Search PubMed⌕ Search

Biomedical subjects

L Shi

Publications and source records attributed to L Shi.

At least 217 records · Page 12Linked to original sources
PubMed1997

Practice environment and the employment of nurse practitioners, physician assistants, and certified nurse midwives by community health centers.

This report examines the relation between state variations in the regulation of nurse practitioners (NPs), physician assistants (PAs), and certified nurse midwives (CNMs), and the employment of these nonphysician providers (NPPs) by community health centers (CHCs). Data for this report came from a 1991-92 survey of CHCs assessing the employment of NPPs, and secondary available data. The dependent variables examined were the numbers of NPPs currently employed by CHCs. Independent variables included 1992 practice environment scores, CHC location, number of CHC physicians, and NPP-to-population ratios. The number of NPs and PAs employed by CHCs was significantly associated with practice environment for these practitioners. NPP-to-population ratios and the number of CHC physicians are also significantly associated with NPP employment by CHCs. State decision makers may reduce legislative and regulatory barriers to practice as a way to improve the practice environment for nonphysician primary care providers, particularly NPs and PAs. Thus, community health centers can employ adequate number of NPPs to fulfill their mission of serving the poor and underserved population.

Certification↗
PubMed1996

Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Animals↗
PubMed1996

Granzyme B induces apoptosis and cyclin A-associated cyclin-dependent kinase activity in all stages of the cell cycle.

Granzyme B rapidly induces apoptosis in the presence of the pore-forming protein perforin. We have examined the cell cycle restriction of this apoptosis by separating Jurkat cells into fractions representing different stages of the cell cycle by centrifugal elutriation. Cells were susceptible to apoptosis from G1 through to G2/M, with no significant resistance detected at any stage. Similarly, cells arrested at G1/S or G2/M with either hydroxyurea or nocodazole were slightly more sensitive than asynchronously growing cells. Granzyme B induces Cdc2 kinase activity and requires its induction for apoptosis. Cyclin-dependent kinase (CDK) activity is regulated by phosphorylation and association with cyclins that also control subcellular localization of the CDK/cyclin complexes. Cdc2 associates with both cyclin A, which is synthesized at G1 and S, and cyclin B, which is produced later during S and G2 before G2/M transition. We find that the CDK activity induced by granzyme B is associated primarily with cyclin A in both asynchronous and G1/S-arrested cells, while cyclin B-associated kinase activity is minimal. Because cyclin A is also able to associate with Cdk2, a kinase that is important for G1/S transition, we examined the activation of this CDK during granzyme B-induced apoptosis and find that Cdk2 is induced as rapidly as Cdc2. In conclusion, we have identified a lack of cell cycle restriction of granzyme B-induced apoptosis and the rapid activation of both cyclin A/Cdc2 and cyclin A/Cdk2 kinase activity.

Apoptosis↗
PubMed1996

Identification and characterization of Ich-3, a member of the interleukin-1beta converting enzyme (ICE)/Ced-3 family and an upstream regulator of ICE.

We report here the isolation and characterization of a new member of the ice/ced-3 family of cell death genes, named ich-3. The predicted amino acid sequence of Ich-3 protein shares 54% identity with murine interleukin-1beta converting enzyme (ICE). Overexpression of ich-3 in Rat-1 and HeLa cells induces apoptosis, which can be inhibited by CrmA and Bcl-2. The mRNA and proteins of ich-3 are dramatically induced in vivo upon stimulation with lipopolysaccharide, an inducer of septic shock. The ich-3 gene product can be cleaved by cytotoxic T cells granule serine protease granzyme B, suggesting that Ich-3 may mediate apoptosis induced by granzyme B. Ich-3 does not process proIL-1beta directly but does promote proIL-1beta processing by ICE. These results suggest that Ich-3 may play a very important role in apoptosis and inflammatory responses and may be an upstream regulator of ICE.

Amino Acid Sequence↗
PubMed1996

The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism.

The major mechanism of cytotoxic lymphocyte killing involves the directed release of granules containing perforin and a number of proteases onto the target cell membrane. One of these proteases, granzyme B, has an unusual substrate site preference for Asp residues, a property that it shares with members of the emerging interleukin-1beta-converting enzyme (ICE)/CED-3 family of proteases. Here we show that granzyme B is sufficient to reproduce rapidly all of the key features of apoptosis, including the degradation of several protein substrates, when introduced into Jurkat cell-free extracts. Granzyme B-induced apoptosis was neutralized by a tetrapeptide inhibitor of the ICE/CED-3 family protease, CPP32, whereas a similar inhibitor of ICE had no effect. Granzyme B was found to convert CPP32, but not ICE, to its active form by cleaving between the large and small subunits of the CPP32 proenzyme, resulting in removal of the prodomain via an autocatalytic step. The cowpox virus protein CrmA, a known inhibitor of ICE family proteases as well as granzyme B, inhibited granzyme B-mediated CPP32 processing and apoptosis. These data demonstrate that CPP32 activation is a key event during apoptosis initiated by granzyme B.

Apoptosis↗
PubMed1996

Conformational characterization of DnaK and its complexes by small-angle X-ray scattering.

DnaK, a member of the 70 kDa heat shock protein (hsp70) family, and its complexes with substrate proteins and nucleotides were characterized by small-angle X-ray scattering (SAXS) and size-exclusion chromatography (SEC) techniques. The SAXS data indicated that DnaK has a dumbbell-shaped structure with a maximum dimension (dmax) of 112 angstrom, which is consistent with the reported two major functional domains [Chappell et al. (1987) J. Biol. Chem. 268, 12730-12735; Flaherty et al. (1990) Nature 346, 623-628]. The data were best fit by a model in which the two domains either are connected by a short hinge region or are just in contact with each other. The radius of gyration (Rg) of DnaK was determined as 37.5 +/- 1.0 angstrom in the absence of nucleotide. Binding of ATP induces a conformational change in DnaK as reflected by the changes in its P(r) function and Kratky plot, the increases (1-2 angstrom) in both its radius of gyration (Rg) and its Stokes radius (Rs), and the increase in its dmax (5-10 angstrom ). SAXS and SEC-HPLC results indicate that the association state of DnaK is very sensitive to the buffer concentration and the presence of substrates, as well as the protein concentration. At high buffer and protein concentrations, DnaK dimerizes, resulting in an increase in its apparent Rg and dmax values. The addition of substrate (unfolded protein or ATP) results in a return to the Rg value of monomeric DnaK, due to the dissociation of DnaK multimers induced by the substrate binding and resultant conformational changes. The DnaK-substrate protein complex gives a smaller Rg than expected, suggesting that the substrate protein binds to a cavity or cleft on DnaK rather than the exterior of the chaperone. The Kratky plot of the Gdn.HCl-induced unfolding intermediate state of DnaK is consistent with a compact, molten globule-like conformation, as previously suggested based on CD, fluorescence, and SEC-HPLC results [Palleros et al. (I 993) Biochemistry 32, 4314-4321].

Bacterial Proteins↗
PubMed1996

Recombinant-baculovirus-expressed PB2 subunit of the influenza A virus RNA polymerase binds cap groups as an isolated subunit.

The influenza A virus RNA-dependent RNA polymerase catalyzes several reactions in transcription and replication of the genome RNA. The first step in viral mRNA synthesis is the recognition of the 5' end cap structure of host cell hnRNA and the cleavage of the RNA substrate between 10 and 14 nucleotides from the 5' end to generate capped primers for initiation of transcription of virus-specific mRNAs. This report describes the use of an in vitro UV crosslinking and protein renaturation assay to identify the polymerase subunits which interact with the 5' end cap structure of an artificial RNA substrate. Our results showed, for the first time, that purified polymerase subunit PB2 expressed by recombinant baculovirus in insect cells possessed cap-binding activity by itself after renaturation by Escherichia coli thioredoxin, whereas cleavage of the artificial capped substrate required the holoenzyme expressed in insect cells triply-infected with baculovirus containing all three polypeptide components, PB1, PB2, and PA. Purified polyclonal anti-PB2 IgG inhibited the binding activity; anti-PB1 and anti-PA IgGs did not.

Animals↗
PubMed1996

Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro.

We have investigated the endonuclease activity of the influenza A virus RNA polymerase in an in vitro assay with an artificial influenza-like mRNA containing a cap structure at its 5' terminus, followed by a 10 nt beta-globin mRNA sequence, and the 5' and 3' conserved termini of a truncated nucleoprotein (NP) cRNA influenza sequence. Results showed that partially purified virion ribonucleoprotein complexes (RNPs) and micrococcal nuclease treated RNPs cleaved the artificial influenza-like mRNA substrate specifically at positions near the 5' terminus to generate capped 14 and 15 nucleotide long RNA fragments which subsequently served as primers to initiate transcription. The endonuclease activity was completely blocked by addition of cap analog and competitively inhibited by added globin mRNA. Furthermore, an in vitro reconstituted influenza RNA transcription reaction containing a truncated NP vRNA as template, micrococcal nuclease treated RNPs and globin mRNA as primer, synthesized capped and uncapped full length (+) sense products. Enzyme kinetics showed that capped RNA was made earlier in the reaction; it reached a peak at 120 min and then declined. However, uncapped cRNA synthesis appeared later and remained as the dominant product later in the reaction. The nature of these products was confirmed by ribonuclease protection assays and by primer extension.

Globins↗
PubMed1996

A Bacillus subtilis secreted phosphodiesterase/alkaline phosphatase is the product of a Pho regulon gene, phoD.

A secreted phosphodiesterase/alkaline phosphatase, APaseD, was purified from a culture of Bacillus subtilis JH646MS. Its phosphodiesterase activity was reminiscent of an APase isolated and characterized previously. Immunoassay and N-terminal sequencing showed the two proteins to be identical. Using the first 20 amino acids of the mature protein, a BLAST search of GenBank was used to find an homologous sequence. An exact match was found but in a putative non-coding region. It was hypothesized that there was a base pair deletion in the phoD gene. A DNA fragment internal to the coding region was generated by PCR using template DNA from a strain which produced APaseD. The PCR fragment was cloned and used to interrupt the gene. Western blot analysis of the parent and the mutated strains showed that APaseD was missing in the mutant. Resequencing of the gene revealed a larger ORF encoding a protein similar in size to the 49 kDa APaseD estimated by SDS-PAGE. The promoter was then cloned, sequenced and used in phoD-lacZ promoter fusions which showed that the gene was phosphate-starvation-induced and dependent on PhoP and PhoR for expression.

Alkaline Phosphatase↗
PubMed1996

Characterization of the Borna disease virus phosphoprotein, p23.

Borna disease virus infection is diagnosed by the presence of serum antibodies reactive with the major viral proteins, p40 and p23. Although p40 and p23 are unrelated in amino acid sequence structure, cross-reactive antibodies are described. Protein fragments and synthetic peptides were analyzed to characterize the specificities of antibodies to p23. Epitope mapping revealed eight continuous epitopes accessible on the surface of a predicted structural model for the monomeric and the disulfide-linked dimeric forms of p23. None of these epitopes was reactive with antibodies to p40. Cross-reactivity with monospecific sera and monoclonal antibodies to p40 was found for one discontinuous epitope located at the amino terminus of p23.

Animals↗
PubMed1996

Influenza A virus RNA-dependent RNA polymerase: analysis of RNA synthesis in vitro.

Influenza A virus RNA-dependent RNA polymerase, purified from virion ribonucleoprotein particles and from which endogenous genomic RNA (vRNA) has been depleted by treatment with micrococcal nuclease, was used to study transcription initiation, elongation, and termination in vitro. Templates that contained either minus- or plus-sense influenza virus nucleoprotein minigenes with conserved 5' and 3' termini and the uridylate tract were constructed. The dinucleotide ApG and alfalfa mosaic virus RNA4 (AlMV4) were used as primers. ApG primed the synthesis of full-length positive-strand or cRNA products and shorter transcripts, depending upon the molar ratio between the nucleoprotein and the vRNA template. Sequence analysis of the ends of these transcripts demonstrated that the 5' termini of both transcripts and the 3' terminus of the full-length product were complementary to the 3' and 5' termini of the vRNA template, respectively, whereas the 3' terminus of the incomplete product corresponded to a sequence located 40 bases downstream from the 5' terminus of the template and was about 20 nucleotides downstream from the uridylate tract, which is the putative signal for polyadenylation. Binding of the cap structure of AlMV4 by the polymerase activated RNA synthesis by ligation-elongation of small genomic RNA fragments which were likely derived from a genome segment protected by the polymerase from micrococcal nuclease digestion. The sequence of these fragments mapped to a region 14 to 28 nucleotides upstream of the 3' terminus of the viral genome. Polymerase subunit involvement in transcription initiation with ApG or AlMV4 was characterized by studying the effect of purified polyclonal antisubunit immunoglobulins of the G class (IgGs) in transcription assays. These results showed that anti-PB2 IgG inhibited transcription initiation in both ApG- and AlMV4-primed reactions, whereas anti-PB1 antibodies also blocked transcription initiated with AlMV4. The differences observed in product size, product sequence, and differential inhibition by antisubunit IgGs are discussed. These observations would support the notion that the influenza virus RNA-dependent RNA polymerase undergoes a conformational change after the binding of the cap structure of host cell heterogeneous nuclear RNA by PB2, which then usually leads to endonucleolytic cleavage of the capped primer 13 nucleotides downstream from the cap.

Animals↗
PubMed1996

Access to care in post-economic reform rural China: results from a 1994 cross-sectional survey.

The purpose of this paper is to assess the current level of access to care in rural China based on results from a 1994 random, cross-sectional survey of three rural counties in China. A total of 722 people were interviewed. The results of the study indicate there is currently a significant gap among rural Chinese communities in terms of access to health care, particularly with regard to availability (e.g., cooperative health plan or other insurance plans) and acceptability (e.g., health care spending burden). Less developed rural communities are placed in double jeopardy: not only are residents paying a higher amount for health care, they have to do so with significantly less income.

Analysis of Variance↗
PubMed1996

[CuZn-SOD determination of sera in patients with rheumatic diseases].

Superoxide anion (O2.-) plays an important part in reactive oxygen species (ROS). In order to explore its effect on the pathogenesis of rheumatic diseases, authors had determined CuZn-SOD contents of sera in 132 subjects involving the patients of rheumatic diseases (SLE, RA, etc), non-rheumatic diseases and normal controls by using enzyme linked immunosorbent assay (ELISA). The results showed the followings: CuZn-SOD contents of 27 normal subjects: 98.80 +/- 20.74 ng/ml (x +/- s); that of 27 non-rheumatic diseases cases: 72.24 +/- 16.60 ng/ml (x +/- s); of 22 SLE cases: 56.56 +/- 19.27 ng/ml (x +/- s); of 27 RA cases: 61.56 +/- 20.53 ng/ml (x +/- s); of 29 other rheumatic diseases cases: 68.97 +/- 17.79 ng/ml (x +/- s). Statistical test was made: both CuZn-SOD contents of rheumatic disease and non-rheumatic disease were lower than that of normal subjects with more significant difference (P < 0.001); compared with that of non-rheumatic diseases patients, SLE cases had significant difference (P < 0.01); RA cases had significant difference (P < 0.05); other cases of rheumatic diseases had no statistical differrence (P > 0.05). Above results suggest that superoxide anion is a non-specific inflammatory mediator which contributes to disorders with inflammatory damages (rheumatic or non-rheumatic diseases), where CuZn-SOD content tested was obviously lower than normal subjects; among the rheumatic disease patients, CuZn-SOD contents of the sera of SLE patients were the lowest because of its more autoimmune antibody, more severe inflammatory and immunological reaction. This work laid the theoretical and experimental foundation for the clinical application of exogenous CuZn-SOD in the treatment of rheumatic diseases. Combined use of CuZn-SOD scavengers may get better result because of the complexibility of ROS inflammatory mechanism.

Adolescent↗
PubMed1996

[Relaxing effects of l-stepholidine on rat resistance arteries].

Bolus injection(i.v.) of l-stepholidine(l-SPD, 3 mg.kg-1) caused hypotension in anaesthetized rats. The blood pressure reduction was sustained for 46.3 +/- 14.1 min. In in vitro experiments, l-SPD induced a concentration dependent relaxation of rat mesenteric, cerebral, renal and coronary resistance arteries preconstricted with 125 mmol.L-1 KCl. l-SPD was least potent with cerebral resistance arteries with the -Log EC50 value being 4.25 +/- 0.22. The relaxation of l-SPD on mesenteric resistance arteries was the most potent with the -Log EC50 value being 4.76 +/- 0.15. The larger influence of l-SPD (100 mumol.L-1) on the contraction induced by KCl in mesenteric than in cerebral arteries was in line with the above results. It is indicated that l-SPD has a direct relaxation on visceral resistance arteries with some degree of selectivity and the relaxation would decrease total peripheral resistance and thus constitute an important cause of its hypotensive action.

Animals↗