Transgenic angiopoietin-like (angptl)4 overexpression and targeted disruption of angptl4 and angptl3: regulation of triglyceride metabolism.

Lipoprotein lipase (LPL) is a key regulator of triglyceride clearance. Its coordinated regulation during feeding and fasting is critical for maintaining lipid homeostasis and energy supply. Angiopoietin-like (Angptl)3 and Angptl4 are secreted proteins that have been demonstrated to regulate triglyceride metabolism by inhibiting LPL.

FGF-21 as a novel metabolic regulator.

Diabetes mellitus is a major health concern, affecting more than 5% of the population. Here we describe a potential novel therapeutic agent for this disease, FGF-21, which was discovered to be a potent regulator of glucose uptake in mouse 3T3-L1 and primary human adipocytes. FGF-21-transgenic mice were viable and resistant to diet-induced obesity.

Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6.

Gene disruption studies of penicillin-binding proteins 1a, 1b, and 2a in Streptococcus pneumoniae.

The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in Streptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed.

16S rRNA is bound to era of Streptococcus pneumoniae.

Era is an essential membrane-associated GTPase that is present in bacteria and mycoplasmas. Era appears to play an important role in the regulation of the bacterial cell cycle. In this study, we expressed the native and glutathione S-transferase (GST) fusion forms of Streptococcus pneumoniae Era in Escherichia coli and purified both proteins to homogeneity.

Penicillin-binding protein 2a of Streptococcus pneumoniae: expression in Escherichia coli and purification and refolding of inclusion bodies into a soluble and enzymatically active enzyme.

Penicillin-binding proteins (PBPs), targets of beta-lactam antibiotics, are membrane-bound enzymes essential for the biosynthesis of the bacterial cell wall. PBPs possess transpeptidase and transglycosylase activities responsible for the final steps of the bacterial cell wall cross-linking and polymerization, respectively. To facilitate our structural studies of PBPs, we constructed a 5'-truncated version (lacking bp from 1 to 231 encoding the N-terminal part of the protein including the transmembrane domain) of the pbp2a gene of Streptococcus pneumoniae and expressed the truncated gene product as a GST fusion protein in Escherichia coli.

Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated.

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S.

Identification, purification, and characterization of transpeptidase and glycosyltransferase domains of Streptococcus pneumoniae penicillin-binding protein 1a.

Resistance to beta-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities.

Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics.

To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically.

Cationic liposome-mediated uptake of human immunodeficiency virus type 1 Tat protein into cells.

The human immunodeficiency virus type 1 (HIV-1) Tat protein strongly transactivates gene expression from the viral long terminal repeat (LTR) and is required for virus efficient replication. Previous studies have shown that cells scrape-loaded in the presence of purified recombinant Tat can absorb the protein in a receptor-independent fashion. Using recombinant Tat in which cysteine residues were blocked by sulfitolysis to prevent disulfide aggregation (S-Tat) we were unable to observe this phenomenon, possibly because of improper protein folding.

Detection of specific human immunodeficiency virus type 1 Tat-TAR complexes in the presence of mild denaturing conditions.

Gene expression from the human immunodeficiency virus 1 (HIV-1) is greatly enhanced by binding of the virally encoded Tat protein to a 59-base RNA stem-loop structure, the Transactivation Responsive Element (TAR), located at the 5'-termini of all viral transcripts. This interaction was investigated in vitro using 32P-labelled TAR and highly purified Tat in which cysteine residues were blocked by sulpitolysis (S-Tat). It is shown that specific complex formation between S-Tat and TAR can occur in the presence of urea, with urea concentrations between 5 and 6 M causing an approximately two-fold increase in the level of binding.

Induction of VanA vancomycin resistance genes in Enterococcus faecalis: use of a promoter fusion to evaluate glycopeptide and nonglycopeptide induction signals.

To characterize induction of VanA resistance a plasmid was constructed in which the gene for firefly luciferase lucA was placed under the control of the promoter for the VanA resistance genes, the vanH promoter. This system afforded convenient quantitative measurement of induction of the VanA genes. Glycopeptide antibiotics and antibiotics representing 19 different mechanisms of action were evaluated for their ability to induce.

Phenethylthiazolylthiourea (PETT) compounds as a new class of HIV-1 reverse transcriptase inhibitors. 2. Synthesis and further structure-activity relationship studies of PETT analogs.

Phenylethylthiazolylthiourea (PETT) derivatives have been identified as a new series of non-nucleoside inhibitors of HIV-1 RT. Structure-activity relationship studies of this class of compounds resulted in the identification of N-[2-(2-pyridyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea hydrochloride (trovirdine; LY300046.HCl) as a highly potent anti-HIV-1 agent.

Phenethylthiazolethiourea (PETT) compounds, a new class of HIV-1 reverse transcriptase inhibitors. 1. Synthesis and basic structure-activity relationship studies of PETT analogs.

A novel series of potent specific HIV-1 inhibitory compounds is described. The lead compound in the series, N-(2-phenethyl)-N'-(2-thiazolyl)thiourea (1), inhibits HIV-1 RT using rCdG as the template with an IC50 of 0.9 microM.

Purification of recombinant human rhinovirus 14 3C protease expressed in Escherichia coli.

A gene encoding the human rhinovirus 14 (HRV14) sequence for expression of the viral polypeptide protein delta 3ABC was inserted into a plasmid driven by the heat-inducible bacteriophage lambda PL promoter. The coding sequence was also inserted into a pET vector for expression in the T7 system to produce 13C, 15N-labeled protein. The expressed HRV14 3C protease (3Cpro) autocatalytically cleaved itself from the polyprotein delta 3ABC, and the mature HRV14 3Cpro partitioned predominantly, in the case of the T7 system, in the insoluble fraction and exclusively, in the case of the PL system, in the insoluble fraction.

The PETT series, a new class of potent nonnucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase.

To identify the minimal structural elements necessary for biological activity, the rigid tricyclic nucleus of the known human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor tetrahydroimidazobenzodiazepinthione was subjected to systematic bond disconnection to obtain simpler structures. A rational selection and testing of modeled analogs containing these potential pharmacophoric moieties led to the discovery of a new series of nonnucleoside inhibitors of RT. The lead compound of this new PETT series of nonnucleoside RT inhibitors, N-(2-phenylethyl)-N'-(2-thiazolyl)thiourea (LY73497), was found to inhibit HIV-1 but not HIV-2 or simian immunodeficiency virus in cell culture at micromolar concentrations.

Effect of fialuridine on replication of mitochondrial DNA in CEM cells and in human hepatoblastoma cells in culture.

Fialuridine (FIAU) is a nucleoside analog with potent activity against hepatitis B virus in vitro and in vivo. In this report, the effect of FIAU on mitochondrial DNA (mtDNA) replication in vitro was investigated. CEM cells, a cell line derived from human T cells, were incubated for 6 days in up to 20 microM FIAU.

Disulfide pairing of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli.

The kringle-2 domain of tissue plasminogen activator, cloned and expressed in Escherichia coli (Wilhelm, O.G., Jaskunas, S.

Mutations in the heparin-binding domains of human basic fibroblast growth factor alter its biological activity.

Eleven structural analogues of human basic fibroblast growth factor (bFGF) have been prepared by site-directed mutagenesis of a synthetic bFGF gene to examine the effect of amino acid substitutions in the three putative heparin-binding domains on FGF's biological activity. After expression in Escherichia coli, the mutant proteins were purified to homogeneity by use of heparin-Sepharose chromatography and analyzed for their ability to stimulate DNA synthesis in human foreskin fibroblasts. Recombinant human bFGF 1-146 and [Ala69,Ser87]bFGF, an analogue where two of the four cysteines had been replaced by alanine and serine, were equipotent to standard bovine basic fibroblast growth factor.

Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli.

The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1.

Recombinant human protein C derivatives: altered response to calcium resulting in enhanced activation by thrombin.

Calcium plays a dual role in the activation of protein C: it inhibits protein C activation by alpha-thrombin, whereas it is required for protein C activation by the thrombomodulin-thrombin complex. Available information suggests that these calcium effects are mediated through calcium induced structural changes in protein C. In this paper, we demonstrate that substitution of Asp167 (located in the activation peptide of human protein C, occupying position P3 relative to the peptide bond Arg169-Leu170 which is susceptible to hydrolysis by thrombin) by either Gly or Phe results in protein C derivatives which are characterized by an altered response to calcium.

Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain.

To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa.

Direct expression of recombinant activated human protein C, a serine protease.

Human protein C, like other serine proteases, is normally secreted as an inactive zymogen. It is converted to its active form extracellularly by limited proteolysis with the thrombin-thrombomodulin complex. This activation results from the removal of a 12-residue activation peptide from the NH2 terminus of the heavy (COOH-terminal) chain.

RecA-independent recombination between direct repeats of IS50.

Recombination between the IS50 sequences that flank Tn5 has been found to occur readily after the transformation of E. coli recA strains with a plasmid that contains direct repeats of these sequences. Analysis of mutants of IS50 indicates that the polypeptide encoded by IS50 that is required for transposition is also required for the recombination.

Role of short regions of homology in intermolecular illegitimate recombination events.

The structures of three recombinants between bacteriophage lambda DNA and plasmid pBR322 that were generated in a recA derivative of Escherichia coli are described. Each resulted from two illegitimate recombination events that resulted in the substitution of part of the lambda genome by part of the plasmid genome. The nucleotide sequences at the six lambda-plasmid junctions were determined and compared with the sequences of the lambda and plasmid genomes before recombination.