Sex pheromone of the cabbage looper: Reactions with antennal proteins in vitro

In the presence of (Z)-7-dodecen-1-ol acetate, the sex attractant of the cabbage looper, Trichoplusia ni, soluble protein from male antennae showed a time-dependent difference spectral absorbance at 280 nm. The change was associated with the enzymatic conversion of the pheromone to (Z)-7-dodecen-1-ol, a potent inhibitor of behavioural responses to the pheromone. In contrast, the response obtained with the inhibitor was indicative of non-enzymatic binding to specific protein(s) in the fraction. GLC analyses of the relative rates of enzymatic hydrolysis of the pheromone by the antennae, haemolymph, and legs revealed 33·9, 10·1, and 6·5 per cent conversion per hour, respectively. These results may provide an insight into the fate of a pheromone in the olfactory process of this insect; however, the significance of the reaction with the inhibitor is not yet known.     

Two related helix-loop-helix proteins participate in separate cell-specific complexes that bind the insulin enhancer.

Cell-specific expression of the insulin gene is dependent on a conserved 8-basepair sequence, GCCATCTG, present in two copies in the 5' flanking DNA of the rat insulin 1 gene (Nir and Far elements). A protein factor with well characterized binding affinities binds to this sequence and is unique to the nuclei of insulin-producing cells. Using the Nir element as a probe to screen a hamster insulinoma cDNA expression library, we cloned two cDNA inserts that encode two related helix-loop-helix DNA-binding proteins: Syrian hamster Pan-1 (shPan-1) and Syrian hamster Pan-2 (shPan-2). These clones have minimal differences from the previously reported human E47/E12 and rat PAN (rPan) DNA-binding proteins. In vitro translated protein products of both clones bound the insulin gene promoter Nir and far elements as well as the E2 elements of the mu heavy chain and kappa light chain immunoglobulin genes. Treating insulinoma cell nuclear extract with antiserum selectively directed to each of the two shPan proteins demonstrated the presence of each form of shPan in separate DNA-binding complexes, which together form the previously described, cell-specific, Nir element-binding complex. We conclude that shPan-1 and shPan-2 are the hamster homologs of the ubiquitous E47/E12 and rPan proteins, but form parts of distinct DNA-binding complexes apparently found only in the nuclei of insulin-producing cells.     

Luteal competency during the resumption of ovarian cyclicity in postpartum Brahman cows

Twenty pluriparous, spring-calving Brahman cows were used to determine luteal competency, as measured by serum progesterone concentrations, during the first and the second postpartum estrous cycles. Prior to and after calving, all cows were maintained in good body condition on Coastal bermudagrass pasture (IFN 1-00-703). The calves were allowed to suckle ad libitum, and sterile marker bulls were maintained with the cow herd as an aid in estrus detection throughout the trial. Cow weight and body condition score were recorded within 24 hours after calving and again at the first behavioral estrus observed. From day 1 through day 14 (day 0 = estrus) of both the first and the second postpartum estrous cycles, blood samples were collected from each cow, processed to yield serum and analyzed by radioimmunoassay for progesterone concentrations. There was a higher incidence of abnormal estrous cycles following the first postpartum estrus (35%) than following the second (5%) postpartum estrus (P<0.05). The abnormal first estrous cycles were characterized by either a short luteal phase (four cows) or by standing estrus behavior without luteal tissue formation (three cows). When serum progesterone concentrations were compared for all cows during the first estrous cycle with those during the second estrous cycle, there was less progesterone released during the cycle (P<0.05) and lower peak progesterone concentrations (P<0.10) during the first estrous cycle. However, if the abnormal cows were excluded from the analyses, there was no difference (P>0.10) in either progesterone concentrations through the 14 days measured or in peak progesterone concentrations between the first and the second postpartum estrous cycles. It can be concluded from this study that the higher incidence of abnormal luteal function following the first postpartum estrus may contribute to the decreased conception rates observed when cows are bred at their first postpartum estrus.     

Isolation and sequence of a rat chymotrypsin B gene

A cDNA clone encoding part of chymotrypsin B was isolated from a cDNA library prepared from rat pancreatic mRNA and used as a probe to isolate the chymotrypsin B gene. The nucleotide sequence of this gene is presented. The 4709-base pair transcribed portion of the isolated gene was inferred from the cDNA and gene sequence, and the 5' border was determined by primer extension on pancreatic polyadenylated RNA. The coding portion of the gene is interrupted by six introns. The active site residues His 57, Asp 102, and Ser 195 are encoded by separate exons. Moreover, two regions of the enzyme which form the substrate-binding pocket are also encoded by separate exons. Thus, the substrate specificity and catalytic activity of the enzyme are produced by joining several exons encoding protein segments that are intrinsically catalytically inactive. The number and location of the intron/exon junctions of the chymotrypsin gene as compared to those of other serine protease genes, as well as the location of the genes on separate chromosomes, suggest that the duplication that resulted in the formation of the chymotrypsin gene was an ancient evolutionary event.     

Mapping polypeptide hormone genes in the mouse: somatostatin, glucagon, calcitonin, and parathyroid hormone

Mouse-Chinese hamster hybrids segregating mouse chromosomes were analyzed by Southern hybridization techniques to map the genes for somatostatin (Smst), glucagon (Gcg), calcitonin (Calc), and parathyroid hormone (Pth). The mouse gene for somatostatin, detected on a 20-kb EcoRI fragment, is located on mouse chromosome 16. Glucagon cDNA hybridized to a 14-kb EcoRI fragment residing on chromosome 2. Calcitonin and parathyroid hormone genes, detected on 7.8-kb HindIII and 6.0-kb BamHI fragments, respectively, were on mouse chromosome 7. The calcitonin and parathyroid hormone genes appear to be part of a larger linkage group which has been conserved in mouse and man.     

Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants

We are probing the determinants of catalytic function and substrate specificity in serine proteases by kinetic and crystallographic characterization of genetically engineered site-directed mutants of rat trypsin. The role of the aspartyl residue at position 102, common to all members of the serine protease family, has been tested by substitution with asparagine. In the native enzyme, Asp102 accepts a hydrogen bond from the catalytic base His57, which facilitates the transfer of a proton from the enzyme nucleophile Ser195 to the substrate leaving group. At neutral pH, the mutant is four orders of magnitude less active than the naturally occurring enzyme, but its binding affinity for model substrates is virtually undiminished. Crystallographic analysis reveals that Asn102 donates a hydrogen bond to His57, forcing it to act as donor to Ser195. Below pH 6, His57 becomes statistically disordered. Presumably, the di-protonated population of histidyl side chains are unable to hydrogen bond to Asn102. Steric conflict may cause His57 to rotate away from the catalytic site. These results suggest that Asp102 not only provides inductive and orientation effects, but also stabilizes the productive tautomer of His57. Three experiments were carried out to alter the substrate specificity of trypsin. Glycine residues at positions 216 and 226 in the substrate-binding cavity were replaced by alanine residues in order to differentially affect lysine and arginine substrate binding. While the rate of catalysis by the mutant enzymes was reduced in the mutant enzymes, their substrate specificity was enhanced relative to trypsin. The increased specificity was caused by differential effects on the catalytic activity towards arginine and lysine substrates. The Gly----Ala substitution at 226 resulted in an altered conformation of the enzyme which is converted to an active trypsin-like conformation upon binding of a substrate analog. In a third experiment, Lys189, at the bottom of the specificity pocket, was replaced with an aspartate with the expectation that specificity of the enzyme might shift to aspartate. The mutant enzyme is not capable of cleaving at Arg and Lys or Asp, but shows an enhanced chymotrypsin-like specificity. Structural investigations of these mutants are in progress.     

Latent-class analysis of recurrence risks for complex phenotypes with selection and measurement error: a twin and family history study of autism.

The use of the family history method to examine the pattern of recurrence risks for complex disorders such as autism is not straightforward. Problems such as uncertain phenotypic definition, unreliable measurement with increased error rates for more distant relatives, and selection due to reduced fertility all complicate the estimation of risk ratios. Using data from a recent family history study of autism, and a similar study of twins, this paper shows how a latent-class approach can be used to tackle these problems. New findings are presented supporting a multiple-locus model of inheritance, with three loci giving the best fit.