Biomedical subjects

H Y Kim

Publications and source records attributed to H Y Kim.

At least 73 records · Page 4Linked to original sources

PubMed2001

Molecular characterization of cycloinulooligosaccharide fructanotransferase from Bacillus macerans.

Cycloinulooligosaccharide fructanotransferase (CFTase) converts inulin into cyclooligosaccharides of beta-(2-->1)-linked D-fructofuranose by catalyzing an intramolecular transfructosylation reaction. The CFTase gene was cloned and characterized from Bacillus macerans CFC1. The CFTase gene encoded a polypeptide of 1,333 amino acids with a calculated Mr of 149,563. Western blot and zymography analyses revealed that the CFTase with a molecular mass of 150 kDa (CFT150) was processed (between Ser389 and Phe390 residue) to form a 107-kDa protein (CFT107) in the B. macerans CFC1 cells. The processed CFT107 was similar in its mass to the previously purified CFTase from B. macerans CFC1. The CFT107 enzyme was produced by B. macerans CFC1 but was not detected from the recombinant Escherichia coli cells, indicating that the processing event occurred in a host-specific manner. The two CFTases (CFT150 and CFT107) exhibited the same enzymatic properties, such as influences of pH and temperature on the enzyme activity, the intermolecular transfructosylation ability, and the ability of hydrolysis of cycloinulooligosaccharides produced by the cyclization reaction. However, the thermal stability of CFT107 was slightly higher than that of CFT150. The most striking difference between the two enzymes was observed in their Km values; the value for CFT150 (1.56 mM) was threefold lower than that for CFT107 (4.76 mM). Thus, the specificity constant (kcat/Km) of CFT150 was about fourfold higher than that of CFT107. These results indicated that the N-terminal 358-residue region of CFT150 played a role in increasing the enzyme's binding affinity to the inulin substrate.

Amino Acid Sequence↗

PubMed2001

Treatment with cyclosporin switching to hydroxychloroquine in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the therapeutic benefit of cyclosporin A (CSA) switching to hydroxychloroquine (HCQ) in patients with rheumatoid arthritis (RA). METHODS: Thirty four patients with RA who displayed residual inflammation and disability despite partial responses to prior maximal tolerated doses of methotrexate, were included. All were treated with a staged approach using CSA for 24 weeks to induce clinical improvement, followed by HCQ for 16 weeks to maintain the improvement. Seven ACR core set measures were evaluated every four to eight weeks. RESULTS: During a 40 week open trial, 27/34 patients completed the study. CSA treatment significantly reduced the tender joints score, swollen joints score, visual analogue pain scale, patient's or doctor's global assessment, patient's self assessed disability, and C reactive protein. Compared with the time of entry into the trial, patients who switched from CSA to HCQ still possessed significantly lower levels of most variables, determined at 28, 32, and 40 weeks. According to the ACR 20% improvement definition, 15/27 (56%) patients had improved at 24 weeks after CSA treatment, and 14/27 (52%) remained improved at 16 weeks after the change to HCQ. Frequent side effects, such as hypertrichosis, gastrointestinal trouble, and hypertension, were noted during CSA treatment, but most of these disappeared after switching to HCQ. The mean levels of blood pressure and serum creatinine were significantly increased during CSA treatment, but returned to normal after changing to HCQ. CONCLUSIONS: The data suggest that CSA switching to HCQ treatment may be an effective strategy for patients with RA partially responding to methotrexate, particularly those with toxicity due to CSA.

Adult↗

PubMed2001

Electrophysiologic and antiarrhythmic effects of the new class III antiarrhythmic drug KCB-328 in experimental canine atrial flutter.

BACKGROUND: The electrophysiologic and antiarrhythmic effects of a new class III antiarrhythmic drug (KCB-328), a delayed rectifier potassium current (IKr) blocker with minimal reverse use-dependent effect on atrial repolarization, were evaluated in the canine night atrial crush-injury model of atrial flutter (AFL). METHODS: Ten anesthetized, open-chest dogs, were studied after right atrial crush-injury. Atrial effective refractory period (ERP), conduction velocity (CV), wavelength, and dispersion of refractoriness were determined during programmed stimulation (S1S2 at S1S1 = 200, 300, 400, and 500 msec) at four sites via a mapping plaque sutured on the right atrial free wall. Right and left ventricular ERP were similarly measured at single sites. Electrophysiological parameters were determined at baseline and following sequential cumulative doses of KCB-328 (10, 30, 100, and 300 microg/kg). RESULTS: Sustained AFL was inducible in 7/10 dogs by rapid pacing following baseline electrophysiologic measurements. KCB-328 significantly prolonged sinus cycle length, but had no effect on PR interval, and prolonged QTc only at the highest dose level. KCB-328 significantly prolonged atrial ERP and wavelength and ventricular ERP, and significantly reduced dispersion of atrial refractoriness. KCB-328 significantly prolonged AFL cycle length, and increasing doses progressively terminated sustained AFL and prevented its reinduction by pacing. No adverse hemodynamic or ventricular proarrhythmic effects were observed. CONCLUSIONS: The electrophysiologic profile of KCB-328 in this canine model of AFL, particularly its lack of reverse use-dependent effect on atrial refractoriness, suggests that it may have significant antiarrhythmic potential in treatment of atrial arrhythmias.

Animals↗

PubMed2001

Thermostable chitosanase from Bacillus sp. strain CK4: its purification, characterization, and reaction patterns.

A thermostable chitosanase, purified 156-fold to homogeneity in an overall yield of 12.4%, has a molecular weight of about 29,000 +/- 2,000, and is composed of monomer. The enzyme degraded soluble chitosan, colloidal chitosan, and glycol chitosan, but did not degrade chitin or other beta-linked polymers. The enzyme activity was increased about 2.5-fold by the addition of 10 mM Co2+ and 1.4-fold by Mn2+. However, Cu2+ ion strongly inhibited the enzyme. Optimum temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable after heat treatment at 80 degrees C for 30 min or 70 degrees C for 60 min and fairly stable in protein denaturants as well. Chitosan was hydrolyzed to (GlcN)4 as a major product, by incubation with the purified enzyme. The effects of ammonium sulfate and organic solvents on the action pattern of the thermostable chitosanase were investigated. The amounts of (GlcN)3-(GlcN)6 were increased about 30% (w/w) in DAC 99 soluble chitosan containing 10% ammonium sulfate, and (GlcN)1 was not produced. The monophasic reaction system consisted of DAC 72 soluble chitosan in 10% EtOH also showed no formation of (GlcN)1, however, the yield of (GlcN)3 approximately (GlcN)6 was lower than DAC 99 soluble chitosan-10% ammonium sulfate. The optimal concentration of ammonium sulfate to be added was 20%. At this concentration, the amount of hexamer was increased by over 12% compared to the water-salt free system.

Ammonium Sulfate↗

PubMed2001

Cloning and sequence analysis of cDNA for heavy-chain ferritin from the Canis familiaris.

Ferritin serves as a storage protein for iron in animals. Complementary DNA encoding a heavy chain ferritin was cloned from the brain of Canis familiaris. The dog ferritin cDNA encodes a 182 amino acid that shows high levels of amino acid identity with vertebrate ferritins (90-98%). Near the cap region of the 5'-untranslated region, the dog H-ferritin mRNA displays a 28-nucleotide sequence that is exactly conserved in the corresponding region of the human and pig H-ferritin mRNA, thus making this sequence a prime candidate for involvement in the known translational regulation of H-ferritin by iron.

Amino Acid Sequence↗

PubMed2001

Diverse profiles of specific IgE response to toluene diisocyanate (TDI)-human serum albumin conjugate in TDI-induced asthma patients.

The prevalence studies on specific IgE to toluene diisocyanate (TDI)-human serum albumin (HSA) conjugate in TDI-induced asthma have shown variable results. In this study, we attempted to compare specific IgE bindings to TDI-HSA conjugate and its specificity using 3 different conjugates. Sera were collected from 20 TDI-induced asthma and 10 controls. Specific IgE were measured by ELISA using three TDI-HSA conjugates; two from Carnegie Mellon (CM; 98 and 99 CM conjugates) and one from Ajou University. To evaluate specificity and cross-reactivity, ELISA inhibition tests were applied. Positive and negative predictive values between Ajou conjugate and 98 CM conjugate were 75% and 100%. Those between Ajou and 99 CM were 100% and 93.8%. One patient showed an isolated positive response to the Ajou with negative responses to the other two conjugates. ELISA inhibition test using this patient's serum revealed the significant inhibitions by the Ajou and minimal inhibitions by the others. On the other hand, another patient showed an isolated positive response to 99 CM with negative responses to the others, and ELISA inhibition test showed significant inhibition by 99 CM with minimal inhibitions by the others. These results suggest that specific IgE bindings to a new antigenic determinant of TDI-HSA conjugate can be heterogeneous and differ from one individual to another.

Asthma↗

PubMed2001

A case of cholestatic autoimmune hepatitis and acute liver failure: an unusual hepatic manifestation of mixed connective tissue disease and Sjögren's syndrome.

Although hepatomegaly is reported to occur occasionally in patients with mixed connective tissue disease (MCTD) or Sjögren's syndrome (SS), autoimmune liver diseases such as primary biliary cirrhosis, sclerosing cholangitis, and autoimmune hepatitis in association with MCTD or SS have rarely been described. We report a case of severe cholestatic autoimmune hepatitis presenting with acute liver failure in a 40-yr-old female patient suffering from MCTD and SS. The diagnosis of MCTD and SS was made at the age of 38. The patient presented severe jaundice and elevation of conjugated bilirubin. The patient denied alcohol and drug use and had no evidence of viral hepatitis. On the 8th day of her hospitalization, the patient developed grade III hepatic encephalopathy. She was diagnosed as autoimmune hepatitis presenting with acute liver failure based on clinical features, positive FANA and anti-smooth muscle antibodies, negative anti-mitochondrial antibodies, high titers of serum globulin, liver biopsy findings, and a good response to corticosteroid therapy, The patient was managed with prednisolone and the clinical symptoms, liver function test results, and liver biopsy findings showed much improvement after steroid therapy.

Adult↗

PubMed2001

Seroepidemiology of HBV infection in South Korea, 1995 through 1999.

BACKGROUND: We analyzed serologic data that were obtained from the Korea Association of Health from 1995 to 1999 to estimate the reliable prevalence of HBV in South Korea. METHODS: 603,375, 639,465, 621,476, 612,705 and 650,398 serum samples were annually tested for HBsAg. Of HBsAg positive persons whose serum samples were available, HBeAg positivity was checked. RESULTS: HBsAg positivities among subjects between 6 and 19 years old were 8.2%, 3.9%, 2.1%, 2.6% and 1.3%. HBsAg positivities among subjects above 20 years old were 8.9%, 6.4%, 5.9%, 5.4% and 5.4%. The positive rates of HBeAg were 39.8 to 62.9% among subjects between 6 and 19 years old, and 18.3 to 37.9% among persons above 20 years old, in each year. In both subgroups, HBsAg positivity in the latter year was significantly lower than that in the former year (p < 0.001). It also showed that HBsAg positivities among subjects between 6 and 19 years old have been significantly lower than those among subjects above 20 years old, but those of HBeAg the exact reverse of HBsAg since 1996 (p < 0.001). CONCLUSIONS: It was observed that prevalence of HBV infection in the late 1990s, especially in the group between 6 and 19 years old, was conspicuously lower than that in the past. The nationwide vaccination programme might be one of the most important contributors to this tendency in Korea.

Adolescent↗

PubMed2001

Isotypes of anti-beta2-glycoprotein I antibodies: association with thrombosis in patients with systemic lupus erythematosus.

OBJECTIVE: To evaluate the association between isotypes of anti-beta2-glycoprotein I antibodies (anti-beta2-GPI) and thrombosis and to identify antiphospholipid antibodies (aPL) that are most associated with thrombosis in patients with systemic lupus erythematosus (SLE). METHODS: IgG anticardiolipin antibody (aCL) and isotypes of anti-beta2-GPI were measured by ELISA, and clinical evidence of thrombosis was analyzed in 270 patients with SLE. RESULTS: IgG, IgM, and IgA anti-beta2-GPI were positive in 38.1, 13.7, and 34.8% of patients, respectively. Patients with a history of thrombosis were significantly more likely to have lupus anticoagulant (LAC), IgG aCL, and the 3 anti-beta2-GPI isotypes. Arterial thrombosis was associated with the presence of IgG aCL and the 3 anti-beta2-GPI isotypes, whereas venous thrombosis was associated with LAC, IgG aCL, and IgA anti-beta2-GPI. In stepwise multivariate logistic regression analysis, the variable that was associated with thrombosis was IgA anti-beta2-GPI. The occurrence of arterial thrombosis was associated with IgG aCL and that of venous thrombosis was related to IgA anti-beta2-GPI in stepwise multivariate analysis. The IgG, IgM, and IgA anti-beta2-GPI titers were closely correlated with IgG aCL titers. The IgA anti-beta2-GPI titers were also significantly correlated with those of IgG and IgM anti-beta2-GPI. CONCLUSION: The results suggest that anti-beta2-GPI isotypes are related to the occurrence of thrombosis, and measurements of IgA anti-beta2-GPI may be useful for predicting thrombotic episodes in patients with SLE.

Antibodies, Antiphospholipid↗

PubMed2001

Clinical significance of the fractional excretion of anions in metabolic acidosis.

The fractional excretion of anions has been proposed as a new index for the differential diagnosis of metabolic acidosis, identifying the properties of the conjugate base by examining the renal handling of the anion. Here, we investigated clinical significance of the fractional excretion of anions in pathophysiologic diagnosis of metabolic acidosis by measuring urine ammonium (NH4+) excretion, the ratio of A plasma anion gap/delta plasma HCO3- concentration (deltaAG/deltaHCO3-), and fractional excretion of anions in three different groups of metabolic acidosis: acid overproduction (8 patients with lactic acidosis, 8 with diabetic ketoacidosis, 3 with hippuric acidosis following glue sniffing), acid underexcretion (10 patients with chronic renal failure) and normal controls (10 normal volunteers who underwent 3-day NH4Cl loading). As expected, urine NH4+ excretion was higher in overproduction acidosis than in acid-loaded normal controls (88.1 +/- 12.3 vs. 54.0 +/- 3.7 mmol/day, p < 0.05), and it was lower in chronic renal failure than in acid-loaded normal controls (12.8 +/- 1.7 vs. 54.0 +/- 3.7 mmol/day, p < 0.05). The fractional excretion of anions had no difference between overproduction acidosis and chronic renal failure (41.2 +/- 42.8% vs. 41.0 +/- 8.1%). However, the fractional excretion of anions showed significant differences between the subgroups in acid overproduction (lactic acidosis, 4.7 +/- 0.3%; diabetic ketoacidosis, 45.8 +/- 3.1%; hippuric acidosis, 126.0 +/- 14.4%; p < 0.05). The ratio of plasma deltaAG/deltaHCO3- also exhibited significant differences between the subgroups in acid overproduction (lactic acidosis, 1.5 +/- 0.1; diabetic ketoacidosis, 1.0 +/- 0.1; hippuric acidosis, 0.3 +/- 0.1; p < 0.05). There was an inverse linear correlation between the fractional excretion of anions and the ratio of plasma deltaAG/deltaHCO3- (r2 =-0.89, p < 0.05). In conclusion, determination of the fractional excretion of anions may provide a useful clue to the differential diagnosis of metabolic acidosis caused by acid overproduction.

Acidosis↗

PubMed2001

The antithrombotic efficacy of AT-1459, a novel, direct thrombin inhibitor, in rat models of venous and arterial thrombosis.

The antithrombotic efficacy of AT-1459, a novel, direct thrombin inhibitor (Ki = 4.9 nM) was evaluated in rat models of venous thrombosis combined with a bleeding time test and arterial thrombosis. After drugs were given by i. v. bolus injection plus a continuous infusion, the ID50, (a dose that exhibits 50% inhibition of thrombus formation over each vehicle group) values of AT-1459, argatroban, and dalteparin were 0.04 mg/kg plus 0.04 mg/kg/h, 0.1 mg/kg plus 0.4 mg/ kg/h, and 13.0 IU/kg plus 26.0 IU/kg/h, respectively, in the venous thrombosis study. The BT2 (a dose that causes 2-fold prolongation of bleeding time over each vehicle group) values of AT-1459, argatroban, and dalteparin were 0.9 mg/kg plus 0.9 mg/kg/h, 1.0 mg/kg plus 0.6 mg/kg/h, and 345.5 IU/kg plus 691.0 IU/kg/h in the rat tail transection model. The ratios of BT2/ID50 of AT-1459, argatroban, and dalteparin were 22.5, 10.0, and 26.6, respectively. In a rat model of arterial thrombosis induced by topical FeCl2 application, intravenous administration of AT-1459, argatroban, and dalteparin improved the vessel patency significantly (P < 0.01) at 0.6 mg/kg plus 0.6 mg/kg/h, 0.6 mg/kg plus 2.4 mg/kg/h, and 300 IU/kg plus 600 IU/kg/h, respectively. The oral antithrombotic effect of AT-1459 lasted for 6 after administering 30 mg/kg and improved the vessel patency significantly 1 h after administering the same dose in venous and arterial thrombosis models, respectively, with a rapid onset of action. Warfarin also inhibited thrombus weight and improved the vessel patency significantly after oral administration of 0.3 mg/kg for three consecutive days in the same study. The antithrombotic and hemorrhagic effects of all drugs studied were correlated with plasma concentration or clotting times. These results suggest that AT-1459 may be clinically useful as an orally available antithrombotic agent for the prevention of venous and arterial thrombosis.

Administration, Oral↗

PubMed2000

Inhibition of neuronal apoptosis by docosahexaenoic acid (22:6n-3). Role of phosphatidylserine in antiapoptotic effect.

Enrichment of Neuro 2A cells with docosahexaenoic acid (22:6n-3) decreased apoptotic cell death induced by serum starvation as evidenced by the reduced DNA fragmentation and caspase-3 activity. The protective effect of 22:6n-3 became evident only after at least 24 h of enrichment before serum starvation and was potentiated as a function of the enrichment period. During enrichment 22:6n-3 incorporated into phosphatidylserine (PS) steadily, resulting in a significant increase in the total PS content. Similar treatment with oleic acid (18:1n-9) neither altered PS content nor resulted in protective effect. Hindering PS accumulation by enriching cells in a serine-free medium diminished the protective effect of 22:6n-3. Membrane translocation of Raf-1 was significantly enhanced by 22:6n-3 enrichment in Neuro 2A cells. Consistently, in vitro biomolecular interaction between PS/phosphatidylethanolamine /phosphatidylcholine liposomes, and Raf-1 increased in a PS concentration-dependent manner. Collectively, enrichment of neuronal cells with 22:6n-3 increases the PS content and Raf-1 translocation, down-regulates caspase-3 activity, and prevents apoptotic cell death. Both the antiapoptotic effect of 22:6n-3 and Raf-1 translocation are sensitive to 22:6n-3 enrichment-induced PS accumulation, strongly suggesting that the protective effect of 22:6n-3 may be mediated at least in part through the promoted accumulation of PS in neuronal membranes.

Animals↗

PubMed2000

Negative regulation of cytosolic phospholipase A(2) by melatonin in the rat pineal gland.

In this paper evidence that supports a new role for melatonin as a negative endogenous regulator of cytosolic phospholipase A(2) (cPLA(2)) is presented. When rat pineal glands were incubated in culture, time-dependent release of arachidonic acid (AA) was observed, which was significantly inhibited by a known 85-kDa cPLA(2) inhibitor, methyl arachidonyl fluorophosphonate. Co-incubation with melatonin inhibited the AA release in a concentration-dependent manner, and this decrease was accompanied by a reduction of cPLA(2) protein and mRNA expression. Melatonin-receptor agonists, 2-iodo-N-butanoyl-5-methoxytryptamine and 5-methoxycarbonylamino-N-acetyltryptamine, also decreased AA release and cPLA(2) protein and mRNA levels, while pre-incubation with the melatonin receptor antagonists luzindole and 2-phenylmelatonin abolished the melatonin effect. In vivo, as melatonin production reflected a typical diurnal variation, endogenous non-esterified AA and cPLA(2) mRNA levels in the rat pineal gland showed an off-phase diurnal pattern in relation to melatonin levels. Intravenous administration of isoproterenol, which has been shown to elevate melatonin production, also decreased the levels of non-esterified AA and cPLA(2) mRNA significantly. Direct administration of melatonin to rats by intravenous injection decreased the levels of non-esterified AA, cPLA(2) protein and mRNA in rat pineal glands. In conclusion, melatonin endogenously down-regulates cPLA(2) expression, presumably through melatonin-receptor-mediated processes.

Amino Acids↗

PubMed2000

CD40 engagement on synovial fibroblast up-regulates production of vascular endothelial growth factor.

We tested the impact of CD40 engagement on the production of vascular endothelial growth factor (VEGF) from rheumatoid synovial fibroblasts. Fibroblast-like synovial cells (FLS) were prepared from the synovial tissues of rheumatoid arthritis patients and cultured in the presence of CD40 ligand-transfected (CD40L+) L cells. VEGF levels were determined in the culture supernatants by ELISA. Stimulation of FLS by CD40L+ L cells increased the production of VEGF by 4.1-fold over the constitutive levels of unstimulated FLS. The CD40L on activated T cells from rheumatoid synovial fluid also up-regulated VEGF production from FLS. Neither indomethacin nor Abs to IL-1beta, TNF-alpha, and TGF-beta did affect CD40L-induced VEGF production. Stimulation of FLS with TNF-alpha, IL-1beta, and TGF-beta increased VEGF production by 1.6-, 2.0-, and 5.2-fold, respectively, and displayed an additive effect on the production of VEGF by CD40L. VEGF mRNA expression was also up-regulated by the stimulation of FLS with membranes from the CD40L+ L cells. Dexamethasone completely abrogated CD40L-induced VEGF production. In addition, pyrrolidine dithiocarbamate partially down-regulated CD40L-induced VEGF production, showing that the NF-kappaB pathway was partly involved in the signaling of CD40L leading to VEGF production. Collectively, these results suggest that the interaction between CD40 on synovial fibroblasts and CD40L expressed on activated T lymphocytes may be directly involved in the neovascularization in rheumatoid synovitis by enhancing the production of VEGF.

Adjuvants, Immunologic↗

PubMed2000

Cloning of a sesquiterpene cyclase and its functional expression by domain swapping strategy.

Sesquiterpene cyclase, the first committed step enzyme from the general isoprenoid building block farnesyl pyrophosphate (FPP) for the synthesis of phytoalexin capsidiol, was isolated from the UV-C treated leaves of Capsicum annuum. This sesquiterpene cyclase, termed as CASC2 showing 77% amino acid identity with the previously cloned sesquiterpene cyclase CASC1, was composed of 560 amino acids with a calculated molecular mass of 64,907. The mRNA expression pattern of CASC2 was very similar to that of CASC1 during the time course of UV-C irradiated leaves of pepper on RNA blot analysis by using each specific probe. The heterologous expression in Escherichia coli using the CASC2 full length failed; however the chimeric construct of CASC2 in which the amino terminal 164 amino acid substituted by the equivalent portion of either CASC1 or tobacco sesquiterpene cyclase was capable of expressing the functional sesquiterpene cyclase activities. The radio-labeled enzymatic products catalyzed by the partially purified chimeric CASC2 were comigrated with authentic radio-labeled sesquiterpene on thin layer chromatography.

Amino Acid Sequence↗

PubMed2000

Clinical implication of altered expression of Mad1 protein in human breast carcinoma.

BACKGROUND: Mad1 protein is known to repress Myc target genes and antagonize Myc function. The authors undertook this study to investigate the clinical implication of Mad1 expression in human breast carcinoma. METHODS: The authors performed immunohistochemical assays for Mad1 and Myc proteins in human breast carcinoma, along with tissues from normal breast and benign diseases. The data from protein assays were analyzed in terms of the clinical and biologic characteristics of the patients. RESULTS: Of 66 patients with invasive ductal carcinoma, Mad1 expression was detected in 22 (33. 3%). Intensity and area of Mad1 expression significantly decreased in DCIS and invasive cancers, whereas high levels of Mad1 expression were persistent in benign breast lesions. Mad1 expression was significantly reduced in poorly differentiated tumors (P < 0.001). Expression of Mad1 was not associated with tumor size, lymph node status, or stage of disease. The authors did not observe any correlation between S-phase and expression status of Myc or Mad1. Mad1 expression was closely linked to differentiation of the cancer cells and inversely correlated with Myc expression (P = 0.042). In survival analysis, Mad1 was a significant factor in predicting recurrence of the disease, but not overall survival after CMF chemotherapy. CONCLUSIONS: In human breast carcinoma cells, expression of Mad1 seems to be down-regulated, whereas expression of Myc is amplified. Altered expression of Mad1 may play a role in the malignant transformation of human mammary epithelial cells and represent an aggressive phenotype in human breast carcinoma.

Adult↗

PubMed2000

Cloning, overexpression and purification of Bacillus subtilis elongation factor Tu in Escherichia coli.

To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His6 tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.

Bacillus subtilis↗

PubMed2000

Profiling neurosteroids in cerebrospinal fluids and plasma by gas chromatography/electron capture negative chemical ionization mass spectrometry.

A quantitative method for the determination of allopregnanolone (5alpha,3alpha-THP) and related neurosteroids in CSF and plasma was established using gas chromatography/electron capture negative chemical ionization mass spectrometry (GC/ECNCI/MS). Neurosteroids were converted to carboxymethoxime, pentafluorobenzyl and trimethylsilyl derivatives and detected as intense (M-181)(-) fragment ions generated under the negative ion chemical ionization process. The response curves constructed using d(4)-dihydrotestosterone (DHT) and d(4)-5alpha,3alpha-THP as internal standards showed linearity in the concentration range of 10-1000 pg/ml. The variation of response ratios determined against internal standards over a 2-month period was less than 10%. Instrumental detection limits for most neurosteroids were in the low picogram range with the exception of progesterone and dihydroprogesterone (DHP) which were detected with approximately 10 times less sensitivity in comparison to other steroids. In conjunction with solid-phase extraction, this method allowed the quantification of at least four neurosteroids, including androsterone, testosterone, 5alpha,3alpha-THP, and pregnenolone in 1-2 ml of human cerebrospinal fluid (CSF). While the level of 5alpha, 3alpha-THP in human CSF was comparable to that in the human plasma, other steroid levels were significantly lower. Although individual CSF and plasma samples showed widely varying neurosteroid levels, species specificity appeared to exist. The levels of 5alpha, 3alpha-THP and pregnenolone in human CSF were higher than those of monkey CSF where these steroids were often not detected with our current detection limit. In comparison to human plasma, rat plasma samples contained considerably lower levels of androsterone and pregnenolone. Among THP stereoisomers, 5beta,3alpha-THP and 5alpha, 3beta-THP were observed only in human plasma, while 5beta,3beta-THP was detected only in rat plasma.

Adult↗