Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Cellular responses to ionizing radiation: effects of interrupting DNA repair with chemical agents

This review is concerned with the influence of different classes of chemical agents on cellular repair of DNA damage induced by ionizing radiation. Single-strand break rejoining is little affected by inhibitors of DNA synthesis; however, such inhibitors do lead to a persistence of double-strand breaks in the DNA, and this correlates with an enhancement of chromosome aberrations and cell killing. Experiments with antagonists of topoisomerase II suggest an intriguing role for this DNA unwinding enzyme in double-strand break repair. Interference with poly(ADP-ribose) synthesis, by means of the inhibitor 3-aminobenzamide, does not have a clear-cut effect on recovery from ionizing radiation damage. Various substances (for example, caffeine and trypsin) affect DNA repair via a modulation of the cell cycle, altering the time available to the cell for repairing potentially lethal DNA damage before such damage is 'fixed' by the process of DNA replication. Finally, disturbing cellular energy metabolism, and depressing the level of ATP, can inhibit the repair of radiation damage.     

The human glutathione S-transferase. Studies on the kinetic, stability and inhibition characteristics of the erythrocyte enzyme

Bromosulphophthalein and N-ethylmaleimide, inhibitors of glutathione S-transferase (EC 2.5.1.18 RX: glutathione R. transferase), have been used to identify variant forms of the erythrocyte enzyme. One 'atypical' sample was detected and was shown to have appreciably different kinetic and stability properties. These inhibitors may be useful in surveys of variation in this group of enzymes.     

A ten-month study of endogenous testosterone levels and behaviour in outdoor-living female rhesus monkeys (Macaca mulatta)

Endogenous testosterone levels were measured in association with sexual, aggressive, and social/affiliative behaviors in 11 outdoor-housed female rhesus monkeys over a ten-month period. Several behaviors (sex directed toward the male, sex received from the male, aggression directed toward the male, submission directed toward the male, submission directed toward the female, and groom another female) were significantly (p<0.05) positively correlated with testosterone in from one to five females. No trends were strong enough across all females to suggest that any of these correlations have species-wide significance. Factor analysis revealed clearcut clusters of behaviors, but elevations in testosterone were not strongly associated with any of these clusters. It is concluded that endogenous testosterone levels have little measurable effect on overt behavior in female rhesus monkeys.     

Sequence similarities between chicken intestinal 110-kDa ATPase and myosin I-like enzymes

We report the partial amino acid sequence of chicken intestinal microvillar 110-kDa protein that, as a complex with calmodulin, has previously been shown to exhibit myosin-like ATPase and actin-binding activities. The sequence shows a high degree of similarity to the sequence of a novel vertebrate myosin I-like heavy chain encoded by a cDNA isolated from bovine intestine. This confirms that the bovine and chicken proteins are the first examples of Acanthamoeba myosin I-like proteins from higher eukaryotes. Comparison of available structural and functional data leads us to postulate that the myosin I family of proteins result from the fusion of a conserved myosin headlike motor domain, with variable COOH-terminal domains responsible for binding to specific intracellular structures.     

Human chromosome-specific DNA libraries: construction and purity analysis

We report the construction of eight human chromosome-specific DNA libraries. Metaphase chromosomes were purified by flow-sorting, and the extracted DNA was cleaved with HindIII before cloning into lamba Charon 21A. There is now a complete digest HindIII library containing greater than five chromosome equivalents for each human chromosome. These are available to the scientific community through the American Type Culture Collection in Rockville, MD. The amount of hamster DNA in libraries in which the chromosome was sorted from human x hamster hybrid cells was estimated by species-specific hybridization. It ranged from 5% to 39%. The sorted chromosomes were examined by fluorescence in situ hybridization with species-specific DNA, and the main source of the hamster DNA contamination was found to be intact hamster chromosomes. In addition, we examined a chromosome 21 library, LL21NS02, for clones that fail to grow on the rec+ host LE392. Less than 0.6% of the recombinant phage exhibited the rec+-inhibited phenotype.     

DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells

Mitomycin C (MMC), a quinone-containing antitumor drug, has been shown to alkylate DNA and to form DNA cross-links. The ability of MMC to alkylate O6-guanine and to form interstrand cross-links (ISC) has been studied using Mer+ and Mer- human embryonic cells. Mer+ (IMR-90) cells have been reported to contain an O6-alkylguanine transferase enzyme and are, in general, more resistant to alkylating agents than the Mer- (VA-13) cell line, which is deficient in the repair of O6-lesions in DNA. Studies reported here show that MMC is more cytotoxic to VA-13 cells compared to IMR-90 cells. The alkaline elution technique was used to quantify MMC-induced ISC, and double strand breaks (DSB) in these cells. The drug-dependent formation of DSB was significantly lower in IMR-90 cells than in VA-13 cells. In contrast, no significant difference in cross-linking could be detected at the end of 2-h drug treatment. Although a small increase in cross-link frequency was observed in the VA-13 cell line relative to the IMR-90 cell line 6 h post drug treatment, it is not clear whether monoalkylated adducts at the O6-position are formed, and contribute to cross-link formation for differential cytotoxicity in VA-13 cells. Electron spin resonance and spin-trapping technique were used to detect the formation of hydroxyl radical from MMC-treated cells. Our studies show that MMC significantly stimulated the formation of hydroxyl radical in VA-13 cells, but not in the IMR-90 cells. The formation of the hydroxyl radical was inhibited by superoxide dismutase (SOD) and catalase. In addition, the presence of these enzymes partially protected VA-13 cells from MMC toxicity but not IMR-90 cells. Further studies indicated that the decreased free radical formation and resistance to MMC may be due to the increased activities of catalase and glutathione transferase in the IMR-90 cell line. These results suggest that MMC-dependent DNA damage (alkylation and DNA DSB) and the stimulation of oxy-radical formation may play critical roles in the determination of MMC-induced cell killing.     

The Tc3 Family of Transposable Genetic Elements in Caenorhabditis Elegans

We describe genetic and molecular properties of Tc3, a family of transposable elements in Caenorhabditis elegans. About 15 Tc3 elements are present in the genomes of several different wild-type varieties of C. elegans, but Tc3 transposition and excision are not detected in these strains. Tc3 transposition and excision occur at high frequencies, however, in strain TR679, a mutant identified because of its highly active Tc1 elements. In TR679, Tc3 is responsible for several spontaneous mutations affecting the unc-22 gene. Tc3-induced mutations are unstable, and revertants result from precise or nearly precise excision of Tc3. Although Tc3 is very active in TR679, it is not detectably active in several other mutator mutants, all of which exhibit high levels of Tc1 activity. Tc3 is 2.5 kilobases long, and except for sequences near its inverted repeat termini, it is unrelated to Tc1. The termini of Tc3 are inverted repeats of at least 70 base pairs; the terminal 8 nucleotides of Tc3 are identical to 8 of the terminal 9 nucleotides of Tc1.     

Choroideremia and deafness with stapes fixation: a contiguous gene deletion syndrome in Xq21.

The study of contiguous gene deletion syndromes by using reverse genetic techniques provides a powerful tool for precisely defining the map location of the genes involved. We have made use of individuals with overlapping deletions producing choroideremia as part of a complex phenotype, to define the boundaries on the X chromosome for this gene, as well as for X-linked mixed deafness with perilymphatic gusher (DFN3). Two patients with deletions and choroideremia are affected by an X-linked mixed conductive/sensorineural deafness; one patient, XL-62, was confirmed at surgery to have DFN3, while the other patient, XL-45, is suspected clinically to have the same disorder. A third choroideremia deletion patient, MBU, has normal hearing. Patient XL-62 has a cytogenetically detectable deletion that was measured to be 7.7% of the X chromosome by dual laser flow cytometry; the other patient, XL-45, has a cytogenetically undetectable deletion that measures only 3.3% of the X chromosome. We have produced a physical map of the X-chromosome region containing choroideremia and DFN3 by using routine Southern blotting, chromosome walking and jumping techniques, and long-range restriction mapping to generate and link anonymous DNA sequences in this region. DXS232 and DXS233 are located within 450 kb of each other on the same SfiI and MluI fragments and share partial SalI fragments of 750 and greater than 1,000 kb but are separated by at least one SalI site. In addition, DXS232, which lies outside the MBU deletion, detects the proximal breakpoint of this deletion. We have isolated two new anonymous DNA sequences by chromosome jumping from DXS233; one of these detects a new SfiI fragment distal to DXS233 in the direction of the choroideremia gene, while the other jump clone is proximal to DXS233 and detects a new polymorphism. These data refine the map around the loci for choroideremia and for mixed deafness with stapes fixation and will provide points from which to isolate candidate gene sequences for these disorders.     

Differential depuration of poliovirus, Escherichia coli, and a coliphage by the common mussel, Mytilus edulis.

The elimination of sewage effluent-associated poliovirus, Escherichia coli, and a 22-nm icosahedral coliphage by the common mussel, Mytilus edulis, was studied. Both laboratory-and commercial-scale recirculating, UV depuration systems were used in this study. In the laboratory system, the logarithms of the poliovirus, E. coli, and coliphage levels were reduced by 1.86, 2.9, and 2.16, respectively, within 52 h of depuration. The relative patterns and rates of elimination of the three organisms suggest that they are eliminated from mussels by different mechanisms during depuration under suitable conditions. Poliovirus was not included in experiments undertaken in the commercial-scale depuration system. The differences in the relative rates and patterns of elimination were maintained for E. coli and coliphage in this system, with the logarithm of the E. coli levels being reduced by 3.18 and the logarithm of the coliphage levels being reduced by 0.87. The results from both depuration systems suggest that E. coli is an inappropriate indicator of the efficiency of virus elimination during depuration. The coliphage used appears to be a more representative indicator. Depuration under stressful conditions appeared to have a negligible affect on poliovirus and coliphage elimination rates from mussels. However, the rate and pattern of E. coli elimination were dramatically affected by these conditions. Therefore, monitoring E. coli counts might prove useful in ensuring that mussels are functioning well during depuration.