Biomedical subjects
Publications and source records attributed to J Shen.
The possibility of using near-infrared reflectance spectroscopy for the evaluating quality of analgini powder pharmaceuticals was investigated. We used the multivariate statistical classification technique to identify true, poor and false phamaceuticals of analgini from their first derivative spectra successfully. The results are satisfactory.
PURPOSE: The present study was carried out to evaluate a new intravascular contrast agent hydroxyethyl-starch-ferrioxamine (HES-FO) for assessment of myocardial perfusion. MATERIAL AND METHODS: Isolated pig hearts were perfused with a crystalloid cardioplegic solution in a Langendorff apparatus. MR images were acquired along the short cardiac axis using T1- and T2*-weighted methods. Gd-DTPA and HES-FO were used as the standard extracellular and test contrast agents, respectively. RESULTS: We found that T1-weighted signal intensity was not significantly affected by HES-FO, but increased significantly in presence of Gd-DTPA. On the other hand, HES-FO resulted in a rapid, transient but significant decrease in T2*-weighted signal intensity. Although Gd-DTPA also decreased T2*-weighted signal intensity considerably, it took much longer for the T2*-weighted signal intensity to return to its initial steady-state with Gd-DTPA than with HES-FO. Moreover, increasing the dose of HES-FO (from 0.0023-0.0138 mmol/kg b.w.) had no effect on the time at which the T2* effect reached its maximum or on the duration of the T2* effect. However, these times and durations were affected significantly by increasing the dose of Gd-DTPA (0.0023-0.027 mmol/kg b.w.). CONCLUSION: The results suggest that HES-FO provides information regarding myocardial vascular flow which cannot be obtained using Gd-DTPA. It is expected that the combined use of intravascular and extracellular type contrast agents will allow more complete assessment of tissue perfusion.
Brequinar sodium (BQR) was originally developed as an antitumor drug and subsequently as an immunosuppressant for controlling transplant rejection. It has been widely accepted that the antitumor and immunosuppressive activities of BQR are dependent on its ability to inhibit the enzymatic activity of dihydroorotate dehydrogenase, the fourth enzyme in the de novo pyrimidine synthesis pathway. Recently, we discovered that BQR has the ability to inhibit protein tyrosine phosphorylation in anti-CD3-stimulated murine T lymphocytes and to inhibit the activity of src-related protein tyrosine kinases, p56lck and p59fyn. We examined the in vivo activities of BQR in MRL-lpr/lpr mice. We report that the dose of BQR (10 mg/kg/day) that induced anemia, controlled lymphadenopathy and inhibited autoantibody production, also selectively reduced the pyrimidine nucleotide levels in the bone marrow and in the lymph nodes. Coadministration of uridine (1000 mg/kg/day) with BQR completely normalized pyrimidine nucleotide levels in the bone marrow and lymph nodes, and prevented BQR-induced anemia. However, coadministration of uridine with BQR only partially reversed the anti-proliferative effects of BQR, and did not antagonize the inhibitory effect of BQR on autoantibody production. Finally, we report that BQR markedly reduced protein tyrosine phosphorylation in lymph nodes of MRL-lpr/lpr mice. These results collectively suggest that the control of lymphadenopathy and autoantibody production in MRL-lpr/lpr mice by BQR is only partially dependent on inhibition of pyrimidine nucleotide synthesis, and suggest a critical role for in vivo inhibition of protein tyrosine phosphorylation.
We stably transfected a gene encoding a dominant negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) into F9 cells and generated cell lines partially deficient in PKA activity (DN16 and DN19). In these cell lines, the retinoic acid (RA) receptor beta and laminin beta(1) chain (LAMB1) genes were regulated normally by RA alone, indicating that in the absence of exogenous modulation of cAMP levels, the PKA signaling pathway does not seem to play a major role in the RA-associated regulation of these genes. However, alterations in gene regulation were observed when the mutant cell lines were treated with a combination of RA and cAMP analogues. Moreover, in the DN16 cell line, which exhibits the lowest PKA activity among the mutant cell lines [22% of wild type (WT) at 1 microM cAMP], there was a significant decrease in the cAMP-associated activation of the LAMB1 gene DNase I hypersensitivity site 2 enhancer, as measured by chloramphenicol acetyl transferase assays. Using electrophoretic mobility shift assays, less protein binding was observed at one of the motifs (C2) within this enhancer region in the DN16 cells as compared to the F9 WT cells after treatment of the cells with RA and cAMP analogues for 24 h. Furthermore, no increase in C2 binding was observed when extracts from RA-treated F9 ST or DN16 cells were subjected to in vitro phosphorylation, suggesting that PKA is involved in the induction of the C2-binding protein in RA-treated cells. In contrast to the results with RA receptor beta and LAMB1, the effects of cAMP analogues on the RA-associated regulation of the bone morphogenetic protein 2 gene were not altered in the cell lines that exhibited reduced PKA activity. These results suggest that a partial reduction in PKA activity is not sufficient to abrogate the effects of cAMP analogues on all of the genes regulated by RA.
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A case-control study including 214 pedigrees (109 cases and 105 controls) was carried out to explore the etiology of family aggregation of arteriosclerotic cerebral infarction in Yi-Xing city, March 1997. The results showed that the family member in both cases and control groups were comparable. The total prevalence rate of the first-degree relatives in cases was 5.12%, significantly higher than (1.63%) that noticed in controls, with chi 2 = 16.40 (P < 0.01). The same results was also found in their parents and siblings. Results from binominal distribution analysis showed that the number of cases in the families of arteriosclerotic cerebral infarction was significantly higher than the theortical range which indicated that there was significant familial aggregation in the occurance of arteriosclerotic cerebral infarction. Logistic regression model also showed that genetic factor was one of the most important risk factors in Xi-xing, Jiangsu. Dose-response was observed in this model. The heritability and the segregation ratio of arteriosclerotic cerebral infarction were the same as seen in other chronic diseases, such as coronary heart disease and esophageal cancer.
An 1:1 matched case-control study including 105 arteriosclerotic cerebral infarction cases and 105 controls was carried out to estimate the etiologic interaction, in Yi-xing city, March 1997. Results showed that the major risk factors were high levels of serum TG, Lp(a) and BMI while serum level of HDL-C was the only protective factor. When several levels were divided on Lp(a) and BMI, a significantly dose response relation was found. Considering the correlation among serum concentration of lipids, we analysed the interaction of different factors, using Log-Linear models. Results showed that there were significant interactions between arteriosclerotic cerebral infarction and TC, TG, HDL-C and Lp(a). There were also interactions between TC, TG, HDL-C and LDL-C. However, the strongest interaction is expected to happen at what level remains further investigations.
The purpose of this study was to investigate the effect of exogenous nitric oxide (NO) on rabbit carotid sinus (CS) baroreceptor (BR) activity in vitro, especially in regard to determining whether the effect might be mediated by cGMP. The carotid sinus nerve (CSN) basal discharge could be divided into two groups: low-amplitude (LA) and high-amplitude (HA) ones. It was found that the HA discharge depended on superfusing-rate, but was not affected by several types of chemoreceptor stimuli, hence indicating a baroreceptive origin of the HA. Secondly, L-arginine (L-arg, 4.6-5.7 mmol/L) and nitroglycerin (NG, 0.07-0.88 mmol/L), precursor or donor of NO, excitated BR in a dose-dependent manner (P < 0.01). Lastly, inhibition of soluble guanylate cyclase (GC) with methylene blue (MB) affected neither the basal discharge of BR nor the response of BR to L-arg or nitroglycerin (NG).
BALB/c-3T3 cell transformation test was used to detect cell carcinogenicity caused by dihydroxyglyoxime (DHG) and determine its carcinogenicity in vitro. Results showed that DHG could induce transformation of BALB/c-3T3 cells and the transformed cells could agglutinate in the presence of Con A and grew in the soft agar. Transformed cells could induce the formation of fibrosarcoma if they were inoculated subcutaneously into the naked mice. It indicated sufficiently that the transformation in BALB/c-3T3 cell culture was malignant, and also predicted its potential carcinogenicity in human beings.
Oxidative modification of LDL increases its atherogenicity, and 15-lipoxygenase (15-LO) has been implicated in the process. To address this issue, we generated transgenic rabbits that expressed 15-LO in a macrophage-specific manner and studied their susceptibility to atherosclerosis development when they were fed a high-fat, high-cholesterol (HFHC) diet (Teklad 0533 rabbit diet 7009 with 10% corn oil and 0.25% cholesterol) for 13.5 wk. Transgenic and nontransgenic rabbits developed similar degrees of hypercholesterolemia and had similar levels of triglyceride, VLDL, LDL, and HDL. Quantitative morphometric analysis of the aortic atherosclerosis indicated that the transgenic animals (n = 19) had significantly smaller lesion areas (9.8+/-6.5%, mean+/-SD) than their littermate controls (n = 14, 17.8+/-15.0%) (P < 0.05). In a subgroup (n = 9) of transgenic rabbits that received the HFHC diet plus the antioxidant N',N '-diphenyl-phenylenediamine (1%), the extent of lesion involvement (9.8+/-7.5%) did not differ from the subgroup (n = 10) that received the regular HFHC diet (9.7+/-5.9%). Since the results were unexpected, we repeated the experiments. Again, we found that the nontransgenic littermates (n = 12) had more extensive lesions (11.6+/-10.6%) than the transgenic rabbits (n = 13; 9.5+/-7.8%), although the difference was not significant. In a third set of experiments, we crossed 15-LO transgenic rabbits with Watanabe heritable hyperlipidemic (WHHL) rabbits and found that the lesion area in the 15-LO transgenic/heterozygous WHHL rabbits (n = 14) was only about one third (7.7+/-5.7%) that found in nontransgenic heterozygous WHHL littermate controls (n = 11, 20.7+/-19.4%) (P < 0.05). These data suggest that overexpression of 15-LO in monocytes/macrophages protects against lipid deposition in the vessel wall during early atherogenesis in these rabbit models of atherosclerosis.
The slowly activating delayed-rectifier K+ current, I(Ks), modulates the repolarization of cardiac action potentials. The molecular structure of the I(Ks) channel is not known, but physiological data indicate that one component of the I(Ks), channel is minK, a 130-amino-acid protein with a single putative transmembrane domain. The size and structure of this protein is such that it is unlikely that minK alone forms functional channels. We have previously used positional cloning techniques to define a new putative K+-channel gene, KVLQT1. Mutations in this gene cause long-QT syndrome, an inherited disorder that increases the risk of sudden death from cardiac arrhythmias. Here we show that KVLQT1 encodes a K+ channel with biophysical properties unlike other known cardiac currents. We considered that K(V)LQT1 might coassemble with another subunit to form functional channels in cardiac myocytes. Coexpression of K(V)LQT1 with minK induced a current that was almost identical to cardiac I(Ks). Therefore, K(V)LQT1 is the subunit that coassembles with minK to form I(Ks) channels and I(Ks) dysfunction is a cause of cardiac arrhythmia.
The inability to provide an adequate supply of human organs for clinical transplantation has created a strong interest in the use of nonhuman, especially nonprimate, organs. The first biological obstacle confronting such discordant transplantations is a series of violent reactions that result in hyperacute rejection of the xenograft. Significant advances in controlling hyperacute rejection have been achieved recently through the generation of transgenic pig donors bearing human complement regulatory proteins. However, when hyperacute rejection is averted, the xenografts are rejected in 2-70 days in spite of high-dose immunosuppression, by a process collectively termed delayed xenograft rejection. Delayed xenograft rejection is characterized by a refractoriness to conventional immunosuppression, extensive xenoreactive antibody deposition, and cellular infiltration that is dominated by macrophages. We have examined the features of extended host and graft response in the concordant hamster-to-rat xenotransplant model, where such features have historically been obscured by early graft destruction. Hamster hearts transplanted into rats do not encounter hyperacute rejection but are rejected within 3-4 days when xenoreactive antibody titers rise exponentially to levels that elicit a classical antibody- and complement-mediated acute xenograft rejection. We have successfully blocked acute xenograft rejection by a combination of immunosuppressive agents, leflunomide, and cyclosporine. Stopping the immunosuppression resulted in graft rejection that is histologically characterized by extensive xenoreactive antibody deposition and cellular infiltration that is predominantly composed of macrophages. We have noted the similarities between the histopathology of rejection of long-surviving concordant xenografts and that described for discordant xenografts and refer to the process of rejection of concordant grafts that have escaped acute xenograft rejection, delayed xenograft rejection.
Glycogen storage disease type HI (GSD-III), an autosomal recessive disease, is caused by deficient glycogen debranching enzyme (GDE) activity. Most GSD-III patients are GDE deficient in both liver and muscle (type IIIa), and some GSD-III patients have GDE absent in liver but retained in muscle (type IIIb). The molecular basis for this enzymatic variability is largely unknown. In the present study, the analysis of the GDE gene in three GSD-IIIb patients by single-strand conformation polymorphism (SSCP), DNA sequencing, restriction analysis, and family studies, revealed each of them as being a compound heterozygote for two different mutations. The first mutant alleles in all three patients involved mutations in exon 3 at amino acid codon 6 of the GDE protein. Two had an AG deletion at nucleotides 17 and 18 of the GDE cDNA (17delAG) which resulted in change of subsequent amino acid sequence and a truncated protein (25X); the other had a C to T transition at nucleotide 16 of the cDNA which changed a Glutamine codon to a stop codon (Q6X). The 17delAG mutation was also found in 8 of the 10 additional GSD-IIIb patients. The Q6X mutation was found in one of the remaining two GSD-IIIb patients. These two mutations were not found in any of the 31 GSD-IIIa patients, 2 GSD-IIId patients, nor 28 unrelated normal controls. The second mutant alleles in each of the three GSD-IIIb patients were R864X, R1228X, and W68OX. The R864X and R1228X were not unique for GSD-IIIb as they were also found in GSD-IIIa patients (frequency of 10.3% and 5.2% in Caucasian patients, respectively). Our data demonstrated that both IIIa and IIIb had mutations in the same GDE gene and established for the first time the molecular basis of GSD-III that differentially expressed in liver and muscle. The striking and specific association of exon 3 mutations with GSD-IIIb may provide insight into mechanisms controlling tissue-specific expression of the GDE gene. The identification of exon 3 mutations has clinical significance as well because it distinguished GSD-IIIb from IIIa hence permitting diagnosis from a blood sample rather than a more invasive muscle biopsy.
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The voltage-gated cardiac sodium channel, SCN5A, is responsible for the initial upstroke of the action potential. Mutations in the human SCN5A gene cause susceptibility to cardiac arrhythmias and sudden death in the long QT syndrome (LQT). In this report we characterize the genomic structure of SCN5A. SCN5A consists of 28 exons spanning approximately 80 kb on chromosome 3p21. We describe the sequences of all intron/exon boundaries and a dinucleotide repeat polymorphism in intron 16. Oligonucleotide primers based on exon-flanking sequences amplify all SCN5A exons by PCR. This work establishes the complete genomic organization of SCN5A and will enable high-resolution analyses of this locus for mutations associated with LQT and other phenotypes for which SCN5A may be a candidate gene.
Granulocyte colony-stimulating factor (G-CSF) administration decreases tumor necrosis factor(TNF) release, an important mechanism in allograft rejection. to study G-CSF's possible antirejection effects, 30 Lewis rats underwent heart transplantation using Brown-Norway donors and were assigned varying dosages of recombinant human G-CSF (0, 20, 100, 250 and 500 microgram/kg/day) for 14 days following the operation. Recipients receiving 250 microgram/kg/day experienced an improvement in graft survival (12.3+/-4 days vs. 7.0+/-0.6 days, P>0.05, Breslow). In a separate cohort, G-CSF-treated recipients (250 microgram/kg/day x 14) killed at 2,4,and 6 days after transplantation revealed improved serial allograft biopsy grading scores versus untreated controls (P<0.001 stratified Wilcoxon). Significant reduction in serum TNF levels was noted in the G-CSF-treated animals (P<0.025, analysis of variance). These data describe a moderate antirejection effect of G-CSF administration. Inhibition of circulating TNF in the G-CSF-treated recipients may describe a marker or possible mechanism of this antirejection effect.
The concentration of high density lipoproteins (HDL) is inversely related to the risk of atherosclerosis. The two major protein components of HDL are apolipoprotein (apo) A-I and apoA-II. To study the role of apoA-II in lipoprotein metabolism and atherosclerosis, we have developed three lines of C57BL/6 transgenic mice expressing human apoA-II (lines 25.3, 21.5, and 11.1). Northern blot experiments showed that human apoA-II mRNA was present only in the liver of transgenic mice. SDS-polyacrylamide gel electrophoresis and Western blot analysis demonstrated a 17.4-kDa human apoA-II in the HDL fraction of the plasma of transgenic mice. After 3 months on a regular chow, the plasma concentrations of human apoA-II were 21 +/- 4 mg/dl in the 25.3 line, 51 +/- 6 mg/dl in the 21.5 line, and 74 +/- 4 mg/dl in the 11.1 line. The concentration of cholesterol in plasma was significantly lower in transgenic mice than in control mice because of a decrease in HDL cholesterol that was greatest in the line that expressed the most apoA-II (23 mg/dl in the 11.1 line versus 63 mg/dl in control mice). There was also a reduction in the plasma concentration of mouse apoA-I (32 +/- 2, 56 +/- 9, 91 +/- 7, and 111 +/- 2 mg/dl for lines 11.1, 21.5, 25.3, and control mice, respectively) that was inversely correlated with the amount of human apoA-II expressed. Additional changes in plasma lipid/lipoprotein profile noted in line 11.1 that expressed the highest level of human apoA-II include elevated triglyceride, increased proportion of total plasma, and HDL free cholesterol and a marked (>10-fold) reduction in mouse apoA-II. Total endogenous plasma lecithin:cholesterol acyltransferase (LCAT) activity was reduced to a level directly correlated with the degree of increased plasma human apoA-II in the transgenic lines. LCAT activity toward exogenous substrate was, however, only slightly decreased. The biochemical changes in the 11.1 line, which is markedly deficient in plasma apoA-I, an activator for LCAT, are reminiscent of those in patients with partial LCAT deficiency. Feeding the transgenic mice a high fat, high cholesterol diet maintained the mouse apoA-I concentration at a normal level (69 +/- 14 mg/dl in line 11.1 compared with 71 +/- 6 mg/dl in nontransgenic controls) and prevented the appearance of HDL deficiency. All this happened in the presence of a persistently high plasma human apoA-II (96 +/- 14 mg/dl). Paradoxical HDL elevation by high fat diets has been observed in humans and is reproduced in human apoA-II overexpressing transgenic mice but not in control mice. Finally, HDL size and morphology varied substantially in the three transgenic lines, indicating the importance of apoA-II concentration in the modulation of HDL formation. The LCAT and HDL deficiencies observed in this study indicate that apoA-II plays a dynamic role in the regulation of plasma HDL metabolism.
The newly developed techniques of peptide libraries have become a conventional and efficient method in screening ligands of proteins of interest. We present here the successful results of selection of trypsin inhibitors in a phage hexapeptide library. After affinity selection and activity assay, peptide sequences, deduced from DNA sequencing of the phage peptides with the most striking trypsin activity, share some common features with trypsin inhibitors reported. All of the phage peptides selected out and those native and synthetic trypsin inhibitors reported are composed of three parts: (a) positively charged part (Arg, Lys or their analogs); (b) polar part that may form hydrogen bonds with Ser in the active site of trypsin; (c) hydrophobic part that interacts with the nonpolar region of trypsin active site.