Biomedical subjects
H Cai
Publications and source records attributed to H Cai.
Human alpha atrial natriuretic peptide fermentation by using a genetically engineered yeast strain.
The genotype of the recombinant yeast strain Y33::YFD71-3 used in this study is alpha, his, leu, ade and suc. Preliminary batch cultures in shaking flasks showed the expression level of atrial natriuretic peptide (ANP) was 1-2 mg/L. Shaking flask cultures were carried out in YG medium which contained glucose, yeast nitrogen base (YNB), and different amounts of adenine, histidine and leucine. When the growth was limited by adenine, protein secreted from Y33::YFD71-3 cells was increased obviously. Adenine became the limiting substrate when the YG medium was supplemented with 5 g/L of casamino acid (CAA) and the level of ANP expression was influenced by the concentrations of adenine, YNB and leucine in the medium. In fed batch cultures carried out in a RIBE-5 fermenter, the cultures were fed with glucose and a mixture of YNB, CAA, adenine, histidine and leucine to improve cell growth and ANP expression, and the maximum ANP concentration in the culture reached 24.8 mg/L.
Dif, a dorsal-related gene that mediates an immune response in Drosophila.
There are striking parallels between the regulation of gene expression along the dorsoventral (DV) axis of Drosophila embryos and lymphoid-restricted expression in the mammalian immune system. Both depend on regulatory factors containing rel domains (dorsal and NF-kappa B) that are controlled at the level of nuclear transport. A novel Rel-containing gene in Drosophila, Dif (dorsal-related immunity factor), provides a potential link between these seemingly disparate processes. Although Dif maps close to dorsal, it does not appear to participate in DV patterning, but instead mediates an immune response in Drosophila larvae. Dif is normally localized in the cytoplasm of the larval fat body, but quickly accumulates in the nucleus upon bacterial infection or injury. Evidence is presented that once in the nucleus, Dif binds to kappa B-like sequence motifs present in promoter regions of immunity genes. These results suggest that mammalian and insect immunity share a common evolutionary origin.
Kinetics of deoxyribonucleotide insertion and extension at abasic template lesions in different sequence contexts using HIV-1 reverse transcriptase.
Deoxyribonucleotide insertion efficiencies were measured opposite site-directed abasic template lesions using human immunodeficiency virus 1 reverse transcriptase (HIV-1RT), and the efficiencies to continue primer synthesis beyond the lesion, by addition of the "next correct" deoxynucleotide, were measured as a function of sequence context. Insertion of purines was favored over pyrimidines, A > G > T approximately C. Primer extension past the lesion occurred by two distinct mechanisms, either by direct or by misalignment extension. An "A-rule" appeared to hold for the case of direct extension, where the abasic template moiety is intrahelical, aligned opposite the primer 3'-terminus. In misalignment extension, the primer terminus is realigned from a site directly opposite the lesion to a new position opposite a neighboring template base 5' to the lesion. Direct extension efficiencies were measured in 16 different configurations, by varying 4 bases at the primer 3'-termini and 4 at the 5'-side (downstream) of the lesion. The predominant order of direct extension was A > G > T approximately C, similar to that observed for insertion. Reduced primer extension rates were not caused by a reduction in HIV-1 RT-DNA binding. Primers terminating in C showed inefficient direct extension, but were readily extended via misaligned configurations. The ratios of direct-to-misalignment extension efficiencies were 27:1, 2.5:1, and 1:25 for A, G, and C opposite the lesion, respectively. For the case of primers terminating in T, misalignment extension was not observed. A striking result was that while primers were extended past an abasic lesion by HIV-1 RT in both direct and misalignment modes, avian myeloblastosis virus RT failed to catalyze significant extension by either mode.
Conversion of a dorsal-dependent silencer into an enhancer: evidence for dorsal corepressors.
In Drosophila, the dorsal (dl) morphogen gradient initiates the differentiation of the embryonic mesoderm and neuroectoderm by activating the expression of regulatory genes (e.g. twist and snail) in a concentration-dependent manner. dl also functions as a repressor that establishes the dorsal epidermis and amnioserosa by restricting regulatory genes such as dpp and zen to dorsal regions of the embryo. The ability of dl to function as both an activator and repressor distinguishes it from the bicoid morphogen, which appears to function solely as an activator. In an effort to determine how dl functions as a repressor we have performed a detailed characterization of a zen silencer element, called the VRE, which mediates ventral repression in response to the dl gradient. A minimal 110 bp VRE sequence is identified, which is able to silence the ventral expression of a heterologous promoter. This sequence contains two dl binding sites as well as binding sites for additional nuclear factors present in early embryos. Mutations in the latter binding sites convert the minimal VRE into an enhancer, which mediates transcriptional activation in ventral regions in response to dl. These results suggest that dl is intrinsically an activator, but is converted into a potent silencer when it interacts with neighboring corepressors.
Continuous percutaneous measurement by laser-Doppler flowmetry of skeletal muscle microcirculation at varying levels of contraction force determined electromyographically.
Laser-Doppler flowmetry (LDF) and electromyography (EMG) were used simultaneously for measuring skeletal muscle blood perfusion in relation to static load and fatigue. Percutaneous single-fibre LDF and bipolar surface EMG of the trapezius muscle were performed continuously during a 10-min series of alternating periods of static contractions and rest, each of 1-min duration. The muscle was exposed to static load expressed as shoulder torque, by keeping the arms straight and elevated at 30, 60, 90 and 135 degrees. On-line computer processing of the LDF and EMG signals made possible the interpretation of the relationship between the perfusion and the activity of the muscle. The LDF and root mean square (rms)-EMG were normalized by using the average value of the serial examinations of each individual as a reference value. Spectrum analyses of EMG showed the lowest variability for median frequency (MDF) in the frequency range 10-1000 Hz and mean power frequency (MPF) at 2-1000 Hz. The LDF power spectrum density during low (muscle rest) and high (high-force muscle contraction) perfusion indicated that disturbances were small when measurements were performed during sustained static contraction with as little movement as possible. Vasomotion, i.e. rhythmic variations in the blood flow, were present and showed a frequency of 5-6 cycles.min-1. Application of a tourniquet to the upper arm caused an arrest of the microcirculation in the distally situated brachioradial muscle which was followed by a postischaemic hyperaemia upon removal of the torniquet.(ABSTRACT TRUNCATED AT 250 WORDS)
Microcirculation in the upper trapezius muscle during varying levels of static contraction, fatigue and recovery in healthy women--a study using percutaneous laser-Doppler flowmetry and surface electromyography.
Microcirculation in the upper portion of the trapezius muscle was measured percutaneously by continuous laser-Doppler flowmetry (LDF) during two 10-min series of alternating 1-min periods of static contraction and rest determined electromyographically (EMG). Stepwise increased contraction was induced by keeping the arms straight and elevated at 30, 60, 90 and 135 degrees, which was repeated with a 1-kg load carried in each hand. Thereafter, fatigue and recovery were recorded while the subject kept her arms straight and elevated at 45 degrees carrying the 1-kg hand load as long as possible, followed by rest with arms hanging and no load. A group of 16 healthy women of different ages was studied. Signal processing was done on line using a 386 SX computer. The LDF- and root-mean-square (rms) EMG signals were normalized. Spectrum analyses of EMG mean power frequency (MPF) and median spectrum frequency were performed. The rms-EMG increased significantly with an increase in the calculated shoulder torque (r = 0.75). Accumulated local fatigue was indicated by a decrease in MPF with increased shoulder angle and added load (r = -0.54). Blood flow increased with increased shoulder angle (r = 0.82, with hand load r = 0.62) and with increased shoulder torque (r = 0.72), and also showed a significant increase with increased EMG activity (r = 0.74). The LDF showed a negative correlation to MPF (r = -0.67), with increased values when MPF was lowered. During the endurance test, a moderate increase of LDF occurred which reached its maximum during the 1st min of recovery.(ABSTRACT TRUNCATED AT 250 WORDS)
The Saccharomyces cerevisiae SDC25 C-domain gene product overcomes the dominant inhibitory activity of Ha-Ras Asn-17.
The carboxy-terminal part of the Saccharomyces cerevisiae SDC25 gene product (SDC25 C domain) can elicit activation of mammalian Ras proteins. Specifically, SDC25 C domain functions as an exchange factor for cellular Ras proteins in CHO cells. In this study, we used the dominant inhibitory Ha-Ras Asn-17 mutant and SDC25 C domain to further investigate the interaction between cellular Ras proteins and their putative endogenous guanine nucleotide-releasing factors. Transcription from the polyomavirus thymidine kinase gene (Py tk) promoter is strongly inhibited by the expression of Ha-Ras Asn-17 in NIH 3T3 cells. Coexpression of SDC25 C domain overcomes the negative effect of the Ras mutant on the Py tk promoter. On the other hand, transactivation of the Ras-responsive element of the Py tk promoter induced by SDC25 C domain is lost upon coexpression of increasing amounts of Ha-Ras Asn-17. In addition, coexpression of SDC25 C domain overcomes the inhibition of proliferation of NIH 3T3 cells caused by Ha-Ras Asn-17. These results are consistent with the idea that the Ha-Ras Asn-17 mutant functions by titrating an upstream activator of cellular Ras proteins.
Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction.
We have investigated the relationship between hydrolysis of phosphatidylcholine (PC) and activation of the Raf-1 protein kinase in Ras-mediated transduction of mitogenic signals. As previously reported, cotransfection of a PC-specific phospholipase C (PC-PLC) expression plasmid bypassed the block to cell proliferation resulting from expression of the dominant inhibitory mutant Ras N-17. In contrast, PC-PLC failed to bypass the inhibitory effect of dominant negative Raf mutants, suggesting that PC-PLC functions downstream of Ras but upstream of Raf. Consistent with this hypothesis, treatment of quiescent cells with exogenous PC-PLC induced Raf activation, even when normal Ras function was blocked by Ras N-17 expression. Further, activation of Raf in response to mitogenic growth factors was blocked by inhibition of endogenous PC-PLC. Taken together, these results indicate that hydrolysis of PC mediates Raf activation in response to mitogenic growth factors.
[Studies of purification, inactivity and immunogenicity of antigenically chimeric poliovirus].
Purified viruses were inactivated with 1:4000 formalin at 37 degrees C for 12d. Before and after inactivation, XF414 with anti-PV-1 and anti-PV-2, XF3 with anti-PV-1 and anti-PV-3 were observed by immunoelectron microscopy and the results were positive. Neutralization inhibition test and D-Ag unit detection were conducted, and the results were as follow: XF414 had type 1 D-Ag 15971 units/ml and type 2 D-Ag 15644 units/ml; XF3 had type 1 D-Ag 13314 units/ml and type 3 D-Ag 15971 units/ml. When rabbits were immunized with XF414 and XF3, they produced bivalent antiserum; i.e. anti-PV-1 and anti-PV-2 induced by XF414 and anti-PV-1 and anti-PV-3 induced by XF3.
[Analysis of fatty acid composition of spotted fever group rickettsiae isolated in China by gas chromatography].
In present paper, fatty acid composition of seven Chinese isolates of SFG rickettsiae and six prototype strains of SFG rickettsiae were analyzed by GC-MS. Tested prototype strains of SFG rickettsiae were R. sibirica (strains 232 and 246), R. conorii (Simko), R. rickettsi (R), R. akari (Kaplan), R. australis (W58); Chinese isolates were An-84, Se-85, W-88 (human strain), MT-84, FT-84 (D. nuttalli strain), TO-85 (ova of nuttalli) and Chinese reference strain -JH-74 (D. nuttalli). They were propagated in yolk sacs of embryonated hen eggs and purified by centrifugation in a 30%-36%-42% discontinuous renografin density gradient. The fatty acid composition of selected strains of SFG rickettsiae was analyzed by gas chromatography, and then comparison being carried out by single linkage on mini-computer. Identification of the strains was performed based on the results obtained from GC-MS. Results showed that the fatty acid profiles of all the isolates from China were quantitatively similar to that of R. sibirica and quite different from other prototype strains of SFG rickettsiae.
Base mispair extension kinetics. Binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini.
We investigate the enzymatic basis for the inefficient extension of single base mismatches by DNA polymerase compared with the extension of correct base pairs. Inefficient mismatch extension could result from either a reduced binding of the enzyme to mispaired versus correctly paired DNA template-primer termini, or from a lowered intrinsic rate of extension of mispairs by a bound enzyme, or from a combination of both factors. Avian myeloblastosis reverse transcriptase is used to measure the affinities (equilibrium dissociation constants) for the four matched and twelve mismatched base pair configurations situated at a primer 3'-terminus. The binding affinities are analyzed by two different assays employing polyacrylamide gels. The first assay uses steady-state kinetics to measure the efficiency of elongating correct and incorrect base pairs and to evaluate the enzyme's dissociation constants for matched and mismatched termini. The estimated KD values obtained in the steady-state analysis fall within a range of approximately 0.1-20 nM. The efficiencies of extending two of the mispairs, G.G and C.C, are too low to allow a determination of KD by the kinetics method. The second assay uses equilibrium binding to measure the ratio of polymerase bound to matched compared with mismatched termini, KDright/KDwrong. The affinity ratios, including values for G.G and C.C mispairs, are in the range of about 0.4-4.2. While around 1 order of magnitude difference is observed in the relative binding affinities of the polymerase for matched and mismatched primer termini, the relative extension efficiencies vary over more than 5 orders of magnitude. Therefore, it appears that inefficient mismatch extension is caused primarily by a kinetic block inhibiting elongation from mispaired primer 3'-termini rather than to a difference in binding.
Towards a Drosophila genome map.
A physical map of the genome of Drosophila melanogaster has been created using 965 yeast artificial chromosome (YAC) clones assigned to locations in the cytogenetic map by in situ hybridization with the polytene salivary gland chromosomes. Clones with insert sizes averaging about 200 kb, totaling 1.7 genome equivalents, have been mapped. More than 80% of the euchromatic genome is included in the mapped clones, and 75% of the euchromatic genome is included in 161 cytological contigs ranging in size up to 2.5 Mb (average size 510 kb). On the other hand, YAC coverage of the one-third of the genome constituting the heterochromatin is incomplete, and clones containing long tracts of highly repetitive simple satellite DNA sequences have not been recovered.
Hydrolysis of phosphatidylcholine is stimulated by Ras proteins during mitogenic signal transduction.
We have used a dominant inhibitory ras mutant (Ha-ras Asn-17) to investigate the relationship of Ras proteins to hydrolysis of phosphatidylcholine (PC) in the transduction of mitogenic signals. Expression of Ha-Ras Asn-17 inhibited NIH 3T3 cell proliferation induced by polypeptide growth factors or phorbol esters. In contrast, the mitogenic activity of PC-specific phospholipase C (PC-PLC) was not inhibited by Ha-Ras Asn-17 expression. Similarly, cotransfection with a cloned PC-PLC gene bypassed the block to NIH 3T3 cell proliferation resulting from expression of the inhibitory ras mutant. Hydrolysis of PC can therefore induce cell proliferation in the absence of normal Ras activity, suggesting that PC-derived second messengers may act downstream of Ras in mitogenic signal transduction. This was substantiated by the finding that Ha-Ras Asn-17 expression inhibited growth factor-stimulated hydrolysis of PC. Taken together, these results indicate that PC hydrolysis is a target of Ras during the transduction of growth factor-initiated mitogenic signals.
[Influence of 5.7-dihydroxytryptamine on electro-acupuncture analgesia and substance P level in central nervous system of the arthralgic rats].
The relation between electroacupuncture (EA) analgesia (A) and substance P (SP) level in the brain stem (BS) and lumbar spinal cord (LSC) of arthralgic rats was investigated. The rats were divided into three groups: 1)5.7-dihydroxytryptamine (5.7-DHT) + EA, 2) vehicle (V) + EA, and 3)5.7-DHT. All the animals were induced arthralgesia by injecting Freund's adjuvant 7 days after cisterna injection of 5.7-DHT or vehicle. The SP level in the BS and LSC was determined by RIA. The results indicated that in V + EA group the EA could prolong tail flick latency by 39.6%, but in other two groups did not. The SP level in LSC of V + EA group (179.1 +/- 11.5 pmol/g) was higher than that in the 5.7-DHT + EA (135.9 +/- 9.3pmol/g) and 5.7-DHT (125.8 +/- 10.0 pmol/g) groups. It suggested that both EA and arthralgia could activate the descending 5-HTergic inhibitory system to inhibit the release of SP in LSC. When the 5-HTergic system was destroyed by 5.7-DHT, the EAA was attenuated, and the SP level in LSC was lowered due to its release was decreased by EA and arthralgia.
[Observations on the changes in plasma pH and kalium content and the effect of acupuncture on them in the rats with hemorrhagic shock].
In the present work, the changes of pH and the kalium content of blood and the effects of acupuncturing "Renzhong" on these changes were observed in rats with hemorrhagic shock. The experimental rats were randomly divided into three groups: surgical control group, shocked control group, shocked acupuncture group. The blood pH was significantly decreased at maintaining shock for one hour (n = 30), P < 0.01; there was no change in surgical control group (n = 15), pH decreased continually with the shock time, (n = 14). But pH tended to going up again after acupuncturing "Renzhong" for 15 min (n = 16). The content of blood kalium was increased at maintainually shock for one hour (n = 26). The content of blood kalium was significantly increased with the prolonging shocked time (n = 12) or acupuncturing "Renzhong" (n = 14), P < 0.05. But there was no clear change in surgical group (n = 15). The results suggested that the acidosis condition was slightly rectified by acupuncture via modulating the respiratory function and reducing the acidic metabolites in blood of the rat with hemorrhagic shock. But the hyperkalemia produced by shock was hardly rectified by acupuncture.
[Pharmacokinetics of liensinine in rabbits].
A method for the determination of plasma concentration of liensinine with HPLC and pharmacokinetic study of liensinine in rabbits were reported. Assay linearity was shown over the range of 0.125-2.5 micrograms/ml serum with regression coefficient of 0.9997. The mean recoveries of liensinine in plasma (within-day and between-days) were all more than 99% of the dose and the coefficients of variation were less than 5%. After a single intravenous injection of liensinine 6mg/kg to rabbit. The pharmacokinetic characteristics were found to fit a two-compartment open model. Important parameters were: t1/2 alpha = 8.303 min t1/2 beta = 129.960min AUC = 132.67 micrograms.min/ml CL = 0.045 L/min Vc = 2.768 L/kg.
Role of Ras in signal transduction from the nerve growth factor receptor: relationship to protein kinase C, calcium and cyclic AMP.
A dominant inhibitory ras mutant (Ha-ras Asn-17) has been used to investigate the role of Ras in nerve growth factor (NGF)-mediated signal transduction in PC12 cells. Expression of Ha-Ras Asn-17 blocks neuronal differentiation of these cells in response to NGF treatment. The Ha-Ras Asn-17 block was bypassed by treatment with NGF plus dibutyryl cAMP or NGF plus the Ca2+ ionophore ionomycin, but not by NGF plus 12-O-tetradecanoyl phorbol acetate (TPA). Direct stimulation of the cAMP or Ca2+ pathways thus appeared to act synergistically with a Ras-independent NGF signaling pathway. This Ras-independent pathway was also distinct from protein kinase C, since its activity was not affected by protein kinase C down-regulation. It thus appears that NGF stimulation generates a Ras-independent intracellular signal that contributes to neuronal differentiation independently of the cAMP, Ca2+ or protein kinase C second messenger systems. Since TPA did not bypass the Ha-Ras Asn-17 block to differentiation, protein kinase C also did not appear to be sufficient for Ras-dependent pathways mediating NGF-induced differentiation. Down-regulation experiments further indicated that protein kinase C was not required for NGF induction of early response genes via either Ras-dependent or Ras-independent pathways. Moreover, the formation of inositol phosphates and mobilization of intracellular calcium in response to NGF was not inhibited in PC12 cells expressing the Ha-Ras Asn-17 protein. Therefore, although calcium was able to bypass the Ha-Ras Asn-17 block to PC12 differentiation, Ras activity was not required for activation of phospholipase C in response to NGF. It thus appears that both Ras-dependent and Ras-independent signaling pathways contribute to NGF-induced PC12 cell differentiation independently of the cAMP, calcium and protein kinase C second messenger systems.