Clarification of chromosomal abnormalities associated with sexual ambiguity by studies with Y-chromosomal DNA sequences

Cases of gonadal dysgenesis, both Turner syndrome and mixed, were studied with Y centromeric and short-arm probes. The Y-centromeric alphoid repeat clone, Y97, allowed sensitive detection of Y-chromosomal material in marker chromosomes or mosaics by in situ analysis or Southern hybridization with purified DNA. The Y short-arm probe, p75/79, allowed detection of sequences normally associated with proximal Yp by Southern analysis. The presence of DNA fragments characteristic of Yp correlates well with partial male sexual differentiation in the cases of mixed gonadal dysgenesis. Thus, the combined use of molecular and cytogenetic techniques has proven to be a powerful approach to the analysis of chromosomal sex disorders.     

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies

We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.     

Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism.

The mechanism by which CD8+ T cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in acutely infected CD4+ T cells was investigated. Cytotoxicity was not involved, as the antiviral activity of the CD8+ cells did not correlate with the ability to lyse HIV-infected or uninfected CD4+ T cells. In addition, the frequency of HIV-infected CD4+ cells increased during coculture with CD8+ T cells even in the absence of detectable levels of virus replication. Moreover, separation of the CD4+ and CD8+ cells by a 0.4-micron-pore-size filter delayed HIV replication, indicating a role, at least in part, for a soluble factor. However, cell contact was required for optimal antiviral activity. These results extend further the observation on the mechanism of antiviral HIV activity by CD8+ cells from infected individuals. They support the conclusion that CD8+ cells can play a major role in preventing development of disease in HIV-infected individuals.     

EMG muscle scanning: comparison to attached surface electrodes.

Electromyographic (EMG) muscle scanning measures brief samples of integrated muscle action potentials from individual muscles using a hand-held scanner with post-style electrodes. This "scanning" technique is widely used by biofeedback practitioners to quickly assess muscle activity in the diagnosis of musculoskeletal disorders. In an effort to compare muscle scanning with the established technique using attached surface electrodes, ten healthy subjects (25-35 years old) were scanned using 2-second sampling at five bilateral muscle sites while simultaneously monitoring the same sites with surface electrodes. This was repeated using 10-second scanning samplings. Pearson's product-moment correlations between scanning for 2 seconds and prolonged surface recording at all sites were 0.54-0.89. Scanning for 10 seconds improved the correlations to 0.68-0.91. EMG scanning for 2 seconds compares favorably with attached surface electrode recording. Comparisons are further improved by 10-second scans.     

Isotope-edited proton NMR study on the structure of a pepsin/inhibitor complex

A general approach is illustrated for providing detailed structural information on large enzyme/inhibitor complexes using NMR spectroscopy. The method involves the use of isotopically labeled ligands to simplify two-dimensional NOE spectra of large molecular complexes by isotope-editing techniques. With this approach, the backbone and side-chain conformations (at the P2 and P3 sites) of a tightly bound inhibitor of porcine pepsin have been determined. In addition, structural information on the active site of pepsin has been obtained. Due to the sequence homology between porcine pepsin and human renin, this structural information may prove useful for modeling renin/inhibitor complexes with the ultimate goal of designing more effective renin inhibitors. Moreover, this general approach can be applied to study other biological systems of interest such as other enzyme/inhibitor complexes, ligands bound to soluble receptors, and enzyme/substrate interactions.     

Changes in parameters of lipoprotein metabolism during rat hepatic development

Maintenance of whole body cholesterol homeostasis is determined in part by the liver. Thus, changes in expression of hepatic parameters important in the regulation of cholesterol metabolism may play key roles in determining how homeostasis is maintained. The expression of hepatic lipoprotein uptake systems was studied during development using as a ligand very-low density lipoproteins rich in apolipoprotein E that had been obtained from hypercholesterolemic adult rats. These lipoproteins can serve as ligands for cell surface receptors recognizing apolipoproteins B and/or E. Uptake was lowest in freshly isolated fetal rat hepatocytes, increased substantially in hepatocytes from neonates and was intermediate in those from adults. Binding of these lipoproteins to liver membranes prepared from fetal, neonatal, suckling, weaned and adult rats was lowest in fetal preparations, while those from suckling, weaned and adult livers behaved similarly. Numbers of binding sites in neonatal liver membranes were similar to those in adult, but showed a different affinity. On the basis of this data, the ability of hepatocytes to recognize and remove apolipoprotein B/E-containing lipoproteins from the plasma appears to be a function of the differential expression or regulation of lipoprotein-uptake systems during development.     

Evidence for protein kinase C regulation of ovarian theca-interstitial cell androgen biosynthesis

The hypothesis that protein kinase C may be an important regulator of ovarian theca-interstitial cell steroidogenesis was tested by using phorbol-12-myristate-13-acetate (PMA) and phorbol-12, 13-dibutyrate (PDB) to directly stimulate protein kinase C activity. Collagenase-dispersed cells (4 x 10(5) viable cells/dish) form ovaries of hypophysectomized immature rats were cultured in serum-free medium in the presence and absence of 0-100 ng/ml of luteinizing hormone (LH), PMA (0-100 nM), and/or PDB (0-100 nM). Treatment with 100 ng/ml LH stimulated androsterone production 100-fold at Day 4 of culture. The presence of 100 nM PMA or PDB had no effect on basal androsterone production; however, treatment with increasing concentrations of PMA or PDB (0-100 nM) caused a dose-related inhibition (maximum 70%) of LH-stimulated androsterone synthesis (ID50 = 1.8 nM and 2.4 nM, respectively). PMA and PDB did not significantly alter DNA, protein, or cell viability, indicating that their inhibitory effects were not due to changes in cell number or viability. Cells treated with LH and 100 nM 4 alpha-phorbol didecanoate (4 alpha-PDD; a phorbol ester that does not activate protein kinase C) failed to show significant decreases in androsterone production. Time-course studies revealed that when PMA treatment was delayed until Day 2 or 4 of culture, dramatic inhibitory effects on LH-stimulated androsterone production were still observed. These results suggest that the biological activity of protein kinase C is retained after the cells have expressed their differentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luteinizing hormone-independent luteinization and ovulation in the hypophysectomized rat: a possible role for the oocyte?

Immature hypophysectomized, estrogen-treated rats were used to study the regulation of luteinization. Particular attention was focused on the potential role of the oocyte in this process. Rats were injected for 2 days with follicle-stimulating hormone (FSH) to stimulate follicular development. Within 48 h following FSH treatment, many follicles became luteinized, as determined by morphometric analysis. This luteinization occurred in the absence of detectable levels of luteinizing hormone (LH). The number of follicles undergoing luteinization was dependent on the FSH dose. In addition, ovulation occurred in some of the animals receiving the highest doses of FSH (3-micrograms or 5-micrograms injections). The majority of follicles undergoing luteinization or ovulation were greater than 400 microns in diameter. Luteinized follicles exhibited positive reactivity for cholesterol side-chain cleavage enzyme, 3 beta-hydroxysteroid dehydrogenase, lipid, and alkaline phosphatase, which was similar to that found in corpora lutea of the cycle. Serum progesterone (P0) and 20 alpha-hydroxypregn-4-en-one levels were elevated in animals with luteinized follicles, especially in those animals that also underwent ovulation. Morphological evaluation of oocytes showed that the majority of luteinized follicles contained a degenerating oocyte. Oocyte degeneration was highly correlated (r = 0.94) to luteinization. These results demonstrate that luteinization and ovulation can occur in the FSH/estrogen-primed hypophysectomized rats in the absence of detectable serum LH. Furthermore, LH-independent luteinization was strongly correlated to degenerative changes in the oocyte. These results provide new evidence to support the concept that the oocyte may be an intraovarian regulator of luteinization.     

Lateral packing of protofibrils in fibrin fibers and fibrinogen polymers

The distinctive transverse banding pattern of fibrin fibers clearly indicates ordering of molecules in the longitudinal direction. In this study we examined the fibers of fibrin clots, as well as two types of fibrinogen polymers, by thin-section electron microscopy. The fibrinogen polymers have a transverse banding pattern identical to that of fibrin fibers—clearly indicating a regular longitudinal repeat—but they are larger in diameter, and show little or no branching. We therefore expected their overall ordering to be better than that of fibrin fibers. Several different fixation protocols were used. We readily observed the typical transverse banding seen previously by negative stain and metal replication techniques. However, only very rarely was any regular lateral lattice seen in any of the samples. X-ray diffraction was used to examine unfixed specimens of the two fibrinogen polymers and, once again, although a longitudinal repeat was evident, only rarely was evidence for lateral crystallinity seen. The electron-microscope and x-ray results showed that the needles and pellet fibers of fibrinogen have essentially the same internal architecture as thick fibrin fibers, and that all three types of polymer, although clearly transversely banded, have almost no crystallinity in their lateral protofibril packing.