Platelet aggregation abnormalities after cardiopulmonary bypass.
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Cardiopulmonary Bypass↗
Biomedical subjects
M Huang
Publications and source records attributed to M Huang.
Tumor necrosis factor-alpha plays a central role in interleukin-2-induced pulmonary vascular leak and lymphocyte accumulation.
Administration of interleukin-2 (IL-2) leads to pulmonary vascular leak. This form of pulmonary edema has previously been postulated to be due to the in vivo induction of tumor necrosis factor-alpha (TNF-alpha). To determine whether TNF-alpha plays a role in IL-2-induced pulmonary vascular leak, we performed in situ hybridization of lung sections and reverse transcriptase-polymerase chain reaction of bronchoalveolar lavage macrophages from IL-2-challenged mice. The results confirm an in situ upregulation of TNF-alpha mRNA expression in the lungs associated with vascular leak. In addition, a significant increase in TNF-alpha protein production was found in the lung following IL-2 administration, as measured by TNF-alpha-specific ELISA of lung supernatants (P = 0.028). Intravenous administration of a soluble TNF receptor significantly diminished IL-2-induced pulmonary vascular leak (P = 0.006). These findings confirm a central role for TNF-alpha in mediating the pulmonary vascular leak associated with IL-2 toxicity.
Animals↗
Interferon-alpha inhibits murine macrophage transforming growth factor-beta mRNA expression.
Transforming growth factor-beta (TGF-beta), a multifunctional polypeptide is produced by a wide variety of cells and regulates a broad array of physiological and pathological functions. TGF-beta appears to play a central role in pulmonary fibrosis and may contribute to tumor-associated immunosuppression. Alveolar macrophages are a rich source of TGF-beta and are intimately involved in lung inflammation. We therefore chose to study TGF-beta regulation in murine alveolar macrophages as well as an immortalized peritoneal macrophage cell line (IC-21). Murine macrophages were incubated with cytokines to evaluate their role in regulating TGF-beta mRNA expression. We conclude that IFN-alpha downregulates TGF-beta mRNA expression in murine macrophages.
Animals↗
Gene synthesis and functional expression of a protein exhibiting monodomain IgG Fc binding.
A gene encoding a bacterial IgG Fc binding domain was designed and synthesized. The synthetic DNA fragment was cloned 3' to an inducible trpE promoter such that expression of the gene in Escherichia coli produced abundant Fc binding protein fused to the first seven amino acids of the trpE protein. The recombinant protein contained a single Fc binding domain and demonstrated efficient binding to human IgG in Western blot analysis. This protein degraded rapidly following cell lysis in the absence of protease inhibitors, but could be effectively protected by the addition of protease inhibitor. After purification of the protein by IgG affinity chromatography, IgG Fc binding ability was retained for at least 24 h at either 23 or 37 degrees C and on heating for 15 min at temperatures up to 65 degrees C. No immunoprecipitation was observed in interactions between the monodomain Fc binding protein and IgG molecules. Unlike staphylococcal protein A, no detectable binding of the monodomain IgG Fc binding protein was observed to either IgM or IgA. Truncated proteins, expressed from a series of 3' deletions of the synthetic gene, were used to estimate the minimum portion of a monodomain Fc binding protein that retained Fc binding ability.
Amino Acid Sequence↗
A single Fc binding domain--alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity.
A recombinant gene fusion was created and cloned using a previously constructed gene encoding a monodomain IgG Fc binding protein and the gene coding for bacterial alkaline phosphatase. The construct was able to express and secrete a fusion protein that exhibited both IgG binding and alkaline phosphatase enzymatic activities. Greater than 60% of the protein demonstrating both biological activities was detected from periplasmic space preparations. Nanogram concentrations of the Fc binding--alkaline phosphatase fusion protein allowed primary IgG antibody detection without the use of conjugated secondary antibodies. Removal of the domain coding for alkaline phosphatase resulted in decreased resistance of the protein to proteolytic degradation and the loss of IgG Fc binding ability. Using affinity-purified fusion protein, the specificity of binding to IgG, IgM and IgA was examined; binding was strong to IgG and barely detectable against IgM or IgA. Affinity for binding of the fusion protein to IgG (Kd = 6.7 x 10(-8) M) was determined to be equal to or greater than previously reported for protein A.
Alkaline Phosphatase↗
pet, a small sequence distal to the pregenome cap site, is required for expression of the duck hepatitis B virus pregenome.
We have found that transcription of the pregenome of an avian hepadnavirus, duck hepatitis B virus (DHBV), is dependent on the presence of a small element in the 5' transcribed region of the pregenome-encoding sequence. This element, which we have named pet (positive effector of transcription), exerts its effect in cis in a position and orientation-dependent manner, suggesting that it may function as part of the nascent pregenome transcript. The requirement for pet depends on the presence in the transcription unit of a region of the DHBV genome located upstream of the envelope promoters, which specifically suppresses transcription of templates lacking pet. In the presence of this region, deletion of pet activates transcription from downstream promoters, suggesting that pregenome transcription complexes fail to reach the downstream promoters. In vitro transcription experiments support the model that pet is required for transcription elongation on the DHBV template. We speculate that pet is required to suppress transcription termination during the first passage of pregenome transcription complexes through a viral termination region on the circular viral DNA.
Animals↗
Murine AIDS is initiated in the lymph nodes draining the site of inoculation, and the infected B cells influence T cells located at distance, in noninfected organs.
The infection of cells which belong to the B-cell lineage is thought to be the primary event leading to the phenotypic and functional alterations seen in the murine AIDS (M. Huang, C. Simard, D. Kay, and P. Jolicoeur, J. Virol. 65:6562-6571, 1991). Using in situ hybridization, we studied the time course of the anatomic distribution of the murine AIDS-infected B cells in C57BL/6 mice inoculated intraperitoneally or in the foot pad with helper-free stocks of the defective murine AIDS virus. The local lymph nodes draining the injection site (the mediastinal or popliteal lymph nodes) were the primary organs in which infected B cells could be detected. From this initial site, the proliferating infected B cells were found to migrate progressively to most of the other lymph nodes and to the spleen. The bone marrow cells (containing the precursor B cells) were not found to be infected by the virus. These results suggest that the defective murine AIDS virus infects mature Ly-1- B cells present in lymph nodes. We compared the concanavalin A response of the T cells at an early time postinoculation, before all lymphoid organs are infiltrated with infected B cells. In lymphoid organs free of infected B cells, T cells were found to be hyperresponsive. In lymphoid organs in which infected B cells were present, T cells were hyporesponsive. These data suggest that infected B cells influence distant T cells, maybe by the release of a circulating factor or through another uninfected cell population activated by the infected B cells.
Animals↗
Myristylation of Pr60gag of the murine AIDS-defective virus is required to induce disease and notably for the expansion of its target cells.
Murine AIDS (MAIDS) is characterized by severe lymphadenopathy and splenomegaly. The proliferation of the infected target B cells is also an important manifestation of the disease (M. Huang, C. Simard, D. G. Kay, and P. Jolicoeur, J. Virol. 65:6562-6571, 1991). The etiologic agent of MAIDS is a defective murine leukemia virus that is deleted of most of its pol and env genes and appears to encode a single protein, the Gag precursor Pr60gag protein. Pr60gag is myristylated and attached to the plasma membrane. To study the role myristylation on the function of Pr60gag, we have generated a myristylation-negative (Myr-) mutant of the MAIDS defective virus. We found that Myr- Pr60gag interacted less tightly with the plasma membrane. In addition, the Myr- MAIDS defective virus mutant was unable to induce expansion of infected cells and was nonpathogenic. These results emphasize the essential role of Pr60gag in the disease process. Our data also suggest that Pr60gag, once recruited to the cell membrane through its myristylation, interacts with other membrane-bound effectors to send signals to induce proliferation of the infected cells and to initiate immune dysfunctions.
Animals↗
The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA and nucleates the assembly of 40S heterogeneous nuclear ribonucleoprotein particles.
A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of 230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt) into a unique 19S triangular complex. In the absence of this unique structure, the basic A- and B-group proteins bind RNA to form several different artifactual structures which are not present in preparations of native hnRNP and which do not function in hnRNP assembly. Three (A2)3B1 tetramers bind the 19S complex to form a 35S assembly intermediate. Following UV irradiation to immobilize the C proteins on the packaged RNA, the 19S triangular complex is recovered as a remnant structure from both native and reconstituted hnRNP particles. C protein-RNA complexes composed of three, six, or nine tetramers (one, two, or three triangular complexes) nucleate the stoichiometric assembly of monomer, dimer, and trimer hnRNP particles. The binding of C-protein tetramers to RNAs longer than 230 nt is through a self-cooperative combinatorial mode. RNA packaged in the 19S complex and in 40S hnRNP particles is efficiently spliced in vitro. These findings demonstrate that formation of the triangular C protein-RNA complex is an obligate first event in the in vitro and probably the in vivo assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.
Binding Sites↗
Systemic and regional hemodynamics after nitric oxide synthase inhibition: role of a neurogenic mechanism.
The study tested the hypothesis that the increase in blood pressure and decrease in cardiac output after nitric oxide (NO) synthase inhibition with N omega-nitro-L-arginine methyl ester (L-NAME) was partially mediated by a neurogenic mechanism. Rats were anesthetized with Inactin (thiobutabarbital), and a control blood pressure was measured for 30 min. Cardiac output and tissue flows were measured with radioactive microspheres. All measurements of pressure and flows were made before and after NO synthase inhibition (20 mg/kg L-NAME) in a group of control animals and in a second group of animals in which the autonomic nervous system was blocked by 20 mg/kg hexamethonium. In this group of animals, an intravenous infusion of norepinephrine (20-140 ng/min) was used to maintain normal blood pressure. L-NAME treatment resulted in a significant increase in mean arterial pressure in both groups. L-NAME treatment decreased cardiac output approximately 50% in both the intact and autonomic blocked animals (P < 0.05). Autonomic blockade alone had no effect on tissue flows. L-NAME treatment caused a significant decrease in renal, hepatic artery, stomach, intestinal, and testicular blood flow in both groups. These results demonstrate that the increase in blood pressure and decreases in cardiac output and tissue flows after L-NAME treatment are not dependent on a neurogenic mechanism.
Amino Acid Oxidoreductases↗
Evaluation of the needle technique for producing an arteriovenous fistula.
The purpose of the present study were to evaluate the needle technique of creating an arteriovenous (a-v) fistula and to quantitatively determine the hemodynamic responses in rats with three different fistula sizes. The fistula was made in male Sprague-Dawley rats between the aorta and vena cava below the renal arteries by using 20-, 18-, and 16-gauge angiocath needles. Five weeks after a sham operation or creation of an a-v fistula, mean arterial blood pressure (MAP), right atrial pressure, shunt flow, cardiac index, systemic flow, individual organ flows, and heart weight were quantitatively determined. All flow measurements were made using radioactive microspheres. The flow to the lungs was used as a measure of shunt flow. The shunt flow in the 20-, 18-, and 16-gauge fistula animals was significantly increased from a value of 2 +/- 1% (SE) to 50.0 +/- 0.1, 78 +/- 3, and 76 +/- 3% of total cardiac output, respectively. Average cardiac index in the 20-, 18-, and 16-gauge fistula animals increased by 105, 270, and 250%, respectively, compared with control. Right atrial pressure and heart weight were increased in proportion to the size of the fistula. MAP in the control and 20-, 18-, and 16-gauge fistula animals was 122 +/- 5, 126 +/- 3, 118 +/- 3, and 111 +/- 4 mmHg, respectively. There were no significant differences in MAP or systemic flow among any of these groups. The calculated total peripheral resistance in all fistula groups was significantly decreased compared with control.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Glucose given after hypoxic ischemia does not affect brain injury in piglets.
BACKGROUND AND PURPOSE: Giving glucose before hypoxic ischemia worsens brain injury in piglets. Does giving glucose after hypoxic ischemia affect severity of injury? METHODS: Forty-three 0- to 3-day-old pigs were used. All piglets received 2 U/kg insulin before injury to prevent stress-induced hyperglycemia. Hypoxic ischemic brain damage was induced by clamping both carotid arteries and reducing arterial blood pressure to two thirds of normal by hemorrhage at time 0. At 15 minutes the fraction of inspired oxygen (FIO2) was reduced to 6%. At 30 minutes FIO2 was increased to 100%, the carotids were released, and the withdrawn blood was reinfused. The piglets were then randomized to receive either 2 mL/kg of 50% dextrose followed by 2 mL/kg per hour for 2 hours or an equal volume of saline. RESULTS: Neurological examination scores (20 is normal, 5 is brain dead, by blinded observer) at 1 day postinjury were similar in the two groups: glucose, median 15.5 (25th percentile, 12.2; 75th percentile, 18); controls, 15.6 (9.3, 18). Piglets were killed at 3 days with brain preservation at death. Pathological examination scores (sum of scores from cortex, hippocampus, and basal ganglia: 30 is normal, 3 is total necrosis) by blinded observer were similar in the two groups: glucose, 26 (18, 28); controls, 25 (16.5, 28); NS. CONCLUSIONS: Although elevated glucose levels during hypoxic ischemic injury worsen brain injury in the piglet, elevated glucose levels after injury do not affect the severity of the injury.
Animals↗
[Effects of monoclonal antibodies on phagocytosis of Entamoeba histolytica to human erythrocytes].
Two monoclonal antibodies 3F7 and 4G6 were produced respectively against plasma membrane and nucleus-cytoplasm of Entamoeba histolytica. The erythrocytophagous quantification and ability of pathogenic strain HM-1:IMSS were evaluated by microscopy immediately at 5 min and 10 min after incubation with McAb 3F7 and McAb 4G6. Sera from normal mice and monoclonal antibody TCE 04 against Trypanosoma cruzi were prepared as negative controls. The McAb 3F7 showed stronger inhibitory action on the erythrocytophagous effect of HM-1:IMSS than the McAb 4G6. The result suggested a significant inhibition (P < 0.001) on erythrocytophagous effect of amoebic trophozoites in the presence of McAb 3F7. This inhibitory action appeared to have a decreasing tendency with the elapse of incubation time.
Animals↗
Down-regulation of macrophage transforming growth factor-beta messenger RNA expression by IL-7.
Transforming growth factor-beta (TGF-beta) is a potent inhibitor of immune responses. Macrophage-derived products, including TGF-beta, have been suggested as inhibitors of the antitumor immune response. We hypothesized that IL-7, a cytokine with antitumor properties, may exert its immunoregulatory effects in part through the down-regulation of TGF-beta. To test this hypothesis we analyzed IL-2-stimulated murine macrophage TGF-beta mRNA expression following exposure to IL-7 both in vitro and in vivo. IL-7 down-regulated IL-2-induced TGF-beta expression by macrophages in vitro, as well as after i.p. injections of IL-2 and IL-7 in vivo. The IL-7-mediated inhibition of TGF-beta mRNA expression did not require new protein synthesis and therefore appears to be a direct effect of IL-7. IL-7 had no significant effect on the stability of TGF-beta mRNA. Nuclear run-on assays revealed that the suppression of IL-2-induced TGF-beta gene expression by IL-7 is mediated at the level of transcription. Also, IL-7 decreased TGF-beta secretion as measured by bioassay. We conclude that IL-7 down-regulates TGF-beta and suggest that some of the proliferative and cytolytic activities mediated by cells exposed to IL-7 may be caused by a decrement in macrophage-derived TGF-beta.
Animals↗
Functional redundancy of octamer elements in the mouse mammary tumor virus promoter.
The promoter of mouse mammary tumor virus contains three overlapping sequence elements related to the octamer consensus (ATGCAAAT) that are largely contained within two 10 bp direct repeats (CTTATGTAAA) separated by a 2 bp spacer between 60 and 39 relative to the start of transcription. Gel electrophoresis mobility shift competition assays demonstrate that the most distal of these octamer-related elements is recognized by a protein that also binds to the most proximal element, while the central octamer-related element is not efficiently recognized. Transient transfection assays with altered promoters reveal that the portion of the 10 bp repeat that is not related to the octamer consensus appears not to be important for transcription. The distal and proximal octamer-related elements are, at least to some extent, functionally redundant. Complete deletion of one element has little or no effect on promoter activity so long as certain spacing constraints among remaining promoter elements are maintained. Systematic variation of such spacing reveals a cyclic effect on promoter activity corresponding to the periodicity of Bform DNA, suggesting functional interactions between proteins bound to adjacent sites.
Animals↗
Mapping of ben genes of Pseudomonas aeruginosa.
Four ben genes responsible for the conversion of benzoate to catechol in Pseudomonas aeruginosa PAO have been mapped to a 4.6 kb KpnI fragment, ben-1 and ben-4 were known to be separate genes but now ben-1508 has been found to be different from ben-2. The two genes were distinguished by Tn5 mutagenesis of a cosmid clone and deletion mapping. It is likely that the four genes mapped (ben-4, ben-2, ben-1508 and ben-1) correspond to the previously characterized benR (regulatory gene) and benABC (benzoate dioxygenase) respectively.
Benzoates↗
Characterization of transcripts from unrearranged V alpha 8 genes in the thymus.
The genes that encode the alpha- and beta-chains of the TCR undergo programmed rearrangement during differentiation of a T cell in the thymus, but it is not known what controls the order and specificity of the rearrangement event. By analogy with Ig genes, it is possible that transcription of an unrearranged V region gene may be necessary for "access" of the recombinase. To begin to address this issue, the six members of the V alpha 8 subfamily (five functional members and a pseudogene) from C57Bl/6 mice were examined. Consistent with the "accessibility" model, we show that unrearanged V alpha 8 genes are specifically expressed in the thymus of adult mice. Each of the functional genes was transcribed, but at different levels in the thymus. The five V alpha 8 genes were identical through the first 50 nucleotides of the 3' flanking region, and each contained an open reading frame that was contiguous with the V coding region. More interestingly, two of these putative proteins ended in Cys-X-Cys, a motif that is known to undergo isoprenoid modification. This finding and the conservation in the region that extends beyond the heptamer-nonamer region raise the possibility that some unrearranged V genes may encode proteins that have a novel function during early T cell development.
Animals↗
Erratum: Scaling theory for the glass transition
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Journal Article↗