Biomedical subjects
Publications and source records attributed to X Lu.
The structure-based design and synthesis of a novel class of 2,4-disubstituted thiazoles as Src SH2 inhibitors is described. Initial results are presented, including the X-ray and NMR analysis of one thiazole inhibitor bound to Lck and Src SH2.
We examined the effects of chronic oral L-arginine treatment on endothelial and cardiovascular function in rats with heart failure induced by coronary artery ligation. Both heart failure and sham-operated rats were treated with either L-arginine in drinking water (12.5 or 50 g/l) or water placebo for 8 weeks following surgery. Plasma L-arginine levels in heart failure rats (153 +/- 11 microM) were lower than sham rats (201 +/- 13 microM, P < 0.05). The lower dose L-arginine treatment improved endothelium-dependent relaxation of isolated aortic rings of heart failure rats, while the higher dose of L-arginine treatment did not. Neither low nor high dose of L-arginine treatment improved hemodynamic parameters in heart failure rats. Thus, chronic oral L-arginine treatment at a dose of 12.5 g/l in drinking water improves endothelium-dependent relaxation, but fails to improve in vivo cardiac function in rats with heart failure.
The negative factor (Nef) from human and simian immunodeficiency viruses is important for the pathogenesis of acquired immune deficiency syndrome. Among other targets, it activates the Nef-associated kinase, which is related to the p21-activated kinase. In this study, we demonstrate that Nef activates Ste20, the homolog of p21-activated kinase in Saccharomyces cerevisiae. Nef binds to the adaptor proteins Bem1 and Ste20 via its proline-rich (PXXP) and diarginine (RR) motifs, respectively. These interactions induce the mitogen-activated protein kinase and increase the rates of budding, sizes of cells, and patterns of mating projections. These effects of Nef depend on the small GTPase Cdc42 and guanine nucleotide exchange factor Cdc24. Thus, studies in S. cerevisiae identified specific interactions between Nef and cellular proteins and their associated signaling cascade.
The Src family of nonreceptor tyrosine kinases has been implicated in many signal transduction pathways. However, due to a possible functional redundancy in vertebrates, there is no genetic loss-of-function evidence that any individual Src family member has a crucial role for receptor tyrosine kinase (RTK) signaling. Here we show that an extragenic suppressor of Raf, Su(Raf)1, encodes a Drosophila Src family gene Src42A. Characterization of Src42A mutations shows that Src42A acts independent of Ras1 and that it is, unexpectedly, a negative regulator of RTK signaling. Our study provides the first evidence that Src42A defines a negative regulatory pathway parallel to Ras1 in the RTK signaling cascade. A possible model for Src42A function is discussed.
Hepatic transcription of insulin-like growth factor binding protein-1 (IGFBP-1) is rapidly downregulated by growth hormone (GH) which is also known to induce expression of c-fos and c-jun. Co-expression of c-fos or c-jun in rat hepatocytes, individually or together, suppresses IGFBP-1 promoter activity by approximately 60%. When hepatic nuclear extracts from sham-operated, hypophysectomized (hypox) and GH-treated hypox rats were analyzed by DNase-1 footprinting, differences in the protection pattern were identified in three regions of the IGFBP-1 promoter. F1 corresponding to - 660 to - 640 bp showed acute changes in response to GH administration. In additional regions, F2 and F3, representing - 758 to - 748 bp and - 477 to - 447 bp, respectively, differences were apparent between nuclear extracts from the hypox and sham-operated rats. When F1 and F2 were removed by deletion of the region from - 824 to - 557 bp, the GH response was lost but suppression by co-expression of c-fos and c-jun was preserved. A putative AP-1 binding site was present in the F3 footprint region, however removal of F3 did not affect the GH responsiveness. These data indicate that several distinct sequences, other than the putative AP-1 site are involved in mediating the GH effects on IGFBP-1 transcription.
The corticospinal tract (CST) plays an important role in the control of voluntary movements. Although the development of the CST has been studied extensively in other species, limited information is available on its development in mice. In the present study, the growth of corticospinal axons was characterized in developing mice using Phaseolus vulgaris leucoagglutinin (PHA-L). Our results indicate that the leading CST axons reach the 8th cervical segment at postnatal day (PD) 2, the 7th thoracic segment at PD4, the 13th thoracic segment at PD7, and the 5th lumbar segment at PD9. The arrival of corticospinal axons at the distal lumbar cord at PD9 was further confirmed by retrograde tracing using fast blue (FB). A waiting period of 2-3 days exists after the leading CST axons pass a particular segment before sending collaterals into the gray matter of that segment. The CST continues to increase in size in lower thoracic and lumbar areas up to PD14 when its adult appearance is achieved. In this study, the date of animal's sacrifice was used as the specific postnatal date to demonstrate the growth of the CST. This definition gives a more reliable indication of the exact location of the CST at a specific developmental time point since the CST continues to grow after tracer injections and since the dye is transported much faster than axonal growth. We suggest that these findings can be used as a template for studies on both normal and transgenic mice where some developmental significance is given to the CST.
The distribution of GAP1(IP4BP), a GTPase-activating protein showing high affinity and stereospecificity for inositol 1,3,4,5-tetrakisphosphate (InsP4), was investigated by Western blot and immunohistochemistry of rodent brain with polyclonal antibodies generated against the carboxy-terminus of the cloned protein. GAP1(IP4BP)-like immunoreactivity was found throughout the brain, most notably in the pyriform cortex, neocortex, hippocampus, striatum, and cerebellar cortex. However, the most striking immunolabeling was consistently localized to area CA1 of the hippocampus and the central, medial, and intercalated nuclei of the amygdala. Western blot analysis of the corresponding brain regions corroborated these immunohistochemical observations. The regionally specific expression of GAP1(IP4BP) provides the prerequisite neuroanatomical substrate toward elucidating the functional role of InsP4 and GAP1(IP4BP) in the central nervous system.
The Kaposi's sarcoma-associated human herpesvirus 8 (KSHV/HHV8) encodes a protein similar to cellular cyclins. This cyclin is most closely related to cellular D-type cyclins, but biochemically it behaves atypically in various respects. Complexes formed between the viral cyclin and the cyclin-dependent kinase subunit, cdk6, can phosphorylate a wider range of substrates and are resistant to cdk inhibitory proteins. We show here that the KSHV-cyclin-cdk6 complex phosphorylates p27(Kip) on a C-terminal threonine that is implicated in destabilization of this cdk inhibitor. Expression of the viral cyclin in tissue culture cells overcomes a cell cycle block by p27(Kip). However, full cell-cycle transit of these cells appears to depend on C-terminal phosphorylation of p27(Kip) and seems to involve transactivation of other cellular cyclin-dependent kinases. A p27(Kip)-phosphorylating cdk6 complex exists in cell lines derived from primary effusion lymphoma and in Kaposi's sarcoma, this indicating that virally induced p27(Kip) degradation may occur in KSHV-associated tumours.
Promethazine and thioridazine were separated and detected by capillary electrophoresis with end-column amperometric detection. The influence of pH value on oxidation potential, the peak current and the resolution were studied and the following conditions was selected: 0.03 M Na2HPO4 and 0.015 M citric acid at pH 3.0, detection potential at 1.10 V. The detection limits of these two substances were in the range of 10(-8) mol/l. The linear range spanned two to three orders of magnitude. This method was applied to the detection of promethazine and thioridazine spiked in urine.
p27, cyclin D1, and retinoblastoma (Rb) protein have been demonstrated using immunohistochemistry in 189 cases of primary breast carcinoma with long-term follow-up. There was a statistically significant association between the expression of p27 and both cyclin D1 and the retinoblastoma gene product (pRb), corresponding to their close interactions in regulating the G1/S transition in the cell cycle. Low levels of p27 were seen in high-grade, rapidly proliferating, oestrogen receptor-negative tumours. In univariate analysis, low p27 expression was associated with a reduced relapse-free and overall survival. In multivariate analysis, p27 was not an independent predictor of survival when either histological grade or proliferative activity (S-phase fraction) was included in the model. When the combined expression of p27 and cyclin D1 was related to survival, patients with high levels of p27, regardless of their cyclin D1 status, did well, whilst those with low p27 had a poor outcome. The only exception, in the latter group, was patients with tumours expressing high levels of cyclin D1, who did as well as the high p27 group. We have shown that in clinical material p27 expression is associated with proliferative activity and while univariate analysis shows it to be a significant indicator of prognosis, this significance is lost in multivariate analysis when traditional prognostic factors are included in the model. The interest in p27 expression in mammary carcinoma lies in its behaviour when examined in combination with other G1 cell cycle regulators.
The relationship between bioconcentration factors and molecular connectivity indices was investigated. A regression model was developed using 80 measured BCFs of nonpolar organic pollutants. The five topological parameters used were 1 chi, 2 chi, 3 chi c, 0 chi v and 2 chi v. Modified jackknife tests were applied to examine the robustness of the model by repeatedly removing a set of or a class of compounds from the database. The model was compared with one using Kow as an independent parameter. The mean absolute errors for the 80 compounds studied were 0.288 and 0.302 log-unit for the two models, respectively.
Recent studies have shown that multiple brain areas contribute to different stages and aspects of procedural learning. On the basis of a series of studies using a sequence-learning task with trial-and-error, we propose a hypothetical scheme in which a sequential procedure is acquired independently by two cortical systems, one using spatial coordinates and the other using motor coordinates. They are active preferentially in the early and late stages of learning, respectively. Both of the two systems are supported by loop circuits formed with the basal ganglia and the cerebellum, the former for reward-based evaluation and the latter for processing of timing. The proposed neural architecture would operate in a flexible manner to acquire and execute multiple sequential procedures.
The binding of RB to MDM2 is shown to be essential for RB to overcome both the antiapoptotic function of MDM2 and the MDM2-dependent degradation of p53. The RB-MDM2 interaction does not prevent MDM2 from inhibiting p53-dependent transcription, but the RB-MDM2 complex still binds to p53. Since RB specifically rescues the apoptotic function but not the transcriptional activity of p53 from negative regulation by MDM2, transactivation by wild-type p53 is not required for the apoptotic function of p53. However, an RB-MDM2-p53 trimeric complex is active in p53-mediated transrepression. These data link directly the function of two tumor suppressor proteins and demonstrate a novel role of RB in regulating the apoptotic function of p53.
The p53 tumour suppressor is stabilised following exposure to genotoxic agents, such as gamma-radiation. Cell responses to p53 stabilisation include induction of apoptosis and/or cell cycle arrest. Several studies have suggested that gamma-radiation stabilises p53 by blocking ubiquitin mediated proteolysis. Here we have compared the biological activities of p53 stabilized following exposure to gamma-radiation or treatment with the proteosome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) in MCF7 cells with wild type p53. Stabilisation of p53 by ALLN was reversible and was not blocked by caffeine. Although ALLN was a more effective p53 stabilising agent than gamma-radiation, ALLN was not as effective at inducing cell cycle arrest/apoptosis as gamma-radiation. Although p53 stabilised by ALLN and gamma-radiation were both able to bind DNA and activate transcription, ALLN did not increase expression of BAX, which is involved in p53-induced apoptosis. Therefore, p53 stabilised by different agents is not always biologically active to the same extent and additional alterations triggered by gamma-radiation may enable p53 to activate a subset of critical target genes, such as BAX, which are required for p53 responses.
Heart failure is associated with increased sympathetic nerve activity. We hypothesized that chronic sympathetic stimulation in heart failure resulted in decreased vascular sympathetic responsiveness. A pithed rat model was employed to evaluate peripheral vascular alpha-adrenoceptor and neuropeptide Y (NPY) receptor responsiveness. Heart failure was induced in Sprague-Dawley rats by coronary artery ligation. Sham operated rats (Sham) served as controls. Two months after this surgical procedure, both heart failure (n = 30) and Sham (n = 30) rats underwent standard pithing procedure. Pressor responses to preganglionic sympathetic nerve stimulation (PNS) and activation of postjunctional alpha1- and alpha2-adrenoceptors as well as Y1 receptors were studied. In response to PNS, cardiac index was similar between heart failure and sham rats (P = n.s.). Mean arterial pressure (MAP) increased in a frequency-dependent fashion after PNS in heart failure rats as well as in control rats. All the agonists used, i.e. the alpha1-adrenoceptor agonist phenylephrine, the alpha2-adrenoceptor agonists clonidine and BHT933 as well as NPY, induced dose-dependent increases in MAP in heart failure and in sham rats. However, in rats with heart failure, the response to all the agonists studied was significantly decreased and the dose response curves were shifted to the right (P < 0.01). We conclude that in vivo vascular response to postjunctional alpha1- and alpha2-adrenoceptors as well as Y1 receptors are decreased in rats with heart failure.
To study the role of the inositol 1,3,4,5-trisphosphate-binding protein GAP1(IP4BP) in store-operated Ca2+ entry, we established a human erythroleukemia (HEL) cell line in which the expression of GAP1(IP4BP) was substantially reduced by transfection with a vector containing antisense DNA under control of a Rous Sarcoma virus promoter and the Escherichia coli LacI repressor (AS-HEL cells). Control cells were transfected with vector lacking antisense DNA (V-HEL cells). GAP1(IP4BP) protein, which is a member of the GTPase-activating protein (GAP1) family, was reduced by 85% in AS-HEL cells and was further reduced by 96% by treatment with isopropylthio-beta-D- galactoside to relieve LacI repression. The loss of GAP1(IP4BP) was associated with both a membrane hyperpolarization and a substantially increased Ca2+ entry induced by thrombin or thapsigargin. The activation of intermediate conductance Ca2+-activated K+ channels in AS-HEL cells (not seen in V-HEL cells) was responsible for the membrane hyperpolarization and the enhanced Ca2+ entry, and both were blocked by charybdotoxin. Stimulated V-HEL cells did not hyperpolarize and basal Ca2+ influx was unaffected by charybdotoxin. In V-HEL cells hyperpolarized by removal of extracellular K+, the thapsigargin-stimulated Ca2+ influx was increased. Expression of mRNA for the human Ca2+-activated intermediate conductance channel KCa4 was equivalent in both AS-HEL and V-HEL cells, suggesting that the specific appearance of calcium-activated potassium current (IK(Ca)) in AS-HEL cells was possibly due to modulation of preexisting channels. Our results demonstrate that GAP1(IP4BP), likely working through a signaling pathway dependent on a small GTP-binding protein, can regulate the function of K(Ca) channels that produce a hyperpolarizing current that substantially enhances the magnitude and time course of Ca2+ entry subsequent to the release of internal Ca2+ stores.
The first documented outbreak of human respiratory disease caused by avian influenza A (H5N1) viruses occurred in Hong Kong in 1997. The kinetics of the antibody response to the avian virus in H5N1-infected persons was similar to that of a primary response to human influenza A viruses; serum neutralizing antibody was detected, in general, >/=14 days after symptom onset. Cohort studies were conducted to assess the risk of human-to-human transmission of the virus. By use of a combination of serologic assays, 6 of 51 household contacts, 1 of 26 tour group members, and none of 47 coworkers exposed to H5N1-infected persons were positive for H5 antibody. One H5 antibody-positive household contact, with no history of poultry exposure, provided evidence that human-to-human transmission of the avian virus may have occurred through close physical contact with H5N1-infected patients. In contrast, social exposure to case patients was not associated with H5N1 infection.
Drosophila Src42A, a close relative of the vertebrate c-Src, has been implicated in the Ras-Mapk signaling cascade. An allele of Src42A, Su(Raf)1, dominantly suppresses the lethality of partial loss-of-function Raf mutations. To isolate genes involved in the same pathway where Src42A functions, we carried out genetic screens for dominant suppressor mutations that prevented Su(Raf)1 from suppressing Raf. Thirty-six mutations representing at least five genetic loci were recovered from the second chromosome. These are Drosophila EGF Receptor (Egfr), rolled, Src42A, and two other new loci, one of which was named semang (sag). During embryogenesis, sag affects the development of the head, tail, and tracheal branches, suggesting that it participates in the pathways of Torso and DFGF-R1 receptor tyrosine kinases. sag also disrupts the embryonic peripheral nervous system. During the development of imaginal discs, sag affects two processes known to require Egfr signaling: the recruitment of photoreceptor cells and wing vein formation. Thus sag functions in several receptor tyrosine kinase (RTK)-mediated processes. In addition, sag dominantly enhances the phenotypes associated with loss-of-function Raf and rl, but suppresses those of activated Ras1(V12) mutation. This work provides the first genetic evidence that both Src42A and sag are modulators of RTK signaling.