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Biomedical subjects

S Xu

Publications and source records attributed to S Xu.

At least 325 records · Page 18Linked to original sources
PubMed1997

Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae.

The class B, type I scavenger receptor, SR-BI, was the first molecularly well defined cell surface high density lipoprotein (HDL) receptor to be described. It mediates transfer of lipid from HDL to cells via selective lipid uptake, a mechanism distinct from receptor-mediated endocytosis via clathrin-coated pits and vesicles. SR-BI is expressed most abundantly in steroidogenic tissues (adrenal gland, ovary), where trophic hormones coordinately regulate its expression with steroidogenesis, and in the liver, where it may participate in reverse cholesterol transport. Here we have used immunochemical methods to study the structure and subcellular localization of murine SR-BI (mSR-BI) expressed either in transfected Chinese hamster ovary cells or in murine adrenocortical Y1-BS1 cells. mSR-BI, an approximately 82-kDa glycoprotein, was initially synthesized with multiple high mannose N-linked oligosaccharide chains, and some, but not all, of these were processed to complex forms during maturation of the protein in the Golgi apparatus. Metabolic labeling with [3H]palmitate and [3H]myristate demonstrated that mSR-BI was fatty acylated, a property shared with CD36, another class B scavenger receptor, and other proteins that concentrate in specialized, cholesterol- and glycolipid-rich plasma membrane microdomains called caveolae. OptiPrep density gradient fractionation of plasma membranes established that mSR-BI copurified with caveolin-1, a constituent of caveolae; and immunofluorescence microscopy demonstrated that mSR-BI colocalized with caveolin-1 in punctate microdomains across the surface of cells and on the edges of cells. Thus, mSR-BI colocalizes with caveolae, and this raises the possibility that the unique properties of these specialized cell surface domains may play a critical role in SR-BI-mediated transfer of lipids between lipoproteins and cells.

Acylation↗
PubMed1997

Reconstitution of mitogen-activated protein kinase phosphorylation cascades in bacteria. Efficient synthesis of active protein kinases.

Mitogen-activated protein (MAP) kinase pathways include a three-kinase cascade terminating in a MAP kinase family member. The middle kinase in the cascade is a MAP/extracellular signal-regulated kinase (ERK) kinase or MEK family member and is highly specific for its MAP kinase target. The first kinase in the cascade, a MEK kinase (MEKK), is characterized by its ability to activate one or more MEK family members. A two-plasmid bacterial expression system was employed to express active forms of the following MEK and MAP kinase family members: ERK1, ERK2, alpha-SAPK, and p38 and their upstream activators, MEK1, -2, -3, and -4. In each kinase module, the upstream activator, a constitutively active mutant of MEK1 or MEKK1, was expressed from a low copy plasmid, while one or two downstream effector kinases were expressed from a high copy plasmid with different antibiotic resistance genes and origins of replication. Consistent with their high activity, ERK1 and ERK2 were doubly phosphorylated on Tyr and Thr, were recognized by an antibody specific to the doubly phosphorylated forms, and were inactivated by either phosphoprotein phosphatase 2A or phosphotyrosine phosphatase type 1. Likewise, activated p38 and alpha-stress-activated protein kinase could also be inactivated by either phosphatase, and alpha-stress-activated protein kinase was recognized by an antibody specific to the doubly phosphorylated forms. These three purified, active MAP kinases have specific activities in the range of 0.6-2.3 micromol/min/mg. Coexpression of protein kinases with their substrates in bacteria is of great value in the preparation of numerous phosphoproteins, heretofore not possible in procaryotic expression systems.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

MAP kinase- and Rho-dependent signals interact to regulate gene expression but not actin morphology in cardiac muscle cells.

Post-natal growth of cardiac muscle cells occurs by hypertrophy rather than division and is associated with changes in gene expression and muscle fiber morphology. We show here that the protein kinase MEKK1 can induce reporter gene expression from the atrial natriuretic factor (ANF) promoter, a genetic marker that is activated during in vivo hypertrophy. MEKK1 induced both stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) activity; however, while the SAPK cascade stimulated ANF expression, activation of the ERK cascade inhibited expression. C3 transferase, a specific inhibitor of the small GTPase Rho, also inhibited both MEKK- and phenylephrine-induced ANF expression, indicating an additional requirement for Rho-dependent signals. Microinjection or transfection of C3 transferase into the same cells did not disrupt actin muscle fiber morphology, indicating that Rho-dependent pathways do not regulate actin morphology in cardiac muscle cells. While active MEKK1 was a potent activator of hypertrophic gene expression, this kinase did not induce actin organization and prevented phenylephrine-induced organization. These data suggest that multiple signals control hypertrophic phenotypes. Positive and negative signals mediated by parallel MAP kinase cascades interact with Rho-dependent pathways to regulate hypertrophic gene expression while other signals induce muscle fiber morphology in cardiac muscle cells.

3T3 Cells↗
PubMed1997

Overexpression of BsoBI restriction endonuclease in E. coli, purification of the recombinant BsoBI, and identification of catalytic residues of BsoBI by random mutagenesis.

BsoBI is a type II restriction enzyme found in Bacillus stearothermophilus JN209 that recognizes the symmetric sequence 5'-CYCGRG-3' (Y=C or T; R=A or G) and cleaves between the first and second base to generate a four-base 5' extension. The cloning and sequencing of BsoBI restriction-modification system has been described by Ruan et al. [Mol. Gen. Genet. 252 (1996) 695-699]. Here we report the overexpression of BsoBI restriction endonuclease gene in E. coli by insertion of the endonuclease gene into an expression vector pRRS. The recombinant BsoBI was purified to homogeneity and its N-terminus sequence was determined. It has the same N-terminal aa sequence as the native enzyme. The constitutive expression of BsoBI from pRRS is lethal to E. coli in the absence of the cognate methylase. The bsoBIR gene was mutagenized with either hydroxylamine or by error-prone polymerase chain reaction in vitro and transferred into E. coli via plasmid vectors in the absence of the cognate methylase. Surviving transformants were selected that carry BsoBI variants which lost endonuclease activity. DNA sequencing of the mutant alleles revealed that G123, D124, D212, D246, E252 and H253 are important residues for enzymatic activity. An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-deficient variants. Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D246N, D246G and E252K can still bind DNA despite the loss of cleavage activity. Thus, residues D124, D212, D246 and E252 may be located near or within the catalytic center, and are likely involved in metal ion binding.

Amino Acid Sequence↗
PubMed1997

NIK is a new Ste20-related kinase that binds NCK and MEKK1 and activates the SAPK/JNK cascade via a conserved regulatory domain.

Nck, an adaptor protein composed of one SH2 and three SH3 domains, is a common target for a variety of cell surface receptors. We have identified a novel mammalian serine/threonine kinase that interacts with the SH3 domains of Nck, termed Nck Interacting Kinase (NIK). This kinase is most homologous to the Sterile 20 (Ste20) family of protein kinases. Of the members of this family, GCK and MSST1 are most similar to NIK in that they bind neither Cdc42 nor Rac and contain an N-terminal kinase domain with a putative C-terminal regulatory domain. Transient overexpression of NIK specifically activates the stress-activated protein kinase (SAPK) pathway. Both the kinase domain and C-terminal regulatory region of NIK are required for full activation of SAPK. NIK likely functions upstream of MEKK1 to activate this pathway; a dominant-negative MEK kinase 1 (MEKK1) blocks activation of SAPK by NIK. MEKK1 and NIK also associate in cells and this interaction is mediated by regulatory domains on both proteins. Two other members of this kinase family, GCK and HPK1, contain C-terminal regulatory domains with homology to that of NIK. These findings indicate that the C-terminal domain of these proteins encodes a new protein domain family and suggests that this domain couples these kinases to the SAPK pathway, possibly by interacting with MEKK1 or related kinases.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Cloning and expression of AatII restriction-modification system in Escherichia coli.

The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli. The nucleotide sequences of aatIIM and aatIIR genes were determined. The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa. The two genes overlap by 4 base pairs and are transcribed in the same orientation. The aatIIRM genes are located next to a putative gene for plasmid mobilization. A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid. The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter. The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns.

Acetobacter↗
PubMed1997

Regulation of cytokine expression in macrophages and the Langerhans cell-like line XS52 by calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP) inhibits antigen presentation by Langerhans cells (LC) and macrophages, and LC are anatomically associated with CGRP-containing epidermal nerves. To determine whether CGRP may produce some of its functional effects through regulation of cytokine expression, we utilized enzyme-linked immunosorbent assay (ELISA) of conditioned supernatants to examine production of interleukin (IL)-10 and IL-1 beta protein in the LC-like cell line XS52 as well as the reverse transcriptase-polymerase chain reaction (RT-PCR) to examine levels of mRNA for IL-10, IL-1 beta, and the 40-kDa subunit (p40) of IL-12. CGRP augmented the lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF) -induced release of IL-10 protein and the induced expression of IL-10 mRNA in these cells. However, it suppressed the induction of release of IL-1 beta protein and the induction of mRNA for IL-12 p40 and IL-1 beta by LPS and GM-CSF. Regulation of cytokine expression in peritoneal macrophages was also examined. By ELISA, the LPS-induced expression of IL-10 was augmented by CGRP, whereas the induction of IL-1 beta was suppressed. Northern analysis demonstrated augmentation of LPS-induced IL-10 mRNA levels and inhibition of LPS-induced IL-1 beta mRNA by CGRP. CGRP inhibited the LPS-induced induction of IL-12 mRNA as assessed by RT-PCR. Up-regulation of B7-2 expression by LPS and GM-CSF was suppressed by CGRP in both XS52 cells and macrophages, as previously reported. This suppression, however, could be abrogated by co-culture with neutralizing antibodies to IL-10. Furthermore, the presence of neutralizing antibodies to IL-10 during exposure of epidermal cells (EC) to CGRP prevented the CGRP-mediated suppression of EC presentation of tumor-associated antigens (from the S1509a spindle cell carcinoma) for elicitation of delayed-type hypersensitivity in S1509a-immune mice. These data suggest that suppression of antigen-presenting function by CGRP is mediated, at least in part, by changes in cytokine expression that favor less robust antigen presentation for cell-mediated immunity.

Animals↗
PubMed1997

Biochemical heterogeneity in hysterectomized uterus measured by 31P NMR using SLIM localization.

Ultrasound and magnetic resonance imaging show contrast between the inner and outer myometrium, which is useful in the diagnosis of gynecological disorders. To determine whether the image contrast is associated with biochemical differences between these myometrial regions, phosphorus metabolite concentrations in the inner one third of the myometrium (the junctional zone; JZ) were compared with the outermost one third of the myometrium (OM) in hysterectomized uteri using 31P spectral localization by imaging (SLIM). The technique was validated by comparing the results of SLIM with the results of standard Fourier-encoded spectroscopic imaging (FSI) analysis using phantoms, and by nonlocalized spectroscopy on biopsies taken from the same hysterectomy specimens. As expected theoretically, SLIM yielded better localization than FSI, as judged by spectral intensity and leakage measurements on phantom compartments of known composition. SLIM localization revealed that the JZ has a higher intracellular phosphomonoester (PME) concentration than does the OM, which was confirmed by nonlocalized spectroscopy, and that there is very little NMR-visible phosphorus in the cervix.

Adult↗
PubMed1997

Additivity in microbial toxicity of nonuniform mixtures of organic chemicals.

Microbial toxicity of nonuniform mixtures of selected synthetic organic chemicals in several proportion is evaluated. Toxicity is quantified by the inhibition of oxygen uptake rate of a surrogate microbial text culture as measured by a respirometer. The joint toxic effects of the chemicals are analyzed for simple addition using toxic units (TU) and similarity parameters (lambda). A new approach is proposed to assign acceptance limits to sigma(TU)i and lambda to account for experiment errors and variances. Based on this approach, the joint toxic effects of 16 chemicals evaluated in this study in 14 different mixtures were found to be simply additive. Predictions of component concentration based on simple additivity agreed with the measured values within an average factor of error of 1.4.

Bacteria↗
PubMed1997

Diffusion measurement in phantoms and tissues using SLIM localization.

A new approach to efficient localized diffusion measurements has been developed and evaluated on phantoms and isolated tissues. The combination of a diffusion-sensitive pulse sequence with SLIM (spectral localization by imaging) makes efficient and accurate localized water and metabolite diffusion measurements possible with a substantial improvement in spatial or time resolution compared to standard methods. Phantom experiments showed that diffusion of substances present in relatively low concentration within small compartments can be measured accurately by this method, suggesting potential applications for diffusion measurements of metabolites in vivo. Experiments on excised rat uterine horns demonstrated the ability of this method to measure localized diffusion of water within irregularly shaped regions of biological samples. Accurate diffusion measurements were achieved in the localized regions with acquisition times less than would have been required by standard diffusion imaging methods.

Animals↗
PubMed1997

Resting cell neutralization assay for HIV-1 primary isolates.

A technique is described for detecting the activity of neutralizing polyclonal or monoclonal antibodies against HIV-1 primary isolates. Most commonly, neutralizing antibody activity for HIV-1 is assessed by quantifying the ability of antibodies to inhibit virus infection in mitogen-activated peripheral blood mononuclear cells or transformed lymphocytes. Because the target of HIV infection in vivo is neither a mitogen-activated nor a transformed cell, an assay using unstimulated peripheral blood mononuclear cells as a more physiologic target cell was developed. This "resting cell assay" mainly utilizes primary HIV-1 isolates that have been carried for only a few passages in vitro. The result is an assay that is more efficient to perform and that detects neutralizing activity with comparable or greater sensitivity than that previously described for assays of primary HIV-1 isolates.

Antibodies, Monoclonal↗
PubMed1997

Investigation on arteriosclerosis among population in a rare earth area in south China.

An ophthalmofunduscope was used to investigate arteriosclerosis among villagers aged 20-40 yr old in two rare earth areas in Ganzhou, Jiangxi Province. It was noted that the occurrence of arteriosclerosis of the fundus aculi was significantly high (P < 0.05-0.01), the detection of serum cholesterol (CHO) was remarkably increased (P < 0.01), and the level of IgM was also elevated. However, high-density lipoprotein (HDL) remained at a low level. The effect of taking rare earth elements (REE) could be direct or indirect, thus causing an increase in cholesterol and interfering with the synthesis of high-density lipoprotein. Furthermore, rare earth could also cause immunogenic damage to the vascular wall. All of these could facilitate the formation of arteriosclerosis.

Adult↗
PubMed1997

Bioelectrical activity of the central nervous system among populations in a rare earth element area.

Auditory brainstem electric response (ABR) and somatosensory evoked potential (SEP) of 21 subjects (41 ears) among villagers in a rare earth element (REE) area in Gan County, Jiangxi, China, were studied. No difference in ABR between the subjects from the REE area and the control group was noted. However, the conduction detected by SEP from the median nerve to the thalamus (P15) was shortened (P < 0.05), especially to the first-grade primary somatosensory responsive region (S1) (P < 0.01) and the amplitude of S1 decreased (P < 0.05), indicating that REE was difficult to accumulate in the brainstem, but it was susceptible to cerebral cortex, thus causing sub-clinical damage. This condition was confirmed in the animal experiment. It was suggested that the toxicity through long-term intake of small doses of REE might not be negligible, and the hazard of REE environments should be investigated.

Acetylcholinesterase↗
PubMed1997

Development and application of an instrument for analysis of bone structure on radiographs.

An instrument used for quantitative assessment of trabecular structure of radius on radiograph including trabecular number and trabecular width was developed using a microdensitometer and a single-chip microcomputer. The device is characterized by its high sensitivity, good reproducibility, convenience and economy. The results obtained with the instrument were significantly correlated to actual bone mineral content. This device can be used for the diagnosis of osteoporosis, fluorosis, rickets and bone damages caused by cadmium.

Adult↗
PubMed1997

X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle.

X-ray diffraction patterns were obtained from skinned rabbit psoas muscle under relaxing and rigor conditions over a wide range of ionic strengths (50-170 mM) and temperatures (1 degree C-30 degrees C). For the first time, an intensification of the first actin-based layer line is observed in the relaxed muscle. The intensification, which increases with decreasing ionic strength at various temperatures, including 30 degrees C, parallels the formation of weakly attached cross-bridges in the relaxed muscle. However, the overall intensities of the actin-based layer lines are low. Furthermore, the level of diffuse scattering, presumably a measure of disorder among the cross-bridges, is little affected by changing ionic strength at a given temperature. The results suggest that the intensification of the first actin layer line is most likely due to the cross-bridges weakly bound to actin, and that the orientations of the weakly attached cross-bridges are hardly distinguishable from the detached cross-bridges. This suggests that the orientations of the weakly attached cross-bridges are not precisely defined with respect to the actin helix, i.e., nonstereospecific. Intensities of the myosin-based layer lines are only marginally affected by changing ionic strength, but markedly by temperature. The results could be explained if in a relaxed muscle the cross-bridges are distributed between a helically ordered and a disordered population with respect to myosin filament structure. Within the disordered population, some are weakly attached to actin and others are detached. The fraction of cross-bridges in the helically ordered assembly is primarily a function of temperature, while the distribution between the weakly attached and the detached within the disordered population is mainly affected by ionic strength. Some other notable features in the diffraction patterns include a approximately 1% decrease in the pitch of the myosin helix as the temperature is raised from 4 degrees C to 20 degrees C.

Actins↗
PubMed1997

Temperature-induced structural changes in the myosin thick filament of skinned rabbit psoas muscle.

By using synchrotron radiation and an imaging plate for recording diffraction patterns, we have obtained high-resolution x-ray patterns from relaxed rabbit psoas muscle at temperatures ranging from 1 degree C to 30 degrees C. This allowed us to obtain intensity profiles of the first six myosin layer lines and apply a model-building approach for structural analysis. At temperatures 20 degrees C and higher, the layer lines are sharp with clearly defined maxima. Modeling based on the data obtained at 20 degrees C reveals that the average center of the cross-bridges is at 135 A from the center of the thick filament and both of the myosin heads appear to wrap around the backbone. At 10 degrees C and lower, the layer lines become very weak and diffuse scattering increases considerably. At 4 degrees C, the peak of the first layer line shifts toward the meridian from 0.0047 to 0.0038 A(-1) and decreases in intensity approximately by a factor of four compared to that at 20 degrees C, although the intensities of higher-order layer lines remain approximately 10-15% of the first layer line. Our modeling suggests that as the temperature is lowered from 20 degrees C to 4 degrees C the center of cross-bridges extends radially away from the center of the filament (135 A to 175 A). Furthermore, the fraction of helically ordered cross-bridges decreases at least by a factor of two, while the isotropic disorder (the temperature factor) remains approximately unchanged. Our results on the order/disordering effects of temperature are in general agreement with earlier results of Wray [Wray, J. 1987. Structure of relaxed myosin filaments in relation to nucleotide state in vertebrate skeletal muscle. J. Muscle Res. Cell Motil. 8:62a (Abstr.)] and Lowy et al. (Lowy, J., D. Popp, and A. A. Stewart. 1991. X-ray studies of order-disorder transitions in the myosin heads of skinned rabbit psoas muscles. Biophys. J. 60:812-824). and support Poulsen and Lowy's hypothesis of coexistence of ordered and disordered cross-bridge populations in muscle (Poulsen, F. R., and J. Lowy. 1983. Small angle scattering from myosin heads in relaxed and rigor frog skeletal muscle. Nature (Lond.). 303:146-152.). However, our results added new insights into the disordered population. Present modeling together with data analysis (Xu, S., S. Malinchik, Th. Kraft, B. Brenner, and L. C. Yu. 1997. X-ray diffraction studies of cross-bridges weakly bound to actin in relaxed skinned fibers of rabbit psoas muscle. Biophys. J. 73:000-000) indicate that in a relaxed muscle, cross-bridges are distributed in three populations: those that are ordered on the thick filament helix and those that are disordered; and within the disordered population, some cross-bridges are detached and some are weakly attached to actin. One critical conclusion of the present study is that the apparent order <--> disorder transition as a function of temperature is not due to an increase/decrease in thermal motion (temperature factor) for the entire population, but a redistribution of cross-bridges among the three populations. Changing the temperature leads to a change in the fraction of cross-bridges located on the helix, while changing the ionic strength at a given temperature affects the disordered population leading to a change in the relative fraction of cross-bridges detached from and weakly attached to actin. Since the redistribution is reversible, we suggest that there is an equilibrium among the three populations of cross-bridges.

Actin Cytoskeleton↗
PubMed1997

Increased incidence of anti-beta-amyloid autoantibodies secreted by Epstein-Barr virus transformed B cell lines from patients with Alzheimer's disease.

During the past several years, evidence has been accumulated to support the thesis that immunological factors may play some role in Alzheimer's disease (AD). We have characterized the reactive antigens detected by certain monoclonal antibodies secreted by Epstein-Barr virus (EBV)-transformed B cell lines from the peripheral blood of AD patients and controls. Autoantibodies against beta-amyloid protein beta-amyloid protein (beta-A) in amyloid plaques and blood vessels and in enzyme-linked immunosorbent assays (ELISA) have been reported in four cell lines derived from an AD patient. In this study, over 3300 EBV-transformed B cell lines from thirteen individuals were tested in ELISAs for antibodies against beta-A peptides. Significantly more autoantibodies against beta-A (1-40) were found in the AD group, 2.26 +/- 0.62% (39/1794 cell lines) than in the control group, 0.28 +/- 0.36% (5/1552 cell lines) with P < 0.005. These new antibodies did not react with plaques or amyloid deposits in blood vessels. In contrast to the four plaque-reactive autoantibodies which reacted better with beta-A (1-40) than with beta-A (1-28), 70% of these anti-beta-A (1-40) antibodies reacted as well or better with beta-A (1-28). Many of them were also reactive with beta-A (1-16). Tested against a panel of cytoskeletal proteins and Hela cells, many of these anti-beta-A (1-40) antibodies appear to be polyreactive. The higher incidence of anti-beta-A antibody secreting B cells in AD patients provides further evidence that autoimmunity may play a role in AD.

Adult↗