Association between sarcopenic obesity and mortality in patients on peritoneal dialysis: a prospective cohort study.

Background: Whether sarcopenic obesity had unfavorable effect on survival of peritoneal dialysis (PD) patients is unknown. We aimed to investigate the association between sarcopenic obesity and survival in PD patients. Methods: This was a prospective observational study. Eligible PD patients from November 2016 to December 2017 were enrolled and followed until August 31, 2023. Sarcopenia was defined following the recommendations of the Asian Working Group for Sarcopenia (AWGS) as low appendicular skeletal muscle mass index (ASMI) and handgrip strength (HGS). Obesity was defined using the percentage of body fat (PBF). Survival analysis was conducted using the Kaplan-Meier and log-rank test. The Cox regression and the cumulative incidence competing risk (CICR) analyzes were used to investigate the association between sarcopenic obesity and all-cause mortality. Results: A total of 223 patients were enrolled with 133 (59.6%) males, a median age of 57.5 (44.6, 65.7) years, a median dialysis vintage of 20.3 (6.4, 57.7) months and 48 (21.5%) who had comorbid diabetes mellitus. Among them, 46 (20.6%) patients were sarcopenic, and 25 (11.2%) patients were diagnosed with sarcopenic obesity. After followed up for 51.6 (25.6, 73.9) months, the Kaplan-Meier curve showed the sarcopenic obesity (log-rank = 13.527, p < 0.001) group had significant lower survival rate compared to the nonsarcopenic non-obesity group. For multivariate analysis, the CICR method showed patients with sarcopenic obesity had significantly higher mortality rate (HR: 2.190, 95% CI: 1.011-4.743, p = 0.047) compared to those with nonsarcopenic non-obesity. Conclusion: Sarcopenia is not uncommon in PD patients, with a considerable proportion having sarcopenic obesity. There is a significant association between sarcopenic obesity and an increased risk of mortality in PD patients.

Discovery of flavone-derivatives as the new skeleton of transient receptor potential vanilloid 3 channel antagonists.

Transient receptor potential vanilloid 3 (TRPV3) channel is a temperature-sensitive and Ca2+-permeable nonselective cation channel, which is abundantly expressed in skin keratinocyte and plays an important role in skin homeostasis and repair. However, only a few TRPV3 inhibitors were reported. Few selective and potent modulators of the TRPV3 channel have hindered the progress of the investigation and clinical application. TRPV3 channel research still faces challenges and requires the new inhibitors. Flavonoids are a kind of natural compounds with various biological and pharmacological activities including anti-inflammatory and anti allergic effects, which is associated with some physiological effects mediated by TRPV3 channel. Herein, our group designed and synthesized a range of flavone derivatives, and investigated their inhibitory properties on the human TRPV3 channel by electrophysiology technique. Then, we identified a new potent TRPV3 antagonist 2d with IC50 of 0.62 µM. It also showed good selectivity on TRPV1, TRPV4, TRPA1 and TRPM8.

Safety, pharmacokinetics and pharmacodynamics of HRS-7535, a novel oral small molecule glucagon-like peptide-1 receptor agonist, in healthy participants: A phase 1, randomized, double-blind, placebo-controlled, single- and multiple-ascending dose, and food effect trial.

AIM: To assess the safety, tolerability, pharmacokinetics (PKs) and pharmacodynamics of HRS-7535, a novel glucagon-like peptide-1 receptor agonist (GLP-1RA), in healthy participants. MATERIALS AND METHODS: This phase 1 trial consisted of single-ascending dose (SAD), food effect (FE) and multiple-ascending dose (MAD) parts. In the SAD part, participants were randomized (6:2) to receive HRS-7535 (at doses of 15, 60 and 120 mg; administered orally once daily) or placebo. In the FE part, participants were randomized (8:2) to receive a single dose of 90-mg HRS-7535 or placebo, in both fed and fasted states. In the MAD part, participants were randomized (18:6) to receive daily HRS-7535 (120 mg [30/60/90/120-mg titration scheme]) or placebo for 28 days. The primary endpoints were safety and tolerability. RESULTS: Nausea and vomiting were the most frequently reported AEs across all three parts. In the SAD part, the median Tmax was 5.98-5.99 hours and the geometric mean t1/2 was 5.28-9.08 hours across the HRS-7535 dosing range. In the MAD part, the median Tmax was 5.98-10.98 hours and the geometric mean t1/2 was 6.48-8.42 hours on day 28 in participants on HRS-7535. PKs were approximately dose-proportional. On day 29 in the MAD part, the mean (percentage) reduction in body weight from baseline was 4.38 kg (6.63%) for participants who received HRS-7535, compared with 0.8 kg (1.18%) for those participants who received a placebo. CONCLUSIONS: HRS-7535 exhibited a safety and tolerability profile consistent with other GLP-1RAs and showed PKs suitable for once-daily dosing. These findings support further clinical development of HRS-7535 for type 2 diabetes.

A multi-epitope subunit vaccine based on CU/ZN-SOD, OMP31 and BP26 against Brucella melitensis infection in BALB/C mice.

Brucellosis, a zoonosis caused by Brucella, is highly detrimental to both humans and animals. Most existing vaccines are live attenuated vaccines with safety flaws for people and animals. Therefore, it is advantageous to design a multi-epitope subunit vaccine (MEV) to prevent Brucella infection. To this end, we applied a reverse vaccinology approach. Six cytotoxic T cell (CTL) epitopes, seven T helper cell (HTL) epitopes, and four linear B cell epitopes from CU/ZN-SOD, Omp31, and BP26 were obtained. We linked the CTL, HTL, B-cell epitopes, the appropriate CTB molecular adjuvant, and the universal T helper lymphocyte epitope, PADRE, with linkers AAY, GPPGG, and KK, respectively. This yielded a 412-amino acid MEV construct, which we named MEVcob. The immunogenicity, stability, safety, and feasibility of the construct were evaluated by bioinformatics tools (including the AlphaFold2 prediction tool, the AlphaFold2 tool, NetMHC-I pan 4.0 server, IEDB MHC-I server, ABCpred service, and C-ImmSim server); the physicochemical properties, secondary and tertiary structures, and binding ability of MEVocb to toll-like receptor 4 (TLR4) was analyzed. Then, codon adaptation and computer cloning studies were performed. MEVocb is highly immunogenic in immunostimulation experiments, The proteins translated by these sequences were relatively stable, exhibiting a high antigenic index. Furthermore, mouse experiments confirmed that the MEVocb construct could raise IFN-γ, IgG, IgG2a, IgG1, IL-2, TNF-α levels in mice, indicating that induced a specific humoral and cellular immune response in BALB/c mice. This vaccine induced a statistically significant level of protection in BALB/c mice when challenged with Brucella melitensis 043 in Xinjiang. Briefly, we utilized immunoinformatic tools to design a novel multi-epitope subunit candidate vaccine against Brucella. This vaccine aims to induce host immune responses and confer specific protective effects. The study results offer a theoretical foundation for the development of a novel Brucella subunit vaccine.

Selective inhibition of overactive warmth-sensitive Ca2+-permeable TRPV3 channels by antispasmodic agent flopropione for alleviation of skin inflammation.

The temperature-sensitive Ca2+-permeable TRPV3 ion channel is robustly expressed in the skin keratinocytes, and its gain-of-function mutations are involved in the pathology of skin lesions. Here, we report the identification of an antispasmodic agent flopropione that alleviates skin inflammation by selective inhibition of TRPV3. In whole-cell patch clamp recordings, flopropione selectively inhibits macroscopic TRPV3 currents in a concentration-dependent manner with an IC50 value of 17.8 ± 3.5 µM. At the single-channel level, flopropione inhibits TRPV3 channel open probability without alteration of its unitary conductance. In an in vivo mouse model of skin inflammation induced by the skin sensitizer DNFB, flopropione also alleviates dorsal skin lesions and ear skin swelling. Further molecular docking combined with site-directed mutagenesis reveals that two residues E501 and I505 in the channel S2-helix are critical for flopropione-mediated inhibition of TRPV3. Taken together, our findings demonstrate that the spasmolytic drug flopropione as a selective inhibitor of TRPV3 channel not only provides a valuable tool molecule for understanding of TRPV3 channel pharmacology but also holds repurposing potential for therapy of skin disorders, such as dermatitis and pruritus.

Integrated bioinformatics analysis and experimental validation reveals hub genes of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by systemic inflammation, especially synovitis, leading to joint damage. It is important to explore potential biomarkers and therapeutic targets to improve the clinical treatment of RA. However, the potential underlying mechanisms of action of available treatments for RA have not yet been fully elucidated. The present study investigated the potential biomarkers of RA and identified specific targets for therapeutic intervention. A comprehensive analysis was performed using mRNA files downloaded from the Gene Expression Omnibus. Differences in gene expression were analyzed and compared between the normal and RA groups. In addition, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on differentially expressed genes (DEGs). A protein-protein interaction network, Molecular Complex Detection and cytoHubba network were evaluated to identify hub genes. Finally, using an experimental RA rat model induced by Freund's complete adjuvant (FCA), the expression of potential biomarkers or target genes in RA were verified through reverse transcription-quantitative PCR. The results of the mRNA dataset processing revealed 195 DEGs in patients with RA when compared with the healthy controls. Moreover, 10 hub genes were identified in patients with RA and four candidate mRNAs were identified, as follows: Discs large homolog-associated protein 5 (DLGAP5), kinesin family member 20A (KIF20A), maternal embryonic leucine zipper kinase (MELK) and nuclear division cycle 80 (NDC80). Finally, the bioinformatics analysis results were validated by quantifying the expression of the DLGAP5, KIF20A, MELK and NDC80 genes in the FCA-induced experimental RA rat model. The findings of the present study suggested that the treatment of RA may be successful through the inhibition of DLGAP5, KIF20A, MELK and NDC80 expression. Therefore, the targeting of these genes may result in more effective treatments for patients with RA.

Crosstalk of Brucella abortus nucleomodulin BspG and host DNA replication process/mitochondrial respiratory pathway promote anti-apoptosis and infection.

The secretory proteins of Brucella mediate the expression of the bacterium in the host, thereby facilitating intracellular parasitism. With the exception of the recently reported BspJ, the Brucella nucleomodulin has not yet been characterized. We defined the Brucella nucleomodulin BspG and verified six proteins (PCBP1, KMT5C, NDUFS6, PCNA, CIAO2B, and SDHB) that interacted with BspG using a yeast two-hybrid assay and co-immunoprecipitation (CO-IP) screening. The deletion of BspG decreased the intracellular proliferation of B. abortus in both in vivo and in vitro experiments. The analysis found that these interacting proteins were related to energy generation, gene expression, and apoptosis of host cells. The crosstalk between B. abortus nucleomodulin BspG and host DNA replication/mitochondrial respiratory pathways promotes anti-apoptosis and infection, but the mechanism needs additional study.

Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host.

BACKGROUND: Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified. OBJECTIVES: To verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles. METHODS: Constructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro. RESULTS: BspJ gene deletion reduced the survival and intracellular proliferation of Brucella at the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1ß, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella. CONCLUSIONS: BspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.

Brucella abortus BspJ Is a Nucleomodulin That Inhibits Macrophage Apoptosis and Promotes Intracellular Survival of Brucella.

To date, a variety of Brucella effector proteins have been found to mediate host cell secretion, autophagy, inflammation, and other signal pathways, but nuclear effector proteins have not yet been reported. We identified the first Brucella nucleomodulin, BspJ, and we screened out the BspJ interaction host proteins NME/NM23 nucleoside diphosphate kinase 2 (NME2) and creatine kinase B (CKB) through yeast two-hybrid and co-immunoprecipitation assays. These proteins are related to the host cell energy synthesis, metabolism, and apoptosis pathways. Brucella nucleomodulin BspJ will decrease the expression level of NME2 and CKB. In addition, BspJ gene deletion strains promoted the apoptosis of macrophages and reduced the intracellular survival of Brucella in host cells. In short, we found nucleomodulin BspJ may directly or indirectly regulate host cell apoptosis through the interaction with NME2 and CKB by mediating energy metabolism pathways in response to the intracellular circulation of Brucella infection, but the mechanism needs further study.

Discovery of [1,2,4]triazolo[4,3-a]pyridines as potent Smoothened inhibitors targeting the Hedgehog pathway with improved antitumor activity in vivo.

Triple-negative breast cancer (TNBC), a subset of breast cancers, have poorer survival than other breast cancer types. Recent studies have demonstrated that the abnormal Hedgehog (Hh) pathway is activated in TNBC and that these treatment-resistant cancers are sensitive to inhibition of the Hh pathway. Smoothened (Smo) protein is a vital constituent in Hh signaling and an attractive drug target. Vismodegib (VIS) is one of the most widely studied Smo inhibitors. But the clinical application of Smo inhibitors is limited to adult patients with BCC and AML, with many side effects. Therefore, it's necessary to develop novel Smo inhibitor with better profiles. Twenty [1,2,4]triazolo[4,3-a]pyridines were designed, synthesized and screened as Smo inhibitors. Four of these novel compounds showed directly bound to Smo protein with stronger binding affinity than VIS. The new compounds showed broad anti-proliferative activity against cancer cell lines in vitro, especially triple-negative breast cancer cells. Mechanistic studies demonstrated that TPB15 markedly induced cell cycle arrest and apoptosis in MDA-MB-468 cells. TPB15 blocked Smo translocation into the cilia and reduced Smo protein and mRNA expression. Furthermore, the expression of the downstream regulatory factor glioma-associated oncogene 1 (Gli1) was significantly inhibited. Finally, TPB15 demonstrated greater anti-tumor activity in our animal models than VIS with lower toxicity. Hence, these results support further optimization of this novel scaffold to develop improved Smo antagonists.

JMY expression by Sertoli cells contributes to mediating spermatogenesis in mice.

Sertoli cells are crucial for spermatogenesis in the seminiferous epithelium because their actin cytoskeleton supports vesicular transport, cell junction formation, protein anchoring, and spermiation. Here, we show that a junction-mediating and actin-regulatory protein (JMY) affects the blood-tissue barrier (BTB) function through remodeling of the Sertoli cell junctional integrity and it also contributes to controlling endocytic vesicle trafficking. These functions are critical for the maintenance of sperm fertility since loss of Sertoli cell-specific Jmy function induced male subfertility in mice. Specifically, these mice have (a) impaired BTB integrity and spermatid adhesion in the seminiferous tubules; (b) high incidence of sperm structural deformity; and (c) reduced sperm count and poor sperm motility. Moreover, the cytoskeletal integrity was compromised along with endocytic vesicular trafficking. These effects impaired junctional protein recycling and reduced Sertoli cell BTB junctional integrity. In addition, JMY interaction with actin-binding protein candidates α-actinin1 and sorbin and SH3 domain containing protein 2 was related to JMY activity, and in turn, actin cytoskeletal organization. In summary, JMY affects the control of spermatogenesis through the regulation of actin filament organization and endocytic vesicle trafficking in Sertoli cells.

Development of an effective microsatellite marker system to determine the genetic structure of Meriones meridianus populations.

Understanding the genetic quality of the gerbil, Meriones meridianus, plays an important role in the study of medical biology. However, no effective system has been established for evaluating a population's genetic diversity to date. In the present study, we established a set of reasonable evaluative systems based on microsatellite markers of the Mongolian gerbil by using the method of cross-amplification of species. Following electrophoresis analysis, short tandem repeat (STR) scanning, and sequencing, 11 microsatellite loci were identified by matching the criteria characteristics and were used to evaluate the genetic diversity of two stocks of Meriones meridianus: Meriones meridianus jei Wang, 1964 (M. m. jei) and Meriones meridianus cryptorhinus Blanford, 1875 (M. m. cryptorhinus) from Xinjiang, China. The microsatellite loci screened were highly polymorphic and were suitable for genetic quality control of Meriones meridianus. In addition, the quality of the non-bred M. m. jei and M. m. cryptorhinus strains in our study is sufficient for them to be promising stocks in the future for the farmed animal industry.

Isolation and identification of respiratory tract and intestinal microflora of Meriones meridianus in a conventional animal facility.

BACKGROUND: Culturable bacterial species from the respiratory tract and ileocecal junction of Meriones meridianus (midday gerbils) captured in the Xinjiang Luntai area were isolated and identified to confirm the microflora and develop approaches for biological purification of laboratory animals and relevant microbial precautions. METHODS: Bacteria from respiratory tracts and ileocecal junctions of 30 wild M. meridianus were harvested and isolated by inoculation into culture media. Isolated strains were confirmed by mass spectrometry and 16S rRNA sequencing. RESULTS: Thirty-nine bacterial species from 20 families and 27 genera were identified and isolated from wild M. meridianus. Typical bacteria were Enterobacteriaceae, Enterococcus, and Staphylococcus aureus, and the most common microflora were Vibrio, Staphylococcus aureus, and Pseudomonas aeruginosa. CONCLUSION: Wild M. meridianus carries multiple bacteria, most of which are pathogenic or conditional pathogens. This study provides a basis for the development of animal models and laboratory animals from wild M. meridianus.

Mononuclear Nickel(II) Complexes with Schiff Base Ligands: Synthesis, Characterization, and Catalytic Activity in Norbornene Polymerization.

The nickel(II) catalyst has manifested higher catalytic activity compared to that of other late transition metal catalysts for norbornene polymerization. Therefore, several structurally similar trans-nickel(II) compounds of N,O-chelate bidentate ligands were synthesized and characterized. Both the electronic effect and the steric hindrance influence polymerization. The molecular structures of 2, 4 and 5 were further confirmed by single-crystal X-ray diffraction.

Risk of cardiovascular morbidity with risperidone or paliperidone treatment: analysis of 64 randomized, double-blind trials.

A post hoc analysis of the risperidone (RIS)/paliperidone (Pali) clinical trials database comprising 64 studies was conducted. Risk of sudden death, cardiovascular (CV), and cerebrovascular events during RIS or Pali treatment was estimated. Treatment emergent CV adverse events were identified using 7 prespecified Standardised MedDRA Queries as follows: embolic/thrombotic events, cerebrovascular disorders, ischemic heart disease, cardiac arrhythmias, cardiac failure, torsades/QT prolongation, and convulsions. Risk in the RIS/Pali pooled group was significantly increased compared to placebo for the following adverse events: syncope, tachycardia, palpitations, edema peripheral, dysarthria, and transient ischemic attack. Incidence of death related to CV events was low and similar across groups. Consistent with the known pharmacologic profile and product information, this analysis of treatment emergent adverse event data from a large, randomized, controlled clinical trials database described increased risk versus placebo for several specific CV events. Apart from events described in existing product labeling, no new safety findings emerged.

Loperamide and P-glycoprotein inhibition: assessment of the clinical relevance.

OBJECTIVES: Loperamide is a peripherally acting mu opioid receptor agonist and an avid substrate for P-glycoprotein. This may give rise to drug-drug interactions and increased risk for central adverse effects. The objective of this study was to re-evaluate the predictability of non-clinical data using loperamide as a probe P-glycoprotein substrate. We searched the literature for papers containing data on drug-drug interactions of loperamide-containing products in humans. We also reviewed the internal worldwide safety database of Johnson & Johnson for spontaneous case reports suggestive of a central opioid effect after coadministration of loperamide with a P-glycoprotein inhibitor or substrate. KEY FINDINGS: Only one of the ten studies in our review supported the finding that inhibition of P-glycoprotein is associated with clinically relevant signs or symptoms of central nervous system (CNS) depression/opioid toxicity of loperamide. None of the 25 spontaneous case reports of interest were suggestive of signs or symptoms of CNS depression/opioid toxicity due to coadministration of loperamide and a P-glycoprotein inhibitor or substrate. SUMMARY: Based on a review of the literature and a cumulative review of the spontaneous case reports, there is insufficient evidence that an interaction between loperamide and a P-glycoprotein inhibitor or substrate is associated with clinical symptoms of CNS depression/opioid toxicity when loperamide is taken at the recommended dose.

Molecular determinants of synapsin targeting to presynaptic terminals.

Although synapsins are abundant synaptic vesicle proteins that are widely used as markers of presynaptic terminals, the mechanisms that target synapsins to presynaptic terminals have not been elucidated. We have addressed this question by imaging the targeting of green fluorescent protein-tagged synapsins in cultured hippocampal neurons. Whereas all synapsin isoforms targeted robustly to presynaptic terminals in wild-type neurons, synapsin Ib scarcely targeted in neurons in which all synapsins were knocked-out. Coexpression of other synapsin isoforms significantly strengthened the targeting of synapsin Ib in knock-out neurons, indicating that heterodimerization is required for synapsin Ib to target. Truncation mutagenesis revealed that synapsin Ia targets via distributed binding sites that include domains B, C, and E. Although domain A was not necessary for targeting, its presence enhanced targeting. Domain D inhibited targeting, but this inhibition was overcome by domain E. Thus, multiple intermolecular and intramolecular interactions are required for synapsins to target to presynaptic terminals.

Regulation of neurotransmitter release by synapsin III.

Synapsin III is the most recently identified member of the synapsin family, a group of synaptic vesicle proteins that play essential roles in neurotransmitter release and neurite outgrowth. Here, through the generation and analysis of synapsin III knock-out mice, we demonstrate that synapsin III regulates neurotransmitter release in a manner that is distinct from that of synapsin I or synapsin II. In mice lacking synapsin III, the size of the recycling pool of synaptic vesicles was increased, and synaptic depression was reduced. The number of vesicles that fuse per action potential was similar between synapsin III knock-out and wild-type mice, and there was no change in the quantal content of EPSCs; however, IPSCs were greatly reduced in synapsin III-deficient neurons. The density and distribution of synaptic vesicles in presynaptic terminals did not appear to be different in synapsin III knock-out mice in comparison to wild-type littermates. In addition to the changes in neurotransmitter release, we observed a specific delay in axon outgrowth in cultured hippocampal neurons from synapsin III knock-out mice. Our data indicate that synapsin III plays unique roles both in early axon outgrowth and in the regulation of synaptic vesicle trafficking.

Differential regulation of exocytosis by alpha- and beta-SNAPs.

We examined the role of SNAPs, soluble proteins that attach N-ethylmaleimide-sensitive factor (NSF), in regulating exocytosis in single rat adrenal chromaffin cells. Whole-cell dialysis of Ca2+-buffered solution or photolysis of caged-Ca2+ was used to manipulate cytosolic Ca2+ concentration ([Ca2+]i), whereas exocytosis was measured via carbon fiber amperometry or membrane capacitance. Buffering [Ca2+]i to approximately 170 nm produced a mean rate of exocytosis of approximately one amperometric event per minute. Including alpha-SNAP (60 or 500 nm) in the intracellular solution dramatically increased the mean rate of exocytosis. The stimulatory action of alpha-SNAP requires ATP hydrolysis mediated via NSF, because this action was blocked by intracellular dialysis of ATP-gamma-S (2 mm) and could not be mimicked by a mutant alpha-SNAP that does not stimulate the ATPase activity of NSF. This action of alpha-SNAP was significant only at [Ca2+]i between 100 and 300 nm and was not shared by beta-SNAP (500 nm), suggesting that alpha-SNAP enhanced a component of exocytosis that is regulated by a high-affinity Ca2+ sensor. In cells dialyzed with both alpha- and beta-SNAP, the rate of exocytosis was smaller than that produced by alpha-SNAP alone, suggesting that alpha- and beta-SNAP interact competitively. Although only alpha-SNAP stimulated exocytosis at [Ca2+]i between 100 and 300 nm, both alpha- and beta-SNAP isoforms equally slowed the time-dependent rundown of the exocytic response. Our results indicate that alpha- and beta-SNAP have different actions in exocytosis. Thus, the ratio of different isoforms of SNAPs can determine release probability at the levels of [Ca2+]i that are involved in regulation of exocytosis.