Changes in connexin 43 protein expression in human endometrial carcinoma

The expression of connexin 43 was studied using immunohistochemical and Western blot analyses on cell lines of endometrial epithelial origin. Connexin proteins were examined because decreases in their expression and function have been correlated with carcinogenesis. The cell lines were chosen to represent increasing grades of endometrial cancer progression starting from FEEC (fetal endometrial epithelial cells; transformed with SV40 large T antigen) to HEC-1A (stage 1A endometrial carcinoma) to RL-95-2 (grade 2 endometrial carcinoma). Parallel studies using connexin 43 polyclonal antibodies for both Western blots and immunofluorescence showed that the levels of connexin 43 expression were normal endometrial stromal cells = FEEC > HEC-1A > RL-95-2. Consequently, we applied the immunofluorescence assay to analyze paraffin-embedded uterine sections from hysterectomy specimens of patients with normal endometrium and from patients diagnosed with grade 1, 2, and 3 endometrial cancer. Using five different cases from each category, we found an inverse correlation between connexin 43 expression and tumor grade. Our data indicate that connexin 43 expression may be useful as an adjunctive marker of progression for endometrial carcinoma.     

Immunodiffusion test for serodiagnosing subcutaneous zygomycosis.

Culture filtrate antigens of Basidiobolus ranarum and Conidiobolus coronatus were analyzed by immunodiffusion (ID) with homologous rabbit antisera. B. ranarum and C. coronatus were each found to have five specific antigens. Results of tests with heterologous antisera indicated that all of the species shared at least one antigen. ID tests incorporating the specific precipitin bands as references were developed for detection of basidiobolomycosis and conidiobolomycosis. These tests were performed with sera from humans and horses with proven basidiobolomycosis and conidiobolomycosis as well as with control sera from humans and animals with and without heterologous mycotic and oomycotic infections. Only sera from cases of basidiobolomycosis and conidiobolomycosis produced lines of identity with the reference precipitates of B. ranarum and C. coronatus, respectively. The ID tests were found to be completely sensitive and specific for determining the etiology of zygomycosis caused by these two species. In addition they appeared useful for monitoring resolution of the infections.     

Risk of localized and widespread endometrial cancer in relation to recent and discontinued use of conjugated estrogens

In a case-control study of the risk of adenocarcinoma of the endometrium in relation to conjugated-estrogen use, we found that 31 per cent of 425 women with endometrial cancer and 15 per cent of 792 controls reported having used conjugated estrogens; the rate-ratio estimate was 3.5 with a 95 per cent confidence interval of 2.6 to 4.7. For use that lasted at least one year, the rate-ratio estimate for Stage I or II cancer was 5.2 (95 per cent confidence interval, 3.7 to 7.2), and for Stages III and IV combined it was 3.1 (1.5 to 6.4). Among women who had used estrogen for at least one year and then discontinued it, the risk of endometrial cancer remained significantly elevated even after estrogen-free intervals of over 10 years. The findings suggest that long-term use of conjugated estrogen increases the risk of both localized and widespread endometrial cancer. The data also suggest that women who have taken conjugated estrogen for one or more years remain at increased risk for at least 10 years after they discontinue use. Such women should be considered for long-term gynecologic surveillance.     

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.      

The effect of ovarian steroids on epithelial ciliary beat frequency in the human Fallopian tube

Using a method that detects variations in light intensity we have studied the effect of ovarian steroids on human Fallopian tube epithelial ciliary beat frequency in vitro. We have found that baseline ciliary beat frequency averages between 5-6 Hz. Cilia from ampullary segments of the Fallopian tube beat significantly faster (5.4 Hz+/-0.2) than those from fimbrial segments (4.8 Hz+/-0.2). There was no significant difference in baseline ciliary beat frequency at any other anatomical site in the Fallopian tube. Incubation with progesterone (10 micromol/l) suppresses human Fallopian tube epithelial ciliary beat frequency by 40-50%. This inhibition was observed at similar magnitudes in all Fallopian tubes studied irrespective of anatomical site. Progesterone-induced reductions in ciliary beat frequency were concentration dependent and prevented by the progesterone receptor antagonist mifepristone (RU486). Oestradiol alone (10 micromol/l) had no effect on ciliary beat frequency at any anatomical site in the Fallopian tube but did prevent the reduction in ciliary beat frequency seen with progesterone when tissues were incubated with these two steroids together.      

A model for antigen-induced T cell unresponsiveness based on autophosphorylative protein tyrosine kinase activity

Helper T cell signaling is initiated by the aggregation of TCRwith the induction of tyrosine kinase activity as one of theearliest consequences. Here, a theoretical model for antigen-inducedunresponsiveness is presented that relies on a cascade of tyrosinephosphorylation- dephoshorylation cycles. A mechanism is describedfor both desensitization in the presence of antigen and persistentlowering of cell responsiveness after stimulus removal. An importantcomponent of the model, leading to bistability, is the presenceof autophosphorylating protein tyrosine kinases in the earlysteps of TCR signaling. One of its predictions is that, followingstimulation, the net phosphorylative activity of these receptor-associatedtyrosine kinases will remain above background level after removalof the antigen. It is proposed that this residual tyrosine kinaseactivity is linked to a deficient signal transduction capacityof the TCR system that leads to a state of prolonged unresponsiveness.In addition, the present analysis defines the notion of a signalingthreshold for hyporesponsiveness induction, associated witha durable switch and amplification of the net tyrosine kinaseactivity. This approach emphasizes the role of tyrosine kinasesin the down-regulation of cellular competence.     

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase epsilon (Pol epsilon), are reduced at stalled replication forks. In contrast, DNA polymerase alpha (Pol alpha) accumulates to higher than normal levels at stalled forks in asf1Delta cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.