Biomedical subjects

J Ren

Publications and source records attributed to J Ren.

At least 163 records · Page 9Linked to original sources

PubMed1999

Sequence and structural selectivity of nucleic acid binding ligands.

The sequence and structural selectivity of 15 different DNA binding agents was explored using a novel, thermodynamically rigorous, competition dialysis procedure. In the competition dialysis method, 13 different nucleic acid structures were dialyzed against a common ligand solution. More ligand accumulated in the dialysis tube containing the structural form with the highest ligand binding affinity. DNA structural forms included in the assay ranged from single-stranded forms, through a variety of duplex forms, to multistranded triplex and tetraplex forms. Left-handed Z-DNA, RNA, and a DNA-RNA hybrid were also represented. Standard intercalators (ethidium, daunorubicin, and actinomycin D) served as control compounds and were found to show structural binding preferences fully consistent with their previously published behavior. Standard groove binding agents (DAPI, distamycin, and netropsin) showed a strong preference for AT-rich duplex DNA forms, along with apparently strong binding to the poly(dA)-[poly(dT)](2) triplex. Thermal denaturation studies revealed the apparent triplex binding to be complex, and perhaps to result from displacement of the third strand. Putative triplex (BePI, coralyne, and berberine) and tetraplex [H(2)TmPyP, 5,10,15, 20-tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine, and N-methyl mesoporphyrin IX] selective agents showed in many cases less dramatic binding selectivity than anticipated from published reports that compared their binding to only a few structural forms. Coralyne was found to bind strongly to single-stranded poly(dA), a novel and previously unreported interaction. Finally, three compounds (berenil, chromomycin A, and pyrenemethylamine) whose structural preferences are largely unknown were examined. Pyrenemethylamine exhibited an unexpected and unprecedented preference for duplex poly(dAdT).

Antibiotics, Antineoplastic↗

PubMed1999

Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: a sequestration mechanism in metabolic regulation.

The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Design of MKC-442 (emivirine) analogues with improved activity against drug-resistant HIV mutants.

Two analogues of the nonnucleoside inhibitor of HIV-1 RT, MKC-442 (emivirine), containing different C6 substituents have been designed to be less susceptible to the commonly found drug-resistance mutation of Tyr181Cys. Compound TNK-6123 had a C6 thiocyclohexyl group designed to have more flexibility in adapting to the mutated drug-binding site. GCA-186 had additional 3',5'-dimethyl substituents aimed at forming close contacts with the conserved residue Trp229. Both compounds showed approximately 30-fold greater inhibitory effect than MKC-442 to the Tyr181Cys mutant virus as well as to the clinically important Lys103Asn virus. X-ray crystallographic structure determination of complexes with HIV-1 RT confirmed the predicted binding modes. These strategies might be used to improve the resilience of other NNRTI series against common drug-resistance mutations.

Animals↗

PubMed1999

Glutamate modulates neurotransmission in the submucosal plexus of guinea-pig small intestine.

Effects of glutamate on synaptic transmission in the submucosal plexus of guinea-pig small intestine were studied with intracellular electrophysiological recording methods. Glutamate suppressed stimulus-evoked slow excitatory postsynaptic potentials (EPSPs) and increased the amplitude of slow inhibitory postsynaptic potentials (IPSPs) in submucosal neurons. The actions of glutamate were mimicked by the group I metabotropic glutamate receptor (mGluRs) agonist DHPG, but not by the group II agonist S-4C3HPG, the group III agonist L-AP4, or selective agonists for ionotropic glutamate receptors (iGluRs). Glutamate actions were suppressed by the selective group I mGluRs antagonist S-4CPG, but not by group II and III mGluRs antagonist CPPG or iGluRs antagonists. Glutamate suppressed substance P- and 5-HT-evoked slow EPSP-like responses and potentiated norepinephrine-induced slow IPSP-like responses. The results suggest that group I mGluRs mediate glutamate-induced suppression of slow EPSPs and potentiation of slow IPSPs in S-type uniaxonal submucosal neurons.

Animals↗

PubMed1999

Crystallographic analysis of the binding modes of thiazoloisoindolinone non-nucleoside inhibitors to HIV-1 reverse transcriptase and comparison with modeling studies.

We have determined the crystal structures of thiazoloisoindolinone non-nucleoside inhibitors in complex with HIV-1 reverse transcriptase to high-resolution limits of 2.7 A (BM +21.1326) and 2. 52 A (BM +50.0934). We find that the binding modes of this series of inhibitors closely resemble that of "two-ring" non-nucleoside reverse transcriptase inhibitors. The structures allow rationalization of stereochemical requirements, structure-activity data, and drug resistance data. Comparisons with our previous structures suggest modifications to the inhibitors that might improve resilience to drug-resistant mutant forms of reverse transcriptase. Comparison with earlier modeling studies reveals that the predicted overlap of thiazoloisoindolinones with TIBO was largely correct, while that with nevirapine was significantly different.

Crystallography, X-Ray↗

PubMed1999

Waglerin-1 inhibits GABA(A) current of neurons in the nucleus accumbens of neonatal rats.

The effect of Waglerin-1, a 22-amino acid peptide purified from the venom of Wagler's pit viper on the whole cell current response (I(GABA)) to gamma-aminobutyric acid (GABA) was examined for neurons freshly isolated from the nucleus accumbens of 3- to 7-day-old rats. Waglerin-1 depressed I(GABA) induced by subsaturating concentrations of GABA; the IC(50) for I(GABA) induced by 10 microM GABA was 2.5 microM Waglerin-1. This concentration of Waglerin-1 shifted the GABA concentration-response curve to the right in a parallel manner, increasing the GABA EC(50) from 12+/-3 to 27+/-5 microM. The depressant effect of Waglerin-1 was greater at negative holding potentials. Zn(2+) also inhibited I(GABA) with an IC(50) of 0.3 microM. Phosphorylation state appeared to modulate GABA(A) receptor sensitivity to the inhibitory effect of Waglerin-1 since dialysis of neurons with N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HCl (H-89), an inhibitor of protein kinase A, prevented inhibition. The data are discussed in terms of developmental influences on the subunit composition of GABA(A) receptors in neurons of the nucleus accumbens.

Amino Acid Sequence↗

PubMed1999

Hypertension augments ethanol-induced depression of cell shortening and intracellular Ca(2+) transients in adult rat ventricular myocytes.

Ethanol, a risk factor for myocardial dysfunction, depresses myocardial contraction. This study was to determine whether ethanol-induced myocardial depression is affected by hypertension. Mechanical properties of ventricular myocytes isolated from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were evaluated using a video edge-detection system. Myocytes were electrically stimulated to contract at 0.5 Hz. Contractile properties analyzed include peak twitch amplitude (PTA), time-to-PTA (TPS), time-to-90% relengthening (TR(90)), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) transients were measured as fura-2 fluorescence intensity (DeltaFFI) changes. Acute ethanol exposure (80-640 mg/dl) caused a concentration-dependent inhibition of PTA and DeltaFFI in both WKY and SHR myocytes. The extent of maximal inhibition of PTA and FFI was significantly greater in SHRs (53.7 and 38.9%) compared to the WKY group (21.0 and 25.4%). Ethanol did not affect TPS but shortened TR(90) and slowed +/-dL/dt at high concentration ranges. Interestingly, the augmented ethanol-induced inhibition of cell shortening in hypertension was greatly attenuated by Ca(2+) channel opener BayK 8644 (1 microM). These results suggest that ethanol-induced myocardial depression may be augmented in hypertension, possibly due to mechanism(s) involving sarcolemmal Ca(2+) channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1999

Regulation of myocardial glucose uptake and transport during ischemia and energetic stress.

Myocardial glucose utilization increases in response to the energetic stress imposed on the heart by exercise, pressure overload, and myocardial ischemia. Recruitment of glucose transport proteins is the cellular mechanism by which the heart increases glucose transport for subsequent metabolism. Moderate regional ischemia leads to the translocation of both glucose transporters, GLUT4 and GLUT1, to the sarcolemma in vivo. Myocardial ischemia also stimulates 5'-adenosine monophosphate-activated protein kinase, which may be a fuel gauge in the heart and other tissues signaling the need to turn on energy-generating metabolic pathways. Pharmacologic stimulation of this kinase increases cardiac glucose uptake and transporter translocation, suggesting that it may play an important role in augmenting glucose entry in the setting of ischemic or energetic stress. Thus, recent work has provided insight into the cellular and molecular mechanisms responsible for glucose uptake during energetic stress, which may lead to new approaches to the treatment of patients with coronary artery disease.

Adenosine Monophosphate↗

PubMed1999

Interaction of diphtheria toxin T domain with molten globule-like proteins and its implications for translocation.

The transmembrane (T) domain of diphtheria toxin has a critical role in the low pH-induced translocation of the catalytic domain (A chain) of the toxin across membranes. Here it is shown that at low pH, addition of proteins in a partly unfolded, molten globule-like conformation converted the T domain from a shallow membrane-inserted form to its transmembrane form. Fluorescence energy transfer demonstrated that molten globule-like proteins bound to the T domain. Thus, the T domain recognizes proteins that are partly unfolded and may function in translocation of the A chain as a transmembrane chaperone.

Antibodies↗

PubMed1999

Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length.

We examined the effect of the length of the hydrophobic core of Lys-flanked poly(Leu) peptides on their behavior when inserted into model membranes. Peptide structure and membrane location were assessed by the fluorescence emission lambdamax of a Trp residue in the center of the peptide sequence, the quenching of Trp fluorescence by nitroxide-labeled lipids (parallax analysis), and circular dichroism. Peptides in which the hydrophobic core varied in length from 11 to 23 residues were found to be largely alpha-helical when inserted into the bilayer. In dioleoylphosphatidylcholine (diC18:1PC) bilayers, a peptide with a 19-residue hydrophobic core exhibited highly blue-shifted fluorescence, an indication of Trp location in a nonpolar environment, and quenching localized the Trp to the bilayer center, an indication of transmembrane structure. A peptide with an 11-residue hydrophobic core exhibited emission that was red-shifted, suggesting a more polar Trp environment, and quenching showed the Trp was significantly displaced from the bilayer center, indicating that this peptide formed a nontransmembranous structure. A peptide with a 23-residue hydrophobic core gave somewhat red-shifted fluorescence, but quenching demonstrated the Trp was still close to the bilayer center, consistent with a transmembrane structure. Analogous behavior was observed when the behavior of individual peptides was examined in model membranes with various bilayer widths. Other experiments demonstrated that in diC18:1PC bilayers the dilution of the membrane concentration of the peptide with a 23-residue hydrophobic core resulted in a blue shift of fluorescence, suggesting the red-shifted fluorescence at higher peptide concentrations was due to helix oligomerization. The intermolecular self-quenching of rhodamine observed when the peptide was rhodamine-labeled, and the concentration dependence of self-quenching, supported this conclusion. These studies indicate that the mismatch between helix length and bilayer width can control membrane location, orientation, and helix-helix interactions, and thus may mismatch control both membrane protein folding and the interactions between membrane proteins.

Circular Dichroism↗

PubMed1999

Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2.

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.

Base Sequence↗

PubMed1999

Localization of verotoxin receptors in nervous system.

We use immunohistochemistry to show the existence of verotoxin receptor in small sensory neurons in DRG of human, rabbit, rat and mouse. In capillary in nervous system, the verotoxin receptor exists in human and rabbit, but the receptor could not be demonstrated in rat and mouse, by this method. The receptors in sensory neuron of rat and in capillary in rabbit brain are determined as galactosylglobotriaosylceramide (GalGb3) and globotriaosylceramide (Gb3,), respectively. Although verotoxin was reported to bind to glycolipid receptors that possess the terminal disaccharide Galalpha1-4Galbeta (galactobiose), the binding to toxin to galabiosylceramide was half of that of GalGb3 which has galactobiose internally.

Animals↗

PubMed1999

Cocaine and lidocaine have additive inhibitory effects on the GABAA current of acutely dissociated hippocampal pyramidal neurons.

Inhibition mediated by gamma-aminobutyric acid (GABA) is a major target for the central actions of cocaine and lidocaine, which can result in seizures, especially when these drugs are abused in combination. In the present study, we investigated how cocaine and lidocaine interact to depress GABA current (IGABA), recorded by the whole-cell technique in freshly isolated rat hippocampal neurons. Cocaine depressed IGABA in a concentration dependent manner, such that cocaine was more potent against lower than higher GABA concentrations: the cocaine IC50 was 0.13, 0.62 and 1.2 mM for GABA at 2, 10 and 100 microM, respectively. Cocaine depressed IGABA to the same extent in the absence and presence of 1 microM tetrodotoxin, indicating that cocaine inhibition of IGABA is distinct from its Na+ channel blocking action. Lidocaine reversibly depressed IGABA evoked by 10 microM GABA, with an IC50 of 9.8 mM. In the presence of 3 mM lidocaine, 0.3 mM cocaine depressed IGABA (10 microM GABA) to 30+/-7%. The significantly greater depression by the combined agents (p<0.05) indicates additive effects on the GABA receptor/channel complex, which are likely to contribute to the additive convulsant effects noted when these drugs are abused in combination.

Anesthetics, Combined↗

PubMed1999

Cocaine decreases the glycine-induced Cl- current of acutely dissociated rat hippocampal neurons.

The effects of cocaine on glycine-induced Cl- current (I(GLY)) of single neurons, freshly isolated from the rat hippocampal CA1 area, were studied with conventional whole-cell recording under voltage-clamp conditions. Cocaine depressed I(GLY) in a concentration-dependent manner, with an IC50 of 0.78 mM. Preincubation with 1 mM cocaine alone had no effect on I(GLY), suggesting that resting glycine channels are insensitive to cocaine. The depression of I(GLY) by cocaine was independent of membrane voltage. Internal cell dialysis with 1 mM cocaine failed to modify I(GLY). Because the depression of I(GLY) was noncompetitive, cocaine may act on the glycine receptor-chloride ionophore complex at a site distinct from that to which glycine binds. The cocaine suppression of I(GLY) was unaffected by 1 microM tetrodotoxin and 1 microM strychnine. Blockers of protein kinase C (Chelerythrine), kinase A (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HCl, (H-89)) and Ca-calmodulin-dependent kinase (1-[N,O-bis(5-isoquinoline-sulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperaz ine (KN-62)) were also ineffective, which suggests that these phosphorylating mechanisms do not modulate cocaine-induced suppressant action on I(GLY). This extracellular, strychnine-independent depression of I(GLY) may contribute to cocaine-induced seizures.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Health literacy among Medicare enrollees in a managed care organization.

CONTEXT: Elderly patients may have limited ability to read and comprehend medical information pertinent to their health. OBJECTIVE: To determine the prevalence of low functional health literacy among community-dwelling Medicare enrollees in a national managed care organization. DESIGN: Cross-sectional survey. SETTING: Four Prudential HealthCare plans (Cleveland, Ohio; Houston, Tex; south Florida; Tampa, Fla). PARTICIPANTS: A total of 3260 new Medicare enrollees aged 65 years or older were interviewed in person between June and December 1997 (853 in Cleveland, 498 in Houston, 975 in south Florida, 934 in Tampa); 2956 spoke English and 304 spoke Spanish as their native language. MAIN OUTCOME MEASURE; Functional health literacy as measured by the Short Test of Functional Health Literacy in Adults. RESULTS: Overall, 33.9% of English-speaking and 53.9% of Spanish-speaking respondents had inadequate or marginal health literacy. The prevalence of inadequate or marginal functional health literacy among English speakers ranged from 26.8% to 44.0%. In multivariate analysis, study location, race/language, age, years of school completed, occupation, and cognitive impairment were significantly associated with inadequate or marginal literacy. Reading ability declined dramatically with age, even after adjusting for years of school completed and cognitive impairment. The adjusted odds ratio for having inadequate or marginal health literacy was 8.62 (95% confidence interval, 5.55-13.38) for enrollees aged 85 years or older compared with individuals aged 65 to 69 years. CONCLUSIONS: Elderly managed care enrollees may not have the literacy skills necessary to function adequately in the health care environment. Low health literacy may impair elderly patients' understanding of health messages and limit their ability to care for their medical problems.

Aged↗

PubMed1999

Differentiation of Lactococcus lactis subspecies lactis and subspecies cremoris strains by their adaptive response to stresses.

Lactococcus lactis subspecies lactis (L. lactis ssp. lactis) and Lactococcus lactis subspecies cremoris (L. lactis ssp. cremoris) were investigated in respect to their response to acid, bile-salt and freezing stresses. First, the sublethal and lethal levels of each stress were determined for both subspecies. For acid stress, the levels were pH 4.5 and 2.5, respectively, for L. lactis ssp. lactis, and pH 5.0 and 3.0, respectively, for L. lactis ssp. cremoris. For bile-salt stress, the levels were 0.03 and 0.1%, respectively, for L. lactis ssp. lactis, and 0.01 and 0.04%, respectively, for L. lactis ssp. cremoris. For freezing stress, 10 degrees C was used as the sublethal temperature and -20 degrees C was used as the lethal temperature for both subspecies. To evaluate the effect of each stress at log phase, a log-phase culture was challenged directly with the appropriate lethal level (control culture) and a second log-phase culture was pre-exposed to the appropriate sublethal level prior to testing survival under normally lethal conditions (test culture). Some, if not most, of the cells were killed in the control cultures for all three stresses. However, in the test cultures, the viability was significantly improved for all of the L. lactis ssp. lactis strains tested, but not for the L. lactis ssp. cremoris strains. It appears, therefore, that L. lactis ssp. lactis is capable of displaying adaptive response to stresses, whereas L. lactis ssp. cremoris seems to lack this phenotype or the response is much weaker in this subspecies. The effect of each stress on stationary-phase cultures was also investigated. Unlike the log-phase cultures, the stationary-phase cultures of both subspecies, challenged directly with the lethal levels, were highly resistant to each of the three stresses tested.

Adaptation, Physiological↗

PubMed1999

Membrane translocation of charged residues at the tips of hydrophobic helices in the T domain of diphtheria toxin.

The low pH triggered membrane insertion of the T domain of diphtheria toxin is a critical step in the translocation of the C domain of the toxin across membranes in vivo. We previously established that the T domain can interact with membranes in two distinct conformations, one in which the TH8/TH9 helical hairpin lies close to the bilayer surface and a second in which it inserts more deeply and appears to be transmembraneous. The loss of charge on residues E349 and D352 due to protonation at low pH has been proposed to be a critical step in transmembrane insertion, because they are within a loop connecting TH8 and TH9, and must cross the membrane upon transmembrane insertion. In this report, the role of these residues was examined by measuring the effect of the double substitution E349K/D352K on the conformation of the TH8/TH9 hairpin through a fluorescent group attached to TH9. At pH 4.5, there was shallower insertion of TH8/TH9 of the E349K/D352K mutant relative to T domain with wild-type residues at 349 and 352. In addition, smaller and/or fewer pores were obtained with the E349K/D352K mutant relative to the wild-type. On the other hand, high T domain concentrations, or further decreasing pH, allowed transmembrane insertion of both the wild-type and the 349K/352K mutant as well as induction of larger and/or more numerous pores. Furthermore, the transmembrane insertion process was rapid for both the mutant and wild-type. This shows that the mutant has the capacity to form a transmembrane structure similar to that of the wild-type T domain and, thus, that introduction of charged groups in membrane-penetrating regions of a protein does not introduce an insurmountable barrier to transmembrane movement. The linkage between the ability of the T domain to form the transmembrane conformation and pores suggests that the effects of these mutations in inhibiting pore formation are likely to partly result from the inability to insert properly. Additionally, the observation that decreasing pH allows the 349K/352K mutant to insert deeply indicates that there are residues other than E349 and D352 whose protonation promotes transmembrane insertion.

Biological Transport↗

PubMed1999

Temperature and pH effects on single-strand conformation polymorphism analysis by capillary electrophoresis.

We investigated the effects of temperature and pH on single strand-conformation polymorphism (SSCP) analyzed by capillary electrophoresis (CE) using short-chain linear polyacrylamide as the sieving medium. Nine different mutations (in factor V, cystathionine beta-synthase, and methylenetetrahydrofolate reductase genes), including both transitions and transversions, were investigated. We confirmed that low temperature in general increased the number of detectable single-strand conformations and thereby the sensitivity of the analysis. The pH effects of the separation matrix on the migration pattern, and thus the assay sensitivity, varied markedly between the different DNA fragments. Seven of nine single point mutations were detected at the ordinary pH of 8.3, whereas the CBS T833C mutation was discriminated at the extreme pH values of 9.0 and 6.4, and the CBS G797A mutation could not be detected at any pH value within the range 6.4--9.0. These data emphasize the importance of the pH of the separation matrix in detecting certain mutations by SSCP.

Base Sequence↗