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L Shi

Publications and source records attributed to L Shi.

At least 19 recordsLinked to original sources
PubMed1998

Defects in regulation of apoptosis in caspase-2-deficient mice.

During embryonic development, a large number of cells die naturally to shape the new organism. Members of the caspase family of proteases are essential intracellular death effectors. Herein, we generated caspase-2-deficient mice to evaluate the requirement for this enzyme in various paradigms of apoptosis. Excess numbers of germ cells were endowed in ovaries of mutant mice and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in caspase-2-deficient B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in caspase-2-deficient mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more effectively than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.

Amyotrophic Lateral Sclerosis↗
PubMed1998

Interactions of the borna disease virus P, N, and X proteins and their functional implications.

Borna disease virus (BDV) causes persistent central nervous system infection and behavioral disturbances in warm-blooded animals. Protein interaction studies were pursued to gain insight into the functions of the putative nucleoprotein (N), phosphoprotein (P), atypical glycoprotein (gp18), and X protein (X) of BDV. Coimmunoprecipitation experiments indicated that N and P, and P and X, form complexes in infected cells. Two-hybrid analyses confirmed interactions between P and P, P and X, and P and N, but not between P and gp18, N and gp18, X and gp18, or X and N. Analysis of P truncation mutants identified three nonoverlapping regions important for oligomerization (amino acids (aa) 135-172), and binding to X (aa 33-115) or N (aa 197-201). Coexpression of X stimulated oligomerization of P but decreased N-P complex formation. Immunocytochemistry of transfected noninfected CHO cells demonstrated that the distribution of X is dependent upon the presence of P-X expressed alone was found predominantly in the cytoplasm whereas coexpression of X and P resulted in nuclear localization. Immunocytochemistry of infected cells revealed nuclear colocalization of P and X. Interactions of P, N, and X may have implications for regulation of BDV transcription/replication and ribonucleoprotein assembly.

Animals↗
PubMed1998

Inhibition of apoptosis in chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation.

We report that chlamydiae, which are obligate intracellular bacterial pathogens, possess a novel antiapoptotic mechanism. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli including the kinase inhibitor staurosporine, the DNA-damaging agent etoposide, and several immunological apoptosis-inducing molecules such as tumor necrosis factor-alpha, Fas antibody, and granzyme B/perforin. The antiapoptotic activity was dependent on chlamydial but not host protein synthesis. These observations suggest that chlamydia may encode factors that interrupt many different host cell apoptotic pathways. We found that activation of the downstream caspase 3 and cleavage of poly (ADP-ribose) polymerase were inhibited in chlamydia-infected cells. Mitochondrial cytochrome c release into the cytosol induced by proapoptotic factors was also prevented by chlamydial infection. These observations suggest that chlamydial proteins may interrupt diverse apoptotic pathways by blocking mitochondrial cytochrome c release, a central step proposed to convert the upstream private pathways into an effector apoptotic pathway for amplification of downstream caspases. Thus, we have identified a chlamydial antiapoptosis mechanism(s) that will help define chlamydial pathogenesis and may also provide information about the central mechanisms regulating host cell apoptosis.

Animals↗
PubMed1997

The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis.

Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

Adenovirus E1B Proteins↗
PubMed1997

Interneuron migration from basal forebrain to neocortex: dependence on Dlx genes.

Although previous analyses indicate that neocortical neurons originate from the cortical proliferative zone, evidence suggests that a subpopulation of neocortical interneurons originates within the subcortical telencephalon. For example, gamma-aminobutyric acid (GABA)-expressing cells migrate in vitro from the subcortical telencephalon into the neocortex. The number of GABA-expressing cells in neocortical slices is reduced by separating the neocortex from the subcortical telencephalon. Finally, mice lacking the homeodomain proteins DLX-1 and DLX-2 show no detectable cell migration from the subcortical telencephalon to the neocortex and also have few GABA-expressing cells in the neocortex.

Animals↗
PubMed1997

Borna disease virus P-protein is phosphorylated by protein kinase Cepsilon and casein kinase II.

Borna disease virus (BDV) is a newly classified nonsegmented negative-strand RNA virus (order of Mononegavirales) that persistently infects specific brain regions and circuits of warm-blooded animals to cause behavioral disturbances. Viruses within the order of Mononegavirales have phosphoproteins that typically serve as transcription factors and are modulated in functional activity through phosphorylation. To identify the kinases involved in BDV phosphoprotein (BDV-P) phosphorylation, in vitro phosphorylation assays were performed using recombinant phosphoprotein produced in Escherichia coli as substrate and cytoplasmic extracts from a rat glioma cell line (C6) or rat brain extracts as sources of kinase activity. These experiments revealed that BDV-P was phosphorylated predominantly by protein kinase C (PKC) and to a lesser extent by casein kinase II. Partial purification of the PKC from rat brain extract suggested that the BDV-P phosphorylating kinase is PKCepsilon. A role for PKC phosphorylation in vivo was confirmed by using the PKC-specific inhibitor GF109203X. Furthermore, peptide mapping studies indicated that BDV-P is phosphorylated at the same sites in vitro as it is in vivo. Mutational analysis identified Ser26 and Ser28 as sites for PKC phosphorylation and Ser70 and Ser86 as sites for casein kinase II phosphorylation. The anatomic distribution of PKCepsilon in the central nervous system may have implications for BDV neurotropism and pathogenesis.

Animals↗
PubMed1997

Activation of caspase-2 in apoptosis.

Members of the CED-3/interleukin-1beta-converting enzyme (ICE) protease (caspase) family are synthesized as proforms, which are proteolytically cleaved and activated during apoptosis. We report here that caspase-2 (ICH-1/NEDD-2), a member of the ICE family, is activated during apoptosis by another ICE member, a caspase-3 (CPP32)-like protease(s). When cells are induced to undergo apoptosis, endogenous caspase-2 is first cleaved into three fragments of 32-33 kDa and 14 kDa, which are then further processed into 18- and 12-kDa active subunits. Up to 50 microM N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), a caspase-3-preferred peptide inhibitor, inhibits caspase-2 activation and DNA fragmentation in vivo, but does not prevent loss of mitochondrial function, while higher concentrations of DEVD-CHO (>50 microM) inhibit both. In comparison, although the activity of caspase-3 is very sensitive to the inhibition of DEVD-CHO (<50 nM), inhibition of caspase-3 activation as marked by processing of the proform requires more than 100 microM DEVD-CHO. Our results suggest that the first cleavage of caspase-2 is accomplished by a caspase-3-like activity, and other ICE-like proteases less sensitive to DEVD-CHO may be responsible for activation of caspase-3 and loss of mitochondrial function.

Apoptosis↗
PubMed1997

Monoclonal antibodies specific to the acute lymphoblastic leukemia t(1;19)-associated E2A/pbx1 chimeric protein: characterization and diagnostic utility.

Nonrandom chromosomal abnormalities are found in most human malignancies, particularly leukemias and lymphomas. A characteristic t(1;19) (q23;p13.3) chromosomal translocation is detected in 5% of childhood acute lymphoblastic leukemia (ALL) cases. This translocation results in the formation of a fusion gene, which leads to the expression of an oncogenic E2A/pbx1 protein. Breakpoints in the E2A gene almost invariably occur within a single intron, and the identical portion of PBX1 is joined consistently to exon 13 of E2A in fusion mRNA. In this article, we report the development of monoclonal antibodies against E2A/pbx1 fusion protein using a specific peptide that corresponds to the junction region of the protein. The obtained antibodies recognize specifically the chimeric E2A/pbx1 fusion protein and lack cross-reactivities with E2A and pbx1. Immunohistochemical staining and flow cytometric studies show that these antibodies can distinguish t(1;19)-positive from t(1;19)-negative leukemic cells. These results indicate that the obtained E2A/pbx1-specific monoclonal antibodies might prove to be valuable diagnostic reagents and important tools for elucidating the mechanisms involved in oncogenesis and progression of t(1;19)-positive childhood ALL.

Amino Acid Sequence↗
PubMed1997

Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization.

Granzyme B (GraB) induces apoptosis in the presence of perforin. Perforin polymerizes in the cell membrane to form a nonspecific ion pore, but it is not known where GraB acts to initiate the events that ultimately lead to apoptosis. It has been hypothesized that GraB enters the target cell through a perforin channel and then initiates apoptosis by cleaving and activating members of the ICE/Ced-3 family of cell death proteases. To determine if GraB can enter the cell, we treated YAC-1 or HeLa cells with FITC-labeled GraB and measured intracellular fluorescence with a high sensitivity CCD camera and image analyzer. GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min. Uptake was inhibited at low temperature (4 degrees C) and by pretreatment with metabolic inhibitors, NaF and DNP, or cytochalasin B, a drug that both blocks microfilament formation, and FITC-GraB remained on the cell membrane localized in patches. With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytoplasmic fluorescence was observed over that found in cells treated only with FITC-GraB. However, FITC-GraB was now detected in the nucleus of apoptotic cells labeling apoptotic bodies and localized areas within and along the nuclear membrane. The ability of GraB to enter cells in the absence of perforin was reexamined using anti-GraB antibody immunogold staining of ultrathin cryosections of cells incubated with GraB. Within 15 min, gold particles were detected both on the plasma membrane and in the cytoplasm of cells with some gold staining adjacent to the nuclear envelope but not in the nucleus. Cells internalizing GraB in the absence of perforin appeared morphologically normal by Hoechst staining and electron microscopy. GraB directly microinjected into the cytoplasm of B16 melanoma cells induced transient plasma membrane blebbing and nuclear coarsening but the cells did not become frankly apoptotic unless perforin was added. We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.

Animals↗
PubMed1997

pp1-cyano2, a protein serine/threonine phosphatase 1 gene from the cyanobacterium Microcystis aeruginosa UTEX 2063.

Polymerase chain reaction (PCR) products similar to protein serine/threonine family I phosphatase genes were identified in five strains of cyanobacteria from three species. The gene for one of these protein phosphatase PCR products, pp1-cyano2 from Microcystis aeruginosa UTEX 2063, was cloned and sequenced. The deduced protein sequence PP1-cyano2 contains 264 amino acid residues ( approximately 30.3 kDa). In its N-terminal region, PP1-cyano2 had a GDXXHG(X)nGDXXDRG(X)nGNHE (nP23) sequence that is well-conserved in all protein serine/threonine family I phosphatases. Of 19 amino acid residues important for either metal binding, structure of the active site, or catalysis in eukaryotic PP1, 18 were present in PP1-cyano2. Reverse-transcription-PCR results showed that pp1-cyano2 was expressed under laboratory culture conditions.

Amino Acid Sequence↗
PubMed1997

A monoclonal antibody shows discrete cellular and subcellular localizations of mGluR1 alpha metabotropic glutamate receptors.

The metabotropic glutamate receptor, mGluR1 alpha, is postsynaptic in excitatory synapses in many populations of neurons and mediates long-term responses. The present study defines the distribution of this receptor using a new, highly specific monoclonal antibody to mGluR1 alpha. Overall distribution of immunostaining was similar to that described previously with polyclonal antibodies, including prominent staining in the olfactory bulb, interneurons of the CA1 hippocampus stratum oriens/alveus, globus pallidus, thalamus, Purkinje cells and in cells of the outer dorsal cochlear nucleus and with little or low staining in principal cells of the cerebral cortex and hippocampus. Interestingly, the well-known association of mGluR1 alpha receptors with neocortical interneurons was even more prevalent than previously noted with polyclonal antibodies. Ultrastructural studies in the hippocampus and cerebellum showed dense immunoperoxidase staining in postsynaptic membranes and densities and in perisynaptic and extrasynaptic membranes, as well as substantial cytoplasmic staining associated with organelles, especially the endoplasmic reticulum.

Animals↗
PubMed1997

Developmental expression of a DNA repair gene in Arabidopsis.

Exposure to alkylating agents results in the formation of a wide variety of DNA damage products. One of these, 3-methyladenine (3-mAde), is lethal if left unrepaired. The 3-methyladenine glycosylase (aMAG) gene of Arabidopsis thaliana is required for base excision repair of this lesion, and probably shares the ability of other 3-mAde glycosylases to recognize and excise a broad spectrum of damaged bases. Given the fact that DNA damage products can act as blocks to both DNA and RNA synthesis, one would expect that this protein should be expressed to some degree in all living cells. Using a DIG-labeled aMAG antisense RNA as a probe, we have investigated the developmental and tissue-specific expression of this repair gene. We found that the gene is preferentially expressed in meristematic tissue, the developing embryo and endosperm, and organ primordia. This pattern of expression is consistent with a requirement for expression in rapidly dividing tissues. However, high levels of expression were also observed in growing leaves, a tissue that is undergoing a relatively low rate of cell division. This result suggests that 3-mAde glycosylase is required not only for DNA replication, but also for cell growth.

Arabidopsis↗
PubMed1997

The STR120 satellite DNA of soybean: organization, evolution and chromosomal specificity.

A highly repeated DNA sequence family, STR120, with tandemly arranged repetitive units (monomers) of approximately 120bp, has been identified in soybean [Glycine max (L.) Merr.]. Five related clones showing tandem repeats of a 120-bp-long monomer were isolated from a soybean genomic library. Results of Southern blotting experiments using three of the clones as probes onto genomic DNA digested with different restriction enzymes were in agreement with a tandem arrangement of these sequences in the genome. A total of 12 monomers were sequenced, showing considerable sequence heterogeneity. A consensus sequence of 126 bp was obtained that exhibits an average similarity of 81% to the sequenced units. In three of the clones identified, neighbouring units are significantly more similar to each other than to units from different clones; in the remaining two clones, however, similarity between the two units observed is low (70%), while the overall similarity between the two clones is high (95%). This indicates that in these cases the repetitive unit may be the dimer rather than the monomer. Based on the presence of direct repeats within each monomer, we suggest that the 120-bp monomer may itself have evolved by duplication of an ancestral 60-bp unit. The STR120 family distribution is limited to annual soybeans and is not found, at least at high-copy number, in related perennial soybeans or other members of the tribe Phaseolae. Fluorescence in situ hybridization (FISH) to metaphase chromosomes using four of the clones as probes shows that the number of chromosomal locations differs depending on the stringency conditions and goes from two to eight when the stringency is progressively lowered. The estimated copy number for one of the clones is from 5000 to 10000, but this may just represent a lower boundary for the whole family in consideration of the high sequence divergence observed within the family. FISH and sequence analysis therefore indicate that different subfamilies as well as higher-order repeat units are present in the STR120 family, very much like those in primate alpha satellite DNA, and that some of the subfamilies seem to exhibit divergence on a chromosomal basis.

Base Sequence↗
PubMed1997

B chromosome behavior in maize pollen as determined by a molecular probe.

The B chromosomes of maize typically undergo nondisjunction during the second microspore division (generative cell division). When the microspore nucleus contains only one B chromosome, two kinds of sperm result, one with two B chromosomes and one with no B chromosomes. The sperm with the B chromosomes preferentially fertilizes the egg cell. Previous studies of these phenomena have been limited to genetic analysis and chromosome spreads. In this study we show that a B chromosome-specific probe can be used with fluorescence in situ hybridization (FISH) analysis to detect the presence, location, and frequency of B chromosomes in intact interphase nuclei within mature pollen of maize. Using genetic line TB-10L18, our results indicate that nondisjunction of the B centromere occurs at an average frequency of 56.6%, based on four plants and 1306 pollen grains analyzed. This is consistent with the results of genetic studies using the same B-A translocation. In addition, our results suggest that B chromosome nondisjunction can occur during the first microspore division. Spatial distribution of the B chromosome-specific probe appears to be largely confined to one tip of the sperm nucleus, and a DNA fragment found outside the pollen nuclei often hybridizes to the B chromosome-specific probe.

Biotinylation↗
PubMed1997

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

Asparagine, the primary assimilation product from N2 fixation in temperate legumes and the predominant nitrogen transport product in many plant species, is synthesized via asparagine synthetase (AS; EC 6.3.5.4). Here, we report the isolation and characterization of a cDNA and a gene encoding the nodule-enhanced form of AS from alfalfa. The AS gene is comprised of 13 exons separated by 12 introns. The 5' flanking region of the AS gene confers nodule-enhanced reporter gene activity in transformed alfalfa. This region also confers enhanced reporter gene activity in dark-treated leaves. These results indicate that the 5' upstream region of the AS gene contains elements that affect expression in root nodules and leaves. Both AS mRNA and enzyme activity increased approximately 10- to 20-fold during the development of effective nodules. Ineffective nodules have strikingly reduced amounts of AS transcript. Alfalfa leaves have quite low levels of AS mRNA and protein; however, exposure to darkness resulted in a considerable increase in both. In situ hybridization with effective nodules and beta-glucuronidase staining of nodules from transgenic plants showed that AS is expressed in both infected and uninfected cells of the nodule symbiotic zone and in the nodule parenchyma. RNA gel blot analysis and in situ hybridization results are consistent with the hypothesis that initial AS expression in nodules is independent of nitrogenase activity.

Adaptation, Physiological↗
PubMed1997

Human TAP1 polymorphisms detected by denaturing gradient gel electrophoresis.

Presentation of endogenous peptides by major histocompatibility complex class I (MHC) molecules is controlled, in part, by the Tap1 and Tap2 genes in the MHC class II region that encode a heterodimeric peptide transporter. Polymorphisms of human Tap1 in normal individuals have now been investigated systematically by denaturing gradient gel electrophoresis (DGGE) analysis of fragments of genomic DNA generated by the polymerase chain reaction. Polymorphisms identified by distinctive DGGE band patterns were confirmed by DNA sequencing. In addition to four previously described polymorphisms in the open reading frame, DGGE detected three new polymorphisms: a G-->T substitution in the promoter region, a 10-bp insert in intron 9, and a G-->T substitution 80-bp downstream of the translation termination codon.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Screening of the TAP1 gene by denaturing gradient gel electrophoresis in insulin-dependent diabetes mellitus: detection and comparison of new polymorphisms between patients and controls.

New protective or disease-associated polymorphisms in the TAP1 gene were sought in insulin-dependent diabetes mellitus (IDDM) patients with the use of denaturing gradient gel electrophoresis (DGGE) screening of genomic DNA. The TAP1 gene is located in the human leukocyte antigen (HLA) class II region of the genome and encodes components of a peptide transporter essential for antigen presentation by HLA class I molecules. Fragments of TAP1 corresponding to the 5' promoter, each of the 11 exons (with portions of adjacent intronic regions) and the 3' flanking region were amplified by the polymerase chain reaction and then subjected to DGGE. DNA fragments of TAP1 yielded DGGE bands with patterns whose frequencies differed between IDDM patients and controls. Specific DGGE band patterns with fragments corresponding to the promoter, exons or introns 3, 6, 7, 8, 9 or 10 of TAP1 were detected exclusively in either patients or controls. Sequencing of TAP1 fragments encompassing exon 7 gave rise to a DGGE band pattern exclusively observed in an IDDM patient and sequencing revealed a previously unidentified polymorphisms at codon 518 (GTC-->ATC, Val-->Ile). Another unique polymorphism uncovered by DGGE revealed by sequencing a polymorphism in intron 2 in a diabetic patient. The genotypes of additional HLA class II matched patients and controls were determined with regard to five exonic and one intronic TAP1 polymorphism. A 10 base pair intronic insertion in intron 9 was exclusively identified in controls and missing from patients (P = 0.017). Further large population-based studies may reveal whether these newly identified at risk or protective TAP1 variants confer markers of statistical risk in diverse population groups.

ATP Binding Cassette Transporter, Subfamily B, Mem↗