Regulation by Ca2+-calmodulin of the actin-bundling activity ofPhysarum 210-kDa protein

Summary From the plasmodia of a lower eukaryote,Physarum polycephalum, we have previously purified a 210-kDa protein that showed similar properties to those of smooth muscle caldesmon. Further characterization of the 210-kDa protein revealed that it bundled actin filaments. This bundling activity was inhibited by calmodulin in the presence of Ca²⁺. Unlike smooth muscle caldesmon, the 210-kDa protein bundled actin filaments whether or not a reducing agent, such as dithiothreitol, was present. The protein was shown to have two (or more) different actin-binding sites which were classified into salt-sensitive and salt-insensitive sites. Electron microscopy revealed that the 210-kDa protein was an elongated molecule (mean length, 97 ± 25 nm) which was bent in the middle. The Stokes radius and sedimentation coefficient of the 210-kDa protein were 130 Å and 2.9 S, respectively. An immunofluorescence study revealed that the 210-kDa protein colocalized with the bundles of actin filaments in thin-spread preparations ofPhysarum plasmodia, suggesting that the 210-kDa protein was regulating the appearance and disappearance of the actin bundles that are associated with the contraction-relaxation cycle of the plasmodia.     

Increased concentrations of soluble tumour necrosis factor receptor (sTNFR) I and II in peritoneal fluid from women with endometriosis

Tumour necrosis factor alpha (TNFalpha), a proapoptotic cytokine, is known to be present in peritoneal fluid from women with endometriosis. An emerging view is that soluble TNF receptors (sTNFR) can modulate the effects of TNFalpha by acting as TNFalpha antagonists. To assess the relevance of sTNFRs in the pathophysiology of endometriosis, concentrations of sTNFR I, sTNFR II and TNFalpha in peritoneal fluid from women with endometriosis (n = 53) and without endometriosis (n = 40) were measured. Concentrations of both sTNFR I and sTNFR II in peritoneal fluid from women with endometriosis were significantly higher than in peritoneal fluid from women without endometriosis, both in the follicular and the luteal phases. TNFalpha concentrations did not differ in patients with and without endometriosis in both phases. When stratified by the stage of the disease, women with both stages I/II and stages III/IV exhibited significantly higher concentrations of sTNFR I and sTNFR II in peritoneal fluid, compared with women without endometriosis, whereas no appreciable difference in the concentrations was detected between stages I/II and stages III/IV. A significant correlation was found between the concentrations of sTNFR I and sTNFR II; while the correlations between TNFalpha and sTNFR I or sTNFR II, were either not significant or were very weak. Furthermore, mRNA for the membrane-associated TNF receptor type 1 and TNF receptor type 2, both of which convey the effects of TNFalpha, were shown to be expressed in endometriotic tissues as well as eutopic endometrium. Together, these findings suggest a possible involvement of sTNFRs in the pathophysiology of endometriosis.     

Increased Antifungal Drug Resistance in Clinical Isolates of Cryptococcus neoformans in Uganda

Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/μl, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC₅₀ of 4 μg/ml and an MIC₉₀ of 8 μg/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC₅₀ to 8 μg/ml and an MIC₉₀ value of 32 μg/ml, with 31% of isolates with a fluconazole MIC of ≥16 μg/ml. We observed an amphotericin B MIC₅₀ of 0.5 μg/ml and an MIC₉₀ of 1 μg/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzed Cryptococcus susceptibility to sertraline, with an MIC₅₀ of 4 μg/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P = 0.52).     

Human papillomavirus DNA in glandular dysplasia and microglandular hyperplasia: presumed precursors of adenocarcinoma of the uterine cervix

Presumed precursors of adenocarcinoma of the uterine cervix were investigated with specific techniques to identify human papillomavirus (HPV) DNA. The presence of HPV DNA in 36 lesions of glandular dysplasia and 16 lesions of microglandular hyperplasia of the uterine cervix was studied by in situ hybridization using 3H-labeled HPV 16 and HPV 18 DNA probes. Only two of 36 lesions (6%) of glandular dysplasia contained HPV 18 DNA, although 64% of coexisting adenocarcinoma in situ, microinvasive adenocarcinoma, and cervical squamous intraepithelial neoplasia III lesions contained HPV 18 and/or HPV 16 DNA. Two lesions of HPV 18 DNA-positive glandular dysplasia coexisted with adenocarcinoma in situ that contained the same type of HPV DNA. None of the microglandular hyperplasia lesions contained HPV 16 DNA or HPV 18 DNA. These results suggest that, if HPV infection is an initial step toward carcinogenesis, it is unlikely that glandular dysplasia and microglandular hyperplasia are precursor lesions of adenocarcinoma of the uterine cervix. A large proportion of glandular dysplasia may represent reactive lesions of endocervical columnar epithelium. Two lesions of HPV 18 DNA-positive glandular dysplasia may represent well-differentiated components of adenocarcinoma in situ of the uterine cervix.     

Incidence of postoperative ileus after paraaortic lymph node dissection in patients with malignant gynecologic tumors

Background Paraaortic lymph node metastasis is an important prognostic factor in gynecologic malignancy. However, paraaortic lymph node dissection (PAND) is not done routinely in Japan because of the difficulty of the procedure and the high incidence of complications. We performed PAND in 217 patients in a 7-year period. In the present study, we focused on the occurrence of postoperative ileus in patients who underwent PAND.Methods Two hundred and seventeen patients with malignant gynecologic tumors were operated on at our hospital between January 1995 and August 2001. All patients underwent PAND and pelvic lymph node dissection (114 patients had a radical hysterectomy; 103 patients had a simple hysterectomy). We evaluated postoperative ileus in three categories of severity.Results The average operation time and blood loss in the patients with radical and simple hysterectomies with PAND were 317 min and 1158 g, and 246 min and 820 g, respectively. The incidence of postoperative ileus was 12.9% (28/217). Although there were no significant differences in the occurrence of ileus between patients with the radical and simple hysterectomies (10.5% vs 15.5%), the incidence of ileus in patients with radical hysterectomy with PAND was significantly higher than that in a control group of patients with radical hysterectomy without PAND (10.5% vs 3.4%). However, in the PAND patients the postoperative ileus was mostly mild or moderate (10 mild cases, 15 moderate cases, and 3 severe cases). Severe ileus occurred in three patients with radical hysterectomy with PAND. Although a repeat operation was necessary for two of these three patients with severe ileus, they recovered uneventfully.Conclusion PAND for malignant gynecologic tumors is a feasible and safe operative procedure, with a low incidence of postoperative ileus.