Biomedical subjects
Publications and source records attributed to Q Li.
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BACKGROUND: Many genetic diseases are caused by mutations in ion channel genes. Because type 2 diabetes is characterized by pancreatic beta-cell insensitivity to glucose, the genes responsible for glucose metabolism and calcium signaling in pancreatic beta-cells are candidate type 2 diabetes susceptibility genes. METHODS: We have examined genomic variations in two ion channel genes relevant to the molecular pathology of diabetes mellitus, the Kir6.2 subunit of the ATP-sensitive potassium channel gene and alpha(1D) subunit of the voltage-dependent calcium channel (VDCC) gene among Japanese type 2 diabetic patients. RESULTS: There are two alleles in the Kir6.2 gene: EI, glutamic acid at codon 23 and isoleucine at codon 337 and KV, lysine at codon 23 and valine at codon 337. The allelic frequencies of these polymorphisms are similar in type 2 diabetic patients and normal subjects. We also detected trinucleotide repeat polymorphisms in the amino terminus and the carboxyl terminal region of the alpha(1D) gene. Expansion of the ATG trinucleotide repeat from seven to eight was detected only in type 2 diabetic patients, but the frequency was low and was similar in type 2 diabetic patients and normal subjects. CONCLUSIONS: Although variations of the Kir6.2 and alpha(1D) genes are not associated with the development of common type 2 diabetes, further studies may determine the role of these genomic variations, especially those in the alpha(1D) VDCC gene, in the pathogenesis of certain subsets of type 2 diabetes, or as a co-factor in the polygenic disorder generally.
Hybrids produced by fusing human fetal erythroblasts (HFE) with mouse erythroleukemia (MEL) cells initially produce predominantly or exclusively human gamma-globin and switch to human beta globin expression as time in culture advances. One explanation for the initially predominant expression of gamma-globin gene in these hybrids is the presence of trans-acting factors that activate gamma-globin gene transcription. We used differential display of hybrids before and after the gamma to beta switch as well as fetal liver and adult erythroblasts to identify cDNAs that could be candidates for potential gamma gene activators. Identically sized amplicons which were present in fetal liver erythroblasts and in the hybrids expressing only gamma-globin but were absent in the adult erythroblasts and in the same hybrids after they had switched to beta globin expression were cloned and sequenced. Fifty pairs of cDNAs fitting these criteria were chosen for further analysis. The sequences of the two members of 48 pairs differed from each other, revealing the low efficiency of this experimental approach. One clone pair coded for human proteosome subunit X. The second pair coded for a protein containing an acidic domain in the N-terminus and three consecutive CDC10/SW16/ankyrin repeats in the C-terminus. Transactivation assays in the yeast hybrid system and transient transfection assays in COS cells showed that a potent trans-activating domain resides in the N-terminus of this protein. Northern blot and RT-PCR assays showed that this gene is expressed in several fetal tissues but not in adult tissues. Stable transfection assays provided evidence that the product of this gene may increase the level of gamma mRNA in HFE x MEL cell hybrids that undergo the gamma to beta switch, suggesting that this new gene encodes a protein that may function as gamma gene activator.
The influence of cocaine exposure on serotonergic neurons and postsynaptic 5-HT1A receptor-mediated responses was evaluated by measuring neuroendocrine responses to a serotonin (5-HT) releaser or a 5-HT1A agonist. Male rats received cocaine (15 mg/kg, i.p.) or saline twice daily for 7 days. Forty-two hr after the final cocaine injection, the 5-HT releaser d-fenfluramine (0, 0.2, 0.6, 2, or 5 mg/kg, i.p.) or the 5-HT1A agonist, 8-OH-DPAT (0, 10, 50, 200 or 500 micrograms/kg, s.c.) were administered. Blood samples were then collected for analysis of plasma ACTH, prolactin, and renin concentrations. The ACTH responses to d-fenfluramine and 8-OH-DPAT were inhibited in cocaine pretreated rats. However, the prolactin responses to d-fenfluramine and 8-OH-DPAT were not significantly modified by cocaine exposure. Additionally, the renin response to d-fenfluramine was unaltered by repeated cocaine administration, while 8-OH-DPAT did not alter renin secretion in either pretreatment group. In contrast to published reports which show that cocaine exposure produces supersensitive 5-HT2A and/or 5-HT2C receptor-mediated responses, the present data suggest that repeated cocaine exposure produces subsensitivity to at least some postsynaptic 5-HT1A receptors. Cocaine-induced deficits in the ACTH response to 5-HT releasers may reflect 5-HT1A receptor subsensitivity, but presynaptic deficits cannot be excluded. Examination of the ACTH response to 5-HT1A agonists may represent a valuable approach to determine deficits in 5-HT function in human cocaine abusers.
Oxygen radicals (OR) generated at the time of reflow of the ischaemic myocardium may cause 'reperfusion injury'. To protect myocardium from this injury, it is important to understand how OR cause their deleterious effects on myocytes. Here we describe the basic electrophysiological alterations caused by OR in patch-clamped ventricular myocytes isolated from the rat heart. Oxygen radicals generated by dihydroxyfumarate (DHF) caused a lengthening of action potential duration (APD) and the appearance of arrhythmogenic alterations such as early and delayed afterdepolarizations. Prolongation of APD was accompanied by a reduction in calcium and potassium currents. When intracellular calcium levels were kept constantly low by 500 microM EGTA in the pipette solution, the effects of DHF on action potential duration and the occurrence of early afterdepolarizations were largely prevented. It is concluded that exposure to OR may induce electrophysiological alterations in isolated myocytes. They are related to changes in specific ionic currents and in levels of intracellular calcium.
OBJECTIVE: CYP3A is the major enzyme responsible for metabolism of the calcineurin inhibitors cyclosporine (CsA) and tacrolimus. Our objective was to determine the relationship between genetic polymorphisms of CYP3A5 with respect to interindividual variability in CsA and tacrolimus pharmacokinetics. METHODS: Kidney transplant recipients receiving CsA (n = 137) or tacrolimus (n = 30) were genotyped for CYP3A5*3 and *6 by a PCR/RFLP method. The patients were grouped according to the CYP3A5 genotype. Dose-adjusted trough levels were correlated with the corresponding genotype. RESULTS: At 3, 6, and 12 months, the tacrolimus dose-adjusted trough levels (dose-adjusted C0) showed a statistically significant difference between the group of CYP3A5*3/*3 (n = 19) and the group of CYP3A5*1 allele carriers. The former was higher than the latter. The CsA dose-adjusted C0 and the actual C0 did not display a significant relation (P < .05) between the group of CYP3A5*3/*3 and the group of CYP3A5*1 allele carriers. CONCLUSION: Patients with the CYP3A5*3/*3 genotype require less tacrolimus to reach target concentrations compared to those with the CYP3A5*1 allele.
In this report, we present the production of a dimeric form of anti-CD19 scFv, the FVS191cys (scFv')2. Anti-CD19 scFv FVS191cys was constructed by engineering a cysteine residue at the C terminus of the V1, domain of scFv FVS191. FVS191cys (scFv')2 was formed through a disulfide bond between two FVS191cys molecules. To optimize the yield of FVS191cys (scFv')2, the effects of oxidation time, buffer pH, and temperature on the formation of dimeric scFv were analyzed. Our study indicates that the formation of FVS191cys (scFv')2 is oxidation time- and buffer pH-dependent; a high pH buffer facilitates the formation of disulfide-linked (scFv')2. The maximum yield of FVS191cys (scFv')2 can be achieved when FVS191cys is air-oxidized at 4 degrees C, in buffer with a pH of 8.5-9. The biological activity of FVS191cys (scFv')2 was analyzed by ELISA and an internalization assay. FVS191cys (scFv')2 has a CD19 binding ability similar to that of its parental mAb B43 and is internalized by CD19 positive Nalm 6 cells. This study indicates that FVS191cys (scFv')2 is a potential candidate for tumor diagnosis or therapy.
Our laboratory utilized two methods to produce the anti-CD19 immunotoxin containing a single-chain Fv (scFv) FVS191 and a ricin A chain (RTA). The first method produced the recombinant protein FVS191CDRTA from a fusing gene containing sequences encoding FVS191, catheptsin D proteinase digestion site (CD), and RTA. FVS191CDRTA did not show CD19 antigen binding and cytotoxic activity. The second method generated a disulfide-linked FVS191cys-dgRTA from a FVS191cys, the FVS191 with an additional C-terminal cysteine, and a deglycosylated RTA (dgRTA). The formation of FVS191cys-dgRTA is efficient; up to 70% of the proteins participating in the reaction had formed FVS191cys-dgRTA when the molar ratio of FVS191cys to dgRTA was 1:1. A competitive ELISA assay indicated that FVS191cys-dgRTA and the parental monoclonal antibody B43 possessed comparable CD19 binding abilities. The protein synthesis inhibition assay revealed that FVS191cys-dgRTA was toxic to CD19 positive cell lines, but it was less potent than the intact antibody-conjugated B43-dgRTA, which had an IC50 = 2 x 10(-11) M. 125I-Labeled FVS191 and 125I-labeled B43 were internalized by Nalm-6 cells at 37 degrees C as demonstrated by internalization studies; this result indicates that cross-linking of CD19 antigen is not required for the endocytosis of CD19 and raises the possibility that the lower cytotoxity of FVS191cys-dgRTA is not due to the monovalent binding of CD19 by FVS191cys-dgRTA. Our study with anti-CD19 scFv immunotoxin indicates that the formation of a disulfide-linked scFv immunotoxin is an alternative to the recombinant method of producing scFv immunotoxin.
A total of 30,000 hospitalized surgical patients in 15 hospitals were screened for chronic ulcers. A total of 489 patients with chronic dermal ulcers were found with their major causes of ulceration including traumatic wounds, infections, diabetes mellitus, and venous diseases. Patients with chronic ulcers following trauma and infection comprised 67.48% of the total patient population. The incidence of diabetic ulcers and venous ulcers was 4.91% and 6.54% respectively. Sites of ulceration differed with different etiological factors. The percentage of chronic dermal ulcers in the lower extremities, upper extremities, thorax and abdomen, back, and head was 63.10%, 17.93%, 7.76%, 4.83% and 6.38% respectively. Of the 489 patients with chronic ulcers, 183 were farmers (37.42%), and 131 were workers (26.79%). Chronic dermal ulcers were more common in men than in women, but there was no significant difference in the sex-related prevalence. According to these data from different hospitals, the incidence of chronic ulcers in patients hospitalized for surgery was 1.5% to 3.0%. These data have primarily shown the prevalence and clinical characteristics of chronic ulcers in hospitalized patients in China. These data may not be consistent with reports from other countries. Significant differences in etiological factors of ulceration, professional distribution of patients with chronic dermal ulcers, and treatment methods were found when compared with reports of studies conducted in developed countries. Our results will benefit not only additional basic research, but these data will also be useful in preventing and managing chronic wounds in developing countries.
The regulatory effects of the thyroid hormone on amphibian metamorphosis is mediated by thyroid hormone receptors. Using Xenopus laevis as a model system, we and others have shown that the mRNA levels of thyroid hormone receptors and 9-cis retinoic acid receptors, which form the functional heterodimers with thyroid hormone receptors, are regulated temporally in a tissue-dependent manner so that high levels of their mRNAs are present in an organ when metamorphosis is occurring. By overexpressing thyroid hormone receptors, 9-cis retinoic acid receptors, or both into developing Xenopus embryos, we have shown that both thyroid hormone receptors and 9-cis retinoic acid receptors are required for mediating the effects of thyroid hormone on embryo development and precocious but specific regulation of the genes, which are normally regulated by thyroid hormone during metamorphosis. Analyses of the developmental expression of one class of thyroid hormone response genes, which encode extracellular matrix-degrading metalloproteinases, suggest that extra cellular remodeling plays an important role during tissue remodeling, including cell death (apoptosis) and cell proliferation and differentiation. This effect of extracellular matrix on cell behavior has been supported directly by in vitro primary cell culture experiments, in which intestinal epithelial cells undergo thyroid hormone-induced apoptosis, just like that during natural metamorphosis.
The intermolecular interaction between two types of non nucleoside reverse transcriptase inhibitors (NNRTIs), HEPT and TIBO, and HIV reverse transcriptase receptor (HIVRT) was investigated. The result of docking study showed that two types of NNRTIs presented similar interaction mechanism with HIVRT. The most active compound of every type of inhibitors could form one hydrogen bond with the residue Lys101 and has hydrophobic interaction with residues Tyr181, Tyr188 and Tyr318, etc. Three 3D-QSAR models including two partial correlation models (one for each family of HEPT and TIBO) and a mixed model gathering two families were constructed. Comparative study of these models indicated that the mixed model offered the strongest prediction ability. For this model, the cross-validated q2 values were 0.720 and 0.675, non-cross-validated r2 values were 0.940 and 0.920 for CoMFA and CoMSIA, respectively. It has been validated by using a test set of 27 inhibitors. Compared with previously reported works, our model showed better prediction ability. It could help us to insight the interaction between NNRTIs and HIVRT, and to design new anti-HIV NNRTIs inhibitors.
Several N-3 substituted carboranyl Thd analogs were synthesized. These agents as well as some non-boronated nucleosides were evaluated in phosphoryl transfer assays with recombinant human TK1 and TK2. For some carboranyl thymidine analogs, TK1 phosphorylation rates approached 38% that of thymidine. Their in vitro cytotoxicty appeared to correlate with the TK1 levels in the tested cells. In some cases increased uptake in tumor cell nuclei compared with the surrounding cytoplasm was detected in vitro.
Mechanical strain reportedly stimulates the synthesis of collagen in vascular smooth muscle cells (SMCs). The present study was designed to investigate a possible involvement of angiotensin II (Ang II) and transforming growth factor (TGF)-beta in stretch-induced collagen synthesis of cultured SMCs derived from the rabbit aortic media. SMCs were cyclically stretched at a rate of 10% elongation and 30 cycles/min for 24 h using the Flexercell strain unit (Flexcell International Corp., McKeesport, Pa.). A two-fold increase in collagen synthesis and a concurrent increase in total protein synthesis were noted in stretched SMCs. Concentration of immunoreactive Ang II in the conditioned medium was elevated under the mechanical strain. Stretch-induced collagen and total protein synthesis were inhibited by either a selective antagonist to Ang II (saralasin), an angiotensin I-converting enzyme inhibitor (captopril) or an antisense oligonucleotide for angiotensinogen mRNA. An elevated secretion of TGF-beta, both active and latent forms, was found in the medium of stretched SMCs. Saralasin inhibited the stretch-induced secretion of TGF-beta from SMCs. Stretch-induced collagen and total protein synthesis was further inhibited by either an anti-TGF-beta1 neutralizing antibody or an adenovirus-mediated transfer of a truncated TGF-beta type II receptor. Elevated expression of collagen alpha1(III) chain and TGF-beta1 mRNAs, and its reversal by saralasin were also demonstrated in stretched SMCs. Results indicate that the stretch-induced collagen and total protein synthesis appears to be mediated via an autocrine-paracrine mechanism of Ang II and TGF-beta released from SMCs.