DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.     

Guanosine 3',5'-cyclic monophosphate stimulates release of actively accumulated calcium in purified disks from rod outer segments of bovine retina

Parallel lines of evidence have suggested that light initiates changes in both cGMP metabolism and calcium levels in rod outer segments (ROS). We report that cGMP stimulates release of a pool of Ca2+ actively accumulated within purified ROS disks. Disks were purified and actively loaded with 45Ca2+ by an associated ATP-dependent calcium uptake activity as previously described [Puckett, K.L., Aronson, E.T., & Goldin, S.M. (1985) Biochemistry 24, 390-400]. Spikes of 45Ca2+ released from disks were observed in a rapid superfusion system. The Ca2+ release was specifically stimulated by physiological levels of cGMP (Kapp approximately 20 microM; Hill coefficient = 1.7). 8-Bromo-cGMP could also activate the release mechanism, but cAMP was ineffective. At cGMP levels of greater than or equal to 100 microM, approximately 20% of the loaded Ca2+ was released. The Ca2+ release rate at saturating cGMP levels reached a maximum within the 10-s time resolution of the assay system. In contrast to other recent reports of cGMP activation of ROS ion conductances, the majority of the release activity terminated in a spontaneous manner, suggestive of an intrinsic inactivation process. The amount of Ca2+ released and the release kinetics were similar to the presence or absence of an unbleached pool of rhodopsin. Cyclic nucleotides did not stimulate release from disks passively equilibrated with 45Ca2+, i.e., in the absence of ATP but otherwise under identical conditions. Preincubation of the disks with cGMP also reduced the level of ATP-dependent Ca2+ uptake (approximately 30%); this apparent inhibition may be due to activation of the release mechanism, rather than direct modulation of the uptake activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crescent mastopexy and augmentation

We have defined a group of patients with a lesser degree of moderate breast ptosis whose ptosis correction is not adequately improved by augmentation alone but requires some elevation of the nipple-areola complex. We have selected the crescent excision mastopexy to provide this additional needed lift. Experience with 26 patients employing this technique has helped to define the indications and limitations for this approach. It seems to adequately provide the additional needed lift when nipple descent has been no more than 1.5 to 2 cm below the inframammary crease. Complications such as scar widening (46 percent) were reviewed, but seemed to be well tolerated by the patients.     

Brain temperature measurements in rats: a comparison of microwave and ambient temperature exposures

In an effort to understand microwave heating better, regional brain and core temperatures of rats exposed to microwave radiation (2450 MHz) or elevated air temperatures were measured in two studies. In general, we have found no substantial evidence for temperature differentials, or "hot spots," in the brain of these animals. In the first study, after a 30-min exposure, no temperature differences between brain regions either after microwave or ambient air exposure were found. However, a highly significant correlation between brain and core temperatures was found and this correlation was the same for both microwave and ambient air heating. In the second study, time-temperature profiles were measured in rats exposed to either 30 mW/cm2 or 36.2 degrees C. In this study, the 30-min exposure period was divided into seven intervals and the change in temperature during each period was analyzed. Only the cortex showed significantly different heating rates between the air heating and microwave heating; however, this difference disappeared after the initial 5 min of exposure.     

Increased incidence of developmental anomalies among descendants of carriers of methylenebisacrylamide-induced balanced reciprocal translocations

N,N'-Methylenebisacrylamide (MBA), a dimer of the monomeric acrylamide, was studied for induction of clastogenic effects in germ cells of male mice. It was found to be effective in inducing dominant-lethal mutations and heritable translocations in maturing sperm. The semisterile translocation carriers and their normal counterparts were used to determine the health impact of transmitted chromosomal rearrangements through anatomical analysis of their immediate descendants in utero. As expected, semisterility resulted primarily from embryonic death during the periimplantation stages presumably caused by sperm segregants with unbalanced chromosome complement fertilizing some of the eggs. Among conceptuses that survived to mid- and late-gestation stages, there was an increased incidence of developmental anomalies including fetal death and phenotypic defects. These anomalies are assumed to be caused by certain types of unbalanced segregants that are compatible with survival beyond the periimplantation period. This class of unbalanced segregants represent in humans a major health problem to the mother and her conceptus.     

Melphalan, a second chemical for which specific-locus mutation induction in the mouse is maximum in early spermatids.

Melphalan (MLP), a bifunctional alkylating agent structurally related to the highly mutagenic chemical chlorambucil (CHL), was found to induce high frequencies of specific-locus mutations in postspermatogonial germ cells of the mouse, and to be one of only a few chemicals that is also mutagenic in spermatogonial stem cells. Productivity patterns following MLP exposures resembled those that had been found for CHL. Mutation rates in successive male germ-cell stages were measured at three MLP-exposure levels in a total of 95,375 offspring. While the induced (experimental minus historical-control) mutation rate is relatively low in stem-cell spermatogonia (1.2 x 10(-5) per locus at a weighted-mean exposure of 7.3 mg/kg), it is about 5 times higher in poststem-cell stages overall, and peaks at 26.7 x 10(-5) per locus in early spermatids at a weighted-mean exposure of only 5.7 mg/kg. This "type-2 pattern" of mutation yield (Russell et al., 1990), i.e., peak sensitivity in early spermatids, has heretofore been found for only one other chemical, CHL. Mutation-rate data earlier reported for CHL (Russell et al., 1989) were augmented in the present study for comparison with MLP-induced rates. Because of the greater toxicity of MLP, average exposures used for this chemical were only about one-half of those for CHL. When MLP and CHL mutation rates are extrapolated to equimolar doses, they appear very similar for poststem-cell stages overall. However, in the case of CHL, a somewhat higher proportion of the mutations is induced in early spermatids than in the case of MLP.     

Purification and properties of nuclease SP.

Single-strand-specific nucleases are a diverse and important group of enzymes that are able to cleave a variety of DNA structures present in duplex molecules. Nuclease SP, an enzyme from spinach, has been purified to apparent homogeneity, allowing for the unambiguous characterization of a number of its physical properties as well as its DNA strand cleavage specificities. The effects of ionic strength, pH, divalent metal cations, and temperature on nuclease SP activity have been examined in detail. Nuclease SP was found to be quite thermostable and could be stimulated by Co2+. In addition, the cleavage of UV-damaged and undamaged supercoiled plasmid substrates under a variety of conditions suggests that at least two types of structures are recognized and processed by nuclease SP: UV photoproduct-induced distortions and unwound "nuclease hypersensitive sites". These studies indicate that nuclease SP is functionally related to other single-strand-specific nucleases and is a potential enzymatic tool for probing and manipulating various types of DNA structures.