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P C Weber

Publications and source records attributed to P C Weber.

At least 19 recordsLinked to original sources
PubMed1992

Crystal structure and ligand-binding studies of a screened peptide complexed with streptavidin.

The thermodynamic binding parameters and crystal structure for streptavidin-peptide complexes where the peptide sequences were obtained by random screening methods are reported. The affinities between streptavidin and two heptapeptides were determined by titrating calorimetric methods [Phe-Ser-His-Pro-Gln-Asn-Thr, Ka = 7944 (+/- 224) M-1, delta G degrees = -5.32 (+/- 0.01) kcal/mol, and delta H degrees = -19.34 (+/- 0.48) kcal/mol; His-Asp-His-Pro-Gln-Asn-Leu, Ka = 3542 (+/- 146) M-1, delta G degrees = -4.84 (+/- 0.03) kcal/mol, and delta H degrees = -19.00 (+/- 0.64) kcal/mol]. The crystal structure of streptavidin complexed with one of these peptides has been determined at 2.0-A resolution. The peptide (Phe-Ser-His-Pro-Gln-Asn-Thr) binds in a turn conformation with the histidine, proline, and glutamine side chains oriented inward at the biotin-binding site. A water molecule is immobilized between the histidine and glutamine side chains of the peptide and an aspartic acid side chain of the protein. Although some of the residues that participate in binding biotin also interact with the screened peptide, the peptide adopts an alternate method of utilizing binding determinants in the biotin-binding site of streptavidin.

Amino Acid Sequence↗
PubMed1992

Oligomerization-mediated activation of plasmid-borne genes in Escherichia coli.

A plasmid carrying a weakly expressed neomycin phosphotransferase (neo) gene from the transposable element Tn5 was found to confer elevated levels of antibiotic resistance on its host cell when it existed in a non-monomeric state. This activation of the neo gene appeared to be a generalized effect which can be exerted on any plasmid-encoded gene, since specific sequences were not required for enhanced neo expression, and the activity of a plasmid-borne chloramphenicol acetyltransferase gene could be similarly induced by oligomerization. The potential role that multiple origins of replication present in such oligomeric plasmids play in these observed increases in gene expression is discussed.

DNA Transposable Elements↗
PubMed1992

Bacterial lipopolysaccharide up-regulates platelet-activating factor-stimulated Ca2+ mobilization and eicosanoid release in human Mono Mac 6 cells.

When human monocytic Mono Mac 6 cells were treated with bacterial LPS (10 ng/ml, 72 h), they showed an increase in phagocytic activity, superoxide anion production, and expression of monocyte/macrophage-associated cell surface Ag. In these more mature (LPS-treated) cells but not in untreated cells, platelet-activating factor (PAF) (100 nM) produced a three- to fourfold increase in cytosolic free Ca2+ concentration. The cytosolic free Ca2+ concentration increase was inhibited by the PAF receptor antagonist L-659,989 (10 microM) and by EGTA (2 mM), indicating receptor-dependent Ca2+ influx. Furthermore, L-659,989 (10 microM), as well as PAF (1 microM), inhibited specific [3H]PAF binding in LPS-treated but not in untreated cells. Consistent with these results, PAF (100 nM) stimulated release of arachidonic acid and thromboxane B2 only in LPS-treated cells, and this could be inhibited by L-659,989 (10 microM) and EGTA (2 mM). Our data indicate that LPS up-regulates PAF-induced Ca2+ influx, resulting in arachidonic acid and eicosanoid release in Mono Mac 6 cells.

Antigens, Surface↗
PubMed1992

In vivo expression of beta-galactosidase in hippocampal neurons by HSV-mediated gene transfer.

Stereotactic inoculation of a herpes simplex virus (HSV) gene transfer vector into the hippocampus and caudate of rat brain resulted in limited and transient viral replication and the establishment of latency. Virus attenuation was achieved by insertional inactivation of a viral gene, Us3. Insertion of a lacZ reporter gene, under the control of the HSV glycoprotein C (gC) late gene promoter, allowed viral replication to be monitored in vivo. Unlike unattenuated virus, the Us3::pgC-lacZ recombinant caused little apparent damage to normal hippocampal morphology. Transient lacZ expression was detected in a considerable population of neurons of the dentate gyrus following hippocampal injection, whereas few positively staining neurons were present within the caudate after injection at that site. Latency-associated transcripts, the hallmark of latent infection, were detected in the brain 10 months after injection. This recombinant virus may be useful as a gene transfer vector for long-term expression of foreign genes in the central nervous system.

Animals↗
PubMed1992

Permuteins of interleukin 1 beta--a simplified approach for the construction of permutated proteins having new termini.

A technique for the rapid and simple generation of permutated versions of the interleukin-1 beta (IL-1 beta) gene is described. In this method, the human IL-1 beta cDNA is twice amplified by the polymerase chain reaction (PCR) and the resulting DNA fragments are ligated in tandem. Between the two genes, the DNA sequence encodes a short four amino acid loop to link the native N- and C-terminal ends of the IL-1 beta protein. By using PCR amplification from this starting template, a new version of the IL-1 beta cDNA was obtained that encodes a permutated form of the IL-1 beta protein where the new N- and C-terminal amino acids correspond to residues 65 and 64 of the native IL-1 beta sequence, respectively. The name 'permutein' is proposed to describe proteins generated by this technology. The molecular profile (IL-1 receptor binding, biologic activity and solution properties) of the IL-1 permutein produced by this technology, permutein 65/64, is shown to be identical to that of native IL-1 beta. The approach should be useful to define further the structural features of this protein that are important for its function.

Amino Acid Sequence↗
PubMed1992

Antiviral properties of a dominant negative mutant of the herpes simplex virus type 1 regulatory protein ICP0.

Dominant negative or trans-dominant mutants of viral proteins represent a new and exciting potential approach to antiviral therapy. Unfortunately, the extreme specificity of a given dominant negative mutant limits its general utility in treating a broad spectrum of viral diseases, since it can typically interfere with the activity of only a single viral polypeptide encoded by a single virus. However, it seems likely that dominant negative mutants of promiscuous viral trans-activator proteins, which by definition would repress rather than activate gene expression, should be able to inhibit infectious virus production for a number of different viruses. One such dominant negative mutant, derived from the herpes simplex virus type 1 (HSV-1) regulatory protein ICP0, was found previously to behave as a powerful repressor of gene expression from an assortment of HSV-1 and non-HSV-1 promoters in transient expression assays. In the present study, this ICP0 mutant was found to be capable of inhibiting the replication of both HSV-1 and a completely unrelated virus, human immunodeficiency virus, in cell culture. The properties of this dominant negative mutant indicate that it may have potential as a means of treating diseases caused by a number of DNA and RNA viruses. Moreover, a truncated form of ICP0 which can hypothetically be created by alternative splicing was found to possess similar inhibitory capabilities, suggesting that a virus-encoded version of this dominant negative mutant may play a role in down-regulating HSV-1 gene expression during infection in vivo.

Antiviral Agents↗
PubMed1992

Identification of dominant-negative mutants of the herpes simplex virus type 1 immediate-early protein ICP0.

ICP0 is a 110,000-molecular-weight immediate-early protein of herpes simplex virus type 1 (HSV-1) which is encoded by three exons. It has been shown to function as a promiscuous transactivator of a variety of different HSV-1 and non-HSV-1 promoters in transient expression assays. Analysis of mutations which truncated the carboxy-terminal end of this 775-amino-acid (aa) protein demonstrated that a polypeptide which contained only aa 1 to 553 still possessed significant transactivation potential. Additional carboxy-terminal truncations which sequentially removed aa 245 to 553 and thus the remainder of the third exon resulted in the eventual loss of transactivation capability in these mutants. However, further analysis of these truncated derivatives demonstrated that they behaved as dominant-negative mutants to the wild-type polypeptide. Moreover, one of the mutants was found to act as a promiscuous repressor, in that it could dramatically inhibit a variety of HSV-1 promoters, non-HSV-1 promoters, and heterologous transactivator proteins in transient expression assays, despite having lost almost the entire third exon. These results indicate that a domain encoded by the first two exons probably interacts with, and can effectively titrate, the unknown cellular factor(s) through which ICP0 mediates transactivation.

Animals↗
PubMed1992

The suprahyoid approach for squamous cell carcinoma of the base of the tongue.

The suprahyoid pharyngotomy is a well-recognized surgical approach to the base of the tongue. However, its use has been widely criticized for squamous cell carcinomas. Some surgeons believe this approach offers limited exposure and that violating the preepiglottic space increases the risk of recurrence. A retrospective study of squamous cell carcinoma of the base of tongue was carried out to address these surgical concerns. Nineteen patients presented with T1 or T2 lesions, 13 of whom underwent suprahyoid pharyngotomy with neck dissection. Those with nodal disease underwent adjunctive irradiation therapy and those with extracapsular spread also received subsequent adjuvant chemotherapy. At present, all patients are disease free. The suprahyoid pharyngotomy is an ideal surgical approach in the management of T1 or T2 squamous cell carcinoma of the base of tongue.

Aged↗
PubMed1992

Endogenous eicosanoids modulate cell growth and the expression of immediate early response genes.

Experiments were performed in rat mesangial cells indicating that endogenous non-cyclooxygenase metabolites of AA act to modulate the mitogenic response. Irrespective of the mitogen used, metabolism of AA by LO and/or CP 450 influences induction of cell growth and expression of the immediate early response genes c-fos and Egr-1. In accordance with other reports (3,4) on the influence of eicosanoids on gene expression, our observations indicate that endogenous eicosanoids may act as intracellular mediators. In the mitogenic response, at least two mechanisms of modulation seem possible: i) AA and its metabolites may act by modulating other 2nd messenger systems or ii) eicosanoids may directly regulate the expression of growth-related genes, e.g. by interacting with a fatty acid response element in the promoter region of respective genes.

Animals↗
PubMed1992

Thromboxane as diagnostic tool and therapeutic target in cardiovascular disease.

Our studies were first directed to improvements in analytical methodology of thromboxane metabolism in man. These methods were then applied in a variety of clinical settings to better define the clinical spectrum and timecourse of platelet related pathomechanisms. They were also applied in studies aiming at optimized therapeutic interventions. Such biochemical guidelines are essential for the optimal planning of future clinical studies.

Blood Platelets↗
PubMed1991

Docosahexaenoic acid inhibits PAF and LTD4 stimulated [Ca2+]i-increase in differentiated monocytic U937 cells.

We investigated the effects of different polyunsaturated fatty acids (PUFAs) of the n-6 and n-3 family on the PAF and LTD4 stimulated increase in cytosolic free Ca(2+)-concentration [Ca2+]i in retinoic acid (RA) differentiated, human monocytic U937 cells. Docosahexaenoic acid (10 microM DHA) reduced the PAF induced increase in [Ca2+]i from 455 +/- 25 nM to 319 +/- 24 nM (P less than 0.01). DHA also significantly attenuated the LTD4 induced increase in [Ca2+]. However [Ca2+]i-increase stimulated by f-MLP, ATP, or ionophore A 23187 was not affected by DHA. Other PUFAs like eicosapentaenoic acid (EPA), alpha-linolenic acid (LnA), arachidonic acid (AA) or gamma-linoleic acid (LA) were ineffective. Cellular differentiation as assessed by nitrobluetetrazolium reduction and enhanced expression of specific PAF binding sites in RA treated cells were not altered by DHA. Fatty acid composition in cellular phospholipids revealed effective incorporation of each PUFA. The DHA-effect on [Ca2+]i was time dependent and occurred at 48 h, whereas the DHA-content in phospholipids reached a plateau already at 24 h. The antioxidant vitamin E, the lipoxygenase inhibitor NDGA and the cytochrome P-450 inhibitor SKF 525A completely prevented the DHA induced reduction of PAF stimulated [Ca2+]i-increase. In contrast, the cyclooxygenase inhibitor indomethacin had no effect. Our results indicate that DHA selectively reduces intracellular [Ca2+]i-increases induced by PAF and LTD4 in RA-treated U937 cells, presumably involving an oxidative modification of DHA.

Animals↗
PubMed1991

Crystallographic refinement of bovine pro-phospholipase A2 at 1.6 A resolution.

Bovine pro-phospholipase A2 (Mr = 14,520), trigonal, P3(1)21, a = b = 46.5, c = 102.0 A, one molecule per asymmetric unit, lambda (Cu K alpha) = 1.54 A. The model incorporating 895 protein atoms, two molecules of 2-methyl-2,4-pentanediol, and 60 solvent water molecules, was refined by restrained least squares to a residual R = 0.194 for 14,667 reflections from 5 to 1.6 A resolution.

Enzyme Precursors↗
PubMed1991

Cholesterol modulates PAF-stimulated Ca2(+)-mobilization in monocytic U937 cells.

We investigated the effect of cellular cholesterol content on platelet activating factor (PAF)-stimulated Ca2+ mobilization in the human monocytic cell line U937. When cholesterol auxotroph U937 cells were depleted of cellular cholesterol by a 48-h incubation in delipidated medium, a 40% reduction in the PAF (100 nM)-stimulated increase in cytosolic Ca2+ concentration was seen. Ca2+ mobilization following stimulation with LTD4 (10 nM) or ATP (10 microM) was not affected. Addition of LDL (100 micrograms/ml, 24 h) to the delipidated medium completely recovered cellular cholesterol content and PAF-induced Ca2+ mobilization. These two LDL effects had very similar time- and dose-dependences. Partial recoveries of PAF-induced Ca2+ mobilization, seen after addition of pure cholesterol dissolved in ethanol (30 micrograms/ml, 24 h) or acetyl-LDL (100 micrograms/ml, 24 h), were associated with partial recoveries of cellular cholesterol content. Our results indicate that cellular cholesterol influences PAF-stimulated events in monocytic cells.

Adenosine Triphosphate↗
PubMed1991

Cooperative ligand reorientations in cytochrome c3: a molecular dynamics simulation.

Molecular dynamics simulations of a tetraheme cytochrome c3 were performed to investigate dynamic aspects of the motion of the axial heme iron ligands. It was found that persistent transitions between alternate axial imidazole orientations of the histidine incorporated in the CXXCH heme binding sequence occurred via correlated motions. The correlated motions involved virtually all of the atoms comprising the polypeptide backbone of the heme binding sequence as well as the histidine imidazole side-chain.

Amino Acid Sequence↗
PubMed1991

Endogenous non-cyclooxygenase metabolites of arachidonic acid modulate growth and mRNA levels of immediate-early response genes in rat mesangial cells.

The role of endogenous arachidonic acid and its metabolites as mediators of cell growth was studied in rat mesangial cells. Inhibitors of the cytochrome P450 monooxygenase and lipoxygenase systems (nordihydroguaiaretic acid (NDGA), SK&F 525A, and ketoconazole) significantly reduced serum-stimulated cell growth as determined by cell counts and incorporation of [3H]thymidine. Inhibition of cyclooxygenase or lipoxygenases alone had no effect on cell growth. Stimulation with arginine vasopressin, epidermal growth factor, or phorbol myristate acetate increased [3H]thymidine incorporation and mRNA levels of the immediate-early response genes c-fos and Egr-1. These increases in [3H]thymidine incorporation and mRNA levels were reduced by NDGA and ketoconazole. NDGA, SK&F 525A, and ketoconazole had no effect on cellular ATP levels. Indomethacin had no effect upon cell growth. 14,15-Epoxyeicosatrienoic acid potentiated the effect of arginine vasopressin to enhance [3H]thymidine incorporation. Reverse-phase high pressure liquid chromatography analysis of lipid extracts from cells prelabeled with [3H]arachidonic acid resulted in the detection of a radioactive peak which eluted with lipoxygenase and monooxygenase products, with the same retention time as vicinal dihydroxyeicosatrienoic acids. This peak increased after stimulation with arginine vasopressin or epidermal growth factor and was reduced by preincubation with NDGA. Furthermore, analysis of unlabeled cell extracts by gas chromatography-mass spectrometry revealed the presence of a compound with epoxyeicosatrienoic acid-like characteristics. These results indicate that mesangial cells in culture likely produce products of the cytochrome P450 monooxygenase system that are important endogenous mediators of the growth response to mitogenic agents.

Animals↗