Serine Protease Inhibitor Research Articles

SPINK13 acts as a tumor suppressor in hepatocellular carcinoma by inhibiting Akt phosphorylation.

The PI3K/Akt pathway is overexpressed in nearly 50% of hepatocellular carcinomas and inhibits apoptosis by promoting the expression of antiapoptotic genes. Serine protease inhibitors have been shown to induce apoptosis in hepatoma cells by downregulating SPINK13 in the PI3K/Akt pathway. In this study, SPINK13 was expressed in lentiviral vectors. Changes in signaling pathway adapter proteins, apoptosis regulatory proteins, cell cycle regulatory proteins, and the biological behavior of hepatocellular carcinoma were observed in cell and nude mouse xenograft models. The underlying mechanism of endogenous SPINK13-induced apoptosis in hepatocellular carcinoma cells was explored via transcriptomics. As a result, endogenous SPINK13 might inhibit the activity of Furin protease, downregulate the Notch1/Hes1 pathway in a binding manner, activate the direct effector PTEN, inhibit Akt phosphorylation, inactivate the downstream PI3K/Akt pathway, and ultimately lead to mitochondrial apoptosis and cell cycle arrest in hepatoma cells. Therefore, the Notch1/Hes1/PTEN pathway may act upstream of SPINK13 to downregulate the PI3K/Akt signaling pathway. Our study helps elucidate the underlying mechanism of SPINK13 in anti-hepatocellular carcinoma and lays a theoretical foundation for the development of novel therapeutic serine protease inhibitors.

Hepatic SerpinA1 improves energy and glucose metabolism through regulation of preadipocyte proliferation and UCP1 expression.

Lipodystrophy and obesity are associated with insulin resistance and metabolic syndrome accompanied by fat tissue dysregulation. Here, we show that serine protease inhibitor A1 (SerpinA1) expression in the liver is increased during recovery from lipodystrophy caused by the adipocyte-specific loss of insulin signaling in mice. SerpinA1 induces the proliferation of white and brown preadipocytes and increases the expression of uncoupling protein 1 (UCP1) to promote mitochondrial activation in mature white and brown adipocytes. Liver-specific SerpinA1 transgenic mice exhibit increased browning of adipose tissues, leading to increased energy expenditure, reduced adiposity and improved glucose tolerance. Conversely, SerpinA1 knockout mice exhibit decreased adipocyte mitochondrial function, impaired thermogenesis, obesity, and systemic insulin resistance. SerpinA1 forms a complex with the Eph receptor B2 and regulates its downstream signaling in adipocytes. These results demonstrate that SerpinA1 is an important hepatokine that improves obesity, energy expenditure and glucose metabolism by promoting preadipocyte proliferation and activating mitochondrial UCP1 expression in adipocytes.

The association between alpha-1 antitrypsin and B-type natriuretic peptide blood levels in healthy African Americans

ObjectiveB-type natriuretic peptide (BNP) is a neurohormone primarily secreted from cardiac ventricles in reaction to increased volume and pressure. The plasma level of BNP is used to measure the mechanical function of the heart and the risk of developing chronic obstructive pulmonary disease (COPD). Alpha-1 antitrypsin (AAT) is a glycoprotein that acts as an inhibitor of serine proteases and plays a crucial role in protecting the lungs against potential harm. AAT deficiency also causes COPD. The data on the prevalence of BNP and AAT in African Americans (AA) are limited and inconsistent. No previous studies have investigated whether any association exists between BNP and AAT. We collected fasting blood samples from 114 AA subjects who provided informed consent. The plasma levels of the biomarkers were measured using ELISA.ResultsOur findings revealed a significant negative correlation between AAT and BNP (r=-0.266, p = 0.01) as well as between AAT and TNF-α (r=-0.242, p = 0.01). Sex hormones (estrogen and testosterone) have a significant relationship with the levels of AAT and BNP. Increasing the AAT and reducing the levels of inflammation can lower the levels of BNP, which in turn could help lower the risk of heart disease in the AA population.

Regulation of melanization in aphids by parasitoid wasp venom proteins enhances mummification.

Interactions between parasitic insects and their hosts demonstrate the complexity of evolutionary processes. Specifically, the parasitoid wasp Aphidius ervi manipulates its host, the pea aphid Acyrthosiphon pisum, through strategic venom injection to enhance mummification. This study explores how this venom affects the aphid's immune system, particularly targeting the activity of the phenoloxidase (PO) enzyme. Following the injection of venom from A. ervi, significant changes were observed in the expression of immune-related genes in A. pisum, especially notable expression changes of ApPPOs and a reduction of PO activity. Multi-omics sequencing identified 74 potential venom proteins in the venom gland of A. ervi, including serine protease homolog 1 (AeSPH1) and serine protease inhibitor (AeSPN1), hypothesized to regulate PO activity. The injection of recombinant protein AeSPH1 and AeSPN1 into the A. pisum hemocoel selectively reduced the expression of ApPPO1, without affecting ApPPO2, and effectively suppressed melanization. Moreover, RNAi targeting AeSPH1 significantly reduced the mummification rate in A. pisum population parasitized by A. ervi. Our findings clarify the complex biochemical mechanisms underlying host-wasp interactions and highlight potential avenues for developing targeted biological control strategies. © 2024 Society of Chemical Industry.

In vitro, in silico and crystallographic-based identification of serine protease inhibitors

Serine proteases are involved in various ailments, including pancreatitis, and colon cancer. Based on substrate recognition serine proteases are classified into different groups. Trypsin and trypsin-like serine proteases are among most studied group of serine proteases. Trypsin is among the chief hydrolysing enzyme involved in the pathogenesis of pancreatitis. Its inhibition can help to manage the disease. Herein, we investigated the trypsin inhibitory effect of some arginine-based small molecules, through in vitro, in silico, and crystallographic methods. Compounds 1-3 were evaluated against bovine pancreatic trypsin (BPT). Compound 1 was found to be active against trypsin with IC50 value of 247.98 ± 2.44 μM. Molecular docking studies were used to investigate the binding energy and binding conformation of inhibitor. All three compounds were subjected to crystallisation with trypsin. Compounds 1-2 were successfully crystallised with BPT The crystal structures of trypsin in complexed with compounds 1, and 2 were determined at 2.30 and 2.50 Å resolution, respectively. Both molecules showed their binding affinity with the active site residues of trypsin. This study will provide insight into the binding mechanism of E-64 and arginine and might be useful in designing effective inhibitors of serine proteases.

Developing a core outcome set for Netherton syndrome: An international multi-stakeholder e-Delphi consensus study.

Netherton syndrome (NS; OMIM#256500) is a rare and severe disorder of epidermal maturation and keratinization caused by pathogenic variants in the serine protease inhibitor Kazal type 5 (SPINK5), leading to severe skin barrier impairment. Although effective treatment is crucial for NS patients, there is a lack of knowledge on what the best treatment options are for these patients. Large heterogeneity in reported outcomes and measurement instruments hinders accurate comparison of treatment results across studies and the development of a treatment guideline. Therefore, we aimed to develop a core outcome set (COS) for NS that can be used in clinical care and research. This study was performed in accordance with the recommendations of the Core Outcome Measures in Effectiveness Trials (COMET) initiative. After identification of outcomes through a literature search and classification based on the International Classification of Functioning and taxonomies published by the COMET initiative, discussion groups were organized at the 2nd International Netherton Congress 2022 to finalize the provisional outcome list. Through a 2-round e-Delphi, 41 stakeholders (patients and family members, professionals, and representatives of industry) from 14 countries rated the importance of the outcomes using a 9-point Likert scale. An online consensus meeting attended by 14 stakeholders finalized the COS. The core outcome set for NS comprised 21 outcomes in 10 domains. These included four 'skin' outcomes, two 'sensation' outcomes, two 'side-effects of treatment' outcomes, one 'vitality' outcome, one 'emotional functioning' outcome, two 'physical development' outcomes, two 'nutrition' outcomes, two 'infections' outcomes, two 'allergies' outcomes, and three 'assessment results' outcomes. In this study, consensus was reached on 21 outcomes to be included in the COS for NS. The selection of outcomes in the COS underlines that NS not only affects the skin, but is a disease requiring a broad multidisciplinary approach in clinical care and research. International implementation of this COS will lead to more uniform reporting, thereby enabling comparison of study results, which may facilitate future treatment guideline development. The next step is to further conceptually define the outcomes and reach consensus on how the measure these.

The Role of the Plasminogen Activator Inhibitor 1 (PAI1) in Ovarian Cancer: Mechanisms and Therapeutic Implications

AbstractOvarian cancer (OC) is one among most significantly fatal gynecological cancers, with late-stage detection and an inadequate prognosis. Plasminogen activator inhibitor-1 (PAI1) gene anticipates negative outcomes in many different kinds of malignancies. Several research investigations are currently being done to examine the biological role of PAI1 in OC and the possible benefits of targeted pharmacotherapies. The PAI1 gene has been linked to the emergence and development of cancer in the ovary. PAI1, an inhibitor of serine protease, influences the fibrinolysis and extracellular matrix remodeling, both of which are crucial for tumor expansion and metastatic growth. PAI1 levels have been discovered to be subsequently more elevated in malignant ovarian tissues than in usual ovarian tissue, demonstrating a potential connection among PAI1 overexpression and OC development. PAI1 promotes tumor cell proliferation, movement, and an invasion by influencing the urokinase-plasminogen activators and through interactions with cell surface receptors. In addition, PAI1 gene contributes to angiogenesis and apoptotic cell death, which contribute to the more hostile phenotypes of OC. The prognostic and therapeutic consequences of focusing on PAI1 in OC are explored, demonstrating PAI1's potential to be a biomarker and emphasizing for novel treatment approaches. The PAI1 gene possesses several functions in OC, affecting tumor development, an invasion, and metastatic growth. Comprehending the complicated interactions and mechanisms that regulate PAI1 in OC may lead to more efficient evaluation and treatment strategies and ultimately enhance patient outcomes.

Serpina3c deficiency aggravates vascular dysfunction and VSMC dysregulation through regulating adamts4 pathway activity in obese mice

Abstract Background Obesity leads to arterial stiffness (AS), which is an independent risk factor for cardiovascular disease. Vascular smooth muscle cell (VSMC) dysfunction is a critical step in the development of AS. Adamts4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) promoting the ECM remodeling can regulate VSMC differentiation. Serpina3c is a serine protease inhibitor that plays a key role in VSMC proliferation and metabolic diseases. Purpose The present study aimed to investigate the function of Serpina3c in high-fat diet (HFD)-induced AS and regulation of VSMC phenotypic switching and possible mechanisms. Methods 8-week-old male Serpina3c-/- and C57BL/6 mice were fed normal diet (ND) or HFD for 12 weeks. The adipocyte-specific Serpina3c overexpression (AAV8-Serpina3c) were constructed and injected in situ into visceral or perivascular adipose tissue (AT) of Serpina3c-/- mice fed a 12-week HFD to test in vivo effect of AT-derived Serpina3c on arterial stiffness. In vivo vascular stiffness was analyzed by obtaining pulse wave velocity (PWV) measurements. Aortic rings were dissected to assess the ex-vivo effects of Serpina3c deprivation on vascular contractile and diastolic function. Elastin van Gieson (EVG) and Hematoxylin-eosin (H&E) staining were implemented to observe the pathological morphology of aortas. IP-Western analysis was performed for identifying Serpina3c-adamts4 interactions. Results The obese WT mice exhibited significantly increased PWV values, compared with ND-fed mice, which were further increased in HFD+Serpina3c-/- mice (Fig. 1A). Phenylephrine-induced vascular constriction was impaired in HFD fed mice and further deteriorated with Serpina3c deficiency (Fig. 1B). No differences were noted in the endothelial-dependent acetylcholine-mediated relaxation in HFD-fed mice. Notably, Serpina3c-/- mice exhibited poorer endothelium-independent sodium nitroprusside induced relaxation (Fig. 1C and D). The HFD+Serpina3c-/- mice showed an increased breakage of elastin fibres and medial thickness compared with the aortas of HFD+WT mice (Fig. 1E and F). Serpina3c deprivation aggravated collagen deposition and matrix metalloproteinases (MMPs, MMP2 and MMP9) accumulation. VSMC contractile gene α smooth muscle actin were upregulated and synthetic phenotype marker osteopontin were downregulated in HFD+WT mice. The effect was more pronounced in aortas with Serpina3c deficiency (Fig. 1G and H). Overexpression of serpina3c in both visceral and perivascular AT or adamts4 deprivation reversed the above phenotypes. Computer simulations and IP-Western results indicate that Serpina3c and adamts4 bind to and maintain the latter low-expression in response to obesity (Fig. 2). Conclusion Serpina3c interacts with adamts4 to maintain contractile phenotype of VSMC and suppress AS development in the context of nutrient excess. Serpina3c is a novel target for the prevention and treatment of obesity associated artery disease.Serpina3c maintains vascular functionSerpina3c interacts with adamts4

Dispensable regulation of brain development and myelination by the immune-related protein Serpina3n.

Serine protease inhibitor clade A member 3n (Serpina3n) or its human orthologue SERPINA3 is a secretory immune-related molecule produced primarily in the liver and brain under homeostatic conditions and up-regulated in response to system inflammation. Yet, it remains elusive regarding its cellular identity and physiological significance in the development of the postnatal brain. Here, we reported that oligodendroglial lineage cells are the major cell population expressing Serpina3n protein in the postnatal murine CNS. Using loss-of-function genetic tools, we found that Serpina3n conditional knockout (cKO) from Olig2-expressing cells does not significantly affect cognitive and motor functions in mice. Serpina3n depletion does not appear to interfere with oligodendrocyte differentiation and developmental myelination nor affects the population of other glial cells and neurons invivo. Interestingly, Serpina3n is significantly up-regulated in response to oxidative stress and its deficiency alleviates oxidative injury and diminishes cell senescence of oligodendrocytes invitro. Together, our data suggest that the immune-related molecule Serpina3n plays a minor role in neural cell development under homeostasis, yet it primes oligodendrocytes for CNS insults and regulates oligodendrocyte health under injured conditions. Our findings raise the interest in pursuing its functional significance in the CNS under disease/injury conditions.

MdSVWC1, a new pattern recognition receptor triggers multiple defense mechanisms against invading bacteria in Musca domestica

BackgroundSingle-domain von Willebrand factor type C (SVWC) constitute a protein family predominantly identified in arthropods, characterized by a SVWC domain and involved in diverse physiological processes such as host defense, stress resistance, and nutrient metabolism. Nevertheless, the physiological mechanisms underlying these functions remain inadequately comprehended.ResultsA massive expansion of the SVWC gene family in Musca domestica (MdSVWC) was discovered, with a count of 35. MdSVWC1 was selected as the representative of the SVWC family for functional analysis, which led to the identification of the immune function of MdSVWC1 as a novel pattern recognition receptor. MdSVWC1 is highly expressed in both the fat body and intestines and displays acute induction upon bacterial infection. Recombinant MdSVWC1 binds to surfaces of both bacteria and yeast through the recognition of multiple pathogen-associated molecular patterns and exhibits Ca2+-dependent agglutination activity. MdSVWC1 mutant flies exhibited elevated mortality and hindered bacterial elimination following bacterial infection as a result of reduced hemocyte phagocytic capability and weakened expression of antimicrobial peptide (AMP) genes. In contrast, administration of recombinant MdSVWC1 provided protection to flies from bacterial challenges by promoting phagocytosis and AMP genes expression, thereby preventing bacterial colonization. MdSPN16, a serine protease inhibitor, was identified as a target protein of MdSVWC1. It was postulated that the interaction of MdSVWC1 with MdSPN16 would result in the activation of an extracellular proteolytic cascade, which would then initiate the Toll signaling pathway and facilitate the expression of AMP genes.ConclusionsMdSVWC1 displays activity as a soluble pattern recognition receptor that regulates cellular and humoral immunity by recognizing microbial components and facilitating host defense.

Clustering-aided prediction of outcomes in patients with idiopathic pulmonary fibrosis

BackgroundBlood biomarkers predictive of the progression of idiopathic pulmonary fibrosis (IPF) would be of value for research and clinical practice. We used data from the IPF-PRO Registry to investigate whether the addition of “omics” data to risk prediction models based on demographic and clinical characteristics improved prediction of the progression of IPF.MethodsThe IPF-PRO Registry enrolled patients with IPF at 46 sites across the US. Patients were followed prospectively. Median follow-up was 27.2 months. Prediction models for disease progression included omics data (proteins and microRNAs [miRNAs]), demographic factors and clinical factors, all assessed at enrollment. Data on proteins and miRNAs were included in the models either as raw values or based on clusters in various combinations. Least absolute shrinkage and selection operator (Lasso) Cox regression was applied for time-to-event composite outcomes and logistic regression with L1 penalty was applied for binary outcomes assessed at 1 year. Model performance was assessed using Harrell’s C-index (for time-to-event outcomes) or area under the curve (for binary outcomes).ResultsData were analyzed from 231 patients. The models based on demographic and clinical factors, with or without omics data, were the top-performing models for prediction of all the time-to-event outcomes. Relative changes in average C-index after incorporating omics data into models based on demographic and clinical factors ranged from 1.7 to 3.2%. Of the blood biomarkers, surfactant protein-D, serine protease inhibitor A7 and matrix metalloproteinase-9 (MMP-9) were among the top predictors of the outcomes. For the binary outcomes, models based on demographics alone and models based on demographics plus omics data had similar performances. Of the blood biomarkers, CC motif chemokine 11, vascular cell adhesion protein-1, adiponectin, carcinoembryonic antigen and MMP-9 were the most important predictors of the binary outcomes.ConclusionsWe identified circulating protein and miRNA biomarkers associated with the progression of IPF. However, the integration of omics data into prediction models that included demographic and clinical factors did not materially improve the performance of the models.Trial registrationClinicalTrials.gov; No: NCT01915511; registered August 5, 2013; URL: www.clinicaltrials.gov.

Characterization of Kunitz-Domain Anticoagulation Peptides Derived from Acinetobacter baumannii Exotoxin Protein F6W77.

Recent studies have revealed that the coagulation system plays a role in mammalian innate defense by entrapping bacteria in clots and generating antibacterial peptides. So, it is very important for the survival of bacteria to defend against the host coagulation system, which suggests that bacterial exotoxins might be a new source of anticoagulants. In this study, we analyzed the genomic sequences of Acinetobacter baumannii and a new bacterial exotoxin protein, F6W77, with five Kunitz-domains, KABP1-5, was identified. Each Kunitz-type domain features a classical six-cysteine framework reticulated by three conserved disulfide bridges, which was obviously similar to animal Kunitz-domain peptides but different from plant Kunitz-domain peptides. Anticoagulation function evaluation showed that towards the intrinsic coagulation pathway, KABP1 and KABP5 had apparently inhibitory activity, KABP4 had weak inhibitory activity, and KBAP2 and KABP3 had no effect even at a high concentration of 20 μg/mL. All five Kunitz-domain peptides, KABP1-5, had no inhibitory activity towards the extrinsic coagulation pathway. Enzyme-inhibitor experiments showed that the high-activity anticoagulant peptide KABP1 had apparently inhibitory activity towards two key coagulation factors, Xa and XIa, which was further confirmed by pull-down experiments that showed that KABP1 can bind to coagulation factors Xa and XIa directly. Structure-function relationship analyses of five Kunitz-type domain peptides showed that the arginine of the P1 site of three new bacterial anticoagulants, KABP1, KABP4 and KABP5, might be the key residue for their anticoagulation activity. In conclusion, with bioinformatics analyses, peptide recombination, and functional evaluation, we firstly found bacterial-exotoxin-derived Kunitz-type serine protease inhibitors with selectively inhibiting activity towards intrinsic coagulation pathways, and highlighted a new interaction between pathogenic bacteria and the human coagulation system.

Aging by autodigestion.

The mechanism that triggers the progressive dysregulation of cell functions, inflammation, and breakdown of tissues during aging is currently unknown. We propose here a previously unknown mechanism due to tissue autodigestion by the digestive enzymes. After synthesis in the pancreas, these powerful enzymes are activated and transported inside the lumen of the small intestine to which they are compartmentalized by the mucin/epithelial barrier. We hypothesize that this barrier leaks active digestive enzymes (e.g. during meals) and leads to their accumulation in tissues outside the gastrointestinal tract. Using immune-histochemistry we provide evidence in young (4 months) and old (24 months) rats for significant accumulation of pancreatic trypsin, elastase, lipase, and amylase in peripheral organs, including liver, lung, heart, kidney, brain, and skin. The mucin layer density on the small intestine barrier is attenuated in the old and trypsin leaks across the tip region of intestinal villi with depleted mucin. The accumulation of digestive enzymes is accompanied in the same tissues of the old by damage to collagen, as detected with collagen fragment hybridizing peptides. We provide evidence that the hyperglycemia in the old is accompanied by proteolytic cleavage of the extracellular domain of the insulin receptor. Blockade of pancreatic trypsin in the old by a two-week oral treatment with a serine protease inhibitor (tranexamic acid) serves to significantly reduce trypsin accumulation in organs outside the intestine, collagen damage, as well as hyperglycemia and insulin receptor cleavage. These results support the hypothesis that the breakdown of tissues in aging is due to autodigestion and a side-effect of the fundamental requirement for digestion.

Impact of Bifidobacterium longum Subspecies infantis on Pediatric Gut Health and Nutrition: Current Evidence and Future Directions.

Background: the intestinal microbiota, a complex community vital to human health, is shaped by microbial competition and host-driven selective pressures. Among these microbes, Bifidobacterium plays a crucial role in early gut colonization during neonatal stages, where Bifidobacterium longum subspecies infantis (B. infantis) predominates and is particularly prevalent in healthy breastfed infants. Objectives: as we embark on a new era in nutrition of the pediatric population, this study seeks to examine the existing understanding regarding B. infantis, encompassing both preclinical insights and clinical evidence. Methods: through a narrative disceptation of the current literature, we focus on its genetic capacity to break down various substances that support its survival and dominance in the intestine. Results: using "omics" technologies, researchers have identified beneficial mechanisms of B. infantis, including the production of short-chain fatty acids, serine protease inhibitors, and polysaccharides. While B. infantis declines with age and in various diseases, it remains a widely used probiotic with documented benefits for infant and child health in numerous studies. Conclusions: the current scientific evidence underscores the importance for ongoing research and clinical trials for a deeper understanding of B. infantis's role in promoting long-term health.

Acylsugars, Nicotine and a Protease Inhibitor Provide Variable Protection for Nicotiana benthamiana in a Natural Setting.

Plants produce an immense diversity of defensive specialized metabolites. However, despite extensive functional characterization, the relative importance of different defensive compounds is rarely examined in natural settings. Here, we compare the efficacy of three Nicotiana benthamiana defensive compounds, nicotine, acylsugars and a serine protease inhibitor, by growing plants with combinations of knockout mutations in a natural setting, quantifying invertebrate interactions and comparing relative plant performance. Among the three tested compounds, acylsugars had the greatest defensive capacity, affecting aphids, leafhoppers, spiders and flies. Nicotine mutants displayed increased leafhopper feeding and aphid colonization. Plants lacking both nicotine and acylsugars were more susceptible to flea beetles and thrips. By contrast, knockout of the serine protease inhibitor did not affect insect herbivory in the field. Complementary experiments under controlled laboratory conditions with caterpillars, grasshoppers and aphids confirmed results obtained in a natural setting. We conclude that the three metabolite groups collectively provide broad-spectrum protection to N. benthamiana. However, there is a gradient in their effects on the interacting invertebrates present in the field. Furthermore, we demonstrate that, even if individual metabolites do not have a measurable defensive benefit on their own, they can have an additive effect when combined with other defensive compounds.

The diversity of AMPA receptor inhibition mechanisms among amidine-containing compounds.

Amidine-containing compounds are primarily known as antiprotozoal agents (pentamidine, diminazene, furamidine) or as serine protease inhibitors (nafamostat, sepimostat, camostat, gabexate). DAPI is widely recognized as a fluorescent DNA stain. Recently, it has been shown that these compounds also act as NMDA receptor inhibitors. In this study, we examined the activity of these compounds and analyzed the mechanisms of action in relation to another important class of ionotropic glutamate receptors-calcium-permeable AMPA receptors (CP-AMPARs) and calcium-impermeable AMPA receptors (CI-AMPARs) - using the whole-cell patch-clamp method on isolated male Wistar rat brain neurons. Gabexate and camostat were found to be inactive. Other compounds preferentially inhibited calcium-permeable AMPA receptors with IC50 values of 30-60µM. DAPI and furamidine were also active against CI-AMPARs with IC50s of 50-60μM, while others showed poor activity. All active compounds acted as channel blockers, which are able for permeating into the cytoplasm on both CP- and CI-AMPARs. Specifically, sepimostat showed trapping in the closed CP-AMPAR channel. Furamidine and DAPI demonstrated a voltage-independent action on CI-AMPARs, indicating binding to an additional superficial site. While the majority of compounds inhibited glutamate-activated steady-state currents as well as kainate-activated currents on CI-AMPARs, pentamidine significantly potentiated glutamate-induced steady-state responses. The potentiating effect of pentamidine resembles the action of the positive allosteric modulator cyclothiazide although the exact binding site remains unclear. Thus, this study, together with our previous research on NMDA receptors, provides a comprehensive overview of this novel group of ionotropic glutamate receptors inhibitors with a complex pharmacological profile, remarkable diversity of effects and mechanisms of action.

Crystallization and crystallographic studies of human serine protease inhibitor (serpin) B9.

Serine protease inhibitor B9 (serpin B9, also known as protease inhibitor 9 or PI9) plays a critical role in regulating the immune response by specifically inhibiting granzyme B, a serine protease found in cytotoxic T lymphocytes and natural killer cells. Despite its potential as an anticancer drug target, the structural details of serpin B9 have remained elusive until now. In this study, a cleaved form of recombinant human serpin B9 was successfully prepared and crystallized. The crystals belonged to space group P212121, with unit-cell parameters a = 68.51, b = 82.32, c = 101.17 Å, and an X-ray diffraction data set was collected at 1.9 Å resolution. The structure shows that serpin B9 adopts a relaxed conformation, with its cleaved reactive-centre loop inserted into the central β-sheet. Unlike other serpins, serpin B9 shows significant structural deviations around helix D, with a larger surface cavity, which could serve as a promising target for small-molecule inhibitors.

Proteolytic degradation of seed proteins of vetch species (Vicia L. subgenus Vicia) of section Peregrinae Kupicha during SDS-electrophoresis and its prevention

Background. Due to its simplicity and good reproducibility, SDS-electrophoresis of seed proteins is widely used for investigating the gene pool of legumes and other plants, for species and varietal identification, analyzing the intraspecific variability, and registering collection material. The data obtained by this method agree well with the DNA analysis results complement them. Typically, legume seed proteins, including representatives of the genus Vicia L., show clear informative SDS electrophoretic profiles. When analyzing seed accessions of vetch species of the section Peregrinae Kupicha using standard approaches previously developed at VIR and approved by ISTA (the International Seed Testing Association), clear electrophoretic protein profiles could not be obtained for many accessions. This called into question the applicability of standard approaches to identifying vetch species in the section Peregrinae. The objective of the work was to clarify the nature of seed proteins degradation in representatives of the Peregrinae section and to find ways to prevent it to ensure the possibility of carrying out species identification and registration of all accessions in the vetch collection using a unified approach. Material and methods. Seed proteins of a number of vetch species Vicia L. from sections Bithynicae (B. Fedtsch.) Maxted, Hypechusa (Alef.) Aschers. et Graebner, Microcarinae Maxted and Peregrinae, members of the subgenus Vicia, were analyzed by SDS-electrophoresis using the standard method of protein extraction from flour with 0.025 M Tris-glycine buffer pH 8.3 at room temperature and its modifications, including heating the extract at 80°C or 100°C with or without the addition of 2-mercaptoethanol, as well as the addition of cysteine and serine protease inhibitors. Results and discussion. An analysis of seed proteins of representatives of most sections of the subgenus Vicia yielded informative species-specific protein profiles, whereas species of the section Peregrinae were characterized by the protein profiles, which indicated protein degradation, and species of this section differed in the frequency of such profile occurrence. While such profiles were obtained for all seeds of seven accessions of V. aintabensis Boiss & Hausskn. ex Boiss differing in geographical origin, year and place of regeneration, and 12 out of 13 of V. peregrine L. accessions demonstrated profiles of partially or completely degraded proteins, complete seed protein profiles were obtained for six out of nine V. michauxii Sprengel accessions. A change in conditions for protein isolation, namely replacement of their extraction from flour with Tris-glycine buffer pH 8.3 at room temperature with extraction in the same buffer by a short-term heating at 100°C in the presence of 2-mercaptoethanol, made it possible to obtain complete protein profiles for all accessions representing the section Peregrinae. The protein profiles of representatives of other vetch sections, as well as the profile of soybean proteins used as a standard for legume species identification, did not differ from the original ones under the modified conditions. Conclusions. The obtained results suggest that protein degradation in species of the Peregrinae section is associated with the abnormal activity of endogenous seed proteases under standard protein extraction conditions, and this trait is determined genotypically. A new modification of the method for isolating proteins from seeds makes it possible to apply the generally accepted approaches based on SDS-electrophoresis in the analysis of the gene pool of the Peregrinae section of the subgenus Vicia, as well as other vetch species.

A-231 A modified Sampson LDL-C equation to improve concordance with the reference method at low levels of LDL-C

Abstract Background Patients at high risk for Atherosclerotic Cardiovascular Disease (ASCVD), who do not achieve adequate lowering of LDL-cholesterol (LDL-C) with maximally tolerated statins or who are statin-intolerant, are often treated with proprotein convertase subtilisin/kexin (type 9) serine protease inhibitor (PCSK9-inh) therapy. The LDL-C reference method, Beta-Quantitation (BQ), is not routinely performed in these patients. Because low values are measured less accurately with current methods, a variety of equations have been developed to calculate LDL-C from the standard lipid panel test results (total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C)). We further modified the Sampson LDL-C (S-LDL-C) equation by a novel approach by combining logistic regression (LR) and linear regression to minimize errors when compared to BQ for low LDL-C. Methods A dataset of BQ results from Mayo Medical Labs was divided randomly into a training set (TG ≤ 1500 mg/dL, TC ≤ 1050 mg/dL, n=19,639) and a validation set (TG ≤ 800 mg/dL, BQ-LDL-C ≤ 130 mg/dL, n=12,910). The training dataset was analyzed by LR, using BQ-LDL-C to classify patients as being above or below the 70 mg/dL cut-point. Dependent variables in the LR model were the S-LDL-C, HDL-C, TC, TG and an interaction term (TG x NonHDL-C). The sum of the exponential terms in the trained LR equation for the dependent variables were then fitted by least squares linear regression to match BQ-LDL-C to develop the modified S-LDL-C equation (mS-LDL-C). We used the validation dataset to compare Friedewald (F-LDL-C), extended Martin (eM-LDL-C), Sampson (S-LDL-C) and modified Sampson (mS-LDL-C) equations by Cohen’s kappa coefficient for agreement with BQ-LDL-C and normalized Matthew’s correlation coefficient (MCC), which considers any imbalances in the number of false high versus false low results. Results The mS-LDL-C equation (mS-LDL-C = (TC/0.9423 - HDL-C/0.9582 - TG/9.397 - ((TGxNonHDL-C)/1305) + TG2/8569) - 5.688), primarily differs from the S-LDL-C equation in the value of the coefficients that include TG as a variable. For S-LDL-C ≤100 mg/dL, mS-LDL-C had the best concordance with BQ-LDL-C in terms of the total percent of patients misclassified as either high or low (mS-LDL-C: 21.4%, S-LDL-C: 24.4%, eM-LDL-C: 25.4% F-LDL-C: 30.8%). mS-LDL-C also had the best kappa score when compared to BQ (mS-LDL-C: 0.833, S-LDL-C: 0.810, eM-LDL-C: 0.789, F-LDL-C: 0.743). MCC was also the highest for mS-LDL-C (mS-LDL-C: 0.916, S-LDL-C: 0.906, eM-LDL-C: 0.894, F-LDL-C: 0.879). Finally, mS-LDL-C also showed the best concordance with BQ-LDL-C when an alternative lower LDL-C therapeutic decision cut-point of 55 mg/dL is used (kappa of 0.766 versus 0.724) but showed an increasingly poorer performance than S-LDL-C for higher LDL-C whenever it exceeded 150 mg/dL. Conclusions A strategy to maximize the overall accuracy of estimated LDL-C could provide an automatic reflex in a laboratory information system to the mS-LDL-C equation whenever S-LDL-C is <100 mg/dL. By doing so, the use of the more accurate mS-LDL-C equation for low LDL-C should improve PCSK9-inh therapeutic decision making over the other LDL-C equations.

Genotypes of carboxypeptidase A1 and gamma-glutamyltransferase 1 may be useful tools for the diagnosis and the predictor of worrisome features of intraductal papillary mucinous neoplasm in Japan.

This study aimed to clarify whether several single-nucleotide polymorphisms (SNPs)-related chronic pancreatitis such as carboxypeptidase A1 (CPA1), carboxypeptidase B1 (CPB1), Gamma-glutamyltransferase 1 (GGT1), G-protein-coupled receptor Class C Group 6 Member A (GPRC6A), and serine protease inhibitor, Kazal type 1 (SPINK-1) genotypes were associated with clinical characteristics of patients with intraductal papillary mucinous neoplasm (IPMN) and worrisome features of IPMN. We enrolled 100 patients with IPMN and 116 patients as a control. Serum p-amylase, lipase, trypsin, phospholipase A2 (PLA2), and elastase-1 levels were measured. An Olympus EUS (GF-UCT 260) was used to perform endosonography in 100 patients with IPMN. Total EUS score was evaluated using endosonography. DNA was isolated from the duodenal tissue using a commercial system and polymerase chain reaction (PCR) was performed on 7500 Fast PCR System. There were no associations between glucose tolerances, lipid levels and genotypes of CPA1, GGT1, GPRC6A, and SPINK-1 in patients with IPMN. CPA1 genotype was significantly associated with the pathophysiology of IPMN. Then, GGT1 genotype was also significantly associated with EUS total score and the size of cyst more than 20 mm and more than 30 mm as one of worrisome features of IPMN. Genotypes of carboxypeptidase A1 and gamma-glutamyltransferase 1 may be useful tools for the diagnosis and the predictor of worrisome features of IPMN.