Time course of hypo-osmotic swellings of human spermatozoa: evidence of ordered transition between swelling subtypes

The hypo-osmotic swelling test (HOST or HOS test) usually takes into consideration the total HOS response value with no emphasis either on the value of the response subtypes or the response evaluation time. This study investigated the time course of HOS responses and analysed their physiological relevance. Raw semen spermatozoa and Percoll washed spermatozoa were used in the experiment. The morphological changes in the sperm tail were monitored by incubating the spermatozoa in the hypo- osmotic solution for 16 different time periods. The HOS reactive spermatozoa and the type of HOS reaction (swelling subtypes) of the samples subjected to different duration of treatment were identified under a phase contrast microscope. Also the fate of individual spermatozoa in a hypo-osmotic environment were monitored for 30 min. In spermatozoa exposed to a hypo-osmotic solution, the motility lasted usually less than 2 min and motility characteristics were uniquely different from that of the spermatozoa under iso-osmotic conditions. The HOS response development was permanent but the motility loss due to hypo-osmotic shock was reversible up to 1 min of incubation. There was an indication of ordered transition among the HOS swelling subtypes apparently initiating with subtype b destined to c, d, e, f and g. Further, the subtypes a and g showed gradual decrease and increase, respectively, while subtype b showed abrupt initial increase and then gradual decrease. Transition from b to g could be direct or via one or more than one subtypes. Ultrastructure based analysis indicated that HOS response subtypes are the apparent reflection of the differences in the cytoskeletal assembly of the sperm tail and thus may be identifying different physiological variants in the sperm population. These results indicate that shorter incubation is essential to document the kinetics of various HOS responses but the conventional HOS test misses these important HOS features because of lengthy incubation. Since the time course of ordered transition of HOS responses will vary more than the total HOS response in semen of different aetiologies, the importance of HOS response subtypes and response evaluation time should be taken into consideration when applying HOS test.      

Redox-linked signal transduction pathways for protein tyrosine kinase activation

The signaling for activation of protein tyrosine kinases (PTKs) is usually started by binding of ligands to cell-surface receptors. However, recent evidence suggests the presence of ligand binding-independent signaling pathways that are mediated by oxidative stress. Oxidation and reduction of protein cysteine sulfhydryl (SH) groups may work as a molecular switch to start or to stop the signaling. It is known that oxidation of cysteine SH groups on protein tyrosine phosphatases switches off the action of protein tyrosine phosphatases. This event may not, however, signal for initial autophosphorylation of previously unphosphorylated PTKs, whereas it certainly prevents dephosphorylation of once-phosphorylated PTKs. We have suggested new mechanisms for oxidative stress-mediated PTK activation. First, cell-surface glycosylphosphatidylinositol-anchoring proteins and a phosphoglycolipid/cholesterol-enriched membrane microdomain termed a "raft" can be the direct targets of oxidative stress for inducing their clustering through an S-S-bonded or S-X-S-bonded crosslinking of cell-surface proteins and subsequent activation of raft-associating Src family PTKs. Second, intracellular specific cysteine SH groups on PTK proteins can be another target of oxidative stress for inducing a conformational change necessary for initial activation of PTKs. A possible relationship between cell-surface and intracellular events is that the former frequently induces superoxide production as the second messenger for the latter.     

An optimized forward-planning technique for intensity modulated radiation therapy

Intensity modulated radiation therapy (IMRT) has stirred considerable excitement in the radiation oncology community. Its objective is to make the dose conform to the tumor and spare other organs. Instead of resorting to the rather complex inverse-planning, the technique described here is an extension of the conventional treatment planning technique. The beam orientation and wedge angles are chosen in the conventional rule-based manner. However, within each conformal beam's eye view (BEV) field including margin, a number of sub-field openings are added. The smaller field openings are designed to irradiate the tumor, while sparing the normal tissue of the organs at risk (OARs) that intrude into the target region in the BEV. As the number of intrusions into the target BEV increases, the number of sub-fields for each beam increases. The Cimmino simultaneous projection method was employed to obtain the optimized weighting for each field of each beam. In cases where the dose constraints for the tumor and for the OARs are reasonable, it is possible to obtain a plan with a fairly small number of beams that satisfies the specified dose objectives. This is illustrated for the treatment of prostate cancer, where the rectum creates a concavity in the planning target volume. An advantage of this technique is that the quality assurance for the delivery of these plans does not require extensive special efforts.     

Genetic and biochemical characterization of Malassezia pachydermatis with particular attention to pigment-producing subgroups.

The aim of the study was the characterization of Malassezia pachydermatis and its pigment-producing subgroup using biochemical tests and RAPD. It was of interest to determine whether particular RAPD patterns could be used to indicate pigment production, as well as a close genetic relatedness to Malassezia furfur. Therefore, 210 strains of M. pachydermatis were examined for morphology, catalase and ss-glucosidase activity, lipid and carbohydrate assimilation and the tryptophan-dependent synthesis of pigments. Of these, 114 strains were subjected to RAPD analyses. A multivariate logistic regression model was applied to classify M. pachydermatis isolates regarding their pigment production by using genetic and biological parameters. Biological and RAPD findings showed a high biological and genetic diversity within the species M. pachydermatis and within pigment producers. RAPD analysis revealed 28 genotypes within 114 strains tested. Pigment producing strains could not be assigned to a common RAPD profile, but a genetic relatedness of pigment-producing M. pachydermatis with M. furfur can be assumed. A particular RAPD pattern allowed statistically significant probability of pigment production (P<0.001) and might be used as a tool to rapidly detect pigment producing M. pachydermatis, e.g. in Malassezia-associated pityriasis versicolor. The reported method is useful for identification of pigment producing M. pachydermatis isolates and has advantages over established tests.     

Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1.

Vibrio cholerae serogroup O139, now considered to be the second organism capable of causing epidemic severe dehydrating cholera, contains a capsular polysaccharide which makes it difficult for it to be used in the conventional vibriocidal antibody assay optimized for V. cholerae O1. After modification of the procedure, which involved the use of specific bacterial strains, a lower bacterial inoculum, and increased amounts of complement, the vibriocidal antibody responses to V. cholerae O139 were measured in acute- and convalescent-phase sera from 33 V. cholerae O139-infected and 18 V. cholerae O1-infected patients and in single serum samples from 20 healthy control subjects. The responses in these individuals to V. cholerae O1 strains were also determined. Significant elevations in the homologous antibody response were found only in the convalescent-phase sera from both groups of patients with cholera. These findings may explain the basis for the lack of heterologous protection between the two serogroups of V. cholerae. Healthy controls had higher background levels of vibriocidal antibody to V. cholerae O1 than to V. cholerae O139.     

Characterization of CD4- CD8- CD3+ T-cell receptor-alphabeta+ T cells in murine cytomegalovirus infection

In this study, we have investigated that after the intraperitoneal infection with murine cytomegalovirus (MCMV), the CD3+ CD4- CD8-(double negative; DN) T-cell receptor (TCR)alphabeta+ T cells increased in peritoneal cavity, liver and spleen in both resistant C57BL/6 and susceptible BALB/c mice. The total cellular population of these cells showed peak levels around day 5 after infection in all the three investigated organs and the following phenotypical and functional characteristics emerged. The peritoneal DN TCRalphabeta+ T cells expressed highly skewed TCRVbeta8 on day 5 after infection compared with the uninfected mice, but those in spleen and liver showed moderate and low skewed TCRVbeta8, respectively. The percentages of NK1.1+ DN TCRalphabeta+ T cells gradually decreased as did modulation of some of their activation markers consistent with an activated cell phenotype. The peritoneal DN TCRalphabeta+ T cells on day 5 after infection expressed the genes of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha, Eta-1 (early T-cell activation-1) and MCP-1 (monocyte chemoattractant protein 1) but lacked expression of interleukin-4 (IL-4). After in vitro stimulation with phorbol 12-myristate 13-acetate and calcium ionophore in the presence of Brefeldin A, higher frequencies of intracellular IFN-gamma+ DN TCRalphabeta+ T cells were detected in all three investigated organs of infected mice compared with those of uninfected mice. Stimulation of peritoneal DN TCRalphabeta+ T cells with plate-bound anti-TCRbeta monoclonal antibodies showed proliferation and also produced IFN-gamma but not IL-4. These results suggest that DN TCRalphabeta+ T cells were activated and may have an antiviral effect through producing IFN-gamma and some macrophage-activating factors during an early phase of MCMV infection.     

Analysis of recombinant human tumour necrosis factor-alpha-induced CD4 expression on human eosinophils.

We examined the hypothesis that one of the pro-inflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha), could induce expression of the adhesion molecule CD4 on human eosinophils. We further examined the effector function of CD4 and the mechanisms regulating CD4 expression. Human eosinophils were cultured with various concentrations of recombinant human TNF-alpha (rhTNF-alpha) with or without various drugs for 24 hr. After culture, eosinophils were stained for CD4 using a monoclonal antibody and then analysed by flow cytometry. Eosinophil-derived neurotoxin (EDN) release as eosinophil degranulation was examined by cross-linking of CD4 on eosinophils. The rhTNF-alpha induced CD4 expression on human eosinophils in a dose- and time-dependent fashion; rhTNF-alpha-induced CD4 expression was significantly inhibited by 10(-6) M cycloheximide, 10(-8) M dexamethasone, or 10(-6) M herbimycin A. Recombinant human interferon-gamma inhibited rhTNF-alpha-induced CD4 expression in a dose-dependent manner. However, cross-linking of CD4 on eosinophils did not evoke EDN release, suggesting that newly expressed CD4 molecules on human eosinophils do not play any role in triggering degranulation. Our data indicate that TNF-alpha-induced CD4 expression on human eosinophils is dependent on protein synthesis and may be dependent on tyrosine kinase activity.     

Production, characterization, and application of monoclonal antibodies to Vibrio cholerae O139 synonym Bengal.

Mouse monoclonal antibodies (MAbs) were derived against acetone-treated whole cells of the newly recognized Vibrio cholerae O139 serogroup which is causing epidemics of cholera-like disease in India and Bangladesh. Four MAbs specifically recognized the lipopolysaccharide antigens of V. cholerae O139. MAbs ICL9 and ICL13 were of the immunoglobulin M (IgM) isotype, ICL11 was of the IgG3 isotype, and ICL12 was of the Ig2b isotype. A fifth MAb, ICL10, of the IgG2b isotype cross-reacted with V. cholerae O91. All five MAbs recognized V. cholerae O139 in an enzyme-linked immunosorbent assay, slide agglutination test, motility inhibition test, and indirect immunofluorescence test. During a 1-month evaluation of these MAbs in our clinical laboratory, all 86 cases diagnosed as V. cholerae O139 by a rabbit polyclonal antiserum were also detected by these MAbs, establishing their utility as highly sensitive and specific diagnostic reagents. With these MAbs, it should now be possible to screen for the V. cholerae O139 serogroup in epidemic and endemic diarrhea cases and in environmental and food samples.     

Evidence of [Ca(2+)]i elevation by anti-calreticulin immunoreactive protein in neurons

Calreticulin is a multifunctional Ca(2+)-binding protein. The effect of anti-calreticulin antibody on intracellular free calcium concentration [Ca(2+)]i was studied in cultured neurons using fura-2 based microfluorometry. Anti-calreticulin increased [Ca(2+)]i in a dose dependent manner. The anti-calreticulin antibody produced a rapid transient [Ca(2+)]i peak followed by a long slowly decaying plateau. Anti-calreticulin induced extracellular Ca(2+) influx in cultured neuron cells was blocked partially by N-methyl-D-aspartate receptor (NMDAR) antagonist 2-amino-5-phosphonovaleric acid (AP5) and spider polyamine toxin JSTX-3, which is recognized as a blocker of glutamatergic nervous system. Furthermore, anti-calreticulin induced intracellular Ca(2+) desensitized NMDAR. Dual immunofluorescent staining studies revealed that NMDAR co-localized with calreticulin in the cultured neurons. Thus, the signal transduction system of NMDA might be closely concerned with the extracellular calreticulin like protein.     

Depression,sleeping pattern,and suicidal ideation among medical students in Bangladesh: a cross-sectional pilot study

Journal of Public Health - Depression is a major morbidity and the most common mental disorder among the medical students in medical schools globally. Undergraduate students suffer stress more due...