Monocots and eudicots have more conservative flower water use strategies than basal angiosperms.

Water balance is crucial for the growth and flowering of plants. However, the mechanisms by which flowers maintain water balance are poorly understood across different angiosperm branches. Here, we investigated 29 floral hydraulic and economic traits in 24 species from ANA grade, magnoliids, monocots, and eudicots. Our main objective was to compare differences in flower water use strategies between basal angiosperms (ANA grade and magnoliids) and derived group (monocots and eudicots). We found that basal angiosperms had richer petal stomatal density, higher pedicel hydraulic diameter, and flower mass per area, but lower pedicel vessel wall reinforcement and epidermal cell thickness compared to monocots and eudicots. We also observed significant trade-offs and coordination among different floral traits. Floral traits associated with reproduction, such as floral longevity and size, were strongly linked with physiological and anatomical traits. Our results systematically reveal the variation in flower economic and hydraulic traits from different angiosperm branches, deepening understanding of flower water use strategies among these plant taxa. We conclude that basal angiosperms maintain water balance with high water supply, whereas monocots and eudicots maintain a more conservative water balance.

[Advances in clinical diagnosis and treatment of drug-induced liver injury in children].

Drug-induced liver injury (DILI) is one of the most common adverse drug reactions that may seriously threaten the health of children and is receiving increasing clinical attention day by day. There is still no independent diagnosis and treatment guideline for DILI in children, but its clinical features are not completely similar to those in adults. This article reviews the epidemiology, clinical features, diagnosis, and treatment progress in order to provide a reference for the management of DILI in children.

[Clinical effect and influencing factors of pegylated interferon alfa-2a and entecavir monotherapy among children with HBeAg-positive chronic hepatitis B based on a real-world study].

Objective: To compare the efficacy, safety, and influencing factors among children with hepatitis B virus e antigen (HBeAg)-positive chronic hepatitis B (CHB) who received short-term therapy with pegylated interferon alfa-2a (Peg-IFNα-2a) or continuous therapy with entecavir (ETV). Methods: Quantitative data were compared using analysis of variance to compare the differences between groups. Enumeration data were compared by χ2 test (or Fisher's exact test). Univariate and multivariate logistic regressions were used to analyze the influencing factors. Results: Peg-IFNα-2a, ETV, and untreated group had HBsAg clearance rates of 46.2%, 5.3%, and 0 after 52 weeks of therapy, respectively. HBsAg clearance in the patients' group with Peg-IFNα-2a and ETV was all accompanied by anti-HBS positive conversion, and the difference was statistically significant (χ2=13.616, P=0.001). Peg-IFNα-2a group was followed-up for 104 weeks. Peg-IFNα-2a, ETV, and the untreated group had HBsAg clearance rates of 46.2%, 10.5%, and 0%, respectively, and the differences were statistically significant (χ2=11.056, P=0.004). Only one of the two children with HBsAg clearance in the ETV group had achieved anti-HBs antibodies, and the difference was statistically significant (χ2=13.616, P=0.001). Univariate and multivariate logistic regression analysis showed that HBsAg clearance was associated with age and antiviral therapy. During treatment, adverse events such as fever (n=4, 30.8%), rash (n=4, 30.8%), fatigue (n=1, 7.7%), leukopenia (n=7, 53.8%), arthritis (n=1, 7.7%), and alopecia (n=3, 23.1%) were observed in the Peg-IFNα-2a group, while none were observed in the ETV group. Conclusion: Peg-IFNα-2a antiviral therapy produced higher HBsAg clearance than ETV in five-year-old and younger children with HBeAg-positive CHB, while ETV had fewer adverse events and was safer than Peg-IFNα-2a.

[Correlation of qAnti-HBc with antiviral efficacy in children with chronic hepatitis B and exploration of its possible immune mechanism].

Objective: To compare the baseline difference in the quantitative hepatitis B core antibody levels (qAnti-HBc) between non-response and response group in children with HBeAg-positive chronic hepatitis B (CHB) who received antiviral therapy, and further explore the proportion and functional activity of CD8 + memory T lymphocyte subsets with different qAnti-HBC levels in peripheral blood of children. Methods: The baseline anti-HBc quantification (qAnti-HBc) levels of 85 children with HBeAg-positive CHB who visited the Department of Infectious Diseases, Children's Hospital of Chongqing Medical University from June 2018 to December 2020 were detected retrospectively. The relationship between the baseline qAnti-HBc level and HBeAg serological response in 37 children who received antiviral therapy was analyzed. The proportion of CD8(+) memory T lymphocyte subsets and the secretion levels of interferon (IFN) γ, and tumor necrosis factor (TNF) α in peripheral blood of 59 children at baseline were detected by flow cytometry. The relationship between qAnti-HBc level and the proportion and functional activity of CD8(+) memory T lymphocyte subsets was analyzed. Pearson's Chi-square test was used to compare the count data. Mann-Whitney U test or Kruskal-Wallis test was used to compare measurement data between two or more groups, and Spearman's rank correlation analysis was used for the correlation between continuous variables. Results: Among 37 children who received entecavir (ETV, 21/37 cases) or pegylated interferon (Peg-IFN, 16/37 cases), 18 cases had developed HBeAg seroconversion (10/ 21 cases in the ETV group, 8/16 cases in the Peg-IFN group). The baseline qAnti-HBc level was significantly higher in the response group [4.71 (4.64~4.81) log(10)IU/ml] than the non-response group children [4.54 (4.45~4.64) log(10)IU/ml, Z = -3.316, P = 0.001]. The proportion of CD8(+) Tem, CD38(+)CD8(+) Tem, CD38(+)CD8(+) Temra cells and the levels of IFNγ and TNFα secreted by CD8(+) T lymphocytes were significantly higher in the high-qAnti-HBc group than the low-qAnti-HBc group (P < 0.05). The proportion of CD8(+) Tem, CD38(+)CD8(+) Tem and CD38(+)CD8(+) Temra cells was significantly higher in ALT > 1× upper limit of normal value (ULN) group than ALT≤1×ULN group (P < 0.05). However, there were no significant differences in the levels of IFNγ and TNFα secreted by CD8(+) T lymphocytes between the two groups (P > 0.05). Spearman's correlation analysis showed that qAnti-HBc was positively correlated with the proportion of CD8(+) Tem, CD38(+)CD8(+) Tem, CD38(+)CD8(+) Temra cells and the level of IFNγ secreted by CD8(+)T lymphocytes (P < 0.05). Additionally, ALT was only positively correlated with the proportion of CD38(+)CD8(+) TEM and CD38(+) CD8(+) Temra cells (P < 0.05). Conclusion: Raised baseline qAnti-HBc level is related to the HBeAg serological response to antiviral therapy in children with CHB. Peripheral blood effector CD8+ T lymphocytes of CHB children with higher qAnti-HBc show stronger phenotype and functional activation characteristics, which may shed some light on the underlying immune mechanism related to antiviral therapy efficacy in children with CHB.

[Analysis of liver pathological characteristics and exploration of noninvasive markers of liver fibrosis in children with chronic hepatitis B].

Objective: To analyze and summarize the characteristics of liver pathology and their relation to clinical markers and further explore noninvasive markers of liver fibrosis in children with chronic hepatitis B. Methods: Data of 80 hospitalized children with chronic hepatitis B who underwent liver biopsy without antiviral treatment from 2011 to 2020 were retrospectively analyzed. Inflammation and liver fibrosis characteristics were analyzed in children of different ages and genders. Variables with good correlation with liver fibrosis stage were selected to establish a non-invasive diagnostic score of liver fibrosis in children. Measurement data was used to compare the t-test or rank sum test. Mantel-Haenszel χ (2) test was used for bidirectional ordered grouping data. Spearman's rank correlation test was used for rank correlation analysis. Receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of the newly established diagnostic score in children with liver fibrosis. Results: The median age of the children was 6.4 years. HBV DNA level was high (P50 = 7.6 log(10) IU/ml), and serum alanine aminotransferase (ALT) in P50 was 171 U/L (< ULN: 5 cases, ULN-2ULN: 10 cases, > 2 ULN: 65 cases). Pathological analysis showed that the incidence of liver tissue inflammation was 97.5%, and the proportion of patients with G≥2 was 42.5%, while S≥2 was 36.3%. The incidence rate of liver fibrosis and liver cirrhosis was 81.3%, and 1.3%, respectively. The changes in liver tissue inflammation and fibrosis were gradually aggravated with the increase of age, and the proportion of high-grade inflammation and liver fibrosis in male children was higher than that in female children. Serum levels of glutamyl transpeptidase (GGT), γ-glutamyltransferase/platelet ratio (GPR) and HBeAg had a good correlation with fibrosis stage (r(s) = 0.397, 0.389, and - 0.311) in children with chronic hepatitis B. The combination of GGT, GPR and HBeAg can establish a non-invasive diagnostic score for evaluating liver fibrosis in children. When the score is less than 1.5, it can be diagnosed as S0, and 1.5 ≤ score < 3.5, it can be diagnosed as S1; 3.5 ≤ score < 5.5, the diagnosis of fibrosis is S2; score≥ 5.5, the diagnosis of fibrosis is S≥3. The sensitivity and specificity were 80%, 83%, 86%, and 53%, 55%, 67%, respectively. Conclusion: The incidence of liver tissue inflammation in children with chronic hepatitis B with elevated and fluctuating transaminase levels is high, and the pathological changes of liver tissue aggravate with the age of the children. GGT, GPR and HBeAg have a good correlation with liver fibrosis in children with chronic hepatitis B. Therefore, combining the above-mentioned markers to establish a new noninvasive diagnostic score has certain diagnostic value for liver fibrosis stage S0-S3 in children with chronic hepatitis B.

[Relationship between phenotype and genotype of ABCB11 deficiency in siblings and literature review].

Objective: To explore the relationship between genotype and phenotype of ABCB11 deficiency. Methods: Clinical data of two siblings with ABCB11 deficiency were retrospectively analyzed. Related literature from PubMed, CNKI and Wangfang databases was reviewed to date (up to August 2017) with 'ABCB11 gene' or 'bile salt export pump', 'cholestasis' and 'child' as key words. Results: The patients were siblings. Both of them presented as jaundice, pruritus and hepatosplenomegaly since 3 days after birth. Significant laboratory findings on admission of the older sister included high total bilirubin, 170 µmol/L;conjugated bilirubin, 115.8 µmol/L;alanine aminotransferase, 168 U/L;total bile acid 186.3 µmol/L and normal gamma-glutamyl transpeptidase. While routine laboratory data of the younger brother were as follows: total bilirubin, 148.8 µmol/L;conjugated bilirubin, 96.3 µmol/L;alanine aminotransferase, 232.8 U/L;total bile acid 226 µmol/L, and normal gamma-glutamyl transpeptidase.Both received ursodeoxycholic acid and fat-soluble vitamins. Liver pathology of the younger brother showed giant hepatocytes with ballooning degeneration, focal necrosis and intrahepatic cholestasis. Both the patients harbor the same compound heterozygous mutations in ABCB11 gene, c.145C>T (p.Q49X) and c.1510G>A (p.E504K). The sister is 9 years old now, with normal liver function. Jaundice faded around 3 months after birth, pruritus relieved at age 5, and medications was stopped since then. The brother progressed to liver failure after an operation on perianal abscess when he was 8-month-old, and received living-related liver transplantation when he was 9 month and 20 days old (from his mother). Now he is 1 year and 5 months old, with normal liver function. Both are under our follow-up. Literature review revealed 18 ABCB11 deficiency patients from 7 families who had apparent different prognoses, within each family the siblings had the same ABCB11 gene mutation. Seven cases relieved after ursodeoxycholic acid therapy and/or partial external biliary diversion, 5 received orthotopic liver transplantation, 2 developed hepatocellular carcinoma and 4 cases died in childhood. Conclusions: The clinical manifestations of ABCB11 deficiency may vary greatly in patients carrying the same genotype, even in siblings. Patients should be managed in individualized maner.

[Etiological analysis and establishment of a discriminant model for lower respiratory tract infections in hospitalized patients].

Objective: To analyze the pathogens of lower respiratory tract infection(LRTI) including bacterial, viral and mixed infection, and to establish a discriminant model based on clinical features in order to predict the pathogens. Methods: A total of 243 hospitalized patients with lower respiratory tract infections were enrolled in Fujian Provincial Hospital from April 2012 to September 2015. The clinical data and airway (sputum and/or bronchoalveolar lavage) samples were collected. Microbes were identified by traditional culture (for bacteria), loop-mediated isothermal amplification(LAMP) and gene sequencing (for bacteria and atypical pathogen), or Real-time quantitative polymerase chain reaction (Real-time PCR)for viruses. Finally, a discriminant model was established by using the discriminant analysis methods to help to predict bacterial, viral and mixed infections. Results: Pathogens were detected in 53.9% (131/243) of the 243 cases.Bacteria accounted for 23.5%(57/243, of which 17 cases with the virus, 1 case with Mycoplasma pneumoniae and virus), mainly Pseudomonas Aeruginosa and Klebsiella Pneumonia. Atypical pathogens for 4.9% (12/243, of which 3 cases with the virus, 1 case of bacteria and viruses), all were mycoplasma pneumonia. Viruses for 34.6% (84/243, of which 17 cases of bacteria, 3 cases with Mycoplasma pneumoniae, 1 case with Mycoplasma pneumoniae and bacteria) of the cases, mainly Influenza A virus and Human Cytomegalovirus, and other virus like adenovirus, human parainfluenza virus, respiratory syncytial virus, human metapneumovirus, human boca virus were also detected fewly. Seven parameters including mental status, using antibiotics prior to admission, complications, abnormal breath sounds, neutrophil alkaline phosphatase (NAP) score, pneumonia severity index (PSI) score and CRUB-65 score were enrolled after univariate analysis, and discriminant analysis was used to establish the discriminant model by applying the identified pathogens as the dependent variable. The total positive predictive value was 64.7%(77/119), with 66.7% for bacterial infection, 78.0% for viral infection and 33.3% for the mixed infection. Conclusions: The mostly detected pathogens were Pseudomonas aeruginosa, atypitcal pathogens, Klebsiella pneumoniae, influenza A virus and human cytomegalovirus in hospitalized patients with LRTI in this hospital. The discriminant diagnostic model established by clinical features may contribute to predict the pathogens of LRTI.

Genetic characteristics of Chinese isolates of the tapeworm Taenia pisiformis based on two mitochondrial genes.

Cysticercosis is caused by infections with embryonated eggs of the tapeworm Taenia pisiformis. Knowledge of the genetic characteristics of T. pisiformis could be applied to study the epidemiology and transmission of this parasite. In this study, 61 isolates of intraperitoneal cysticerci from eight geographically distinct regions in Sichuan province, China, were subjected to a molecular analysis in order to determine their intra-regional genetic characteristics. Partial sequences of the mitochondrial cytochrome c oxidase subunit I (cox1, 1427 bp) and NADH dehydrogenase 1 (nad1, 738 bp) were concatenated. Five haplotypes were identified, and 89.04% of total genetic variation was found in collections of T. pisiformis isolates from a single region. According to the phylogenetic reconstruction, the T. pisiformis isolates from eight regions did not form geographical clusters. Our study highlights the genetic characteristics of T. pisiformis with the aim of accelerating the genetic research and control of cysticercosis.

Protection against Taenia pisiformis larval infection induced by a recombinant oncosphere antigen vaccine.

Taenia pisiformis larvae cause significant health problems to rabbits. At present, it is not known whether the recombinant antigen from the T. pisiformis oncosphere is able to confer protective immunity against T. pisiformis larval infection. The full-length cDNA was cloned into a pET32a (+) vector, and the recombinant protein was then expressed in BL21 (DE3) cells. Vaccination with the purified rTpUbc2 coupled with QuilA was carried out in New Zealand rabbits to evaluate the immunoprotective effect against T. pisiformis infection. The full-length open reading frame of the TpUbc2 gene was 444 bp, and encoded a 16.63-kDa protein. Finally, rTpUbc2 was used to evaluate the ability to induce immunoprotective responses in rabbits. A 79.3-90.8% reduction (P < 0.01) in the recovery of larvae was observed in the experimental group compared to the control group. Specific anti-rTpUbc2 antibodies from immunized rabbits had significantly higher levels of IgG (P < 0.01) compared to the control group; however, no significant difference in IgA levels was found between groups (P > 0.05). Our data support the use of rTpUbc2 as a potential candidate to develop a vaccine against T. pisiformis larvae.

Synonymous codon usage patterns in different parasitic platyhelminth mitochondrial genomes.

We analyzed synonymous codon usage patterns of the mitochondrial genomes of 43 parasitic platyhelminth species. The relative synonymous codon usage, the effective number of codons (NC) and the frequency of G+C at the third synonymously variable coding position were calculated. Correspondence analysis was used to determine the major variation trends shaping the codon usage patterns. Among the mitochondrial genomes of 19 trematode species, the GC content of third codon positions varied from 0.151 to 0.592, with a mean of 0.295 ± 0.116. In cestodes, the mean GC content of third codon positions was 0.254 ± 0.044. A comparison of the nucleotide composition at 4-fold synonymous sites revealed that, on average, there was a greater abundance of codons ending on U (51.9%) or A (22.7%) than on C (6.3%) or G (19.14%). Twenty-two codons, including UUU, UUA and UUG, were frequently used. In the NC-plot, most of points were distributed well below or around the expected NC curve. In addition to compositional constraints, the degree of hydrophobicity and the aromatic amino acids also influenced codon usage in the mitochondrial genomes of these 43 parasitic platyhelminth species.

The diabetes autoantigen ICA69 and its Caenorhabditis elegans homologue, ric-19, are conserved regulators of neuroendocrine secretion.

ICA69 is a diabetes autoantigen with no homologue of known function. Given that most diabetes autoantigens are associated with neuroendocrine secretory vesicles, we sought to determine if this is also the case for ICA69 and whether this protein participates in the process of neuroendocrine secretion. Western blot analysis of ICA69 tissue distribution in the mouse revealed a correlation between expression levels and secretory activity, with the highest expression levels in brain, pancreas, and stomach mucosa. Subcellular fractionation of mouse brain revealed that although most of the ICA69 pool is cytosolic and soluble, a subpopulation is membrane-bound and coenriched with synaptic vesicles. We used immunostaining in the HIT insulin-secreting beta-cell line to show that ICA69 localizes in a punctate manner distinct from the insulin granules, suggesting an association with the synaptic-like microvesicles found in these cells. To pursue functional studies on ICA69, we chose to use the model organism Caenorhabditis elegans, for which a homologue of ICA69 exists. We show that the promoter of the C. elegans ICA69 homologue is specifically expressed in all neurons and specialized secretory cells. A deletion mutant was isolated and found to exhibit resistance to the drug aldicarb (an inhibitor of acetylcholinesterase), suggesting defective neurotransmitter secretion in the mutant. On the basis of the aldicarb resistance phenotype, we named the gene ric-19 (resistance to inhibitors of cholinesterase-19). The resistance to aldicarb was rescued by introducing a ric-19 transgene into the ric-19 mutant background. This is the first study aimed at dissecting ICA69 function, and our results are consistent with the interpretation that ICA69/RIC-19 is an evolutionarily conserved cytosolic protein participating in the process of neuroendocrine secretion via association with certain secretory vesicles.

VAMP2, but not VAMP3/cellubrevin, mediates insulin-dependent incorporation of GLUT4 into the plasma membrane of L6 myoblasts.

Like neuronal synaptic vesicles, intracellular GLUT4-containing vesicles must dock and fuse with the plasma membrane, thereby facilitating insulin-regulated glucose uptake into muscle and fat cells. GLUT4 colocalizes in part with the vesicle SNAREs VAMP2 and VAMP3. In this study, we used a single-cell fluorescence-based assay to compare the functional involvement of VAMP2 and VAMP3 in GLUT4 translocation. Transient transfection of proteolytically active tetanus toxin light chain cleaved both VAMP2 and VAMP3 proteins in L6 myoblasts stably expressing exofacially myc-tagged GLUT4 protein and inhibited insulin-stimulated GLUT4 translocation. Tetanus toxin also caused accumulation of the remaining C-terminal VAMP2 and VAMP3 portions in Golgi elements. This behavior was exclusive to these proteins, because the localization of intracellular myc-tagged GLUT4 protein was not affected by the toxin. Upon cotransfection of tetanus toxin with individual vesicle SNARE constructs, only toxin-resistant VAMP2 rescued the inhibition of insulin-dependent GLUT4 translocation by tetanus toxin. Moreover, insulin caused a cortical actin filament reorganization in which GLUT4 and VAMP2, but not VAMP3, were clustered. We propose that VAMP2 is a resident protein of the insulin-sensitive GLUT4 compartment and that the integrity of this protein is required for GLUT4 vesicle incorporation into the cell surface in response to insulin.

Focal exocytosis of VAMP3-containing vesicles at sites of phagosome formation.

Phagocytosis involves the receptor-mediated extension of plasmalemmal protrusions, called pseudopods, which fuse at their tip to engulf a particle. Actin polymerizes under the nascent phagosome and may propel the protrusion of pseudopods. Alternatively, membrane extension could result from the localized insertion of intracellular membranes into the plasmalemma next to the particle. Here we show focal accumulation of VAMP3-containing vesicles, likely derived from recycling endosomes, in the vicinity of the nascent phagosome. Using green fluorescent protein (GFP) as both a fluorescent indicator and an exofacial epitope tag, we show that polarized fusion of VAMP3 vesicles precedes phagosome sealing. It is therefore likely that targeted delivery of endomembranes contributes to the elongation of pseudopods. In addition to mediating pseudopod formation, receptor-triggered focal secretion of endosomes may contribute to polarized membrane extension in processes such as lamellipodial elongation or chemotaxis.

Prolactin regulation of tissue type plasminogen activator and plasminogen activator inhibitor type-I gene expression in eCG-primed rat granulosa cells in culture.

The present study was designed to investigate the effect of prolactin (PRL) on plasminogen activator inhibitor-I (PAI-I) and tissue type plasminogen activator (tPA) gene expression in eCG-primed granulosa cells in vitro. At 46 h after the hormone treatment, ovaries were removed, and granulosa cells were prepared for culture. Cells were incubated for various times in serum-free medium in the presence or absence of LH and PRL alone or in combination. tPA and PAI-I activities in the media were assayed by fibrin overlay and reverse fibrin autograph, respectively. Cytoplasmic RNA from granulosa cells was prepared using the NP-40 method and was assayed for PAI-I and tPA mRNA levels. We demonstrated the following. 1) PRL increased PAI-I mRNA production in cultured granulosa cells. Inclusion of LH with PRL had a synergistic effect on increasing PAI-I mRNA levels. After 48-h culture, 3-fold increases in PAI-I mRNA levels were seen with LH in combination with PRL as compared with PRL alone. The synergistic increase in PAI-I mRNA levels occurred in a dose- and time-dependent manner. 2) The increase in PAI-I mRNA synthesis by PRL alone, or by PRL in combination with LH, was well correlated with the changes in PAI-I activity and antigen levels in the conditioned media. 3) PRL in the culture also dramatically decreased LH-induced tPA mRNA and activity in a dose- and time-dependent fashion. The decrease in the tPA activity by PRL was also correlated with an increase in the amount of PA-PAI-I complexes in the cell-conditioned media. 4) In situ hybridization of tPA and PAI-I mRNAs in the cultured granulosa cells also showed that PRL was capable of enhancing PAI-I mRNA while diminishing tPA mRNA production induced by LH. This suggests that the dose- and time-dependent decrease in the gonadotropin-induced tPA activity in the culture by the presence of PRL may be due to decreasing tPA mRNA synthesis on one hand and to neutralization of the tPA activity by the increased PAI-I activity on the other.

Effect of mutations in vesicle-associated membrane protein (VAMP) on the assembly of multimeric protein complexes.

The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.

Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.

Prolactin delays gonadotrophin-induced ovulation and down-regulates expression of plasminogen-activator system in ovary.

This study was conducted to determine whether prolactin (PRL) suppresses gonadotrophin-induced ovulation and disturbs the co-ordinated gene expression of tissue type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) in rat ovary. Immature female rats were injected with 10 IU pregnant mare's serum gonadotrophin to stimulate follicle growth, and 48 h received different doses of prolactin followed by 7 IU human chorionic gonadotrophin (HCG). The oviducts were examined for the presence of ova, and the amounts of tPA and PAI-1 mRNA present in the ovary were measured at various times after the hormone treatment. PRL had no significant effect on ovarian weight but caused a dose-dependent decrease in ovulation number. In the control animals receiving HCG alone, 13.3 +/- 1.3 (mean +/- SEM) ova/oviduct were found; while in animals receiving HCG plus 50, 100 or 200 microg PRL, the ovulation number was dose-dependently suppressed by 53.6, 66.9 and 76% respectively at 18 h after treatment. PRL suppression of HCG-induced ovulation was time-dependent. By 24 h after treatment, the number of ova in the oviducts in HCG- and HCG plus PRL-treated groups was not significantly different. PRL also suppressed HCG-induced tPA gene expression in a dose- and time-dependent manner. At all time points examined, tPA mRNA content of whole ovaries and granulosa cells (GC) in PRL-treated groups was lower than in the HCG-treated controls. The activities of PAI-1 in ovarian extracellular fluid (OEF) and PAI-1 mRNA in the theca-interstitial cells (TI) in the PRL-treated groups were higher than in the HCG-treated controls. The highest stimulation by PRL of PAI-1 activity in OEF and of PAI-1 mRNA in TI was observed at 9 h and 6 h after HCG treatment respectively. The localization of tPA and PAI-1 antigens in the ovaries was consistent with changes in the mRNA and activity levels. These data suggest that PRL temporarily delays, but does not completely inhibit, HCG-induced ovulation, which may be caused by a suppression of PA-mediated proteolysis.

Ovulation efficiency is reduced in mice that lack plasminogen activator gene function: functional redundancy among physiological plasminogen activators.

Several lines of indirect evidence suggest that plasminogen activation plays a crucial role in degradation of the follicular wall during ovulation. However, single-deficient mice lacking tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), or PA inhibitor type 1(PAI-1) gene function were recently found to have normal reproduction, although mice with a combined deficiency of tPA and uPA were significantly less fertile. To investigate whether the reduced fertility of mice lacking PA gene function is due to a reduced ovulation mechanism, we have determined the ovulation efficiency in 25-day-old mice during gonadotropin-induced ovulation. Our results reveal that ovulation efficiency is normal in mice with a single deficiency of tPA or uPA but reduced by 26% in mice lacking both physiological PAs. This result suggests that plasminogen activation plays a role in ovulatory response, although neither tPA nor uPA individually or in combination is obligatory for ovulation. The loss of an individual PA seems to be functionally complemented by the remaining PA but this compensation does not appear to involve any compensatory up-regulation. Our data imply that a functionally redundant mechanism for plasmin formation operates during gonadotropin-induced ovulation and that PAs together with other proteases generate the proteolytic activity required for follicular wall degradation.

Secretion of plasminogen activator inhibitor type 1 by cultured ovarian cells obtained from gonadotropin-treated immature rats.

It is demonstrated that i) theca-interstitial compartment synthesizes the majority of plasminogen activator inhibitor type 1 (PAI-1) in the ovary before ovulation, and the follicular wall may therefore serve as a specific barrier with the presence of PAI-1 activity to prevent the secretion of tPA into the extrafollicular compartments; ii) granulosa cells secrete only a small amount of ovarian PAI-1, but synthesize the most of tissue-type plasminogen activator tPA involved in the processes leading to ovulation; iii) since only matured cumulus-oocyte complexes secrete a large amount of tPA and PAI-1, both tPA and PAI-1 activity in the conditioned medium may be used as reliable markers for evaluating oocyte quality for in vitro fertilization.