Different subfamilies of alphoid repetitive DNA are present on the human and chimpanzee homologous chromosomes 21 and 22.

The alphoid repeat DNA on chimpanzee chromosome 22 was compared with alphoid repeat DNA on its human homologue, chromosome 21. Hybridization of different alphoid probes under various conditions of stringency show that the alphoid repeats of chimpanzee chromosome 22 are not closely related to those of human chromosome 21. Sequence analysis of cloned dimer and tetramer EcoRI fragments from chimpanzee chromosome 22 confirm the low overall level of homology, but reveal the presence of several nucleotide changes which are exclusive to the chromosome 21 subfamily of human alphoid DNA. Southern blot analysis of alphoid repeat DNA on the chimpanzee X chromosome suggests this subfamily has been strongly conserved during and since the separation of chimpanzee and man although the two subfamilies can be distinguished on the basis of Taq I restriction fragments.     

Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells.

The close association of human papillomavirus type 16 DNA with a majority of cervical carcinomas implies some role for the virus in this type of cancer. To define the transforming properties of HPV-16 DNA in vitro we have now performed transfection experiments on baby rat kidney cells using HPV-16 DNA in conjunction with an activated ras gene. We have demonstrated that a 6.6-kb DNA fragment, containing the early genes of HPV-16 under the control of Moloney murine leukaemia virus long terminal repeats (MoMuLV-LTRs), cooperates with EJ-ras in transforming these cells. Both DNAs are required and neither alone is effective. The cooperating activity appears to reside in a protein or proteins derived from the E6/E7 region of the HPV-16 genome.     

Elimination of the non-specific binding of avidin to tissue sections

Summary A simple procedure is described for eliminating non-specific staining with avidin—peroxidase conjugates. Murine ovaries were embedded in either paraffin wax or epoxy resin and, after blocking endogenous peroxidase activity, were treated with 10 µg/ml biotinylatedPisum sativum agglutinin. Avidin—peroxidase conjugates (5 µg/ml), diluted in standard 0.05m tris-buffered saline, pH 7.6, containing 0.139m NaCl, produced considerable background coloration and intense mast cell staining in controls without the lectin. This background diminished as the ionic strength of the buffer was raised. At 0.125m Tris-buffered saline (containing 0.347m NaCl) the background was completely unstained, with elimination of all binding to mast cells and only minimal loss of specific lectin binding.     

Seasonal changes in the effects of arginine vasotocin and stretch on Anolis uterine contractions in vitro

We determined the effects of acute stretch on spontaneous and arginine vasotocin (AVT)-driven contractions of the Anolis carolinensis uterus in vitro. Whole uteri from reproductively inactive females (October) were placed in a bath of oxygenated 32 degrees C Anolis "Ringer's." Two initial tensions were utilized, 1.5 g or 15 g, the latter being an estimate of the tension on the wall of a uterine compartment. Uteri were then exposed to either saline or AVT (50 ng/ml), and spontaneous or AVT-driven contractions were recorded for 20 min with the use of a strain gauge and physiograph. A similar experiment was performed on uteri from reproductively active females in the summer (June). Our results indicate that the effects of acute stretch and AVT on uterine contractility were qualitatively similar in summer and fall. That is, AVT induced a tonic contraction; stretch decreased the duration of the tonic contraction; the saline-treated uteri exhibited spontaneous rhythmic contractions; AVT increased the amplitude of the rhythmic contractions, but only at the lower tension; there were no effects of AVT on the timing (contraction interval, duration, rest interval) of the rhythmic contractions; and stretch increased the frequency of the rhythmic contractions. Season greatly influenced the magnitude of these contractile phenomena. Uteri tested during the breeding season exhibited greater distensibility, an increase in the amplitude and duration of the AVT-driven tonic contraction, and an increase in the frequency of both spontaneous and AVT-driven rhythmic contractions because of a decrease in both contraction duration and rest interval.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle

The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2+-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was approximately 35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be three- to fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogold-labeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.     

Communication between two globular domains of calmodulin in the presence of mastoparan or caldesmon fragment. Ca2+ binding and 1H NMR

Ca2+ binding to calmodulin was measured in the presence of mastoparan or caldesmon fragment. Mastoparan and caldesmon fragment were used as model compounds of enzymes and cytoskeleton proteins, respectively, working as the target of calmodulin. Although the Ca2+ bindings of the two globular domains of calmodulin occur independently in the absence of the target peptide (or proteins), mastoparan and caldesmon fragment increased the affinity of Ca2+ and, at the same time, produced the positive cooperative Ca2+ bindings between the two domains. The result of Ca2+ binding was compared with 1H NMR spectra of calmodulin in the presence of equimolar concentration of mastoparan. It is known that a conformation change of the C-terminal half-region (C-domain) occurs by the Ca2+ binding to C-domain. A further change in conformation of C-domain was demonstrated by the Ca2+ binding to the N-terminal half-region (N-domain) in the presence of mastoparan. It indicates that the two domains of calmodulin get into communication with each other in the associated state with the target, and we concluded that the Ca2+ binding to the N-domain is responsive to the development of calmodulin function.     

Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation

Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.     

2-Phenylethylamine catabolism by Escherichia coli K12

Escherichia coli K12 grows on 2-phenylethylamine as sole carbon and energy source by converting it, via phenylacetaldehyde, to phenylacetic acid. Phenylacetaldehyde was formed by the action of an inducible amine oxidase and catalase activity was increased sixfold, presumably to ensure removal of the H2O2 that was expected to be a product of the amine oxidation. The phenylacetaldehyde was oxidized to phenylacetic acid by an inducible NAD+-dependent dehydrogenase. Mutants defective in phenylacetaldehyde dehydrogenase cannot grow on 2-phenylethylamine as carbon and energy source but can still use it as a nitrogen source.