Search PubMed⌕ Search

Biomedical subjects

L Shi

Publications and source records attributed to L Shi.

At least 127 records · Page 7Linked to original sources
PubMed1999

Differential expression of peroxiredoxin subtypes in human brain cell types.

The peroxiredoxin (Prx) protein is expressed widely in animal tissues and serves an antioxidant function associated with removal of cellular peroxides. We have cloned two Prx genes and observed differential expression of Prx-I and Prx-II (formerly NKEF-A and NKEF-B) in purified rat brain cell cultures (Sarafian et al. [1998] Mol. Chem. Neuropathol. 34:39-51). We have examined regional and cell-type-specific expression of Prx-I and Prx-II in paraffin sections of human brain using immunohistochemical methods. These studies revealed a clear segregation of expression of these two gene products in different brain cell types. In the cerebral cortex, cerebellum, basal ganglia, substantia nigra, and spinal cord, Prx-I was expressed primarily in astrocytes, while Prx-II was expressed exclusively in neurons. Prx-I was also prominently expressed in ependymal cells and subependymal matrix of substantia nigra and basal ganglia. Prx-II was not expressed at uniform density in all neurons. In general, small neurons such as cerebellar granule neurons displayed little or no staining, while large neurons, such as hippocampal pyramidal and Purkinje neurons were heavily stained. The absence of expression of Prx-I in neurons and the selective expression of Prx-II in large neurons suggest that these antioxidant enzymes serve distinct functional roles that may reflect the different functions and biochemical activities of these cell types. Restricted expression of these genes may also contribute to the selective vulnerability of these cells to a wide variety of neuropathologic conditions.

Adult↗
PubMed1999

Cyanobacterial PPP family protein phosphatases possess multifunctional capabilities and are resistant to microcystin-LR.

The structural gene for a putative PPP family protein-serine/threonine phosphatase from the microcystin-producing cyanobacterium Microcystis aeruginosa PCC 7820, pp1-cyano1, was cloned. The sequence of the predicted gene product, PP1-cyano1, was 98% identical to that of the predicted product of an open reading frame, pp1-cyano2, from a cyanobacterium that does not produce microcystins, M. aeruginosa UTEX 2063. By contrast, PP1-cyano1 displayed less than 20% identity with other PPP family protein phosphatases from eukaryotic, archaeal, or other bacterial organisms. PP1-cyano1 and PP1-cyano2 were expressed in Escherichia coli and purified to homogeneity. Both enzymes exhibited divalent metal dependent phosphohydrolase activity in vitro toward phosphoserine- and phosphotyrosine-containing proteins and 3-phosphohistidine- and phospholysine-containing amino acid homopolymers. This multifunctional potential also was apparent in samples of PP1-cyano1 and PP1-cyano2 isolated from M. aeruginosa. Catalytic activity was insensitive to okadaic acid or the cyanobacterially produced cyclic heptapeptide, microcystin-LR, both potent inhibitors of mammalian PP1 and PP2A. PP1-cyano1 and PP1-cyano2 displayed diadenosine tetraphosphatase activity in vitro. Diadenosine tetraphosphatases share conserved sequence features with PPP family protein phosphatases. The diadenosine tetraphosphatase activity of PP1-cyano1 and PP1-cyano2 confirms that these enzymes share a common catalytic mechanism.

Adenine Nucleotides↗
PubMed1999

[Preparation of standard mixture of volatile organic compounds].

This article presented a simple, convenient and flexible device for the preparation of standard mixtures of VOCs. By the aid of a constant pressure on the surface of a liquid with specific composition of VOCs, the liquid passed through a long capillary tube at a definite flow rate. A standard VOCs source was thus formed. The mean relative standard deviation (RSD) of flow rate was 3%. As the outlet of capillary tube inserted in a stream of zero air, the components of liquid vaporized and eventually became a homogeneous gas mixture. The concentration of VOCs in the mixture varied with the pressure applied and the flow rate of dilution air. The mean RSD of concentration of total VOCs in the standard mixture determined by GC method was 5.7%, and the absolute error was 8%. No significient difference was found between the determined and calculated concentrations of the mixture. This method is suitable for preparation of standard mixtures of VOCs with boiling points lower than 150 degrees C.

Air Pollutants↗
PubMed1999

Novel splicing of the human MHC-encoded peptide transporter confers unique properties.

Presentation of intracellularly derived antigenic peptides to T cells requires their assembly together with MHC class I molecules in the endoplasmic reticulum (ER). Such peptides are delivered to the ER by an MHC-encoded transporter composed of TAP1 and TAP2 protein delivery. Here, the first alternative splicing of Tap2 is described. The human splice variant, termed Tap2iso, lacks exon 11 and original 3' untranslated region and contains a newly identified exon 12 and 3' untranslated region. The full-length Tap2iso cDNA (2496 bp) predicts a protein of 653 amino acids. Tap2iso mRNA was normally coexpressed with Tap2 mRNA in all human lymphocyte cell lines examined. Function of TAP2iso was evaluated at multilevel in TAP1/2iso and TAP1/2 cotransfected T2 cells, a mutant cell line deplete of endogenous Tap gene products. The TAP1-TAP2iso transporter facilitated the maturation of MHC class I molecules in the ER and restored surface expression of class I. Importantly, TAP1-TAP2iso transporters expressed in T2 cells exhibited distinct and opposing influences on peptide selectivities, at times exceeding 30-fold differences in competition experiments and attributable to diversity in the 3'-COOH tail. The common coexpression of an alternative splice product of the Tap2 gene may contribute to broaden immune diversity, a mechanism previously described to occur predominantly at the level of the TCR and MHC class I gene products.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis.

Granzyme B (GraB) is required for the efficient activation of apoptosis by cytotoxic T lymphocytes and natural killer cells. We find that GraB and perforin induce severe mitochondrial perturbation as evidenced by the release of cytochrome c into the cytosol and suppression of transmembrane potential (Deltapsi). The earliest mitochondrial event was the release of cytochrome c, which occurred at the same time as caspase 3 processing and consistently before the activation of apoptosis. Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release. However, they suppressed Deltapsi and increased reactive oxygen species generation, indicating that mitochondrial dysfunction is also associated with this nonapoptotic cell death. Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria. Of additional interest, Deltapsi suppression mediated by GraK or GraB and perforin was not affected by zVAD-FMK and thus was caspase independent. Overexpression of Bcl-2 and Bcl-XL suppressed caspase activation, mitochondrial cytochrome c release, Deltapsi suppression, and apoptosis and cell death induced by GraB, GraK, or perforin. In an in vitro cell free system, GraB activates nuclear apoptosis in S-100 cytosol at high doses, however the addition of mitochondria amplified GraB activity over 15-fold. GraB- induced caspase 3 processing to p17 in S-100 cytosol was increased only threefold in the presence of mitochondria, suggesting that another caspase(s) participates in the mitochondrial amplification of GraB apoptosis. We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.

Animals↗
PubMed1999

Nix and Nip3 form a subfamily of pro-apoptotic mitochondrial proteins.

We have identified Nix, a homolog of the E1B 19K/Bcl-2 binding and pro-apoptotic protein Nip3. Human and murine Nix have a 56 and 53% amino acid identity to human and murine Nip3, respectively. The carboxyl terminus of Nix, including a transmembrane domain, is highly homologous to Nip3 but it bears a longer and distinct asparagine/proline-rich N terminus. Human Nip3 maps to chromosome 14q11.2-q12, whereas Nix/BNip3L was found on 8q21. Nix encodes a 23. 8-kDa protein but it is expressed as a 48-kDa protein, suggesting that it homodimerizes similarly to Nip3. Following transfection, Nix protein undergoes progressive proteolysis to an 11-kDa C-terminal fragment, which is blocked by the proteasome inhibitor lactacystin. Nix colocalizes with the mitochondrial matrix protein HSP60, and removal of the putative transmembrane domain (TM) results in general cytoplasmic and nuclear expression. When transiently expressed, Nix and Nip3 but not TM deletion mutants rapidly activate apoptosis. Nix can overcome the suppressers Bcl-2 and Bcl-XL, although high levels of Bcl-XL expression will inhibit apoptosis. We propose that Nix and Nip3 form a new subfamily of pro-apoptotic mitochondrial proteins.

Amino Acid Sequence↗
PubMed1999

A real time quantitative PCR-based method for the detection and quantification of simian virus 40.

A real time quantitative PCR-based simian virus 40 (SV40) detection and quantification method has been developed. This method takes advantage of the 5' to 3'-exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for direct monitoring of PCR product accumulation through a dual-labelled fluorogenic probe. This method provides accurate, precise and reproducible quantification of SV40 DNA over a linear dynamic range of at least 100,000-fold with a minimum detection level of 6.4 copy equivalents/microL of SV40 viral particle in test samples. The sample preparation procedure employed allows for efficient and consistent recovery of SV40 DNA from test samples. High concentrations of protein and cellular DNA presenting in test samples have been demonstrated to have no impact on SV40 quantification. This method offers significant advantages over other PCR methods and cell-based infectivity assays currently available for SV40 detection and quantification. The availability of this method should greatly facilitate the pathogenic investigation of SV40, as well as viral clearance evaluations required for the development of new biological products.

DNA, Viral↗
PubMed1999

Real time quantitative PCR as a method to evaluate simian virus 40 removal during pharmaceutical protein purification.

Continuous cell lines used for pharmaceutical protein manufacturing have the potential to be contaminated by viruses. To ensure the safety of pharmaceutical proteins derived from continuous cell lines, validation of the ability of the manufacturing process to clear potential contaminating viruses is required for product registration. In this paper, a real time quantitative PCR method has been applied to the evaluation of simian virus 40 (SV40) removal during chromatography and filtration procedures. This method takes advantage of the 5'-3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for automated SV40 DNA quantification through a dual-labeled fluorogenic probe. This method provides accurate and reproducible quantification of SV40 DNA. The SV40 clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. This method offers significant advantages over cell-based infectivity assays, such as higher sensitivity, greater reliability, higher sample throughput and lower cost. This method can be potentially used to evaluate the clearance of all model viruses during chromatography and filtration procedures. This method can be used to substitute cell-based infectivity assays for process validation of viral removal procedures and the availability of this method should greatly facilitate and reduce the cost of viral clearance evaluations required for new biologic product development.

Antibodies, Monoclonal↗
PubMed1999

Expression of human gelatinase B in Pichia pastoris.

Full-length human gelatinase B (FLGelB) and its C-terminal truncated form (dGelB) were expressed in Pichia pastoris strain GS115, using the Saccharomyces cerevisiae Mat alpha signal peptide. In both cases, a high level of the secreted protein could be detected by SDS-PAGE. The truncated gene was also expressed using the human gelatinase B native signal peptide. Secretion using the Mat alpha signal peptide was significantly greater than that from the native signal peptide. The recombinant products were purified and characterized biochemically. The recombinant proteins, FLGelB and dGelB, were found to have similar biochemical properties and activity to that of the human gelatinase B native protein.

Base Sequence↗
PubMed1999

The cytoplasmic kinase domain of PhoR is sufficient for the low phosphate-inducible expression of pho regulon genes in Bacillus subtilis.

PhoP-PhoR, one of three two-component systems known to be required to regulate the pho regulon in Bacillus subtilis, directly regulates the alkaline phosphatase genes that are used as pho reporters. Biochemical studies showed that B. subtilis PhoR, purified from Escherichia coli, was autophosphorylated in vitro in the presence of ATP. Phosphorylated PhoR showed stability under basic conditions but not acidic conditions, indicating that the phosphorylation probably occurs on a conserved histidine residue. Phospho-PhoR phosphorylated its cognate response regulator, PhoP in vitro. B. subtilis phoR was placed in the Bacillus chromosome under the control of the Pspac promoter, which is IPTG inducible. The wild-type phoR, under either native promoter or Pspac promoter with IPTG induction, resulted in a similar level of alkaline phosphatase production. Under high phosphate conditions, strains containing wild-type phoR, or phoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR mutant gene products that lacked either the periplasmic domain, or both N-terminal transmembrane PhoR sequences or various extended N-terminal sequences, showed no significant APase production. Under phosphate starvation conditions, in the presence of IPTG, all strains containing mutated phoR genes showed alkaline phosphatase induction patterns similar to that of the wild-type strain, although the fully induced level was lower in the mutants. The decrease in total alkaline phosphatase production in these mutant strains can be compensated completely or partially by increasing the copy number of the mutant phoR gene. These in vivo results suggest that the C-terminal kinase domain of PhoR is sufficient for the induction of alkaline phosphatase expression under phosphate-limited conditions, and that the regulation for repression of APase under phosphate-replete conditions remains intact.

Bacillus subtilis↗
PubMed1999

Genetic control of cortical regionalization and connectivity.

Herein, the genetic control of regionalization and connectivity of the neocortex are reviewed. Evidence is accumulating which suggests that intrinsic mechanisms have a central role in controlling cortical regional specification and differentiation. Expression patterns of several genes (Id-2, Tbr-1, cadherin-6, cadherin-8, neuropilin-2, Wnt-7b, Eph-A7 and RZR-beta) are described; the expressions of these genes have regional boundaries which demarcate distinct functional areas of the cerebral cortex in neonatal mice.

Animals↗
PubMed1999

Money, mission, and medicine: an innovative managed care partnership between the community health centers of Maryland and Johns Hopkins University.

Across the nation, states are placing their faith in managed care as the solution to the rising health care costs associated with the Medicaid program. Historical providers of care to the vulnerable and uninsured are competing in this new price-sensitive market while struggling to remain faithful to their missions. In Maryland, a unique partnership between an academic health center (Johns Hopkins HealthCare) and a coalition of community health centers has emerged as a model that promises to preserve the financial and philosophical goals of historical providers.

Academic Medical Centers↗
PubMed1999

Experience of primary care by racial and ethnic groups in the United States.

OBJECTIVES: The purpose of this study was to examine the experience of primary care by racial and ethnic groups and identify aspects of primary care where significant disparities in experience exist across racial and ethnic groups. METHODS: Data for this study came from the Household Component of the 1997-1998 Medical Expenditure Panel Survey (MEPS), a nationally representative survey of the civilian noninstitutionalized population of the United States. Measures were identified within MEPS that denote race, ethnicity, experience of primary care, and socioeconomic covariates associated with access to care. RESULTS: Racial and ethnic minorities experienced worse primary care, particularly in the first-contact aspect, than did white Americans. Their usual sources of care were more likely to be hospital settings than private clinics. They faced greater barriers accessing their usual source of care (USC), finding it more difficult to get an appointment and waiting longer during an appointment. Many of the significant differences persist after adjustment for sociodemographic and health-status characteristics. CONCLUSIONS: Racial and ethnic disparity in primary care experience is not simply a reflection of sociodemographic and health-status differences across racial/ethnic groups. Efforts must be made to reduce nonfinancial as well as financial barriers to care and ensure that quality primary care is provided in all settings, public as well as private, and to individuals of all colors.

Adolescent↗
PubMed1999

Gastrointestinal symptoms and use of medical care associated with child day care and health care plan among preschool children.

OBJECTIVES: This study examined whether out-of-home day care increases the risk of gastrointestinal symptoms for children <6 years of age and whether there is an association between gastrointestinal symptoms and medical care utilization. METHODS: The study population comprised members of 2 health plans residing in 2 South Carolina counties: 417 families from an health maintenance organization (HMO) plan; and 643 families from a Medicaid plan. Participants were followed for 18 months by either bimonthly telephone interviews or personal interviews. The survey/interview collected familial, personal, day-care and disease-related information and medical care utilization. The analyses controlled for both family characteristics and the type of health care plan. RESULTS: Children attending out-of-home day care had higher incidence rates for most gastrointestinal symptoms studied than did children staying at home (2.51 vs. 1.61 episodes of mild gastrointestinal symptoms and 1.63 vs. 1.08 episodes of moderate gastrointestinal symptoms for the HMO group; 1.72 vs. 1.28 episodes of moderate gastrointestinal symptoms for the Medicaid group). Nearly one-fourth of the annual physician visits by children younger than 6 years were attributed to gastrointestinal symptoms, and more than one-fourth of the children with gastrointestinal symptoms were prescribed antibiotics. When displaying gastrointestinal symptoms, children in the Medicaid group were significantly more likely to visit a doctor and receive antibiotics than those in the HMO group. CONCLUSIONS: Although the type of health plan has negligible influence on the frequency of reported gastrointestinal symptoms, it exerts a strong influence on medical care utilization.

Child Care↗
PubMed1999

Patient characteristics associated with hospitalizations for ambulatory care sensitive conditions in South Carolina.

BACKGROUND: Data for this study came from South Carolina 1995 Hospital Inpatient Encounter Data Base. The population figures were 1995 South Carolina population estimates. METHODS: We examined patient sociodemographic characteristics and costs associated with adult and pediatric ambulatory care sensitive condition (ACSC) hospitalization in South Carolina. Bivariate statistical comparisons were done to test the differences between ACSC and non-ACSC groups in specific sociodemographic characteristics. Logistic regression determined the relative significance of individual characteristics to hospitalizations for ACSC. Cost/charge ratios were used to estimate costs associated with ACSC hospitalizations. RESULTS: Nonwhites, low-income individuals, and those residing in more rural areas had significantly more ACSC hospitalizations than their respective counterparts (i.e., whites, high-income individuals, and those residing in metropolitan statistical areas). Individuals without a primary care physician were more likely to be hospitalized with ACSC. Ambulatory care sensitive condition hospitalization charges were 12% of total hospital charges for adults and 20% for children. The average cost per ACSC hospitalization was $3,929 for adults and $1,818 for children. CONCLUSION: Providers can rapidly assess needs and barriers faced by vulnerable population groups by using regularly collected hospital discharge data at the national, state, and community level.

Adolescent↗
PubMed1999

The icfG gene cluster of Synechocystis sp. strain PCC 6803 encodes an Rsb/Spo-like protein kinase, protein phosphatase, and two phosphoproteins.

A set of open reading frames (ORFs) potentially encoding signal transduction proteins are clustered around icfG, a gene implicated in the regulation of carbon metabolism, in the genome of Synechocystis sp. strain PCC 6803. slr1860 is the ORF for icfG, whose predicted product resembles the protein phosphatases SpoIIE, RsbU, and RsbX from Bacillus subtilis. Bracketing slr1860/icfG are (i) ORF slr1861, whose predicted product resembles the SpoIIAB, RsbT, and RsbW protein kinases from B. subtilis, and (ii) ORFs slr1856 and slr1859, whose predicted products resemble the respective phosphoprotein substrates for the B. subtilis protein kinases: SpoIIAA, RsbS, and RsbV. In order to determine whether the protein products encoded by these ORFs possessed the functional capabilities suggested by sequence comparisons, each was expressed in Escherichia coli as a histidine-tagged fusion protein and analyzed for its ability to participate in protein phosphorylation-dephosphorylation processes in vitro. It was observed that ORF slr1861 encoded an ATP-dependent protein kinase capable of phosphorylating Slr1856 and, albeit with noticeably lower efficiency, Slr1859. Site-directed mutagenesis suggests that Slr1861 phosphorylated these proteins on Ser-54 and Ser-57, respectively. Slr1860 exhibited divalent metal ion-dependent protein-serine phosphatase activity. It catalyzed the dephosphorylation of Slr1856, but not Slr1859, in vitro.

Adenosine Triphosphate↗
PubMed1999

Changes in cervical resistance and collagen fluorescence during gestation in rats.

During pregnancy the role of the cervix shifts between two opposing functions. Throughout most of gestation, the cervix is rigid and resists tension in order to maintain the products of conception inside the uterus. At term, however, cervical function changes drastically in order to accommodate stretch and delivery. The events that control cervical function are not known. The aim of this study was to characterize changes in cervical resistance and collagen content during pregnancy in the rat. To determine the change in cervical resistance, non-pregnant and timed pregnant Sprague Dawley rats were sacrificed at various times. Their cervices were isolated and suspended in organ baths connected to a cervimeter for measurement of the stretch-tension relationship. In a different group of animals, cervical collagen content was measured using light-induced fluorescence in non-pregnant and, longitudinally, in pregnant rats. Cervical resistance and collagen content decreased progressively during pregnancy. The changes in cervical resistance mirrored those in cervical collagen content and the nadir in both occurred about two days prior to the onset of labor. Our study suggests that cervical preparation for delivery does not occur acutely at the time of labor and that cervical collagen content determines cervical resistance.

Animals↗