Biomedical subjects
Publications and source records attributed to M Lu.
Five flavonoids were isolated from Epimedium wanshanense and identified as sagittatoside B, anhydroicaritin-3-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-rhamnopyranoside, sagittatoside A, ikarisoside B and desmethylanhydroicaritin-3-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L- rhamnopyranoside by means of IR, UV, 1HNMR, 13CNMR, MS and chemical evidence. They were obtained from this plant for the first time.
Enzymatic methylation of adenine underlies a variety of biological regulatory mechanisms in Escherichia coli. We present here structural and thermodynamic characterization of a non-self-complementary DNA decamer duplex containing the dam sequence 5'-GATC in the unmethylated, hemimethylated (both forms), and methylated states. Differential scanning calorimetry measurements show that the free energies for adenine methylation of the decamer duplex are +1.1 and +2.0 kcal/mol for hemimethylation, respectively, and +3.3 kcal/mol for full methylation. In all cases, a large unfavorable enthalpy change is partially compensated by a favorable entropy term. CD spectroscopy indicates an overall conformational difference between the unmethylated decamer duplex and its methylated analogs. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced in the vicinity of the methylation site of the duplex, consistent with loosening of base pairing at this site. Comparison of the scission patterns of these decamer duplexes by the reactive probes methidiumpropyl-EDTA.FeII [MPE.FeII] and CuI(o-phenanthroline)2 [(OP)2CuI] indicates that the methylation site of the decamer duplex represents a site of enhanced reactivity for these agents. On the basis of these thermodynamics and structural features, we suggest that the methylated base pair exists in two different helical states, which require local transient opening of the duplex for interconversion.
Previous attempts to define the oligomeric state of the HIV and SIV envelope glycoproteins have yielded conflicting results. We have produced in Escherichia coli a recombinant model for the ectodomain of the SIV envelope protein gp41 and have identified a small, trimeric subdomain by proteolytic digestion of this gp41 fragment. The subdomain assembles from two peptide fragments, spanning residues 28-80 (N28-80) and residues 107-149 (C107-149) of SIV gp41. Each of these peptides contains a 4,3-hydrophobic repeat, the hallmark of coiled-coil sequences. Upon mixing, the peptides form a highly helical, trimeric complex [3(N+C)] that resists proteolysis and has a melting temperature (Tm) above 90 degrees C in physiological buffer. The N- and C-terminal fragments are antiparallel to each other in the complex, as judged by the observation that digestion of a variant recombinant protein truncated at the amino terminus yields a C-terminal fragment shortened at its carboxy terminus. The N28-80 peptide contains more positions within the heptad repeat than C107-149 that are predominantly hydrophobic, suggesting that N28-80 is buried in the interior of the complex. We propose that the complex consists of a parallel, trimeric coiled-coil of the N-terminal peptide, encircled by three C-terminal peptide helices arranged in an antiparallel fashion, and that this complex forms a core within the gp41 extracellular domain.
Explore the source record for details and available documents.
The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.
Ribosomal protein L11 and an antibiotic, thiostrepton, bind to the same highly conserved region of large subunit ribosomal RNA and stabilize a set of NH4(+)-dependent tertiary interactions within the domain. In vitro selection from partially randomized pools of RNA sequences has been used to ask what aspects of RNA structure are recognized by the ligands. L11-selected RNAs showed little sequence variation over the entire 70 nucleotide randomized region, while thiostrepton required a slightly smaller 58 nucleotide domain. All the selected mutations preserved or stabilized the known secondary and tertiary structure of the RNA. L11-selected RNAs from a pool mutagenized only around a junction structure yielded a very different consensus sequence, in which the RNA tertiary structure was substantially destabilized and L11 binding was no longer dependent on NH4+. We propose that L11 can bind the RNA in two different 'modes', depending on the presence or absence of the NH4(+)-dependent tertiary structure, while thiostrepton can only recognize the RNA tertiary structure. The different RNA recognition mechanisms for the two ligands may be relevant to their different effects on protein synthesis.
In 1978-79 a single-source outbreak of hepatitis C occurred in 2533 women who had received virus-contaminated anti-D immunoglobulin. Children and husbands of 74 women with self-limited, and of 86 women with chronic, hepatitis C were followed up for over 10-15 years. In 3 of 231 investigated children (1.3%) serological evidence for HCV infection was found. However, none of the children developed an apparent or chronic hepatitis. Serum samples of the 94 husbands investigated showed no HCV antibodies or HCV RNA. We consider the risk of intrauterine or perinatal transmission of HCV, as well as that of transmission through close family contacts, to be low. No evidence was found for sexual transmission for women to men.
HOX-11 (TCL-3) is a homeobox proto-oncogene isolated from the breakpoint region of the t(10;14) chromosomal translocation associated with pediatric T-cell acute leukemia. To better understand the transcriptional regulation of the HOX-11 gene in response to extracellular signals, the levels of HOX-11 RNA were examined in normal and leukemic human T cells upon phytohemagglutinin and hematopoietic growth factor stimulation. While individual hematopoietic growth factors tested did not show any effect on HOX-11 gene expression, a drastic increase in HOX-11 RNA was observed under the induction of phytohemagglutinin. In the presence of cycloheximide, a protein synthesis inhibitor, phytohemagglutinin-induced HOX-11 up-regulation was suppressed, indicating that HOX-11 acts as a delayed early response gene which requires protein synthesis. The HOX-11 gene expression was also suppressed by the tyrosine kinase inhibitors tryphostin and lavendustin A. Our data therefore suggest that the delayed early response of HOX-11 up-regulation in T cells requires a tyrosine phosphorylation signal.
Infection with HIV-1 is initiated by fusion of cellular and viral membranes. The gp41 subunit of the HIV-1 envelope plays a major role in this process, but the structure of gp41 is unknown. We have identified a stable, proteinase-resistant structure comprising two peptides, N-51 and C-43, derived from a recombinant protein fragment of the gp41 ectodomain. In isolation, N-51 is predominantly aggregated and C-43 is unfolded. When mixed, however, these peptides associate to form a stable, alpha-helical, discrete trimer of heterodimers. Proteolysis experiments indicate that the relative orientation of the N-51 and C-43 helices in the complex is antiparallel. We propose that N-51 forms an interior, parallel, homotrimeric, coiled-coil core, against which three C-43 helices pack in an antiparallel fashion. We suggest that this alpha-helical, trimeric complex is the core of the fusion-competent state of the HIV-1 envelope.
We have used patch-clamp techniques to study the effects of arachidonic acid (AA) on the activity of the 70-pS K+ channel, the predominant type of the two apical K+ channels operating under physiological conditions in the thick ascending limb (TAL) of the rat kidney. Addition of 5-10 microM AA blocked the activity of the 70-pS K+ channel in both cell-attached and inside-out patches. The inhibitory effect of AA was specific, because application of 10 microM linoleic acid, oleic acid, or palmitic acid failed to mimic the effect of AA. The effect of AA could not be blocked by pretreatment of the TAL tubules with either 5 microM indomethacin (inhibitor of cyclooxygenase) or 4 microM cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC) (inhibitor of lipooxygenase). In contrast, addition of 5 microM 17-octadecynoic acid (17-ODYA), an inhibitor of P450 monooxygenases, abolised the effect of AA on the channel activity, indicating that the effect was mediated by cytochrome P450 metabolites of AA. Addition of 10 nM 20-hydroxyeicosatetraenoic acid (20-HETE), the main metabolite of the cytochrome P450 metabolic pathway in the medullary TAL, mimicked the inhibitory effect of 10 microM AA. However, addition of 100 nM 19-HETE or 17-HETE had no significant effects and 100 nM 20-carboxy AA (20-COOH) reduced the channel activity by only 20%, indicating that the inhibitory effect of 20-HETE was specific and responsible for the action of AA. Inhibition of the P450 metabolic pathway by either 5 microM 17-ODYA or 12, 12-dibromododec-11-enoic acid (DBDD) dramatically increased the channel activity by 280% in cell-attached patches. The stimulatory effect of 17-ODYA or DBDD was not observed in inside-out patches. The results strongly indicate that 20-HETE is a specific inhibitor for the 70-pS K+ channel and may play an important role in the regulation of the K+ channel activity in the TAL.
OBJECTIVE: The effects of perindopril treatment on hypertension development and the lifespan of adult spontaneously hypertensive rats (SHR) were studied. DESIGN: Adult male SHR (aged 15 weeks) were given once a day treatment with 4 mg/kg perindopril by gavage for 12 weeks. Littermates given distilled water were used as controls. The blood pressure and lifespan of these rats were studied. METHODS: The systolic blood pressure (SBP), heart rate and body weight of these rats were measured at regular intervals until they died from natural causes. At necropsy macroscopic and microscopic examinations were made of various organs to determine the cause of death. Serum levels of creatinine, urea and protein were also measured. RESULTS: Perindopril treatment resulted in the normalization of SBP after 2 weeks of treatment. Withdrawal of the treatment after 12 weeks of treatment caused an elevation of SBP, but the blood pressure of the treated SHR had remained in the normotensive range (< or = 150 mmHg). The heart rate and body weight of the SHR were not affected by the treatment. The average lifespan of the SHR was increased by 12 weeks compared with the control rats. The heart weight, brain lesions and arterial lesions were reduced by the treatment. CONCLUSION: A 12-week treatment of adult SHR with perindopril was effective in causing a permanent prevention of hypertension, amelioration of some of the tissue damage associated with hypertension and an increase in the lifespan of these rats.
Hepatitis C virus (HCV) forms complex quasispecies populations which consist of a large number of closely related genetic variants. This genetic heterogeneity may cause antigenic variation or drug resistance. We used heteroduplex analysis by temperature gradient gel electrophoresis (TGGE) to characterize genetic variants of HCV. The high resolution of TGGE was proven by comparison of DNA sequence data of different cDNA clones from the HCV 5'NCR with their corresponding migration pattern in TGGE. Using this method we were able to identify virus variants of the HCV 5'NCR even if they only differed from each other by a single base. HCV populations from three patients with chronic hepatitis C were found to consist of genetic variants, although the degree of the heterogeneity varied. In addition, we compared the genetic heterogeneity of the core and E2 regions of the HCV genome in one patient. Our results demonstrate that TGGE is a useful tool for characterization of the genetic heterogeneity of virus populations in vivo.
Winter flounder renal proximal tubule primary monolayer cultures mounted in Ussing chambers were used to determine the effect of salmon somatolactin (sSL) on transepithelial Pi and Ca2+ transport. sSL stimulated Pi reabsorption in a dose-dependent manner at physiological levels of the hormone (12.5 ng/ml). Net Pi transport was significantly altered by sSL (200 ng/ml) within 2 h after the initial exposure. Ca2+ fluxes were unchanged by the addition of 200 ng/ml sSL. The sSL-induced Pi reabsorption was abolished by 10 microM H-89, a highly specific protein kinase A inhibitor. Moreover the production and release of adenosine 3',5'-cyclic monophosphate were significantly increased after 1 and 2 h of exposure to sSL. The data indicate that sSL directly stimulates net renal Pi reabsorption by an adenosine 3',5'-cyclic monophosphate-dependent pathway. In addition to sSL, flounder SL and rat prolactin greatly, and salmon growth hormone (2.3 micrograms/ml) slightly, increased net Pi reabsorptive flux, whereas salmon prolactin had no effect.
The protective effect of ischemic preconditioning has been confirmed in animal models. In this study we analyzed the clinical data of 110 cases of acute myocardial infarction (77 male, 33 female). Sixty-nine cases (group A) had ischemic manifestations prior to myocardial infarction, while forty one (group B) did not have. Our data showed that the clinical features in group B were quite different as compared with those in group A: (1) larger infarct size (ratio between necrotic size and ischemic size: 71.55 +/- 3.70 to 41.65 +/- 3.96, P < 0.0001); (2) higher peak level of serum cardiac enzymes (CPK: 2085.78 +/- 265.57 to 1329.80 +/- 189.44, P < 0.01; CK-MB: 102.73 +/- 12.47 to 47.38 +/- 8.83, P < 0.01); (3) poorer cardiac function [LVEF < 0.45: 12/41 (29.3%) to 6/69 (8.7%), P < 0.05]; (4) higher incidence of left ventricular aneurysm [9/41 (22.0%) to 3/69 (4.3%), P < 0.05] and (5) higher mortality rate in 4 weeks [6/41 (14.6%) to 2/69 (2.9%), P < 0.05]. The difference between the two groups is statistically significant. In addition, the effect of the duration of preconditioning on protection of myocardium, the potential mechanism of preconditioning and its clinical significance were discussed.
Fourteen patients with aorto-ilio-femoral or ilio-femoral atherosclerotic occlusive disease were treated with aorto-profunda femoris bypass grafting. Excellent results were confirmed after a mean follow-up period of 15 months. Ankle-brachial index raised from 0.1 +/- 0.12 before operation to 0.64 +/- 0.26 months after operation. The authors considered that the profunda femoral artery plays an important role in aorto-femoral arterial reconstruction. It is a good outflow providing arterial blood to severely ischemic legs with occlusion of aorto-ilio-superficial femoral or ilio-superficial femoral arteries.
The growth of plants may be controlled by clipping the aerial part. This may increase the yield of Bupleurum falcatum. Different methods of cultivation may result in different outputs of crude drugs. Compared with land plotting, deep ploughing and high ridging may increase the root weight of one-year-old plant by 28% and 50% respectively.
The effect of electrical stimulation of afferent renal nerve (ARN) on cardiovascular response, the synthesis and release of vasopressin were studied in rabbits. During the course of the experiment, the pathway of ARN to central nerve system was also analyzed. The results showed that electrical stimulation of ARN elicited significantly decrease of mean arteral blood pressure and heart rate as well as inhibition of cervical sympathetic nerve activity. In the event of the above physiological changes, the AVP concentration in supraoptic nucleus (SON), paraventricular nucleus (PVN) and plasma was increased, but that in hypophysis was decreased. Injection of sodium nitropruside (SNP) or AVPa indicated that increase of AVP release was due directly to stimulation of ARN. Nodose ganglionectomy or transversal section of spinal cord at different levels suggested that the main afferent pathway of ARN to higher level of central nerve system entered into spinal cord at T5-L2.
We have previously shown that platelet factor 4 (PF 4) is a potent inhibitor of megakaryocytopoiesis and that it may protect stem cells from 5-fluorouracil (5-FU) cytotoxicity. In the present work, the effects of human PF 4 on megakaryocyte (MK) growth from human CD34+ cord blood (CB) cells were studied in comparison with transforming growth factor beta 1 (TGF-beta 1). Development of MK from CD34+ cells in both plasma clot culture and liquid culture was significantly inhibited by PF 4 (5 micrograms/ml) and TGF beta 1 (1 ng/ml). Inhibition of cell growth by PF 4 was reversible judging from the fact that the CD34+ cells preincubated with PF 4 could regenerate colonies after washing and replating into the cultures. By contrast, TGF-beta 1 pretreated CD34+ cells gave rise to few colonies following replating. Moreover, incubation of CD34+ cells with PF 4 in liquid culture caused an increase in the number of both stem cell factor (SCF)-binding cells and CD34 antigen-bearing cells, and exhibited greater capacity to form MK colonies than control after the treatment of 5-FU. In vivo in mice, twice injections of PF 4 at 40 micrograms/kg with an interval of 6 h followed by one injection of 5-FU at 150 mg/kg resulted in a significant increase in the number of colony-forming cells with high proliferative potential (HPP-CFC) and colony-forming unit-megakaryocyte (CFU-MK) in bone marrow. In exponentially growing human erythroleukemia cells (HEL), the addition of PF 4 prolonged cell cycle progression and therefore resulted in an increased cell population in S phase, as determined by flow cytometric analysis. Different from PF 4, TGF-beta 1 blocked more cells in G 1 phase. These results demonstrate that PF 4 and TFG-beta 1 inhibit MK development from CD34+ CB cells by different mechanisms and suggest that PF 4, unlike TGF-beta 1, exerts its inhibitory effect on cell growth in a reversible and S phasespecific manner by which it protects stem cells and MK progenitor cells from 5-FU cytotoxicity.