CABIOS REVIEW: The GenBank nucleic acid sequence database

‘The GenBank’^* nucleic acid sequence database isa computer-based collection of all published DNA and RNA sequences;it contains over five million bases in close to six thousandsequence entries drawn from four thousand five hundred publishedarticles. Each sequence is accompanied by relevant biologicalannotation. The database is available either on magnetic tape,on floppy diskettes, on-line or in hardcopy form. We discussthe structure of the database, the extent of the data and theimplications of the database for research on nucleic acids.     

Highly potent side chain–main chain cyclized dermorphin–deltorphin analogues: An integrated approach including synthesis,bioassays, NMR spectroscopy and molecular modelling

Our continuing efforts to study structure–activity relationships of peptide opioids have resulted in the synthesis of a series of cyclic opioids related to dermorphins and deltorphins. The biological activities of the compounds have been determined and the conformational analyses carried out using ¹H-NMR spectroscopy and molecular modelling. The three compounds in the series Tyr-c[D -Orn-Phe-Ala], Tyr-c[D -Lys-Phe-Ala], and Tyr-c[A₂Bu-Phe-Ala-Leu] are cyclized via a lactam bridge from the side-chain of the residue at the second position with the carboxyl terminus of each compound. The molecules incorporate 12-, 13- and 14-membered rings, respectively. They include a phenylalanine at the third position which is a distinguishing characteristic of dermorphins and deltorphins. The guinea pig ileum and mouse vas deferens assays show that the compounds are highly active at both μ- and δ-opioid receptors. The compounds are all highly effective antinociceptive agents as measured by the intrathecal rat hot plate test. Conformational analyses of the molecules indicate that they can adopt topochemical arrays required for bioactivity at both μ- and δ-receptors which explains their high activity in both guinea pig ileum and mouse vas deferens in vitro assays. The results support our models for μ- and δ-receptor activity for constrained peptide opioids.     

Evidence that cyclic adenosine 3',5'-monophosphate-dependent protein kinase activation causes pig ovarian granulosa cell differentiation, including increases in two type II subclasses of this kinase

Agents that elevated intracellular cyclic adenosine 3',5'-monophosphate (cAMP) caused a 3- to 10-fold increase in the luteinizing hormone (LH) receptor level and in progesterone biosynthesis in primary cultures of pig ovarian granulosa cells. Associated with these effects was a 2- to 4-fold increase in the total activity of the catalytic subunit of cAMP-dependent protein kinase in the tissue. From quantitation by [3H]cAMP binding and changes in the specific labeling with the photoaffinity analog [32P]-8-azido-cAMP, these agents were found to cause a concomitant 5- to 15-fold increase in two isoforms of the type II R-subunit (Mr = 54,000 and 56,000) of the protein kinase. Since the two intrasubunit cAMP binding sites of the protein kinase have been found to be positively cooperative, the addition of a combination of an analog selective for site 1 and an analog selective for site 2 causes synergistic increases in protein kinase activation in vitro and synergistic increases in intact cell responses if mediated by the cAMP-dependent protein kinase. In the present study, the addition of such a combination of site 1- and site 2-selective analogs to granulosa cells caused a synergistic increase in LH receptor induction and progesterone production. For both responses, synergism did not occur when two analogs selective for the same site were combined. The results indicated that these responses are mediated by either of the two major isozyme types of cAMP-dependent protein kinase.     

Overview of the LiMB database.

The rapidly increasing number of databases relevant to molecular biology has given rise to a need for a coordinated effort to identify, characterize, and link them. The LiMB database, which contains information about molecular biology and related databases, is a step in that direction. It serves molecular biologists seeking data sets containing information relevant to their research, and is also intended to anticipate the needs of database designers and managers building software links for related data sets. We present an abbreviated version of the database here; the full database is available free of charge as described below.     

Metabolism of sugars to polyols in the boll weevil

When fed to starved adults of Anthonomus grandis, several pentoses and hexoses were metabolized to the corresponding polyols (sugar alcohols). Xylitol, galactitol, arabitol, ribitol, rhamnitol, mannitol, and sorbitol were metabolites of d-xylose, d-galactose and lactose, d-arabinose, d-ribose, l-rhamnose, d-mannose, and d-glucose and d-fructose, respectively. l-Sorbose was not metabolized to a polyol. Large quantities of xylitol and galactitol and intermediate amounts of arabitol, ribitol, and rhamnitol accumulated while only small amounts or traces of mannitol and sorbitol were detected. The limited accumulation of sorbitol in the glucose- and fructose-fed weevils probably was caused by the rapid metabolism of sorbitol to glucose, fructose, trehalose, and glycogen. Each of the ingested sugars, the corresponding polyols, and trehalose were present in the weevil haemolymph. Most of the polyols had never before been detected as metabolites in an insect.     

Synthesis and biological evaluation of N alpha-hydroxysulfonyl-[Nle28,31]-CCK26-33

Two analogues of [Nle28,31]-CCK26-33 containing an N-terminal acetyl or N-terminal hydroxysulfonyl moiety were prepared and characterized. Both analogues were equipotent to native CCK26-33 in four bioassays, demonstrating that N-terminal sulfation of CCK26-33 analogues is compatible with full biological activity.     

Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction.

Mammalian fertilization involves interactions of sperm surface receptors with ligands of the zona pellucida, an extracellular matrix surrounding the ovulated egg. In mouse, the zona is composed of three glycoproteins. One of them, ZP3, participates in primary sperm binding and in the subsequent triggering of the sperm's acrosome reaction. Considerable evidence suggests that carbohydrate determinants of ZP3 are responsible for binding to sperm and may be important for acrosomal exocytosis. A full-length cDNA encoding mouse ZP3 was assembled and cloned into expression vectors that contained either a cytomegalovirus (CMV) or a vaccinia (P11) promoter. Mouse L-929 cells were stably transformed with the pZP3-CMV constructs, and green monkey CV-1 cells were infected with a recombinant vaccinia virus containing ZP3. rZP3 was affinity purified from culture media and detected on Western blots as a single 60- to 70-kDa band, which differed in molecular weight from native ZP3 (mean, 83 kDa). Nevertheless, rZP3 is biologically active. rZP3 decreases sperm-zona binding with a potency equivalent to that of native zona pellucida and, like native ZP3, rZP3 triggers acrosomal exocytosis in capacitated mouse sperm. Thus, rZP3 isolated from both rodent and primate cells appears to contain those carbohydrate and protein structures necessary for ZP3's dual role in fertilization.     

Influence of peptidase inhibitors on the apparent agonist potency of delta selective opioid peptides in vitro.

Several peptides of diverse structure, reported to possess high affinity and selectivity for the delta opioid receptor, were studied using the mouse isolated vas deferens preparation to determine the effect of peptidase inhibition on their apparent potency. The peptides evaluated included [Leu5] enkephalin, the cyclic enkephalin analogs [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,p-F-Phe4,D-Pen5]enkephalin (F-DPDPE), the linear enkephalin analogs [D-Ala2,D-Leu5]enkephalin (DADLE) and [D-Ser2(O-tBu), Leu5,Thr6]enkephalin (DSTBULET), and the naturally occurring amphibian peptides Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (dermenkephalin), Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin II). Concentration-response curves were determined for each peptide in the absence and presence of a combination of the peptidase-inhibiting agents bacitracin, bestatin, and captopril. A wide range of potencies was observed, both in the control state and in the presence of peptidase inhibition. The synthetic enkephalin analogs demonstrated small increases in potency with peptidase inhibition (no increase in the case of DPDPE), whereas the naturally occurring peptides were markedly increased in potency, up to as much as 123-fold for dermenkephalin. In the presence of peptidase inhibition, deltorphin II was the most potent peptide tested (IC50 = 1.13 x 10(-10) molar), and as such is the most potent delta opioid agonist reported to date. Stability to metabolism must be considered in the design and evaluation of in vitro experiments using peptides of this type.     

Gene sequence and predicted amino acid sequence of the motA protein, a membrane-associated protein required for flagellar rotation in Escherichia coli.

The motA and motB gene products of Escherichia coli are integral membrane proteins necessary for flagellar rotation. We determined the DNA sequence of the region containing the motA gene and its promoter. Within this sequence, there is an open reading frame of 885 nucleotides, which with high probability (98% confidence level) meets criteria for a coding sequence. The 295-residue amino acid translation product had a molecular weight of 31,974, in good agreement with the value determined experimentally by gel electrophoresis. The amino acid sequence, which was quite hydrophobic, was subjected to a theoretical analysis designed to predict membrane-spanning alpha-helical segments of integral membrane proteins; four such hydrophobic helices were predicted by this treatment. Additional amphipathic helices may also be present. A remarkable feature of the sequence is the existence of two segments of high uncompensated charge density, one positive and the other negative. Possible organization of the protein in the membrane is discussed. Asymmetry in the amino acid composition of translated DNA sequences was used to distinguish between two possible initiation codons. The use of this method as a criterion for authentication of coding regions is described briefly in an Appendix.