Biomedical subjects

B He

Publications and source records attributed to B He.

At least 181 records · Page 10Linked to original sources

PubMed1998

Dissociation of creatine kinase under different denaturation conditions.

Dissociation of dimeric creatine kinase under different denaturation conditions was investigated using the cleavable cross-linker 3, 3;-dithiobis(succinimidyl propionate). The results show that at low denaturant concentrations or at low denaturation temperatures the creatine kinase was mostly inactivated, but the enzyme was still either in the dimeric state or very slightly dissociated. It appears, therefore, that for several denaturation conditions, inactivation of the enzyme is not due to the dissociation of the active dimer.

Animals↗

PubMed1997

Recovery of infectious SV5 from cloned DNA and expression of a foreign gene.

A complete cDNA clone of the genome (15,246 nucleotides) of the paramyxovirus SV5 was constructed from cDNAs such that an anti-genome RNA could be transcribed by T7 RNA polymerase and the correct 3' end generated by cleavage using hepatitis delta virus ribozyme. The plasmid encoding the antigenome sequence was transfected into cells previously infected with recombinant vaccinia virus that expressed T7 RNA polymerase, together with helper plasmids that expressed the viral replication proteins, NP, P, and L, under the control of the T7 polymerase promoter. Rescue of the RNA genome from DNA was demonstrated by recovering SV5 with the tag restriction sites introduced into the DNA clone, using RT-PCR of the genome RNA and nucleotide sequencing. Rescue of SV5 from DNA did not require expression of the viral V protein as a helper plasmid, suggesting that V protein is not essential for initial replication. The infectious cDNA of SV5 was also manipulated to express green fluorescent protein (GFP) under the control of SV5 transcriptional start and stop signals introduced between the HN and L genes. The amount of GFP that was expressed varied depending on the nature of the newly introduced transcription signals.

DNA, Complementary↗

PubMed1997

Mutant bacteriophage T7 RNA polymerases with altered termination properties.

We have identified mutants of bacteriophage T7 RNA polymerase (RNAP) that are altered in their ability to pause or terminate at a variety of signals. These signals include a terminator found fortuitously in the human preproparathyroid hormone (PTH) gene, a pause site found in the concatamer junction (CJ) of replicating T7 DNA, and termination signals that are also utilized by Escherichia coli RNAP (e.g. rrnB T1 and T2). Whereas the mutant enzymes terminate normally at the late terminator in T7 DNA (T(phi)) and rrnB T2, they fail to terminate at one of the termination sites of rrnB T1, and also fail to recognize the PTH and CJ signals. The mutant enzymes exhibit normal processivity on linear templates, but show a slightly reduced processivity on supercoiled templates and terminate more efficiently when synthesizing poly(U) tracts. The mutant enzymes also show a decreased tendency to produce aberrant transcription products from DNA templates having protruding 3' ends. T7 lysozyme (an inhibitor of T7 RNAP) has been shown to exert its action by preventing the transition of the RNAP from an unstable initiation complex (IC) to a stable elongation complex (EC). We have found that T7 lysozyme enhances recognition of CJ by wild-type T7 RNAP, and that mutant T7 RNAPs that show increased sensitivity to lysozyme show enhanced recognition of this signal, even in the absence of lysozyme. These results, together with the observation that the mutations that result in the termination-deficient phenotype affect a region of the RNAP that has been implicated in RNA binding and upstream promoter contacts, support the hypothesis that, in some cases, termination represents a reversal of the events that occur during initiation.

Amino Acid Sequence↗

PubMed1997

Elevated hypothalamic neuropeptide Y levels in rats with dorsomedial hindbrain lesions.

Lesions centered on the area postrema (AP) and adjacent nucleus of the solitary tract (AP/mNTS-lesions) are reported to result in increased consumption of highly palatable diets. Recent studies suggest that neuropeptide Y (NPY) may cause a preference for carbohydrate-rich diets. Thus, it is possible that NPY may play a role in the enhanced intake of highly palatable diets by AP/mNTS-lesioned rats. In the studies reported here, we found that lesions centered on the AP result in increased levels of NPY-immunoreactivity in the paraventricular nucleus of the hypothalamus. Additionally, steady-state NPY mRNA in the basomedial hypothalamus including the arcuate nucleus was elevated. Enhanced NPY was not found throughout the hypothalamus however, as NPY-immunoreactivity was not elevated in the lateral hypothalamus or the tissue bordering the anteroventral third ventricle. These data suggest the possibility that elevated hypothalamic NPY, particularly in the arcuate and paraventricular nuclei, may contribute to the altered food intake and energy balance observed in rats with lesions centered on the AP.

Animals↗

PubMed1997

Conserved and sexually dimorphic behavioral responses to biogenic amines in decapitated Drosophila.

A preparation of decapitated Drosophila melanogaster has been used for direct application of drugs to the nerve cord. Serotonin, dopamine, and octopamine stimulate locomotion and grooming, showing distinguishable effects that often are potentiated by addition of the vertebrate monoamine oxidase-inhibitor hydrazaline. Many of the hydrazaline-induced effects are sexually dimorphic, with males showing greater responses than females. Behaviors similar to those induced by dopamine can be induced by application of the vertebrate dopamine D2-like receptor agonist quinpirole, whose effects are also sexually dimorphic. In contrast, vertebrate D2-like and D1-like dopamine antagonists result in akinesic states, and D1-like agonists selectively stimulate grooming. These data indicate that Drosophila nerve cord amine receptors are coupled to reflexive behaviors similar to those stimulated by brain dopamine receptors in vertebrates.

Animals↗

PubMed1997

The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase.

In human cells infected with herpes simplex virus 1 the double-stranded RNA-dependent protein kinase (PKR) is activated but phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2) and total shutoff of protein synthesis is observed only in cells infected with gamma(1)z34.5- mutants. The carboxyl-terminal 64 aa of gamma(1)34.5 protein are homologous to the corresponding domain of MyD116, the murine growth arrest and DNA damage gene 34 (GADD34) protein and the two domains are functionally interchangeable in infected cells. This report shows that (i) the carboxyl terminus of MyD116 interacts with protein phosphatase 1alpha in yeast, and both MyD116 and gamma(1)34.5 interact with protein phosphatase 1alpha in vitro; (ii) protein synthesis in infected cells is strongly inhibited by okadaic acid, a phosphatase 1 inhibitor; and (iii) the alpha subunit in purified eIF-2 phosphorylated in vitro is specifically dephosphorylated by S10 fractions of wild-type infected cells at a rate 3000 times that of mock-infected cells, whereas the eIF-2alpha-P phosphatase activity of gamma(1)34.5- virus infected cells is lower than that of mock-infected cells. The eIF-2alpha-P phosphatase activities are sensitive to inhibitor 2. In contrast to eIF-2alpha-P phosphatase activity, extracts of mock-infected cells exhibit a 2-fold higher phosphatase activity on [32P]phosphorylase than extracts of infected cells. These results indicate that in infected cells, gamma(1)34.5 interacts with and redirects phosphatase to dephosphorylate eIF-2alpha to enable continued protein synthesis despite the presence of activated PKR. The GADD34 protein may have a similar function in eukaryotic cells. The proposed mechanism for maintenance of protein synthesis in the face of double-stranded RNA accumulation is different from that described for viruses examined to date.

Animals↗

PubMed1997

A mutant T7 RNA polymerase that is defective in RNA binding and blocked in the early stages of transcription.

We have identified a mutation (E148A) in T7 RNA polymerase (RNAP) that results in an enzyme which aborts transcription primarily when the nascent RNA achieves a length of 5 nt. This phenomenon is observed at a consensus promoter, but is even more strongly observed at promoters that are altered in the initiation region. Although the abortive product is of a fixed length (5 nt), the positions of the base substitutions in the initiation region that enhance this effect do not appear to be fixed, and we have observed the effect with a variety of initiation-region promoter variants. The phenomenon is also observed during promoter-independent transcription when transcribing a homopolymeric template such as poly(dC). Under conditions where the active site of the RNAP cannot extend beyond the third nucleotide in the template strand and the maximum length of the RNA:DNA hybrid cannot exceed three base-pairs (i.e. when synthesizing oligoG products due to transcript slippage at a promoter that initiates with the sequence +1 GGG...) the mutant RNAP gives rise to a normal spectrum of products 2 to 14 nt in length with no evidence of a block at 5 nt. Neither promoter binding nor promoter melting appears to be involved in this phenotype, as the mutant RNAP binds normally to promoter sequences and the behavior of the enzyme is unaffected by removal of the non-template strand in the initiation region of the promoter or on a supercoiled template. Importantly, the mutant RNAP is defective in binding single strand oligomers of RNA. These results suggest that the affected region of the RNAP may form part of the RNA product binding site and may be involved in the transition from an unstable initiation complex to a stable elongation complex, perhaps by sensing the presence of a nascent RNA and/or RNA:DNA hybrid.

Amino Acids↗

PubMed1997

Rapid mutagenesis and purification of phage RNA polymerases.

We have developed plasmid-based expression systems that encode modified forms of T7 RNA polymerase (RNAP) having 6-12 histidine residues fused to the amino terminus. The histidine-tagged RNAPs (His-T7 RNAPS) are indistinguishable from the wild-type (WT) enzyme in nearly all biochemical assays. Similar plasmids that encode His-tagged T3 and SP6 RNAPs have also been constructed. To facilitate site-directed mutagenesis of the RNAP gene, the size of the target plasmid was minimized by using T7 RNAP itself as a selectable marker. BL21 (DCAT4) cells (which carry a chromosomal copy of the chloramphenicol acetyltransferase cat gene under control of a T7 promoter) are resistant to chloramphenicol when functional T7 RNAP is expressed, thus allowing the selection and maintenance of the target plasmid in these cells. Mutagenesis is accomplished by denaturing the plasmid, annealing mutagenic DNA primers, and repairing the plasmid with T4 DNA polymerase. Two DNA primers are used: one corrects a defect in the bla gene, the other introduces the desired mutation into the RNAP gene; 30-85% of the ampicillin-resistant transformants carry the desired mutation in the RNAP gene. By using BL21 (DCAT4) cells as a recipient for transformation the functional integrity of the RNAP gene may conveniently be monitored by assessing the level of chloramphenicol resistance in vivo. Methods for rapid, simultaneous purification of multiple samples of modified (His-tagged) and conventional RNAPs are described. Together, these developments greatly enhance our ability to characterize this important class of enzymes.

Amino Acid Sequence↗

PubMed1997

A comparison of volume conductor effects on body surface Laplacian and potential ECGS: a model study.

The objective of this investigation is to study, using a computer model, the torso volume conductor effects on body surface potential electrograms and body surface Laplacian electrograms. A spherical volume conductor model was used to approximate the torso and the heart. Myocardial electrical events were approximated by two distributed dipole-layers representing activation wavefronts propagating from the endocardium to the epicardium. The present computer simulation results indicate that the body surface Laplacian maps provide enhanced performance over the body surface potential maps in resolving the configurations of two activation wavefronts over the anterior wall of the heart.

Body Surface Potential Mapping↗

PubMed1997

Comparison of inactivation and conformational changes of aminoacylase during denaturation in lithium dodecylsulphate solutions.

The denaturation of aminoacylase in LDS solutions of different concentrations has been studied by following the changes in the ultraviolet absorbance, circular dichroism and intrinsic fluorescence. The results obtained show that the denaturation of the enzyme results in negative peaks at 287 and 295 nm in the denatured minus native enzyme difference spectrum. The fluorescence emission intensify of the enzyme decreases with no red shift of emission maximum in LDS solutions of increasing concentrations. In the LDS concentration regions employed in present study, no marked changes of secondary structure of the enzyme have been observed by following the changes in far ultraviolet CD spectra. The inactivation of this enzyme has been followed and compared with the unfolding observed during denaturation in LDS solutions. A marked inactivation is already evident at low LDS concentrations before signification conformational changes can be detected by ultraviolet absorbance and fluorescence changes. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by the kinetics method of the substrate reaction in the presence of inactivator previously described by Tsou. At the same LDS concentrations, the inactivation rate constants of the enzyme are a order of magnitude faster than the rate constants of conformational changes at least. The above results show that the active sites of metal enzyme containing Zn2+ are also situated in a limited and flexible region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.

Amidohydrolases↗

PubMed1997

Comparison of inactivation and unfolding of yeast alcohol dehydrogenase during thermal denaturation.

It has been reported that inactivation occurs before noticeable conformational change can be detected during denaturation of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) and other enzymes by guanidinium chloride or urea. It has therefore been suggested that enzyme active sites may display more conformational flexibility than the enzyme molecules as a whole. The present paper compares the inactivation and unfolding of yeast alcohol dehydrogenase during thermal denaturation. Under identical conditions, inactivation takes place before noticeable conformational changes. Kinetics of unfolding can be resolved into two phases. For a given temperature, the fast phase rates are about one order of magnitude slower than the inactivation rates of the free enzyme and approximately the same magnitude as the inactivation rates of enzyme-substrate complexes. This is general accord with the suggestion made previously by Tsou, indicating that the active sites of metal enzymes are situated in a region more flexible than the molecules as a whole.

Alcohol Dehydrogenase↗

PubMed1997

A bioelectric inverse imaging technique based on surface Laplacians.

A new approach is proposed to solve bioelectric inverse problems by employing the surface Laplacian of the bioelectrical potential. A theoretical investigation was conducted to test the feasibility of epicardial inverse imaging of cardiac electrical activity. A two-sphere homogeneous volume conductor model, where the inner sphere represents the epicardium and the outer sphere the body surface, was used. Radial and tangential current dipoles were used to approximate localized wavefronts propagating from the endocardium to the epicardium, and ectopic myocardial activities. The epicardial potential distribution was reconstructed from the body surface Laplacians with the aid of the Tikhonov zero-order regularization technique, which then was compared with the results obtained from the body surface potentials using the same regularization scheme. The two inverse solutions were compared qualitatively via visual inspection of the reconstructed epicardial potential maps, and quantitatively by examining relative errors and correlation coefficients between the "true" and the reconstructed epicardial potentials. Both qualitative and quantitative results indicate that the surface Laplacians play a positive role in improving the ill-posed nature of the bioelectric inverse problem, which would enhance our capability of reconstructing important epicardial events such as extrema in the epicardial potential distribution. The present theoretical study suggests that the Laplacian-based inverse imaging technique may have important applications to epicardial inverse imaging and other bioelectric inverse imaging.

Artifacts↗

PubMed1997

No linkage or association of a VNTR marker in the junction region of the immunoglobulin heavy chain genes in multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the central nervous system. Autoantibodies are though to participate in the pathogenesis. Previous reports on the role of immunoglobulin (Ig) variable gene segments in MS are contradictory. Here, by using a highly polymorphic variable number tandem repeat (VNTR) marker located in the centre of the IgH chain locus, we demonstrate a lack of linkage and association with MS in 34 multiplex families and 113 sporadic MS patients in Sweden. Stratification for the presence or absence of the MS-associated HLA-Dw2 haplotype did not influence the negative outcome. We conclude that the IgH chain genes are unlikely to play a role in genetic susceptibility to MS in the Swedish population.

Chromosomes, Human, Pair 14↗

PubMed1997

Suppression of the phenotype of gamma(1)34.5- herpes simplex virus 1: failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the alpha47 gene.

Earlier studies have shown that infection of human cells by herpes simplex virus 1 (HSV-1) results in the activation of RNA-dependent protein kinase (PKR) but that the alpha subunit of eIF-2 is not phosphorylated and that protein synthesis is unaffected. In the absence of the viral gamma(1)34.5 gene, eIF-2alpha is phosphorylated and protein synthesis is prematurely shut off (J. Chou, J. J. Chen, M. Gross, and B. Roizman, Proc. Natl. Acad. Sci. USA 92:10516-10520, 1995). A second recent paper reported the selection of second-site suppressor mutants characterized by near-wild-type protein synthesis in cells infected with gamma(1)34.5- mutants (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). Here, we report the properties of the spontaneous HSV-1 suppressor mutant Sup-1, which is characterized by spontaneous deletion of 503 bp encompassing the domain of the alpha47 gene and junction with the inverted repeats flanking the unique short (U(S)) sequence of the HSV-1 DNA resulting in the juxtaposition of the alpha47 promoter to the coding domain of the U(S)11 gene. This mutant does not exhibit the shutoff of protein synthesis characteristic of the gamma(1)34.5- virus. Specifically, Sup-1 in SK-N-SH human neuroblastoma cells (i) did not exhibit the function of the alpha47 gene characterized by a reduction in the transport of peptides across the endoplasmic reticulum of permealized cells consistent with the absence of alpha47 gene sequences, (ii) accumulated U(S)11 protein at levels analogous to those of the wild-type parent but the protein was made at earlier times after infection, as would be expected from a change in the promoter, and (iii) activated PKR like that of the parent, gamma(1)34.5- virus, but (iv) did not cause premature shutoff of protein synthesis and therefore was similar to the wild-type parent virus rather than the gamma(1)34.5- virus from which it was derived. We conclude that the mechanism by which Sup-1 blocks the shutoff of protein synthesis associated with phosphorylation of eIF-2alpha by the activated PKR is not readily explainable by a secondary mutation characterized by a deletion.

Animals↗

PubMed1997

Cytomegalovirus modulates transcription factors necessary for the activation of the tumor necrosis factor-alpha promoter.

Several studies have demonstrated that cytomegalovirus (CMV) infection increases expression of the tumor necrosis factor (TNF) gene. This effect is mediated, in part, by an effect of the CMV immediate early 1 (IE1) gene product on the TNF promoter. To further analyze these interactions, we used plasmids with TNF promoter truncations to determine the site within the promoter where the CMV IE1 gene product mediates its effect. The site was localized to a 40-base pair segment that contains a cAMP response element (CREB). Deletion of the cAMP response element increased basal promoter activation but had little effect on IE1-induced activation. Additional studies demonstrated that the cAMP element is flanked 5' by a PU.1 site and 3' by an NF-kappa B site, both of which increase expression of the TNF promoter. These sequences demonstrated IE1 responsiveness. We next determined the relevance of these observations for normal human cells by infecting human alveolar macrophages with CMV. In these studies we evaluated expression of NF-kappa B, PU.1 and CREB by gel shift assay at immediate early times after infection. We found that CMV infection increased the binding activity of NF-kappa B and PU.1 and decreased the binding activity of CREB. CMV infection also increased expression of the TNF gene in alveolar macrophages. These observations suggest that CMV increases TNF gene expression, in part, by altering the binding activity of transcription factors that regulate gene expression.

Base Sequence↗

PubMed1997

[The therapeutic effect of TGF-beta monoclonal antibody to bleomycin-induced pulmonary fibrosis in rats].

OBJECTIVE: Transforming growth factor-beta (TGF-beta) plays an important role in the pathogenesis of interstitial lung fibrosis. It can stimulate indirectly the mitosis of lung fibroblasts (LFb) as well as the synthesis and disposition of extracellular matrix. The purpose of this study was to evaluate the effects of TGF-beta monoclonal antibody on bleomycin-induced interstitial pulmonary fibrosis. METHOD: The effect of TGF-beta monoclonal antibody to bleomycin-induced pulmonary fibrosis in rat was studied either in vivo or in vitro. The proliferation of lung fibroblasts was studied by measuring the incorporation rate of 3H-TdR. Northern hybridization was used to detect mRNA level of procollagen A1 (I) and procollagen A1 (II) in cultured LFbs and lung tissue. RESULT: The results showed that the incubation supernatant of alveolar macrophages from rats with pulmonary fibrosis had the ability to stimulate fibroblast proliferation as well as their procollagen expression. The monoclonal antibody could inhibit the proliferation of LFbs in a concentration-dependent manner. The highest concentration, 100 micrograms/ml, of TGF-beta antibody could inhibit incorporation rate from 1749 +/- 322 of the fibrosis group to only 833 +/- 277 (P < 0.01). The mRNA level of procollagen I and II was decreased by 44% and 28% respectively after the antibody treatment. In vivo, procollagen I and II mRNA level were decreased by 40% and 12% after the administration of TGF-beta antibody though it could only slightly alleviate the extent of fibrosis and alveolitis in lung tissue. CONCLUSION: TGF-beta is a key factor stimulating the proliferation and collagen synthesis. TGF-beta antibody can partially neutralize its action. This may suggest that the antibody might become a new therapeutic choice for pulmonary fibrosis in the future.

Animals↗

PubMed1997

[The results of questionnaire of basic concepts about asthma management and prevention for respiratory professionals in Beijing's hospitals at different levels].

OBJECTIVE: To evaluate the information of basic concepts about asthma management and prevention collected from respiralogy professionals in Beijing's hospitals at different levels and provide the basis of drawing up a work plan for Asthma Management and Prevention Committee. METHODS: Fifty-five hospitals including municipal hospitals, urban district hospitals and county hospitals of suburban district were involved and four hundred and thirty-six respiralogy professionals consisting of resident physician and physician-in-charge were asked using questionnaire method. RESULTS: The respiralogy professionals at different level hospitals have some confused ideas in the definition of asthma, the importance of inhalation and anti-inflammatory therapy and the value of peak flow meter. The results of quetionnaire of respiralogy professionals in municipal hospitals were superior to that in urban district hospitals and county hospitals of suburban district. CONCLUSION: The physician education of asthma management and prevention is an important task. Special attention should be paid on the hospitals at basic level.

Asthma↗

PubMed1997

[An experiment study of reversed pulmonary hypertension with inhaled nitric oxide on smoke inhalation injury].

We evaluated the effect and mechanisms of reversed pulmonary hypertension with inhaled nitric oxide (NO) on smoke inhalation injury in the dog model, 21 dogs were divided into 3 groups randomly. Following smoke inhalation, the control group (n = 8) inhaled O2 (FiO2, 0.45) and the treated group (n = 9) inhaled O2 and 0.0045% (45 ppm) NO. Hemodynamics was serially measured for 12 hours. In addition, 4 dogs without smoke inhalation were used to study the normal lung histomophologic findings. The data were analyzed by ANOVA. After inhalation of NO, the mean pulmonary artery pressure (mPAP), pulmonary minute vessels pressure (Pmv), and pulmonary vascular resistance (PVR) were decreased significantly (P < 0.05), while the mean aortic pressure (mAP) and total peripheral resistance (TRP) were not remarkably changed (P > 0.05). The levels of cyclic guanosine monophosphate (cGMP) were increased significantly (P < 0.01). Inhaled NO may reverse pulmonary hypertension with smoke inhalation injury in dogs. The mechanism of selective pulmonary circulation was increased cGMP level in smooth cells. Inhaled NO may be recommended for clinical application.

Administration, Inhalation↗