Direct coupling between meiotic DNA replication and recombination initiation.

During meiosis in Saccharomyces cerevisiae, DNA replication occurs 1. 5 to 2 hours before recombination initiates by DNA double-strand break formation. We show that replication and recombination initiation are directly linked. Blocking meiotic replication prevented double-strand break formation in a replication-checkpoint-independent manner, and delaying replication of a chromosome segment specifically delayed break formation in that segment. Consequently, the time between replication and break formation was held constant in all regions. We suggest that double-strand break formation occurs as part of a process initiated by DNA replication, which thus determines when meiotic recombination initiates on a regional rather than a cell-wide basis.

Diphenylhydantoin: an alternative ligand of a glucocorticoid receptor affecting prostaglandin generation in A/J mice.

Evidence for the binding of 5,5-diphenylhydantoin and glucocorticoids to a common receptor is presented for pulmonary and hepatic cytosols and thymocytes of A/J female mice. The 5,5-diphenylhydantoin-protein complex is absorbed by DNA cellulose, and is incorporated into nuclei, 5,5-Diphenylhydantoin, like glucocorticoids, inhibits the production of prostaglandins in thymocytes. Thus a common receptor is probably responsible for the inhibitory and teratogenic effects of these drugs.

Persistent testicular delta5-isomerase-3beta-hydroxysteroid dehydrogenase (delta5-3beta-HSD) deficiency in the delta5-3beta-HSD form of congenital adrenal hyperplasia.

A partial testicular defect in testosterone secretion has been documented in a pubertal male with a congenital adrenal hyperplasia due to hereditary deficiency of the delta5-isomerase-3beta-hydroxysteroid dehydrogenase enzyme complex (delta5-3beta-HSD). Diagnosis of the enzymatic defect is based on the clinical picture of ambiguous genitalia and salt-losing crisis in infancy, together with high urinary delta5-pregnenetriol and plasma dehydroepiandrosterone when the patient was taken off replacement corticoid treatment. No hormonal response to ACTH or salt deprivation was demonstrable. In addition, in vivo studies revealed a partial enzymatic defect in the testis. Although plasma testosterone was low-normal (250 ng/100 ml), plasma delta5-androstenediol was markedly elevated and rose to a greater extent than testosterone after human chorionic gonadotropin administration. In vitro testicular incubation studies suggested a testicular delta5-3beta-HSD enzyme defect with less delta4 products formed from delta5 precursors than in a control testis. Histochemical studies of the testis were also consistent with this defect. Testicular biopsy revealed spermatogenic arrest, generally diminished Leydig cells, but with focal areas of Leydig cell hyperplasia as well as benign Leydig cell hyperplasia as well as benign Leudig cell nodules within the spermatic cord. In vivo studies of steroid metabolism suggested intact peripheral or hepatic delta5-3beta-HSD activity. These studies imply that delta5-3beta-HSD activity differs in the gonad, adrenal, and peripheral organs. These findings are compatible with the concept that the enzyme complex consists of subunits and/or that enzymes in these organs are under different genetic control.

Missense mutation in exon 7 of the common gamma chain gene causes a moderate form of X-linked combined immunodeficiency.

Clinical and immunologic features of a recently recognized X-linked combined immunodeficiency disease (XCID) suggested that XCID and X-linked severe combined immunodeficiency (XSCID) might arise from different genetic defects. The recent discovery of mutations in the common gamma chain (gamma c) gene, a constituent of several cytokine receptors, in XSCID provided an opportunity to test directly whether a previously unrecognized mutation in this same gene was responsible for XCID. The status of X chromosome inactivation in blood leukocytes from obligate carriers of XCID was determined from the polymorphic, short tandem repeats (CAG), in the androgen receptor gene, which also contains a methylation-sensitive HpaII site. As in XSCID, X-chromosome inactivation in obligate carriers of XCID was nonrandom in T and B lymphocytes. In addition, X chromosome inactivation in PMNs was variable. Findings from this analysis prompted sequencing of the gamma c gene in this pedigree. A missense mutation in the region coding for the cytoplasmic portion of the gamma c gene was found in three affected males but not in a normal brother. Therefore, this point mutation in the gamma c gene leads to a less severe degree of deficiency in cellular and humoral immunity than that seen in XSCID.

A novel X-linked combined immunodeficiency disease.

A novel X-linked combined immunodeficiency disease was found in five living males in an extended family in the United States. The age of the affected males ranged from 2.5 to 34 yr. The most prominent clinical abnormalities were a paucity of lymphoid tissue; recurrent sinusitis, otitis media, bronchitis, and pneumonia; severe varicella; and chronic papillomavirus infections. The principal immunologic features of the disorder were normal concentrations of serum immunoglobulins but restricted formation of IgG antibodies to immunogens; normal numbers of B cells and NK cells but decreased numbers of CD4+ and CD8+ T lymphocytes, particularly the CD45RA+ subpopulations; diminished proliferative responses of blood T cells to allogeneic cells, mitogens and antigens; and decreased production of IL-2 by mitogen stimulated blood lymphocytes. Thus, affected males in this family carry an abnormal gene on their X chromosome that results in a combined immunodeficiency that is distinct from previously reported disorders.

Electrochemical and chemical routes to hydride loss from an iridium dihydride.

With a view towards replacing sacrificial hydrogen acceptors in alkane dehydrogenation catalysis, electrochemical methods for oxidative activation of a pincer-ligated iridium hydride intermediate were explored. A 1H(+)/2e(-) oxidation process was observed in THF solvent, with net hydride loss leading to a reactive cationic intermediate that can be trapped by chloride. Analogous reactivity was observed with the concerted hydride transfer reagent Ph3C(+), connecting chemical and electrochemical hydride loss pathways.

An H-2 linked difference in the binding of dexamethasone to murine hepatic cytosol receptor. A possible role of endogenous modifier(s).

The binding of dexamethasone to its receptor in hepatic cytosol preparations from pregnant mice of four congenic and recombinant strains, C57BL/10, B10.A, B10.A(2R) and B10.A(5R), which have almost identical genetic backgrounds other than the H-2 complex, on day 12 of gestation was analyzed by plotting the binding of ligand against cytosol concentration. The plots of C57BL/10 and B10.A(5R) mice were straight lines, but those of the strains B10.A and B10.A(2R) were upward concave curves. The curvature probably did not result from denaturation of the receptor, as indicated by the time course of the dexamethasone binding and by the fact that at a lower concentration of the ligand, at which the receptor would be less stable, there was less curvature than at a higher concentration of the ligand. The curvature can be explained by the presence of endogenous modifier(s) using an analogy from enzymology. Mathematical analysis, partial removal of the modifier(s) by gel filtration, and mixing of the cytosols from the two types of strains indicated the presence of an unsaturated amount of a modifier(s) in the cytosol of the B10.A and B10.A(2R) strains, and the presence of a saturated amount of the cytosol of the C57BL/10 and B10.A(5R) strains. Thus, the H-2 complex contains a gene(s) which regulates the level of a modifier(s) in hepatic cytosol which affects the binding of glucocorticoid to its hepatic cytosolic receptor.

Malformations in infants of diabetic mothers occur before the seventh gestational week. Implications for treatment.

In the present study we used a developmental morphologic approach to fix the latest time in development at which the malformations commonly reported in infants of diabetic mothers could occur. Developmental morphologic dating shows that the significantly more common congenital malformations in infants of diabetic mothers occur before the seventh week of gestation. This suggests that any therapeutic intervention aimed at decreasing the incidence of congenital malformations must be instituted during the critical early period.

PGE2 prevents anomalies induced by hyperglycemia or diabetic serum in mouse embryos.

Both a high level of D-glucose in the medium and serum from a diabetic rat can induce neural-tube fusion defects and growth retardation in cultured mouse and rat embryos. To test our hypothesis that a deficiency of PGs may be involved in the mechanism of hyperglycemia- and diabetic serum-induced teratogenesis and growth retardation, we added PGE2 to the medium of a whole mouse embryo culture containing either normal rat serum and 52.7 mM D-glucose (hyperglycemic) or diabetic rat serum and 22.2 mM D-glucose (diabetic). After a 24-h culture, 94% of hyperglycemic embryos and 81% of diabetic embryos had neural-tube fusion defects; in addition, the number of somites, the morphological score, and the protein content of the embryos were significantly lower than those of controls. Supplementing the medium with PGE2 at concentrations of 0.028-28.4 nM (hyperglycemic) or 28.4 nM (diabetic) significantly reduced the incidence of neural-tube defects and increased the number of somites, the morphological score, and the protein content. These results strongly support the hypothesis that the teratogenicity of diabetic serum, as well as the teratogenic action of hyperglycemic culture, are mediated through a deficiency of PGs.

Complications of pregnancy and fetal development.

Although the outcome of pregnancy for women with diabetes mellitus has improved in recent years, the infant of the diabetic mother has an increased risk of major clinical problems, particularly in the early neonatal period. These include birth injury due to macrosomia, neonatal hypoglycemia, respiratory distress syndrome, and serious congenital anomalies. Because of the great difficulties encountered during attempts to investigate these problems in clinical research protocols, there is a continuing need to establish appropriate animal models of the diabetic pregnancy. Studies carried out over the past decade, primarily with chemically-induced diabetes have suggested techniques which might be useful. In general, the choice of the animal to be studied will depend on the hypotheses being addressed. For instance, small animals such as rabbits and rats made diabetic with streptozotocin have been successfully used for investigation of fetal lung development. Furthermore, the rat model has been helpful for evaluation of fetal anomalies associated with maldevelopment of the spine and central nervous system. Larger animals, such as the nonhuman primate, are more appropriate for studying placental function and amniotic fluid composition in diabetic pregnancies. The task group on pregnancy and fetal development recommends that animal models of diabetes mellitus be used for a more extensive hormonal and metabolic characterization of diabetic mothers during pregnancy, for investigation of placental physiology with respect to the transfer of substrates from mother to fetus, for systematic and comprehensive study of mechanisms controlling fetal lung development, and for delineation of the pathophysiology of neonatal hypoglycemia. It is further recommended that animal models of spontaneous diabetes such as the BB/W rat be used in future studies dealing with pregnancy and fetal development. Because females with spontaneous diabetes show reduced conception rates, there is a pressing need to enhance the fertility of these animals in order to intensify studies on fetal development.

The efficiency of meiotic recombination between dispersed sequences in Saccharomyces cerevisiae depends upon their chromosomal location.

To examine constrains imposed on meiotic recombination by homologue pairing, we measured the frequency of recombination between mutant alleles of the ARG4 gene contained in pBR322-based inserts. Inserts were located at identical loci on homologues (allelic recombination) or at different loci on either homologous or heterologous chromosomes (ectopic recombination). Ectopic recombination between interstitially located inserts on heterologous chromosomes had an efficiency of 6-12% compared to allelic recombination. By contrast, ectopic recombination between interstitial inserts located on homologues had relative efficiencies of 47-99%. These findings suggest that when meiotic ectopic recombination occurs, homologous chromosomes are already colocalized. The efficiency of ectopic recombination between inserts on homologues decreased as the physical distance between insert sites was increased. This result is consistent with the suggestion that during meiotic recombination, homologues are not only close to each other, but also are aligned end to end. Finally, the efficiency of ectopic recombination between inserts near telomeres (within 16 kb) was significantly greater than that observed with inserts > 50 kb from the nearest telomere. Thus, at the time of recombination, there may be a special relationship between the ends of chromosomes not shared with interstitial regions.

Activated neutrophils and neutrophil activators in human milk: increased expression of CD11b and decreased expression of L-selectin.

Human milk neutrophils and macrophages were examined by flow cytometry to determine whether they displayed phenotypic markers of activation. The markers were CD11b and L-selectin, which are increased or shed, respectively, from the surface of activated neutrophils. Phenotypic features of milk neutrophils and macrophages were similar to blood neutrophils stimulated with fMLP: plasma membrane expression of CD11b was increased and L-selectin was decreased. After blood neutrophils were incubated in acellular milk, their expression of CD11b increased and L-selectin decreased. The activation was not affected by trypsin but was significantly decreased by treating acellular milk with chloroform or ether. Sedimentation studies suggested that particulate fractions of milk were active. Further, the activation was partly blocked by treating target blood neutrophils with cytochalasin B. Thus, human milk neutrophils are activated, and the activation may be due partly to phagocytosis of membranous structures in milk.

Testicular maintenance and masculinized development of the vagina of the androgen-insensitive male rat pseudohermaphrodite.

The vaginae of about one-third of androgen-insensitive male rat pseudohermaphrodites never open nor can they be opened by the administration of estrogen, progesterone or andorgens. The vaginae of the remaining pseudohermaphrodites open significantly later than normal females, and, once open, require the presence of the testes or exogenous estrogen to maintain their patency. These defects in the vaginal development of the pseudohermaphrodite may be due to some perinatal masculinization of the perineum, unlike most of the phenotypically female development exhibited by this animal.

Role of testosterone and its metabolites in the differentiation of the mammary gland in rats.

The capacity of various androgens to virilize the differentiating mammary gland in the female rat fetus has been determined. Testosterone, 5alpha-androstane-3alpha, 17beta-diol (3alpha-diol), and dihydrotestosterone (DHT) virilize the anlagen of the mammary gland by suppressing nipple formation but 5alpha-androstane-3beta, 17beta-diol, androsterone, and dehydroepiandrosterone sulfate do not affect female mammary differentiation. However, unlike the genitalia and wolffian ducts of the female rat fetus in which the masculinizing potency of DHT and 3alpha-diol is greater than that of testosterone, testosterone is more potent than its metabolites DHT and 3alpha-diol, in virilizing the mammary gland. The results suggest that testosterone is the fetal androgen mediating masculine development of the mammary gland.

Masculine-liky hypothalamic-pituitary axis in the androgen-insensitive genetically male rat pseudohermaphrodite.

The hypothalamic-pituitary sex of the androgen insensitive, genetically male rat pseudohermaphrodite was studied by examining its vaginal cytology, response to ovarian transplants and urinary steroidal excretion patterns. More than half the pseudohermaphrodites studied were in constant vaginal estrus, while the remaining rats displayed either persistent diestrus or irregular cyclicity tending towards lengthened estrus. Following gonadectomy and ovarian transplantation, normal females displayed regular 14-day cycles while pseudohermaphrodites remained in constant vaginal estrus. In pseudohermaphrodites with ovarian transplants, only C19 steroids were detected in the urine while females excreted both C21 and C19 steroids. Indicative of the urinary findings, transplants in females had corpora lutea and maturing follicles while grafts from pseudohermaphrodites and males contained follicular cysts and luteinized theca. In addition, distribution and activity of histochemical 3beta-hydroxy-delta5 steroid oxidoreductase were similar in the grafts from pseudohermaphrodites and males, but unlike the females. Although previous reports have shown that much of the sex-dependent differentiation of the genetic male rat pseudohermaphrodite is phenotypically female, our results suggest that the phenotype of the hypothalamic-pituitary axis of this animal is, at least in certain respects, male.

Prepubertal diagnosis of steroid 5 alpha-reductase deficiency.

The diagnosis of 5 alpha-reductase deficiency was proven in two prepubertal patients with male pseudohermaphroditism (MPH). Both had a 46-XY karyotype and were reared as females; one child had been castrated in infancy. Clitoromegaly, urogenital sinus, and short vaginal pouch were present in both; inguinal gonads were palpable in one. The diagnosis was made biochemically by observing characteristic changes in five parameters: 1) abnormally high testosterone to dihydrotestosterone (T:DHT) ratio after hCG stimulation (35 and 53 vs. normal, 11 +/- 3), 2) abnormally high 5 beta-T metabolites in urine (8.1 and 6.0 vs. normal, less than 1),3) deficient conversion of T to DHT during [3H] T infusion (0.3 and 0.4% vs. normal, 5.3 +/- 3), 4) deficient conversion of [14 C] T to 5 alpha-reduced metabolites by nongenital skin fibroblasts (2.2 and 1.9 pmol/microgram DNA/nmol substrate vs. 68.4+/- 7.8 Pmol/microgram DNA/nmol substrate in normal controls), and 5) deficient conversion of [14C]T to DHT in genital skin slices. The fact that this syndrome represents a defect in T metabolism rather in in T binding is demonstrated by the observation that binding of [3H]DHT to cytosol of skin fibroblasts was normal (4.2 dpm/micrograms DNA vs. normal male values of 3.7 +/- 0.64). Thus, the present report suggests that 5 alpha-reductase deficiency can be diagnosed during childhood and even after castration by metabolic studies of nongenital skin fibroblasts and determination of the conversion ratio of [3H]T to [3H]DHT in plasma.

Analysis of the primary sex ratio, sex chromosome aneuploidy and diploidy in human sperm using dual-colour fluorescence in situ hybridisation.

In situ hybridisation technology provides a new tool for chromosome analysis of human spermatozoa. We have used dual-colour fluorescence in situ with probes specific for the X and Y chromosomes and chromosomes 1 and 12 to (a) identify the primary male gametic sex chromosome ratio; (b) assess the number of numerical sex chromosome abnormalities, and (c) quantify the incidence of diploid sperm. We have examined over 60,000 sperm from three normal males and found the primary sex ratio to be indistinguishable from unity. The frequency of hyperhaploid sperm was 0.8, 1.03 and 2.27 per thousand for XX, YY and XY respectively, whilst 1.67 per thousand sperm were diploid. A comparison of our results with estimates of sex chromosome aneuploidy in human populations suggests that sperm carrying two sex chromosomes may be at a selective disadvantage.

Postpartum maternal blood helper T (CD3+CD4+) and cytotoxic T (CD3+CD8+) cells: correlations with iron status, parity, supplement use, and lactation status.

Iron deficiency reduces T cell counts; however, iron sufficiency is difficult to maintain during pregnancy and to reestablish in the early postpartum period. This cross-sectional study examined relations among postpartum maternal iron status, parity, lactation, supplement use, and maternal blood T cell populations. Sixty lactating and 41 nonlactating postpartum (NLPP) women at 1-2 wk and 1, 2, 4, or 8 mo postpartum and 13 nulliparous women were studied. Among multiparous women, multiple linear regression showed that relative percentages and absolute numbers of CD3+CD8+ cells were correlated positively with maternal serum transferrin saturation. In a separate multiple linear regression model, multiparous NLPP women who did not use multivitamin and mineral supplements had lower CD3+CD4+ cell percentages in the first month postpartum than did nulliparous control women. Lactating women who used supplements, however, had reduced CD3+CD4+ percentages 4-8 mo postpartum compared with control women. CD3+CD4+ percentages did not differ among control women, NLPP women who used supplements, or lactating women who did not use them. These results suggest that nutritional factors such as maternal iron status and use of dietary supplements play a role in a mother's postpartum immune status.

Progressive dystonia masking ataxia in ataxia-telangiectasia.

A 10-year-old boy with ataxia-telangiectasia had severe progressive dystonic posturing that masked the ataxia until treatment relieved the dystonia. A younger sister had mor classical neurologic manifestations of the disease. However, both children had telangiectasia, immunologic abnormalities, and other features of ataxia-telangiectasia. The pathologic changes that have been found in the basal ganglia at autopsy and the occurrence of choreoathetosis, oculomotor disturbances, and now dystonia indicate that the function of the basal ganglia in patients with ataxia-telangiectasia is abnormal. Children who have basal ganglial abnormalities should be studied for ataxia-telangiectasia.

Use of C-reactive protein in differentiation between acute bacterial and viral otitis media.

OBJECTIVES: The objectives of this investigation were: (1) to determine degree of elevation of serum C-reactive protein (CRP) in uncomplicated acute otitis media (AOM); (2) to compare serum CRP levels in bacterial and viral otitis media; and (3) to determine whether a single serum CRP level, obtained early in the course of AOM, could be used to differentiate between viral and bacterial otitis media. DESIGN AND METHODS: Sera were obtained from otherwise healthy infants and children with AOM who were 3 months to 7 years of age between 1989 and 1991. Tympanocentesis, bacterial and viral studies of the middle ear fluids, virologic studies of nasal wash specimens, measurements of serum antibody titers to respiratory viruses, blood counts, and quantitation of serum CRP concentrations were performed. After the initial tympanocentesis, an oral antibiotic was given for the next 10 days. The patients were clinically reevaluated over next 4 weeks. OUTCOME MEASURES: Serum CRP concentrations were compared among subjects with AOM who were divided into four groups based on the results of bacteriologic and virologic studies: group I, Bacterial infection (n = 82); group II, bacterial and viral infections (n = 69); group III, viral infection (n = 12); and group IV, no identifiable pathogen (n = 22). RESULTS: There was no statistical difference in serum CRP values among the four groups. The ranges of CRP were less than 0.6 to 22.8, less than 0.6 to 17.8, less than 0.6 to 2.0, and less than 0.6 to 6.8 mg/dL in groups I through IV, respectively. However, when CRP values in bacteria-positive cases were compared with CRP concentrations in bacteria-negative cases (1.58 +/- 3.16 vs 0.64 +/- 1.24 mg/dL), the difference was statistically significant. Furthermore, a significantly higher proportion of bacteria-positive cases had serum CRP concentrations greater than 2 mg/dL, compared with those in bacteria-negative cases. There was no correlation between initial CRP values and clinical findings and/or the clearance of bacteria from the middle ear. After 10 days of antibiotic treatment, CRP values returned to normal (< 0.6 mg/dL) in all cases. CONCLUSION: In AOM, the range of serum CRP varied from less than 0.6 to 22.8 mg/dL. High CRP values (> 2.0 mg/dL) were associated with 22% of cases of bacterial AOM but only with 6% of nonbacterial AOM. High levels of serum CRP were found to be very specific in detecting bacterial AOM, and no cases of viral AOM without a concurrent bacterial infection were found to exhibit high serum levels of CRP.