Binding and aggregation of the 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis to cell membranes and alteration by monoclonal antibodies and amino acid modifiers

The 25-kilodalton toxin of Bacillus thuringiensis subsp. israelensis binds irreversibly to Aedes albopictus cells, Choristoneura fumiferana cells, and erythrocytes. The binding to cells increased with both toxin concentration and time and when the cells were first preincubated with unlabeled toxin. Binding data indicated a two- to threefold increase in the rate of binding after the amount of the membrane-bound toxin reached approximately 3.5 fmol/3 x 10(5) A. albopictus cells or 3.3. fmol/2 x 10(5) C. fumiferana cells. When this level of bound toxin was reached, the toxins also began forming aggregates at the cell membrane. The toxin aggregates were extracted with 10% Triton X-100 and separated from the monomers with a 5 to 20% sucrose density gradient. The toxin aggregates isolated from A. albopictus and C. fumiferana cell membranes were ca. 400 kilodaltons, while those isolated from human erythrocytes were significantly smaller. The proportion of the toxin found in aggregate form increased rapidly with the amount of toxin bound; however, the molecular size of the aggregates remained constant. Eleven monoclonal antibodies raised against the native form of the toxin blocked 80 to 97% of the toxin binding to cells. The epitope of one of these monoclonal antibodies was mapped to a domain which included the cysteine, suggesting the importance of the domain around this amino acid to binding. Toxin binding and cell lysis were also inhibited by treating the toxin with HgCl2, further indicating the importance of the C-terminal hydrophobic cysteine-containing domain in cytolytic activity of the 25-kilodalton protein.     

Unusual genetic phenomena associated with Tn5 mutagenesis in Alcaligenes eutrophus strain H1

Tn5 was introduced into Alcaligenes eutrophus strain H1 by a suicide vector pSUP1011. Physical characterization of mutants obtained after Tn5 mutagenesis revealed a relatively high frequency of plasmid curing, or deletion of a 50 kb plasmid DNA segment. Results of Southern hybridization and chromosomal walking indicate that the same continuous stretch of plasmid DNA (designated as D region of plasmid) is deleted in four independent isolates. Moreover, the same deletion of plasmid DNA is also observed in a mitomycin C-generated mutant strain H1-4.Journal Paper No. J-12095 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2607, supported in part by a grant from the Iowa High Technology Council     

Characterization of the human gene for a polypeptide that acts both as the beta subunit of prolyl 4-hydroxylase and as protein disulfide isomerase

A single polypeptide acts as the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide isomerase and may also function as a cellular thyroid hormone binding protein. We report here that the human gene for this polypeptide is about 18 kilobase pairs and consists of 11 exons. The two thioredoxin-like regions are coded by exons 1-2 and 8-9, respectively. The codons for the two presumed active sites of protein disulfide isomerase, each a Cys-Gly-His-Cys sequence, are located 12 base pairs from the beginning of exons 2 and 9. The last 3 amino acids coded by exons 1 and 8 and the first 9 amino acids coded by exons 2 and 9, including a broken codon for Tyr, are identical in the respective exon-intron junctions. These regions are also highly homologous to the active sites of bacterial thioredoxins. The data suggest that evolution of this gene has involved exon shuffling and duplication of a two-exon unit, in which the internal exon-intron junctions have been entirely conserved. The region between exons 1-2 and 8-9 appears to contain other duplications. The 5' flanking sequences contain a TATA box, six CCAAT boxes, and other elements which may be involved in regulation of the cellular amounts of this polypeptide.     

Human glutathione S-transferases. The Ha multigene family encodes products of different but overlapping substrate specificities

The human glutathione S-transferase cDNAs encoding subunits 1 and 2 contain intrinsic ribosome-binding sites in their 5'-untranslated regions for direct expression in Escherichia coli. We show that functional human GSH S-transferases 1-1 and 2-2 are synthesized from lambda gt11 cDNA clones lambda GTH1 and lambda GTH2 in phage lysates of E. coli Y1090, in lysogens of E. coli Y1089, and from the plasmid expression constructs in pKK223-3. The E. coli-expressed human GHS S-transferases 1-1 and 2-2 do not have blocked N termini in contrast to those directly purified from human livers. These two isozymes, with 11 amino acid substitutions between them, are similar in their Km values for GSH and 1-chloro-2,4-dinitrobenzene and Kcat values for this conjugation reaction. The human GSH S-transferase 2-2, however, is a more active GSH peroxidase than transferase 1-1 toward cumene hydroperoxide and t-butyl hydroperoxide. Our results indicate that different members of a GSH S-transferase gene family with limited amino acid substitutions have different with limited amino acid substitutions have different but overlapping substrate specificities. We propose that accumulation of single amino acid replacements may be an important mechanism for generating diversity in GSH S-transferases with various xenobiotic substrates. In situ chromosomal hybridization results show that the GSH transferase Ha genes are located in the region of 6p12.     

Lung hyperinflation in isolated dog lungs during high-frequency oscillation

To study the phenomenon of lung hyperinflation (LHI), i.e., an increase in lung volume without a concomitant rise in airway pressure, we measured lung volume changes in isolated dog lungs during high-frequency oscillation (HFO) with air, He, and SF6 and with mean tracheal pressure controlled at 2.5, 5.0, and 7.5 cmH2O. The tidal volume and frequency used were 1.5 ml/kg body wt and 20 Hz, respectively. LHI was observed during HFO in all cases except for a few trials with He. The degree of LHI was inversely related to mean tracheal pressure and varied directly with gas density. Maximum expiratory flow rate (Vmax) was measured during forced expiration induced by a vacuum source (-150 cmH2O) at the trachea. Vmax was consistently higher than the peak oscillatory flow rate (Vosc) during HFO, demonstrating that overall expiratory flow limitation did not cause LHI in isolated dog lungs. Asymmetry of inspiratory and expiratory impedances seems to be one cause of LHI, although other factors are involved.     

The influence of prior ovipositional experience on host selection in four species of aphidiid wasps (Hymenoptera: Aphidiidae)

We evaluated the influence of prior ovipositional experience on host selection in four solitary parasitoids, Aphidius erviHaliday, A. pisivorusSmith, A. smithiSharma & Subba Rao, and Praon pequodorumViereck (Hymenoptera: Aphidiidae). When provided simultaneously with equal numbers of two species of aphids, Acyrthosiphon pisum(Harris) and Macrosiphum creeliiDavis (Homoptera: Aphididae), all four parasitoids showed a moderate to strong preference for A. pisum.This preference did not increase after sampling, except in P. pequodorum.Learning did not alter host selection behavior in A. erviand P. pequodorum.However, females of A. pisivorusconditioned on A. pisumselected fewer M. creeliithan unconditioned wasps and wasps conditioned on M. creelii.It is suggested that prior ovipositional experience influences a parasitoid's expectations of the kinds of hosts available, but it does not alter the (innate) rank order of hosts.     

Oxytocin and an oxytocin agonist administered centrally decrease food intake in rats.

Intracerebroventricular administration of oxytocin (OT) and an OT agonist significantly decreased food intake in a dose-related manner in fasted rats. Central administration of an OT antagonist by itself (up to doses of 8 nmol) did not potentiate deprivation-induced food intake, but pretreatment with the OT receptor antagonist prevented the expected inhibition of food intake produced by OT and the OT agonist. Once-daily ICV injections of OT led to the development of tolerance to the inhibitory effects on food intake by the third day of treatment, but daily pretreatment with the OT antagonist prevented the development of this tolerance. In addition to causing decreased food intake, ICV administration of OT significantly increased grooming behavior but produced no dyskinesias. The inhibitory effect of OT on food intake was characterized by decreased amounts of food intake but a normal pattern of ingestion. The anorexia produced was central in nature and was not associated with altered plasma levels of hormones involved in caloric homeostasis or with changes in blood glucose. The OT agonist had relatively little effect on water intake when given in doses that significantly inhibited food intake. These results support the hypothesis that specific OT receptors within the central nervous system participate in the inhibition of feeding under certain conditions in rats.