Adenovirus type 5 virions can be assembled in vivo in the absence of detectable polypeptide IX.

Mutant dl313 is an adenovirus type 5 deletion mutant which lacks 2,307 base pairs, including the 5' portion of the polypeptide IX gene. Mutant virions did not contain detectable levels of this polypeptide. They were substantially more thermolabile than wild-type particles and did not produce hexon nonomers upon pyridine disruption.     

Cytosolic factors which affect microsomal lipid peroxidation in lung and liver

Studies were carried out to determine the effects of lung and liver cytosol on pulmonary and hepatic mierosomal lipid peroxidation, to determine the cytosolic concentrations of various substances which affect lipid peroxidation, and to determine which of these substances is responsible for the effects of the cytosol on lipid peroxidation. Lung cytosol inhibits both enzymatic (NADPH-induced) and nonenzymatic (Fe²⁺-induced) lung microsomal lipid peroxidation. In contrast, liver cytosol stimulates lipid peroxidation in hepatic microsomes during incubation alone, enhances Fe²⁺-stimulated lipid peroxidation, and has no effect on the NADPH-induced response. Substances which are known to be involved in inhibition of lipid peroxidation, including glutathione, glutathione reductase, glutathione peroxidase, and superoxide dismutase, are found in greater concentrations in liver cytosol than in lung cytosol. However, ascorbate is found in approximately equal concentrations in pulmonary and hepatic cytosol. Most of the effects of the cytosol on lipid peroxidation seem to be due to ascorbate and glutathione. For example, ascorbate, in concentrations found in lung cytosol, inhibits lung microsomal lipid peroxidation to about the same extent as the cytosol. The effects of liver cytosol on hepatic microsomal lipid peroxidation can be duplicated by concentrations of ascorbate and glutathione normally found in the cytosol; i.e., ascorbate stimulates and glutathione inhibits lipid peroxidation with the net effect being similar to that of liver cytosol. The results indicate that ascorbate has opposite effects on pulmonary and hepatic microsomal lipid peroxidation and suggest that ascorbate plays a major role in protecting pulmonary tissue against the harmful effects of lipid peroxidation.     

Direct shoot organogenesis and plant regeneration from leaves of grape (Vitis spp.)

Adventitious shoots developed from in vitro-grown leaves of Vitis vinifera cultivars Cabernet Sauvignon, French Colombard, Grenache, Thompson Seedless (syn. Sultana) and White Riesling, V. rupestris cv. St. George (syn. du Lot) and V. vinifera × rupestris cv. Ganzin 1. Leaf explants less than 15 mm long were excised from nodal cultures and cultured on Murashige and Skoog or Nitsch and Nitsch-based regeneration media with 0, 1, 2 or 4 mgl^-1 6-benzylaminopurine (BAP). Adventitious shoots developed within 4 weeks at the petiolar stub and occasionally from wounded lamina tissues. Shoot organogenesis occurred only on media containing BAP and at a higher frequency with 2 mgl^-1 than with 1 or 4 mgl^-1. On media containing 2 mgl^-1 BAP, 47, 67, 60, and 42%, respectively, of leaf explants of Cabernet Sauvignon, French Colombard, Thompson Seedless, and White Riesling produced adventitious shoots compared to 14, 14, and 29%, respectively, for Grenache, St. George, and Ganzin 1. Solid culture medium was superior to liquid medium and transfer frequency on solid medium did not affect the regeneration frequency. Further shoot growth was promoted by the transfer of regenerating tissues to fresh regeneration medium. More than 80% of explants initially producing adventitious buds exhibited further shoot growth, developing an average of more than 6 shoots each. Shoots rooted easily and the resulting plants appeared morphologically identical to parent vines.     

Spironolactone inhibition of cortisol production by guinea pig adrenocortical cells

Prior investigations with adrenal subcellular fractions demonstrated that the diuretic, spironolactone (SL), was converted to a reactive metabolite by adrenal microsomes, resulting in the degradation of microsomal cytochrome(s) P-450. Studies were done to evaluate the effects of SL and 7 alpha-thio-SL, a putative intermediate in the activation pathway, on cortisol production by intact guinea pig adrenocortical cells. Preincubation of adrenal cells with SL or 7 alpha-thio-SL caused time-dependent and concentration-dependent decreases in subsequent ACTH-stimulated cortisol production. 7 alpha-Thio-SL was a far more potent inhibitor than SL. In the absence of a preincubation period, neither SL nor 7 alpha-thio-SL affected cortisol production. The results indicate that the effects of SL on adrenal microsomal cytochrome(s) P-450 compromise steroid synthesis by intact adrenal cells and lend support to the hypothesis that metabolism of the drug is required for the inhibition of steroidogenesis.     

Agrobacterium rhizogenes mutants that fail to bind to plant cells.

Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria to bind to carrot cells. The parent strain bound to carrot cells in greatest numbers in low-ionic-strength media such as 4% sucrose but still showed significant binding in Murashige-Skoog tissue culture medium. All of the mutants showed reduced binding in 4% sucrose after 2 h of incubation with carrot cells. One mutant was delayed in binding in 4% sucrose. This mutant and one other mutant also showed reduced binding to carrot cells in Murashige-Skoog medium. To determine whether the Tn5 insertion was responsible for the mutant phenotype, DNA containing the Tn5 insertion was cloned from the mutant bacteria and used to introduce Tn5 into the parent strain in the same location as in the original mutant by marker exchange. The resulting transconjugants had the same avirulent, nonattaching phenotype as the original mutants, suggesting that the mutant phenotype was due to the Tn5 insertion. The cloned DNA containing the Tn5 insertion was also tested for homology to DNA of known genes that affect attachment of Agrobacterium tumefaciens to plant cells by DNA hybridization. No homology to chv, att, or pscA clones was observed. In addition, cloned chv, att, and pscA genes from A. tumefaciens were unable to complement the attachment-minus A. rhizogenes mutants. Thus, the A. rhizogenes nonattaching mutants appear to be different from the previously described A. tumefaciens mutants.     

Analysis of fibrinogen A alpha-fusion proteins. Mutants which inhibit thrombin equivalently are not equally good substrates

We have examined the interaction of thrombin with fibrinogen A alpha chain residues 7-16. Using genetically engineered constructions, we have synthesized in Escherichia coli a fibrinogen A alpha 1-50 fusion protein and seven mutant proteins with single amino acid substitutions. These are: Asp7----Ala, Phe8----Tyr, Glu11----Ala, Gly12----Val, Gly13----Val, Gly14----Val, and Arg16----Leu. Competitive immunoassay of cell lysates showed that all the mutations but one, Arg16----Leu, altered the structure of the protein such that cross-reactivity with the A alpha-specific monoclonal antibody, Y18, was significantly reduced. The fusion proteins were purified and analyzed as thrombin inhibitors and substrates. All the fusion proteins are competitive inhibitors of the amidolytic hydrolysis of Spectrozyme TH, a thrombin-specific chromogenic substrate, with inhibition constants corresponding to that for fibrinogen. We conclude that these 7 amino acid substitutions do not alter thrombin binding to the fusion proteins. The fusion proteins were tested as substrates by monitoring thrombin-dependent peptide release. The natural sequence and three mutants, Asp7----Ala, Glu11----Ala, and Gly14----Val, are good substrates. The other mutants are either poor substrates or are not cleaved by thrombin within A alpha 1-50. These results indicate that residues between Asp7 and Arg16 are critical to efficient peptide hydrolysis, whereas residues outside this region are critical to thrombin binding.     

The effect of serum on the transport and phosphorylation of 2-deoxyglucose by untransformed and transformed mouse 3T3 cells.

Serum starvation of growing and nongrowing (density-inhibited) mouse 3T3 cells resulted in decreased phosphorylation of 2-deoxy--D-glucose, while the time course of transport of this sugar remained unchanged. Serum starvation of SV40 transformed 3T3 cells (SV101) and spontaneously transformed 3T6 cells did not alter either the time course of transport, or phosphorylation of the sugar. Treatment of SV101 cells with 10(-4) M dibutyryl adenosine cyclic 3':5' monophosphate and 10(-3) M theophylline did not restore the capacity to regulate 2-deoxy-D-glucose phosphorylation when these cells were serum deprived. We conclude that serum factors are involved in the modulation of phosphorylation of 2-deoxy-D-glucose in 3T3 cells rather than its transport. This regulation is operative both in growing as well as nongrowing 3T3 cells. In contrast, transformed cells do not respond to this regulation of 2-deoxy-D-glucose phosphorylation.     

Assimilation of exogenous fructose, aspartate and some organic acids during the growth of methylotrophs.

The percentage of bacterial carbon that was derived from exogenous labelled compounds present in the medium during the growth of some methylotrophs on trimethylamine or on non-C1 compounds was determined. Less than 10% of bacterial carbon was derived from acetate during the growth of the obligate methylotrophs 4B6 and C2A1, and of the restricted facultative methylotroph PM6; the other restricted facultative methylotroph W3A1 gave a value of 18%. Corresponding values for three typical facultative methylotrophs growing on trimethylamine were 41, 42 and 52%. Aspartate, fructose, pyruvate and succinate made only a small percentage contribution (0-4 to 12%) to bacterial carbon in 4B6, C2A1, W3A1 and PM6. Washed suspensions of 4B6, C2A1, W3A1 and PM6, all grown on trimethylamine, assimilated labelled acetate only in the presence of trimethylamine and there was a linear relationship between the amount of acetate assimilated and the amount of trimethylamine oxidized. Organisms 4B6, C2A1, W3A1 and PM6 assimilated 14C from labelled acetate predominantly into lipid (except PM6), glutamate, arginine, proline and leucine, whereas the typical facultative methylotrophs assimilated 14C from acetate into lipid, nucleic acid and all the protein amino acids. These results are consistent with the presence of a deficient tricarboxylic acid cycle in the obligate methylotrophs and in the restricted facultative methylotrophs.     

Cephalometric analysis of the craniofacial region of the northern Foxe Basin Eskimo

Past investigations of the Eskimo have indicated that there are marked morphological differences in the craniofacial skeleton of this relatively isolated ethnic group compared to other ethnic and racial groups. This study, using cephalometric radiography, attempted to characterize the craniofacial phenotype of the Eskimo living in the northern Foxe Basin, Northwest Territories, Canada. Age changes were examined on a cross-sectional basis with comparisons being made with a Winnipeg Caucasian group. This investigation indicates that the Igloolik Eskimo has a phenotype, established early in life, and is distinct from the Winnipeg group. The overall size of the Eskimo craniofacial complex was significantly larger at three years of age and remained larger through the ages studied. Development of the craniofacial region, however, was fairly similar in rate and direction for both populations. The greatest differences between the Eskimo and Caucasian groups were found in the linear measurements assessing cranial width, facial width, mandibular length, facial height, protrusion of the incisors, chin point development, and nasal morphology. Differences between the two groups in the morphological relationships of the component structures include the angular relationships of the maxilla and nasal bones to the anterior cranial base, the gonial angle of the mandible, and the angle of facial convexity.     

Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs.

Dromedary (Camelus dromedarius) RNAase (ribonuclease) was isolated from pancreatic tissue by affinity chromatography. Peptides obtained by digestion with different proteolytic enzymes and CNBr were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. Peptides were sequenced by the dansyl-Edman method. All peptide bonds were overlapped by one or more peptides. The polypeptide chain consists of 123 amino acids. A deletion (position 39) was observed in an external loop of the polypeptide chain (residues 35-40), as was found earlier to horse RNAase (Scheffer & Beintema, 1974). A heterogeneity was found at position 103 (glutamine and lysine). Dromedary RNAase differs at 23-32% of the positions from all other pancreatic RNAases sequenced to date. In evolutionary terms this indicates that dromedary RNAase has evolved independently during the larger part of the evolution of the mammals. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50046 (14 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.