction and ionic strength.
• Larionova, N. I., Balabushevich, N. G., Gladysheva, I. P., Moroz, N. A., Kazanskaia, N. F., Polekhina, O. V., & Donetskiĭ, I. A. (1993). [Natural proteinase inhibitors as a basis for creating new drugs]. Voprosy meditsinskoi khimii, 40(3), 25-31.
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  Isolation of the proteinases inhibitors, available for medicinal purposes, was described, where the inhibitor of the Kunitz type was obtained from bovine pancreas and the inhibitor of the Bowman-Birk type from soybeans. Screening of the immobilization procedures was carried out, which enabled the authors to produce the polymeric conjugates of the proteinase inhibitors exhibiting the maximal rate of activity against pancreatic proteinases and granulocyte elastases. Pharmacokinetics of the proteinase inhibitors obtained was studied. High molecular derivatives of the inhibitors from the bovine pancreas circulated in rat blood in larger quantities and longer, their total clearance was 5 times than native inhibitor preparations. The preparations containing these inhibitors from bovine pancreas exhibited a high therapeutic efficiency in treatment of rats with hemorrhagic pancreatitis and acute liver failure in rabbits.
• Larionova, N. I., Gladysheva, I. P., Tikhonova, T. V., & Kazanskaia, N. F. (1993). [Inhibition of cathepsin G and elastase from human granulocytes by multiple forms of the Bowman-Birk type of soy inhibitor]. Biokhimiia (Moscow, Russia), 58(9), 1437-44.
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  A classical soybean inhibitor (Bowman-Birk inhibitor, BBI 2-IV) and two high molecular weight glycine-enriched inhibitors of the same type (3-II and 4-II) have been isolated, purified to homogeneity and characterized. All of the BBI isoforms have been found to effectively inhibit cathepsin G and human granulocyte elastase. The constants for leucocyte cathepsin G inhibition by classical BBI 2-IV (Ki = 1.2 x 10(-9) M) and high molecular mass BBI 3-II (Ki = 8.0 x 10(-8) M) as well as for leucocyte elastase inhibition by high molecular mass BBI 3-II (Ki = 1.1 x 10(-7) M) have been determined.
• Larionova, N. I., Gladysheva, I. P., Topchieva, I. N., & Kazanskaia, N. F. (1993). [Conjugation of classic Bowman-Birk soy inhibitor with a copolymer of ethylene oxide and propylene oxide]. Biokhimiia (Moscow, Russia), 58(10), 1658-64.
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  A classical soybean inhibitor of the Bowman-Birk type (BBI) with a copolymer of ethylene oxide and propylene oxide (PE) has been synthesized. The BBI-PE conjugate contain five covalently bound polymeric chains per one protein molecule and retains its capacity to inhibit trypsin (Ki = 10(-10) M), alpha-chymotrypsin (Ki = 7 x 10(-8) M) and human granulocyte elastase (Ki = 3 x 10(-8) M). The preservation of the antiproteinase activity in the antichymotrypsin center creates a prerequisite for the manifestation of the anticarcinogenic effect of the inhibitor.
• Larionova, N. I., Gladysheva, I. P., Topchieva, I. N., & Kazanskaya, N. F. (1993). CONJUGATION OF THE CLASSICAL SOYBEAN BAUMAN-BIRK INHIBITOR WITH A COPOLYMER OF ETHYLENE OXIDE AND PROPYLENE OXIDE. Biochemistry (Moscow), 58(10), 1220-1225.
• Tikhonova, T. V., Larionova, N. I., Gladysheva, I. P., & Kazanskaia, N. F. (1993). [Bowman-Birk soy inhibitor as an affinity ligand for isolating leukocyte elastase. Inhibition of elastin hydrolysis, catalyzed by leukocyte elastase]. Biokhimiia (Moscow, Russia), 58(11), 1669-76.
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  A one-step procedure for human leukocyte elastase purification using an affinity adsorbent based on protein soybean Bowman-Birk proteinase inhibitor has been developed. The leukocyte elastase was purified 70-fold with a 70-100% yield. The enzyme preparations did not contain cathepsin G and displayed a high specific activity. The soybean Bowman-Birk type inhibitor effectively inhibited the elastin hydrolysis by leukocyte elastase both when the enzyme and the inhibitor were simultaneously added to the substrate and after preliminary elastase adsorption on elastin. The inhibitory effect was preserved at high degrees of elastin hydrolysis.
• Tikhonova, T. V., Larionova, N. I., Gladysheva, I. P., & Kazanskaya, N. F. (1993). BOWMAN-BIRK SOYBEAN INHIBITOR AS AN AFFINITY LIGAND FOR ISOLATION OF LEUKOCYTE ELASTASE : INHIBITION OF LEUKOCYTE ELASTASE-MEDIATED ELASTIN HYDROLYSIS. Biochemistry (Moscow), 58(11), 1227-1233.

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• Gladysheva, I. P. (2018, November). Pathophysiologic mechanisms defining progression of dilated cardiomyopathy and its transition to symptomatic heart failure. Translational Cardiovascular Research Collaborative. Biomedical Sciences Partnership Building: UA COM-Phoenix..