Solution structure of the chicken cysteine-rich protein, CRP1, a double-LIM protein implicated in muscle differentiation.

The mechanism by which the contractile machinery of muscle is assembled and maintained is not well-understood. Members of the cysteine-rich protein (CRP) family have been implicated in these processes. Three vertebrate CRPs (CRP1-3) that exhibit developmentally regulated muscle-specific expression have been identified.

Genes coding for phosphotransacetylase and acetate kinase in Sinorhizobium meliloti are in an operon that is inducible by phosphate stress and controlled by phoB.

Recent work in this laboratory has shown that the gene coding for acetate kinase (ackA) in Sinorhizobium meliloti is up-regulated in response to phosphate limitation. Characterization of the region surrounding ackA revealed that it is adjacent to pta, which codes for phosphotransacetylase, and that these two genes are part of an operon composed of at least two additional genes in the following order: an open reading frame (orfA), pta, ackA, and the partial sequence of a gene with an inferred peptide that has a high degree of homology to enoyl-ACP reductase (fabI). Experiments combining enzyme assays, a chromosomal lacZ::ackA transcriptional fusion, complementation analysis with cosmid subclones, and the creation of mutations in pta and ackA all indicated that the orfA-pta-ackA-fabI genes are cotranscribed in response to phosphate starvation.

Comparison of the backbone dynamics of the apo- and holo-carboxy-terminal domain of the biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase.

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of post-translational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post-translational attachment of biotin to a single lysine residue on BCCP.

Structural biology of HIV.

The human immunodeficiency virus (HIV) genome encodes a total of three structural proteins, two envelope proteins, three enzymes, and six accessory proteins. Studies over the past ten years have provided high-resolution three-dimensional structural information for all of the viral enzymes, structural proteins and envelope proteins, as well as for three of the accessory proteins. In some cases it has been possible to solve the structures of the intact, native proteins, but in most cases structural data were obtained for isolated protein domains, peptidic fragments, or mutants.

Agricultural Chemical Safety Training Materials for Farmworkers: Review and Annotated Bibliography.

Preventing or reducing exposure to agricultural chemicals is an important focus for health educators serving migrant and seasonal farmworkers and their families. The development of the US Environmental Protection Agency Worker Protection Standard requiring that farmworkers receive pesticide-related training created a demand for culturally appropriate and effective training materials, yet no compendium of such materials exists. This paper reports the results of a search for and evaluation of training materials designed for use with farmworkers.

Expression and regulation of phosphate stress inducible genes in Sinorhizobium meliloti.

Sinorhizobium meliloti 104A14 was mutated with transposon Tn5B22, which creates lacZ transcriptional fusions when inserted in the correct orientation relative to the promoter. This promoter reporter allowed us to identify six phosphate stress inducible (psi) genes in S. meliloti that are up-regulated in response to inorganic phosphate (Pi) starvation.

The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.

Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box.

NMR structure and comparison of the archaeal histone HFoB from the mesophile Methanobacterium formicicum with HMfB from the hyperthermophile Methanothermus fervidus.

The solution-state structure of the recombinant archaeal histone rHFoB, from the mesophile Methanobacterium formicicum, has been determined by two- and three-dimensional (3D) proton homonuclear correlated nuclear magnetic resonance (NMR) methods. On the basis of 951 nuclear Overhauser effect (NOE)-derived distance restraints, rHFoB monomers form the histone fold and assemble into symmetric (rHFoB)2 dimers that have a structure consistent with assembly into archaeal nucleosomes. rHFoB exhibits approximately 78% sequence homology with rHMfB from the hyperthermophile Methanothermus fervidus, and the results obtained demonstrate that these two proteins have very similar 3D structures, with a root-mean-square deviation for backbone atoms of 0.

Solution structure of a zinc domain conserved in yeast copper-regulated transcription factors.

The three dimensional structure of the N-terminal domain (residues 1-42) of the copper-responsive transcription factor Amtl from Candida glabrata has been determined by two-dimensional 1H-correlated nuclear magnetic resonance (NMR) methods. The domain contains an array of zinc-binding residues (Cys-X2-Cys-X8-Cys-X-His) that is conserved among a family of Cu-responsive transcription factors. The structure is unlike those of previously characterized zinc finger motifs, and consists of a three-stranded antiparallel beta-sheet with two short helical segments that project from one end of the beta-sheet.

Dynamical behavior of the HIV-1 nucleocapsid protein.

The HIV-1 nucleocapsid protein (NC) contains two CCHC-type zinc knuckle domains that are essential for genome recognition, packaging and infectivity. The solution structure of the protein has been determined independently by three groups. Although the structures of the individual zinc knuckle domains are similar, two of the studies indicated that the knuckles behave as independently folded, non-interacting domains connected by a flexible tether, whereas one study revealed the presence of interknuckle NOE cross-peaks, which were interpreted in terms of a more compact structure in which the knuckles are in close proximity.

Cloning and characterization of a Rhizobium meliloti nonspecific acid phosphatase.

Nodulated legumes require high levels of phosphorus for optimal symbiotic performance. However, the basis for this elevated phosphorus requirement is poorly understood, and very little information regarding bacteroid phosphorus metabolism is available. To develop an understanding of the relative importance of organic and inorganic phosphorus sources for bacteroids, we investigated phosphatase activity in Rhizobium meliloti.

Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase.

Baculovirus infection results in the induction of membrane structures within the nucleoplasm of the host cells. The source of these membranes is unclear; however, using the normal dynamics of cellular membranes and the nuclear envelope as a model, it is possible that the cell cycle might play a role in the regulation of formation of these intranuclear membranes. Therefore, one goal of this study was to investigate the effect of baculovirus infection on the cell cycle of Sf9 host cells.

Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element.

The three-dimensional structure of the human immunodeficiency virus-type 1 (HIV-1) nucleocapsid protein (NC) bound to the SL3 stem-loop recognition element of the genomic Psi RNA packaging signal has been determined by heteronuclear magnetic resonance spectroscopy. Tight binding (dissociation constant, approximately 100 nM) is mediated by specific interactions between the amino- and carboxyl-terminal CCHC-type zinc knuckles of the NC protein and the G7 and G9 nucleotide bases, respectively, of the G6-G7-A8-G9 RNA tetraloop. A8 packs against the amino-terminal knuckle and forms a hydrogen bond with conserved Arg32, and residues Lys3 to Arg10 of NC form a 310 helix that binds to the major groove of the RNA stem and also packs against the amino-terminal zinc knuckle.

Autographa californica nuclear polyhedrosis virus: subcellular localization and protein trafficking of BV/ODV-E26 to intranuclear membranes and viral envelopes.

The Autographa californica nuclear polyhedrosis virus da26 gene codes for an envelope protein of both budded virus (BV) and occlusion derived virus (ODV). Western blot and temporal analysis of infected cell extracts detected a protein of 26 kDa by 4 h postinfection (p.i.

Structure of the carboxy-terminal fragment of the apo-biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase.

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle the biotin carboxyl carrier protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of posttranslational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the posttranslational attachment of biotin to an essential lysine residue on BCCP.

Zinc ejection as a new rationale for the use of cystamine and related disulfide-containing antiviral agents in the treatment of AIDS.

The highly conserved and mutationally intolerant retroviral zinc finger motif of the HIV-1 nucleocapsid protein (NC) is an attractive target for drug therapy due to its participation in multiple stages of the viral replication cycle. A literature search identified cystamine, thiamine disulfide, and disulfiram as compounds that have been shown to inhibit HIV-1 replication by poorly defined mechanisms and that have electrophilic functional groups that might react with the metal-coordinating sulfur atoms of the retroviral zinc fingers and cause zinc ejection. 1H NMR studies reveal that these compounds readily eject zinc from synthetic peptides with sequences corresponding to the HIV-1 NC zinc fingers, as well as from the intact HIV-1 NC protein.

Administration of DL-2-amino-5-phosphonovaleric acid (AP5) induces transient inhibition of reminder-activated memory retrieval in day-old chicks.

DL-2-Amino-5-phosphonovaleric acid (50 microM) administered immediately after a visual reminder presented to day-old chickens between 7.5 min and 24 h following a single trial passive avoidance learning task produced transient losses of memory on retention test, an effect not observed in the absence of a reminder or when the reminder was given 48 h post learning. The duration of the transient deficits decreased with increasing interval between training and the reminder trial.

Solution structure of 3-oxo-delta5-steroid isomerase.

The three-dimensional structure of the enzyme 3-oxo-delta5-steroid isomerase (E.C. 5.

N-terminal sequences from Autographa californica nuclear polyhedrosis virus envelope proteins ODV-E66 and ODV-E25 are sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the envelope of occlusion derived virus.

Baculovirus occlusion-derived virus (ODV) derives its envelope from an intranuclear membrane source. N-terminal amino acid sequences of the Autographa californica nuclear polyhedrosis virus (AcMNPV) envelope proteins, ODV-E66 and ODV-E25 (23 and 24 amino acids, respectively) are highly hydrophobic. Recombinant viruses that express the two N-terminal amino acid sequences fused to green fluorescent protein (23GFP or 24GFP) provided visual markers to follow protein transport and localization within the nucleus during infection.

Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos.

The effect of replacing bovine serum albumin (BSA) in a simple defined medium (KSOM) with polyvinyl alcohol (PVA) and/or amino acids on the percentages of mouse zygotes that develop to at least the blastocyst stage and that hatch at least partially or completely is reported. Blastocysts could form when BSA was replaced with only PVA, but at a moderately reduced rate; however, partial hatching, and hence complete hatching, were severely impaired when BSA was replaced with only PVA. The substitution of BSA with amino acids alone resulted in a high rate of blastocyst formation and moderate impairment of hatching.

Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein.

Nucleocapsid p7 (NCp7) proteins of human immunodeficiency virus type 1 (HIV-1) contain two zinc binding domains of the sequence Cys-(X)2-Cys-(X)4-His-(X)4-Cys (CCHC). The spacing pattern and metal-chelating residues (3 Cys, 1 His) of these nucleocapside CCHC zinc fingers are highly conserved among retroviruses. These CCHC domains are required during both the early and late phases of retroviral replication, making them attractive targets for antiviral agents.

Three-dimensional structure of the HTLV-II matrix protein and comparative analysis of matrix proteins from the different classes of pathogenic human retroviruses.

The matrix protein performs similar roles in all retroviruses, initially directing membrane localization of the assembling viral particle and subsequently forming a stable structural shell associated with the inner surface of the mature viral membrane. Although conserved structural elements are likely to perform these functions in all retroviral matrix proteins, invariant motifs are not evident at the primary sequence level and three-dimensional structures have been available for only the primate lentiviral matrix proteins. We have therefore used NMR spectroscopy to determine the structure of the matrix protein from human T-cell leukemia virus type II (HTLV-II), a member of the human oncovirus subclass of retroviruses.

Synergistic antitumour activity of vitamins C and K3 against human prostate carcinoma cell lines.

Vitamins C, K3 (VC, VK3) and a VC/VK3 combination with a VC:VK3 ratio of 100:1 were assayed for their antitumour activity against two human prostatic carcinoma cell lines. Co-administration of the vitamins enhanced the antitumour activity 5- to 20-fold even with a 1 h exposure time. While exogenous catalase destroyed the antitumour activity, hydrogen peroxide-induced lipid peroxidation was negligible.

Comparison of the NMR and X-ray structures of the HIV-1 matrix protein: evidence for conformational changes during viral assembly.

The three-dimensional solution- and solid-state structures of the human immunodeficiency virus type-1 (HIV-1) matrix protein have been determined recently in our laboratories by NMR and X-ray crystallographic methods (Massiah et al. 1994. J Mol Biol 244:198-223; Hill et al.