Carboxypeptidase Research Articles

Prevalence, toxin-typing, and antimicrobial susceptibility of Clostridium perfringens from retail meats in Seoul, Korea

Clostridium perfringens is a ubiquitous, Gram-positive, spore-forming bacterium. It can contaminate many types of retail meat products and cause food poisoning by producing enterotoxins in the small intestines of humans and domestic animals. We investigated the prevalence, toxin-encoding gene profile, and antimicrobial resistance of C. perfringens in beef, chicken, and pork meat purchased from retail markets in Seoul, Korea. C. perfringens was detected according to the International Organization for Standardization 7937, with some modifications, and confirmed using the Vitek 2 system. In total, 38 C. perfringens strains were isolated from 200 meat samples (38/200, 19%; thirty-three from chicken, and five from beef). Among the six toxins evaluated, including alpha, beta, epsilon, iota, enterotoxin (encoded in the cpe gene), and netB, only the cpa gene was detected in all isolates by polymerase chain reaction (PCR) amplification. The antimicrobial resistance of the isolates was evaluated using the agar dilution method and resistance to ampicillin (12/38, 31.6%), tetracycline (38/38, 100%), chloramphenicol (26/38, 68.4%), metronidazole (13/38, 34.2%), and imipenem (27/38, 71%) was observed. Interestingly, 30 of the 38 isolates (78.9%) were multiple-drug resistant, showing resistance to more than three different antimicrobial classes.

The prevalence of Clostridium perfringens in retail meat of Mardan, Pakistan

The most prevailing bacteria in distribution is Clostridium perfringens C. perfringens which is ranked 1st among fatal diseases in small ruminants. This rapidly growing food borne pathogen is present in water, food, soil, air, and humans and animals intestinal tract. In the present study, a cross-sectional study was performed to sort out the prevalence of C. perfringens types estimation in various retail meats and areas of Mardan district, Pakistan. 600 fresh meat samples equally from cows, sheep and goats were collected from retail shops. C. perfringens was diagnosed in various types of meat however highest prevalence was found in goats 174/200 87% followed by sheep 103/200 51.5% while the least prevalence was in cow meat 89/200 44.5% . All obtained isolates were further genotyped through multiplex PCR indicating 93/174 53.45% C. perfringens type A, 16/174 9.19% type B, 3/174 1.72% type C and 62/174 35.63% type D in goat isolates. 53/103 51.46% C. perfringens type A, 11/103 10.68% type B, 3/103 2.91% type C and 36/103 34.95% type D in sheep isolates. 73/89 82.02% C. perfringens type A, 9/89 10.11% type D and 7/89 7.86% type E in cow isolates. Out of 89 cow meat isolates 4 4.49% carried cpe gene, 16 17.98% contained cpb2 gene and 1 1.12% carried both cpe and cpb2 genes. In 103 sheep meat isolates, 2 1.94% carried cpe gene, 8 7.77% carried cpb2 gene and 3 2.91% carried both cpe and cpb2 genes. In 174 goat?s meat isolates, 3 1.72% carried cpe gene, 18 10.34% carried cpb2 gene while 1.72% carried both cpe and cpb2 genes. While analyzing prevalence in months and area wise, no significance was observed through chi-square test. It indicates that this pathogen is prevalent in various meat sources in Mardan and could be a threat to public health.

Identification and expression characterization of a novel carboxypeptidase A-like gene (RvCPA) during early developmental stages in the gastropod Rapana venosa (Muricidae)

Rapana venosa is an economically important aquaculture species in China. Carboxypeptidases (CPs) A and B (CPA and CPB, respectively) hydrolyze peptides and proteins and play critical roles in the development of the digestive system in animals. In this study, a full-length CPA-like cDNA sequence from R. venosa (RvCPA) was isolated using RACE, and RvCPA mRNA expression levels were analyzed in different tissues and developmental stages by quantitative real-time PCR. Additionally, the expression patterns of RvCPA under different diets were investigated in competent larvae of R. venosa. The full-length cDNA of the RvCPA gene was 1,810 bp and consisted of a 1,296-bp open reading frame (ORF) encoding 431 aa. Phylogenetic analysis revealed high homology of RvCPA with CPs from Biomphalaria glabrata, Pomacea canaliculata, and Ampullaria crossean. Our results showed that RvCPA had higher expression in the salivary gland than in other tissues and that the expression of RvCPA was dramatically increased in the juvenile (postlarval) stage compared with other stages, indicating that functional changes occur in the larval digestive system during the food habit transition. Furthermore, we found that an animal diet could significantly increase the expression of RvCPA, which may suggest that animal diets can promote the development of the digestive system. This information on a novel CP in R. venosa that participates in digestion and is related to the food habit transition may broaden our understanding of digestive system development during metamorphosis in carnivorous gastropods.

Synaptic Vesicles Dynamics in Neocortical Epilepsy

Neuronal hyperexcitability often results from an unbalance between excitatory and inhibitory neurotransmission, but the synaptic alterations leading to enhanced seizure propensity are only partly understood. Here we assess changes in functional synaptic pools in mouse visual cortex made epileptic by tetanus neurotoxin (TeNT) injection. Using an ultrastructural measure of synaptic activity, we quantified functional differences at excitatory and inhibitory synapses. We found homeostatic changes in epileptic networks, expressed as an early onset lengthening of active zones at inhibitory synapses, followed by spatial reorganization of recycled vesicles at excitatory synapses. A proteomic analysis of synaptic content revealed an upregulation of Carboxypeptidase E (CPE) following TeNT injection; remarkably, its inhibition rapidly decreased network discharges in vivo. Altogether, our analyses reveal a complex landscape of changes affecting the epileptic synaptic release machinery, unveiling presynaptic mechanisms which may impact vesicles’ release timing rather than synaptic strength. Our results indicate i) altered positioning of release-competent vesicles as a novel signature of epileptic networks and ii) inhibiting CPE a potential therapeutic strategy to counteract epileptic discharges.

SUN-260 Dual Role of Carboxypeptidase E in Prohormone Processing and a Novel Neurotrophic Factor Mediating Neuroprotection and Cognitive Functions in Hippocampal CA3 Neurons in Mice

Stress causes release of glucocorticoids from the adrenals which then circulate to the brain. High concentrations glucocorticoid from chronic severe stress results in pathophysiology in the brain, including neuronal degeneration, cell death and cognitive dysfunction, leading to diseases such as Alzheimer Disease and Major Depressive Disorders. Neurotrophic/growth factors such as BDNF, NGF and NT3 have been linked to these pathological conditions. Carboxypeptidase E (CPE), a proneuropeptide/prohormone processing enzyme, also named neurotrophic factor-α1(NFα1) is highly expressed in the stress-vulnerable hippocampal CA3 neurons, and was shown to have neuroprotective activity from in vitro studies. Here we investigated if CPE-NFα1 functions in vivo, independent of its enzymatic activity, and the mechanism underlying its action. We generated knock-in mice expressing a non-enzymatic form of CPE, CPE-E342Q, but not wild-type CPE. The CPE-E342Q mice showed significantly decreased neuropeptide content and exhibited obesity, diabetes and infertility due to lack of prohormone processing activity, similar to CPE-KO mice. However, they showed no hippocampal CA3 degeneration, exhibited neurogenesis in the dentate gyrus, and displayed normal spatial learning and memory, similar to CPE wild-type mice, after weaning stress; unlike CPE-KO mice which showed hippocampal CA3 neuronal degeneration and cognitive deficits. Binding studies showed that radiolabeled CPE bound hippocampal cell membrane specifically, in a saturable manner. Binding of CPE and CPE-E342Q to hippocampal neurons activated Erk signaling and pre-treatment with either of these proteins protected neurons against H2O2- or glutamate-induced neurotoxcity by increasing BCL2 expression. In vitro and in vivo inhibitor studies demonstrated that this neuroprotective effect was independent of tyrosine kinase receptor signaling. Taken together, the data provide evidence that CPE-NFα1 is a unique neurotrophic factor which acts through a non-tyrosine kinase receptor to activate Erk-BCL2 signaling to protect hippocampal CA3 neurons against stress-induced neurodegeneration and maintaining normal cognitive functions in mice.

OR19-06 Sepsis-Induced Critical Illness in Mice Alters Key Regulators of ACTH Production and Secretion Within the Anterior Pituitary Gland

IntroductionCritical illness is hallmarked by high plasma cortisol without elevated ACTH. This is partly explained by low cortisol-binding proteins and reduced binding affinity and by suppressed cortisol metabolism, 3 peripheral drivers of increased free cortisol (corticosterone (CORT) in rodents).1 We hypothesized that lack of elevated plasma ACTH is explained by CORT-induced feed-back inhibition either due to suppressed CRH or AVP pituitary signaling or to direct glucocorticoid receptor (GR)-mediated action within corticotrophs. To test this hypothesis, we documented alterations in ACTH precursors and key enzymes controlling ACTH maturation and secretion in a mouse model of sepsis-induced critical illness.MethodsC57Bl/6 mice were randomly allocated to a healthy control group or to 4 critically ill groups sacrificed after increasing illness duration (30 hours (H), 3 days (D), 5D or 7D). Critical illness was induced by sepsis brought about by cecal-ligation and puncture followed by fluid-resuscitation and antibiotics treatment. The study was continued until 15 surviving animals per time cohort were reached (n=120).We quantified pituitary pro-opiomelanocortin (POMC) gene/protein expression and POMC plasma concentrations, pituitary POMC intracellular trafficking and cleavage via intracellular POMC sorting/trafficking receptor Carboxypeptidase E (CPE) and prohormone convertase 1 (PC1/3) gene/protein expression. Gene expression of Annexin A1, an inhibitor of mature ACTH secretion, was quantified as marker of GR-mediated CORT-induced feedback inhibition at corticotroph level.ResultsPlasma CORT concentrations were median 3-fold increased during critical illness (p<0.001 for all time cohorts) in the face of normal (for 30H, 3D and 5D cohorts) to low (7D time cohort; p=0.01) plasma ACTH concentrations. Plasma POMC concentrations were higher in critically ill than in control mice (p=0.05). POMC gene expression (but not protein, P=0.8) was a median 55% higher in critically ill mice than in controls (p<0.05 for all time cohorts). In contrast, pituitary mature ACTH protein concentration was median 61% lower in critically ill than in control mice (p<0.01). CPE gene expression was only increased in 30H time cohort (p<0.001). PC1/3 gene and protein expression were positively correlated (R2 0.1; p=0.001) and were reduced (by 37% and 43%, respectively) during the entire course of critical illness (p<0.01). Annexin A1 gene expression was increased during critical illness (p<0.05 for all time cohorts).ConclusionSuppressed CRH or AVP signaling and GR-mediated action within corticotrophs explained lack of elevated plasma ACTH in critical illness, as indicated by impaired POMC processing and ACTH maturation. However, increased POMC gene expression suggests ongoing corticotroph activation, the driver of which needs to be identified. 1. Teblick A et al. Nat Rev Endocrinol 2019

Extracellular interaction between Neurotrophic factor‐α1 and HTR1E serotonin receptor promotes cell survival

Neurotrophic factor α1 (NFα1), classically known as carboxypeptidase E (CPE) has a non‐enzymatic role in the survival of neurons and cancer cells via binding to a putative receptor. We have identified 5‐Hydroxy tryptamine receptor 1E (HTR1E), a serotonin receptor with unknown function, as a binding partner that interacts with NFα1/CPE extracellularly to mediate cell survival. Co‐immunoprecipitation using a human LN18 cells and pull‐down assay with NFα1/CPE and HTR1E expressed in HEK293 cells confirmed interaction of these two molecules. 125I NFα1/CPE binding studies demonstrated saturable, high affinity binding to HTR1E. Molecular docking studies revealed the surface interaction between NFa1/CPE and HTR1E via 3 salt bridges, further stabilized by hydrogen bonding. We also found that HTR1E was able to activate ERK/CREB signaling upon treatment with NFα1/CPE. Pretreatment of HTR1E stable cells with NFα1/CPE followed by H2O2‐induced oxidative stress prevented a decrease in pro‐survival protein BCL2 and reduced cytotoxicity. SiRNA knock‐down of HTR1E in U118 glioblastoma cells resulted in significant inhibition of these cancer cells survival. Immunocytochemical analysis of human hippocampus indicated co‐expression of HTR1E with NFα1/CPE in CA1‐3 and dentate gyrus, and cellular co‐localization at the cell membrane in CA3 neurons. This study has uncovered a novel role for HTR1E as a binding partner for NFα1/CPE, and their interaction activates the ERK‐CREB‐BCL2 pathway to promote cell survival.Support or Funding InformationDivision of intramural research, NICHD, NIH, Bethesda, MD, USA

Isolation and characterization of Clostridium perfringens strains isolated from ostriches (Struthio camelus) in Vietnam.

Background and Aim:Clostridium perfringens can cause enteritis in ostriches. The toxin release is believed to play a major role in determining pathogenesis properties of these pathogenic bacteria. This study was conducted to isolate and characterize C. perfringens strains from ostriches in Vietnam for identifying if particular virulence factors of these pathogenic bacteria are associated with enteritis progress in ostriches.Materials and Methods:The prevalence of cpa, cpb, iA, etx, cpe, and cpb2 genes among C. perfringens isolates was determined by a multiplex polymerase chain reaction (PCR) method. The NetB toxin-encoding gene was detected by PCR and then sequenced to observe their variation. The expression of NetB toxin was checked by SDS-PAGE.Results:A total of 116 C. perfringens isolates were obtained from 318 fecal samples and 105 intestinal organs. Of 80 isolates from fecal samples, 33 isolates were from healthy and 47 isolates were from diseased ostriches. The results of multiplex PCR showed that all 116 C. perfringens strains from healthy and enteric disordered ostriches were positive for the alpha toxin-encoding gene (cpa). The cpe and cpb2 genes were found in only one and five diseased ostriches, respectively. The netB gene was detected in 1/33 (3.03%) C. perfringens isolates from healthy ostriches, in 8/47 (17.05%) isolates from feces, and in 7/36 (19.44%) intestinal contents of diseased ostriches. The full-length sequences of 5 out of 15 netB-positive isolates from diseased ostriches showed 100% identity to each other as well as to the netB sequences available in GenBank. All of these five isolates produced NetB toxin in vitro.Conclusion:Type A is the most prevalent among C. perfringens isolates from ostriches in Vietnam. Especially, the study provides data emphasizing the role of NetB toxin in causing necrotic enteritis by C. perfringens in ostriches.

Proteomic and Genomic Endocrine Responses in Oxytocin Knockout Mice

Gene knockout animal models have proven extremely useful for studying the effects of a single gene product in systems where human studies are not an option. The nine amino acid peptide, oxytocin, has been shown to play dual roles as a peptide hormone in parturition and milk production; and as a neuropeptide in social and maternal behavior. In this study, we investigated the effect of the absence or presence of a functioning oxytocin allele on other endocrine genes. Taking advantage of an oxytocin-null murine model (OTKO-/-), gene expression levels were assayed for the prohormones; pro-oxytocin, pro-vasopressin, and proopiomelanocortin; processing enzymes, prohormone convertase 1, prohormone convertase 2 and carboxypeptidase E; and transcription factors, c-Fos, and c-Jun in OT+/+ and OTKO-/- animals. Significant changes in gene expression levels were found to occur in cortex and hypothalamic tissues of OTKO-/- mice. Specifically, there was an up-regulation of POMC, PC2, c-Fos and c-Jun in hypothalamus. PC2 was also found to be up-regulated in the cortex. Western blot analysis of the affected tissues showed similar results. We conclude that the absence of the peptide hormone oxytocin can affect other peptide hormone systems, including processing enzymes and transcription factors.

Analysis of different proteins in primary open angle glaucoma based on aqueous proteome

Objective To analyze the changes of protein expression in aqueous humor of patients with primary open angle glaucoma (POAG), and to explore the biological markers and potential therapeutic targets associated with disease occurrence. Methods A retrospective case-control study was adopted.Ten patients with age-related cataract and 10 patients with POAG combined with cataract.were collected at the Tianjin Medical University Eye Hospital from October 2016 to December 2017.In the process of surgery, 100 μl of aqueous humor were collected with 1 ml syringe and No.1 needle through the surgical access.Those proteins extracted from aqueous humor were analyzed by quantitative proteomic mass spectrometry.The differential significance test was performed by Maxquant significances A. The differential proteins of the two groups were screened and determined with the conditions of P 2.The biological big data was annotated by the function of GO and KEGG.The study was approved by the Ethics Committee of the Tianjin Medical University Eye Hospital (No.2013KY[L]-10). Patients and their guardians all signed the informed consent. Results Ninty-seven differential proteins were detected in this proteomic analysis, including 48 up-regulated proteins and 49 down-regulated proteins.GO analysis significantly differs in protein function involved in many aspects, some different proteins including lipopolysaccharide-binding protein (LBP), cluster of Differentiation 163(CD163), C-reactive protein (CRP), annexin A1 (ANXA1) were involved in inflammation; redox-related proteins were glutathione S-transferase P (GSTP1), thioredoxin (TXN), some different proteins related with cell adhesion movement were cartilage oligomeric matrix protein (COMP), desmocollin-2 (DSC2) and laminin subunit beta-2 (LAMB2); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1), solid protein (tenascin, TNC) are associated with fibrosis; some different proteins related to nerve growth were reelin (RELN), semaphorin-3F(SEMA3F), semaphorin-4B(SEMA4B). Metabolism-related proteins included pyruvate kinase (PKM), carb oxypeptidase N subunit 2 (CPN2) and so on.KEGG analysis indicated that the expression of NrF2/ERK signaling pathway and TGF-β/NF-κB signaling pathway were different between the two groups. Conclusions The expression of GSTP1 and TXN in the aqueous humor of POAG is significantly decreased, which may regulate cell adhesion and activity and expression of extracellular matrix by regulating NrF2/ERK1/2 and TGF-β/NF-κB signaling pathways.GSTP1 and TXN may serve as a potential biomarker and therapeutic target of POAG. Key words: Primary open angle glaucoma; Aqueous humor; Label free quantitative proteome; Glutathione S-transferase P; Thioredoxin; Transforming growth factor-β; nuclear factor-kappa B; Nuclear factor erythroid 2-related factor 2; extracellular regulated protein kinases

Potential Role Of Clostridium Difficile And Clostridium Perfringens As A Cause Of Diarrhea In Horses

Diarrhea is a major clinical problem affecting foals and horses at different ages which can be fatal and cause great economic losses particularly in foals. The aim of this study was to identify Clostridium perfringens and Clostridium difficile involved in enteric infections and the associated virulence factors in diarrheic foals and horses. Thirty fecal samples were examined for identification of C.perfringens and C.difficile (bacteriological culturing, microscopical examination, count and molecular detection). C.perfringens was isolated from young horses less than 6 months age in an incidence of 46.7% with count ranged from 1.3×104- 5.6×108 cfu/g, while from horses of 6 months to one year, it was 20% with count ranged from 1.7×104- 1.9×106 cfu/g, on the other hand, 13.3% with count ranged from 1.4×103- 2.5×104 cfu/g was the incidence of C.perfringens in horse more than one year. C. difficile was not detected in all examined samples. C.perfringens type A was the most predominant type detected in an incidence of 58.3% then type B (4.7%). All C.perfringens isolates were sensitive to penicillin, nitromedazole, fluramfenicole and ceftiofur and all are resistant to oxytetracycline. Based on the high count, eight C.perfringens isolates were chosen to be molecular characterized for the presence of some virulence factor genes which included ; cpa, cpb2, cpe and netF toxin genes. All examined isolates have cpa (alpha) and cpb2 (beta 2) toxin genes, 5/8 isolates were found to have netF gene while only 2 isolates harbored cpe (enterotoxin gene). C. difficile tpi gene was not detected in all examined samples (10). This study may throw the light on the synergistic effect of β2 and net F toxins together with alpha toxin on induction of enterocolitis in horse.

Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens in England and Wales over a 7-year period indicates distribution of clonal toxigenic strains in multiple outbreaks and extensive involvement of enterotoxin-encoding (CPE) plasmids.

Clostridium perfringens is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, only limited whole-genome sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of outbreak-associated C. perfringens strains. We performed phylogenomic analysis on 109 C. perfringens isolates (human and food) obtained from disease cases in England and Wales between 2011 and 2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak C. perfringens strains, when compared to the current Public Health England referencing laboratory technique of fluorescent amplified fragment length polymorphism analysis. Further analysis identified that isogenic C. perfringens strains were associated with nine distinct care-home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected, the enterotoxin cpe gene was encoded in all but 4 isolates (96.3 %; 105/109), with virulence plasmids encoding cpe (particularly pCPF5603 and pCPF4969 plasmids) extensively distributed (82.6 %; 90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7 %; 51/109), and genes encoding phage proteins were also frequently identified. Overall, this large-scale genomic study of gastroenteritis-associated C. perfringens suggested that three major cpe-encoding (toxinotype F) genotypes underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid and (3) chromosomal-cpe strains. Our findings substantially expanded our knowledge on type F C. perfringens involved in human-associated gastroenteritis, with further studies required to fully probe the dissemination and regional reservoirs of this enteric pathogen, which may help devise effective prevention strategies to reduce the food-poisoning disease burden in vulnerable patients, such as the elderly.

Impacts of the prostate stem cell antigen (PSCA) and Clostridium perfringens enterotoxin (CPE) on the apoptosis and cell cycle regulatory genes in PC3

Clostridium perfringens enterotoxin (CPE) has anti-prostate cancer effects and the prostate stem cell antigen (PSCA) has been used as a plasmid-based vaccine. So we expressed both of them in the PC3 cells to evaluate their effects on cell cycling and apoptosis. The PC3 cells were transfected either by the pBudCE4.1-CPE-PSCA or empty plasmid. The expression of the cpe and PSCA genes in transfected PC3 was evaluated. The apoptosis genes (Fas, P53, Bak, and Bax) as well as cell cycling genes (cyclin D1 and E) expression was evaluated by qPCR. Successful expression of cpe and PSCA in PC3 cells was confirmed. The flow cytometry results showed the cellular death rates of 62.6% and 21.8% for PC3 cells transformed with recombinant and empty plasmids respectively. Bak, Fas, Bax and P53 genes were significantly upregulated in PC3 cells transformed with pBudCE4.1-CPE-PSCA, while cyclin D1 and E were downregulated when compared with the pBudCE4.1-transfected PC3 and normal cells (p < .05). The results showed the lethal consequences of cpe and PSCA genes expression on PC3 transfected cells. Expression of the cpe and PSCA genes affects the PC3 cell death so it could be a suitable candidate for further researches in prostate cancer vaccine development.

Why don't corticotroph tumors always produce Cushing's disease?

Silent corticotroph tumors are a pituitary neuroendocrine tumor subtype of corticotroph lineage that do not clinically express Cushing's disease. The silencing of this type of tumor is not fully understood. The aim of the present study was to delve into the lack of secretory activity, studying the post-transcriptional and post-translational regulation of POMC/ACTH in a series of molecularly identified functioning and silent corticotroph tumors. We analyzed 24 silent corticotroph, 23 functioning corticotroph and 25 silent gonadotroph tumors. We used Sanger sequencing, quantitative real-time PCR and Western blot to analyze genetic alterations in POMC, gene expression of TBX19, NEUROD1, POMC, PCSK1, PCSK2, CPE and PAM and protein expression of POMC, PC1/3, PC2, CPE and PAM. We found different polymorphisms in the POMC gene of corticotroph tumors, some of them related to deficiency of proopiomelanocortin. Silent corticotroph tumors showed lower PC1/3 gene and protein expression than functioning ones, especially compared to micro-functioning corticotroph tumors (all P < 0.05). Moreover, we found a positive correlation between PC2 and CPE gene and protein expression (rho ≥ 0.670, P < 0.009) in silent corticotroph tumors compared with functioning ones. By studying the post-transcriptional and post-translational processing of POMC and ACTH, respectively, in a large series of silent and functioning corticotroph tumors, we found that the lack of secretory activity of these tumors is related to an impaired processing of POMC and a high degradation of ACTH, with the macro-functioning corticotroph tumor behaving as an intermediate state between micro-functioning and silent corticotroph tumors.

Small size apolipoprotein(a) isoforms enhance inflammatory and proteolytic potential of collagen-primed monocytes

BackgroundAtherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease.Design and methodsThe aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen.ResultsApo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites.ConclusionsThese results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis.

Application of blood purification in acute lymphoblastic leukemia pediatric patients with hypermethotrexemia accompanied with acute kidney injury

Objective To investigate the efficacy of blood purification for acute lymphoblastic leukemia pediatric patients with high-dose methotrexate(MTX)-induced hypermethotrexemia and acute kidney injury (AKI). Methods The clinical data of 50 acute lymphoblastic leukemia pediatric patients with hypermethotrexemia (the 45th hour MTX blood concentration >20 μmol/L) and AKI who were admitted to Beijing Children's Hospital Capital Medical University from May 2010 to August 2018 were collected. After the treatment of blood purification, the declining rate of MTX concentration, the incidence of drug-related side effects and the clinical transition were analyzed retrospectively. Results The median MTX blood concentration at the 45th hour after high-dose MTX chemotherapy was 31.5 μmol/L (20.0-80.3 μmol/L). After blood purification treatment, 48 patients (96%) survived, 1 patient (2%) died, and 1 patient (2%) gave up treatment. It costed 10.0 days (7.0-15.0 days) to decline the MTX concentration to the normal level by using blood purification. The median time of purification was 32.5 hours (2.0-168.0 hours), and the days of dialysis were 3.0 days (1.0-9.0 days). The AKI occurred in approximately 96% (48/50) of patients, which was the main side effect. The time of declining the high MTX concentration to the normal was positively correlated with the increase times of serum creatinine (r = 0.371, P = 0.009) and urea nitrogen (r = 0.486, P = 0.001), and the value of the alanine aminotransferase (r = 0.364, P = 0.010) and gamma glutamyl transpeptidase (r = 0.344, P = 0.010), and the days of dialysis (r = 0.532, P < 0.01), but there was no relationship with the 45th hour MTX blood concentration (r=0.110, P = 0.248). The reduction of MTX blood concentration from the 45th hour to the 69th hour after high-dose MTX chemotherapy was negatively correlated with the increase times of urea nitrogen (r = -0.336, P = 0.009) and serum creatinine (r = -0.260, P = 0.035). Conclusion When the MTX blood concentration of patients with hypermethotrexemia and AKI couldn't be declined to the normal level by using high-dose leucovorin, hydration and alkalization, and without the effective detoxification drug (carboxypeptidase G2), they should be offered blood purification, especially continuous renal replacement therapy as soon as possible, which can reduce the blood concentration of MTX quickly and decrease the incidence of side effects effectively. Key words: Leukemia, lymphoblastic, acute; Methotrexate; Blood purification

Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate.

Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well-characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of carboxypeptidase D (dCPD) in global neuropeptide processing and selected peptide-regulated behaviours in Drosophila. We found that a deficiency in dCPD results in C-terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD-encoding gene silver in the larva causes lethality, and leads to deficits in starvation-induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing and peptide-regulated behaviour in Drosophila. dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE.

Abnormalities in proinsulin processing in islets from individuals with longstanding T1D

We recently described the persistence of detectable serum proinsulin in a large majority of individuals with longstanding type 1 diabetes (T1D), including individuals with undetectable serum C-peptide. Here, we sought to further explore the mechanistic etiologies of persistent proinsulin secretion in T1D at the level of the islet, using tissues obtained from human donors. Immunostaining for proinsulin and insulin was performed on human pancreatic sections from the Network for Pancreatic Organ Donors with Diabetes (nPOD) collection (n = 24). Differential proinsulin processing enzyme expression was analyzed using mass spectrometry analysis of human islets isolated from pancreatic sections with laser capture microdissection (n = 6). Proinsulin processing enzyme mRNA levels were assessed using quantitative real-time PCR in isolated human islets (n = 10) treated with or without inflammatory cytokines. Compared to nondiabetic controls, immunostaining among a subset (4/9) of insulin positive T1D donor islets revealed increased numbers of cells with proinsulin-enriched, insulin-poor staining. T1D donor islets also exhibited increased proinsulin fluorescence intensity relative to insulin fluorescence intensity. Laser capture microdissection followed by mass spectrometry revealed reductions in the proinsulin processing enzymes prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE) in T1D donors. Twenty-four hour treatment of human islets with inflammatory cytokines reduced mRNA expression of the processing enzymes PC1/3, PC2, and CPE. Taken together, these data provide new mechanistic insight into altered proinsulin processing in long-duration T1D and suggest that reduced β cell prohormone processing is associated with proinflammatory cytokine-induced reductions in proinsulin processing enzyme expression.

EIF4G1 and carboxypeptidase E axis dysregulation in O-GlcNAc transferase–deficient pancreatic β-cells contributes to hyperproinsulinemia in mice

An early hallmark of type 2 diabetes is a failure of proinsulin-to-insulin processing in pancreatic β-cells, resulting in hyperproinsulinemia. Proinsulin processing is quite sensitive to nutrient flux, and β-cell-specific deletion of the nutrient-sensing protein modifier OGlcNAc transferase (βOGTKO) causes β-cell failure and diabetes, including early development of hyperproinsulinemia. The mechanisms underlying this latter defect are unknown. Here, using several approaches, including site-directed mutagenesis, Click O-GlcNAc labeling, immunoblotting, and immunofluorescence and EM imaging, we provide the first evidence for a relationship between the O-GlcNAcylation of eukaryotic translation initiation factor 4γ1 (eIF4G1) and carboxypeptidase E (CPE)-dependent proinsulin processing in βOGTKO mice. We first established that βOGTKO hyperproinsulinemia is independent of age, sex, glucose levels, and endoplasmic reticulum-CCAAT enhancer-binding protein homologous protein (CHOP)-mediated stress status. Of note, OGT loss was associated with a reduction in β-cell-resident CPE, and genetic reconstitution of CPE in βOGTKO islets rescued the dysfunctional proinsulin-to-insulin ratio. We show that although CPE is not directly OGlcNAc modified in islets, overexpression of the suspected OGT target eIF4G1, previously shown to regulate CPE translation in β-cells, increases islet CPE levels, and fully reverses βOGTKO islet-induced hyperproinsulinemia. Furthermore, our results reveal that OGT O-GlcNAc-modifies eIF4G1 at Ser-61 and that this modification is critical for eIF4G1 protein stability. Together, these results indicate a direct link between nutrient-sensitive OGT and insulin processing, underscoring the importance of post-translational O-GlcNAc modification in general cell physiology.

The Dual α-Amidation System in Scorpion Venom Glands.

Many peptides in scorpion venoms are amidated at their C-termini. This post-translational modification is paramount for the correct biological function of ion channel toxins and antimicrobial peptides, among others. The discovery of canonical amidation sequences in transcriptome-derived scorpion proproteins suggests that a conserved enzymatic α-amidation system must be responsible for this modification of scorpion peptides. A transcriptomic approach was employed to identify sequences putatively encoding enzymes of the α-amidation pathway. A dual enzymatic α-amidation system was found, consisting of the membrane-anchored, bifunctional, peptidylglycine α-amidating monooxygenase (PAM) and its paralogs, soluble monofunctional peptidylglycine α-hydroxylating monooxygenase (PHMm) and peptidyl-α-hydroxyglycine α-amidating lyase (PALm). Independent genes encode these three enzymes. Amino acid residues responsible for ion coordination and enzymatic activity are conserved in these sequences, suggesting that the enzymes are functional. Potential endoproteolytic recognition sites for proprotein convertases in the PAM sequence indicate that PAM-derived soluble isoforms may also be expressed. Sequences potentially encoding proprotein convertases (PC1 and PC2), carboxypeptidase E (CPE), and other enzymes of the α-amidation pathway, were also found, confirming the presence of this pathway in scorpions.