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Biomedical subjects

J Shi

Publications and source records attributed to J Shi.

At least 289 records · Page 16Linked to original sources
PubMed1996

Apoptosis of neutrophils and their elimination by Kupffer cells in rat liver.

The fate of neutrophils in the peripheral circulation is poorly understood. In this study, the role of Kupffer cells in eliminating aged and apoptotic neutrophils was investigated. Liver, spleen, lung, and blood samples from Wistar rats were examined by light and electron microscopy, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method, and immunohistochemistry after the intravenous injection of OK-432, a streptococcal preparation. Neutrophils were trapped predominantly in the periportal and midzonal regions of hepatic lobules and were in contact with endothelial cells and Kupffer cells, or were surrounded by Kupffer cells. The trapping of neutrophils peaked after 6 hours. Apoptotic neutrophils, with or without buds, were found in the lumen of hepatic sinusoids as early as 6 hours, reached maximal levels after 12 hours, and represented greater than 60% of the total number of neutrophils in the liver. The presence of apoptotic neutrophils was correlated with the degree of neutrophil phagocytosis. Double-staining showed that TUNEL-positive neutrophils were phagocytosed or encircled by ED1- or ED2-positive Kupffer cells. In contrast, apoptosis and phagocytosis of neutrophils were rare in the spleen, lung, and peripheral blood. These results suggested that the appearance of apoptotic neutrophils in the hepatic sinusoids and their rapid clearance by Kupffer cells occurs after the invasion of bacteria (i.e., bacteremia or bacteriotoxemia) or the release of inflammatory mediators into the blood stream. These findings have important implications for the regulation of neutrophil homeostasis, the limitation of inflammation and tissue injury, and provide insight into the physiological removal of circulating, senescent neutrophils.

Animals↗
PubMed1996

Antibacterial activity of a synthetic peptide (PR-26) derived from PR-39, a proline-arginine-rich neutrophil antimicrobial peptide.

PR-39 is a proline-arginine-rich (PR) neutrophil antibacterial peptide originally identified and purified from the porcine small intestine. We report on the synthesis of a functional antibacterial domain of PR-39, the first 26 amino acid residues of the NH2 terminus. PR-26 was as potent as or more potent than PR-39 against enteric gram-negative bacteria. This truncated form of PR-39 potentiated neutrophil phagocytosis of Salmonella choleraesuis and decreased the level of S. typhimurium invasion into intestinal epithelial cells. Scanning electron microscopy confirmed that these peptides did not lyse cells by pore-forming mechanisms; however, they potentiated the antibacterial capabilities of a pore-forming peptide, magainin A. In addition, PR-26 was not toxic to epithelial cells at concentrations several times greater than its bactericidal concentration. These data suggest that PR-39 and its functional domain, PR-26, may potentiate the host's defense capabilities against gram-negative infections.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of nnrR, whose product is required for the expression of proteins involved in nitric oxide metabolism in Rhodobacter sphaeroides 2.4.3.

During denitrification, the production and consumption of nitric oxide (NO), an obligatory and freely diffusible intermediate, must be tightly regulated in order to prevent accumulation of this highly reactive nitrogen oxide. Sequencing upstream of norCB, the structural genes for NO reductase, in the denitrifying bacterium Rhodobacter sphaeroides 2.4.3, we have identified a gene, designated nnrR, which encodes a protein that is a member of the cyclic AMP receptor family of transcriptional regulators. Insertional inactivation of nnrR prevents growth on nitrite, as well as the reduction of nitrite and NO, but has no effect on reduction of nitrate or photosynthetic growth. By using nirK-lacZ and norB-lacZ fusions, we have shown that NnrR is a positive transcriptional regulator of these genes. nnrR is expressed at a low constitutive level throughout the growth of R. sphaeroides 2.4.3. These results show that NnrR is not a global regulator but is instead a regulator of genes whose products are directly responsible for production and reduction of NO. Evidence is also presented suggesting that an NnrR homolog may be present in the nondenitrifying bacterium R. sphaeroides 2.4.1. The likely effector of NnrR activity, as determined on the basis of work detailed in this paper and other studies, is discussed.

Amino Acid Sequence↗
PubMed1996

[The establishment of a three dimensional PCR screening system and quality evaluation of human YAC library].

In order to screen out YAC clones in the CEPH YAC library related to a definite gene, DNA fragment or locus, we need to establish a screening system. This paper includes the establishment of this system and the evaluation of its feasibility. According to the experiments, we suggest that it is better to screen the Mega YAC part of the library at first and put five culture plates as a group. Meanwhile, FISH experiments to assess the quality of CEPH YAC library and the first generation physical map of human genome, Were conducted FISH experiments on 26 YAC clones were carried out. There were 13 clones that were chimeric (about 50%), 6 clones that were mapped by mistake (about 23%). So, there were only 9 clones that were mapped correctly and were not chimeric (about 35%).

Chromosomes, Artificial, Yeast↗
PubMed1996

[Effects of capsules on attachment of human periodontal ligament fibroblasts to root surface].

Inhibition of attachment of human periodontal ligament fibroblasts to root surface by three capsules were determined with the methods of trypan blue and L*a*b* color difference measurement. The results showed that capsule from porphyromonas gingivalis was the strongest one in inhibiting attachment and followed by the one from provotalla intermedia. Capsule from porphyromonas endodontolis was the weakest one. The values of color difference from that of control root surface were 11, 13, 8.28 and 4.09 respectively. Inhibitory mechanism of capsules may include a direct growth inhibition of fibroblasts and a decrease in attachment due to changes of root surface properties. There were a few fibroblasts attached to root surface with progressive periodontitis, this was a sharp contrast with that of control root surface. We deduce that long term affections of toxins including capsules and other substances led to changes of root surface with periodontitis, which made fibroblasts difficult to attach.

Bacterial Capsules↗
PubMed1996

[A study of gingivain from extracellular vesicles of Porphyromonas gingivalis W50: purification and cytotoxic characterization].

Gingivain was purified from extracellular vesicles of Porphyromonas gingivalis W50 with high performance liquid chromatography (HPLC) and its cytotoxic characterization on L929 cell line was examined. The result showed that gingivain might belong to systeine proteinases. The molecular weight of gingivain was about 27,800 unit by HPLC in which there might be one or two peptides being 10,900 unit, the enzyme was activated by Ca2+ and thiol-containing agents such as dithiothycitol and inhibited by Na-4-tosyl-L-lysine chioromethyl and EDTA. The maximum activity of the enzyme were found at pH 7.5, purified gingivain was toxic to L929 cell line at the content of 10 micrograms/ml. The study indicates that gingivain carried by vesicles might be more important in bacterial toxic activities.

Bacterial Proteins↗
PubMed1996

[Experience with reoperation on mitral valve prostheses].

Heart valve replacement after closed mitral commissurotomy and mitral valve replacement with bioprostheses may result in complication and higher mortality and morbidity. Mitral valve replacement was performed in 32 cases after closed mitral commissurotomy and 12 cases after mitral valve replacement with bioprostheses from April 1986 to May 1994 in our hospital. The eraly mortality was 6.8% (3/42). The choice of operation and methods, myocardial preservation, measurement of heart resuscitation, and prevention of perioperative hemorrhage were discussed.

Adult↗
PubMed1996

[Application of monoclonal antibody immuno-colloide gold probe in the study of porphyromonas gingivalis].

Immuno-electron localization of McAb BGG9 was specific for Porphyromonas gingivalis labeled with colloide gold. The monoclonal antibody immuno-colloide gold probe reacted with Porphyromonas gingivalis and 5 species of black pigmented bacteroides groups. The bacteria were fixed and embbed in Epon. sections were examined under transmission electron microscope, and the gold granular was observed near Porphyromonas gingivalis cell membrane. Our conclusion suggests that monoclonal antibody immuno-colloide gold probe is useful tool for studying Porphyromonas gingivalis.

Antibodies, Bacterial↗
PubMed1996

[The inhibitory effect of TNF-alpha on alkaline phosphatase activity of human periodontal ligament fibroblasts].

The present investigation was to identify the impact of recombinant human tumor necrosis factor-alpha (TNF-ALPHA) on alkaline phosphatase (ALP) activity of cultured human periodontal ligament fibroblasts (HPLF). It was found that, after treatment with TNF-alpha, HPLF in the experimental groups showed significantly lower ALP activity than in the blank control groups (P < 0.01). Further, the ALP activity was observed to decrease progressively with increasing TNF-alpha concentrations. These findings indicate that TNF-alpha possesses a remarkable concentration-dependent inhibitory effect on human periodontal ligament fibroblasts.

Adolescent↗
PubMed1996

[Endoscopic sinus surgery for the treatment of inverted papilloma of nasal cavity and paranasal sinuses].

Fourteen patients suffering from inverted papilloma of the nasal cavity and sinus were operated by the intranasal endoscopic approach. The period of follow-up were 24-60 months. Only one case had recurrence after three months and was then cured by a combined treatment of external nasal approach and radiotherapy. The other 13 cases had no recurrence in the follow up period. The intranasal endoscopic surgical technique was proved a better method for treating inverted papilloma of nasal cavity and sinus even when the lesions had affected the posterior ethmoid and spenoidal sinuses. It seems that this approrch would be a new way for removing inverted papilloma completely and the major advantage of which is an incision of the face can be avoided, while the curative rate might be the same or even better than that of the external approach.

Adult↗
PubMed1995

CD28 delivers costimulatory signals independently of its association with phosphatidylinositol 3-kinase.

CD28, a cell-surface molecule expressed by T cells, delivers costimulatory signals during the activation of T cells by Ag. Stimulation of CD28 induces its association with phosphatidylinositol 3'-kinase (PI3-K), raising the possibility that PI3-K plays a critical role in CD28 signaling. We find, however, that wortmannin, a potent inhibitor of PI3-K, does not block CD28-mediated costimulation of Jurkat (a human T cell line) or of murine CD4+ T cells. To address further the role of PI3-K in CD28-mediated signaling, we expressed mutant murine CD28 molecules in Jurkat cells. Mutation of Tyr 170 of murine CD28 to Phe abrogates the association of murine CD28 with PI3-K but does not affect the ability of murine CD28 to augment IL-2 production by Jurkat cells in response to the combination of ionomycin and PMA. Conversely, a mutant of murine CD28 that has a Tyr at position 170 but has Phe substitutions at the remaining three cytoplasmic tyrosines retains the ability to associate with PI3-K and has an impaired ability to deliver a costimulus that augments IL-2 production. CD28, therefore, can deliver costimulatory signals independently of its interaction with PI3-K, and association with PI3-K is insufficient to mediate the full effector function of CD28. Optimal signaling by CD28 requires the integrity of one or more of the carboxyl-terminal three Tyr residues.

Androstadienes↗
PubMed1995

A versatile low-copy-number cloning vector derived from plasmid F.

We have constructed a cloning vector based on plasmid mini-F for use in Escherichia coli. Plasmid pZC320 consists of the ori-2 replication unit of F that confers very low copy number (lcn), and includes the sop partition functions to insure stable plasmid maintenance in the absence of selection. A multiple cloning site (MCS) containing 16 unique restriction sites is located within the 5' end of the lacZ alpha gene. Expression of lacZ alpha is under the control of the wild-type lactose operator/promoter (lacOP) region and is efficiently repressed by the lacI repressor. Clones containing inserts can be detected using the blue/white screen for beta-galactosidase (beta Gal). A T7 promoter allows transcription of cloned inserts in the presence of T7 RNA polymerase. We have demonstrated the use of this lcn vector for cloning the regulated tetracycline-resistance genes from Tn10, which confer only low-level resistance when present at high copy number.

Antiporters↗
PubMed1995

Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants.

Expansins are unusual proteins discovered by virtue of their ability to mediate cell wall extension in plants. We identified cDNA clones for two cucumber expansins on the basis of peptide sequences of proteins purified from cucumber hypocotyls. The expansin cDNAs encode related proteins with signal peptides predicted to direct protein secretion to the cell wall. Northern blot analysis showed moderate transcript abundance in the growing region of the hypocotyl and no detectable transcripts in the nongrowing region. Rice and Arabidopsis expansin cDNAs were identified from collections of anonymous cDNAs (expressed sequence tags). Sequence comparisons indicate at least four distinct expansin cDNAs in rice and at least six in Arabidopsis. Expansins are highly conserved in size and sequence (60-87% amino acid sequence identity and 75-95% similarity between any pairwise comparison), and phylogenetic trees indicate that this multigene family formed before the evolutionary divergence of monocotyledons and dicotyledons. Sequence and motif analyses show no similarities to known functional domains that might account for expansin action on wall extension. A series of highly conserved tryptophans may function in expansin binding to cellulose or other glycans. The high conservation of this multigene family indicates that the mechanism by which expansins promote wall extensin tolerates little variation in protein structure.

Amino Acid Sequence↗
PubMed1995

Establishment of micrometastatic carcinoma cell lines: a novel source of tumor cell vaccines.

BACKGROUND: Cancer cells of microscopic metastases can be envisaged as ideal constituents for the development of a genetically modified, autologous tumor cell vaccine. However, their extremely low number has thus far blocked this approach. PURPOSE: The aim of this study was to culture micrometastatic tumor cells present in bone marrow of patients with various forms of epithelial cancer and to thereby establish immortalized cell lines. METHODS: Bone marrow aspirates from the upper iliac crest of 152 patients with cancer of the prostate, kidney, lung, breast, or colorectum were cultured at 1 x 10(7) to 6 x 10(7) mononuclear cells (MNC) per flask in fetal calf serum-containing RPMI-1640 medium supplemented with 10 ng/mL epidermal growth factor and 10 ng/mL basic fibroblast growth factor. The proliferation of epithelial cells on extracellular matrix-coated plates was monitored by sampling and staining aliquots with cytokeratin-specific antibodies. After 3-6 weeks in culture, the cells were transferred to Petri dishes, and 200-300 epithelial cells per plate were microinjected with DNA encoding for the simian virus 40 (SV40) large T antigen. Cells were screened at various time points for expression of large T antigen and epithelial markers, such as cytokeratins, prostate-specific antigen, prolactin-inducible protein, or intestinal-specific annexin; their bone marrow-seeking potential was tested in immunodeficient SCID (i.e., severe combined immunodeficiency) mice given subcutaneous transplants of the immortalized cells. RESULTS: Prior to culture, more than 90% of all samples presented with fewer than 10 tumor cells per 8 x 10(5) MNC. In 68 cases (44.7%), the established culture conditions allowed a two to four log transient expansion of these cells with rather small differences among the tumor types studied. Epidermal growth factor and basic fibroblast growth factor were found to be essential for this culture system. After microinjection of the propagated cells with T-antigen DNA, permanent cell lines were obtained; some of these cell lines (prostate and lung cancer cell lines) are now beyond culture passage 80. The cells showed no notable changes in the pattern of expressed epithelial antigens and were able to disseminate into bone marrow in SCID mice. CONCLUSIONS: This procedure allows the selective immortalization of micrometastatic carcinoma cells. Integration of SV40 DNA and expression of T antigen did not substantially change the epithelial phenotype of the propagated cells. IMPLICATIONS: The established system will allow an in-depth molecular analysis of human micrometastatic cancer cells and could become a useful source for the generation of autologous tumor cell vaccines.

Animals↗
PubMed1995

Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein.

Amino Acid Sequence↗
PubMed1995

Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean.

Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian delta-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian delta-type PI-PLCs and yeast PI-PLC1, the putative Ca(2+)-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.

Amino Acid Sequence↗