Localization of tubulin in the mitotic apparatus of mammalian cells by immunofluorescence and immunoelectron microscopy

Antitubulin antibody was used as an immunofluorescent and immunoelectron microscopic probe to localize tubulin in components of the mitotic apparatus of rat kangaroo (strain PtK₁) cells in vitro. In addition to the detection of tubulin in the spindle microtubules and centrioles, other structures were found to display specific staining including kinetochores, amorphous pericentriolar material and small virus-like particles associated with the centrioles. The kinetochores consisted of a densely stained outer layer about 400 Å thick which is separated from an inner layer of the same dimension by a lightly staining middle layer. Microtubules were primarily associated with the outermost plate of the kinetochore but tubulin was uniformly distributed in both outer and inner plates. Colcemid treatment prevented the assembly of spindle microtubules and resulted in specific alterations of the kinetochore but failed to diminish the staining of the kinetochores. These observations suggest that tubulin molecules may comprise an important structural component of the kinetochore.     

Testosterone and sex-related behavior and morphology: Relationship during maturation and in the adult Japanese Quail

Hormonal, behavioral, and morphological aspects of reproduction were monitored in maturing and in adult male Japanese Quail. In Experiment I, animals were observed daily for crowing, tested for mating, and measured for cloacal gland area. Blood samples were taken on the day the animal first crowed and on the day the animal first mated. Animals initiated crowing significantly earlier than mating (Day 32 vs. Day 35, respectively; P < .05). The concentration of testosterone and the cloacal gland area increased significantly between the onset of crowing and mating (p < .01). In the second experiment, animals were observed, tested, and sampled between 36 and 94 days of age. Hormonal, behavioral, and morphological variables increased rapidly in time to reach a maximum about which subsequent values oscillated. Attainment of these maxima followed the sequence observed at the onset of sexual behavior and increases in serum testosterone and cloacal gland area. All variables were highly positively correlated in time. Maxima were attained at 44 days for testosterone concentrations, 49 days for crowing, 50 days for cloacal gland area, 52 days for mating attempts, and 81 days for cloacal contacts. This similar sequence in the onset and maxima of sex-related hormonal, behavioral, and morphological characteristics implies a differential activation of target areas which regulate these variables.     

Isolation and characterization of new strains of cholesterol-reducing bacteria from baboons.

We isolated and characterized nine new strains of cholesterol-reducing bacteria from feces and intestinal contents of baboons. Cholesterol-brain agar was used for the primary isolation, and subsequent biochemical tests were done in a lecithin-cholesterol broth containing plasmenylethanolamine and various substrates. All strains had similar colony and cell morphology, hydrolyzed the beta-glucosides esculin and amygdalin, metabolized pyruvate, and produced acetate and acetoin. Unlike previously reported strains, the nine new strains did not require cholesterol and an alkenyl ether lipid (e.g., plasmalogen) for growth; however, only two strains reduced cholesterol in the absence of the plasmalogen. These two strains also produced succinate as an end product. Carbohydrate fermentation was variable; some strains produced weak acid (pH 5.5 to 6.0) from only a few carbohydrates, whereas other strains produced strong acid reactions (pH less than or equal to 5.5) from a wide variety of carbohydrates.     

Effect of Miracil D on Marker Frequency Ratio and Cytotoxicity in Bacillus subtilis

When Miracil D was added to mid-log phase Bacillus subtilis cells, the rate of growth decreased immediately, and the turbidity of the culture began to decrease within 15 to 20 min after addition of the drug. At this stage, ghostlike cells were observed under phase-contrast microscopy. The viable count also began to decrease rapidly after 15 min in the presence of the drug, and within 60 min there was a 10,000-fold reduction in viability. Incorporation of (3)H-thymidine into deoxyribonucleic acid (DNA) proceeded normally up to 20 min of exposure to the drug, after which incorporation ceased. Cells which were prelabeled with (3)H-thymidine and exposed to the drug released labeled DNA into the medium after 15 to 20 min. Results of transformation analyses with donor DNA from cells grown in the presence of Miracil show a decrease with time in the ratio of origin markers to terminus markers. Electron micrographs of sectioned cells grown for 5, 10, and 15 min in the presence of Miracil D show profound cytotoxic effects. The most striking aspect of such cells is the very condensed appearance of the nucleoid. In those cells about to divide, the nucleoid appears not to be properly separating, being strung out in the region of the developing septum. Finally, the mesosome is very poorly defined or entirely absent in Miracil-treated cells.     

Temperature tolerance and thyroid activity of the white perch Roccus (=Morone) americanus

The seasonaliy of temperature tolerance using thiourea-treated, normal and thyroxine treated white perch was determined after laboratory acclimation and LD50 values calculated by normit analysis. Treatment effects, reflected in slight shifts in LD50 values were evident. However, these did not fit a consistent pattern of thyroidal involvement in temperature tolerance.
When specimens of known thyroidal functional state were used in tolerance tests, results, similar to the seasonal tolerance tests, were obtained. Thus, although the white perch has a functioning thyroid capable of forming radiohormone and this capability was experimentally varied in these tests, no tolerance shifts were noted.
Data obtained in tolerance testing were statistically adjusted to partition out variation due to differences in acclimation temperatures (season) and to test for treatment (thyroidal functional state) effects. Treatment difference was not significant while that due to season was significant (P<0.01). Thyroidal functional state was thus unrelated to tolerance.     

Elevated soluble lectin-like oxidized LDL receptor-1 (sLOX-1) levels in obese postmenopausal women

We investigated the association between soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) levels and obesity in older women. Fifty-one postmenopausal women (10 lean, 22 overweight, and 19 obese) were included in this small retrospective analysis. Plasma sLOX-1 levels were measured using a chemiluminescent enzyme-linked immunoassay. Plasma levels of sLOX-1 were significantly higher in obese women (55.33 +/- 4.49 pg/ml) compared to lean (30.91 +/- 6.19 pg/ml, P = 0.002) and overweight women (38.31 +/- 4.18 pg/ml, P = 0.017). Plasma sLOX-1 levels were positively associated with body weight, BMI, total body fat, and trunk fat. The relationship between sLOX-1 and BMI was attenuated after adjustment for age, hormone replacement therapy, and body fat. In conclusion, obese women have higher sLOX-1 levels, which may reflect increased LOX-1 expression in adipose tissue.     

Selecting representative model micro-organisms

Background

The sexually transmitted disease, gonorrhea, is a serious health problem in developed as well as in developing countries, for which treatment continues to be a challenge. The recent completion of the genome sequence of the causative agent, Neisseria gonorrhoeae, opens up an entirely new set of approaches for studying this organism and the diseases it causes. Here, we describe the initial phases of the construction of an expression-capable clone set representing the protein-coding ORFs of the gonococcal genome using a recombination-based cloning system.

Results

The clone set thus far includes 1672 of the 2250 predicted ORFs of the N. gonorrhoeae genome, of which 1393 (83%) are sequence-validated. Included in this set are 48 of the 61 ORFs of the gonococcal genetic island of strain MS11, not present in the sequenced genome of strain FA1090. L-arabinose-inducible glutathione-S-transferase (GST)-fusions were constructed from random clones and each was shown to express a fusion protein of the predicted size following induction, demonstrating the use of the recombination cloning system. PCR amplicons of each ORF used in the cloning reactions were spotted onto glass slides to produce DNA microarrays representing 2035 genes of the gonococcal genome. Pilot experiments indicate that these arrays are suitable for the analysis of global gene expression in gonococci.

Conclusion

This archived set of Gateway^® entry clones will facilitate high-throughput genomic and proteomic studies of gonococcal genes using a variety of expression and analysis systems. In addition, the DNA arrays produced will allow us to generate gene expression profiles of gonococci grown in a wide variety of conditions. Together, the resources produced in this work will facilitate experiments to dissect the molecular mechanisms of gonococcal pathogenesis on a global scale, and ultimately lead to the determination of the functions of unknown genes in the genome.     

Infectious circular DNA of human adenovirus type 5: regeneration of viral DNA termini from molecules lacking terminal sequences.

A series of plasmids containing the entire human adenovirus genome with viral DNA termini joined 'head to tail' has been isolated. Several plasmids were able to generate infectious virus following transfection of human cells in spite of having small deletions and rearrangements at the junctions of termini. One plasmid has lost 2 bp of DNA from one end of the viral genome and 11 bp from the other end yet produced viruses with complete wild-type sequences at both ends of the genome. We propose a model for replication of viral DNA off circular templates in which regeneration of terminal information involves translocation of primer and polymerase during initiation of DNA replication. The model suggests a novel mechanism for extension of the 5' ends of linear DNA molecules which could be applicable to chromosomal telomeres.