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P Schultz

Publications and source records attributed to P Schultz.

At least 55 records · Page 3Linked to original sources
PubMed1996

Linker histone-dependent DNA structure in linear mononucleosomes.

We have examined the binding of the linker histone H5 (LH) to mononucleosomes. Mononucleosomes reconstituted on short DNA fragments display a series of discrete bands on a gel corresponding to various nucleosome positions along the DNA. When a series of engineered H5s with differing extents of the C-terminal tail are bound to these mononucleosomes, the electrophoretic mobilities of the resulting complexes are altered. Not only is there a general increase in mobility upon complex formation, but there is a reduction in the differences in mobility of the most distal nucleosomes. The complexes were also visualized by electronmicroscopy. From these two complementary studies, we conclude the following. (1) Entering and exiting DNAs are uncrossed in the LH-free particles, despite a DNA wrapping of 1.65 to 1.7 turns around the histone core. This results from a bending of the entering and exiting DNA away from each other and the histone surface, presumably as a consequence of electrostatic repulsion. This confirms and extends conclusions derived from our recent examination of the same particles in 3D through cryo-electron microscopy. (2) Binding of the globular domain of H5 increases DNA wrapping to 1.8 to 1.9 turns, but fails to induce a crossing due to an accentuation of the bends. (3) The C-terminal tail of H5 bridges entering and exiting DNAs together into a four-stranded stem over a distance of about 30 bp. The occurrence of such a stem may introduce constraints on models of the 30 nm chromatin fiber.

Animals↗
PubMed1996

The value of continuous blockade of the lumbar plexus as an adjunct to acetylsalicyclic acid for pain relief after surgery for femoral neck fractures.

In a randomized, double-blind investigation the analgesic effect of continuous blockade of the lumbar plexus as an adjunct to acetylsalicyclic acid by suppository after surgery for femoral neck fractures under spinal anaesthesia was examined in 20 patients. Before surgery, a catheter was inserted into the femoral nerve sheath. The patients were allocated randomly to receive bupivacaine or saline by bolus and then continuous infusion, started immediately after the operation. No statistically significant differences in additional morphine requirements, visual analogue pain scores or adverse effects were observed between the two treatment groups. It is concluded that continuous blockade of the lumbar plexus as an adjunct to rectal acetylsalicyclic acid offers no major additional pain relief after surgery for femoral neck fractures under spinal anaesthesia.

Administration, Rectal↗
PubMed1996

Relevance of RET proto-oncogene mutations in sporadic medullary thyroid carcinoma.

Analysis of peripheral blood or tumor DNA samples from 101 patients with apparent sporadic medullary thyroid carcinoma (MTC) was performed to assess the frequency of RET proto-oncogene mutations in this patient population. Peripheral blood and/or tumor DNA was amplified by polymerase chain reaction. DNA sequence or restriction enzyme analysis was performed to detect mutations of RET proto-oncogene codons 609, 611, 618, 620, 634, 768, and 918. Six of 101 patients with apparent sporadic MTC had peripheral blood DNA mutations more commonly associated with hereditary MTC. In 4 patients, these mutations led to the identification of previously unrecognized kindreds. The remaining 2 patients were examples of de novo mutations. A codon 918 mutation was found in 14 of 57 (approximately 25%) tumor DNA samples. Mutations were not identified in the remaining patients. In this large cancer center population, approximately 6% of patients with sporadic MTC carry peripheral blood DNA mutations, either inherited or de novo, more commonly associated with MEN 2A or familial MTC. Seven additional gene carriers were identified as a direct result of these studies, a 2-fold multiplying effect. We conclude routine application of RET proto-oncogene testing should be included in all cases of apparent sporadic MTC.

Base Sequence↗
PubMed1995

Structural analysis of a MIP family protein from the digestive tract of Cicadella viridis.

Homopteran insects, and especially Cicadella viridis, display in their digestive tract a specialized epithelial differentiation, the filter chamber (FC) acting as a water-shunting complex. The main intrinsic membrane protein of the FC is a 25,000-Da polypeptide (P25). In this paper we demonstrate that this P25 polypeptide is a member of the MIP family of membrane channel proteins, and that P25 forms homotetramers in the native membranes. Using polymerase chain reaction, a 360-base pair cDNA, named cic, was isolated from RNA of the FC. cic encodes a 119-amino acid polypeptide (CIC) whose homologies with MIP26, AQP1 (CHIP), AQP2, and gamma-TIP are 38, 38, 34, and 20%, respectively. Using a specific antibody raised against a 15-amino acid peptide from the CIC sequence, we concluded that CIC and P25 are identical entities, and hence that P25 belongs to the MIP family. We investigated the quaternary structure of P25 in the membranes of the FC using biophysical analysis of P25 nondenaturing detergent micelles, scanning transmission electron microscopy, and image processing of conventional transmission electron microscopic images. All those different approaches converged to the conclusion that P25 exists as an homotetramer forming a regular two-dimensional array in the membranes.

Amino Acid Sequence↗
PubMed1994

Three-dimensional model of Escherichia coli gyrase B subunit crystallized in two-dimensions on novobiocin-linked phospholipid films.

Two-dimensional crystals of the Escherichia coli DNA gyrase B subunit were obtained upon specific interactions with novobiocin linked phospholipid films. A three-dimensional surface model of the protein was generated by analysing images of tilted negatively stained crystals. The structure showed, at 2.5 to 3.0 nm resolution, two elongated arms organised as a V-shaped protein: the bottom of the V contains the novobiocin binding site, and the extremities of the arms mediate protein-protein interactions between the two monomers in the unit cell. Image analysis of frozen hydrated two-dimensional crystals resulted in a 1.0 nm resolution projection map that shows structural elements not revealed with negative staining. Electron microscopic structural data were compared with the crystallographic structure of the 43 kDa N-terminal fragment of the B subunit complexed with a non hydrolysable ATP analogue.

Binding Sites↗
PubMed1994

Proficiency of internal medicine residents in outpatient care of patients with HIV infection.

To determine internal medicine residents' knowledge of HIV care, the authors conducted a survey of residents from four internal medicine programs in the San Francisco Bay area. On a knowledge test, the mean score was 42.4/55, 77% correct. The residents performed relatively worse on questions regarding didanosine and zalcitabine, tuberculosis prophylaxis, and risk of cervical neoplasia in HIV-infected women. Predictors of greater knowledge were specific residency program, higher postgraduate year, primary care residency track, and more extensive HIV experience. Primary care internal medicine residencies and programs with more exposure to HIV patients are most effective in producing knowledgeable residents.

Ambulatory Care↗
PubMed1994

Mass analysis of biological macromolecular complexes by STEM.

Scanning transmission electron microscopy (STEM) provides a superbly versatile method of measuring the masses of macromolecular complexes ranging in size from single protein subunits to large virus particles. The physical basis of the method is the elastic scattering of electrons by the component atoms of the specimen. Unstained molecules yield a dark-field signal that is proportional to their local mass density, thus allowing direct measurements of the total mass of an individual particle, as well as of the masses of its resolved domains by integrating over appropriate regions of the image. In this review, we present an introduction to the STEM method of mass analysis from a practical standpoint, stressing the essential points of specimen preparation, as well as the scope and current limitations of the method. Its potentialities are illustrated by applications to several classes of macromolecules: isolated oligomeric proteins (the envelope glycoprotein of HIV), nucleoprotein complexes (SV40 minichromosome, transcription factor TFIIIC), membranous specimens (clathrin-coated membranes, the VDAC channel), and viruses (vesicular stomatitis virus; herpes simplex virus). In the case of multicomponent complexes, STEM mass measurements of both the intact complex and of defined biochemical derivatives (for instance, after extraction of specific components), allow one to compile complete and precise molecular inventories. Finally, we briefly anticipate future advances that should allow even more precise and detailed mass mappings, the labelling of specific sites with heavy atom clusters, and elemental mapping based on weak inelastic signals acquired in parallel with the relatively intense dark-field signals that have been so successfully exploited to date.

Animals↗
PubMed1993

Basal promoter and enhancer element of yeast U6 snRNA gene.

The yeast U6 snRNA gene, SNR6, transcribed by RNA polymerase III or C, is shown to have a mixed promoter with upstream, intragenic and downstream elements. The distant downstream B block behaves as a typical enhancer element. Required in vivo, and for transcription of chromatin templates in vitro, it was also active in reversed orientation. As shown by footprinting and electron microscopy, the factor TFIIIC, or tau, bound the B block in an oriented manner and was able to induce DNA looping. The factor TFIIIC appeared to act via a weak A block located at position +21. This A block-related motif was essential in vivo and with chromatin templates. When changed into a consensus A block it favored DNA looping by TFIIIC firmly anchored on the B block, and activated a B block lacking gene in vivo and in vitro. The role of the TATA box at -30 was most apparent using a purified transcription system. With the A block, it appeared to contribute to start site selection. The results suggest a model where three weak promoter elements collaborate to assemble the transcription complex by DNA looping and synergistic protein-DNA interactions.

Base Sequence↗
PubMed1993

On the subunit composition, stoichiometry, and phosphorylation of the yeast transcription factor TFIIIC/tau.

Saccharomyces cerevisiae transcription factor IIIC/tau is a multisubunit DNA-binding protein that plays key roles in tRNA and 5 S rRNA gene activation. Subunit composition, stoichiometry, and in vivo phosphorylation of TFIIIC/tau factor were investigated using factor prepared from strains carrying modified forms of TFC1, the gene encoding the 95-kDa TFIIIC/tau subunit (tau 95). Using an epitope-tagged TFC1 as well as a TFC1-lacZ fusion, TFIIIC was shown to contain a single 95-kDa subunit, which was localized by electron microscopy into tau A, the A block-binding domain of TFIIIC/tau. Three 35S-labeled polypeptides (at 138, 131, and 91 kDa) coimmunoprecipitated with a tau 95-beta-galactosidase fusion protein. The coprecipitation of the 91-kDa polypeptide makes it a likely subunit of the factor. Immunoprecipitation from 32P-labeled extracts revealed that three of the subunits (138, 131, and 95 kDa), but not the 91-kDa component, are phosphorylated in vivo.

Base Sequence↗
PubMed1993

Increasing the chemical potential of the germ-line antibody repertoire.

To augment the chemical potential of the immunological repertoire, a metal ion-binding light chain has been introduced into the murine genome. Mice containing the transgene were subsequently immunized with a fluorescein conjugate. The transgenic light chain was found at a high frequency in the anti-fluorescein memory B-cell compartment. This general method should be applicable to other cofactors and small molecules and should lead to generation of antibodies with unique catalytic activities.

Animals↗
PubMed1993

Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals.

Two-dimensional crystals of yeast RNA polymerase I dimers were obtained upon interaction with positively charged lipid layers. A three-dimensional surface model of the enzyme was determined by analyzing tilted crystalline areas and by taking advantage of the non-crystallographic internal symmetry of the dimer to correct for the missing viewing directions. The structure shows, at approximately 3 nm resolution, an irregularly shaped molecule 11 nm x 11 nm x 15 nm in size characterized by a 3 nm wide and 10 nm long groove which constitutes a putative DNA binding site. The overall structure is similar to the Escherichia coli holo enzyme and the yeast RNA polymerase II delta 4/7 structures. The most remarkable structural feature is a finger-shaped stalk which partially occludes the entrance of the groove and forms a 2.5 nm wide channel. We discuss the possible location of the catalytic centre and of the carboxy-terminal region of the beta-like subunit in the channel. The interference of different DNA fragments with RNA polymerase dimerization and crystallization indicates the orientation of the template in the putative DNA binding groove.

Amino Acid Sequence↗
PubMed1992

Modified nucleosides in human serum.

Methylated purines and pyrimidines derived from the degradation of transfer ribonucleic acid have been shown to be excreted in abnormal amounts in the urine of patients with cancer. Recent technology developed by Gehrke and Kuo has allowed the separation and quantification of modified nucleosides in serum using reversed-phase high-performance liquid chromatography with diode-array measurement. Serum levels of ten modified nucleosides were measured in 37 normal healthy adults to establish normal values and to correlate activity with age and sex. In addition, serum levels of patients with several malignancies were measured to determine activity in these diseases. Levels of modified nucleosides in normal individuals were consistently reproducible and showed no significant variation among males versus females or with age. Patients with malignant diseases showed consistent elevations and these were highest in patients with more advanced disease. The evidence of no significant differences in the mean levels of modified nucleosides in serum with age or sex in normal adults and elevations in patients with malignancies demonstrate the potential value of modified nucleosides as cancer biomarkers.

Adult↗
PubMed1992

Complementation of formyl peptide receptor-mediated signal transduction in Xenopus laevis oocytes.

Formyl-methionine-containing peptides (e.g. fMet-Leu-Phe) stimulate a variety of neutrophil functions by interacting with specific cell surface receptors which are coupled via G-proteins to stimulation of phospholipase C. Two markedly distinct cDNAs coding for formyl peptide receptors have recently been isolated from a rabbit and a human cDNA library respectively. To examine the hitherto unknown signal transduction properties of the formyl peptide receptor encoded by the human cDNA, we have used the PCR to clone this cDNA from poly(A)+ RNA of myeloid differentiated human leukaemia (HL-60) cells, and have injected the cDNA-derived receptor cRNA into Xenopus laevis oocytes. Receptor activity was determined electrophysiologically by measuring the agonist-dependent opening of intracellular Ca2+ concentration ([Ca2+]i)-independent Cl- channels. Injection of pure formyl peptide receptor cRNA did not lead to peptide-dependent changes in membrane current. In contrast, marked alterations of membrane current were observed in response to formyl peptides when the receptor cRNA was supplemented with poly(A)+ RNA isolated from undifferentiated HL-60 cells. Injection of the latter RNA did not lead to formyl-peptide-dependent alterations of membrane current. Binding studies using a radioiodinated formyl peptide revealed that injection of formyl peptide receptor cRNA alone led to expression of the formyl peptide receptor on the oocyte surface, and that co-injection of poly(A)+ RNA from undifferentiated HL-60 cells did not alter the level of receptor expression. Size fractionation of poly(A)+ RNA from undifferentiated HL-60 cells showed that the mRNA required to complement formyl-peptide-dependent signal transduction in oocytes had a size of approx. 3-3.5 kb. These results strongly suggest that the human formyl peptide receptor requires a specific cofactor(s), which is lacking in Xenopus oocytes but is present in undifferentiated HL-60 cells, to activate the second messenger pathway in oocytes. Identification of this factor will provide important information about the molecular mechanisms by which G-protein-coupled granulocyte-activating receptors stimulate phospholipase C.

Amino Acid Sequence↗
PubMed1992

Framework multipoint map of the long arm of human chromosome 4 and telomeric localization of the gene for FSHD.

Mapping the long arm of Chromosome (Chr) 4 has assumed medical relevance with the establishment of linkage of facioscapulohumeral muscular dystrophy (FSHD) to distal 4q markers. We have constructed a multipoint linkage map using DNA markers that map to the long arm of Chr 4. Segregation data were collected for 17 DNA markers on the multigenerational CEPH mapping families, and data for one marker were taken from the published CEPH database. Genotypic information for six of these markers was also collected from a set of 24 families that exhibited inheritance of FSHD. Multipoint analyses allowed us to construct a map of 12 loci, connecting two previously separate linkage groups. Significant sex-specific differences in recombination were found for some genetic intervals. Four loci from the distal region of this map showed linkage with FSHD. A map using these terminal markers gave the strongest support for FSHD in the most distal position over all other possible positions.

Chromosome Mapping↗
PubMed1992

Functional reconstitution of a receptor-activated signal transduction pathway in Xenopus laevis oocytes using the cloned human C5a receptor.

We have used the polymerase chain reaction to isolate and clone the cDNA encoding the human C5a receptor, and have injected the cDNA-derived receptor cRNA into Xenopus laevis oocytes for functional characterization of the receptor protein. Receptor activity was determined either electrophysiologically by measuring the agonist-dependent opening of [Ca2+]i-dependent Cl- channels, or by analysing the agonist-dependent efflux of 45Ca2+ from the oocytes. Using both methodologies, injection of pure C5a receptor cRNA failed to confer C5a sensitivity on the oocytes. In contrast, marked responses to C5a were observed when the receptor cRNA was supplemented with poly(A)+ RNA isolated from undifferentiated HL-60 cells, which is devoid of C5a receptor mRNA. Binding studies using radioiodinated C5a revealed that the C5a receptor polypeptide was in fact synthesized and targeted to the oocyte plasma membrane in oocytes injected with receptor cRNA alone, and that the level of receptor expression was not influenced by coinjection of poly(A)+ RNA from undifferentiated HL-60 cells. These results strongly suggest that the human C5a receptor requires a specific cofactor(s) lacking in Xenopus oocytes but present in undifferentiated HL-60 cells, to generate intracellular signals in oocytes. Identification and characterization of this factor will provide important information about the molecular mechanisms by which G-protein-coupled receptors activate phospholipase C.

Animals↗
PubMed1992

Increased urinary loss of uric acid in adults with acute respiratory failure requiring mechanical ventilation.

INTRODUCTION: The purpose of this study was to test a hypothesis of increased urinary excretion of uric acid as an indicator of adenosine triphosphate (ATP) degradation in adult patients with acute respiratory failure, and to look for a correlation to the clinical outcome. STUDY DESIGN: Prospectively 31 patients with acute respiratory failure were studied. The patients were divided into two groups according to the clinical outcome: the need for solely supplemental oxygen (group 1), death or mechanical ventilation (group 2). METHODS: Uric acid was determined by spectrophotometry. RESULTS: Mean uric acid excretion was 39 mumol/kg (range, 7 to 92 mumol/kg) body weight/per 24 h in group 1 (16 patients) compared with 65 mumol/kg/24 h (range, 8 to 253 mumol/kg/24 h) in group 2 (13 patients were mechanically ventilated, and two patients died). The difference was highly significant (p less than 0.0001). CONCLUSION: Increased amount of urinary uric acid was related to the severity of acute respiratory failure in adults.

Acute Disease↗
PubMed1992

Linkage analyses of five chromosome 4 markers localizes the facioscapulohumeral muscular dystrophy (FSHD) gene to distal 4q35.

The genetic locus for facioscapulohumeral muscular dystrophy (FSHD) has been mapped to chromosome 4. We have examined linkage to five chromosome 4q DNA markers in 22 multigenerational FSHD families. Multipoint linkage analyses of the segregation of four markers in the FSHD families and in 40 multigenerational mapping families from the Centre d'Etude du Polymorphisme Humaine enabled these loci and FSHD to be placed in the following order: cen-D4S171-factor XI-D4S163-D4S139-FSHD-qter. One interval, D4S171-FSHD, showed significant sex-specific differences in recombination. Homogeneity tests supported linkage of FSHD to these 4q DNA markers in all of the families we studied. The position of FSHD is consistent with that generated by other groups as members of an international FSHD consortium.

Chromosome Mapping↗