DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144.     

The effects of S9 mix from rat oesophagus, salivary gland, and liver on the mutagenicity of the rat oesophageal carcinogen N-nitroso-N-methylaniline in the Salmonella typhimurium assay

The present study examines the feasibility of using the Salmonella typhimurium plate-incorporation assay of Ames for detecting target-organ specificity with N-nitroso-N-methylaniline (NMA), a compound for which the target site for tumour formation in the rat is the oesophagus. Thus it was anticipated that the oesophagus would bioactivate this compound. The compound has been investigated using S9 from Aroclor- and NMA-induced rat oesophagus, salivary gland and liver in the presence and absence of the co-mutagen, norharman. No response to NMA was seen with oesophageal S9 even though benzo[a]pyrene produced a dose-related increase in revertants in strain TA98 and TA100. No response to NMA was seen with salivary-gland S9. However, a response was produced with Aroclor-induced rat-liver S9 in the presence of norharman and with NMA-induced rat-liver S9 in the absence of norharman.     

Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

Parasite specific antibodies in three strains of mice after infection with Trypanosoma cruzi

Three inbred strains of mice (BALB/cJ, C3H/HeJ and NZB/BInJ) were infected with trypomastigotes of Trypanosoma cruzi. Sera were taken at different times after infection and radioimmunoprecipitation assays were used to detect antibodies against individual T. cruzi epimastigote and trypomastigote antigens. The mouse strains differed in regard to the spectrum of antibodies and the time after infection when the various epimastigote specific antibody species appeared. NZB mice had antibodies against at least 25 polypeptides ranging in molecular weight from 20,000 to 90,000 D at 3 wk after infection, and these persisted until at least 10 wk post-infection. C3H and BALB/c had antibodies against fewer than 5 antigens at 3 wk after infection; whereas by week 10, antibodies against at least 25 polypeptides were detected. C3H mice that were most susceptible to infection (but not NZB or BALB/c mice) had antibodies against a 25,000 D molecular weight epimastigote antigen. The antibody response against trypomastigote polypeptides was more uniform. Sera from all mouse strains at 3 wk after infection precipitated the same polypeptides and the radioimmunoprecipitation patterns did not change as a function of time after infection.     

Construction and expression of synthetic DNA fragments coding for polypeptides with elevated levels of essential amino acids

Summary Polypeptides, with elevated levels of essential amino acids, could be useful as partial protein supplements to food and feeds. To obtain DNA fragments coding for these polymers, oligonucleotides were constructed by random synthesis of a mixture of appropriate codon pairs and inserted into a bacterial plasmid in E. coli. Two of the isolated fragments were subjected to DNA sequence analysis and theoretically code for polypeptides containing up to 23% lysine, 12% tryptophan, 12% methionine, 6% isoleucine, and 6% threonine. These five amino acids make up 60% of the total amino acid content of the peptide, compared with 25% for the same amino acids in lactalbumin, a milk protein considered to be high in essential amino acids. These fragments, when fused to an active bacterial promoter, which directs the synthesis of chloramphenicol acetyl transferase (CAT), cause bacteria, harboring these modified genes, to take up more lysine as compared to control cells and produce commensurately larger CAT polypeptides. This method of gene synthesis may permit production of polypeptides with a specified amino acid composition to supplement specific diets low in the essential amino acids.     

Metabolism and gastric acid secretion. Substrate-dependency of aminopyrine accumulation in isolated rat parietal cells.

The substrate-dependency of gastric acid secretion was investigated in isolated rat parietal cells by using the accumulation of the weak base aminopyrine as an index of acid secretion. Exogenous substrates enhanced accumulation of aminopyrine in rat parietal cells stimulated by secretagogues, and this effect was probably directly related to the provision of energy for acid secretion. At physiological concentrations, certain of the substrates (glucose, oleate, lactate, D-3-hydroxybutyrate, L-isoleucine, L-valine and acetoacetate) could support acid secretion, with glucose being the most effective. L-Leucine and acetate were only effective stimulators of parietal-cell aminopyrine accumulation at high concentrations (5mM). L-Glutamine was unable to stimulate aminopyrine accumulation even at high concentrations, and glutaminase activity in parietal cells was estimated to be low by comparison with small-intestinal epithelial cells. Variation in the concentrations of D-3-hydroxybutyrate and L-isoleucine, but not of glucose, within the physiological range affected their ability to support aminopyrine accumulation. The presence of 5 mM-L-isoleucine, 5 mM-lactate and combinations of certain substrates at physiological concentrations produced aminopyrine accumulation in stimulated parietal cells that was greater than that obtained in cells incubated with 5 mM-glucose alone. In conclusion, fulfillment of the metabolic requirements of the acid-secreting parietal cell under physiological circumstances requires a combination of substrates, and integration of the results with previous data [Anderson & Hanson (1983) Biochem. J. 210, 451-455; 212, 875-879] suggests that after overnight starvation in vivo metabolism of glucose, D-3-hydroxybutyrate and L-isoleucine may be of particular importance.     

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.     

Dexamethasone and indomethacin modify endotoxin-induced respiratory failure in pigs

We studied the porcine pulmonary response to endotoxemia before and after administration of nonsteroidal antiinflammatory drugs (NSAID, i.e., indomethacin or flunixin meglumine) or dexamethasone (DEX). Escherichia coli endotoxin was infused intravenously into anesthetized 10- to 12-wk old pigs for 4.5 h. In endotoxemic pigs, the phase 1 (i.e., 0-2 h) increases in pulmonary arterial pressure, pulmonary vascular resistance (PVR), and alveolar-arterial O2 gradient and the decreases in cardiac index (CI) and lung dynamic compliance (Cdyn) were blocked by NSAID. Thus phase 1 changes were cyclooxygenase dependent. Furthermore, these effects were blocked or greatly attenuated by DEX. During phase 2 of endotoxemia (i.e., 2-4.5 h), the increased PVR and decreased CI and Cdyn were not blocked by NSAID but were attenuated by DEX, suggesting the presence of cyclooxygenase-independent metabolites. Both NSAID and DEX blocked the endotoxin-induced increases in lung water, bronchoalveolar lavage (BAL) neutrophil, and BAL albumin content. The fall in plasma proteins persisted in NSAID but not DEX-treated pigs. We conclude that endotoxemia in the pig causes severe acute respiratory failure largely mediated by cyclooxygenase and possibly lipoxygenase products of arachidonic acid metabolism.     

Impact of whooping cough on patients and their families.

The effects of whooping cough were studied in 21 children admitted to hospital with the disease and in their families. The illness caused considerable distress to both child and family. Parents suffered especially from fears for the life and health of their child and from serious loss of sleep. Two months after admission the child''s behaviour was still disturbed, but in most cases the rest of the family had returned to normal. There was much misunderstanding and misinformation about whooping cough among both parents and doctors.