GABAB Receptor-Mediated Enhancement of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Production in Slices of Rat Cerebral Cortex

Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose-related enhancement of the VIP response. The ability of either GABA or (-)-baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors.     

Characterization of an early intermediate in the folding of the alpha subunit of tryptophan synthase by hydrogen exchange measurement

The development of the hydrogen bonding network in the early stages of the folding of the alpha subunit of tryptophan synthase was monitored with a hydrogen exchange technique. The orders of magnitude difference between the rapid conversions of the unfolded forms to two stable intermediates (milliseconds) and the subsequent slow conversions of the intermediates to the native form (greater than 100 s) was used to selectively label with tritium the hydrogen bonds that form in the first 30 s of folding at 0 degree C. Rapid removal of the tritiated solvent by gel filtration ensured that hydrogen bonds formed in subsequent folding reactions would be unlabeled. Limited proteolysis and separation of peptides by high-pressure liquid chromatography permitted the determination of the amount of label retained in individual peptides by scintillation counting. Peptides 1-70 and 71-188, which when covalently linked comprise the stable amino domain in the native conformation, retain 91% and 93%, respectively, of the label retained when the protein is allowed to completely refold in tritiated solvent. Peptide 189-268, the marginally stable carboxyl domain, only retains 43% of the label. The striking difference in retention of label confirms the independent folding of these two domains and shows that the kinetic intermediates that appear in the folding of alpha subunit correspond to structural domains in the native conformation. The near-equality of the labeling of the two peptides comprising the amino domain shows that this domain folds as a single entity and that subdomain folding is unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of zona binding proteins of rabbit, pig, human, and mouse spermatozoa on nitrocellulose blots

Mammalian fertilization is a multi-step process with different requirements for specificity at each step. In the present report we have examined the binding of spermatozoa to homologous and heterologous zonae pellucidae. The homologous zona binding proteins (ZBP) of ejaculated rabbit, pig and human spermatozoa and epididymal mouse spermatozoa have been identified. The rabbit's ZBPs have relative molecular weights (MW) of 32K, 18K, 16K and 14K; the pig's major ZBP is 16K while human spermatozoa bind human zona protein at 17K and 18K. Mouse sperm ZBPs are 19K, 18K and 16K.     

Comparison of goose-type,chicken-type,and phage-type lysozymes illustrates the changes that occur in both amino acid sequence and three-dimensional structure during evolution

Summary The three-dimensional structure of goose-type lysozyme (GEWL), determined by x-ray crystallography and refined at high resolution, has similarities to the structures of hen (chicken) eggwhite lysozyme (HEWL) and bacteriophage T4 lysozyme (T4L). The nature of the structural correspondence suggests that all three classes of lysozyme diverged from a common evolutionary precursor, even though their amino acid sequences appear to be unrelated (Grütter et al. 1983).In this paper we make detailed comparisons of goose-type, chicken-type, and phage-type lysozymes. The lysozymes have undergone conformational changes at both the blobal and the local level. As in the globins, there are corresponding -helices that have rigid-body displacements relative to each other, but in some cases corresponding helices have increased or decreased in length, and in other cases there are helices in one structure that have no counterpart in another.Independent of the overall structural correspondence among the three lysozyme backbones is another, distinct correspondence between a set of three consecutive -helices in GEWL and three consecutive -helices in T4L. This structural correspondence could be due, in part, to a common energetically favorable contact between the first and the third helices.There are similarities in the active sites of the three lysozymes, but also one striking difference. Glu 73 (GEWL) spatially corresponds to Glu 35 (HEWL) and to Glu 11 (T4L). On the other hand, there are two aspartates in the GEWL active site, Asp 86 and Asp 97, neither of which corresponds exactly to Asp 52 (HEWL) or Asp 20 (T4L). (The discrepancy in the location of the carboxyl groups is about 10 Å for Asp 86 and 4 Å for Asp 97.) This lack of structural correspondence may reflect some differences in the mechanisms of action of three lysozymes. When the amino acid sequences of the three lysozyme types are aligned according to their structural correspondence, there is still no apparent relationship between the sequences except for possible weak matching in the vicinity of the active sites.     

Physical and biotic determinants of space utilization by the Galapagos land iguana (Conolophus pallidus)

Summary Home ranges of the Galapagos land iguana (Conolophus pallidus) were examined with respect to food availability and the thermal environment. Activity patterns, the amount of space used per day, and time required to use the entire home range were also investigated. The effects of, and the relationships between, these factors vary seasonally, as do home range sizes and preferred body temperatures.Food supplementation experiments resulted in only temporary reductions in use of space. Home range sizes were not different between the seasons with the least (Fall) and the most (Hot) food availalble, but home ranges were significantly smaller in Garua when food supplies were low, but not as low as in Fall. Calculations of metabolic expenditures in each season suggests that food availability alone does not explain seasonal patterns of home range size in this species.The thermal environment within each home range was characterized by microclimatic measurements and measurements of the area of sun, shade, and semi-shade. An index with units of m²h was used to quantify the thermal quality of each home range. Iguanas exploited optimal (with respect to body temperature) conditions more than would be expected from random use of their home ranges. Thermal transients (due to large body size) and optimal conditions were exploited to the largest degree in Fall.During Garua, low metabolic rates and time constraints imposed by an abundance of stressful thermal environments may result in small home ranges. In Fall, increased temperatures cause higher metabolic rates and allow more time for exploitation of the cooler portions of the home range, hence, home range sizes increase. In the Hot season, there is abundant food and optimal thermal conditions, but home ranges remain large. Searching for preferred foods may cause the large home ranges in this season.     

Oxalate digestibility in Neotoma albigula and Neotoma mexicana

Summary The cactus specialist, Neotoma albigula, tolerates high concentrations of potentially harmful oxalate compounds in its diet. Previous research has shown that oxalate compounds are broken down by intestinal micro-organisms. Thus the ability of N. albigula to utilize a diet high in oxalates may be a consequence of the adaptation of the microflora rather than its own evolution. To test this hypothesis, the oxalate degradation ability of N. albigula was compared with that of N. mexicana, a generalist herbivore. Apparent oxalate digestibility was not significantly different in the two species, when tested using field-acclimated individuals. Analysis of scats recovered from traps indicated that both species were consuming oxalates in the wild. I conclude that the ability of these herbivores to tolerate oxalates is a natural consequence of the utilization of microbial fermentation to degrade the structural carbohydrates of plants coupled with the high adaptive and evolutionary potential of the microflora.     

Dihydrofolate reductase. The stereochemistry of inhibitor selectivity

X-ray structural results are reported for 10 triazine and pyrimidine inhibitors of dihydrofolate reductase, each one studied as a ternary complex with NADPH and chicken dihydrofolate reductase. Analysis of these data and comparison with structural results from the preceding paper (Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B. T., Beddell, C.R., Champness, J.N., Stammers, D.K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391) in which we contrasted binding of the antibiotic trimethoprim (TMP) to chicken dihydrofolate reductase on the one hand with its binding to Escherichia coli dihydrofolate reductase on the other, permit identification of differences that are important in accounting for TMP's selectivity. The crystallographic evidence strongly suggests that loss of a potential hydrogen bond between the 4-amino group of TMP and the backbone carbonyl of Val-115 when TMP binds to chicken dihydrofolate reductase but not when it binds to the E. coli reductase is the major factor responsible for this drug's more potent inhibition of bacterial dihydrofolate reductase. A key finding of the current study which is important in understanding why TMP binds differently to chicken and E. coli dihydrofolate reductases is that residues on opposite sides of the active-site cleft in chicken dihydrofolate reductase are about 1.5-2.0 A further apart than are structurally equivalent residues in the E. coli enzyme.     

Alanine and inter-organ relationships in branched-chain amino and 2-oxo acid metabolism

Branched-chain amino acid metabolism in skeletal muscte promotes the production of alanine, an important precursor in hepatic gluconeogenesis. There is controversy concerning the origin of the carbon skeleton of alanine produced in muscle, specifically whether it is derived from carbohydrate via glycolysis (the glucose-alanine cycle) or from amino acid precursors (viz. glutamate, valine, isoleucine, methionine, aspartate, asparagine) via a pathway involving phosphoenolpyruvate (PEP) carboxykinase and pyruvate kinase, or NADP-malate dehydrogenase (malic enzyme). The relevant literature is reviewed and it is concluded that neogenic flux from amino acids is unlikely to be of major quantitative importance for provision of the carbon skeleton of alanine either in vitro or in vivo. Evidence is presented that branched-chain amino acid oxidation in muscle is incomplete and that the branched-chain 2-oxo acids and the products of their partial oxidation (including glutamine) are released. The role of these metabolites is discussed in the context of fuel homeostasis in starvation.     

Propagation in vitro of the apple rootstock M4: effect of phytohormones on shoot quality

The quality of shoots in cultures of the apple rootstock, M4, was used as a criterion for the selection of an optimum medium. The frequency of shoots in defined shoot clases was monitored for each of five media, which differed in the type and concentration of phytohormone. Media containing BA (1.15 mg l^-1) and IBA (either 0.15 or 0.20 mg l^-1) produced the maximum number of shoots that were desirable for transplantation and acclimatization.     

Queuosine modification of the wobble base in tRNAHis influences ''in vivo'' decoding properties.

The 'in vivo' decoding properties of four tRNAHis isoacceptors, two from Drosophila melanogaster and two from brewer's yeast, were studied after their microinjection, along with turnip yellow mosaic virus (TYMV) coat protein mRNA, into Xenopus laevis oocytes. The two Drosophila isoacceptors are identical besides containing either a guanosine (G) or the hypermodified nucleoside queuosine (Q) in the wobble position. The brewer's yeast isoacceptors differ by four bases in the anticodon stem, and by one base in the amino acceptor stem. Our results show that, under competing 'in vivo' conditions, the Drosophila tRNAHis with the anticodon GUG clearly prefers the histidine codon CAC to the codon CAU, whereas little preference is observed for the tRNAHis with the anticodon QUG for the codon CAU, and no preference for either codon by the two yeast isoacceptors. Hence, it can be concluded that the presence of the Q-base clearly affects the choice of the codon. This is the first demonstration of an 'in vivo' codon preference by tRNA isoacceptors differing in the modification of the wobble base during the elongation step of protein synthesis. These results imply that one function of the Q-base is at the translational level.