Search PubMed⌕ Search

Biomedical subjects

J Gao

Publications and source records attributed to J Gao.

At least 127 records · Page 7Linked to original sources
PubMed2000

Determination of morphine by capillary electrophoresis immunoassay in thermally reversible hydrogel-modified buffer and laser-induced fluorescence detection.

In this paper thermally reversible hydrogel used as a replaceable packed material for capillary electrophoresis was examined. A simple and rapid method of detecting morphine was developed, which demonstrated the potential of strong affinity antibodies as a selector for immunologically-based separations in serum by capillary electrophoresis. Polyclonal antibodies were linked to hydrogel and applied to the separation of free fluorescein isothiocyanate (FITC)-labeled antigen and bound FITC antigen. The separation was monitored with laser-induced fluorescence detection. Different separation conditions were studied. The results indicated that poly-N-isopropylacrylamide hydrogel (PNIPA) is a kind of steady, replaceable gel. The specific determination of morphine did not require a long incubation time and PNIPA hydrogel-modified antibodies can be stockpiled at 4 degrees C before assay. It can be used to determine morphine with good precision and a detection limit lower than 8.5 ng/ml. Details of the preparation of hydrogel cross-linked polyclonal antibody and of typical separations of bound and free antigen are presented.

Antibody Specificity↗
PubMed2000

A new lectin with highly potent antihepatoma and antisarcoma activities from the oyster mushroom Pleurotus ostreatus.

A dimeric lectin, composed of subunits with a molecular weight of 40 and 41 kDa, respectively, and demonstrating similarity in N-terminal sequence to each other and to Aleuria aurantia lectin, was isolated from fresh fruiting bodies of the edible mushroom Pleurotus ostreatus. The lectin was unadsorbed on DEAE-cellulose in 10 mmol/L phosphate buffer (pH 7.5), subsequently adsorbed on CM-cellulose in 10 mmol/L NH(4)OAc (pH 5.4), and came off in the first peak from a Superose 12 column during fast protein liquid chromatography. The lectin was acid-labile, alkali-labile, and heat-labile. Its hemagglutinating activity was sensitive to inhibition by CaCl(2), MgCl(2), MnCL(2) and FeCl(3) and potentiation by AlCl(3). Melibiose, lactose, d-galactose, alpha-methyl-d-galactopyranoside, N-acetylneuraminic acid, raffinose, and inulin were capable of inhibiting its hemagglutinating activity, with melibiose being the most potent. The lectin exerted potent antitumor activity in mice bearing sarcoma S-180 and hepatoma H-22. Survival in these mice was prolonged and body weight increase reduced after lectin treatment.

Amino Acid Sequence↗
PubMed2000

Cloning and characterization of human DDX24 and mouse Ddx24, two novel putative DEAD-Box proteins, and mapping DDX24 to human chromosome 14q32.

DEAD-box proteins are a large group of putative RNA helicases that exist ubiquitously in organisms ranging from bacteria to humans. They are likely to play important roles in many different RNA metabolic processes. In this paper, we report the cloning of human DDX24, a putative DEAD-box protein, and its ortholog, Ddx24 in mouse. The deduced proteins encoded by these two cDNAs share 78.7% identity at the amino acid level and possess all the well-conserved motifs of DEAD-box proteins. However, little homology can be found between them and other DEAD-box proteins, even in their core region (identity <40%). Northern blot analysis showed that a 3.0-kb transcript of human DDX24 exists ubiquitously in the 16 human tissues examined and was most abundant in heart and brain, but with lowest levels in thymus and small intestine. The mouse Ddx24, whose transcript is 4.0 kb, was also expressed widely in 10 tissues tested with the highest level in heart and testis. By radiation hybrid mapping, the human DDX24 gene was localized to human chromosome 14q32 between the markers D14S81 and D14S265. Moreover, the gene structure of DDX24 was determined by comparing its cDNA and genomic sequence from BAC R-1089B7, which showed that the gene spanned a 30-kb region and consisted of at least nine exons.

Amino Acid Sequence↗
PubMed2000

Cloning and mapping of human PKIB and PKIG, and comparison of tissue expression patterns of three members of the protein kinase inhibitor family, including PKIA.

Two novel members of the human cAMP-dependent protein kinase inhibitor (PKI) gene family, PKIB and PKIG, were cloned. The deduced proteins showed 70% and 90% identity with mouse PKIbeta and PKIgamma respectively. Both the already identified pseudosubstrate site and leucine-rich nuclear export signal motifs were defined from the 11 PKIs of different species. The PKIB and PKIG genes were mapped respectively to chromosome 6q21-22.1, using a radiation hybrid GB4 panel, and to chromosome 20q13.12-13.13, using a Stanford G3 panel. Northern-blot analysis of three PKI isoforms, including the PKIA identified previously, revealed significant differences in their expression patterns. PKIB had two transcripts of 1.9 kb and 1.4 kb. The former transcript was abundant in both placenta and brain and the latter was expressed most abundantly in placenta, highly in brain, heart, liver, pancreas, moderately in kidney, skeletal muscle and colon, and very little in the other eight tissues tested. PKIG was widely expressed as a 1.5-kb transcript with the highest level in heart, hardly detectable in thymus and peripheral blood leucocytes and was moderately expressed in the other tissues, with slightly different levels. However, PKIA was specifically expressed as two transcripts of 3.3 kb and 1.5 kb in heart and skeletal muscle. The distinct expression patterns of the three PKIs suggest that their roles in various tissues are probably different.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Anilides of (R)-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

The optimization of a series of anilide derivatives of (R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase (PDHK) is described that started from N-phenyl-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide 1 (IC(50) = 35 +/- 1.4 microM). It was found that small electron-withdrawing groups on the ortho position of the anilide, i.e., chloro, acetyl, or bromo, increased potency 20-40-fold. The oral bioavailability of the compounds in this series is optimal (as measured by AUC) when the anilide is substituted at the 4-position with an electron-withdrawing group (i.e., carboxyl, carboxyamide, and sulfoxyamide). N-(2-Chloro-4-isobutylsulfamoylphenyl)-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropionamide (10a) inhibits PDHK in the primary enzymatic assay with an IC(50) of 13 +/- 1.5 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts, lowers blood lactate levels significantly 2.5 and 5 h after oral doses as low as 30 micromol/kg, and increases the ex vivo activity of PDH in muscle, kidney, liver, and heart tissues. However, in contrast to sodium dichloroacetate (DCA), these PDHK inhibitors did not lower blood glucose levels. Nevertheless, they are effective at increasing the utilization and disposal of lactate and could be of utility to ameliorate conditions of inappropriate blood lactate elevation.

Anilides↗
PubMed2000

[Contrast test using three kinds of air-microorganism samplers LWC-I, CA6 and CA2 in public places].

A sampling experiment was conducted using three kinds of air-samplers, i.e. LWC-1, CA6 and CA2, on bacteria and fungus in the air under the same conditions and at the same time yet in three different public places. The results showed that there was no significant difference in the total number of bacteria and fungus colony forming units in the air in different public places using the above mentioned three air-samplers. In comparison, the LWC-1 air-sampler demonstrated a feature of small size, light weight, simple structure, and little noise, therefore, it was easy in carrying and requires no external power source. Such being the case, the LWC-1 air-sampler was more suitable for monitoring microorganism in the air in public places.

Air Microbiology↗
PubMed2000

Effects of the renin-angiotensin system on the current I(to) in epicardial and endocardial ventricular myocytes from the canine heart.

The Ca(2+)-independent portion of transient outward K(+) current (I(to)) exhibits a transmural gradient in ventricle. To investigate control mechanisms for this gradient, we studied canine epicardial and endocardial ventricular myocytes with use of the whole-cell patch-clamp technique. I(to) was larger in amplitude, had a more negative voltage threshold for activation, and had a more negative midpoint of inactivation in epicardium. Recovery from inactivation was >10-fold slower in endocardium. Incubation of epicardial myocytes with angiotensin II for 2 to 52 hours altered I(to) to resemble unincubated endocardium and reduced the amplitude of the phase 1 notch of the action potential. In contrast, incubation of endocardial myocytes with losartan for 2 to 52 hours altered I(to) to resemble unincubated epicardium and induced a phase 1 notch in the action potential. With RNase protection assays, we determined that incubations with angiotensin II or losartan did not alter mRNA levels for either Kv4.3 or Kv1.4; thus, a change in the alpha subunit for I(to) is unlikely to be responsible. To test whether posttranslational modification produced the effects of angiotensin II, we coexpressed Kv4.3 and the angiotensin II type 1a receptor in Xenopus oocytes. Incubation with angiotensin II increased the time constant for recovery from inactivation of the expressed current by 2-fold with an incubation time constant of 3.7 hours. No effect on activation or inactivation voltage dependence was observed. These results demonstrate that the properties of I(to) in endocardium and epicardium are plastic and likely under the tonic-differing influence of the renin-angiotensin system.

Action Potentials↗
PubMed2000

Eden et al. reply:

Explore the source record for details and available documents.

Journal Article↗
PubMed2000

New C=C bond formation via nonstoichiometric titanium(IV) halide mediated vicinal difunctionalization of alpha,beta-unsaturated acyclic ketones

[reaction: see text] Highly stereoselective vicinal difuctionalization of alpha,beta-unsaturated ketones for the synthesis of multifunctionalized trisubstituted alkenes is described. The new reaction employs titanium(IV) halides (0.5 equiv) as promoters and inexpensive commercial chemicals as starting materials. The reaction can be performed at room temperature in a convenient vial without the protection of inert gases. Good to excellent yields and high Z/E stereoselectivity have been realized in most cases presented (16 examples).

Journal Article↗
PubMed2000

Electrostatic stress in catalysis: structure and mechanism of the enzyme orotidine monophosphate decarboxylase.

Orotidine 5'-monophosphate decarboxylase catalyzes the conversion of orotidine 5'-monophosphate to uridine 5'-monophosphate, the last step in biosynthesis of pyrimidine nucleotides. As part of a Structural Genomics Initiative, the crystal structures of the ligand-free and the6-azauridine 5'-monophosphate-complexed forms have been determined at 1.8 and 1.5 A, respectively. The protein assumes a TIM-barrel fold with one side of the barrel closed off and the other side binding the inhibitor. A unique array of alternating charges (Lys-Asp-Lys-Asp) in the active site prompted us to apply quantum mechanical and molecular dynamics calculations to analyze the relative contributions of ground state destabilization and transition state stabilization to catalysis. The remarkable catalytic power of orotidine 5'-monophosphate decarboxylase is almost exclusively achieved via destabilization of the reactive part of the substrate, which is compensated for by strong binding of the phosphate and ribose groups. The computational results are consistent with a catalytic mechanism that is characterized by Jencks's Circe effect.

Amino Acid Sequence↗
PubMed2000

Amido-(propyl and allyl)-hydroxybenzamidines: development of achiral inhibitors of factor Xa.

The design, synthesis and SAR of amido-(propyl and allyl)-hydroxybenzamidine coagulation factor Xa inhibitors is described. These achiral inhibitors are selective for fXa vis a vis structurally related serine proteases and are readily prepared in 6-7 linear steps. The most potent member 9j (fXa Ki = 0.75 nM) is selective (>1000-fold) and an effective anticoagulant in mammalian plasma.

Animals↗
PubMed2000

Secondary amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid as inhibitors of pyruvate dehydrogenase kinase.

N'-methyl-N-(4-tert-butyl-1,2,5,6-tetrahydropyridine)thiourea, SDZ048-619 (1), is a modest inhibitor (IC(50) = 180 microM) of pyruvate dehydrogenase kinase (PDHK). In an optimization of the N-methylcarbothioamide moiety of 1, it was discovered that amides with a small acyl group, in particular appropriately substituted amides of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid, are inhibitors of PDHK. Utilizing this acyl moiety, herein is reported the rationale leading to the optimization of a series of acylated piperazine derivatives. Methyl substitution of the piperazine at the 2- and 5-positions (with S and R absolute stereochemistry) markedly increased the potency of the lead compound (>1,000-fold). Oral bioavailability of the compounds in this series is good and is optimal (as measured by AUC) when the 4-position of the piperazine is substituted with an electron-poor benzoyl moiety. (+)-1-N-[2,5-(S, R)-Dimethyl-4-N-(4-cyanobenzoyl)piperazine]-(R)-3,3, 3-trifluoro-2-hydroxy-2-methylpropanamide (14e) inhibits PDHK in the primary enzymatic assay with an IC(50) of 16 +/- 2 nM, enhances the oxidation of [(14)C]lactate into (14)CO(2) in human fibroblasts with an EC(50) of 57 +/- 13 nM, diminishes lactate significantly 2.5 h post-oral-dose at doses as low as 1 micromol/kg, and increases the ex vivo activity of PDH in muscle, liver, and fat tissues in normal Sprague-Dawley rats. These PDHK inhibitors, however, do not lower glucose in diabetic animal models.

Amides↗
PubMed2000

Partial characterization of the CCAAT box in the promoter of the hLGFBP-1 gene: interaction with negatively acting transcription factors in decidualized human endometrial stromal cells.

The CCAAT cis-element and its adjacent DNA sequence (-82 to -52 bp) in the human insulin-like growth factor binding protein-1 gene (IGFBP-1) promoter are active in both decidualized human endometrial stromal cells and HepG2 cells. In HepG2 cells, CCAAT activity is mediated by interacting with hepatocyte nuclear factor, HNF-1. In endometrial cells, this region is protected by the nuclear extracts of endometrial decidual cells, however, the transactivator which interacts with the region has not been identified. This study was carried out to characterize and identify the stromal/decidual nuclear proteins that interact with the IGFBP-1 CCAAT motif. Gel shift analysis showed that the CCAAT motif (-82 to -52 bp) formed three specific complexes (CI, CII, and CIII) by extracts from human endometrial decidual or stromal cells. The intensity of CIII formed by the nuclear extracts of decidual cells was less compared to that formed by stromal cells whereas CI/CII was found to be opposite. To evaluate the transcription factors that bind to this region, a number of known CCAAT binding proteins were tested. Among them, the CCAAT binding proteins NF-Y (alpha2(1) collagen promoter CCAAT binding protein) and CBF (hsp70 promoter CCAAT binding protein), were characterized by the gel shift assay. The NF-Y consensus binding sequence (the alpha2(1) collagen promoter) and NF-YA,B antibody abolished or shifted CIII. Although the CBF consensus binding sequence (the hsp70 promoter) eliminated all three complexes, the antibody to CBF had no effect on all three complexes. The nuclear extracts of the endometrial stromal/decidual cells did not form a band corresponding to the HNF-1/CCAAT complex. These results indicate that the CCAAT motif binds to NF-Y and the CI/CII binding protein (remains to be identified) but not HNF-1 in endometrium. Systematic mutation in the CCAAT motif showed that NF-Y(CIII binding protein) bound to the 12 bp sequence GGCGCTGCCAAT(-79 to -68 bp) and the CI/CII binding protein bound to 9 bp, TGCCAATCA(-74 to -66 bp). These findings indicate that the CCAAT motif is a composite element. The CCAAT mediated function was analyzed in decidualized endometrial stromal cells. Mutations in the CCAAT motif increased the promoter activity. The maximum activity was found in mutants which abolished the NF-Y complex. The CCAAT core sequence mutants in which both CIII and CI/CII were abolished, also increased the promoter activity. Results indicated that NF-Y and the CI/CII binding protein, yet to be identified, interact with the composite CCAAT element in the IGFBP-1 promoter to repress the promoter activity in endometrial decidual cells.

Base Sequence↗
PubMed2000

Stimulation of adipose differentiation related protein (ADRP) expression in adipocyte precursors by long-chain fatty acids.

Adipose differentiation related protein (ADRP) is a 50-kDa protein expressed in adipocytes and transcriptionally activated when adipocyte precursors differentiate into mature adipocytes. Recent experiments have demonstrated that ADRP is a fatty acid binding protein that specifically facilitates the uptake of long-chain fatty acids. The present investigation provides evidence that ADRP mRNA and protein expression in preadipocytes is stimulated by fatty acids in a time- and dose-dependent fashion. ADRP mRNA expression was maximally stimulated at fatty acid concentrations of or above 10(-5) M. Stimulation of ADRP expression was observed with the nonmetabolizable fatty acid 2-bromopalmitate and with natural fatty acids. Stimulation of ADRP mRNA expression by fatty acids peaked between 5 and 8 hr and decreased by 24 hr. Stimulation of ADRP expression by fatty acids was completely inhibited by treatment with actinomycin D, suggesting that fatty acid stimulates ADRP gene expression at the transcriptional level. Comparison of the effect of several fatty acids with varying carbon chain lengths indicated that long-chain fatty acids were active in stimulating ADRP, whereas short-chain fatty acids such as caproate and 2-bromooctanoate had no effect. The degree of saturation of fatty acids did not influence their ability to stimulate ADRP expression. These studies provide new information on the regulation of ADRP and identify a new target regulated by fatty acids during adipose differentiation.

Adipocytes↗
PubMed2000

Immunolocalization of types I, II, and X collagen in the tibial insertion sites of the medial meniscus.

The medial meniscus of the rabbit knee joint attaches to the tibial plateau via anterior and posterior insertions. Intact meniscal tibial insertions are essential for meniscal function. In the present study the distributions of types I, II, and X collagen in meniscal tibial insertions were investigated by indirect immunohistochemistry in a rabbit model. Four tissue zones were histologically identified in the anterior insertion site, including the ligamentous zone, uncalcified and calcified fibrocartilaginous zones and bone; the ligamentous zone was not observed in the posterior insertion site. Labeling for type I collagen was found to be strong in the ligament tissue and bone, and weak in the fibrocartilages which were also labeled for type II collagen. Tissues positive for different types of collagen overlapped and formed an irregular interface with various angles and depths, especially at the interface between the calcified fibrocartilage and bone. Positive labeling for type X collagen was identified only in the calcified fibrocartilage zone. The coexistence of types I and II collagen in the meniscal tibial insertions may indicate that this structural unit is subjected to both compressive and tensile loads. Type X collagen may play a role in maintaining the calcifying status of this tissue zone, so that its mechanical stiffness is kept between that of uncalcified fibrocartilage and hard bone. Restoration of the insertional structure including the distinct collagen distribution should be considered for a functional meniscal substitution.

Animals↗
PubMed2000

Cochlear findings in the white spotting (Ws) rat.

White spotting (Ws) rats possess a c-kit gene mutation at the W locus, resulting in a variety of characteristics including a lack of intermediate cells of the stria vascularis. The present study employs a light microscope (LM), scanning (SEM) and transmission electron microscopes (TEM), diaminobenzidine (DAB) staining techniques and auditory brainstem response (ABR) to investigate the structure and function of the cochlea in 26 homozygous Ws/Ws rats aged 1-6 months. A slight thinning of the stria vascularis and moderate elevation of ABR threshold were about the only defects noted in 1 month animals, while older animals displayed various defects that tended to worsen with age. At 3 months LM revealed pigment granules in the basal turn of most animals, with a loss of pigmentation in the upper turns. The stria vascularis and organ of Corti tended to be well preserved in the lower, pigmented portion, while the upper, unpigmented portion showed severe strial degeneration and some outer hair cell loss. DAB staining revealed a well developed strial capillary net throughout the pigmented portion of the cochlea, with severe degradation in the unpigmented apical portion. ABR thresholds were slightly elevated over 1 month values. At 6 months great differences in degeneration were noted between right and left ears of the same animal.

3,3'-Diaminobenzidine↗
PubMed2000

An investigation of the impulse functions for the nonlinear BOLD response in functional MRI.

Functional MRI (fMRI) based on blood oxygenation level dependent (BOLD) contrast can be used to detect hemodynamic responses to a broad range of stimuli. It however remains unclear in what fashion the BOLD response is a linear system, and how the impulse function differs with stimulation of varying duration. To address this question, fMRI using visual stimulation with a wide range of duration (0.5-12 s) was performed in six human volunteers. A strong linear correlation was shown on the full width at half maximum (r = 0.998) of the BOLD response curves and the area under the curves (r = 0.999) to the duration of stimulation. However, comparing the errors of the measured and predicted response curves, our results showed a poorer linearity at stimuli of shorter duration. By examining the impulse functions derived from different stimuli, based on the assumption that a linear convolution relationship existed, a higher differentiation was shown in the experiments with shorter stimuli (<3 s). Compared to the area under the impulse function derived from 12 s stimulation, with that obtained from 0.5, 1, 2, 3, 4, 8 s stimuli resulted in differences of 66.2, 33.5, 15.1, 5.4, 0.9, 7.9%, respectively. This study suggests a higher degree of nonlinearity in the BOLD signal changes due to stimuli of shorter duration, in agreement with earlier work.

Brain↗