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Biomedical subjects

X Lu

Publications and source records attributed to X Lu.

At least 145 records · Page 8Linked to original sources

TRH regulates Kv1.5 gene expression through a Galphaq-mediated PLC-independent pathway.

Thyrotropin-releasing hormone (TRH) decreases transcription of the Kv1.5 K(+) channel gene in GH(3) pituitary cells. Here, we examine whether TRH utilizes Gq activated phospholipase C, Gs or Gi to produce this response. We report that expression of constitutively active Galphaq mimicked and occluded the TRH effect. In contrast, expression of activated Galpha(S) or Galpha(i2) had no effect on Kv1. 5 mRNA expression. Furthermore, pertussis and cholera toxins failed to block the TRH-induced decrease in channel mRNA. Surprisingly, despite the role of Gq, the phospholipase C inhibitor U73122 did not alter down-regulation of channel mRNA by TRH, although it abolished the TRH-induced increase in intracellular [Ca(2+)] and up-regulation of c-fos mRNA. Furthermore, depletion of an intracellular Ca(2+) pool or inhibition of protein kinase C did not block the TRH-induced decrease in Kv1.5 mRNA. These results indicate that TRH-induced down-regulation of Kv1.5 gene expression is mediated by Galphaq proteins, but does not require PLC activation.

Animals↗

Calpain-mediated degradation of PSD-95 in developing and adult rat brain.

PSD-95 is a major postsynaptic density protein that is degraded as a result of synaptic activity. We used four different methods to test the hypothesis that calpain is involved in PSD-95 turnover. Treatment of synaptic membranes with purified calpain resulted in a decrease in immunoreactivity of the native 95 kDa protein and the appearance of two smaller molecular weight species, migrating at 50 and 36 kDa, respectively. Calcium treatment of frozen-thawed brain sections produced an identical digestion pattern, an effect blocked by calpain inhibitors. N-methyl-D-aspartate treatment of organotypic hippocampal cultures produced truncation of PSD-95 and accumulation of the 36 kDa species. Finally, calpain-generated degradation products of PSD95 were prominent in neonatal hippocampus, and disappeared with postnatal development. Our data suggest that PSD-95 is a substrate for calpain, and that calpain-mediated truncation contributes to PSD-95 turnover.

Age Factors↗

Cloning of human Ca2+ homoeostasis endoplasmic reticulum protein (CHERP): regulated expression of antisense cDNA depletes CHERP, inhibits intracellular Ca2+ mobilization and decreases cell proliferation.

A monoclonal antibody which blocks InsP(3)-induced Ca(2+) release from isolated endoplasmic reticulum was used to isolate a novel 4.0 kb cDNA from a human erythroleukaemia (HEL) cell cDNA expression library. A corresponding mRNA transcript of approx. 4.2 kb was present in all human cell lines and tissues examined, but cardiac and skeletal muscle had an additional transcript of 6.4 kb. The identification in GenBank(R) of homologous expressed sequence tags from many tissues and organisms suggests that the gene is ubiquitously expressed in higher eukaryotes. The gene was mapped to human chromosome 19p13.1. The cDNA predicts a 100 kDa protein, designated Ca(2+) homoeostasis endoplasmic reticulum protein (CHERP), with two putative transmembrane domains, multiple consensus phosphorylation sites, a polyglutamine tract of 12 repeats and regions of imperfect tryptophan and histadine octa- and nona-peptide repeats. In vitro translation of the full-length cDNA produced proteins of M(r) 128000 and 100000, corresponding to protein bands detected by Western blotting of many cell types. CHERP was co-localized in HEL cells with the InsP(3) receptor by two-colour immunofluorescence. Transfection of HEL cells with antisense cDNA led to an 80% decline in CHERP within 5 days of antisense induction, with markedly decreased intracellular Ca(2+) mobilization by thrombin, decreased DNA synthesis and growth arrest, indicating that the protein has an important function in Ca(2+) homoeostasis, growth and proliferation.

Amino Acid Sequence↗

Stress signals induce transcriptionally inactive E2F-1 independently of p53 and Rb.

One of the common features of cellular response to stress is cell cycle arrest or apoptosis. E2F is one of the key factors which controls cell cycle progression. Overexpression of E2F-1 can also induce apoptosis. In order to understand the role of E2F-1 in cellular response to stress, we studied the E2F-1 response in various cell lines to different types of stress signals including UV irradiation, cisplatin, etoposide and hypoxia. We showed here that the expression level of E2F-1 can be up regulated by the treatment of DNA damage agents as well as hypoxia. The kinetics of E2F-1 increase was dependent on the types of inducer and was similar to that of p53. However, stress signals can induce E2F-1 expression independently of p53 and Rb. Furthermore, the induced E2F-1 was transcriptionally inactive. All these results suggested that E2F-1 may play a very important role in cellular response to stress and this novel role of E2F-1 is independent of its transactivation function.

Carrier Proteins↗

Calpain-mediated truncation of rat brain AMPA receptors increases their Triton X-100 solubility.

Previous studies have indicated that calpain activation results in the truncation of the C-terminal domains of AMPA and NMDA receptor subunits. The present study determined the distribution of the truncated species of the subunits between Triton-soluble and -insoluble fractions. Western blots were performed with various antibodies to quantify the amounts of the various species of GluR1, GluR2, GluR3 and NR2B subunits. The results indicate that calpain activation decreased the amount of all the intact subunits in Triton-insoluble fractions. Calpain-generated truncated forms of GluR1 and GluR2, but not NR2B, were absent in these fractions, and were recovered in Triton-soluble fractions. These findings suggest that calpain-mediated truncation of AMPA but not NMDA receptor C-terminal domains results in modifications of the interactions between the receptors and postsynaptic densities, and that this mechanism could be involved in activity-dependent changes in the subcellular distribution of AMPA receptors.

Animals↗

A nonionic block co-polymer adjuvant (CRL1005) enhances the immunogenicity and protective efficacy of inactivated influenza vaccine in young and aged mice.

The use of adjuvants is one approach to improve influenza vaccine immunogenicity and efficacy, particularly in aged populations. The response of BALB/c mice to subcutaneously administered formalin-inactivated whole influenza virus vaccine in the presence or absence of a nonionic block copolymer adjuvant CRL1005 was evaluated. In young adult naïve mice, the copolymer adjuvant significantly enhanced virus-specific IgG and hemagglutination-inhibition (HI) antibody responses and augmented the production of IL-2 following vaccination. Influenza vaccine formulated with 2.5 mg CRL1005 significantly enhanced the protective efficacy of the inactivated vaccine in the upper and lower respiratory tract. In mice previously infected with influenza virus or naïve aged mice, inactivated vaccine administered with the copolymer adjuvant substantially enhanced the serum HI antibody response to inactivated influenza vaccine and significantly reduced lung virus titers following subsequent challenge with live virus compared with mice administered vaccine alone. These results suggest that the copolymer adjuvant warrants further investigation as a potential adjuvant for use in human vaccination against influenza.

Adjuvants, Immunologic↗

Phosphorescent oxygen sensors utilizing sulfur-nitrogen-phosphorous polymer matrixes: synthesis, characterization, and evaluation of poly(thionylphosphazene)-b-poly(tetrahydrofuran) block copolymers

We examine the use of thionylphosphazene-based block copolymers as matrixes for oxygen sensor applications. Poly(aminothionylphosphazene)-b-poly(tetrahydrofuran) (PATPy-PTHFx) block copolymers were prepared via reaction of ring-opened poly(chlorothionylphosphazene) with THF and subsequently with excess n-butylamine (to form PBATPy-PTHFx) or methylamine (to form PMATPy-PTHFx). The block copolymers were characterized by NMR, gel permeation chromatography, and differential scanning calorimetry. Films of PBATPy-PTHFx block copolymers containing platinum octaethylporphyrin or [Ru(dpp)3]Cl2 (dpp = 4,7-diphenyl-1,10-phenanthroline) as the oxygen-sensitive chromophore were prepared, and time-scan experiments were carried out to determine the diffusion coefficients, Do2, and solubilities, So2, of oxygen therein. Despite microphase separation, the data fit well to a simple Fick's law description of oxygen diffusion and gave Do2 values smaller than that for the n-butylamino-substituted PBATP635. For films freshly annealed above the melting point of PTHFx, the Do2 values were 35-50% (dye-dependent) larger than after aging 3 days at room temperature. Films with [Ru(dpp)3]Cl2 as the dye were evaluated as media for phosphorescent pressure-sensing. The dye-containing polymer films exhibit linear Stern-Volmer-like plots, even at high dye concentrations, as well as good photostability, and significantly higher sensitivity to oxygen quenching than simple mixtures of the analogous homopolymers.

Journal Article↗

An artificial transcriptional activating region with unusual properties.

We describe a series of transcriptional activators generated by adding amino acids (eight in one case, six in another) to fragments of the yeast Saccharomyces cerevisiae activator Gal4 that dimerize and bind DNA. One of the novel activating regions identified by this procedure is unusual, compared with previously characterized yeast activating regions, in the following ways: it works more strongly than does Gal4's natural activating region as assayed in yeast; it is devoid of acidic residues; and several lines of evidence suggest that it sees targets in the yeast transcriptional machinery at least partially distinct from those seen by Gal4's activating region.

Base Sequence↗

Membrane fusion proteins are required for oskar mRNA localization in the Drosophila egg chamber.

We used a genetic screen in Drosophila to identify mutations which disrupt the localization of oskar mRNA during oogenesis. Based on the hypothesis that some cytoskeletal components which are required during the mitotic divisions will also be required for oskar mRNA localization during oogenesis, we designed the following genetic screen. We screened for P-element insertions in genes which slow down the blastoderm mitotic divisions. A secondary genetic screen was to generate female germ-line clones of these potential cell division cycle genes and to identify those which cause the mislocalization of oskar mRNA. We identified mutations in ter94 which disrupt the localization of oskar mRNA to the posterior pole of the oocyte. Ter94 is a member of the CDC48p/VCP subfamily of AAA proteins which are involved in homotypic fusion of the endoplasmic reticulum during mitosis. Consistent with the function of the yeast ortholog, ter94-mutant egg chambers are defective in the assembly of the endoplasmic reticulum. We tested whether other membrane biosynthesis genes are required for localizing oskar mRNA during oogenesis. We found that ovaries that are mutant for syntaxin-1a, rop, and synaptotagmin are also defective in oskar mRNA localization during oogenesis. We suggest a pathway for the role of membrane assembly proteins on oskar mRNA localization.

Animals↗

Differential recognition of snake venom proteins expressing specific Arg-Gly-Asp (RGD) sequence motifs by wild-type and variant integrin alphaIIbbeta3: further evidence for distinct sites of RGD ligand recognition exhibiting negative allostery.

Several studies have demonstrated that the amino acid residues flanking the Arg-Gly-Asp (RGD) sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. Because of the absence of structural data for integrin complexes with bound ligand, the molecular basis for this specificity modulation remains obscure. In a previous paper [Rahman, Lu, Kakkar and Authi (1995) Biochem. J. 312, 223-232] we demonstrated that two genetically distinct venom-derived RGD proteins, kistrin and dendroaspin (both containing the sequence PRGDMP), were simple competitors, indicating the recognition of an identical binding site on the alpha(IIb)beta(3) complex. Furthermore, both kistrin and dendroaspin inhibited the binding of the disintegrin elegantin (containing the sequence ARGDNP) via a non-competitive mechanism, suggesting that the binding of elegantin to the alpha(IIb)beta(3) complex was at a remote site and down-regulated via an allosteric mechanism. Here we present further evidence for distinct RGD ligand recognition sites on the alpha(IIb)beta(3) complex that exhibit a negative allosteric relationship. A panel of well-characterized recombinant dendroaspin and elegantin derivatives were employed for this study. These recombinant molecules were constructed as glutathione S-transferase fusion proteins with either an Ala or Pro residue N-terminal to the RGD sequence in combination with either a Met or an Asn residue immediately C-terminal. Equilibrium competition experiments showed that elegantin binding to ADP-treated platelets was inhibited by derivatives Eleg. AM (ARGDMP) and Eleg. PM (PRGDMP) via an allosteric competitive mechanism, providing direct evidence that modulation of the RGD motif can alter competitive behaviour. In addition, recombinant kistrin and dendroaspin both inhibited elegantin binding via a non-competitive mechanism, confirming our previous observations. Further evidence for distinct binding sites employing an independent approach was obtained by analysing the binding of the panel of venom proteins to the functionally defective heterodimer alpha(IIb)beta(3) Ser(123)-->Ala expressed on Chinese hamster ovary cells. These studies demonstrated that simple competitors kistrin and dendroaspin bound with high affinity to the variant integrin complex. In contrast, the binding of elegantin and most significantly, recombinant Dendro. PN (PRGDNP) and Dendro. AN (ARGDNP) were abolished. These observations, taken together, are consistent with a model depicting the presence of distinct sites of RGD ligand recognition on the alpha(IIb)beta(3) complex that show the preferential recognition of specific RGD motifs. Competition experiments demonstrate a negative allosteric relationship between these RGD recognition sites.

Alanine↗

Protein interaction mapping in C. elegans using proteins involved in vulval development.

Protein interaction mapping using large-scale two-hybrid analysis has been proposed as a way to functionally annotate large numbers of uncharacterized proteins predicted by complete genome sequences. This approach was examined in Caenorhabditis elegans, starting with 27 proteins involved in vulval development. The resulting map reveals both known and new potential interactions and provides a functional annotation for approximately 100 uncharacterized gene products. A protein interaction mapping project is now feasible for C. elegans on a genome-wide scale and should contribute to the understanding of molecular mechanisms in this organism and in human diseases.

Animals↗

A controlled-notch specimen to study fatigue crack initiation in bone cement.

Despite the extensive literature on the mechanical characteristics and failure properties of poly(methyl methacrylate) bone cement, little is known of its fatigue crack initiation process. The most likely in vivo bone cement fatigue crack initiation sites are internal flaws and irregularities on the bone cement surface. The stress concentration created by a flaw, and subsequently the stress state at that flaw, depends on the flaw geometry. To model the fatigue crack initiation process of a flaw, it is necessary to reproduce the stress state at that flaw. In this study, a special mold was designed to introduce notches with specific tip radii into fatigue specimens. The notch was molded into the specimen to simulate the in vivo flaw formation process. The molding method allows control of the stress concentration by specifying the notch tip radius. We created notched specimens where the tip radii of the notches ranged from "sharp" (< 3 microm) to 400 microm. The results demonstrated that notched specimens created by the special mold satisfied two necessary requirements for fatigue crack initiation studies: (1) the material microstructure at the notch tip must not be disrupted by the notching process, and (2) the notch tip stress field, determined by the notch tip geometry, must be reproducible.

Biocompatible Materials↗

Excretion pattern investigation of urinary normal and modified nucleosides of breast cancer patients by RP-HPLC and factor analysis method.

Modified nucleosides, formed post-transcriptionally in RNA by a number of modification enzymes, are excreted in abnormal levels in the urine of patients with malignant tumors. To test their usefulness as tumor markers, and to compare them with the conventional tumor markers, a reversed-phase high-performance liquid chromatographic (RP-HPLC) method and a factor analysis method have been used to study the excretion pattern of nucleosides of breast cancer patients. A clear cut differentiation of the breast cancer group and the healthy individuals in two clusters without overlapping was obtained.

Adult↗

cDNA cloning and functional expression of growth hormone receptor from soft-shelled turtle (Pelodiscus sinensis japonicus).

The growth hormone receptor (GHR) cDNA was cloned from the liver of soft-shelled turtle (Pelodiscus sinensis japonicus) using the polymerase chain reaction (PCR). Although GHR has been cloned from several mammalian and avian species, this is the first description of the reptilian receptor. As deduced from the nucleotide sequence, the precursor GHR of soft-shelled turtle (tGHR) is a protein of 615 amino acids which presents 72% identity with the chicken receptor and 57-64% identity with GHRs of several mammals. The tGHR expressed in COS-7 cells specifically bound human growth hormone (hGH) and was able to transduce an activation of transcription in the transfected cells. Binding of (125)I-hGH to the expressed receptor was decreased by the addition of excess unlabeled hGH, pig GH, and bream GH but not by pig insulin. The open reading frame of tGHR cDNA was inserted into the pSINrep/gfp (green fluorescence protein) vector and the tGHR-gfp fusion protein was stably expressed in baby hamster kidney (BHK) cells. Confocal imaging showed that tGHR-gfp was largely concentrated on the plasma membrane. Western blot analysis and deglycosylation treatment with PNGase F demonstrated that tGHR was a glycoprotein in BHK cells.

Amino Acid Sequence↗

Characterization of a class of cationic peptides able to facilitate efficient protein transduction in vitro and in vivo.

Protein transduction domains (PTDs), such as the third helix of the Drosophila Antennapedia homeobox gene (Antp) and the HIV TAT PTD, possess a characteristic positive charge on the basis of their enrichment for arginine and lysine residues. To determine whether cationic peptides are able to function as protein transduction domains, 12-mer peptide sequences from an M13 phage library were selected for synthesis on the basis of their varying cationic charge content. In addition, polylysine and polyarginine peptides were synthesized in order to assess the effect of charge contribution in protein transduction. Coupling of the biotinylated peptides to avidin-beta-galactosidase facilitated transduction in a wide variety of cell lines and primary cells, including islet beta-cells, synovial cells, polarized airway epithelial cells, dendritic cells, myoblasts, and tumor cells. Two of the peptides, PTD-4 and PTD-5, mediated transduction nearly 600-fold more efficiently than a random control peptide, but with an efficiency similar to the TAT PTD and the 12 mers of polylysine and polyarginine. Furthermore, confocal analysis of biotinylated peptide-streptavidin-Cy3 conjugates demonstrated that the internalized PTDs are found in both the nuclei and the cytoplasm of treated cells. When tested in vivo, the PTDs were able to facilitate efficient and rapid protein delivery into rabbit synovium and mouse solid tumors following intraarticular and intratumoral administration, respectively. These novel PTDs can be used to transfer therapeutic proteins and DNA for the treatment of a wide variety of diseases, including arthritis and cancer.

Amino Acid Sequence↗

The relationship between apoptosis and the expression of proliferating cell nuclear antigen and the clinical stages in gastric carcinoma.

The relationship between the apoptosis and the expression of proliferating cell nuclear antigen (PCNA) and the clinical stages in gastric cancers was studied. By using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique and PCNA immunohistochemical staining, the apoptosis and the expression of PCNA in tissue of gastric carcinoma were assayed in situ, the index of apoptosis (AI), index of PCNA (PI) and the rate of AI/PI were calculated. AI and PI in gastric cancer tissues were (6.5 +/- 3.7)% and (49.8 +/- 15.9)% respectively, and the rate of AI/PI was 0.13 +/- 0.05, which were obviously different from those of normal gastric mucosa in paragastric cancer (P < 0.01). With the advanced TNM stages of gastric carcinoma, the AI was decreased, PI was increased and the rate of AI/PI decreased in gastric carcinoma. There was significant difference in them between the gastric cancer tissues and normal gastric mucosa in pericarcinoma in TNM stage II to IV (P < 0.05). It was suggested that the decreased apoptotic cells and the increased proliferating cells were obviously related to the tumor genesis and tumor progression in gastric carcinoma. The AI, PI and the rate of AI/PI would become the prognostic factors in advanced gastric carcinoma.

Adenocarcinoma↗

Fatty acids modulate protein kinase C activation in porcine vascular smooth muscle cells independently of their effect on de novo diacylglycerol synthesis.

AIMS/HYPOTHESIS: Diabetes-induced activation of protein kinase C has been associated with the development of vascular complications. Elevated de novo diacylglycerol synthesis has been postulated to underlie this protein kinase C activation. Diabetes also increases the circulating concentrations of non-esterified fatty acids, which are immediate precursors of diacylglycerol through the de novo pathway. We hypothesized that increased fatty acids contribute to de novo diacylglycerol synthesis and activation of protein kinase C in vascular cells. METHODS: Primary cultures of porcine carotid smooth muscle cells were exposed to fatty acids, bound to albumin in physiologic ratios. Diacylglycerol and triacylglycerol were measured in extracts of these cells. Protein kinase C activation was measured as membrane translocation with isoform-specific antibodies. RESULTS: Saturated fatty acids caused considerable accumulation of diacylglycerol through de novo synthesis. Unsaturated fatty acids increased triacylglycerol, but not diacylglycerol. Platelet-derived growth factor activated the alpha, epsilon and zeta protein kinase C isoforms. Activation of the alpha and zeta isoforms was amplified by oleate pretreatment but inhibited by palmitate. In the absence of growth factor stimulation, neither palmitate nor oleate had any effect on the membrane/cytosol distribution of any protein kinase C isoform. CONCLUSION/INTERPRETATION: Saturated fatty acids elicited de novo diacylglycerol synthesis in vascular smooth muscle cells without activating protein kinase C. Effects of fatty acids on protein kinase C activation by platelet-derived growth factor did not correlate with the effects on de novo diacylglycerol synthesis. These results indicate that de novo diacylglycerol synthesis is, by itself, insufficient to activate protein kinase C.

Animals↗

Characteristics of sequential movements during early learning period in monkeys.

We previously demonstrated that the organization of a learned sequential movement, after long-term practice, is based on the entire sequence and that the information pertaining to the sequence is largely specific to the hand used for practice. However, it remained unknown whether these characteristics are present from the beginning of learning. To answer the question, we examined the performance of four monkeys for the same sequential procedure in the early stage of learning. The monkeys' task was to press five consecutive pairs of buttons (which were illuminated), in a correct order for every pair, which they had to find by trial-and-error during a block of trials. We first examined whether the memory of a sequential procedure that was learned once was specific to the hand used for practice. The second time that the monkeys attempted to learn a novel sequence, they were required to use either the same hand they used the first time or the opposite hand. The number of errors decreased to a similar degree in the same-hand condition and in the opposite-hand condition. The performance time decreased in the same-hand condition, but not in the opposite-hand condition. The results suggest that, in the early stage of learning, memory of the correct performance of a sequential procedure is not specific to the hand originally used to perform the sequence (unlike the well-learned stage, where the transfer was incomplete), whereas memory of the fast performance of a sequential procedure is relatively specific to the hand used for practice (like the well-learned stage). We then examined whether memory of a sequential procedure depends on the entire sequence, not individual stimulus sets. For the second learning block, we had the monkey learn the sequence in the same or reversed order. In the reversed order, the order within each set was identical, but the order of sets was reversed. The number of errors decreased in both the same-order and reversed-order conditions to a similar degree for two out of four monkeys; the decrease was larger in the same-order condition for the other two monkeys. For all monkeys, the performance time decreased in the same-order condition, but not in the reversed-order condition. The results suggest that the memory structure for correct performance varies among monkeys in the early stage of learning (unlike the well-learned stage, where the memory of individual sets was consistently absent). On the other hand, memory of the fast performance of a sequential procedure is relatively specific to the learned order used for practice (like the well-learned stage).

Animals↗