Protein threading by recursive dynamic programming.

We present the recursive dynamic programming (RDP) method for the threading approach to three-dimensional protein structure prediction. RDP is based on the divide-and-conquer paradigm and maps the protein sequence whose backbone structure is to be found (the protein target) onto the known backbone structure of a model protein (the protein template) in a stepwise fashion, a technique that is similar to computing local alignments but utilising different cost functions. We begin by mapping parts of the target onto the template that show statistically significant similarity with the template sequence. After mapping, the template structure is modified in order to account for the mapped target residues. Then significant similarities between the yet unmapped parts of the target and the modified template are searched, and the resulting segments of the target are mapped onto the template. This recursive process of identifying segments in the target to be mapped onto the template and modifying the template is continued until no significant similarities between the remaining parts of target and template are found. Those parts which are left unmapped by the procedure are interpreted as gaps.The RDP method is robust in the sense that different local alignment methods can be used, several alternatives of mapping parts of the target onto the template can be handled and compared in the process, and the cost functions can be dynamically adapted to biological needs.Our computer experiments show that the RDP procedure is efficient and effective. We can thread a typical protein sequence against a database of 887 template domains in about 12 hours even on a low-cost workstation (SUN Ultra 5). In statistical evaluations on databases of known protein structures, RDP significantly outperforms competing methods. RDP has been especially valuable in providing accurate alignments for modeling active sites of proteins.RDP is part of the ToPLign system (GMD Toolbox for protein alignment) and can be accessed via the WWW independently or in concert with other ToPLign tools at http://cartan.gmd.de/ToPLign.html.     

PCO(2) threshold for CNS oxygen toxicity in rats in the low range of hyperbaric PO(2).

Central nervous system (CNS) oxygen toxicity, as manifested by the first electrical discharge (FED) in the electroencephalogram, can occur as convulsions and loss of consciousness. CO(2) potentiates this risk by vasodilation and pH reduction. We suggest that CO(2) can produce CNS oxygen toxicity at a PO(2) that does not on its own ultimately cause FED. We searched for the CO(2) threshold that will result in the appearance of FED at a PO(2) between 507 and 253 kPa. Rats were exposed to a PO(2) and an inspired PCO(2) in 1-kPa steps to define the threshold for FED. The results confirmed our assumption that each rat has its own PCO(2) threshold, any PCO(2) above which will cause FED but below which no FED will occur. As PO(2) decreased from 507 to 456, 405, and 355 kPa, the percentage of rats that exhibited FED without the addition of CO(2) (F(0)) dropped from 91 to 62, to 8 and 0%, respectively. The percentage of rats (F) having FED as a function of PCO(2) was sigmoid in shape and displaced toward high PCO(2) with the reduction in PO(2). The following formula is suggested to express risk as a function of PCO(2) and PO(2) [abstract: see text] where P(50) is the PCO(2)for the half response and N is power. A small increase in PCO(2) at a PO(2) that does not cause CNS oxygen toxicity may shift an entire population into the risk zone. Closed-circuit divers who are CO(2)retainers or divers who have elevated inspired CO(2)are at increased risk of CNS oxygen toxicity.     

Use of pre-embedding ultrastructural immunocytochemistry in the localization of a secretory product and membrane proteins in cultured prolactin cells

A pre-embedding immunoperoxidase procedure performed directly on cultured cells in situ was used to localize several intracellular antigens at the electron-microscope level. With this procedure, we compared the effect of various fixatives, with or without saponin permeabilization, on the immunoreactivity of a secretory product (prolactin) and membrane proteins in cultured prolactin cells. Prolactin was detected within all compartments of its intracellular secretory pathway. Membrane antigens of the rough endoplasmic reticulum, the Golgi apparatus, and lysosomes were localized in distinct intracellular compartments. These immunocytochemical results are discussed in relationship to others in the literature that describe the localization of similar types of antigens. The technique, here described, which preserves ultrastructural detail and antigenicity, should be applicable for the localization of other intracellular antigens in cultured cells.     

The effects of monocyte-conditioned medium and interleukin 1 on the synthesis of collagenous and non-collagenous proteins by mouse bone and human bone cells in vitro

Cultural adherent human mononuclear cells produce factor(s) which stimulate the release of calcium from new-born mouse calvaria in organ culture. This stimulation of bone resorption is accompanied by an inhibition of the incorporation of [³H]proline into collagen which is independent of increased prostaglandin production by the bone. When human osteoblast-like cells are treated with conditioned medium from human mononuclear cells, collagen accounts for a decreased proportion of the protein synthesised. This effect on matrix synthesis is not accompanied by an inhibitory action of the monocyte-conditioned medium preparations on net cell proliferation. In human osteoblast-like cell cultures, partially purified human interleukin 1 also inhibits the production of the bone-specific protein osteocalcin in a dose-dependent fashion. These observations are consistent with the hypothesis that products of human monocytes similar to, or identical with, human interleukin 1 may be important regulators of bone metabolism and may contribute to the bone loss seen in diseases such as chronic rheumatoid arthritis.     

Cloning and expression of the 3' portion of the T4 denV gene as a lacZ fusion gene

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage lambda gt11 T4 dC DNA library was screened for phage which produced a beta-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25% of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within lambda gt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native beta-galactosidase.     

Peripheral receptor populations involved in the regulation of gastrointestinal motility and the pharmacological actions of metoclopramide-like drugs

This minireview is concerned with a re-examination of the locus of action and the possible peripheral mechanisms involved in the gastrointestinal (GI) stimulant effects of metoclopramide. Such a re-evaluation is opportune given the increasing use of this drug in the therapy of certain GI tract disorders. To provide an orientation on this subject the location in the GI tract and function of several relevant receptor types have been reviewed. In the past metoclopramide has been reported to enhance contractions of a variety of GI preparations to electrical stimulation, acetylcholine, carbachol and ganglion stimulants, to inhibit responses to alpha 2-adrenoreceptor agonists and 5-hydroxytryptamine, as well as blocking those to dopamine. Also in such preparations metoclopramide facilitates the release of acetylcholine to transmural stimulation. One important question is whether this effect is mediated via a specific prejunctional receptor. In this respect 2 suggestions have been made. Firstly that the drug may act as a preferential, prejunctional muscarinic antagonist thus inhibiting the negative feedback inhibition of acetylcholine release and secondly that metoclopramide may be a prejunctional agonist (partial) at 5-hydroxy-tryptamine receptors. Although the latter possibility appears most tenable at present, the involvement of a specific receptor remains to be confirmed. The important finding that dopamine receptors are probably not involved in the local stimulant effects of metoclopramide has important implications for future research orientated towards the discovery of a new generation of GI drugs lacking the side effects associated with central dopamine receptor blockade. Several compounds (cinitapride, BRL 20627A and cisapride) are now in the early stages of clinical evaluation.     

Is prostaglandin E the neural mediator of the febrile response? The case against a proven obligatory role.

We have reviewed the evidence in favor of a prostaglandin mediator of the thermal responses in fever and found that PGE injected into the hypothalamus does not always cause fever, that cerebrospinal fluid concentrations of PGE are not reliable reflections of hypothalamic events, and that antipyretic drugs may act in ways other than inhibiting PGE synthesis. Fever is not blocked by prostaglandin antagonists, nor by ablation of PGE-sensitive areas of the brain. There is poor correlation between the effects of pyrogens and of PGE on cerebral neurons. There is evidence that at least one prostanoid other than prostaglandin is a mediator of fever, but the prostanoid has not been identified yet. We conclude that PGE may contribute to the neural responses in fever but is not essential.