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Biomedical subjects

J Ren

Publications and source records attributed to J Ren.

At least 109 records · Page 6Linked to original sources
PubMed2001

[An experimental study of the effects of early enteral feeding of L--arginine enriched food on postburn intestinal mucosal proliferation in rats].

OBJECTIVE: To investigate the effects of early enteral feeding of L -- arginine enriched food on postburn intestinal mucosal proliferation in rats. METHODS: Twenty Wistar rats prepared with gastrostomy 7 days before the experiment were inflicted by 30% TBSA flame burn, and were divided into early enteral feeding (EF), L -- arginine enriched early enteral feeding group (AEF) receiving feeding from 2 PBHs and delayed feeding (DF) group, receiving feeding from 48 PBHs. The changes in intestinal mucosal DNA content and intestinal mucosal proliferating cell nuclear antigen labeling index (PCNALI) were determined on the 1st, 3rd, 6th and 9th postburn days (PBDs) respectively in rats. RESULTS: There were rare intestinal mucosal PCNA positive cells on the 1st PBD in all groups. Compared with that in DF group, the intesinal mucosal PCNALI in AEF group increased obviously on 3 PBD, while that in EF group increased evidently on 6 PBD. (P < 0.05). CONCLUSION: There existed intestinal mucosal injury postburn as shown by the inhibition of intestinal epithelial proliferation. Early enteral feeding with L -- arginine enriched food could not only ameliorate intestinal mucosal injury, but also enhance the proliferation of intestinal mucosal epithelium.

Animals↗
PubMed2001

Loss of cardiac contractile response to tetrahydropapaveroline with advanced age and hypertension.

Tetrahydropapaveroline (THP), a condensation product of ethanol-derived acetaldehyde, potentiates cardiac function through beta-adrenoceptor. We have recently shown that THP-induced cardiac contractile action is likely due to its action at the single myocyte level, and is markedly diminished during early hypertension. Cardiac function alters with advanced age reminiscent of hypertension. This study was to examine cardiac contractile response to THP with advanced age and hypertension. Left ventricular papillary muscles and myocytes were isolated from normotensive (WKY) or hypertensive (SHR) rats, and stimulated to contract at 0.5 Hz. Mechanical parameters evaluated include: peak tension developed (PTD)/peak shortening (PS), time-to-PTD/PS (TPT/TPS), time-to-90% relaxation/relengthening (RT90/TR90), and maximal velocities of contraction/relaxation (+/- VT/+/- dLdt). Intracellular Ca2+ transients were measured as fura-2 fluorescence intensity changes (AFFI). THP (0.1-100 microM) increased PTD in 10- but not 36-wk-old WKY rat myocardium. THP elicited positive, negative or no response on PS in myocytes from 10-wk WKY, 36-wk WKY, and 36-wk SHR groups, respectively. Interestingly, THP elicited discrepant response on intracellular Ca2+ transient compared with that of myocyte shortening. THP increased AFFI in 10-wk WKY and 36-wk SHR myocytes while exhibiting a significant inhibiting action in 36-wk WKY myocytes. Lastly, THP shortened TPT/TPS, RT90/TR90 and increased +VT in all animal groups. These results indicate that the THP-induced myocardial contractile response is altered in advanced age and hypertension, in a manner similar to early stage of hypertension. It is possible that altered intracellular Ca2+ responsiveness may be involved in THP-induced action.

Aging↗
PubMed2001

[The recombinant vaccinia viruses expressing HPV type 16 wild or mutant E7 protein elicit immunity against tumor cells in mice].

OBJECTIVE: To select mutants of HPV 16 E7 suitable for vaccine development. METHODS: Several recombinant vaccinia viruses (VwE7, VmE7-1, and VmE7-2) expressing wild E7 or its mutants, ME7-1 (24G26G) and ME7-2 (24G26G91G) respectively, were employed to study their antigenicity and anti tumor activity. RESULTS: Both VmE7-1 and VwE7 could elicit cytotoxic T lymphocytes (CTL)and protect mice against tumor cells challenge, but VmE7-2 could not induce the immune reactions compared with VmE7-1 and VwE7. CONCLUSIONS: It suggested that ME7-1 could be used to develop a therapeutic vaccine for cervical cancer.

Animals↗
PubMed2001

[Expression of baculovirus recombinant HPV16L1 and L2 capsid proteins in insect cells].

BACKGROUND: To express capsid proteins of HPV16 in insect cells system. METHODS: Recombinant baculovirus stocks were generated by cotransfection with baculovirus DNA and baculovirus recombinant transfer plasmids. The expression of aimed proteins in insect cells were confirmed by using SDS-PAGE and Western blot. RESULTS: Two stable strains of recombinant baculovirus expressing HPV16L1 protein alone and co-expressing HPV16L1 plus L2 proteins were obtained. The L1 and L2 ORFs of HPV16 produced proteins of 57000 and 97000, respectively. The yield of L1 protein was about 25%-30% of the total proteins of the insect cell. CONCLUSIONS: L1 and L2 proteins of HPV16 could be expressed with high efficiency in insect cells via recombinant baculoviruses.

Animals↗
PubMed2001

[Expression of the human papillomavirus type 58L1 capsid protein in E.coli and preparation of the HPV58 L1 mouse antiserum].

BACKGROUND: Human papillomavirus type 58 (HPV58) has been shown to be one of the most important highly-oncogenic, risky HPV types. The present study aimed at investigating the expression of the HPV58 L1 major capsid protein by E.coli and preparation of HPV58 L1 antiserum. METHODS: The full length L1 coden region of HPV58 L1 was amplified by PCR, the cloned, sequenced. The expression vector pRSETB58 L1 was constructed to produce HPV58 L1 protein. The protein expressed was purified by SDS-PAGE, and used to immunize BALB/c mice. RESULTS: HPV58 L1 protein was expressed in E. coli, which has cross reaction with anti-HPV16 L1 antibody. The mouse anti-HPV58 L1 antibody was obtained and used to test the HPV58 L1 protein expressed in insect cell. CONCLUSIONS: HPV58 L1 major capsid protein was efficiently expressed in E. coli. The mouse anti-HPV58 L1 specific antibody was prepared, which can be used to test the HPV58 L1 protein expressed in eukaryotic cell.

Animals↗
PubMed2001

[Study of blood dynamics of ophthalmic artery in patients with sudden sensorineural hearing loss using color Doppler flow imaging].

OBJECTIVE: To investigate the relationship between the pathogenesis of sudden sensorineural hearing loss (SSHL) and the disorder of blood circulation in inner ear. METHOD: Blood dynamics of the ophthalmic artery were studied quantitatively using color doppler imaging in 34 patients with SSHL. RESULT: Compared with 34 self-controls and 15 normal controls, 28 patients (82.4%) with SSHL had significantly lower blood flow velocities and higher resistance indices (P < 0.05), and there was no significant difference between the self-control group and the normal control group (P > 0.05). CONCLUSION: The study suggested that the blood situations--the decreased blood flow velocities and perfusion and increased resistance of ophthalmic artery in patients with SSHL maybe play a role in the pathogenesis of SSHL.

Adolescent↗
PubMed2000

13C/31P NMR assessment of mitochondrial energy coupling in skeletal muscle of awake fed and fasted rats. Relationship with uncoupling protein 3 expression.

To examine the relationship between mitochondrial energy coupling in skeletal muscle and change in uncoupling protein 3 (UCP3) expression during the transition from the fed to fasted state, we used a novel noninvasive (31)P/(13)C NMR spectroscopic approach to measure the degree of mitochondrial energy coupling in the hind limb muscles of awake rats before and after a 48-h fast. Compared with fed levels, UCP3 mRNA and protein levels in the gastrocnemius increased 1.7- (p < 0.01) and 2.9-fold (p < 0.001), respectively, following a 48-h fast. Tricarboxylic acid cycle flux measured using (13)C NMR as an index of mitochondrial substrate oxidation was 212 +/- 23 and 173 +/- 25 nmol/g/min (p not significant) in the fed and 48-h fasted groups, respectively. Unidirectional ATP synthesis flux measured using (31)P NMR was 79 +/- 15 and 57 +/- 9 nmol/g/s (p not significant) in the fed and 48-h fasted groups, respectively. Mitochondrial energy coupling as expressed by the ratio of ATP synthesis to tricarboxylic acid cycle flux was not different between the fed and fasted states. To test the hypothesis that UCP3 may be involved in the translocation of long chain free fatty acids (FFA) into the mitochondrial matrix under conditions of elevated FFA availability, [U-(13)C]palmitate/albumin was administered in a separate group of rats with (+) or without (-) etomoxir (an inhibitor of carnitine palmitoyltransferase I). The ratio of glutamate enrichment ((+) etomoxir/(-) etomoxir) in the hind limb muscles was the same between groups, indicating that UCP3 does not appear to function as a translocator for long chain FFA in skeletal muscle following a 48-h fast. In summary, these data demonstrate that despite a 2-3-fold increase in UCP3 mRNA and protein expression in skeletal muscle during the transition from the fed to fasted state, mitochondrial energy coupling does not change. Furthermore, UCP3 does not appear to have a major role in FFA translocation into the mitochondria. The physiological role of UCP3 following a 48-h fast in skeletal muscle remains to be elucidated.

Adenosine Triphosphate↗
PubMed2000

Formaldehyde-induced alkylation of a 2'-aminoglucose rebeccamycin derivative to both A.T and G.C base pairs in DNA.

Rebeccamycin derivatives represent a promising class of antitumor agents. In this series, two glycosylated indolocarbazoles, NB-506 and NSC-655649, are currently undergoing clinical trials. Their anticancer activities are associated with their capacities to interact with DNA and to inhibit DNA topoisomerases. Previous studies revealed that the planar indolocarbazole chromophore can intercalate into DNA, locating the appended carbohydrate residue in one of the two helical grooves, probably the minor groove as is the case with the anthracyclines and other DNA-binding antibiotics. The sugar residue contributes significantly to the DNA binding free energy of NB-506. However, the exact positioning of the glycosyl residue of rebeccamycin derivatives in the drug-DNA complex remains poorly understood. To better understand how glycosylated indolocarbazoles interact with DNA, we investigated the interaction of a rebeccamycin derivative (85) bearing a 2'-amino group on the sugar residue. We show that the presence of the 2'-amino function permits the formation of covalent drug-DNA complexes in the presence of formaldehyde. Complementary biochemical and spectroscopic measurements attest that 85 reacts covalently with the 2-amino group of guanines exposed in the minor groove of the double helix, as is the case with daunomycin. In contrast to daunomycin, 85 also forms cross-links with an oligonucleotide containing only A.T base pairs. The covalent binding to A.T base pairs was detected using a gel mobility shift assay and was independently confirmed by thermal denaturation studies and by fluorescence measurements using a series of synthetic polynucleotides. The HCHO-mediated alkylation reaction of the drug with A.T base pairs apparently involves the 6-amino group of adenines exposed in the major groove whereas the covalent attachment to G.C base pairs implicates the 2-amino group of guanines situated in the opposite minor groove. Therefore, the results suggest that either the drug is able to switch grooves in response to sequence or it can simultaneously bind to both the minor and major grooves of the double helix. This study will help to guide the rational design of new DNA-binding antitumor indolocarbazole drugs and also provides a general experimental approach for probing minor versus major groove interactions between small molecules and DNA.

Alkylation↗
PubMed2000

Inhibition of Na(+)/H(+) exchange by 5-(N-ethyl-N-isopropyl)-amiloride reduces free fatty acid efflux from the ischemic reperfused rat cerebral cortex.

Brain tissue acidosis is considered to be a contributor to ischemic brain injury. The deleterious effects of marked acidosis may be associated with reperfusion and an excessive entry of Na(+) into cerebral neurons and glia as intracellular pH is restored by Na(+)/H(+) exchange. Normalization of pH, with activation of many calcium-dependent and other phospholipases and proteases with pH optima in the neutral or alkaline range, could account for the pronounced elevation in extracellular levels of free fatty acids which occurs during reperfusion following cerebral ischemia. In the present investigation we evaluated the effects of inhibition of Na(+)/H(+) exchange with N-(N-ethyl-N-isopropyl)-amiloride (EIPA; 25 microM) applied topically onto the rat cerebral cortex prior to and during ischemia. Free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high pressure liquid chromatography. EIPA application effectively inhibited the increases in arachidonic and linoleic acid release observed in the control rats during reperfusion, and non-significantly depressed that of palmitic and oleic acids. Superfusate levels of glucose, which decline to near zero levels during ischemia and then rebound during reperfusion, were not affected by EIPA administration. Lactate levels in cortical superfusates from EIPA-treated animals rose more rapidly during reperfusion than did those in the control rats and then significantly declined towards basal levels. The data indicate that inhibition of Na(+)/H(+) exchange prevented the activation of phospholipases that usually occurs during reperfusion following a cerebral ischemic episode. These results are the first demonstration of such an effect and may provide an explanation for the cerebroprotective effects that have been observed in stroked animals following administration of Na(+)/H(+) exchange inhibitors.

Acidosis↗
PubMed2000

Structural basis for the resilience of efavirenz (DMP-266) to drug resistance mutations in HIV-1 reverse transcriptase.

BACKGROUND: Efavirenz is a second-generation non-nucleoside inhibitor of HIV-1 reverse transcriptase (RT) that has recently been approved for use against HIV-1 infection. Compared with first-generation drugs such as nevirapine, efavirenz shows greater resilience to drug resistance mutations within HIV-1 RT. In order to understand the basis for this resilience at the molecular level and to help the design of further-improved anti-AIDS drugs, we have determined crystal structures of efavirenz and nevirapine with wild-type RT and the clinically important K103N mutant. RESULTS: The relatively compact efavirenz molecule binds, as expected, within the non-nucleoside inhibitor binding pocket of RT. There are significant rearrangements of the drug binding site within the mutant RT compared with the wild-type enzyme. These changes, which lead to the repositioning of the inhibitor, are not seen in the interaction with the first-generation drug nevirapine. CONCLUSIONS: The repositioning of efavirenz within the drug binding pocket of the mutant RT, together with conformational rearrangements in the protein, could represent a general mechanism whereby certain second-generation non-nucleoside inhibitors are able to reduce the effect of drug-resistance mutations on binding potency.

Alkynes↗
PubMed2000

XIAP regulates DNA damage-induced apoptosis downstream of caspase-9 cleavage.

The IAP (inhibitor of apoptosis) family of anti-apoptotic proteins regulates programmed cell death. Of the six known human IAP-related proteins, XIAP is the most potent inhibitor. To study the mechanistic effects of XIAP on DNA damage-induced apoptosis, we prepared U-937 cells that stably overexpress XIAP. The results demonstrate that XIAP inhibits apoptosis induced by 1-[beta-d-arabinofuranosyl]cytosine (ara-C) and other genotoxic agents. XIAP had no detectable effect on ara-C-induced release of mitochondrial cytochrome c and attenuated cleavage of procaspase-9. In addition, we show that ara-C induces the association of XIAP with the cleaved fragments of caspase-9 and thereby inhibition of caspase-9 activity. The results also demonstrate that ara-C induces cleavage of procaspase-3 by a caspase-8-dependent mechanism and that XIAP inhibits caspase-3 activity. These results demonstrate that XIAP functions downstream of procaspase-9 cleavage as an inhibitor of both proteolytically processed caspase-9 and -3 in the cellular response to genotoxic stress.

Apoptosis↗
PubMed2000

The effects of polar and/or ionizable residues in the core and flanking regions of hydrophobic helices on transmembrane conformation and oligomerization.

To explore the influence of amino acid composition on the behavior of membrane-inserted alpha-helices, we examined the behavior of Lys-flanked polyleucyl (pLeu) helices containing a single polar/ionizable residue within their hydrophobic core. To evaluate the location of the helices within the membrane by fluorescence, each contained a Trp residue at the center of the sequence. When incorporated into dioleoylphosphatidylcholine (DOPC) model membrane vesicles, pLeu helices with or without a single Ser, Asn, Lys, or Asp residue in the hydrophobic core maintained a transmembrane state (named the N state) at neutral and acidic pH. In this state, the central Trp exhibited highly blue-shifted fluorescence, and fluorescence quenching by nitroxide-labeled lipids showed it located at the bilayer center. A state in which Trp fluorescence red-shifted by several nanometers (named the B state) was observed above pH 10-11. B state formation appears to result from deprotonation of the flanking Lys residues. Despite the red shift in Trp emission, fluorescence quenching showed that in the B state the Trp at most is only slightly shallower than in the N state, suggesting the B state also is a transmembrane or near-transmembrane structure. The B state is characterized by increased helix oligomerization, as shown by the dependence of Trp lambda(max) on the concentration of the peptide within the bilayer at high pH. The pLeu peptide with a Asp residue in the core underwent a pH-dependent transition at a lower pH than the other peptides (pH 8-9). At high pH, it exhibited both a more highly red-shifted fluorescence and shallower Trp location than the other peptides. This state (named the S state) did not exhibit a concentration-dependent Trp lambda(max). We attribute S state behavior to the formation of a charged Asp residue at high pH, and a consequent movement of the Asp toward the membrane surface, resulting in the formation of a nontransmembrane state. We conclude that a polar or ionizable residue can readily be tolerated in a single transmembrane helix, but that the charges on ionizable residues in the core and regions flanking the helix significantly modulate the stability of transmembrane insertion and/or helix-helix association.

Amino Acids↗
PubMed2000

Energetics of DNA intercalation reactions.

Isothermal titration calorimetry has been used to determine the binding enthalpy and heat capacity change (DeltaC(p)()) for a series of DNA intercalators, including ethidium, propidium, daunorubicin, and adriamycin. Temperature-dependent binding enthalpies were measured directly for the ligands, from which DeltaC(p)() values of -140 to -160 cal mol(-)(1) K(-)(1) were calculated. Published van't Hoff plots were reanalyzed to obtain DeltaC(p)() values of -337 to -423 cal mol(-)(1) K(-)(1) for the binding of actinomycin D to several DNA oligonucleotide duplexes with defined sequences. Heat capacity changes for DNA intercalation were found to correlate with the alterations in solvent-accessible surface area calculated from available high-resolution structural data. Multiple linear regression was used to derive the relationship DeltaC(p)() = 0. 382(+/-0.026)DeltaA(np) - 0.121(+/-0.077)DeltaA(p) cal mol(-)(1) K(-)(1), where DeltaA(np) and DeltaA(p) are the binding-induced changes in nonpolar and polar solvent-accessible surface areas (in square angstroms), respectively. The DeltaC(p)() terms were used to estimate the hydrophobic contribution to intercalative binding free energies, yielding values that ranged from -11.2 (ethidium) to -30 kcal mol(-)(1) (actinomycin D). An attempt was made to parse the observed binding free energies of ethidium and propidium into five underlying contributions. Such analysis showed that the DNA binding behavior of these simple intercalators is driven almost equally by hydrophobic effects and van der Waals contacts within the intercalation site.

Animals↗
PubMed2000

Human gamma-globin gene promoter element regulates human beta-globin gene developmental specificity.

The persistence of fetal hemoglobin in many patients with deletion type beta thalassemias and the expression patterns of human globin genes in transgenic mice suggest that gamma- to beta-globin gene switching results primarily from competition of gamma- and beta-globin genes for interaction with the beta-globin locus control region (LCR). To define regulatory sequences that are essential for the competitive advantage of the gamma gene at early developmental stages, stable transgenic mouse lines were produced with LCR gamma-beta constructs containing deletions of gamma 5'-flanking DNA. All constructs contained the full 22 kb LCR, a 4.1 kb beta-globin gene and a gamma-globin gene with 1348, 383, 202, 130, 72 or 52 bp of 5'-flanking sequence. Primer extension analysis of yolk sac, fetal liver and blood RNA from these lines demonstrated that a region between -202 and -130 of the human gamma-globin gene promoter was required to suppress beta-globin gene expression at early developmental stages. Four transcription factor binding sites within this region [GATA(p), Oct1, GATA(d) and CACCC] were mutated independently in LCR gamma-beta constructs and transgenic mouse lines were produced. Only the gamma CACCC box mutation resulted in high levels of beta-globin gene expression in early embryos. These results demonstrate that the CACCC box of the human gamma-globin gene plays a critical role in human beta-globin gene developmental specificity. The data also suggest that gamma CACCC box binding factors mediate LCR-gamma interactions which normally enhance gamma-globin and suppress beta-globin gene expression in fetal erythroid cells.

Animals↗
PubMed2000

The effect of intravenous insulin on accumulation of excitotoxic and other amino acids in the ischemic rat cerebral cortex.

Insulin has been reported to be neuroprotective during cerebral ischemia/reperfusion. However, it may also increase the sensitivity of cultured cortical neurons to glutamate toxicity. The experiments described here utilized a rat four-vessel occlusion model with cerebral cortical windows to determine the effects of intravenous insulin, alone (I) or combined with glucose (IG) to maintain physiologic blood glucose levels, on the extracellular accumulation of amino acids in superfusates of the cerebral cortex. Aspartate, phosphoethanolamine, taurine and gamma-aminobutyric acid were increased in the I and IG groups and glutamate was increased in the IG group compared to controls during ischemia/reperfusion. Insulin treatment attenuated the rebound in cortical superfusate glucose levels in both groups of animals during reperfusion. The increases in amino acid release during reperfusion may be due to a lack of glycolytically derived energy available for amino acid uptake systems and ionic pumps.

Animals↗
PubMed2000

Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with DL-threo-beta-benzyloxyaspartate.

Elevated levels of the excitotoxic amino acids, glutamate and aspartate, have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the contribution of reversed high-affinity, Na(+)-dependent, glutamate transport to the ischemia-evoked release of glutamate and aspartate using DL-threo-beta-benzyloxyaspartate (DL-TBOA), a newly developed competitive, non-transported blocker of the EAAT 1-3 transporters. Changes in the extracellular levels of these and other amino acids, and of glucose and lactate in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical window technique. Basal and ischemia-evoked amino acid, glucose and lactate efflux were compared in control versus DL-TBOA (100 microM; applied topically for 35 min prior to ischemia) animals. Twenty minutes of ischemia caused large increases in aspartate, glutamate, GABA and taurine effluxes into cortical superfusates, with non-significant effects on the efflux of glycine, glutamine, alanine and serine. Application of DL-TBOA caused a 2-fold increase in basal, preischemic, extracellular glutamate levels, but did not affect those of the other compounds. In the presence of DL-TBOA, ischemia-evoked release of aspartate, glutamate, taurine and glutamine was significantly reduced; that of the other amino acids was not affected. The ischemia-evoked declines in glucose were significantly attenuated, and lactate release was enhanced above that in control animals. The amino acid data are interpreted as indicating that aspartate and glutamate releases were reduced as a consequence of DL-TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers, predominantly involving the glial EAAT 2 transporter. The reduction in ischemia-evoked taurine release is interpreted as being due to a decrease in cell swelling prior to and during the initial phase of ischemia due to reduced entry of the Na(+), and other ions, associated with a decreased glutamate uptake. Glucose-sparing and availability for lactate formation would also result from a reduced glutamate/Na(+) uptake. These results indicate that reversed transport, primarily from glial cells by the EAAT 2 carrier, is responsible for a substantial (42 and 56%) portion of the ischemia-evoked increase in extracellular glutamate and aspartate levels, respectively. As a potent, competitive, non-transported blocker of high-affinity, Na(+)-dependent, glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms.

ATP-Binding Cassette Transporters↗