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Biomedical subjects

B Yan

Publications and source records attributed to B Yan.

At least 73 records · Page 4Linked to original sources
PubMed1997

Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters.

Induction of islet neogenesis by cellophane wrapping (CW) reverses streptozotocin-induced (STZ) diabetes. Administration of Ilotropin, a protein extract isolated from CW pancreata, causes recapitulation of normal islet ontogeny and reverses STZ diabetes, reducing mortality by 50%. We investigated the hypothesis that a novel gene encoding a constituent of Ilotropin was expressed in the hamster pancreas undergoing islet neogenesis. Islet neogenesis associated protein (INGAP) is a product of a novel gene expressed in regenerating hamster pancreas. Northern blot analysis showed a strong single transcript of 850 bp at 1 and 2 d after CW that disappeared by the 6th day and was absent from untreated control pancreata. INGAP gene is expressed in acinar cells, but not in islets. Western blot analysis demonstrated the presence of INGAP in Ilotropin but not in extracts from control pancreata. A synthetic pentadecapeptide, corresponding to a region unique to INGAP, stimulated a 2.4-fold increase in [3H]thymidine incorporation into hamster duct epithelium in primary culture and a rat pancreatic duct cell line but had no effect on a hamster insulinoma tumor cell line. A portion of human INGAP gene was cloned and appears to be highly homologous to the hamster gene. This data suggests that the INGAP gene is a novel pancreatic gene expressed during islet neogenesis whose protein product is a constituent of Ilotropin and is capable of initiating duct cell proliferation, a prerequisite for islet neogenesis.

Amino Acid Sequence↗
PubMed1997

Cytokines (IL-1beta and TNFalpha) in relation to biochemical and immunological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rats.

Previous studies in different strains of rats and mice have shown that the inhibition of gluconeogenesis as a result of reduced liver phosphoenolpyruvate carboxykinase (PEPCK) activity together with appetite suppression play critical roles in the acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recent immunological studies in rats demonstrated that exposure to low doses of TCDD resulted in an early and enhanced IgG response to immunization with sheep red blood cells (SRBC) and an enhanced delayed-type hypersensitivity (DTH) reaction as well as a positive popliteal lymph node (PLN) response. However, high doses of TCDD suppressed the DTH reaction. This study aimed at examining the involvement of cytokines (IL-1 and TNF) in mediating the above effects. Liver samples from a previous dose-response study on DTH reaction were investigated, in which rats were treated with TCDD (1, 3, 10, 30 and 90 microg/kg) and immunized with an antigen. mRNA levels of IL-1beta were elevated begining at the 1 microg/kg (non-lethal) dosage group with a maximum increase of about 5-fold above controls in the 90 microg/kg (lethal) dosage group. mRNA levels of TNFalpha were also significantly elevated begining at the 30 microg/kg dosage group. These results suggest that at low doses of TCDD, increased IL-1beta could be responsible for immune function stimulation, whereas at high doses of TCDD, greatly elevated TNFalpha and IL-1beta levles may exacerbate or mediate acute toxicity including immune suppression and related biochemical effects. A time course study (60 microg TCDD/kg without immunization) revealed that liver mRNA levels of TNFalpha were significantly elevated starting 24 h, and reaching a maximum 48 h after dosing with TCDD. This change was accompanied by a transient increase of mRNA levels of IL-1beta at day 4 after TCDD dosage. Thus, these data demonstrated that TCDD alone (without immunization) can cause transient increases of mRNA levels of TNFalpha and IL-1beta in liver. Results from these experiments suggest that TCDD-induced cytokine changes may play important roles in various effects of TCDD.

Animals↗
PubMed1997

Transcriptional initiation sites in sorghum mitochondrial DNA indicate conserved and variable features.

Transcriptional initiation and processing was examined for three sorghum mitochondrial DNA genes (atp6-1, atp6-2, urf209) and two open reading frames (orf265/130, orf107) to characterize sequences associated with initiation and other transcriptional strategies for this species. The 5' termini of ten transcripts were determined by primer extension, and mtRNA was capped with guanylyl transferase and annealed to anti-sense riboprobes to identify transcriptional initiation regions. Eight transcript termini were suitable substrates for guanylyl transferase, indicating the presence of one (atp6-1, atp6-2, urf209), two (orf265/130), or three (orf107) promoters for the five examples. The majority of the putative promoters were associated with single primer-extension termini, while two examples exhibited two transcript-initiation sites within the promoter. Four examples were characterized by initiated transcripts without subsequent processing, indicating that processing is not obligatory. Each of the putative promoter regions included significant A/T-rich 5' regions, consistent with previous examples, but four exceptions to a consensus core YRTA sequence were identified. The anomalies (AATA, CTTA) suggest plasticity in the primary structure of the core region of higher-plant mitochondrial DNA promoters.

Base Sequence↗
PubMed1997

Different distribution of fluorinated anesthetics and nonanesthetics in model membrane: a 19F NMR study.

Despite their structural resemblance, a pair of cyclic halogenated compounds, 1-chloro-1,2,2-trifluorocyclobutane (F3) and 1,2-dichlorohexafluorocyclobutane (F6), exhibit completely different anesthetic properties. Whereas the former is a potent general anesthetic, the latter produces no anesthesia. Two linear compounds, isoflurane and 2,3-dichlorooctofluorobutane (F8), although not a structural pair, also show the same anesthetic discrepancy. Using 19F nuclear magnetic spectroscopy, we investigated the time-averaged submolecular distribution of these compounds in a vesicle suspension of phosphatidylcholine lipids. A two-site exchange model was used to interpret the observed changes in resonance frequencies as a function of the solubilization of these compounds in membrane and in water. At clinically relevant concentrations, the anesthetics F3 and isoflurane distributed preferentially to regions of the membrane that permit easy contact with water. The frequency changes of these two anesthetics can be well characterized by the two-site exchange model. In contrast, the nonanesthetics F6 and F8 solubilized deeply into the lipid core, and their frequency change significantly deviated from the prediction of the model. It is concluded that although anesthetics and nonanesthetics may show similar hydrophobicity in bulk solvents such as olive oil, their distributions in various regions in biomembranes, and hence their effective concentrations at different submolecular sites, may differ significantly.

Anesthetics↗
PubMed1997

Transcription initiation sites for sorghum mitochondrial atp9 are positioned immediately 3' to trnfM.

Sorghum mitochondrial atp9 is polymorphic among male-sterile cytoplasms, but each cytoplasm is characterized by a major 650 nt transcript, regardless of fertility status. The gene is positioned 323 bp 3' to trnfM. Primer extension revealed multiple atp9 5' transcript termini, distributed from +1 to +28 3' to trnfM; the termini could be labeled with polynucleotide kinase, suggesting that they result from the maturation of trnfM. Guanylyltransferase experiments, however, showed that four of the termini were capable. The juxtaposition of a putative promoter 3' to trnfM results in a unique atp9 transcript population consisting of primary and processed transcripts.

Amino Acid Sequence↗
PubMed1997

Circular dichroism studies in conformation of cellobiohydrolase and endoglucanase from Trichoderma pseudokiningii S-38: effects of pH and ligand binding.

Effects of pH and ligand binding upon the conformation of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from Trichoderma pseudokiningii S-38 have been studied by circular dichroism measurements. In the high-pH range (6-9), increasing pH resulted in a similar conformational change occurring in free CBHI and EGI, while such treatment gave different changes of the two enzyme conformations in the presence of cellobiose. On the other hand, in the low-pH region, with both CBHI an EGI in the active form, decreasing pH resulted in a large conformational change of free EGI compared to that of free CBHI, whereas ligand binding resulted in a similar change of both CBHI and EGI, independent of pH change.

Cellobiose↗
PubMed1997

Three-dimensional model of sensory rhodopsin I reveals important restraints between the protein and the chromophore.

A structural model is constructed for the integral membrane protein, sensory rhodopsin I (SRI), the phototaxis receptor of the archaeon Halobacterium salinarium. The model is built on the template of the homologous bacteriorhodopsin (BR). The modeling procedure includes sequence alignment, a side chain rotamer search and simulated annealing by restricted molecular dynamics. The structure is in general agreement with previous results from mutagenesis experiments, chromophore substitution and room and cryogenic temperature spectroscopy. In particular, a residue near the beta-ionone ring of the retinylidene chromophore is found to be critical in maintaining the proper isomeric conformation of the chromophore; a layer of residues lying on the cytoplasmic side of the chromophore pocket is found to modulate the restraints around the C13 region of the chromophore, affecting the isomerizations around its 13 = 14 bond that are important to the protein's activity. The restraints in these regions are more stringent in SRI than in BR. The tightened restraints are chiefly due to van der Waals interactions, where the attractive and repulsive components play separable roles. Aromatic residues account for a majority of the restrictive interactions. It is hypothesized that the enhanced barriers due to these restrictions regulate the progress of SRI's photocycle, so that it can couple with the phototaxis reaction chain in the bacterium. A possibility is also suggested that conformational changes of the protein provide the signal recognized by the transducer.

Amino Acid Sequence↗
PubMed1997

Beta-adrenoceptor activation-induced placental prorenin secretion is mediated by increased renin messenger RNA and protein synthesis.

Activation of beta-adrenoceptors has been shown to promote renin secretion in both human kidney and placenta. In kidney, the enhanced secretion is immediately observed, and mobilization of renin in the storage granules accounts for such a rapid response. In contrast, the enhanced secretion in placenta is delayed for 6-12 hr after receptor activation and consists almost entirely of the renin precursor prorenin. It is hypothesized that newly synthesized rather than stored enzyme is responsible for the enhanced secretion in human placenta. To test this hypothesis, placental explants were cultured in the presence or absence of the protein synthesis inhibitor cycloheximide, and prorenin concentrations in the tissue and medium were measured. Dobutamine and terbutaline, beta1- and beta2-adrenoceptor agonists, evoked 17- and 5-fold increases in secretion, respectively. Tissue content of prorenin in response to the treatment was increased by a similar magnitude, yet values were consistently <10% of medium concentrations. The increases in prorenin concentrations in both medium and tissue, however, were markedly attenuated by cycloheximide, suggesting that prorenin synthesis in response to beta-adrenoceptor activation is required. Reverse transcription coupled with polymerase chain reaction revealed that renin mRNA levels were increased by 3-8-fold and occurred before increases in tissue and medium prorenin, indicating that increased renin mRNA levels are responsible for the increased synthesis of prorenin. Explants cultured in the presence of actinomycin D, an inhibitor of transcription, did not show the agonist-induced prorenin mRNA levels or enhancement of its secretion. The peak levels of renin mRNA were reached after 6 hr of incubation, were sustained at similar levels after 24 hr, and were not affected by cycloheximide. These findings are consistent with the notion that enhancement of renin mRNA and de novo protein synthesis are required for prorenin secretion induced by activation of placental beta-adrenoceptors.

Adrenergic beta-Agonists↗
PubMed1997

[Construction of mycobacterium tuberculosis genomic library].

OBJECTIVE: To provide effective tools for identification and characterization of M. tuberculosis genes/antigenes and to evaluate their roles in diagnosis, vaccination, drug resistance, and pathogenesis. METHODS: M. tuberculosis genomic DNA obtained from H 37 Ra strain was partially digested with DNase I. The DNA fragments ranging from 4-8 kb were isolated from agarose gel and ligated to EcoR I adaptor, and the products were linked to lamda gt11 arms and packaged using an in vitro packaging extract. The different diluted bacteriophages were used to transfect bacteria Y1090. RESULTS: The efficiency and titer of the library were 85% and 3 x 10(5) pfu/ml, respectively. The library contained 1.3 x 10(5) individual recombinant phage whose foreign DNA inserted fragment size was 3.5 kb on average. CONCLUSION: The genomic DNA library constructed here can provide sufficient clone to cover H37Ra gene.

Genome, Bacterial↗
PubMed1996

Solid-state 13C-NMR spectroscopy of adduction products of 2,5-hexanedione with ribonuclease, albumin, and rat neurofilament protein.

The Paal-Knorr condensation reaction between the gamma-diketone 2,5-hexanedione (2,5-HD) and epsilon-amine moieties of proteins of various molecular weight, including ribonuclease (RNase), bovine serum albumin (BSA) and rat neurofilament (NF), has been investigated by solid-state 13C-NMR spectroscopy. These proteins all reacted with 2,5-HD with the formation of 2,5-dimethylpyrrole (2,5-DMP) derivatives. The size and complexity of the protein affected the rate of formation of 2,5-DMP derivatives. Using the selective reducing reagent NaCNBH3, the Paal-Knorr reaction intermediates were trapped by conversion into amines, which were identified by solid-state NMR spectroscopy. The secondary autoxidation reaction following the formation of 2,5-DMP derivatives was also studied by solid-state NMR spectroscopy.

Animals↗
PubMed1996

Sorghum mitochondrial orf25 and a related chimeric configuration of a male-sterile cytoplasm.

We describe fundamental characteristics of sorghum mitochondrial orf25, urf209, and a related chimeric configuration, orf265/130, which is restricted to the IS1112C source of cytoplasmic male sterility in sorghum. Transcripts of urf209 are edited at ten nucleotides, resulting in nine amino-acid changes predicted from genomic sequences. The cDNA-predicted polypeptide product is 23.6 kDa, while Western blot analyses identify a product of 20k Da. Transcription of urf209 is characterized by one or two transcripts, dependent on nuclear background, but this difference is not related to male fertility status. The orf265/130 chimeric region includes 288 bp 95% identical to sequences 5' to maize T-cytoplasm T-urf13 and atp6, which includes a common transcription initiation site, and terminates with a recombinational event involving urf209. The urf209 similarity extends 189 bp, followed by sequences duplicated 5' to sorghum atp6-2. Sequences immediately 3' to the atp6-2 similarity include a second in-frame start codon, defining orf130. Structural features 5' to orf130 are shared with motifs found 5' to several translated mitochondrial open reading frames. The orf265/orf130 configuration is uniquely transcribed, and transcripts of orf130 exhibit one silent RNA editing event. Transcription in somatic cells is not altered by male fertility status.

Amino Acid Sequence↗
PubMed1996

Solid 13C CPMAS NMR spectroscopy studies of biosynthesis in whole cells of Methanosphaera stadtmanae.

Whole cells of Methanosphaera stadtmanae were grown in media containing [13C]CO2, [2-13C]acetate, [1-13C]acetate, [3-13C]serine and [1-13C]formate. The label incorporation was determined using solid state 13C CPMAS NMR spectroscopy. The incorporation of serine hydroxymethyl carbon into the purine rings of nucleic acids and most probably the methyl group of thymine is demonstrated. The one carbon atom pathway shown in our previous work is operative in the biosynthesis of purines and pyrimidines. In addition, these studies clearly identified signals not observed in solution NMR spectroscopy and revealed an important pathway not previously known. The reversibility of formyl-containing one carbon atom carriers is demonstrated. The pattern of labeled carbon atoms in sugars confirms the biosynthetic route from pyruvate, which is formed from acetic acid and carbon dioxide. Finally, a preliminary lipid assignment is indicated. The solid state 13C CPMAS NMR of these intact cells proved to be a facile method to follow specific pathways.

Acetic Acid↗
PubMed1996

Transcript processing internal to a mitochondrial open reading frame is correlated with fertility restoration in male-sterile sorghum.

A chimeric mitochondrial DNA (mtDNA) configuration of the cytoplasmic male-sterile (cms) sorghum line IS1112C includes a 321 bp open reading frame designated orf107, encoding a predicted polypeptide product of 11.85 kDa. The open reading frame, similar to several other genes associated with cms, consists of amino-terminal sequences derived from an obligate gene. Unlike other examples to date, however, the carboxy-terminal sequences are highly similar to the carboxy terminus of an open reading frame implicated in cms of rice, orf79. The amino-terminal 31 residues of orf107 are 84% similar to atp9, and the carboxy-terminal 49 residues are 57% identical and 80% similar to the carboxy terminus of orf79. Transcripts of orf107 are edited, with four C-to-U changes that alter amino acids. Sorghum lines partially or fully restored to fertility exhibit a high-efficiency internal-orf107 transcript processing activity, precluding abundant whole-length transcripts, while male-sterile lines exhibit only a trace of the activity. Previous data on the abundance of a 12kDa in organello-synthesized polypeptide in male-sterile versus male-fertile lines are correlated with differential orf107 transcript processing activity of these lines. Examinations of backcross and F2 lines suggest a gametophytic mode of restoration, and indicate that enhanced transcript processing activity is necessary, but not sufficient, to restore full fertility. These novel observations indicate that mitochondrial open reading frames associated with cms in different species can include highly similar motifs, and that fertility restoration could involve a mechanism by which synthesis of a cms-associated gene product may be precluded through internal transcript cleavage.

Amino Acid Sequence↗