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Biomedical subjects

L He

Publications and source records attributed to L He.

At least 343 records · Page 19Linked to original sources
PubMed1995

Automated linkage analysis in psychiatric disorders.

A genome-wide search for linkage of microsatellite markers to chromosomal loci containing genes responsible for the major psychoses is a laborious task which can be carried out with greater speed and economy by introducing automation to several steps in the procedure. We describe the use of the Automated Linkage Preprocessor (ALP) program for the computer analysis of the waveform generated by fluorescein-labelled markers after electrophoretic separation. (To obtain a copy send a request to A.F. Brown at the below MRC address or use Anonymous FTP to ftp.hgu.mrc.ac.uk. Software is in directory pub/ALP). The program runs on a PC in the Microsoft Windows environment, and is used in conjunction with an automated laser fluorescence (ALF) sequencer (Pharmacia) and its Fragment Manager software to detect and size the PCR products, filter out peaks of fluorescence due to nonallele fragments, and generate genotypes in a format suitable for direct input to standard linkage analysis programs. The method should offer the advantages of speed, accuracy, and reduced cost. Its use in linkage studies in a large family with manic-depressive illness is discussed.

Automation↗
PubMed1995

The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase do not localize to the same intracellular vesicles in rat skeletal muscle.

The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase of rat skeletal muscle are two proteins which redistribute from intracellular membranes to plasma membranes following in vivo insulin stimulation. Here we show that although both proteins co-segregate after subcellular fractionation of unstimulated rat hindlimb muscles, they do not share the same intracellular residence inside the muscle fibre. By immunogold single- and double-labeling on ultrathin muscle cryosections with specific antibodies, the GLUT4 glucose transporter and the Na+,K(+)-ATPase alpha 2 subunit were observed on different vesicular structures within the cell. GLUT4 was detected on subsarcolemmal and perinuclear membranes, and at the junction between myofibrillar A and I bands where triads are localized. The alpha 2 subunit of the Na+,K(+)-ATPase was observed at the plasma membrane and in distinct subsarcolemmal vesicles and intermyofibrillar membranes. Quantitative analysis of double-labeling of GLUT4 and Na+,K(+)-ATPase alpha 2 subunit revealed that less than 6% of the two proteins co-localize in the same continuous vesicular structures. The differential intracellular localization of the two proteins was further confirmed by immunopurification of GLUT4-containing membranes from muscle homogenates, in which the alpha 2 subunit of the Na+,K(+)-ATPase was found only at the same extent as the alpha 1 subunit of the enzyme, a protein exclusively present at the plasma membrane.

Animals↗
PubMed1995

Cellubrevin is a resident protein of insulin-sensitive GLUT4 glucose transporter vesicles in 3T3-L1 adipocytes.

Insulin stimulates glucose transport in muscle and fat cells by inducing translocation of GLUT4 glucose transporters from a storage site to the cell surface. The mechanism of this translocation and the identity of the storage site are unknown, but it has been hypothesized that transporters recycle between an insulin-sensitive pool, endosomes, and the cell surface. Upon cell homogenization and fractionation, the storage site migrates with light microsomes (LDM) separate from the plasma membrane fraction (PM). Cellubrevin is a recently identified endosomal protein that may be involved in the reexocytosis of recycling endosomes. Here we describe that cellubrevin is expressed in 3T3-L1 adipocytes and is more abundant in the LDM than in the PM. Cellubrevin was markedly induced during differentiation of 3T3-L1 fibroblasts into adipocytes, in parallel with GLUT4, and the development of insulin regulated traffic. In response to insulin, the cellubrevin content decreased in the LDM and increased in the PM, suggesting translocation akin to that of the GLUT4 glucose transporter. Vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin-II, a protein associated with regulated exocytosis in secretory cells, also redistributed in response to insulin. Both cellubrevin and VAMP-2 were susceptible to cleavage by tetanus toxin. Immunopurified GLUT4-containing vesicles contained cellubrevin and VAMP-2, and immunopurified cellubrevin-containing vesicles contained GLUT4 protein, but undiscernible amounts of VAMP-2. These observations suggest that cellubrevin and VAMP-2 are constituents of the insulin-regulated pathway of membrane traffic. These results are the first demonstration that cellubrevin is present in a regulated intracellular compartment. We hypothesize that cellubrevin and VAMP-2 may be present in different subsets of GLUT4-containing vesicles.

3T3 Cells↗
PubMed1995

Phosphorylation of the C-terminal domain of RNA polymerase II by the extracellular-signal-regulated protein kinase ERK2.

Rat ERK2, an extracellular-signal-regulated protein kinase family member, phosphorylates RNA polymerase II in vitro. Phosphorylation occurs within the heptapeptide repeats of the C-terminal domain of the largest subunit, in a region important for regulation of transcriptional activity. Analysis of deletion mutants and synthetic peptides showed that ERK2 phosphorylation occurs at multiple serine residues throughout the C-terminal domain, with no marked preference for consensus repeats versus naturally occurring variants. Our results are consistent with the idea that protein kinases in the extracellular-signal-regulated protein kinase family regulate transcription by direct phosphorylation of RNA polymerase II, but do not support a model where particular portions of the C-terminal domain are special targets of ERK phosphorylation.

Amino Acid Sequence↗
PubMed1995

Cell cycle variation of Hsp70 levels in HeLa cells at 37 degrees C and after a heat shock.

The expression of the 72 kD inducible heat shock protein (hsp72) has been reported to be cell cycle associated in unheated, synchronized HeLa cells. In this study, flow cytometry was used to investigate hsp72 levels through the cell cycle in HeLa cells by dual labeling with propidium iodide and antibodies against hsp72. The entire cell cycle distribution of hsp72 could be measured in a single sample of asynchronously growing cells. For unheated cells, the level of hsp72 increased about 30% from G1 to S phase, with about a 65% increase in G2/M, probably due to cell size differences. Neither mitotic selection nor serum stimulation induced a higher level of hsp72 than in the control cells. Western blot analysis of hsp72 from Hoechst-stained cells sorted from G1, mid-S, or G2/M showed that G1 cells had the lowest level of hsp72, with about a 30% increase in S phase and a 60% increase in G2/M, in good agreement with the flow cytometry results. These data conflict with previous reports by other laboratories which showed a 3-fold higher level of hsp72 in S phase than in G1 or G2. In contrast, heat shock (both acute and chronic) led to a non-uniform increase in hsp72 through the cell cycle. Most cells in mid S phase had high levels of hsp72, and a larger range in the levels of hsp72 were found in G1 and late S/G2/M phase cells.

Blotting, Western↗
PubMed1995

Three-dimensional reconstruction of the rubrocerebellar premotor network of the turtle.

Neuroanatomical studies have demonstrated that the organization of the reptilian rubrocerebellar limb premotor network is similar to that of mammals. This network is composed of prominent recurrent connections among the red nucleus, lateral cerebellar nucleus and lateral reticular nucleus. In this paper the rubrocerebellar system of the turtle was three-dimensionally reconstructed to permit detailed examination of its anatomical organization. Each nucleus and its major efferent pathway was imaged and reconstructed from separate anatomical cases. Section images were used to draw tissue boundaries, mark cell positions and locate axonal trajectories. For each nucleus, drawings of section images containing labeled cells were stacked in the rostrocaudal direction using anatomical landmarks, and a graphic model of the surface was constructed using the method of triangulation. An ellipsoid of equal concentration was computed for each nucleus to ascertain their three-dimensional boundaries and location within the brainstem. To examine the entire rubrocerebellar network, a template of the turtle brainstem and cerebellum was constructed. The component nuclei of the rubrocerebellar network and their axonal projections were then spatially warped onto the template reconstruction on a section by section basis. The final three-dimensional reconstruction of the turtle rubrocerebellar limb premotor network could be rotated in space, allowing proper visualization of the anatomical details of this system. Furthermore, we were able to mathematically section through the reconstruction to obtain brainstem slices with differing orientations and thickness.

Animals↗
PubMed1995

Induction of geranyl pyrophosphate pyrophosphatase activity by cholesterol-suppressive isoprenoids.

Diets supplemented (1 mmol/kg) with thymol, carvacrol, and beta-ionone significantly decreased the serum cholesterol levels of cockerels. These mevalonate-derived end products of plant secondary metabolism (isoprenoids) had no impact on two cytosolic prenyl alcohol (and ethanol) dehydrogenase activities; each treatment increased microsomal geranyl pyrophosphate pyrophosphatase activity by greater than twofold. The structural diversity of the isoprenoids which suppress cholesterol synthesis may be reconciled by their ability to increase pyrophosphatase activity, thus leading to the production of the endogenous, post-transcriptional regulator of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity.

Animals↗
PubMed1995

The C-terminal domain of NifL is sufficient to inhibit NifA activity.

In Klebsiella pneumoniae, transcription of all nif (nitrogen fixation) operons except the regulatory nifLA operon itself is regulated by the proteins NifA and NifL. NifA, an enhancer-binding protein, activates transcription by RNA polymerase containing the alternative sigma factor sigma 54. The central catalytic domain of NifA is sufficient for transcriptional activation, which can occur from solution. In vivo, NifL antagonizes the action of NifA in the presence of molecular oxygen or combined nitrogen. Inhibition has also been shown in vitro, but it was not responsive to environmental signals. Assuming a two-domain structure of NifL, we localized inhibition by NifL to its carboxy (C)-terminal domain, which is more soluble than the intact protein. The first line of evidence for this is that internal deletions of NifL containing an intact C-terminal domain were able to inhibit transcriptional activation by NifA in a coupled transcription-translation system. The second line of evidence is that the isolated C-terminal domain of NifL (assayed as a fusion to the soluble maltose-binding protein [MBP]) was sufficient to inhibit transcriptional activation by the central domain of NifA in a purified transcription system. The final line of evidence is that an MBP fusion to the C-terminal domain of NifL inhibited transcriptional activation by NifA in vivo. On the basis of these data, we postulate that the inhibitory function of NifL lies in its C-terminal domain and hence infer that this domain is responsible for interaction with NifA. Gel filtration experiments with MBP-NifL fusion derivatives lacking portions of the N- or C-terminal domain of the protein revealed that the C-terminal domain is the most soluble part of NifL. Up to 50% of two MBP-NifL truncations containing only the C-terminal domain appeared to be in a defined dimeric state.

Bacterial Proteins↗
PubMed1995

[Cloning and sequencing of ntrBC genes from Azospirillum brasilense].

A gene library of Azospirillum brasilense Yu62 was constructed in EMBL3. The library was screened with PCR amplified fragment as a special probe. Ten positive plaques (EA1-EA10) were selected. Detection results showed they contained two different types of clones, representing as EA4 and EA9 respectively. Southern hybridization of EA4 displayed that target gene was located in a 2.9kb EcoRI fragment. Sequence of this fragment had allowed the position and identification of ntrC gene, which encoding a protein of 53469, consisted of 480 amino acids. In the upstream of ntrC, a complete ntrB coding region was also found, which encoding a protein of 43487, consisted of 400 amino acids. Homologous analysis of the deduced amino acid sequences of ntrC and ntrB from different bacteria demonstrated that A. brasilense was closer to Rhizobia than to other free-living diazotrophs.

Azospirillum brasilense↗
PubMed1995

Dynorphin A modulates acute and chronic opioid effects.

A single dose of dynorphin A-(1-13) [dyn A(1-13)] is effective in suppressing the expression of opioid withdrawal and tolerance in morphine-dependent mice. In addition, this modulatory activity is retained by the corresponding non-opioid [des-Tyr1]-dynorphin A peptide [dynA(2-17)]. We have further investigated the non-opioid nature of this activity by comparing the efficacies of dyn A(1-13) and (2-17) under different experimental protocols with a variety of dosing regimens. The effect of dyn A(1-13) on withdrawal and tolerance expression was dose-dependent and could be enhanced by repeated dosing. Thus, the ED50 of naloxone to precipitate withdrawal jumping was increased 1.8-fold when morphine-dependent mice were treated with 4.2 mumol/kg dyn A(1-13) on the fourth day after pellet implantation and 2.4-fold on the sixth day with continued daily dyn A(1-13) treatment. The maximal effect was observed on day 6 when the ED50 of mice treated with 8.4 mumol/kg of dyn A(1-13) was increased nearly 6-fold over that of saline controls. Dyn A(2-17) proved to be nearly as effective as dyn A(1-13).

Animals↗
PubMed1995

[Des-Tyr1]dynorphin A-(2-17) has naloxone-insensitive antinociceptive effect in the writhing assay.

The dynorphin family of peptides stands out among the opioids in that its members are not antinociceptive after central administration in the common antinociceptive assays. In addition, reports of spinal antinociception have been conflicting. We have tested the antinociceptive activity of i.v. dynorphin A-(1-13) in the writhing assay and have found it to be very potent, with an ED50 of 1.0 (0.99-1.02) mumol/kg. Remarkably, [des-tyr1]dyn A-(2-17) was equally active with an ED50 of 1.1 (0.99-1.20). This activity was also retained by several smaller, non-opioid dynorphin A fragments and was not affected by the presence of either 50 mumol/kg naloxone or 20 mumol/kg Nor-BNI. Further, ED50 values were not different in morphine-dependent mice. The peak effect of dyn A-(1-13) and A-(2-17) was observed 5 min after administration and the effect of dyn A-(1-13) or dyn A-(2-17) was still measurable 1 hr after i.v. administration with a 5- to 6-fold increase in ED50 at this time. The ED50 values after i.c.v. and i.t. administration of dyn A-(1-13) were similar to those reported previously. Dyn A(2-17) was also effective by these routes with ED50 values not significantly different from those of dyn A-(1-13). Both dyn A-(1-13) and A-(2-17) were also active when injected i.p., whereas ED50 values increased substantially after s.c. administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics, Opioid↗
PubMed1995

P-glycoprotein expression in primary breast cancer.

In a retrospective study, liquid nitrogen preserved specimens from 50 women with primary breast cancer, who underwent surgery at the Beijing Institute for Cancer Research between June, 1986 and September, 1988, were investigated. All patients under this study were staged in TNM II or later, involved with axillary lymph node metastasis, and treated with systemic postoperative adjuvant chemotherapy. The median length of follow-up was 69 months. The expression of P-glycoprotein was investigated by means of immunohistochemistry, using a monoclonal antibody C219 specifically against P-glycoprotein and avidin-biotin peroxidase method. Positive staining for P-glycoprotein was found in 23 (46%) of the 50 patients. The P-glycoprotein expression negative group fared better than the group that was P-glycoprotein positive in overall survival curves (p = 0.0008, by the generalized Wilcoxon test). The prognostic effect of P-glycoprotein expression remained statistically significant (p = 0.0007) after adjustment by multivariate analysis (Cox's model) for other prognostic factors. It is demonstrated that P-glycoprotein expression is a significant and independent predictor of postoperative survival in breast cancer patients. The results of the present study suggest that P-glycoprotein expression might also influence the biological behavior of breast cancers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

[Anisodamine (654-2) improves impaired cognitive function induced by experimental brain damage].

Surgery of brain injury was established in rats with medial frontal cortex lesion or acute cerebral ischemia and reperfusion. Learning and memory deficits were induced. These rats can be served as the animal model of dementia. Intraperitoneal injection of 654-2 (30 mg/kg per day) started from the day of surgery until a total of 10 shots were given. We first reported that 654-2 improved the deficits of learning and memory in avoidance response of rats after the two types of brain damage. Further more, the results showed that 654-2 enhanced SOD activity and inhibited the elevation of superoxides (MDA) in forebrain of cortex-lesioned rats. results suggest that 654-2 probably has the effect of protection of the brain cell against the injury of free radicals.

Animals↗