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Biomedical subjects

L F Wang

Publications and source records attributed to L F Wang.

At least 145 records · Page 8Linked to original sources
PubMed1990

Site directed mutagenesis of subunit 8 of yeast mitochondrial ATP synthase. Functional and import properties of a series of C-terminally truncated forms.

The function of the positively charged C-terminal region of mitochondrially encoded subunit 8 of yeast mitochondrial ATP synthase was investigated using derivatives truncated at each of the 3 positively charged residues (Arg37, Arg42 and Lys47). Each construct, allotopically expressed in the nucleus, was tested for its ability to import and assemble functionally into ATP synthase in yeast cells unable to synthesize mitochondrial subunit 8. The efficiency of import of each construct into isolated wild-type yeast mitochondria was also determined. One construct truncated at the penultimate residue of subunit 8 (Lys47) functions in vivo and shows efficient import in vitro. Thus subunit 8 can function with only two positively charged residues. The remainder of the subunit 8 variants failed to rescue in vivo. Since they all show greatly reduced or undetectable import in vitro, presumably because of the increased hydrophobic character of the subunit 8 moiety in the chimaeric precursors, the status of these variants as regards assembly and function is not clear.

Base Sequence↗
PubMed1990

Expression of a sperm protein gene during spermatogenesis in mammalian testis: an in situ hybridization study.

In previous work a specific membrane protein with an estimated Mr of 20.1 kDa was purified from rabbit sperm tails and designated as rSMP-B protein. Antibodies were raised against rSMP-B protein and used to isolate and identify the cDNA coding the rSMP-B protein from a rat testis lambda gt11 expression library. The nucleotide sequence of the cDNA was determined in a previous study. Single-stranded 35S-labeled RNA probes were prepared. With the techniques of in situ hybridization, rSMP-B mRNA was detected in spermatids of rat and rabbit testis. The present results support our previous observation that immunization of male rabbits with the rSMP-B protein results in the arrest of spermatogenesis at the spermatid stage. Overall, rSMP-B protein appears to be involved in spermiogenesis, and the synthesis of the mRNA encoding the protein occurs in germ cells during the postmeiotic haploid phase of spermatogenesis.

Animals↗
PubMed1990

Characterization of cDNA encoding a human sperm membrane protein related to A4 amyloid protein.

A rat testis lambda gt11 cDNA library was screened with a monoclonal antibody raised against a human sperm membrane protein designated YWK-II. A clone was found with a cDNA insert composed of 1837 base pairs that contained an open reading frame coding for 191 amino acid residues. The deduced polypeptide contained a segment with high homology to the transmembrane-cytoplasmic domains of the A4 amyloid protein found in brain plaques of Alzheimer disease patients. A sequence of basic amino acid residues, Arg-Lys-Arg, was found instead of Lys-Lys-Lys at the probable membrane-cytoplasmic junction that may be a unique property of sperm membrane proteins.

Amino Acid Sequence↗
PubMed1990

A seral epidemiological study of HIV transmitted through human seral gamma-globulin preparations.

In order to study the potential risk of transferring HIV through human seral gamma-globulin preparations (immunoglobulin), indirect immunofluorescent antibody test (IFA) and Western Blot (WB) assay were applied to 343 random samples (sera) with previous injection of imported human seral gamma-globulins (Ig) positive for Human Immunodeficiency Virus (HIV) antibodies between 1981-1987 for the detection of HIV antibodies. All results were negative and tests on all 23 controls who had previously received Ig made in China also gave negative results. However all 12 batches of imported Ig collected from the above-mentioned users, were positive for HIV antibodies when tested by WB and IFA. This study shows that under normal conditions, human seral gamma-globulin does not transmit HIV.

Adolescent↗
PubMed1990

Neutrophil activation--an important cause of tissue damage during liver allograft rejection?

Although neutrophils have been implicated as mediators of tissue damage in a variety of conditions, their role in graft rejection has not previously been studied. Peripheral neutrophil activation was assessed sequentially in 16 patients following liver transplantation by measuring the ability of neutrophils to respond to the chemotactic peptide FMLP, to release superoxide radicals, and to cause extracellular proteolysis. All three functions increased 1-2 days before episodes of acute rejection and only returned to normal after treatment with high-dose corticosteroids. Three patients who did not develop rejection showed no increase in neutrophil function. Lymphocytes isolated from patients with acute rejection produced factors that were both chemotactic for and capable of activating normal neutrophils. This study shows that neutrophil activation occurs during acute liver allograft rejection, possibly in response to lymphokine secretion, and suggests an important, and hitherto unrecognized, mechanism of graft damage during rejection.

Adult↗
PubMed1990

Inhibition of leukocyte chemotaxis by immunosuppressive agents. Specific inhibition of lymphocyte chemotaxis by cyclosporine.

Since chemotaxis is an important mechanism by which leukocytes are recruited to allografts during rejection, the effect of immunosuppressant agents on in vitro chemotactic responses of neutrophils, monocytes, and lymphocytes was studied. Cyclosporine caused profound inhibition of lymphocyte chemotaxis to three different chemotactic factors at pharmacologic levels but had no effect on neutrophil or monocyte chemotaxis. Methylprednisolone and azathioprine both inhibited chemotactic responses of neutrophils and monocytes as well as lymphocytes. These data suggest that immunosuppressant agents are potent inhibitors of leukocyte chemotaxis and that drugs differ as to which leukocyte cell type they inhibit. The selective action of cyclosporine might explain why this drug is ineffective in reversing established rejection episodes in which monocytes and neutrophils are also involved. Inhibition of leukocyte chemotaxis may be an important mode of action of immunosuppressant drugs, and drug regimes might be adjusted to cause selective inhibition of particular cell types.

Azathioprine↗
PubMed1990

Complex character of senS, a novel gene regulating expression of extracellular-protein genes of Bacillus subtilis.

The senS gene of Bacillus subtilis, which in high copy number stimulates the expression of several extracellular-protein genes, has been cloned, genetically mapped, and sequenced. The gene codes for a highly charged basic protein containing 65 amino acid residues. The gene is characterized by the presence of a transcription terminator (attenuator) located between the promoter and open reading frame, a strong ribosome-binding site, and a strong transcription terminator at the 3' end of this monocistronic gene. The amino acid sequence of SenS showed partial homology with the N-terminal core binding domain region of bacterial RNA polymerase sigma factors and a helix-turn-helix motif found in DNA-binding proteins. The gene can be deleted without any effect on growth or sporulation.

Amino Acid Sequence↗
PubMed1990

Expression in Escherichia coli of the Bacillus subtilis neutral protease gene (NPRE) lacking its ribosome binding site.

Bacillus subtilis neutral protease (NprE) is first produced as a precursor, pre-pro-NprE, which consists of a signal peptide or prepeptide for secretion (27 amino acid residues) and a pro-peptide (194 amino acid residues) between the signal peptide and the mature protease. While the wildtype nprE gene could not be maintained in Escherichia coli, we have been able to show that expression and secretion of the neutral protease can be achieved from the nprE gene when its ribosome binding site (RBS) is removed. The results suggest that the failure to observe expression of the wildtype nprE gene is due to the lytic effect of the nprE gene product on E. coli host cells and that translation initiation in E. coli can be achieved even in the absence of a classical ribosome binding site.

Bacillus subtilis↗
PubMed1990

[Effect of drug volume on spinal anesthesia with isobaric tetracaine].

The effects of drug volume on spinal anesthesia with 10 mg tetracaine in 1 ml or 2 ml isobaric solution were studied in 100 demographically compatible patients in a randomized double-blind manner. Identical technique was used for every patient in the study except for the volume of anesthetic solutions. Drugs were administered in a lateral decubitus position and patients remained horizontal during the study. Hoghest cephalad spread of analgesia varied from T12-T3, with a median sensory level of T7 in those who received 2 ml solutions, compared with T12-T5 and a median level of T10 in those who received 1 ml solutions. The time for maximum spread was longer in 2 ml--group (7.54 +/- 1.76 min vs 4.85 +/- 1.54 min). The 2-segment regression times were 180 +/- 53.44 min (mean) with a range of 75-270 min, and 219 +/- 34.11 min (mean) with a range of 150-300 min respectively for the 2 ml--group and the 1 ml--group. The differences were statistically significant. Complete motor blockade of the lower extremities as assessed by the modified Bromage scale was achieved in 35 out of 50 patients given 2 ml solutions, while all those who received 1 ml solutions achieved complete motor blockade. The onset times of maximum blockade were 5.42 +/- 1.59 min (mean) with a range of 2-10 min, and 5.98 +/- 1.79 min (mean) with a range of 2-12 min respectively. The difference between the degree of maximum motor blockade was also statistically significant but was insignificant regarding the time to maximum motor blockade.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1989

Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis.

A 1.6 kb cDNA fragment encoding the mature part of the human tissue-type plasminogen activator (t-PA) was subcloned into a Bacillus subtilis dual plasmid expression system [Le Grice et al., Gene 55 (1987) 95-103]. Expression of the tPA gene in this vector was regulated by the inducible Escherichia coli lac elements, as well as a strong phage-T5-derived promoter and ribosome-binding site preceding the polylinker. The 5' end of the tPA gene corresponding to the N terminus of mature t-PA was fused in phase to the third codon present in the polylinker region of the expression vector, p602/22, to form p602-t-PA. B. subtilis containing p602-t-PA, when induced with isopropyl-beta-D-thiogalactopyranoside, produced large amounts of immunoreactive t-PA (approx. 20 micrograms/ml). As expected, t-PA was not secreted into the culture media, but was localized in intracellular inclusion bodies and was found to be enzymatically inactive. However, enzymatic activity could be regained following complete reduction followed by slow oxidation of the solubilized inclusion bodies. The recombinant t-PA (rt-PA) showed, after purification, a smaller molecular size than melanoma t-PA, probably due to lack of glycosylation in the Bacillus system. Like melanoma t-PA, rt-PA exhibited tremendous stimulation of plasminogen activation in the presence of fibrin. Our results illustrate that B. subtilis, when supplied with the proper transcriptional/translational regulatory elements, can be an effective system for expression of heterologous gene products.

Bacillus subtilis↗
PubMed1989

Bluetongue virus-17 fusion protein ns1 expressed in Escherichia coli by pUC vectors.

The cDNA coding for the major nonstructural protein, NS1, of bluetongue serotype 17 (BTV-17) was cloned previously. Using pUC plasmids, we have successfully expressed the NS1 protein in Escherichia coli as a LacZ-NS1 fusion protein. The recombinant NS1 protein reacted with rabbit anti-BTV-17 antiserum, and was thus immunologically indistinguishable from the native BTV-17 NS1 protein. This was the first bluetongue viral protein to be produced in a bacterial system.

Blotting, Western↗
PubMed1989

Bacillus subtilis subtilisin gene (aprE) is expressed from a sigma A (sigma 43) promoter in vitro and in vivo.

In vitro studies demonstrated that the Bacillus subtilis subtilisin gene (aprE) could be transcribed by RNA polymerase holoenzyme reconstituted from core and sigma A factor obtained from vegetative cells. Upstream deletions (from -45) reduced the amount of transcription from the promoter. A deletion downstream of the promoter that overlapped a putative downstream minor promoter did not affect transcription from the sigma A promoter, which indicated that the putative downstream promoter is not utilized in vivo. S1 nuclease mapping studies showed that there was a low level of transcription from the subtilisin promoter during the growth phase and that the site of transcription initiation was the same during log and stationary phases. We conclude from these findings that there is only one promoter for the subtilisin gene and that it can be transcribed by the sigma A form of RNA polymerase in vitro.

Bacillus subtilis↗
PubMed1989

[Mechanism of active oxygen in cytocidal activity of tetrabromofluorescein].

Photosensitization induced by tetrabromofluorescein (eosin, 3.8 mumol/L) in ascites hepatoma cells or in normal kidney cells of mice was found to be significant. The cytocidal activity increased in proportion to the concentration of fluorescein as well as with irradiation time. ESR signals were not detected using a trapping agent, 2,2,6,6-tetramethyltetrahydroxy-piperidine (TMHP) which functions as a singlet oxygen probe. No effect on photosensitization by superoxide dismutase (SOD), NaN3, histidine, mannitol or beta-carotene were observed. However, catalase did decrease photosensitization. These results indicate that cytocidal activity is not related to 1O2, O2-. or OH., but is related to H2O2. The cytocidal activity of tetrabromofluorescein in ascites hepatoma cells is stronger than that in normal kidney cells.

Animals↗