Src-family kinases are modular signaling proteins involved in a diverse array of cellular processes. All members of the Src family share the same domain organization, with modular SH3, SH2 and kinase ...domains followed by a C-terminal negative regulatory tail. X-ray crystallographic analyses of several Src family members have revealed critical roles for the SH3 and SH2 domains in the down-regulation of the kinase domain. This review focuses on biological, biophysical, and computational studies that reveal conformationally distinct active states within this unique kinase family.
In this study, we characterized the glycome of cervical-vaginal fluid, collected with a Catamenial cup. We quantified: glycosidase levels; sialic acid and high mannose specific lectin binding; ...mucins, MUC1, MUC4, MUC5AC, MUC7; and albumin in the samples collected. These data were analyzed in the context of hormonal status (day of menstrual cycle, hormonal contraception use) and role, if any, of the type of the vaginal microflora present. When the Nugent score was used to stratify the subjects by microflora as normal, intermediate, or bacterial vaginosis, several important differences were observed. The activities of four of six glycosidases in the samples from women with bacterial vaginosis were significantly increased when compared to normal or intermediate women: sialidase, P = <0.001; α-galactosidase, P = 0.006; β-galactosidase, P = 0.005; α-glucosidase, P = 0.056. Sialic acid binding sites as measured by two lectins, Maackia amurensis and Sambucus nigra binding, were significantly lower in women with BV compared to women with normal and intermediate scores (P = <0.0001 and 0.008 respectively). High mannose binding sites, a measure of innate immunity were also significantly lower in women with BV (P = <0.001). Additionally, we observed significant increases in MUC1, MUC4, MUC5AC, and MUC7 concentrations in women with BV (P = <0.001, 0.001, <0.001, 0.02 respectively). Among normal women we found that the membrane bound mucin MUC4 and the secreted MUC5AC were decreased in postmenopausal women (P = 0.02 and 0.07 respectively), while MUC7 (secreted) was decreased in women using levonorgestrel-containing IUDs (P = 0.02). The number of sialic acid binding sites was lower in the postmenopausal group (P = 0.04), but the number of high mannose binding sites, measured with Griffithsin, was not significantly different among the 6 hormonal groups. The glycosidase levels in the cervical-vaginal mucus were rather low in the groups, with exception of α-glucosidase activity that was much lower in the postmenopausal group (P<0.001). These studies present compelling evidence that the vaginal ecosystem responds to the presence of different vaginal microorganisms. These effects were so influential that it required us to remove subjects with BV for data interpretation of the impact of hormones. We also suggest that certain changes occurring in vaginal/cervical proteins are due to bacteria or their products. Therefore, the quantitation of vaginal mucins and lectin binding offers a new method to monitor bacteria-host interactions in the female reproductive tract. The data suggest that some of the changes in these components are the result of host processing, such as the increases in mucin content, while the microflora is responsible for the increases in glycosidases and the decreases in lectin binding. The methods should be considered a valid marker for insult to the female genital tract.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
OBJECTIVE:The reported incidence of acute pelvic inflammatory disease (PID) has decreased but rates of tubal infertility have not, suggesting that a large proportion of PID leading to infertility may ...be undetected. Subclinical PID is common in women with uncomplicated chlamydial or gonococcal cervicitis or with bacterial vaginosis. We assessed whether women with subclinical PID are at an increased risk for infertility.
METHODS:A prospective observational cohort of 418 women with or at risk for gonorrhea or chlamydia or with bacterial vaginosis was recruited. Women with acute PID were excluded. An endometrial biopsy was performed to identify endometritis (subclinical PID). After provision of therapy for gonorrhea, chlamydia and bacterial vaginosis participants were followed-up for fertility outcomes.
RESULTS:There were 146 incident pregnancies during follow-up, 50 pregnancies in 120 (42%) women with subclinical PID and 96 in 187 (51%) women without subclinical PID. Women with subclinical PID diagnosed at enrollment had a 40% reduced incidence of pregnancy compared with women without subclinical PID (hazard ratio 0.6, 95% confidence interval 0.4–0.8). Women with Neisseria gonorrhoeae or Chlamydia trachomatis, in the absence of subclinical PID, were not at increased risk for infertility.
CONCLUSION:Subclinical PID decreases subsequent fertility despite provision of treatment for sexually transmitted diseases. These findings suggest that a proportion of female infertility is attributable to subclinical PID and indicate that current therapies for sexually transmitted diseases are inadequate for prevention of infertility.
LEVEL OF EVIDENCE:II
Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, and Fyn) ...induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family kinases impacts Bcr-Abl protein conformation and signaling.
A longitudinal cohort study of sexually active women 18–30 years of age was conducted to identify variables associated with the acquisition of herpes simplex virus type 2 (HSV-2) infections. Six ...hundred seventy HSV-2–seronegative women were followed up at 4-month intervals for 1 year; acquisition of HSV-2 antibodies was detected in 32 of these women. Black race, ⩽12 years of education, having a new sex partner, and bacterial vaginosis (BV) were associated with HSV-2 seroconversion on univariate analysis. Antecedent HSV-1 infection was not protective against HSV-2 acquisition. After controlling for other identified risk factors in multivariable models, the diagnosis of BV remained associated with an increased risk of acquiring HSV-2 infection (hazard ratio, 2.1; 95% confidence interval, 1.0–4.5; P = .05). In this study, the population attributable risk of BV for HSV-2 seroconversion was 21%. Additional studies are needed to determine whether screening and treatment of BV could reduce susceptibility to the acquisition of HSV-2 in women.
Hepatitis C virus (HCV) infection is increasing among pregnant women because of the opioid epidemic, yet there are no interventions to reduce perinatal HCV transmission or to treat HCV during ...pregnancy. Physiological changes in pregnancy alter the pharmacokinetics of some medications; thus, our aim was to compare the pharmacokinetic parameters of ledipasvir 90 mg plus sofosbuvir 400 mg during pregnancy with non-pregnant women.
This was an open-label, phase 1 study of pregnant women with genotype 1 HCV infection and their infants. A reference group of women who had participated in pharmacokinetic studies of ledipasvir–sofosbuvir during phase 2 and 3 trials was used. Participants were enrolled at Magee-Womens Hospital (Pittsburgh, PA, USA) between 23 and 24 weeks' gestation and had a 12-week course of oral ledipasvir–sofosbuvir (daily 90 mg ledipasvir plus 400 mg sofosbuvir). Three 12-h intensive pharmacokinetic visits were done at 25–26, 29–30, and 33–34 weeks' gestation and individual pharmacokinetics were summarised by geometric mean across the three visits. The primary outcome, analysed in all participants without suspected dosing errors, was the ledipasvir–sofosbuvir area under the concentration–time curve of the dosing interval (AUCtau) during pregnancy compared with the reference group by geometric mean ratio. This study is registered with ClinicalTrials.gov, NCT02683005.
From Oct 1, 2016, to Sept 30, 2018, 29 pregnant women were screened and nine (31%) were enrolled. Eight (89%) women were included in the primary analysis. Ledipasvir and sofosbuvir exposures were similar in the pregnant women versus the non-pregnant reference group (geometric mean ratio of AUCtau ledipasvir 89·3% 90% CI 68·7–116·1; sofosbuvir 91·1% 78·0–106·3).
Ledipasvir–sofosbuvir was safe and effective without clinically meaningful differences in drug exposure among pregnant versus non-pregnant women.
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Institutes of Health/Office of Research on Women's Health, and Gilead Sciences.
The purpose of this study was to determine factors that are associated with recurrent prolapse.
Of 389 women who underwent vaginal prolapse and incontinence between June 1996 and May 1999, 176 women ...had 1-year follow-up evaluations. Recurrent prolapse was analyzed by both pelvic organ prolapse quantification stage and centimeter measurements that were relative to the hymen. Logistic regression was used to determine odds ratios and 95% CI for factors that were associated with recurrent prolapse.
One year after surgery, 102 women (58%) had recurrent prolapse (≥stage II). Seventeen women (10%) had prolapse ≥1 cm beyond the hymen. Age <60 years (odds ratio, 3.2; 95% CI, 1.6-6.4;
P
=
.001) and preoperative pelvic organ prolapse quantification stage III or IV (odds ratio, 2.7; 95% CI, 1.3-5.3;
P
=
.005) were associated with a greater likelihood of recurrent prolapse (≥stage II) at 1 year.
Younger women and women with more advanced prolapse are more likely to experience recurrent prolapse after vaginal repair.
The objective of this study was to evaluate the impact of hormonal status and bacterial vaginosis (BV) on the glycosidases present and glycosylation changes as assessed by lectin binding to ...cervicovaginal lavage constituents. Frozen cervicovaginal lavage samples from a completed study examining the impact of reproductive hormones on the physicochemical properties of vaginal fluid were utilized for the present study. In the parent study, 165 women were characterized as having BV, intermediate or normal microflora using the Nugent criteria. The presence of glycosidases in the samples was determined using quantitative 4-methyl-umbelliferone based assays, and glycosylation was assessed using enzyme linked lectin assays (ELLA). Women with BV had elevated sialidase, α-galactosidase, β-galactosidase and α-glucosidase activities compared to intermediate or normal women (P<0.001, 0.003, 0.006 and 0.042 respectively). The amount of sialic acid (Sambucus nigra, P = 0.003) and high mannose (griffithsin, P<0.001) were reduced, as evaluated by lectin binding, in women with BV. When the data were stratified according to hormonal status, α-glucosidase and griffithsin binding were decreased among postmenopausal women (P<0.02) when compared to premenopausal groups. These data suggest that both hormonal status and BV impact the glycosidases and lectin binding sites present in vaginal fluid. The sialidases present at increased levels in women with BV likely reduce the number of sialic acid binding sites. Other enzymes likely reduce griffithsin binding. The alterations in the glycosidase content, high mannose and sialic acid binding sites in the cervicovaginal fluid associated with bacterial vaginosis may impact susceptibility to viruses, such as HIV, that utilize glycans as a portal of entry.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Self-renewal and differentiation of embryonic stem (ES) cells are regulated by cytokines and growth factors through tyrosine
kinase-dependent signaling pathways. In murine ES cells, signals for ...self-renewal are generated by the leukemia inhibitory
factor (LIF). LIF and other growth factors are linked to the activation of the Src family of cytoplasmic protein-tyrosine
kinases (SFKs), which consists of eight members having shared structural architecture. In this article, we show that murine
ES cells express seven SFKs, three of which (Hck, Src, and Fyn) exhibit constitutive activity in self-renewing ES cells. Differentiation
of ES cells to embryoid bodies was associated with rapid transcriptional silencing of Hck and Lck and with the loss of the
corresponding kinase proteins. The expression of other family members remained relatively constant, although some loss of
Fgr and Lyn proteins was observed during differentiation. Like ES cells, embryoid bodies maintained constitutive Src and Fyn
kinase activity. Partial inhibition of endogenous SFK activity with the ATP-competitive inhibitors 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo3,4- d pyrimidine or Src kinase inhibitor-1 induced differentiation of ES cells in the presence of LIF. In contrast, suppression
of all SFK activity with higher concentrations of these inhibitors, or with the more potent compound A-419259 ( Bioorg Med Chem Lett 12: 1683-1686, 2002) blocked differentiation in response to LIF withdrawal. It is surprising that these inhibitor-treated cells
remained pluripotent despite the absence of LIF. Our results implicate individual members of the Src kinase family in distinct
ES cell renewal and differentiation pathways and show that small-molecule SFK inhibitors can control ES cell fate.
Multiple Src family kinases (SFKs) are present in murine embryonic stem (mES) cells. Whereas complete inhibition of SFK activity blocks mES cell differentiation, sole inhibition of the SFK member ...c-Yes induces differentiation. Thus, individual SFKs may have opposing roles in the regulation of mES cell fate. To test this possibility, we generated SFK mutants with engineered resistance to a nonselective SFK inhibitor. The presence of an inhibitor-resistant c-Src mutant, but not analogous mutants of Hck, Lck, c-Yes, or Fyn, reversed the differentiation block associated with inhibitor treatment, resulting in the formation of cells with properties of primitive ectoderm. These results show that distinct SFK signaling pathways regulate mES cell fate and demonstrate that the formation of primitive ectoderm is regulated by the activity of c-Src.
PDF
