Biomedical subjects

J Guo

Publications and source records attributed to J Guo.

At least 19 recordsLinked to original sources

PubMed2000

Potentiation of benzo[a]pyrene-induced pulmonary and forestomach tumorigenesis in mice by D,L-buthionine-S,R-sulfoximine-mediated tissue glutathione depletion.

In vitro studies have suggested that the glutathione (GSH) S-transferase (GST)-catalyzed GSH conjugation is an important mechanism for the detoxification of (+)-anti-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the activated form of the widespread environmental pollutant benzo[a]pyrene (BP). However, in vivo experimental evidence for the importance of GSH/GST system in defense against carcinogenic effects of BP is lacking. We hypothesized that if GSH/GST were to play an important role in the detoxification of (+)-anti-BPDE, the tumorigenic activity of BP would be increased by depleting the levels of GSH, which is the required nucleophilic substrate for GST-catalyzed conjugation reactions. In the present study, we have tested the above hypothesis by determining the effect of D, L-buthionine-S,R-sulfoximine (BSO)-mediated tissue GSH depletion on BP-induced tumorigenesis of the lung and forestomach in female A/J mice. Treatment of mice with three i.p. injections of 2.5 mmol BSO/kg (12 h apart) plus 20 mM BSO in drinking water, resulted in a statistically significant reduction in hepatic, pulmonary and forestomach GSH levels. At the same time, BSO-administration caused a statistically significant increase in BP-induced pulmonary and forestomach tumor multiplicity. To the best of our knowledge, the present study is the first report that provides in vivo experimental evidence for the importance of GSH/GST system in cellular protection against carcinogenic effects of BP.

Animals↗

PubMed2000

Decreased vaginal disease in J-chain-deficient mice following herpes simplex type 2 genital infection.

J-chain-deficient (Jch(-/-)) mice were used to study the role of polymeric IgA (pIgA) in primary disease and protective immunity following genital herpes simplex type 2 (HSV-2) infection. Vaginal IgA in the Jch(-/-) mice was composed primarily of monomeric IgA and was not associated with secretory component (SC). In contrast, vaginal IgA in wild-type (WT) mice was predominantly polymeric and bound to SC. Following HSV-2 genital infection, the Jch(-/-) mice consistently exhibited fewer vaginal symptoms (P = 0.010) and mortality (P = 0.075) than did the WT mice. The variation in disease expression could not be explained by differences in local viral replication, since titers in vaginal wash fluid were comparable. To assess the effect of J chain deficiency on protective immunity, WT and Jch(-/-) mice were immunized intravaginally with attenuated HSV-2, challenged intravaginally with wild-type virus 5 weeks later, and evaluated for vaginal infection and neurological disease. Although the Jch(-/-) mice had reduced vaginal HSV-specific IgA and IgG levels following immunization, both WT and Jch(-/-) mice were protected from symptoms following wild-type virus challenge. We conclude that pIgA is not required for protective immunity against genital HSV-2 disease and that J chain deficiency offers some protection against symptoms following primary HSV-2 genital infection.

Animals↗

PubMed2000

Analysis of domains in the IKKalpha and IKKbeta proteins that regulate their kinase activity.

The IkappaB kinases IKKalpha and IKKbeta are critical in activating the NF-kappaB pathway. Although these proteins have a similar structure that includes kinase, leucine zipper, and helix-loop-helix domains, they exhibit marked differences in their kinase activity and functional properties. For example, IKKbeta has a 10-20-fold higher level of kinase activity for IkappaBalpha than does IKKalpha. Furthermore, disruption of the murine IKKbeta gene, but not the IKKalpha gene, results in severe defects in activating the NF-kappaB pathway. Mice lacking IKKbeta succumb to severe hepatic apoptosis because of failure to activate the NF-kappaB pathway, whereas mice deficient in IKKalpha exhibit skin and skeletal abnormalities and an embryonic lethal phenotype. To better characterize differences in the functional properties of these kinases, hybrid IKK proteins were constructed by domain swapping, and their kinase activity was assayed. These studies demonstrated that differences in the IKKalpha and IKKbeta helix-loop-helix domains are primarily responsible for differences in their kinase activity. In contrast, their kinase and leucine zipper domains exhibited relatively conserved function. These studies further define the properties of IKKalpha and IKKbeta, which are involved in their unique regulatory roles.

Animals↗

PubMed2000

Identification and functional characterization of a conserved, nuclear factor 1-like element in the proximal promoter region of CYP1A2 gene specifically expressed in the liver and olfactory mucosa.

CYP1A2 is a major cytochrome P-450 isoform in the liver and the olfactory mucosa but is essentially not expressed in other tissues. A nuclear factor 1 (NF-1) -like element was identified in the proximal promoter region of rat, mouse, rabbit, and human CYP1A2 genes through data base analysis. In vitro DNase I footprinting with a -211 to +81 probe from the rat CYP1A2 gene and nuclear extracts from rat liver and olfactory mucosa revealed a single protected region corresponding to the NF-1-like element at -129 to -111. Protein binding to this NF-1-like element was tissue-selective and was confirmed by in vivo footprinting in native chromatin from rat liver. Multiple DNA-binding complexes were detected in gel-shift assays using the CYP1A2 NF-1-like element and nuclear extracts from liver and olfactory mucosa, all of which were supershifted in the presence of an anti-NF1 antibody. The NF-1-like element was essential for transcriptional activity of the CYP1A2 gene in an in vitro transcription assay using nuclear extracts from the two tissues. Thus, members of the NF-1 family of transcription factors may play an important role in the tissue-selective expression of the CYP1A2 gene in the liver and olfactory mucosa.

Animals↗

PubMed2000

Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection.

P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm.

Antibodies, Monoclonal↗

PubMed2000

Identification of a human brain-specific isoform of mammalian STE20-like kinase 3 that is regulated by cAMP-dependent protein kinase.

A novel isoform of mammalian STE20-like kinase 3 (MST3) with a different 5' coding region from MST3, termed MST3b, was identified by searching through expressed sequence tag data base and obtained by rapid amplification of cDNA 5'-ends. MST3b was assigned to the long arm of human chromosome 13, D13S159-D13S280, by use of the National Center for Biotechnology Information sequence-tagged sites data base. Reverse transcription-polymerase chain reaction and Northern blot analysis with a probe derived from 5' distinct sequence of MST3b revealed that the expression of MST3b mRNA is restricted to the brain, in contrast to ubiquitous distribution of MST3 transcript. Western analysis confirmed the brain-specific expression of MST3b protein. In situ hybridization of rat brain sections with a MST3b-specific probe indicated that MST3b is widely expressed in different brain regions, with especially high expression in hippocampus and cerebral cortex. When expressed in human embryonic kidney 293 (HEK293) cells, MST3b effectively phosphorylated myelin basic protein, as well as undergoing autophosphorylation. Interestingly, expression of MST3, but not MST3b, in HEK293 cells was able to activate the endogenous p42/44 mitogen-activated protein kinase (MAPK) up to 4-fold, whereas neither isoform activated p38 MAPK under the same conditions. Further experiments demonstrated that MST3b, but not MST3, was effectively phosphorylated by activation of cyclic AMP-dependent protein kinase (PKA) in both in vivo and in vitro assays. The mutation of Thr-18 into Ala in MST3b (T18A), a putative PKA phosphorylation site that is absent in MST3, abolished its phosphorylation by PKA. Consequently, expression of the T18A mutant in HEK293 cells led to partial activation of p42/44 MAPK, indicating that MST3b is under the regulation of PKA. Taken together, our data provide evidence that the two isoforms of STE20-like kinase 3 are differentially distributed and regulated.

Animals↗

PubMed2000

Lecithin vesicular carriers for transdermal delivery of cyclosporin A.

Two kinds of vesicles with and without the presence of sodium cholate (flexible vesicles and conventional vesicles) were prepared, using cyclosporin A as model drug. When applied onto the excised abdominal skin of mice non-occlusively, the enhancing effects of vesicles on the penetration of cyclosporin A were assessed by an in vitro permeation technique. The effect of sodium cholate micelles was also studied. In vivo study was carried out by topical application of vesicles onto the mice skin and drug serum concentration was detected. Results showed that after 8 h of administration, flexible vesicles transported 1.16 microg of cyclosporin A through per cm(2) mice skin and amounted to 1.88 microg 24 h later. The residual amount in the skin was 1.78+/-0.51 microg/cm(2). However, flexible vesicles failed to transport measurable amount of drug through pre-hydrated skin while deposited 2.39+/-0.26 microg/cm(2) into the skin. Conventional vesicles failed to transfer cyclosporin A into the receiver while accumulated 0. 72+/-0.19 microg/cm(2) of drug in the skin. Furthermore, 1 and 40% sodium cholate micelles precluded the transport of cyclosporin A. In vivo studies indicated that with the application of flexible vesicles, serum drug concentration of 53.43+/-9.24 ng/ml was detected 2 h later. After the stratum corneum of mouse skin has been destroyed by shaving, flexible vesicles transferred large amount of drug into blood, up to 187.32+/-53.21 ng/ml after 1 h of application. Conventional vesicles failed to deliver measurable amount of drug into the blood under normal skin condition. In conclusion, flexible vesicle is better than conventional vesicle as the carrier for transdermal delivery of cyclosporin A. Penetration and fusion have been suggested to be two major functional mechanisms. Hydration is detrimental to the enhancement effect. Stratum corneum constitutes main barrier to the transport of lipophilic cyclosporin A.

Administration, Cutaneous↗

PubMed2000

New strategies for the synthesis of biologically important tetrapyrroles. The "B,C + D + A" approach to linear tetrapyrroles.

Linear tetrapyrroles related to phytochrome (1) were prepared in enantiospecific fashion by a new strategy beginning with ring-B,C synthons of type 19 (bis-iododipyrrins). Rings A and D were elaborated by Pd(0)-mediated coupling of 19a with the appropriate alkyne acid or amide derivatives 9 and 20, followed by intramolecular cyclization (method C: BC + D + A --> ABCD).

Magnetic Resonance Spectroscopy↗

PubMed2000

Absolute stereochemistry of the strevertenes.

The CD exciton chirality method was applied to determine the absolute stereochemistry of the strevertenes, antifungal pentaene macrolides produced by Streptoverticillium sp. LL-30F848. The CD difference spectrum of strevertene A methyl ester 15-dimethylaminobenzoate showed a positive couplet between the dimethylaminobenzoate and the pentaene chromophores, and therefore established the 15R configuration. Thus, by considering the relative configurations of the remaining stereogenic centers as derived from X-ray crystallography and ROESY experiments, the absolute stereochemistry of the strevertenes is established as 2R, 3S, 5S, 7S, 11R, 13R, 14R, 15R, 26S and 27R.

Anti-Bacterial Agents↗

PubMed2000

Role of MHC class I expression and CD8(+) T cells in the evolution of iodine-induced thyroiditis in NOD-H2(h4) and NOD mice.

Dietary iodine has long been known to influence the development of human autoimmune thyroid disease. In nonobese diabetic (NOD) and NOD-H2(h4) mice elevated dietary iodine has been shown to induce autoimmune thyroid disease. Immune responses to thyroid antigens can be detected in these mouse strains, including T cell responses in the NOD-H2(h4) mouse to thyroid peroxidase. Cell transfer studies and antibody depletion experiments reveal a requirement for both CD4(+) T cells and CD8(+) T cells in the development of thyroid autoimmunity. Histological analyses of the thyroids show that following 1 week of iodine administration MHC class I expression is elevated on thyroid follicular cells and CD4(+) and CD8(+) T cells have begun to infiltrate the gland. Although MHC class II expression on thyroid epithelial cells was also elevated, the tempo of expression was slower and the extent of expression was far less than that for MHC class I. Depletion of CD8(+) T cells at early stages of disease induction inhibited not only thyroid infiltration and autoantibody production but also reduced the levels of MHC expression in the thyroid, suggesting that cytokine production by infiltrating lymphocytes was responsible for the increased MHC expression.

Adoptive Transfer↗

PubMed2000

Amphiphysin IIm, a novel amphiphysin II isoform, is required for macrophage phagocytosis.

Phagocytosis of pathogens by macrophages initiates the innate immune response, which in turn orchestrates the adaptive immune response. Amphiphysin II participates in receptor-mediated endocytosis, in part, by recruiting the GTPase dynamin to the nascent endosome. We demonstrate here that a novel isoform of amphiphysin II associates with early phagosomes in macrophages. We have ablated the dynamin-binding site of this protein and shown that this mutant form of amphiphysin II inhibits phagocytosis at the stage of membrane extension around the bound particles. We define a signaling cascade in which PI3K is required to recruit amphiphysin II to the phagosome, and amphiphysin II in turn recruits dynamin. Thus, amphiphysin II facilitates a critical initial step in host response to infection.

Amino Acid Sequence↗

PubMed2000

Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II.

Injection of AKR/N mice with fibroblasts co-expressing MHC class II and TPO in the absence of adjuvant induces IgG-class TPO antibodies that resemble spontaneously arising human thyroid autoantibodies. We have used this model to examine the effect of iodide on TPO antibody induction as well as to analyse the interaction between T and B cells. Despite its importance as a major environmental factor in thyroid autoimmunity, variable iodide intake had no detectable effects on TPO antibody levels, lymphocytic infiltration of the thyroid or thyroid hormone levels. In terms of T cell responsiveness, splenocytes from TPO fibroblast-injected mice, but not from control mice, proliferated in response to TPO. Intriguingly, B cell-depleted splenocytes (mainly T cells without reduction of macrophages) proliferated in response to TPO only when co-cultured with irradiated autologous splenocytes from TPO fibroblast-injected mice but not from control mice. These data suggest that TPO-specific B cells are involved in antigen presentation to sensitized T cells and are supported by the ability of spleen cells from TPO cell-injected (but not control) mice to secrete TPO antibodies spontaneously in culture. In conclusion, we provide the first evidence for the presence of thyroid autoantigen-specific B cells and their ability to present their autoantigen to sensitized T cells in mice induced to develop TPO antibodies resembling autoantibodies in humans.

Animals↗

PubMed2000

A locus for brachydactyly type A-1 maps to chromosome 2q35-q36.

Brachydactyly type A-1 (BDA1) was, in 1903, the first recorded example of a human anomaly with Mendelian autosomal dominant inheritance. Two large families, the affected members of which were radiographed, were recruited in the study we describe here. Two-point linkage analysis for pedigree 1 (maximum LOD score [Zmax] 6.59 at recombination fraction [theta] 0.00) and for pedigree 2 (Zmax=5.53 at straight theta=0.00) mapped the locus for BDA1 in the two families to chromosome 2q. Haplotype analysis of pedigree 1 confined the locus for family 1 within an interval of <8.1 cM flanked by markers D2S2248 and D2S360, which was mapped to chromosome 2q35-q36 on the cytogenetic map. Haplotype analysis of pedigree 2 confined the locus for family 2 within an interval of <28. 8 cM flanked by markers GATA30E06 and D2S427, which was localized to chromosome 2q35-q37. The two families had no identical haplotype within the defined region, which suggests that the two families were not related.

Adolescent↗

PubMed2000

Analysis of maxillopedia expression pattern and larval cuticular phenotype in wild-type and mutant tribolium.

The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

Alleles↗

PubMed2000

Characterization of the interaction between the interferon-induced protein P56 and the Int6 protein encoded by a locus of insertion of the mouse mammary tumor virus.

For determining cellular functions of the interferon-inducible human cytoplasmic protein P56, we undertook a Saccharomyces cerevisiae two-hybrid screen that identified Int6 as a P56-interacting protein. That the interaction also occurs in human cells was confirmed by coimmunoprecipitation and the observed cytoplasmic displacement of nuclear Int6 upon coexpression of P56. Because Int6 has been claimed to be both a cytoplasmic and a nuclear protein, we investigated the structural basis of this discrepancy. By mutational analyses, we showed that the Int6 protein contains a bipartite nuclear localization signal and a nuclear export signal at the far end of the amino terminus. The 20 amino-terminal residues of Int6, when they were attached to a different nuclear protein, were sufficient to translocate that protein to the cytoplasm. Within this region, replacement of any of the three leucine residues with alanine destroyed the function of the export signal. The specific domain of P56 that is required for its interaction with Int6 was mapped using the yeast two-hybrid assay and a mammalian coimmunoprecipitation assay. Both assays demonstrated that the C-terminal region of P56 containing three specific tetratricopeptide motifs is required for this interaction. In contrast, removal of an internal domain of P56 enhanced the interaction, as quantified by the two-hybrid assay.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Domains in the SPT5 protein that modulate its transcriptional regulatory properties.

SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.

Amino Acid Sequence↗

PubMed2000

Therapy with antisense TGF-beta1 oligodeoxynucleotides reduces kidney weight and matrix mRNAs in diabetic mice.

Inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) relies on their ability to bind complementary mRNA sequences and prevent translation. The proximal tubule is a suitable target for ODN therapy in vivo because circulating ODNs accumulate in the proximal tubule in high concentrations. Because increased proximal tubular transforming growth factor- beta1 (TGF-beta1) expression may mediate diabetic renal hypertrophy, we investigated the effects of antisense TGF-beta1 ODN on the high-glucose-induced proximal tubular epithelial cell hypertrophy in tissue culture and on diabetic renal hypertrophy in vivo. Mouse proximal tubular cells grown in 25 mM D-glucose and exposed to sense ODN as control (1 microM) exhibited increased (3)[H]leucine incorporation by 120% and total TGF-beta1 protein by 50% vs. culture in 5.5 mM D-glucose. Antisense ODN significantly decreased the high-glucose-stimulated TGF-beta1 secretion and leucine incorporation. Continuous infusion for 10 days of ODN (100 microg/day) was achieved via osmotic minipumps in diabetic and nondiabetic mice. Sense ODN-treated streptozotocin-diabetic mice had 15.3% increase in kidney weight, 70% increase in alpha1(IV) collagen and 46% increase in fibronectin mRNA levels compared with nondiabetic mice. Treatment of diabetic mice with antisense ODN partially but significantly decreased kidney TGF-beta1 protein levels and attenuated the increase in kidney weight and the alpha1(IV) collagen and fibronectin mRNAs. In conclusion, therapy with antisense TGF-beta1 ODN decreases TGF-beta1 production and attenuates high-glucose-induced proximal tubular cell hypertrophy in vitro and partially prevents the increase in kidney weight and extracellular matrix expression in diabetic mice.

Animals↗

PubMed2000

Ocular nonnephropathic cystinosis: clinical, biochemical, and molecular correlations.

Ocular nonnephropathic cystinosis, a variant of the classic nephropathic type of cystinosis, is an autosomal recessive lysosomal storage disorder characterized by photophobia due to corneal cystine crystals but absence of renal disease. We determined the molecular basis for ocular cystinosis in four individuals. All had mutations in the cystinosis gene CTNS, indicating that ocular cystinosis is allelic with classic nephropathic cystinosis. The ocular cystinosis patients each had one severe mutation and one mild mutation, the latter consisting of either a 928 G-->A (G197R) mutation or an IVS10-3 C-->G splicing mutation resulting in the insertion of 182 bp of IVS10 into the CTNS mRNA. The mild mutations appear to allow for residual CTNS mRNA production, significant amounts of lysosomal cystine transport, and lower levels of cellular cystine compared with those in nephropathic cystinosis. The lack of kidney involvement in ocular cystinosis may be explained by two different mechanisms. On the one hand (e.g. the G197R mutation), significant residual cystinosin activity may be present in every tissue. On the other hand (e.g. the IVS 10-3 C-->G mutation), substantial cystinosin activity may exist in the kidney because of that tissue's specific expression of factors that promote splicing of a normal CTNS transcript. Each of these mechanisms could result in minimally reduced lysosomal cystine transport in the kidneys.

Amino Acid Transport Systems, Neutral↗