[Study on the expression of T, B lymphocyte antigen and platelet antibodies in patients with platelet transfusion refractoriness].

OBJECTIVE: To explore whether T lymphocytes subgroup, B lymphocytes, platelet antigen CD41a, CD61 or platelet antibodies are involved in the platelet transfusion refractoriness. METHODS: Forty-seven patients diagnosed as platelet transfusion refractoriness and 32 patients that achieved effective platelet therapy were ennolled in this study. Flow cytometry was performed to detect the proportion of cytotoxic T cell (CD3(+)CD4(-)CD8(+)), helper T cell (CD3(+)CD4(+)CD8(-)) and B lymphocytes (CD19 (+) ), and the expression of platelet glycoproteins, including CD41a and CD61, while platelet antibodies were also measured by solid-phase agglutination. RESULTS: The significant lower level of helper T cell (36.60% vs 48.53%), higher level of cytotoxic T cell (53.26% vs 44.02%) and lower cytotoxic/helper T cell ratio (0.85 vs 1.31) were observed in platelet refractoriness group when compared with effective platelet therapy group (P<0.05). However, the significant difference was not observed in B lymphocytes between the two group (3.02% vs 2.85%, P>0.05). Platelet glycoproteins CD41a and CD61 and antibodies were both expressed at high levels in platelet refractoriness group (88.10% vs 51.69%, 88.36% vs 51.83%, 85.37% vs 14.82%, respectively, P<0.05). CONCLUSIONS: Activation of cytotoxic T cells, suppression of helper T cells, higher expression level of platelet glycoproteins CD41a and CD61 as well as the development of anti-platelet antibodies are involved in the immunologic mechanism of platelet refractoriness.

16,17-dihydroxycyclooctatin, a new diterpene from Streptomyces sp. LZ35.

Genome sequence analysis of Streptomyces sp. LZ35 has revealed a large number of secondary metabolite pathways, including a complete gene cluster for the biosynthesis of cyclooctatin. This cluster contains four genes, cotB1-4, located in a 5-kb region. Optimization of fermentation medium for LZ35Δheng (SR107) led to the identification of cyclooctatin (1) and 16,17-dihydroxycyclooctatin (2), a new diterpene. The structures of these substances were elucidated on the basis of 1D-, 2D-NMR, and HRESIMS data. Cytotoxicity against MDA-MB-231 and A549 cell lines was also evaluated. Results demonstrated that gene cluster and pathway analysis are key to guided isolation of new natural products.

Linkage of hypertension to chromosome 2q14-q23 in Chinese families.

OBJECTIVES: To identify chromosome regions containing hypertension susceptibility genes in Chinese. SUBJECTS AND METHODS: A three-stage study was carried out in Chinese siblings ascertained through outpatient clinics. In the first stage, 283 affected sib-pairs from 79 nuclear families were subjected to a genome-wide scan with 240 microsatellite marker loci. The second stage focused on chromosome 2 with additional markers resulting in an average distance of 5 cM and used an independent sample of 637 affected sib-pairs from 161 families. In the third stage, a fine-scale mapping study on the suggestive region was performed in an independent set of 777 affected sib-pairs from 106 families. Fourteen markers were used with an average distance less than 2 cM. Non-parametric linkage analyses (NPL), parametric linkage analyses and transmission-disequilibrium tests were used to assess evidence for linkage and association. RESULTS: Three markers (D2S168 at 27.06 cM, D2S151 at 152.04 cM and D2S142 at 161.26 cM) on chromosome 2 with suggestive linkage to hypertension susceptibility genes were identified in the genome-wide scan. In stage II, the suggestive region around D2S151 and D2S142 was replicated, while the linkage around D2S168 was not. In the stage III fine-scale mapping study, multipoint linkage analyses showed LOD scores greater than 2.0 throughout a region between 157.16 cM and 162.46 cM (all P < 0.001) with a maximum peak of 2.24 (P= 0.00067) at 160.52 cM. We also observed a NPL Z-score peak of 3.27 at 157.55 cM (P= 0.00086). CONCLUSIONS: The results of a suggestive region on chromosome 2q14-q23 (D2S112-D2S2370) were consistent between each of the three studies. Interestingly, this region overlaps a syntenic region that contains blood pressure quantitative trait loci identified in rat models of hypertension. These data suggest that the region near D2S142 and D2S151 deserves to be further screened for hypertension susceptibility genes.

[Determination of sotalol hydrochloride by reversed-phase high performance liquid chromatography].

An RP-HPLC method for determination and detection of sotalol hydrochloride is described. The baseline separation was achieved on ODS column within 15 minutes by using 0.1% HAc-acetonitrile (80:20, V/V) as mobile phase at a flow rate 1.5 mL/min, and the wavelength was set at 227 nm. The linear range was 5-45 mg/L (r = 0.9991) and the limit of detection was 1 mg/L(S/N > 3). The intra-day and inter-day RSDs were 0.20% and 0.93% respectively.

L-phenylalanine and smooth muscle cell proliferation from SHR and WKY rats.

Cell growth and proliferation were evaluated in cultured vascular smooth muscle cells (VSMCs) isolated from spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto (WKY) rat aortae by measuring [3H]-thymidine incorporation into newly synthesized DNA and by determining cell number, respectively. The results showed that in cultures from both rat strains (1) serum-, basic fibroblast growth factor (bFGF)- and thrombin-induced DNA synthesis were inhibited by L-phenylalanine dose-dependently; (2) L-phenylalanine inhibited cell proliferation in response to serum in a concentration-dependent manner; (3) L-phenylalanine inhibited serum-induced proto-oncogene c-fos and c-myc expression; (4) L-tyrosine, L-histidine and D-phenylalanine failed to mimic the inhibitory effect of L-phenylalanine. All these data demonstrate that L-phenylalanine could exert a direct and specific antiproliferative effect on VSMCs suggesting that such effect can account for the antihypertensive action of this amino acid observed in SHR.

Effects of osthole on isolated guinea pig heart atria.

AIM: To study the effects of osthole (Ost) on the isolated guinea pig atria and the relationship between Ost effect and Ca2+. METHODS: Contractions of left atria were induced by electric stimulations. The contractile amplitude of left atria pre- and post-treated with Ost was measured according to the cumulative concentration method, the drug being added at 15 min intervals, the pA2 or pD2' were calculated. It were measured that the effects of Ost to the positive staircase and to the post-rest potential enhancement. The contractile responses were recorded via an auto-equiolibration recording instrument. RESULTS: Ost 10-300 mumol.L-1 and Ver 0.1-30 mumol.L-1 decreased the contractile force and inhibited the isoprenaline-induced restoration of contractile response in the left atria rendered inexcitable by KCl 25 mmol.L-1. Ost and Ver antagonized the CaCl2- and isoprenaline-induced positive inotropic response noncompetively, the pD2' values to Ost were 4.41 +/- 0.13 and 4.90 +/- 0.15, to Ver were 6.53 +/- 0.22 and 6.91 +/- 0.17, respectively. Both of them inhibited the contraction of the left atrium and reversed the frequency-contraction response from positive to negative staircase in the higher dosage (500 and 1 mumol.L-1), but they showed only slight inhibitory effect on post-rest potentiation. CONCLUSION: Ost was similar to, but much less potent than Ver in inhibiting the isolated guinea pig atria.

Expression of protein kinase C gene in the brain and heart of spontaneously hypertensive rats.

1. The aim of this study was to investigate protein kinase C (PKC) gene expression in spontaneously hypertensive rats (SHR). 2. Using the PKC oligodeoxyribonucleotide probes (gamma, epsilon), we detected PKC isoforms gene expression in the heart and brain of 4 and 20 week old SHR with those of age-matched Wistar-Kyoto (WKY) rats by northern blot analysis. 3. In the cerebral cortex, there were significantly increased levels of expression of the Ca2+-dependent isoform PKC-gamma in 4 and 20 weeks SHR compared with that of WKY, while Ca2+ -independent isoform PKC-epsilon did not differ between SHR and WKY. 4. In ventricular myocytes, there was a significant expression of the Ca2+-independent isoform PKC-epsilon in 4 and 20 week old SHR compared with that of WKY, while Ca2+ -dependent isoform PKC-gamma could not be detected in the same extracts of SHR or WKY. 5. We conclude that both of the Ca2+ -dependent and Ca2+ -independent PKC could be involved in the pathogenesis of SHR. Ca2+ -dependent PKC-gamma may be mainly involved in the modulation of blood pressure in the level of the central nervous system, while Ca2+ -independent PKC-epsilon could be related to the genetic myocardial hypertrophy.

[Effect of ligustrazine on isolated myocardial ischemic reperfusion injury in rats].

In recent years, it is believed by some scholars that the injury of myocardial ischemic reperfusion is correlated to the thromboxane A2 (TXA2) released by platelets. In order to explore that whether the myocardial and hemangio-endothelial cells participate in the TXA2 production during the process of reperfusion, the modified Langendorff method was used to establish the model of reperfusing the isolated rat heart. On the other hand, this experiment was also intended to observe the effect of ligustrazine on the injury of myocardial ischemic reperfusion. The results revealed that the level of thromboxane B2 (metabolite of TXA2) and lactic dehydrogenase (LDH) in coronary sinus reflux fluid increased during the process of reperfusion, while the level of 6-keto-PGF1 alpha in the same fluid relatively decreased (P < 0.05). The ratio of TXB2/6-keto-PGF1 alpha was raised. The ligustrazine inhibited the release of TXB2 and LDH, but promoted the production of 6-keto-PGF1 alpha (P < 0.05). The results also proved that the myocardial and hemangio-endothelial cells could synthesize TXA2, and the amount of TXA2 released increased during the reperfusion of ischemic myocardium, which was likely to be the major factor of the injury of ischemic myocardial reperfusion. Ligustrazine plays an important role in protecting the myocardium.

[Effects of osthole on the isolated guinea-pig ileum and taeniae coli].

Effects of osthole (Ost) and verapamil (Ver) were investigated in the isolated guinea-pig ileum and taeniae coli. In the isolated guinea-pig ileum or taeniae coli, Ost and Ver were both found to inhibit the contractions induced by acetylcholine (ACh), histamine (His) and KCl in a dose-dependent manner; and noncompetitively antagonize the CaCl2 cumulative dose-response curves, with pD2 values of 4.41 +/- 0.15 and 7.0 +/- 0.2, respectively. Ost at dose of 100 mumol/L and Ver at 1 mumol/L inhibited CaCl2 0.2 mmol/L induced the contractions of the isolated guinea-pig taeniae coli, and the contractions can be abolished by adding CaCl2 2 mmol/L in Ca(2+)-free medium; both of them showed pronounced inhibitions on intracellular calcium-dependent contractions induced by ACh, but showing no effects on the extracellular calcium-dependent contractions. These results indicate that Ost has calcium antagonistic effect and is similar to Ver in mechanism.

[A study of the effect of fish oils on the risk factors of cardiovascular and cerebrovascular diseases].

In this study 22 normotensive (NT) and 12 essential hypertensive (EHT) subjects were included to test the possibility that fish oils might influence the blood pressure, plasma lipids and fibrinogen as well as the erythrocytic membrane lipid composition and pump activity. Each EHT subject was given capsules containing fish oils (6 g/d) for 18 days. At the end of the study, SBP fell (P < 0.05) and plasma fibrinogen level decreased (P < 0.05), while DBP, TG, TC, and HDL-C showed no significant change. The fatty acid composition of erythrocyte phosphatidyl ethanolamine (PE) and phosphatidyl choline (PC) showed the following changes: in PE, C22:6, C20:5 increased (P < 0.05; P < 0.01), CI18:1 decreased (P < 0.01); in PC C22:6, C22:4 increased (P < 0.05; P < 0.01). The activity of Na+, Ca2+ max, CaM-stimulated Ca2+ pumps increased (P < 0.05; P < 0.01; P < 0.01). As compared to NT, EHT subjects had higher C20:4 in both PE (P < 0.05) and PC (P < 0.01) and lower C18:2 in PC. It is also shown in this study that EHT subjects have higher C20:4 in both PE (P < 0.05) and PC (P < 0.01) and lower C18:2 in PC as compared to NT ones.

[Determination of m-nifedipine and its pharmacokinetic study in rabbits by high-pressure liquid chromatography].

A high-pressure liquid chromatographic method was developed for determination of m-nifedipine in plasma using a chemical bonded C-18 phase column (YWG-C18 10 microns, made in China) with nitrendipine as internal standard. To increase life of the YWG-C18 column a mixture of methanol and 5 mmol.L-1 phosphate buffer (70:30 vol/vol) was selected as mobile phase with a flow rate of 0.8 ml.min-1. The method was sensitive to m-nifedipine 3 ng.ml-1 plasma and the standard curve was linear from 10 to 1000 ng.ml-1 with correlation coefficient of 0.99. The within-day and day-to-day precisions (CV) of this method were 4.5% and 7.0%, respectively, with recoveries of 95-102% (10-1000 ng.ml-1). There was no interference with nifedipine, amiodarone, propranol, and verapamil. A pharmacokinetic study on m-nifedipine was carried out in 8 rabbits. A better computer fitted to a two-compartment model was observed using 3P87 program. The parameters obtained were as follow: Vc 6.3 L.kg-1, Cl 0.021 L.kg-1.min-1, T1/2 alpha 30 min, T1/2 beta 230 min, AUC 102 micrograms.min.ml-1.

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph of Escherichia coli. The gene was highly expressed in E. coli due to the use of the high-copy number vector pUC18. The P. aeruginosa cyclohexadienyl dehydratase expressed in E. coli was purified to electrophoretic homogeneity. The latter enzyme exhibited identical physical and biochemical properties as those obtained for cyclohexadienyl dehydratase purified from P. aeruginosa. The activity ratios of prephenate dehydratase to arogenate dehydratase remained constant (about 3.3-fold) throughout purification, thus demonstrating a single protein having broad substrate specificity. The cyclohexadienyl dehydratase exhibited Km values of 0.42 mM for prephenate and 0.22 mM for L-arogenate, respectively. The pheC gene was 807 base pairs in length, encoding a protein with a calculated molecular mass of 30,480 daltons. This compares with a molecular mass value of 29.5 kDa determined for the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the native molecular mass determined by gel filtration was 72 kDa, the enzyme probably is a homodimer. Comparison of the deduced amino acid sequence of pheC from P. aeruginosa with those of the prephenate dehydratases of Corynebacterium glutamicum, Bacillus subtilis, E. coli, and Pseudomonas stutzeri by standard pairwise alignments did not establish obvious homology. However, a more detailed analysis revealed a conserved motif (containing a threonine residue known to be essential for catalysis) that was shared by all of the dehydratase proteins.

Effects of isocorydine on action potentials in isolated canine Purkinje fibers and ventricular muscles.

Standard microelectrode techniques were used to study the effects of isocorydine (Isoc) on potential characteristics of canine cardiac Purkinje fibers (PF) and ventricular myocardium (VM) in vitro. In PF, the action potential durations (APD), APD50, and APD90, were prolonged at 3 mumol.L-1 but shortened at 30 mumol.L-1 by Isoc. The action potential amplitude (APA) and the maximal upstroke velocity (Vmax) were decreased at 100 mumol.L-1. In VM, the action potential characteristics were changed by Isoc at above 30 mumol.L-1. APD50 was shortened by APD90 was prolonged. Vmax were decreased at 30 mumol.L-1. The effective refractory period (ERP) was prolonged by Isoc in PF and VM. The results suggest that Isoc may interfere with K+, Na+, and Ca2+ currents in myocardiac cell membrane at different concentrations.

A single cyclohexadienyl dehydratase specifies the prephenate dehydratase and arogenate dehydratase components of one of two independent pathways to L-phenylalanine in Erwinia herbicola.

Dual biosynthetic pathways diverge from prephenate to L-phenylalanine in Erwinia herbicola, the unique intermediates of these pathways being phenylpyruvate and L-arogenate. After separation from the bifunctional P-protein (one component of which has prephenate dehydratase activity), the remaining prephenate dehydratase activity could not be separated from arogenate dehydratase activity throughout fractionation steps yielding a purification of more than 1200-fold. The ratio of activities was constant after removal of the P-protein, and the two dehydratase activities were stable during purification. Hence, the enzyme is a cyclohexadienyl dehydratase. The native enzyme has a molecular mass of 73 kDa and is a tetramer made up of identical 18-kDa subunits. Km values of 0.17 mM and 0.09 mM were calculated for prephenate and L-arogenate, respectively. L-Arogenate inhibited prephenate dehydratase competitively with respect to prephenate, whereas prephenate inhibited arogenate dehydratase competitively with respect to L-arogenate. Thus, the enzyme has a common catalytic site for utilization of prephenate or L-arogenate as alternative substrates. This is the first characterization of a purified monofunctional cyclohexadienyl dehydratase.

[Effects of metoclopramide on slow response action potentials of myocardium].

The effects of metoclopramide (Met) on the action potential of rabbit sinus node cells and slow response action potential of guinea pig papillary muscles were studied with the intracellular microelectrodes. Met 10 mumol.L-1 prolonged the action potential duration at 90% repolarization (APD90) of SA node cell and sinus cycle length (SCL). Met 100 mumol.L-1 caused a decrease in action potential amplitude (APA), Vmax and the slope of phase 4 of action potential of SA node cell. APD90 and SCL were prolonged further. For slow response action potential induced by KCl (25 mmol.L-1), Met 100 mumol.L-1 produced a decrease in APA and depolarization rate. Met 10 mumol.L-1 began to suppress the spontaneous electrical activities induced by barium ion. These findings suggest that Met probably has the effect of blocking calcium inward current in myocardium.

[Effects of metoclopramide on Vmax of action potentials in guinea pig papillary muscles].

The effects of metoclopramide (MCP) on Vmax of action potentials of guinea pig papillary muscles were studied with intracellular microelectrodes. MCP 10-550 mumol/L abbreviated the action potential duration at 90% repolarization (APD90) and decreased the maximal rate of rise of action potential (Vmax) dose-dependently. MCP caused rate-dependent and voltage-dependent reductions of Vmax and retardation of the recovery of Vmax. The results present the possible causes of depressant effect of MCP on Na channel.

[Effects of isocorydine on contraction of the rabbit oviduct smooth muscle].

On isolated isthmus of oviduct of the rabbit, Isocorydine (Isoc) (3 mu mol/L) decreased significantly the frequency of spontaneous contraction and muscle tension, but not the amplitude of contraction. The tension and frequency of spontaneous contraction can be suppressed by Isoc at concentrations from 3 to 300 mumol/L. The amplitude of contraction was decreased only at 300 mu mol/L. It is suggested that the frequency and tension of spontaneous contraction of isthmic muscle are more sensitive to Isoc than the amplitude. Isoc antagonized the norepinephrine-induced contraction of the oviduct. The transport of ova through the oviduct reduced by hCG could be delayed by Isoc.

Relationship between dietary factors and blood pressure in China. The Sino-Japan CARDIAC Cooperative Research Group.

As part of the international cooperative Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study, we carried out surveys for the relationship of dietary factors to blood pressure (BP) in 10 areas in China, (Altai, Beijing, Guangzhou, Guiyang, Hetian, Lhasa, Shanghai, Shijiazhuang, Tulufan, and Urumqi). Systolic BP and diastolic BP were significantly positively associated with salt excretion and body mass index. However, 3-methylhistidine divided by creatinine, and taurine divided by urea nitrogen in 24-h urine were significantly negatively associated with both BPs. These results suggest that meat protein intake may beneficially influence BP, whereas salt may adversely affect BP.

[Free calcium in platelets and the serum in essential hypertension].

Intracellular free calcium of platelets was studied in 45 patients with essential hypertension and in 6 patient's normotensive offspring. Intracellular free calcium of platelets in hypertensive patients was significantly higher than those in normotensive subjects (223 +/- 26 vs 170 +/- 28 nmol/lL, P less than 0.001). There was a positive correlation between intracellular free calcium of platelets and blood pressure (r = 0.653 approximately 0.729, P less than 0.001). No increase of intracellular free calcium of platelets was found in patient's normotensive offspring. 33 patients also had serum free calcium measured simultaneously. There was no difference between patients and the normotensive control (1.10 +/- 0.15 vs 1.09 +/- 0.10 mmol/L, P greater than 0.05). However, a positive correlation between serum free calcium and blood pressure (r = 0.363, P less than 0.05) and a weak positive correlation between serum free calcium and intracellular free calcium of platelets (r = 0.337, r 0.05 = 0.344) were found. These results indicated that intracellular free calcium was involved in the regulation of blood pressure, it was postulated that the intracellular free calcium of smooth muscle cell in the peripheral arteriole with resistance might have the similar changes seen in platelets. The changes of intracellular free calcium of platelets might be valuable in the prevention and treatment of hypertension and its vascular complications.

[Hemodynamic effects of zinc sulfate on anesthetized open-chest dogs].

ZnSO4 3-5 mg/kg iv decreased systolic blood pressure, diastolic blood pressure and mean arterial blood pressure, +dP/dtmax, -dP/dtmax, VCE- +dP/dtmax, and left ventricular systolic pressure. However, ZnSO4 had no significant effects on T values and total peripheral resistance. In addition, ZnSO4 obviously decreased heart rate and cardiac index. These results suggest that iv ZnSO4 may cause hypotension via cardiac inhibition.