Biomedical subjects

X C Wu

Publications and source records attributed to X C Wu.

At least 37 records · Page 2Linked to original sources

PubMed1999

Relationship between erythropoietin and nitric oxide in the contraction of rat renal arcuate arteries and human umbilical vein endothelial cells.

We have investigated the effects of recombinant human erythropoietin (EPO) on the responses of rat renal arcuate arteries to dopamine, noradrenaline and acetylcholine and on the release of NO from human umbilical vein endothelial cells (HUVEC) in culture. Noradrenaline induced a concentration-dependent constriction and acetylcholine a concentration-dependent relaxation of the vessels. The effects of dopamine were concentration-dependent, leading to relaxation of the vessels at low concentrations and contraction of the vessels at high concentrations. N(G)-Nitro-L-arginine methyl ester (L-NAME; 0.1 mM) did not change the vasoconstrictor responses to noradrenaline and dopamine, but inhibited the acetylcholine- and dopamine-induced vasorelaxation. Neither 0.1 nor 20 units.ml(-1) EPO affected noradrenaline-induced constriction, or dopamine- or acetylcholine-induced relaxation, of the vessels. EPO at 20 units. ml(-1) attenuated dopamine-induced constriction of the vessels. This effect was blunted by application of L-NAME, suggesting that EPO may stimulate dopamine-mediated NO release from these vessels. EPO stimulated NO release from the resting HUVEC in a concentration- and time-dependent manner, an effect that was inhibited by the presence of N(G)-nitro-L-arginine. These data suggest that, in vitro, EPO is able to stimulate NO release from rat renal arcuate arteries and HUVEC in culture. Whether these acute short-term actions can be related to the longer-term actions of EPO remains to be resolved.

Acetylcholine↗

PubMed1999

The influence of erythropoietin on the vascular responses of rat resistance arteries.

This study examined the effect of erythropoietin (EPO) on resting tension and on the responses of rat mesenteric and renal arcuate arteries in vitro to a number of agonists as a possible cause of its blood pressure elevating properties when used therapeutically. Noradrenaline and potassium chloride induced concentration-dependent vasoconstrictions in both vessel types but the basal tension, maximum tension, and the -log concentration producing half-maximal response (pEC50) were altered in the presence of 0.1 or 20 U ml-1 EPO. The thromboxane A2 receptor agonist U46619 induced a constriction of the renal arcuate arteries which was enhanced by EPO at 20 U ml-1, from 1.68 +/- 0.34 to 2.64 +/- 0.39 mN mm-1 (P < 0.01), but which was unchanged by NG-nitro-L-arginine methyl ester (10-4 m). Serotonin (10-9-10-5 M) caused a concentration-related vasoconstriction in renal arcuate arteries which was shifted to the right in the time control study (P < 0.001) but this was abolished by both 0.1 and 20 U ml-1 of EPO. Acetylcholine induced a relaxation of precontracted mesenteric arteries, by 95.4 +/- 1.64 % with an EC50 of 7.08 +/- 0.08 M which was reduced (P < 0.001) by 20 U ml-1 EPO to 81.7 +/- 3.56 % and 6.10 +/- 0.11 M, respectively. The sodium nitroprusside-induced relaxations were unaffected by EPO. The acetylcholine-mediated relaxations in renal arcuate arteries were unchanged by EPO. Bradykinin-induced relaxations in mesenteric and renal arcuate arteries were unaffected by both EPO concentrations. Together these data showed that EPO over a large concentration range had only minor effects on basal tension and the vascular responsiveness of both mesenteric and renal arcuate arteries. The mechanism whereby EPO causes a chronic elevation in blood pressure is unlikely to be due to acute interactions with agonist-mediated responses.

Animals↗

PubMed1999

Role of erythropoietin and nitric oxide in modulating the tone of human renal interlobular and subcutaneous arteries from uraemic subjects.

This study investigated potential reasons why erythropoietin (EPO) given therapeutically to patients with renal failure may increase peripheral, but not renal, vascular resistance. This was done by comparing the effects of EPO on resting tension in normal renal interlobular and subcutaneous vessels from uraemic patients. In human subcutaneous arteries from uraemic subjects, noradrenaline- and KCl-induced vasoconstrictions were enhanced when nitric oxide (NO) production was blocked with N(G)-nitro-L-arginine methyl ester (L-NAME), but were unaffected by EPO, while acetylcholine- and bradykinin-induced concentration-dependent relaxations were markedly attenuated by L-NAME, but not by EPO. The noradrenaline- and KCl-induced vasoconstrictions of human renal interlobular arteries were unaffected by the presence of L-NAME, but were attenuated by EPO (20 units.ml(-1)) by some 33% (P<0.01); this effect was enhanced by the co-administration of L-NAME. Acetylcholine and bradykinin caused comparable dilatations of the interlobular arteries; the response to the former was attenuated by L-NAME, but none of these responses were changed by EPO. EPO given alone, at a concentration of either 0.1 or 20 units.ml(-1), had no effect on basal resting tone. NO production mediated both acetylcholine- and bradykinin-induced relaxation in this vessel type. In contrast, in the interlobular arteries there was no indication of NO modulating the level of vasoconstriction, and it only mediated acetylcholine-induced dilation. These acute responses to EPO only partially explain its differential effects on the vasculature in renal failure.

Acetylcholine↗

PubMed1998

Engineering of plasmin-resistant forms of streptokinase and their production in Bacillus subtilis: streptokinase with longer functional half-life.

The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilis WB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.

Bacillus subtilis↗

PubMed1998

Enhanced secretory production of a single-chain antibody fragment from Bacillus subtilis by coproduction of molecular chaperones.

Formation of inclusion bodies is a major limiting factor for secretory production of an antidigoxin single-chain antibody (SCA) fragment from Bacillus subtilis. To address this problem, three new strains with enhanced production of molecular chaperones were constructed. WB600BHM constitutively produces the major intracellular molecular chaperones in an appropriate ratio without any heat shock treatment. This strain reduced the formation of insoluble SCA by 45% and increased the secretory production yield by 60%. The second strain, WB600B[pEPP], overproduces an extracytoplasmic molecular chaperone, PrsA. An increase in the total yield of SCA was observed. The third strain, WB600BHM[pEPP], coproduces both intracellular and extracytoplasmic molecular chaperones. This led to a further reduction in inclusion body formation and a 2.5-fold increase in the secretory production yield. SCA fragments secreted by this strain were biologically active and showed affinity to digoxin comparable to the affinity of those secreted by strains without overproduction of molecular chaperones. Interestingly, accumulation of a pool of periplasmic SCA was observed in the PrsA-overproducing strains. This pool is suggested to represent the secreted folding intermediates in the process of achieving their final configuration.

Antigen-Antibody Reactions↗

PubMed1998

Cancer incidence in the industrial corridor: an update.

Because of the high density of industries along the Lower Mississippi River, there is a concern about adverse impact on health, including cancer, among residents in these parishes. This study provides an update of cancer incidence in the Industrial Corridor for the period 1989-93. Age-adjusted cancer incidence rates were calculated for the seven-parish study area from Baton Rouge down to, but not including, New Orleans. Rates were also computed for the entire state of Louisiana and for the combined Surveillance, Epidemiology and End Results (SEER) program. Cancer incidence rates for the Industrial Corridor are either similar to, or lower than, the combined SEER rates for most of the common cancers as well as for rare tumors. The only two exceptions are lung cancer in white males and kidney cancer in white females that are significantly elevated when compared to the SEER averages. Significantly lower rates are found among white males for cancers of kidney, brain, and nervous system, and melanoma; among black males, cancers of all sites combined, oral cavity, stomach, rectum, and prostate, Hodgkin's disease, and non-Hodgkin's lymphoma; among white females, cancers of all sites combined, cervix, uterine corpus, ovary, bladder, and melanoma; and among black females, cancers of all sites combined, oral cavity, lung, breast, ovary, and melanoma. The persistent excess of lung cancer has led to the development of a multi-agency project to evaluate the impact of potential environmental exposures, genetic susceptibility, and their interactions on lung cancer risk. The findings also confirm the urgent need to include and strengthen tobacco prevention and cessation programs in our cancer control activities.

Environmental Exposure↗

PubMed1997

Highlights of cancer incidence in Louisiana, 1988-1992.

This paper highlights the major findings from the recently released volume of the Louisiana Tumor Registry monograph series: Cancer Incidence in Louisiana, 1988-1992. One out of three Louisiana residents will develop cancer in his or her lifetime. Lung cancer remains the most common cancer for all races, both genders combined. Lung cancer rates for women continue to rise substantially (20% over the previous 5-year period) and African-American men in the Acadiana Region have the state's highest lung cancer rate. The number of prostate cancer cases has surpassed that of lung cancer for the first time in Louisiana men; the sizable increase since 1983 (about 50%) in prostate cancer likely reflects the recent aggressive screening by the PSA test. Breast cancer remains the most common cancer among Louisiana women and incidence rates have increased about 20% over the previous 5-year period. Geographic comparisons show that the New Orleans Region continues to have rates higher than state averages, and a clear pattern of high cancer risk has emerged for the Acadiana region. The Central Louisiana and the Baton Rouge Regions have rates lower than state averages.

Adult↗

PubMed1997

Aggressiveness of colon carcinoma in blacks and whites. National Cancer Institute Black/White Cancer Survival Study Group.

Black patients with colon cancer in the Black/White Cancer Survival Study were found to have a poorer survival than white patients. More advanced-stage disease at diagnosis was the primary determinant, accounting for 60% of the excess mortality. After adjusting for stage, factors such as poverty, other socioeconomic conditions, and treatment did not further explain the remaining survival deficit. This study examined the aggressiveness of colon tumors in blacks and whites to explore its role in the racial survival differences. Tumor characteristics of 703 cases of newly diagnosed invasive colon adenocarcinoma were centrally evaluated by a gastrointestinal pathologist, blinded in regard to the age, race, and sex of the patients. Blacks were less likely to have poorly differentiated (grade 3) tumors [odds ratio (OR), 0.44; 95% confidence interval, 0.22-0.88] and lymphoid reaction (OR, 0.49; 95% confidence interval, 0.26-0.90) when compared with whites. These black/white (B/W) differences remained statistically significant after adjusting for age, sex, metropolitan area, summary stage, socioeconomic status, body mass index, and health care access and utilization. In addition, blacks were less likely to have high-grade (grade 3) nuclear atypia, mitotic activity, and tubule formation, although these ORs did not reach a statistical significance level of 0.05. Similar B/W differences were observed for patients with advanced disease but not with early stage. Comparison by anatomical subsite showed that blacks had statistically significantly better differentiated tumors for cancers of the proximal and transverse colon but not for the distal. No racial differences were found for blood vessel and lymphatic invasion, necrosis, fibrosis, and mucinous type of histology. The findings, therefore, are the opposite of those hypothesized. After adjusting for stage, more aggressive tumor characteristics do not explain the adverse survival differential in blacks. This suggests that there may be racial differences in environmental exposure, and that the intensity and mode of delivery of carcinogen insult as well as host susceptibility may differ by race and anatomical subsite. Future studies should explore the B/W differences in tumor biology using molecular markers that precede the conventional histological parameters evaluated here.

Adult↗

PubMed1997

Patterns of DNA ploidy and S-phase fraction associated with breast cancer survival in blacks and whites.

A significant survival difference between black and white breast cancer patients has been observed in the United States. Evaluation of the prognostic value of DNA ploidy and S-phase fraction (SPF) in black and white breast cancer patients may contribute to our understanding of the mechanisms of racial disparity in survival. A sample of 98 patients (50 blacks and 48 whites) who participated in the Black/White Cancer Survival Study was selected for DNA flow cytometry analysis. Patients were followed between 4.5 and 6.5 years. The impacts of DNA ploidy and SPF on breast cancer survival were examined. Kaplan-Meier survival curves, log rank statistics, and Cox proportional hazards regression were used for survival analyses. Black patients were more likely than white patients to have tumors with high SPF (P < 0.05), but there was no difference in DNA ploidy (P = 0.79). Because there were significant interactions of both DNA ploidy and SPF with race, survival was examined separately for blacks and whites. Significantly poorer survival was observed for white patients with class A ploidy (hypodiploidy, hypotetraploidy, and hypertetraploidy; P = 0.001) and with high SPF (P = 0.025). The elevated hazard ratios remained significant after adjusting for age and stage. Further adjustment for adjuvant therapy and histopathological characteristics of tumor reduced the hazard ratios of SPF to a nonsignificant level. No significant associations were found between survival and DNA ploidy or SPF among blacks. DNA ploidy and SPF are prognostic factors for breast cancer survival in white patients but not in blacks. This may have clinical implication in breast cancer management.

Black People↗

PubMed1996

[Clinical and experimental study on sustained release tablet of Tripterygium wilfordii in treating rheumatoid arthritis].

Adopt to the prospective, multi-center, random, single-blind, equal rank-control methods, 226 patients of rheumatoid arthritis diagnosed according to the ARA criteria, were divided into 2 groups. One hundred and fourteen patients of test group were treated with sustained release tablets of Tripterygium wilfordii (TW-SR) orally, 2 tablets, twice a day for 4 weeks, 112 patients of control group received tablets of Tripterygium wilfordii (TW) orally, 2 tablets 3 times per day for 4 weeks. Results showed that the total effective rate of the two groups were 92.11% and 90.65%, respectively (P > 0.05). The adverse reaction rate of TW-SR group was 20.18%, which was lowered than that of TW group (70.54%, P < 0.01). Results of pre-clinical pharmacologic experimental study showed that the TW-SR has obvious anti-inflammatory, analgesia and immunosuppress'ive action as the TW has, while its toxicity was less than the latter significantly.

Adult↗

PubMed1995

Racial disparity in the association of p53 gene alterations with breast cancer survival.

A significant black/white difference in breast cancer prognosis has been observed in the United States. Alterations of p53 tumor suppressor gene in breast cancer have been associated with poor prognosis. This study was designed to test the hypothesis that p53 gene alterations are related to the difference in prognosis between black and white breast cancer patients. Formalin-fixed paraffin-embedded breast tissue blocks were available from 45 black and 47 white patients for PCR-single strand conformation polymorphism analysis and DNA sequencing. The types of p53 gene alterations were compared between blacks and whites. Associations between p53 gene alterations and survival were also evaluated. Three missense, 2 nonsense, 1 microdeletion, 1 intron, and 4 silent mutations were detected in blacks, while 7 missense, 1 microdeletion, 1 silent mutation, and 3 polymorphisms were observed in whites. Among the point mutations, G:C to A:T transitions at non-CpG sites were found in 80.0% of blacks (8 of 10) and 62.5% of whites (5 of 8). Significantly poorer survival associated with p53 gene alterations was observed for blacks (P = 0.012), but not for whites. Black patients with p53 alterations had a significant 4-5-fold excess risk of death from breast cancer than those without p53 alterations. Adjustment for stage, age, tumor histopathology, receptor status, and adjuvant treatment did not change the excess risk. The findings suggest that the types of p53 gene alterations may contribute to the racial difference in breast cancer survival.

Adult↗

PubMed1994

Interdependence of contractile responses of rat small mesenteric arteries on nitric oxide and cyclo-oxygenase and lipoxygenase products of arachidonic acid.

1. We have examined the effects of nitric oxide inhibition, indomethacin and the dual lipoxygenase/cyclo-oxygenase inhibitor, 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW755C), on the responses of small mesenteric arteries of Wistar rats, with and without endothelium, to noradrenaline, potassium chloride, endothelin-1, acetylcholine and sodium nitroprusside. 2. Noradrenaline, potassium chloride and endothelin-1 caused concentration-dependent contraction of small mesenteric arteries. Indomethacin (14 microM) attenuated the contractile response to both noradrenaline and potassium chloride. The inhibitory action of indomethacin persisted in vessels treated with CHAPS. 3. Acetylcholine produced concentration-dependent relaxation in these vessels. Indomethacin (14 microM) had no significant effect on the acetylcholine concentration-response relationship. 4. NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) potentiated the contractile response to both noradrenaline and potassium chloride and inhibited acetylcholine-induced relaxation. Indomethacin attenuated the effects of L-NAME. 5. BW755C inhibited the contractile response to noradrenaline and potassium chloride but not to endothelin-1. The inhibitory effects of BW755C persisted in the presence of indomethacin and in vessels treated with CHAPS. 6. BW755C enhanced endothelium-dependent relaxation, as assessed by the response to acetylcholine. In the presence of indomethacin, BW755C produced a shift to the right of the concentration-response curve to acetylcholine. 7. Inhibition of nitric oxide synthase with L-NAME, reversed the inhibitory effect of BW755C on noradrenaline- and potassium-induced contraction. L-NAME and BW755C in combination resulted in a shift to the right of the concentration-response curve to acetylcholine. 8. Sodium nitroprusside produced concentration-dependent relaxation of the vessels. Endothelium removal reduced the maximum relaxation to nitroprusside. BW755C did not alter the response to sodium nitroprusside in vessels with or without endothelium.9 .These data support the existence of two vasoconstrictor products of arachidonic acid released during contraction of small mesenteric arteries with noradrenaline and potassium chloride: a cyclo-oxygenase product and a lipoxygenase product both of which appear to be largely endothelium-independent.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1994

Relative roles of nitric oxide and cyclo-oxygenase and lipoxygenase products of arachidonic acid in the contractile responses of rat renal arcuate arteries.

1. We have examined the effects of inhibition of nitric oxide synthase, cyclo-oxygenase and lipoxygenase on the responses of renal arcuate arteries of Wistar rats, with and without endothelium, to noradrenaline, potassium chloride, endothelin-1, acetylcholine and sodium nitroprusside. 2. Noradrenaline, potassium chloride and endothelin-1 caused concentration-dependent contraction of the vessels. Indomethacin (14 microM) attenuated the contractile response to noradrenaline and to potassium chloride. The inhibitory effect of indomethacin persisted following endothelial removal. 3. Acetylcholine produced concentration-dependent relaxation of the vessels which was potentiated by indomethacin (14 microM). 4. NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) did not affect the contractile response to either noradrenaline or potassium chloride but abolished relaxation to acetylcholine. In addition, L-NAME abolished the affects of indomethacin on acetylcholine-induced relaxation and noradrenaline- and potassium chloride-induced contraction. 5. BWC755C attenuated noradrenaline and potassium chloride-induced contraction. This effect persisted in the presence of indomethacin. 6. In vessels pretreated with CHAPS, BW755C inhibited both noradrenaline and potassium chloride-induced contraction. In these vessels BW755C had no additional inhibitory effect to indomethacin on noradrenaline- and potassium-induced contraction. 7. Inhibition of nitric oxide synthase with L-NAME (100 microM) attenuated the effect of BW755C on noradrenaline- and potassium-induced contraction. 8. BW755C alone did not affect endothelium-dependent relaxation as assessed by the response to acetylcholine. However, in the presence of indomethacin, BW755C inhibited acetylcholine-induced relaxation. 9. BW755C did not affect endothelium-independent relaxation as assessed by the response to sodium nitroprusside in vessels with or without endothelium. 10. These data support the existence of two vasoconstrictor products of arachidonic acid released during contraction of renal arcuate arteries with noradrenaline and potassium chloride. A cyclooxygenase product which appears to be endothelium-independent and the other an endothelium dependent lipoxygenase product.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1994

Vascular effects of 17 beta-estradiol in male Sprague-Dawley rats.

Bolus intravenous injections of 100 micrograms/kg 17 beta-estradiol significantly decreased the pressor responses to norepinephrine (NE; 0.3 microgram/kg) at the fourth, fifth, and sixth hour in anesthetized male Sprague-Dawley rats. At doses of 10(-6) to 3 x 10(-5) M, 17 beta-estradiol relaxed the sustained phase of contraction in male Sprague-Dawley rat tail artery helical strips precontracted in vitro by [Arg8]vasopressin (AVP), KCl, or NE. The effect was dose dependent. At doses of 3 x 10(-6) to 3 x 10(-5) M, it also decreased the initial phase of tension generation and extracellular Ca(2+)-dependent vasoconstriction induced by NE, AVP, or KCl in a dose-dependent manner in male Sprague-Dawley rat tail artery helical strips. 17 beta-Estradiol (2 x 10(-8) to 2 x 10(-6) M) decreased the voltage-dependent inward Ca2+ current and the intracellular free Ca2+ concentration ([Ca2+]i) increment induced by 15 mM KCl in a dose-dependent manner (3.6 x 10(-8) to 3.6 x 10(-6) M) in vascular smooth muscle cells (VSMC) isolated from male Sprague-Dawley rat tail arteries. We suggest that, at pharmacological doses, estrogen has a direct vasodilating effect on the rat tail artery that is mediated by its inhibitory effect on Ca2+ influx through voltage-dependent Ca2+ channels. The inhibitory effect of estrogen on the pressor responses to NE or AVP may be correlated with its modulation of VSMC [Ca2+]i through its actions on membrane Ca2+ channels.

Animals↗

PubMed1994

Histological characteristics of breast carcinoma in blacks and whites.

Tumor characteristics of 963 newly diagnosed invasive breast cancer cases from the population-based Black/White Cancer Survival Study were evaluated. Representative slides of the tumors were requested from all participating hospitals of three metropolitan areas and reviewed by one expert pathologist, blinded in regard to the age and race of patients. Nine tumor characteristics were evaluated for black and white patients. After adjusting for age, stage, and metropolitan area, blacks were significantly more likely to have high grade nuclear atypia [odds ratio (OR) = 1.97, 95% confidence interval (CI) = 1.27-3.04]; high mitotic activity (OR = 2.05, 95% CI = 1.34-3.14), grade 3 tumors (OR = 1.58, 95% CI = 1.02-2.45), and more necrosis (OR = 1.51, 95% CI = 1.16-1.98); and less likely to have well defined tubular formation (OR = 0.57, 95% CI = 0.42-0.77), marked fibrosis (OR = 0.65, 95% CI = 0.45-0.94), and positive estrogen receptor status (OR = 0.78, 95% CI = 0.58-1.05). These black/white differences remained after controlling for socioeconomic status (SES), body mass index, use of alcohol and tobacco, reproductive experience, and health care access and utilization. No significant racial differences were found for blood vessel invasion and lymphatic invasion. Although white women of high SES had more favorable tumors than those of low SES, the same pattern was not observed for blacks. High SES black women had statistically nonsignificant elevated ORs of a high mitotic index and tumor grade. These racial differences in tumor biology may have etiological and clinical implications.

Adult↗

PubMed1994

Advanced stage at diagnosis: an explanation for higher than expected cancer death rates in Louisiana?

Data from the Louisiana Tumor Registry, Louisiana Vital Statistics Division, and the SEER program of the National Cancer Institute were explored to evaluate reasons for elevated cancer death rates in the absence of elevated cancer incidence in Louisiana. The results indicate that cancer patients in South Louisiana are more likely to be diagnosed with advanced stage disease than SEER patients. The Louisiana-SEER differences are greater for blacks than whites, and black patients are more likely to have advanced stage cancer than whites in Louisiana as well as in the SEER program. Of particular interest and concern is the finding that the increased risk of advanced stage disease in Louisiana is significant for cancers which can be detected early and, if so, effectively treated: colon, breast, and cervix. The findings reinforce the need for cancer control programs.

Adult↗

PubMed1993

Efficient production of a functional single-chain antidigoxin antibody via an engineered Bacillus subtilis expression-secretion system.

We have applied a Bacillus subtilis expression-secretion system to produce a functional antidigoxin SCA (single-chain antibody consisting of VL-linker-VH) and the individual variable domains of light (VL) and heavy (VH) chains. The secreted antidigoxin SCA can be affinity purified in one step by applying the culture supernatant directly to a ouabain-Sepharose column. N-terminal sequence determination indicated that the protein has the expected N-terminus with the signal peptide properly processed. Affinity and ligand specificity studies demonstrated that the engineered antidigoxin SCA has almost identical properties as those of the parental monoclonal antibody. The use of B. subtilis WB600, an engineered, six-extracellular protease-deficient strain, is vital for the production of antidigoxin SCA in high quality and quantity (5 mg/liter in a shake flask culture). All the secreted SCAs are biologically active. The ability to produce secreted SCAs by the B. subtilis expression system provides a simple and efficient means to analyze the binding properties of engineered antibodies generated through rational design or site-directed mutagenesis.

Amino Acid Sequence↗

PubMed1992

Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning.

Cloning of the sorbitol dehydrogenase gene (gutB) from Bacillus subtilis offers an excellent system for studying zinc binding, substrate specificity, and catalytic mechanism of this enzyme through protein engineering. As a first step to clone gutB, B. subtilis sorbitol dehydrogenase has been purified to homogeneity and characterized. It is a tetrameric enzyme with a molecular mass of 38 kDa for each subunit. Atomic absorption analysis shows the presence of 1 mol of zinc atom/subunit. Substrate specificity and stereospecificity of the enzyme toward C-2 and C-4 of hexitols were established. Sequence of the first 31 amino acids was determined, and a set of oligonucleotide probes was designed for gene cloning. A positive clone carrying a 5-kilobase pair HindIII insert was isolated and sequenced. Sequence alignment indicated that the deduced amino acid sequence of B. subtilis sorbitol dehydrogenase shows 36% identity in sequence with the liver sorbitol dehydrogenase from sheep, rat, and human. In reference to the sequence of alcohol dehydrogenase, two potential zinc binding sites were identified. Sequence information related to the structure-function relationships of the enzyme is discussed.

Amino Acid Sequence↗