Biomedical subjects
Publications and source records attributed to G S Yu.
A snake venom gene TSV-PA was inserted into the donor plasmids pFastBacHTa and expressed in Tn-5B1-4 cells. SDS-PAGE analysis revealed that the molecular weight of expressed product of TSV-PA were 33 kD. It was also proved by Western blot. The result of enzyme activity showed that TSV-PA protein expressed in insect cells had a higher activity.
Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-determining step in mitochondrial fatty acid beta-oxidation. CPT-I has two structural genes (alpha and beta) that are differentially expressed among tissues. Our CPT-Ibeta isolates from a human cardiac cDNA library contained two different extreme 5'-sequences derived from short alternative first untranslated exons that utilize a common splice acceptor site in exon 2. Primer extension identified single dominant start sites for each transcript, and ribonuclease protection assays showed the presence of one 5'-exon in liver, muscle, and heart mRNAs, indicating that the cognate promoter U (upstream/ubiquitous) is active in each of these tissues. By contrast, mRNAs containing the alternative 5'-exon were present only in muscle and heart, indicating a muscle-specific promoter M (muscle). CPT-Ibeta mRNA levels increased markedly in tissues of fasted rats, when circulating free fatty acid concentrations are elevated. Using CPT-Ibeta promoter/reporter transient transfection of murine C2C12 myotubes and HepG2 hepatocytes, fatty acids were found to increase promoter activity in a peroxisome proliferator-activated receptor alpha (PPARalpha)-dependent fashion. A promoter fatty acid response element (FARE) was mapped, mutation of which ablated fatty acid-mediated production of both transcripts. PPARalpha/retinoid X receptor alpha formed specific complexes with oligonucleotides containing the FARE, and anti-PPARalpha antibody shifted nuclear protein-DNA complexes, confirming the role of this factor in regulating the expression of this critical metabolic enzyme gene. The constitutive repressor chicken ovalbumin upstream promoter transcription factor competitively binds at the FARE and modulates fatty acid induction of the promoters.
Midkine (MK) is a neurotrophic and angiogenic growth factor whose expression occurs mainly in fetus. It was reported that MK was present in senile plaques of Alzheimer's disease (AD). To investigate the role of MK during amyloid plaques formation in AD, we examined the in vitro effect of MK on Abeta aggregation and Abeta-induced cytotoxicity. We found that incubation of MK with Abeta resulted in the formation of MK/Abeta complexes. The C-terminus of MK (60-121) played a similar role as the full length MK in complex formation. This interaction of MK and Abeta demonstrated significant inhibition on Abeta self-aggregation. MK also inhibited the cytotoxicity of Abeta on PC12h cells. These findings suggest that MK protects the cells from Abeta-induced cytotoxicity through its complex formation with Abeta. MK is probably expressed to prevent cell death in AD.
Carnitine palmitoyltransferase I (CPT-I) catalyses the rate-determining step in mitochondrial fatty acid beta-oxidation. The enzyme has two cognate structural genes that are preferentially expressed in liver (alpha) or fat and muscle (beta). We hypothesized the existence of additional isoforms in heart to account for unique kinetic characteristics of enzyme activity in this tissue. Hybridization and PCR screening of a human cardiac cDNA library revealed the expression of two novel CPT-I isoforms generated by alternative splicing of the CPT-Ibeta transcript, in addition to the beta and alpha cDNA species previously described. Ribonuclease protection and reverse transcriptase-mediated PCR assays confirmed the presence of mRNA species of each splicing variant in heart, skeletal muscle and liver, with differing relative concentrations in the tissues. The novel splicing variants omit exons or utilize a cryptic splice donor site within an exon. Deduced polypeptide sequences of the novel enzymes include omissions in the region of putative membrane-spanning and malonyl-CoA regulatory domains compared with the previously described CPT-Is, implying that the encoded enzymes will exhibit unique features with respect to outer mitochondrial membrane topology and response to physiological and pharmacological inhibitors.
Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-determining step in mitochondrial fatty acid beta-oxidation. The enzyme has two cognate structural genes (alpha and beta) that are differentially expressed in tissues. We show multiple mature mRNAs in rat heart derived from alternative splicing of CPT-Ibeta transcripts. Two novel messages are deleted for regions of the previously described mRNA that encode membrane-spanning and regulatory domains, suggesting that the cognate isozymes will exhibit unique kinetic characteristics.
A 20-year-old man with a large, asymptomatic mediastinal mass was found to have desmoid-type fibromatosis (DF) by needle biopsy. The tumor arose from the internal periosteum of the sternum and mimicked an anterior mediastinal mass. A wide resection of the sternum, including portions of the clavicles and costal cartilages, and reconstruction with a Gore-Tex soft tissue patch were performed. Although extremely rare, desmoid tumor of the sternum should be considered in the differential diagnosis of anterior mediastinal tumors.
We report a case of a synchronous primary small-cell carcinoma and adenocarcinoma of the esophagus with ectopic gastrin and calcitonin production. The double primary carcinomas appeared to arise from dysplastic Barrett's mucosa in a 43-year-old woman with a long history of reflux esophagitis. She is one of the few reported long-term survivors treated by surgical resection and combination chemotherapy. The histopathology and treatment of this rare tumor are briefly discussed.
We microanalyzed 2,3-dinor-6-keto-prostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha 1) and 11-dehydrothromboxane B2 (11-dehydro-TXB2, 2) in human urine. Samples containing a [2H4]-analogue as an internal standard were extracted by chromatography using Sep Pak tC18 and silica gel. The compounds were then analysed by means of the lactone ring opening reaction and dimethylisopropylsilylation. The conversion of 1 to 1-methyl ester (ME)-propylamide (PA)-9, 12, 15-dimethylisopropylsilyl (DMIPS) ether derivative and of 2 to 1-ME-6-methoxime (MO)-9, 12, 15-tris-DMIPS ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using a DB-5 column. We were able to detect 1 (222-1031 pg/mg creatinine) and 2 (18-155 pg/mg creatinine) in human urine. Furthermore, the thromboxane/prostacyclin (IX/PGI) ratio in the urine of diabetics was higher than that of healthy volunteers. This method can be used to determine the TX/PGI balance in human urine.
We have previously demonstrated that prolonged simulated microgravity (tail-suspension) leads to cardiac alterations with increased resting heart rate, myocardial degradation changes and attenuated myocardial contractility. The present study investigated the potential role of adrenoceptor mechanisms underlying them. Changes of myocardial alpha 1-adrenoceptor (alpha 1-AR) and beta 1-adrenoceptor (beta-AR) in 90-day tail-suspended rats was investigated by the method of radioligand binding assay and application of Scatchard's method. The results showed significantly decreased quantity of specific binding of 125I-BE[2-beta-(4-hydroxy-3-[125I]indophenyl)-ethylaminomethyltetralone] to alpha 1-AR present in membrane derived from ventricular myocardium of the suspended animals, despite the affinity of the alpha 1-AR to 125I-Be was unchanged. But neither the quantity nor the affinity of beta-AR binding to 125I-Pindolol was significantly altered. In addition, the spontaneously beating rate of isolated right atria from tail-suspended animals showed little change in sensitivity and reactivity to the stimulations of graded phenylephrine (alpha-agonist, measured in the presence of beta-antagonist propranolol) and isoproterenol (beta-agonist), compared with the control rats. There were also no obvious differences of the effects of the isoproterenol on the contractility of isolated left ventricular papillary muscles between the two groups. Since myocardial alpha 1-AR mediated-effects include production of cardiac hypertrophy and enhancement of myocardial glucose uptake and glycolysis, the down-regulation of the alpha 1-AR may be a contributor to the cardiac cellular accumulation and the myocardial degradation changes as found in our tail-suspended rats. The data from this study also suggest that the myocardial beta-adrenoceptors are not affected by the prolonged tail-suspension.
A mutation at nucleotide 3243 in the mitochondrial DNA (mtDNA) specifying tRNALeu(UUR) has been shown to be related with diabetes mellitus. Since mtDNA shows heteroplasmy and its composition would change among various tissues, it is important to quantitate the percentage of mutant mtDNA precisely. Here we report a rapid and sensitive method to determine the mutant mtDNA. A fragment of mtDNA containing nucleotide 3243 was amplified by PCR using a rhodamine-labeled primer and the product was digested with ApaI. The PCR product from normal gene was resistant to ApaI digestion, while a mutation from A to G generated an ApaI site in the mutant mtDNA and its PCR product was cleaved into two fragments. The digested product was separated by acrylamide gel electrophoresis and each product was quantitated by a fluorescence analyzer. PCR analysis using standard mixtures of normal and mutant mtDNAs indicated that this method can be used to detect as little as 1% mutant mtDNA.
The effects of long-term beta 1-AR selective antagonist atenolol treatment on beta-adrenoceptor subtypes were studied by radioligand binding assay, function determination and cAMP accumulation measerment in rat heart. The reasults showed that during long-term administration of atenolol: (1) The density of total beta-AR was increased by approximately 57%; the positive inotropic response and cAMP formation induced by activation of beta-AR were also enhanced. (2) The 125I-pindol competitive inhibition curve for CGP20712A showed that there were no significant difference in the percentage of beta 1- and beta 2-AR sites between the atenolol treated rats and the control rats; pA2 values for selective beta 1-AR antagonist CGP20712A and pKB values for selective beta 1-AR antagonist ICI 118, 551 were not significantly different in the two groups. The results suggested that beta 1- and beta 2-adrenoceptors were upregulated not only in density but also in positive inotropic effect to the same extent.
AIM: To determine alterations of subtypes of myocardial adrenoceptors in senescence. METHODS: Heart membrane preparations were made from 3- and 25-month old Wistar rats. Alpha 1- and beta-adrenoceptors were measured by radioligand, 125I-BE2254 and 125I-pindolol, binding assays, respectively. RESULTS: In the old rat heart, alpha 1- and beta-adrenoceptor densities were declined from the young rats of 119 +/- 4 and 45.9 +/- 1.9 pmol L-1 to 70 +/- 6 and 36.4 +/- 1.6 pmol L-1 (P < 0.01), with a greater change in alpha 1-AR and in beta-AR. The ratio of alpha 1A/alpha 1B subtypes was decreased from the young rats of 39/61 to the old rats of 26/74 (P < 0.05). CONCLUSION: The cardiac adrenoceptors are decreased with different extents in the different subtypes in old rats.
The microdetermination of 2,3-dinor-6-ketoprostaglandin F1 alpha (I) in human urine is described. Samples to which the [2H4]-analogue was added as an internal standard were extracted by chromatographic sample preparation using a Bond Elut C18 cartridge and a silica gel column. Conversion of the extracted I into the 1-methyl ester-6-methoxime-9,11,15-trisdimethylisopropylsilyl ether derivative was followed by gas chromatography-high-resolution selected-ion monitoring (GC-HR-SIM). Interfering substances from the urine matrix were eliminated during GC-HR-SIM analysis using a DB-1 column. A good linear response over the range 10 pg-100 ng per tube was demonstrated. Compound I could be detected in the range 26-375 pg/ml of human urine. The proposed method can be applied to the determination of I in human urine.
Natural vibrational optical activity consists of two principal forms. The IR form is known as vibrational circular dichroism (VCD) and is simply the extension of electronic circular dichroism into the IR vibrational region of the spectrum. The Raman form, known as Raman optical activity (ROA), is a new form of optical activity that has no counterpart in the classical forms of optical activity. In this paper, the similarities and differences of the IR and Raman forms of vibrational optical activity will be examined. Although both VCD and ROA were discovered and confirmed in the period from 1973 to 1975, each field has evolved independently with key advances in theoretical description, instrumentation and application coming at different times over the past 20 years. The current relative strengths and weaknesses of VCD and ROA will be discussed, and specific examples of VOA spectra of (-)-alpha-pinene and the amino acid L-alanine, for which overlapping VCD and ROA data are available, will be presented.
We determined the distribution of alpha 1A- and alpha 1B-adrenoceptor subtypes and their functional roles in phenylephrine (PE)-induced positive inotropic responses in rat atrium. Radioligand binding assays in membrane preparations of rat atria showed that 62% of [125I]BE 2254 binding sites were irreversibly inactivated by pretreatment with 20 microM chloroethylclonidine (CEC). Inhibition curves for WB 4101 and 5-methyl-urapidil (5-MU) were better fit by a two-site model, comprising 29-35% high-affinity sites and 65-71% low-affinity sites, suggesting that rat atria contains both alpha 1A and alpha 1B subtypes in a ratio of approximately 1:2. In isolated perfused atria, pretreatment with CEC inhibited the maximum PE-induced positive inotropic response by 55%, and pA2 values for WB 4101 and 5-MU in inhibiting this response were 8.26 +/- 0.4 and 7.85 +/- 0.07, respectively, between the KD values for alpha 1A and alpha 1B subtypes. PE shifted the concentration-contractile response curve for Ca2+ to the left and upward. Pretreatment with CEC completely abolished whereas 1 nM WB 4101 did not alter, the effect of PE on Ca2+ sensitivity. These results demonstrate that both alpha 1A- and alpha 1B- adrenoceptor subtypes are involved in the PE-induced positive inotropic response. Only activation of the alpha 1B subtype potentiates the positive inotropic effect induced by increasing extracellular Ca2+, however, which suggests that the mechanisms involved in the action of the two subtypes may differ at least in part.
The distribution of the alpha 1-adrenoceptor (alpha 1-AR) subtypes and the effects of activation of alpha 1-AR subtypes on the beta-adrenoceptor (beta-AR) mediated positive inotropic response were investigated. The radioligand binding assays indicated that the Bmax and Kd values were 11.7 +/- 18 fmol/mg.protein and 86.0 +/- 9.6 pmol/L respectively. Pretreatment of the preparations with 20 mumol/L chloroethylclonidine (CEC) which inactivated alpha 1B subtype, decreased the Bmax to 45.7 +/- 5.2 fmol/mg.protein (P < 0.01). The inhibition curves of 5-methyl-urapidil were best fitted to two site model and indicated that alpha 1A subtype took 28.5% of total 125IBE specific binding sites. In the functional experiments, norepinephrine (NE) induced a positive inotropic response in a concentration dependent manner by activation of both beta- and alpha 1-AR. The concentration-response curves (CRC) for NE were shifted rightward after the pretreatment of the preparations with 20 mumol/L CEC, but leftward in the presence of 1 nmol/L WB4101. In the presence of 10 mumol/L phentolamine which inactivated both alpha 1-AR subtypes, the CRC for NE were shifted leftward. When alpha 1-AR was activated by phenylephrine the CRC for isoproterenol (selective beta-AR agonist) were shifted rightward. The results suggested that the alpha 1B subtype enhanced while the alpha 1A subtype inhibited the beta-AR mediated positive inotropic response. When both alpha 1A and alpha 1B subtypes were activated simultaneously the alpha 1A subtype showed a dominate role.
In the present study, we found that a novel alpha 1-adrenergic receptor subtype existed in rat heart by radioligand binding assay. This new alpha 1-adrenergic receptor subtype was different from alpha 1A-, alpha 1B-, alpha 1C-, and alpha 1D-subtypes reported recently. It had high affinity for WB4101, as the same of alpha 1A-subtype, but low affinity for 5-methyl-urapidil, and was insensitive to chlorethylclonidine.
Peliosis involving solid internal organs is a rare entity, and it has been reported in association with chronic debilitating diseases. Bacillary angiomatosis (BA), on the other hand, is a recently identified lesion found virtually only in individuals infected by the human immunodeficiency virus. We describe herein two cases of visceral BA and peliosis in human immunodeficiency virus-infected patients. Based on light and electron microscopic findings, we conclude that (1) BA bacilli present in the hepatic sinusoidal endothelial cells, in a suitable host milieu, may be the causative agents of peliosis hepatis; (2) BA bacilli can be found both intracellularly and extracellularly; and (3) peliosis is also identified in association with BA in abdominal lymph nodes.