Biomedical subjects

J Gao

Publications and source records attributed to J Gao.

At least 253 records · Page 14Linked to original sources

PubMed1998

Surface hydrolysis of poly(glycolic acid) meshes increases the seeding density of vascular smooth muscle cells.

A procedure for surface hydrolysis of poly(glycolic acid) (PGA) meshes was developed to increase cell seeding density and improve attachment of vascular smooth muscle cells. Hydrolysis of PGA in 1N NaOH transformed ester groups on the surface of PGA fibers to carboxylic acid and hydroxyl groups. After hydrolysis, the polymer scaffold retained its original gross appearance and dimensions while the fiber diameter decreased. A plot of fiber diameter versus the hydrolysis time showed a linear relationship, with a rate of decrease in fiber diameter of 0.65 microm/min. The molecular weight and thermal properties of the polymer did not change significantly following surface hydrolysis. In cell seeding experiments, surface-hydrolyzed mesh was seeded with more than twice as many cells as unmodified PGA mesh. Vascular smooth muscle cells attached to the surface-hydrolyzed PGA mesh both as individual cells and as cell aggregates while only cell aggregates were observed on the unmodified mesh. Control experiments indicated that adsorption of serum proteins onto the surface-hydrolyzed PGA fibers was correlated with the increase in cell seeding density. These results demonstrate that optimization of biomaterial-cell interactions provides a strategy for increasing the initial cell seeding density for the engineering of tissues of high cell density.

Animals↗

PubMed1998

Specific local histone-DNA sequence contacts facilitate high-affinity, non-cooperative nucleosome binding of both adf-1 and GAGA factor.

Sequence-specific transcription factors need to gain access to regulatory sequences in chromatin. Previous studies utilizing model systems have suggested many mechanisms involved in this process. It is unclear however how these findings relate to natural promoters. The Drosophila alcohol dehydrogenase ( Adh ) gene distal promoter is organized into an ordered nucleosome array before multiple transcription factors recognize their sites within this nucleosomal context and activate transcription. Here we used a purified in vitro system to study the binding of the ubiquitous Drosophila transcription factors Adf-1 and GAGA factor to the Adh distal promoter in chromatin. Several nucleosome core particles were assembled on 150 bp DNA fragments containing the Adh distal cis -acting elements in the natural promoter context but different DNA-histone environments. We found that the Adh distal promoter regulatory sequences can position nucleosomes in the same rotational setting as observed in vivo. In one particular nucleosome position, the wrapping of the Adf-1 and adjacent GAGA factor binding sitesaround the histone octamer creates a unique local DNA conformation. High-affinity but non-cooperative nucleosome binding of Adf-1 and GAGA factortherefore occurs, in contrast to the inhibition of Adf-1 and GAGA factor binding in other nucleosome positions. Thus, local histone-DNA sequence contact giving rise to a specific asymmetric nucleosome structure may play important roles in modulating the affinities of transcription factors for their nucleosomal sites.

Alcohol Dehydrogenase↗

PubMed1998

Decrease in Ca-ATPase activity in aged synaptosomal membranes is not associated with changes in fatty acyl chain dynamics.

We have examined lipid peroxidation (LPO) and fatty acid acyl chain dynamics in synaptosomal membranes isolated from aged rat (Fischer 344 x Brown Norway F1 hybrids) brains, correlating these results with measurements of enzymatic activity of the synaptic plasma membrane Ca2(+)-ATPase (PMCA). Calcium-dependent ATPase activity in these membranes exhibits progressive decreases with a maximal loss of activity with age of approximately 35%. The sensitivity of this membrane-bound ion transporter to the lipid composition of the surrounding membrane, as well as the high abundance of oxidatively sensitive polyunsaturated fatty acyl chains in synaptosomal membranes, suggests that this age-related loss in catalytic turnover may result from LPO-mediated protein modification and/or changes in the physical structure of the bilayer. However, high-performance liquid chromatography analysis of 2,4-dinitrophenylhydrazone derivatives reveals no significant age-related increases in the content of reactive aldehydes (malondialdehyde, formaldehyde, acetaldehyde or acetone) which comprise breakdown products of lipid peroxidation. Electron paramagnetic resonance measurements employing 5- and 12-stearic acid spin labels with the nitroxide reporter groups at two depths in the bilayer were used to assess the fatty acyl chain dynamics (fluidity) of synaptosomal membranes. The resulting spectra demonstrate anisotropic lipid dynamics of two populations of lipids, i.e. lipids in direct association with membrane proteins (boundary lipids) and bulk lipids that do not directly associate with proteins. The nanosecond dynamics of both lipid populations is unaltered with age indicating that any compositional changes occurring with age are insufficient to result in alterations in bilayer fluidity relevant to PMCA activity. Thus, the observed age-related decline in PMCA activity may be explained by direct modification of membrane protein.

Aging↗

PubMed1998

Substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones as hypoglycemic agents.

A series of substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones was synthesized from amino alcohols or amino thiols and keto acids. A pharmacological model based on the results obtained with these compounds led to the synthesis and evaluation of a series of isoxazoles and other monocyclic compounds. These were evaluated for their ability to enhance glucose utilization in cultured L6 myocytes. The in vivo hypoglycemic efficacy and potency of these compounds were evaluated in a model of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), the ob/ob mouse. 25a(2S) (SDZ PGU 693) was selected for further pharmacological studies.

Animals↗

PubMed1998

Comparison of carcinoembryonic antigen promoter regions isolated from human colorectal carcinoma and normal adjacent mucosa to induce strong tumor-selective gene expression.

To establish in vivo gene therapy against cancer, it is requisite to induce strong, cancer cell-selective expression of a therapeutic gene. Comparison of the promoter activity of 5' flanking regions of the carcinoembryonic antigen (CEA) gene isolated from various origins is therefore of considerable interest. The 5' flanking region of the CEA gene between -135 and +69 bp upstream from the transcriptional start site, which is recognized as the core promoter region, was isolated from CEA-producing human colorectal carcinoma (CRC), normal adjacent mucosa, CEA-producing cell lines and CEA-non-producing cell lines. No mutations were observed by single-strand conformation polymorphism in the CEA promoter regions. Subsequent sequence analysis revealed that there were no mutations in the CEA promoter regions isolated from CEA-producing CRC and normal adjacent mucosa. Furthermore, nuclear extracts prepared from CEA-producing human CRC cells could equally bind to both the CEA promoter fragments isolated from CEA-producing CRC and normal mucosa. Both CEA promoter regions could direct 5- to 20-fold higher expression of a luciferase reporter gene in CEA-producing cells than in CEA-non-producing cells. Therefore, we suggest that the use of either CEA promoter region isolated from CRC or normal mucosa is equally effective to induce strong, CEA-producing cancer-selective expression of a therapeutic gene.

Base Sequence↗

PubMed1998

Development of an androgen receptor-null model for identifying the initiation site for androgen stimulation of proliferation and suppression of programmed (apoptotic) death of PC-82 human prostate cancer cells.

Whether androgen regulates the proliferation and survival of androgen-responsive prostate cancer cells directly or indirectly via a paracrine pathway initiated in androgen receptor (AR)-expressing stromal cells is unknown. To resolve this issue, female mice heterozygous for the testicular feminized male loss of function mutation in their X-linked AR genes were cross-bred to T cell-defective homozygous male nude mice. Using a PCR-based restriction enzyme digestion method, the resulting AR/tfm, Nu/nu F1 hybrid females were identified and back-crossed to homozygous male nude mice to produce AR-null male nude mice lacking both AR and T-cell function. Androgen-responsive PC-82 human prostate cancers were xenografted into these AR-null versus AR-wild-type male nude mice. In both backgrounds, the cancer cells did not grow in nonandrogenized hosts. In contrast, PC-82 prostate cancer cells grew with identical characteristics (i.e., take rate, morphology, PSA expression, growth rate, and percentage of cell proliferating or dying) in androgenized hosts of both backgrounds. Likewise, in both backgrounds, androgen ablation of mice bearing growing PC-82 cancers resulted in the inhibition of proliferation and activation of programmed (apoptotic) cell death of the cancer cells. These results demonstrate that both the androgen-stimulated proliferation and the suppression of programmed cell death of PC-82 human prostate cancer cells are initiated by the AR pathway directly within these cancer cells themselves and do not involve initiation by AR-expressing stromal cells in a paracrine manner.

Androgens↗

PubMed1998

Progressive decline in the ability of calmodulin isolated from aged brain to activate the plasma membrane Ca-ATPase.

To identify possible relationships between the loss of calcium homeostasis in brain associated with aging and alterations in the function of key calcium regulatory proteins, we have purified calmodulin (CaM) from the brains of Fischer 344 rats of different ages and have assessed age-related alterations in (i) the secondary and tertiary structure of CaM and (ii) the ability of CaM to activate one of its target proteins, the plasma membrane (PM) Ca-ATPase. There is a progressive, age-dependent reduction in the ability of CaM to activate the PM-Ca-ATPase, which correlates with the oxidative modification of multiple methionines to their corresponding methionine sulfoxides. No other detectable age-related posttranslational modifications occur in the primary sequence of CaM, suggesting that the reduced ability of CaM to activate the PM-Ca-ATPase is the result of methionine oxidation. Corresponding age-related changes in the secondary and tertiary structure of CaM occur, resulting in alterations in the relative mobility of CaM on polyacrylamide gels, differences in the intrinsic fluorescence intensity and solvent accessibility of Tyr99 and Tyr138, and a reduction in the average alpha-helical content of CaM at 20 degreesC. Shifts in the calcium- and CaM-dependent activation of the PM-Ca-ATPase are observed for CaM isolated from senescent brain, which respectively requires larger concentrations of either calcium or CaM to activate the PM-Ca-ATPase. The observation that the oxidative modification of CaM during normal biological aging results in a reduced calcium sensitivity of the PM-Ca-ATPase, a lower affinity between CaM and the PM-Ca-ATPase, and the reduction in the maximal velocity of the PM-Ca-ATPase is consistent with earlier results that indicate the calcium handling capacity of a range of tissues including brain, heart, and erythrocytes isolated from aged animals declines, resulting in both longer calcium transients and elevated basal levels of intracellular calcium. Thus, the oxidative modification of selected methionines in CaM may explain aspects of the loss of calcium homeostasis associated with the aging process.

Aging↗

PubMed1998

Identification of the CD8 DE loop as a surface functional epitope. Implications for major histocompatibility complex class I binding and CD8 inhibitor design.

We used an approach of protein surface epitope mapping by synthetic peptides to analyze the surface structure-function relationship of the CD8 protein. Small synthetic peptide mimics of the CD8 DE loop were shown to effectively block CD8 binding to major histocompatibility complex (MHC) class I molecules and possess significant inhibitory activity on in vitro CD8(+) T cell function. These results suggested that the DE loop region of the CD8 protein is an important functional epitope mediating CD8-MHC class I interaction and the activation of CD8(+) T cells, a finding that is consistent with the recently reported crystal structure of the CD8-MHC class I complex. The structural basis for the biological activity of the DE loop peptide was further analyzed in a series of analogs containing alanine substitutions. This study provides support for the concept of bioactive peptide design based on protein surface epitopes and suggests that such an approach may be applicable to other protein-protein complexes, particularly those of immunoglobulin superfamily molecules.

Amino Acid Sequence↗

PubMed1998

Enzymatic activation of a doxorubicin-peptide prodrug by prostate-specific antigen.

New approaches to target cytotoxic therapy specifically to metastatic prostate cancer sites are urgently needed. As such an approach, an inactive prodrug was synthesized by coupling the primary amine of doxorubicin to the COOH-terminal carboxyl of a seven-amino acid peptide carrier (i.e., Mu-His-Ser-Ser-Lys-Leu-Gln-Leu). The seven-amino acid peptide was documented to be hydrolyzable specifically by the serine protease prostate-specific antigen (PSA) to liberate the active cytotoxin L-leucyl-doxorubicin. Primary cultures of PC-82 human prostate cancer cells secreted high levels of enzymatically active PSA (i.e., 70 +/- 5 ng of enzymatically active PSA/10(6) cells/24 h), whereas LNCaP human prostate cancer cells produced lower levels of enzymatically active PSA (i.e., 2.3 +/- 1 ng/10(6) cells/24 h). LNCaP cells, however, secreted sufficient amounts of enzymatically active PSA to activate the doxorubicin prodrug to a cytotoxic form in vitro. The specificity of the cytotoxic response to the prodrug was demonstrated by the fact that 70 nM of the prodrug killed 50% of the PSA-producing LNCaP cells, whereas doses as high as 1 microM had no cytotoxic effect on PSA-nonproducing TSU human prostate cancer cells in vitro.

Antibiotics, Antineoplastic↗

PubMed1998

Photochemically induced focal cochlear lesions in the guinea pig: I. DAB staining and SEM study.

A photochemical reaction was used to produce focal microcirculation disorders in the guinea pig cochlea. Temporal bones were removed at various intervals between 5 minutes and 1 month after infusion of rose bengal (RB) and illumination. Infused but unilluminated contralateral cochleae served as controls. Dissected cochlear structures were stained by 3,3'-diaminobenzidine (DAB) peroxidase substrate medium. After observation by light microscopy (LM), the same specimens were processed and observed by scanning electron microscopy (SEM). Dilation of strial capillaries and destruction of strial epithelial cells became apparent at 1 hour after illumination. Tightly packed red blood cells were found filling the severed end of markedly dilated strial capillaries at 24 hours after the procedure. DAB staining of the osseous spiral lamina indicated vascular change with vessel dilation in the illuminated area. At 1 week after illumination, the lesion area of the stria vascularis and spiral prominence was replaced by a layer of larger, flat cells. At 1 month after illumination, all vascular supply to the cochlear lateral wall disappeared at the site of illumination. All lesions remained focal and showed no sign of expansion or reduction throughout the observation period. The cochlear duct of the guinea pig appears to be segmentally nourished by the capillary system. Observation of DAB stained tissue by LM and SEM proved to be informative in the study of microcirculation disorders of the inner ear.

3,3'-Diaminobenzidine↗

PubMed1998

alpha-Adrenergic effects on Na+-K+ pump current in guinea-pig ventricular myocytes.

1. The whole-cell patch clamp was employed to study Na+-K+ pump current (Ip) in acutely isolated myocytes. alpha-Adrenergic receptors were activated with noradrenaline (NA) after blocking beta-adrenergic receptors with propranolol. Ip was measured as the current blocked by strophanthidin (Str). 2. Activation of alpha-receptors by NA increased Ip in a concentration-dependent manner. The K0.5 depended on intracellular calcium ([Ca2+]i), however maximal stimulation did not. At 15 nM [Ca2+]i the K0.5 was 219 nM NA whereas at 1.4 microM [Ca2+]i it was 3 nM. 3. The voltage dependence of Ip was not shifted by NA at either high or low [Ca2+]i. At each voltage, maximal stimulation of Ip was 14-15 %. 4. Staurosporine (St), an inhibitor of protein kinase C (PKC), eliminated the alpha-receptor-mediated stimulation of Ip at either high or low[Ca2+]i. 5. The stimulation of Ip was independent of changes in intracellular sodium or external potassium concentrations, and did not reflect a change in affinity for Str. 6. Phenylephrine, methoxamine and metaraminol, three selective alpha1-adrenergic agonists, stimulate Ip in a similar manner to NA. Stimulation of Ip by NA was eliminated by prazosin, an alpha1-antagonist, but was unaffected by yohimbine, an alpha2-antagonist. 7. We conclude noradrenaline activates ventricular alpha1-receptors, which are specifically coupled via PKC to increase Na+-K+ pump current. The sensitivity of the coupling is [Ca2+]i dependent, however the maximal increase in pump current is [Ca2+]i and voltage independent.

Adrenergic alpha-Agonists↗

PubMed1998

A novel site, Mt, in the human desmin enhancer is necessary for maximal expression in skeletal muscle.

Previous investigations have shown that expression of the muscle-specific intermediate filament desmin gene in skeletal muscle is controlled in part by a 5' muscle-specific enhancer. This enhancer activity can be divided into myoblast-specific and myotube-specific activation domains. The myotube-specific region contains a MyoD and MEF2 sites, whereas the myoblast-specific region contains Sp1, Krox, and Mb sites. In the present study, we designed mutations in the conserved portion of the myotube-specific region; transfection analysis of these mutations showed that a novel site located between the MyoD and MEF2 sites, named Mt (GGTATTT), is required for full transcriptional activity of the desmin enhancer in skeletal muscle. Although gel mobility shift assays demonstrate that myotube, myoblast, fibroblast, and HeLa nuclear extracts contain a nuclear factor that binds specifically to Mt, four copies of the Mt site function as the native enhancer only in myotubes. Functional synergism among the MyoD, MEF2, and Mt sites in myotubes has been demonstrated. These results show that the novel Mt site cooperates with MyoD and MEF2 to give maximal expression of the desmin gene.

Base Sequence↗

PubMed1998

Effects of calcitonin gene-related peptide on somatostatin and gastrin gene expression in rat antrum.

The ability of exogenous calcitonin gene-related peptide (CGRP) to regulate gastric somatostatin and gastrin messenger RNA was studied in vitro in rat antral mucosal/submucosal tissues. Somatostatin and gastrin mRNA were quantified by Northern and dot blot hybridization and regulatory peptides were measured by radioimmunoassay. Incubation of antral tissues in the presence of CGRP [1 x 10(-7) M] for 60 min resulted in a reciprocal increase in somatostatin and a decrease in gastrin release: 214.7+/-28.5 vs. control of 81.7+/-5.9 pg somatostatin per gram of tissue and 2.2+/-0.3 vs. control of 5.5+/-0.7 ng gastrin per gram of tissue (P < 0.001). CGRP caused parallel changes in somatostatin and gastrin mRNA levels: somatostatin mRNA increased by 212% from 0.40+/-0.02 to 1.25+/-0.09 absorbance units (AU) (P < 0.001) and gastrin mRNA decreased by 73% from 0.55+/-0.08 to 0.15+/-0.02 AU (P < 0.001). Somatostatin monoclonal antibody prevented CGRP-mediated inhibition of both gastrin release and gastrin mRNA levels. In conclusion, CGRP is capable of modulating both the secretion and gene expression of regulatory peptides from antral G and D cells. Somatostatin immunoneutralization studies suggest that the actions of CGRP on gastrin release and gene expression are indirect and mediated through the paracrine influences of somatostatin.

Animals↗

PubMed1998

The relationship of galanin immunoreactive nerve fibers to glandular cells in the anterior pituitary in the monkey.

The anterior pituitary is known to be regulated by hypothalamic hormones via the portal system. However, our recent studies have demonstrated the presence of a substantial amount of substance P (SP)-, calcitonin gene-related peptide (CGRP)- and galanin (GAL)- immunoreactive (ir) nerve fibers with numerous varicosities in the anterior pituitaries of the Macaca mulatta monkey. The present study investigated the relationship of the GAL-ir nerve fibers to the glandular cells. The M. mulatta monkeys were used and sections of the anterior pituitary were double immunostained. GAL-ir nerve fibers and/or varicosities were found in proximity to contact directly with corticotropes, somatotropes, lactotropes, gonadotropes and thyrotropes without any exception. These findings indicate that a direct neural factor may be involved in the regulation of adenohypophyseal secretion.

Animals↗

PubMed1998