Flash labelling of S-phase cells in short-term organ culture of normal and pathological human endometrium using bromodeoxyuridine and tritiated thymidine

Normal hyperplastic and malignant endometrial specimens were labelled in vitro with bromodeoxyuridine (BrdU). S-phase cells were stained after DNA denaturation, using a monoclonal antibody to BrdU and an indirect immunoperoxidase method. Various conditions for BrdU uptake, DNA denaturation, and staining were tested. BrdU labelling was compared with autoradiography using tritiated thymidine, with good correlation. Glandular labelling indices of proliferative endometrium were significantly higher than both secretory and hyperplastic endometrium but were similar to carcinoma. Stromal labelling showed the same trend but the differences were not statistically significant.     

Detection and characterisation of the genes encoding glyoxalase I and II from Neisseria meningitidis

Glyoxalase enzymes I and II are involved in a detoxification process consisting of conversion of reactive dicarbonyl compounds (e.g., methylglyoxal) to less reactive hydroxy acids. The structural gene for meningococcal glyoxalase I (gloA) was identified by screening an expression library with a rabbit antiserum. The meningococcal gloA gene consisted of 138 deduced amino acids, with a calculated mol. wt of 15.7 kDa. The DNA and deduced protein sequence of gloA was compared to known sequences of glyoxalase I enzymes and showed high homology with gloA of several eukaryotic and prokaryotic species. Insertion of a gloA-containing plasmid in Escherichia coli increased the host organism's tolerance to methylglyoxal from <2 mM to >4 mM, thus demonstrating its functional identity. A databank search also revealed the presence of a putative gloB gene, encoding glyoxalase II (GlxII), in the recently released genomic sequences of Neisseria meningitidis and N. gonorrhoeae.     

The mouse Mid1 gene: implications for the pathogenesis of Opitz syndrome and the evolution of the mammalian pseudoautosomal region

We have recently reported isolation of the gene responsible for X- linked Opitz G/BBB syndrome, a defect of midline development. MID1 is located on the distal short arm of the human X chromosome (Xp22. 3) and encodes a novel member of the B box family of zinc finger proteins. We have now cloned the murine homolog of MID1 and performed preliminary expression studies during development. Mid1 expression in undifferentiated cells in the central nervous, gastrointestinal and urogenital systems suggests that abnormal cell proliferation may underlie the defect in midline development characteristic of Opitz syndrome. We have also found that Mid1 is located within the mouse pseudoautosomal region (PAR) in Mus musculus , while it seems to be X- specific in Mus spretus. Therefore, Mid1 is likely to be a recent acquisition of the M. musculus PAR. Genetic and FISH analyses also demonstrated a high frequency of unequal crossovers in the murine PAR, creating spontaneous deletion/duplication events involving Mid1. These data provide evidence for the first time that genetic instability of the PAR may affect functionally important genes. In addition, we show that MID1 is the first example of a gene subject to X-inactivation in man while escaping it in mouse. These data contribute to a better understanding of the molecular content and evolution of the rodent PAR.      

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.      

The androgen receptor of the prostate plasma membrane - an hypothesis

The hypothesis that the prostatic plasma membrane sodium pump apparatus functions as a non-genomic androgen receptor is based upon a number of its properties: (1) Androgen enhances the uptake of K(+) into minced rat prostate. (2) Ouabain, a specific inhibitor of Na/K-ATPase activity, strongly opposes the androgenic effect. (3) In non-genomic microsomes, ouabain sensitivity of the enzyme is enhanced by androgen. (4) Kinetic studies show that androgen significantly increases Vmax, Km and energy of activation of the enzyme. (5) Enzyme, treated with [gamma-(32)P]-ATP and then subjected to SDS-PAGE electrophoresis, binds only to its alpha-subunit, but, if treated with [(3)H]-DHT, shows isotope binding to the beta-subunit. (6) [(3)H]-ouabain binding to androgenized enzyme is 5.5 times greater than to the non-androgenized enzyme. (7) Treatment of the enzyme with 10(-9) M DHT enhances by 40% the binding of the ouabain derivative, anthroyl ouabain (AO). (8) Fluorescent spectra appears to show that, upon phosphorylation of the androgenized enzyme, there is a 14% approximation of the two subunits to each other. (9) Except for neuroepithelium, only the epithelium of the prostate has apically located Na/K-ATPase. Preliminary work in other labs suggests that the beta-subunit of the Na/K-ATPase may be required for establishing the polarity of some epithelial cells.     

A longitudinal study of maternal serum inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC and follistatin during pregnancy

Maternal serum concentrations of inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC-related inhibin forms, total follistatin, steroids and gonadotrophins were measured longitudinally in six normal singleton pregnancies. Maternal venous blood was collected randomly during a spontaneous follicular phase prior to donor insemination, at 5, 7, 9, 11, 16, 20, 24, 28, 32 and 36 weeks after the first missed menses and in the early puerperium. Steroid and gonadotrophin profiles conformed to previous reports. While at week 5 of gestation inhibin-A, activin-A and follistatin concentrations were similar to those at the follicular phase, all three increased progressively (P < 0.001) to maximal concentrations in week 36: approximately 48-fold (3740 +/- 1349 ng inhibin-A/ml), approximately 22-fold (6109 +/- 1443 ng activin-A/ml) and approximately 10-fold (3563 +/- 418 ng follistatin/ml) higher. Pro- alphaC concentrations reached a maximum in weeks 5 (approximately 5- fold, P < 0.001) and 36 (1027 +/- 174 pg/ml, P < 0.01). Inhibin-B (71 +/- 23 pg/ml prior to pregnancy) was undetectable (<12 pg/ml) between week 5-16 of gestation but increased slightly in the third trimester (26 +/- 7 pg/ml in week 36). Activin-AB was undetectable throughout pregnancy. Post-partum concentrations of inhibin-A (41 +/- 12 ng/ml), inhibin-B (<12 pg/ml), activin-A (950 +/- 149 pg/ml), pro-alphaC (128 +/- 22 pg/ml) and follistatin (990 +/- 79 ng/ml) were substantially lower than at week 36 of gestation. The activin-A:follistatin ratio increased from 0.5 in week 5 to 1.8 in week 36, suggesting that more free activin-A is available in the maternal circulation during late pregnancy.      

Persistent and selective effects of inflammation on smooth muscle cell contractility in rat colitis

Intestinal inflammation affects smooth muscle contractility contributing to altered motility, but changes to the individual smooth muscle cells are not well described. We used video microscopy to study the contractility of circular smooth muscle cells (CSMC) isolated from the rat mid-descending colon throughout the course of TNBS-induced colitis, measuring their shortening response to carbachol (CCh), 5-HT, histamine or high K⁺. In control CSMC, CCh caused a maximal shortening response of 28 (2%), similar to that for 5-HT of 27 (1%), but by day 4 of colitis, these responses were decreased by 35% and 37%, respectively. By day 36, all aspects of cholinergic contraction returned to control levels, while 5-HT-induced contraction remained significantly attenuated. In contrast, the contractile responses to histamine remained similar at all time points. K⁺-induced contraction was impaired only on day 4, and the maximal response remained substantially greater than CCh or 5-HT. Colitis caused a 121% increase in CSMC length by day 2 that persisted through day 36, independent evidence for phenotypic change. We conclude that impaired CSMC contractility at both the receptor and non-receptor levels contribute to altered smooth muscle function during colitis. Persistent changes in contractile response remained detectable after resolution of inflammation, and similar events may occur in post-enteritis syndromes seen in humans.     

Development and Evaluation of an Interactive CD-ROM Refusal Skills Program to Prevent Youth Substance Use: “Refuse to Use”

An interactive CD-ROM program designed to reduce adolescent substance use was developed and evaluated. The program uses video vignettes to teach refusal skills and socially acceptable responses to substance use situations, specifically offers of marijuana. In a randomized pretest-to-posttest experiment with 74 public school students from six classes in three high schools, significant changes were observed at posttest on (1) the adolescent's personal efficacy to refuse the offer of marijuana, (2) the adolescent's intention to refuse marijuana if offered, and (3) the adolescent's perceptions of the social norms associated with substance use and the importance of respecting another's decision to refuse a drug offer. In addition, adolescents in the treatment condition were able to recall approximately 50% of the portrayed refusal strategies. Findings are discussed with regard to the potential benefits of an interactive multimedia approach for conducting substance use interventions.     

Passive immunity in calf rotavirus infections: maternal vaccination increases and prolongs immunoglobulin G1 antibody secretion in milk.

Ten heifers were inoculated on two occasions with an inactivated preparation of tissue culture-grown calf rotavirus, and a further ten heifers received a placebo vaccine. Serum anti-rotavirus antibody titers were significantly increased throughout pregnancy in the vaccinated group. After calving, the mean neutralizing antibody titer of colostral whey in control cows was 100, associated with immunoglobulins A and G1. No antibody was detected in the milk of these cows after the 4th day postpartum. The colostral whey from the vaccinated cows had a mean antibody titer of 20,452; 28 days after calving, the mean milk antibody titer was 320, associated mainly with immunoglobulin G1. Calves were challenged with a large oral inoculum of calf rotavirus at the 7th day of age. There was significant lengthening of the incubation and prepatent periods in calves born to vaccinated dams, but rotavirus-associated diarrhea of equal severity occurred in both groups. Evidence is presented which suggests that rotavirus antibody in milk can protect against a smaller challenge dose. Maternal immunization against rotavirus may be a practical proposition.