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Biomedical subjects

W Sun

Publications and source records attributed to W Sun.

At least 163 records · Page 9Linked to original sources
PubMed1998

ATP cross-linked to Escherichia coli single-strand DNA-binding protein can be utilized by the catalytic center of primase as initiating nucleotide for primer RNA synthesis on phage G4oric template.

We report a new observation of the role of Escherichia coli single-strand DNA binding protein (SSB) in synthesis of primer RNA (pRNA) catalyzed by.E.coli primase on the SSB-coated phage G4oric template. Using a set of ATP priming substrates with reactive groups attached to the 5' gamma-phosphate on different length "arms", we have demonstrated that, in the primase/SSB/G4oric pRNA synthesis complex, ATP cross-linked to both primase and SSB could be equally utilized as initiating nucleotide for pRNA synthesis. The distance between SSB surface and alpha-phosphorus of the priming substrate was estimated to be less than 7 A. ATP cross-linked to primase and SSB can be further elongated in the presence of other NTPs, giving almost identical patterns of covalently attached pRNAs of up to 12 nucleotides in length. The regions of primase and SSB with cross-linked ATP that can be used for pRNA synthesis are, therefore, arranged in a similar way relative to the active center of pRNA synthesis. The pRNA covalently linked to SSB was localized, mapping between Met48 and Trp88. This observation raises the possibility that SSB may play an active role in the initiation of pRNA synthesis in this system.

Adenine Nucleotides↗
PubMed1998

Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4oric complex required for primer RNA synthesis.

Escherichia coli primase/SSB/single-stranded phage G4oric is a simple system to study how primase interacts with DNA template to synthesize primer RNA for initiation of DNA replication. By a strategy of deletion analysis and antisense oligonucleotide protection on small single-stranded G4oric fragments, we have identified the DNA sequences required for binding primase and the critical location of single-strand DNA-binding (SSB) protein. Together with the previous data, we have defined the structure of the primase/SSB/G4oric priming complex. Two SSB tetramers bind to the G4oric secondary structure, which dictates the spacing of 3' and 5' bound adjacent SSB tetramers and leaves SSB-free regions on both sides of the stem-loop structure. Two primase molecules then bind separately to specific DNA sequences in the 3' and 5' SSB-free G4oric regions. Binding of the 3' SSB tetramer, upstream of the primer RNA initiation site, is also necessary for priming. The generation of a primase-recognition target by SSB phasing at DNA hairpin structures may be applicable to the binding of initiator proteins in other single-stranded DNA priming systems. Novel techniques used in this study include antisense oligonucleotide protection and RNA synthesis on an SSB-melted, double-stranded DNA template.

Base Sequence↗
PubMed1998

Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo.

NMDA receptors, a class of glutamate-gated cation channels with high Ca2+ conductance, mediate fast transmission and plasticity of central excitatory synapses. We show here that gene-targeted mice expressing NMDA receptors without the large intracellular C-terminal domain of any one of three NR2 subunits phenotypically resemble mice made deficient in that particular subunit. Mice expressing the NR2B subunit in a C-terminally truncated form (NR2B(deltaC/deltaC) mice) die perinatally. NR2A(deltaC/deltaC) mice are viable but exhibit impaired synaptic plasticity and contextual memory. These and NR2C(deltaC/deltaC) mice display deficits in motor coordination. C-terminal truncation of NR2 subunits does not interfere with the formation of gateable receptor channels that can be synaptically activated. Thus, the phenotypes of our mutants appear to reflect defective intracellular signaling.

Amino Acid Sequence↗
PubMed1998

Rapid analysis of steroidal saponin mixture using electrospray ionization mass spectrometry combined with sequential tandem mass spectrometry.

Using electrospray ionization mass spectrometry (MS) combined with sequential tandem MS(ESI-MSn), two major steroidal saponins extracted from Tribulus terrestris were studied, and considerable useful structural information was obtained. The structure of the proposed known steroidal saponin was verified, and the structure of the unknown saponin was investigated using MSn experiments. Some special fragment ions were also observed, and the corresponding fragmentation mechanisms were investigated which are characteristic for steroidal saponins and can give some information on the linkage position of some sugar groups in saponins. This methodology has been established as a powerful tool for the rapid, comparative analysis of mixtures such as crude plant extracts.

Carbohydrate Sequence↗
PubMed1998

Phosphorylation sensitizes microtubule-associated protein tau to Al(3+)-induced aggregation.

In Alzheimer's disease the microtubule-associated protein tau becomes hyperphosphorylated and aggregates into paired helical filaments (PHFs). Although the biochemical basis of the aggregation of tau into PHFs is not very clear, Al3+ has been suggested to play some role. Previous studies have shown that Al3+ alters the phosphorylation state and causes aggregation of tau in experimental animals and cultured neurons. In this study Al3+ inhibited phosphorylation of tau by neuronal cdc2-like kinase and dephosphorylation of phosphorylated tau by phosphatase 2B. These inhibitions are very likely due to Al(3+)-induced aggregations of various proteins present in phosphorylation/dephosphorylation assay mixtures since Al3+ caused aggregations of all proteins examined. Furthermore, compared to other proteins, tau displayed only an average sensitivity towards Al(3+)-induced aggregation. However upon phosphorylation, tau's sensitivity towards Al3+ increased 3.5 fold. In the presence of the metal chelator EDTA, Al(3+)-induced aggregates of tau became soluble, whereas Al(3+)-induced phosphorylated tau aggregates were insoluble in the buffer containing EDTA and remained insensitive to proteolysis. Our data suggest that phosphorylation sensitizes tau to Al3+ and phosphorylated tau transforms irreversibly into a phosphatase and protease resistant aggregate in presence of this metal ion.

Alum Compounds↗
PubMed1998

Neural network mosaic model for pupillary responses to spatial stimuli.

A neural network mosaic model was developed to investigate the spatial-temporal properties of the human pupillary control system. It was based on the double-layer neural network model developed by Cannon and Robinson and the pupillary dual-path model developed by Sun and Stark. The neural network portion of the model received its input from a sensor array and consisted of a retina-like two-dimensional neuronal layer. The dual-path portion of the model was composed of interconnections of the neurons that formed a mosaic of AC transient and DC sustained paths. The spatial aggregates of the AC and DC signals were input to the AC and DC summing neurons, respectively. Finally, the weighted sum of the aggregate AC and DC signals provided the output for driving the pupillary response. An important property of the model was that it could adaptively learn from training samples by adjustment of the weights. The neural network mosaic model showed excellent performance in simulating both the traditional pupillary phenomena and the new spatial stimulation findings such as responses to change in stimulus pattern and shift of light spot. Moreover, the model could also be used for the diagnosis of clinical deficits and image processing in machine vision.

Brain Stem↗
PubMed1998

Structural determinants of the blocker binding site in glutamate and NMDA receptor channels.

Glutamate receptor channels of the NMDA-type (N-methyl-D-aspartate) and non-NMDA-type (GluR) differ in their pore properties. The N-site in the M2 transmembrane segment of NMDA receptors (NMDAR), or the corresponding Q/R-site in GluRs, is a pivotal structural determinant of their permeation and blockade characteristics. Substitutions at a second site in M2, the L-site (L577) in GluR1, drastically alter the receptor selectivity to divalent cations. Here we report that M2 mutants carrying an asparagine or a threonine residue at the Q-site of GluR1, along with a tryptophan residue at the L-site, form homomeric GluR1 channels that are highly sensitive to structurally diverse, uncompetitive NMDA antagonists such as arylcyclohexylamines, dibenzocycloheptenimines, and to morphinian and adamantane derivatives. Analysis of the voltage dependence of channel blockade locates the blocker binding site approximately 0.65 partway into the transmembrane electric field in both GluR1 mutants and NMDAR channels. Our results suggest that the homomeric GluR1 double mutants, L577W/Q582N and L577W/Q582T, fairly approximate the pore properties of the heteromeric NMDA receptor and support the structural kinship of their permeation pathways.

Amino Acid Substitution↗
PubMed1998

Helicobacter pylori infection induces cyclooxygenase-2 expression in human gastric mucosa.

Recent studies indicate that expression of mitogen-inducible cyclooxygenase-2 (COX-2) occurs in gastrointestinal tumors. We investigated the effects of Helicobacter pylori (H. pylori) infection, a class I carcinogen for the human stomach, on gastric COX-2 expression using immunohistochemistry. Human subjects without macroscopic lesions, as determined by endoscopic screening, were biopsied for H. pylori infection. The biopsy samples were immunohistochemically examined for COX-2 expression. COX-2 was expressed in gastric epithelia and subepithelial inflammatory cells in all H. pylori-infected subjects. There was no expression of COX-2 in the gastric mucosa of H. pylori-negative subjects. COX-2 expression has been reported in gastrointestinal carcinomas, gastrointestinal cancer cell-lines, and in the gut after carcinogenic treatment. The present study demonstrates that H. pylori infection leads to gastric mucosal expression of COX-2, indicating that the enzyme is involved in H. pylori-related gastric pathology in humans.

Adult↗
PubMed1998

Analysis of local structure in the D2/S1-S2 region of the rat skeletal muscle type 1 sodium channel using insertional mutagenesis.

A reporter epitope was inserted at 11 positions in a region encompassing proposed transmembrane segments S1 and S2 in the second repeat domain (D2) of the rat skeletal muscle type 1 sodium channel. All mutations produced full-length membrane-associated protein following transfection into cultured cells, although the level of expression varied with insertion position. Characterization of cognate cRNAs for each mutation in Xenopus oocytes by two-electrode voltage clamp defined a permissive region between the proposed transmembrane regions in which these large insertions did not interfere with channel function. Two of the mutations, in which the point of insertion was within the proposed S1-S2 loop, demonstrated extracellular membrane labeling when studied either by antibody binding in oocytes or by confocal analysis following transfection into primary muscle cells. Our results define the likely boundaries of an extramembrane region linking the S1 and S2 transmembrane segments in D2 and confirm the extracellular location of this S1-S2 loop predicted by current models of channel tertiary structure.

Amino Acid Sequence↗
PubMed1998

Granulocytic ehrlichiosis in the laboratory mouse.

C3H mice that were inoculated with ehrlichiae isolated from a patient with human granulocytic ehrlichiosis (HGE) developed anemia and leukopenia, but by day 24, they returned to normal values. Granulocytic morulae were present in peripheral blood and spleen smears on days 5 and 10, and there was a reduction in morulae on day 17. Ehrlichiae were present in HL-60 cell cultures of blood and spleen from all mice at all intervals. Pathogenicity, but not infectivity, waned with mouse passage but could be resurrected by SCID mouse passage. Various methods were tested for their relative sensitivity in detecting infection: blood smears, HL-60 cell cultures, polymerase chain reaction (PCR) amplification of a 16S recombinant DNA target, and a mouse infectivity assay. All assays detected the HGE agent in blood during early infection, but PCR and the mouse infectivity assay were most sensitive during late infection. Xenodiagnosis demonstrated that mice remain persistently infected through 55 days.

Age Factors↗
PubMed1998

Cloning of the gene encoding the 44-kilodalton antigen of the agent of human granulocytic ehrlichiosis and characterization of the humoral response.

Antibodies in the sera of patients with human granulocytic ehrlichiosis (HGE) commonly recognize a 44-kDa antigen. We cloned the gene encoding the 44-kDa protein of the agent of HGE (aoHGE) by probing an aoHGE lambda ZAP II genomic DNA expression library with sera from aoHGE-infected mice. The gene, hge-44, is part of a multigene family, with sequence similarity to the Anaplasma marginale msp-2 genes. RNA-PCR studies confirmed that hge-44 is expressed by aoHGE cultured in HL-60 cells and by aoHGE during murine infection. Recombinant HGE-44, expressed and purified as a glutathione transferase fusion protein, was used as the substrate in immunoblots to help diagnose HGE. Antibodies in eight sera from eight patients with HGE and in two sera from two aoHGE-infected mice bound recombinant HGE-44. Antibodies in the sera of healthy individuals or patients with Ehrlichia chaffeensis or Borrelia burgdorferi infection did not recognize HGE-44. We conclude that hge-44 is a member of a multigene family and that hge-44 is expressed and elicits specific antibodies during infection.

Amino Acid Sequence↗
PubMed1998

Activation of bacteriophage Mu mom transcription by C protein does not require specific interaction with the carboxyl-terminal region of the alpha or sigma 70 subunit of Escherichia coli RNA polymerase.

Late in its growth cycle, transcription of the phage Mu mom Promoter (Pmom) is activated by the phage gene product, C, a site-specific DNA binding protein. In vitro transcription analyses showed that this activation does not require specific contacts between C and the carboxyl-terminal region of the alpha or sigma 70 subunit of Escherichia coli RNA polymerase. Unexpectedly, these results are in contrast to those known for another Mu-encoded transcriptional activator, Mor, which has a high degree of sequence identity with C and appears to interact with the carboxyl termini of both alpha and sigma 70.

Bacteriophage mu↗
PubMed1998

Molecular cloning and characterization of a new member of the rat placental prolactin (PRL) family, PRL-like protein H.

The rat placental PRL family consists of molecules structurally similar to PRL and GH, and to date nine members have been identified. In the course of investigating late stage specific placental PRL family expression by differential display, we have isolated a complementary DNA encoding a new molecule that is highly homologous to PRL-like protein C (PLP-C) and PLP-D, and named this molecule PLP-H. The complementary DNA encoded a mature protein of 239 amino acids, including a 31-amino acid signal sequence. Sequence comparison between PLP-H and other members of the placental PRL family showed that PLP-H is highly homologous to PLP-C and PLP-D (78% and 67% homology at the amino acid level, respectively). Expression of PLP-H was similar to that of PLP-C and PLP-D; PLP-H messenger RNA (mRNA) first appeared on day 14 of pregnancy, and its expression increased until term. RT-PCR analysis showed that PLP-H as well as PLP-C and PLP-D are expressed in all rat strains examined, confirming that PLP diversity is not due to strain differences. In situ hybridization analysis indicated that PLP-H mRNA is specifically expressed in spongiotrophoblast cells and in trophoblast giant cells of the placental junctional zone. Differentiated Rcho-1 cells also expressed PLP-H mRNA, whereas undifferentiated Rcho-1 cells did not. PLP-H seems to exist as a secretory protein because its N-terminal sequence is identical to that of GH/PRL-like molecule secreted from placental explants. PLP-H contains two putative N-glycosylation sites and eight cysteine residues, of which six are highly conserved in the placental PRL family. We prepared a recombinant protein for PLP-H together with PLP-D using a COS7 transfection system. Purified PLP-H showed two bands with molecular masses of 27 and 29 kDa. Only the 27-kDa protein was detected after N-glycosidase treatment, indicating that PLP-H is a glycoprotein. PLP-H and PLP-D did not stimulate the proliferation of Nb2 lymphoma cells or the phosphorylation of Janus kinase-2 and signal transducer and activator of transcription-5. These data indicate that PLP-H and PLP-D are nonlactogenic hormones. Thus, we have cloned a new member of the PLP subfamily, PLP-H, which has features in common with PLP-C and PLP-D.

Amino Acid Sequence↗
PubMed1998

Childhood diabetes in China. Enormous variation by place and ethnic group.

OBJECTIVE: To investigate the incidence rate of IDDM in China. RESEARCH DESIGN AND METHODS: The Chinese IDDM registry was established in 1991 as part of the World Health Organization's Multinational Project for Childhood Diabetes (DiaMond) project. Twenty-two centers were developed to monitor the incidence of IDDM in children < 15 years of age. The population under investigation includes > 20 million individuals, representing approximately 7% of the children in China. Capture-recapture methods were used to estimate the ascertainment. RESULTS: The overall ascertainment-corrected IDDM incidence rate in China was 0.51 per 100,000, the lowest rate ever reported. There was a 12-fold geographic variation (0.13-1.61 per 100,000). In general, the incidence rate was higher in the north and the east. There was a sixfold difference among ethnic groups (highest: Mongol group, 1.82 per 100,000; lowest: Zhuang group, 0.32 per 100,000). CONCLUSIONS: China has an extremely low overall IDDM incidence rate. China also has the greatest geographic and ethnic variation seen for any country.

Adolescent↗
PubMed1998

Laboratory diagnosis of acute dengue fever during the United Nations Mission in Haiti, 1995-1996.

We evaluated laboratory methods to confirm a clinical diagnosis of dengue. Acute sera were collected from personnel (n = 414) supporting the United Nations Mission in Haiti and presenting with febrile illness consistent with dengue fever or no apparent underlying cause. Dengue virus was recovered from 161 of 379 acute sera by inoculation into C6/36 cell culture. While 93 of 414 acute sera had detectable IgM antibodies, the IgM capture ELISA (MAC ELISA) had a sensitivity of only 13% compared with the virus isolation gold standard. If presumptive dengue fever cases were identified by both virus isolation and the presence of IgM, virus isolation and the MAC ELISA had clinical sensitivities of 69% and 40%, respectively. This study suggests that a combination of laboratory methods that target virus or subviral components as well as anti-viral IgM antibodies may be necessary for sensitive laboratory diagnosis with acute sera.

Acute Disease↗
PubMed1998

[Clinical analysis of 19 cases of scleroma respiratorium treated surgically].

Nineteen patients with scleroma respiratorium treated surgically were studied retrospectively. The results suggested that the surgical operation combined with antibiotic therapy were beneficial to the curtive effect of the patients in the granulomatous stage with nasal or pharyngeal obstruction or nasal sinuses involvement due to the serious proliferation of the lesions; tracheotomy should be considered in the patients with laryngeal obstruction up to the second degree or above; plastic operations were necessary for the patients with cicatrical stenosis or imperforation remained of the nasal cavity, pharynx, larynx or trachea.

Adult↗
PubMed1998

[Changes of activities of MLCK and dephosphatase in different arterial vessels from hypertensive rats].

The changes of activities of myosin light chain kinase (MLCK) and Ca2+/CaM-PP in different arterial vessels from hypertensive and normotensive rats were studied. The results were as follows. The MLCK activity of different arteries of spontaneous hypertensive rats (SHR) was different with the order of aorta (A) >> caudal artery (CA) >> mesenteric artery (MA), while in WKY rats the order of activity among different arteries is A << CA and MA and MA Ca2+/CaM-PP is obviously higher than in SHR. In renal hypertensive rats the activities of Ca2+/CaM-PP in different arteries are not quite different from those of the Wistar rats. The above results suggest that higher activities of MLCK or/and lower activity might be related to vasocontraction and hypertension.

Animals↗