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Biomedical subjects

J J Liu

Publications and source records attributed to J J Liu.

At least 55 records · Page 3Linked to original sources
PubMed1998

The recombinant nucleocapsid protein of classical swine fever virus can act as a transcriptional regulator.

The cDNA of the nucleocapsid (core) protein of classical swine fever virus (CSFV) was generated by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a eukaryotic expression vector. The effect of the recombinant core protein on the transcriptional regulation of cellular as well as viral promoters was studied. Using transient transfection assay, our results demonstrated that the core protein can activate the promoter of human heat shock protein 70 gene, and suppressed the SV40 early promoter. These findings indicate that the core protein appears to function not only as a viral structural protein but also as a regulator of gene expression. The implications of core proteins on the viral maturation are discussed.

Animals↗
PubMed1998

Cloning, expression and sequence analysis of the classical swine fever virus nucleocapsid protein.

The DNA complementary to the 5'-terminal 1929 nucleotides of classical swine fever virus (CSFV; alias hog cholera virus, HCV) LPC vaccine strain RNA was cloned and sequenced. The sequence encompasses a 5'-noncoding region (NCR) of 264 nucleotides and an open reading frame (ORF) of 1665 nucleotides. The cloned sequence contains genes of four viral proteins, P23, nucleocapsid (core) protein, E0 and part of E1 proteins. Alignment of the 5'-terminal 1929 nucleotides of LPC strain with other strains of CSFV showed well conservation and a homology as high as 84-95% was found between these strains. The cDNA of CSFV-LPC core was cloned into an expression vector, and a fusion protein of 38.5 kDa was obtained which reacted strongly to CSFV antiserum. Purification of the core fusion protein was achieved by a single-step affinity chromatography and the purified product could be recognized by the sera of CSFV-infected swine in ELISA assay. Phylogenetic analysis of the 5'-terminal 1929 nucleotides between pestiviruses revealed that the 5'-end region seems to be suitable for differentiation of different strains of CSFV.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and nucleotide sequence of 3'-terminal region of classical swine fever virus LPC vaccine strain.

A cDNA of the 3'-terminus of classical swine fever virus (LPC vaccine strain) was cloned and sequenced. The 3431 nucleotides and deduced amino acid sequences were compared with those of other pestiviruses, and the similarity of nucleotide sequences and deduced amino acid sequences were found to be 84-95% and 95-98%, respectively. Similar to other isolates of classical swine fever virus, the sequenced region included the non-structural gene p58 (NS5A) and part of p76 (NS5B) gene. The p76 gene of LPC vaccine strain also contained a highly conserved motif G-D-D (Gly-Asp-Asp) that is present in the RNA replicase of positive-stranded RNA viruses. With the sequence data currently available, we carried out a phylogenetic analysis and obtained a genealogical relationship among members of the classical swine fever virus.

Amino Acid Sequence↗
PubMed1998

Augmented effects of methionine and cholesterol in decreasing the elastic lamina while thickening the aortic wall in the rat aorta.

1. Patients with an elevated plasma level of either homocysteine or cholesterol are at increased risk of cardiovascular disease. Both methionine, the precursor of homocysteine, and cholesterol are found primarily in the same foods; therefore we investigated the effect of methionine feeding alone, cholesterol feeding alone, and both, on the thickness of the aortic wall and the aortic elastic lamina of normotensive animals.2. Twenty normotensive rats were divided into four groups of five animals. The following diet was administered for 15 weeks: normal chow; normal chow supplemented with 2% methionine; normal chow supplemented with 2% cholesterol; normal chow supplemented with 2% methionine+2% cholesterol.3. The results showed a 3-fold decrease (P<0.003) in the aortic elastic lamina in the 2% methionine group and a 2.5-fold decrease in the 2% cholesterol group compared with the normal chow group. There was a 9-fold (P<0.0003) decrease in the 2% methionine+2% cholesterol group compared with the normal chow group. Furthermore, feeding with methionine plus cholesterol significantly increased aortic wall thickness compared with the methionine group, cholesterol group or control.4. These results demonstrate an augmented effect of cholesterol plus methionine in the deterioration of the aortic elastic lamina, and furthermore, the combination of these two agents increases the thickness of the aortic wall. The results indicate a more important role for these two agents in combination than for either agent alone.

Analysis of Variance↗
PubMed1998

A novel immunohistochemical semiquantitative technique for endothelial constitutive nitric oxide synthase immunoreactivity in rat coronary artery.

It has been difficult to quantify protein production in small pathological specimens by conventional techniques. We describe a new method for semiquantification of immunohistochemical staining, which involves application of the enzyme-labeled avidin (LAB) technique, coupled with an ultra-sensitive and fast chemiluminescent substrate for alkaline phosphatase. The entire procedure can be completed in less than 3 hr. The final step involves X-ray film exposure for 30 min, and the optical density of the subsequent images is examined with a microcomputer imaging device. The optical densities are translated into relative protein concentrations by a reference standard curve, obtained via an immunoblot. To establish a model for semiquantification of endothelial constitutive nitric oxide synthase (eNOS) protein, we compared the coronary arteries of WKY rats fed a normal chow diet to the coronary arteries of WKY rats fed a cholesterol diet. Using this technique, we have found a relative 130-fold decrease in eNOS in the cholesterol-fed group compared to the normal chow-fed group.

Animals↗
PubMed1998

Expression of the glycoprotein E2 of the classical swine fever virus in Escherichia coli.

The glycoprotein E2 sequences of classical swine fever virus (strain p97) were cloned, sequenced and expressed in E. coli. Result from SDS-polyacrylamide gel electrophoresis analysis of expressed proteins revealed the presence of a prominently stained band corresponding to a molecular mass of 61 kDa, which is in agreement with the predicted size from the DNA sequence. The recombinant E2 protein contained an aminoterminal tag of six histidines that could be used for purification by the nickel chelate affinity chromatography. The elution fractions of the expressed protein also contain additional bands of 40 and 35 kDa proteins, indicating proteolytic cleavages might occur. Our Western blotting result also supported that the expression of the recombinant E2 protein of the classical swine fever virus were accomplished.

Animals↗
PubMed1998

Prognostic factors in patients with methanol poisoning.

OBJECTIVE: To identify prognostic factors in methanol poisoning and determine the effect of medical interventions on clinical outcome. METHODS: Retrospective review of all patients treated for methanol poisoning from 1982 through 1992 at The Toronto Hospital. Presenting history, physical examination, results of laboratory tests, medical interventions, and final outcomes after hemodialysis were abstracted. RESULTS: Of 50 patients treated for methanol poisoning, 18 (36%) died, 32 (64%) survived. Seven of the 32 survivors sustained visual sequelae (22%), the remaining 25 (78%) recovered completely. Patients presenting with coma or seizure had 84% (16/19) mortality compared to 6% (2/31) in those without (p < 0.001). Initial arterial pH < 7 was also associated with significantly higher mortality (17/19, 89% vs 1/31, 3%, p < 0.001). There were no differences in time from presentation to dialysis between survivors and fatalities (8.4 +/- 3.6 vs 7.6 +/- 3.5 hours, p = 0.47). The deceased patients had higher mean methanol concentration than the survivors (83 +/- 53 vs 41 +/- 25 mmol/L, p = 0.004). Subgroup analysis of 19 patients presenting with visual symptoms who survived showed prolonged acidosis (5.4 +/- 2.3 vs 3.0 +/- 2.1 hours, p = 0.06) in those with persistent visual sequelae. CONCLUSIONS: Coma or seizure on presentation and severe metabolic acidosis, in particular initial arterial pH < 7, are poor prognostic indicators in methanol poisoning. Survivors presented with lower methanol concentrations. Patients with residual visual sequelae had more prolonged acidosis than those with complete recovery. Future studies will be needed to confirm the effect of correction of acidosis on final clinical outcome.

Acidosis↗
PubMed1998

Characterization of a soluble KGF receptor cDNA from human corneal and breast epithelial cells.

PURPOSE: Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family FGF-7. It exhibits potent mitogenic activity for epithelial cells, including corneal and mammary epithelial cells. A messenger RNA has been reported that is generated by alternative splicing of bek that putatively codes only for the extracellular ligand-binding domain of KGF receptor (soluble KGF receptor). In the present study, the expression of the mRNA coding for this alternative bek transcript was examined and the corresponding protein characterized. METHODS: Alternative messenger RNA transcripts were detected in various cell lines or tissues using reverse transcription-polymerase chain reaction (RT-PCR) and RNase protection assay. NIH/3T3 fibroblast cells and 293 kidney embryonic epithelial cells were stably transfected with soluble KGF receptor cDNA and transmembrane KGF receptor cDNA. Soluble KGF receptor protein was produced using a baculovirus-insect expression system. Soluble KGF receptor protein was detected using western and dot blot analyses. Binding assays and cross-linking labeling were used to determine the affinity and specificity of soluble KGF receptor. A mitogenic assay was performed to examine the function of the soluble KGF receptor. RESULTS: The soluble KGF receptor mRNA was primarily expressed in epithelial cells, including cells from the cornea and breast. Cross-linking labeling and affinity-binding assays with 125I-KGF showed that the soluble KGF receptor bound KGF (FGF-7) but not FGF-1 or FGF-2. Soluble KGF receptor was detected in the culture medium of cells stably transfected with soluble KGF receptor cDNA but not with transmembrane KGF receptor cDNA, suggesting that the soluble receptor was generated by mRNA splicing and probably not by proteolysis or posttranslational processing. Soluble KGF receptor inhibited KGF binding to transmembrane KGF receptor and DNA synthesis in BALB/MK epidermal keratinocytes in response to KGF, suggesting that soluble KGF receptor expression could provide a mechanism for the cell to downregulate responses to KGF. CONCLUSIONS: A truncated soluble KGF receptor expressed in corneal and other epithelial cells probably functions to downregulate the response of the cell to KGF.

3T3 Cells↗
PubMed1997

Protein memory through altered folding mediated by intramolecular chaperones.

The 77-residue propeptide of subtilisin acts as an intramolecular chaperone that organizes the correct folding of its own protease domain. Similar folding mechanisms are used by several prokaryotic and eukaryotic proteins, including prohormone-convertases. Here we show that the intramolecular chaperone of subtilisin facilitates folding by acting as a template for its protease domain, although it does not form part of that domain. Subtilisin E folded by an intramolecular chaperone with an Ile(-48)-to-Val mutation acquires an 'altered' enzymatically active conformation that differs from wild-type subtilisin E. Although both the altered and wild-type subtilisins have identical amino-acid sequences, as determined by amino-terminal sequencing and mass spectrometry, they bind their cognate intramolecular chaperones with 4.5-fold greater affinity than non-cognate intramolecular chaperones, when added in trans. The two subtilisins also have different secondary structures, thermostability and substrate specificities. Our results indicate that an identical polypeptide can fold into an altered conformation through a mutated intramolecular chaperone and maintains memory of the folding process. Such a phenomenon, which we term 'protein memory', may be important in investigations of protein folding.

Binding Sites↗
PubMed1997

Perindopril chronically inhibits angiotensin-converting enzyme in both the endothelium and adventitia of the internal mammary artery in patients with ischemic heart disease.

BACKGROUND: ACE inhibitors are widely used in treating hypertension and heart failure, but the sites and mechanisms of ACE inhibition in human blood vessels are not understood. The present study was undertaken to assess the sites and extent of in vivo inhibition of ACE by long-term perindopril treatment in different layers of the internal mammary artery in patients with ischemic heart disease. METHODS AND RESULTS: Sixteen patients with ischemic heart disease were treated either with perindopril (4 mg/d PO) for up to 36 days before surgery (n = 9) or without the inhibitor as control subjects (n = 7). The segments of the internal mammary artery were collected for measurement of vascular free and total ACE by quantitative in vitro autoradiography with 125I-351A binding. The patients treated with perindopril had lower plasma ACE (P < .001) and plasma angiotensin (Ang) II-to-Ang I ratio (P < .05). In the internal mammary artery, free ACE was similarly inhibited by perindopril in the endothelium (P < .05) and adventitia (P < .05), and the free ACE-to-total ACE ratio, an index of ACE inhibition, was markedly decreased by perindopril in parallel in the endothelium (P < .001) and adventitia (P < .001). Moreover, plasma ACE correlated highly with vascular ACE in the endothelium (r = .85, P < .001) or adventitia (r = .78, P < .001), and mean arterial pressure correlated significantly with free ACE in the endothelium (r = .52, P < .05) or adventitia (r = .53, P < .05) and with the plasma Ang II-to-Ang I ratio (r = .53, P < .05). Light microscopic autoradiographs of 125I-351A binding revealed a marked inhibition of ACE by perindopril in both layers of the vascular wall. CONCLUSIONS: The present demonstrates that long-term administration of perindopril potently inhibits both endothelial and adventitial ACE to a comparable degree in the human internal mammary artery. These results indicate that perindopril effectively penetrates the vascular wall to inhibit ACE in the adventitia, thus providing evidence that perindopril may be beneficial in inhibiting both circulating Ang II and its local formation in the vascular wall.

Aged↗
PubMed1997

Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae.

The [PSI+] factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a "protein-only" seeded polymerization model for the inheritance of [PSI+].

Fungal Proteins↗
PubMed1997

Direct vasodilator effect of milrinone, an inotropic drug, on arterial coronary bypass grafts. FANZCA .

Milrinone is an inotropic drug with vasodilator activity that has been shown to be useful in increasing cardiac output and decreasing wedge pressure. Despite these advantages, it is unknown whether this drug can be used for the treatment of perioperative spasm of coronary bypass grafts. This study was undertaken to investigate the in vitro vascular effect of milrinone on internal thoracic arteries obtained from patients undergoing coronary artery bypass grafting. The results showed that milrinone produced a potent, concentration-dependent, preventive effect on the norepinephrine-induced contraction of internal thoracic arteries, as well as reversing contraction of internal thoracic arteries by receptor-dependent agents, including the thromboxane A2 mimetic U46619, the vasoconstrictor peptide endothelin-1, and the alpha1-adrenal receptor agonist phenylephrine. The relaxing effect of milrinone was weaker, however, on internal thoracic arteries contracted with 25 mmol/L potassium chloride. Comparison of milrinone with other vasodilators, including papaverine, nitroprusside, and glyceryl trinitrate, showed milrinone to be more potent than papaverine but less potent than nitroprusside and glyceryl trinitrate. The inhibitory effect of milrinone on internal thoracic artery contraction appeared as a reduction in contractile force, not as an increase in the values of concentrations of the agonists causing 50% maximal contraction, which indicates that milrinone exerts its vasodilator effect directly on the smooth muscles, not on the membrane receptors. The results also showed no significant difference in relaxing effect between internal thoracic artery rings with and without endothelium. In conclusion, this study provides experimental evidence that milrinone is a potent, endothelium-independent, direct vasodilator of the human internal thoracic artery and provides the scientific rationale for a future clinical trial with this drug for the perioperative treatment of internal thoracic artery spasm in cardiac surgical patients.

Coronary Artery Bypass↗
PubMed1997

In vivo gene transfer by intravenous administration of stable cationic lipid/DNA complex.

PURPOSE: A stable cationic lipid/DNA complex has been developed for in vivo gene transfer. The formulation capitalizes on a previously described procedure to obtain stable lipid/DNA complexes for in vitro gene transfer (1). METHODS: Conditions for DNA/lipid complex formation were modified to yield a DNA concentration of 1 mg/ml. Heat stable alkaline phosphatase (AP) under a CMV promoter was used as a reporter gene. RESULTS: The resulting complex was completely insensitive to serum inactivation. Tail vein injection of a 80 micrograms DNA into Balb C mice yielded significant levels of reporter enzyme activity in the lung, heart, spleen, muscle, and liver. Less AP activity was observed in the kidney. No AP activity was observed in blood, bone marrow or brain. A titration of the lipid (DOSPA) to DNA-nucleotide ratio showed the optimal molar ratio for in vivo gene transfer to be 1/1. Using this ratio in a dose response study showed approximately 80 micrograms of DNA/mouse yielded the highest level of gene expression. Using this dose at a 1/1 lipid to DNA nucleotide ratio, the time course for alkaline phosphatase activity was determined. Maximal AP activity was observed 24 hours after injection for all tissues. By day 5, the activity dropped approximately 10 fold for all tissues. By day 7, residual activity was detected in the lung, heart, and muscle. Histology of the lung showed both interstitial and endothelial cells to be transfected. In all other tissues, however, endothelial cells were the only transfected cell type. CONCLUSIONS: These results demonstrate that reformulation of an existing cationic lipid can result in the formation of a stable lipid/DNA complex, which is able to reproducibly transfect lung, heart, spleen, and liver upon intravenous administration.

3T3 Cells↗
PubMed1997

Histologic liver abnormalities in an autopsy series of patients with rheumatoid arthritis.

A retrospective review was performed on 188 autopsied cases of rheumatoid arthritis at our institutions during 1958-1985, prior to the widespread use of methotrexate. Hepatic histology was reported in 182 cases. All available microscopic liver slides from cases in which the autopsy report described portal tract inflammation, fibrosis, cirrhosis, tumour, amyloid, vasculitis, or infections involving the liver were examined and graded by a hepatic pathologist blinded to the original diagnosis, along with a representative sample of cases with reports describing fatty change or no hepatic pathologic abnormalities. Ninety normal and abnormal cases were reviewed from the 182 for which hepatic histology was available. Fifteen cases of diffuse fibrosis were identified upon blinded review. Two cases were graded as severe fibrosis (grades 3 or 4 on a scale of 0-4) without an identifiable pathologic cause, in both of which the liver disease was suspected premortem (alcohol abuse and viral hepatitis). Although the incidence of fibrosis in this series is slightly higher than that previously described, serious fibrotic liver disease was rare. These results support the current practice of limiting pre-treatment liver biopsies prior to methotrexate therapy to patients with suspected liver disease.

Adult↗
PubMed1997

Regulation of acetylcholine release by presynaptic nicotinic receptors at developing neuromuscular synapses.

Autoregulation of synaptic transmission in the nervous system is one of the homeostatic processes by which the transmission can be regulated according to varied physiological conditions. The neuromuscular cocultures of Xenopus laevis embryos were used to investigate the role of presynaptic nicotinic receptors in the autoregulation of developing motoneurons. The bath application of 2 microM nicotine had no significant effect on the frequency of spontaneous synaptic currents (SSCs). However, nicotine markedly increased the SSC frequency in the presence of low concentrations of glutamate (2 microM) or ATP (0.15 mM) or high K+ (8 mM), which only slightly increased the frequency of spontaneous acetylcholine (ACh) secretion. Carbachol but not oxotremorine was similar to nicotine in the positive regulation of spontaneous ACh release. Treatment with alpha-bungarotoxin, hexamethonium, d-tubocurarine, or mecamylamine, which only slightly inhibited the SSC amplitude, effectively antagonized the increasing effect of nicotine plus glutamate on SSC frequency. Local perfusion of isolated neurons with nicotine induced an inward current at nerve terminal but not at soma, suggesting that nicotinic receptors localize at nerve terminals. Both d-tubocurarine and hexamethonium, which produced tetanic fade in adult neuromuscular preparations, did not show tetanic fade at embryonic neuromuscular junction. The bath application of alpha-bungarotoxin or hexamethonium but not 6-cyano-2,3-dihydroxy-7-nitroquinoxaline inhibited the frequency of SSCs at high-activity (>3 Hz) synapses. A P2-purinoceptor antagonist, suramin, or desensitizing P2-purinoceptor with alpha, beta-methylene ATP also reduced the frequency of SSCs at these high-activity synapses. These results suggest that nicotinic receptors, P2-purinoceptors and glutamate, receptors coexist at nerve terminals of developing motoneurons. The activation of presynaptic nicotinic receptors, which cooperates with either P2-purinoceptors or glutamate receptors, may greatly increase the spontaneous ACh secretion. Endogenously released ACh and ATP are both involved in the positive regulation of spontaneous transmitter secretion at developing neuromuscular synapses.

Acetylcholine↗
PubMed1997

Glucan-induced disease resistance in tiger shrimp (Penaeus monodon).

Non-specific disease resistance induced by yeast cell wall extract, beta-1,3-1,6-glucan, was demonstrated in the tiger shrimp. In this study beta-1,3-1,6-glucan was administered to shrimps by immersion before culturing and orally during the culturing period. Challenge of the treated shrimps with the virulent pathogens, Vibrio vulnificus and viral agents extracted from the white spot syndrome victims, yielded promising results. The tolerance of glucan-treated shrimps was slightly enhanced to stresses including catching, transport and ammonia. The growth and survival rates of treated and untreated shrimps were not significantly different. Therefore, we suggest that beta-1,3-1,6-glucan can be used as an immuno-stimulant of cultured shrimps and may benefit shrimp farmers.

Adjuvants, Immunologic↗