Impact of smoking on the surgical outcome of Crohn's disease: a propensity-score matched National Surgical Quality Improvement Program analysis.

AIM: Smoking is known to have a deleterious effect on Crohn's disease (CD). The present study addressed the specific impact of smoking on the outcome of surgery for CD. METHOD: A review of the National Surgical Quality Improvement Program (NSQIP) database (2005-2012) identified 7631 patients with CD who underwent surgical resection. Patients were stratified based on smoking status and were compared with univariate statistical tests. Generalized linear regression and multiple logistic regressions were used to model the impact of smoking on the surgical outcome [length of stay (LOS), mortality, postoperative complications and readmission]. To confirm the validity of the regression models and to evaluate the influence of smoking in comparable patient cohorts, a propensity score match was also performed. RESULTS: There were 2047 (26.8%) patients with CD identified as current smokers, and 5584 (74.2%) identified as non- or ex-smokers. Smokers were more likely to have a pulmonary comorbidity, preoperative weight loss and a higher American Society of Anesthesiologists classification. No differences in mortality were observed between smokers and non- or ex-smokers in univariate analysis. In multivariate analysis, smoking status was not significantly associated with LOS. Morbidity (OR 1.20, P = 0.003), particularly infectious (OR 1.30, P < 0.001) and pulmonary (OR 1.87, P < 0.001) complications, and readmission (OR 1.58, P = 0.004) were significantly associated with smoking status. These findings were validated on propensity-score matching analysis. CONCLUSION: In patients with CD, the detrimental effects of smoking on surgical outcomes are driven by infectious and pulmonary complications, and by an increased likelihood of readmission.

Hernia incidence following single-site vs standard laparoscopic colorectal surgery.

AIM: Compared with standard laparoscopic (SDL) approaches, less is known about the incidence of hernias after single-site laparoscopic (SSL) colorectal surgery. This study hypothesized that SSL colorectal surgery was associated with an increased risk of hernia development. METHOD: Institutional retrospective chart review (September 2008-June 2013) identified 276 evaluable patients who underwent laparoscopic colorectal procedures. The following data were collected: demographic data, risk factors for the development of a hernia, operative details and postoperative course including the development of a hernia. Patients were stratified by laparoscopic technique to compare the characteristics of those undergoing SDL and SSL. Patients were subsequently stratified by the presence or absence of a hernia to identify associated factors. RESULTS: One hundred and nineteen patients (43.1%) underwent SDL and 157 patients (56.9%) underwent SSL surgery. The development of an incisional hernia was observed in 7.6% (9/119) of SDL patients compared with 17.0% (18/106) of SSL patients (P = 0.03) over a median 18-month follow-up. Similar proportions of patients developed parastomal hernias in both groups [SDL 16.7% (10/60) vs SSL 15.9% (13/80)]. Hernias were diagnosed at a median of 8.1 (SDL) and 6.5 (SSL) months following the index operation and were less likely to be incarcerated in the SSL group [SDL 38.9% (7/18) vs SSL 6.5% (2/31), P = 0.01]. CONCLUSION: SSL colorectal surgery is associated with an increase in the incidence of incisional hernias but not parastomal hernias. Site of specimen extraction in SSL may contribute to the development of an incisional hernia.

Laparoscopic colectomy is associated with a lower incidence of postoperative complications than open colectomy: a propensity score-matched cohort analysis.

AIM: Elective laparoscopic colectomy (LC) has been shown to provide short-term results comparable with open colectomy (OC), but there is potential selection bias whereby LC patients may be healthier and therefore more likely to have a superior outcome. The aim of this study was to compare the incidence of postoperative complications between matched laparoscopic and open colectomy cohorts, while controlling for differences in comorbidity. METHOD: A retrospective cohort study (2005-2010) using National Surgical Quality Improvement Program data was performed, identifying laparoscopic and open partial colectomy patients through common procedural terminology codes. Patient having rectal resection were excluded. The cohorts were matched 1:1 on a propensity score to control for observable selection bias due to patient characteristics, comparing overall complication rates, length of hospital stay (LOS), the incidence of superficial (S-SSI) surgical site infection, urinary tract infection (UTI) and deep-venous thrombosis (DVT). RESULTS: We analysed 37 249 patients. After propensity score matching the LC group had a significantly lower overall incidence of postoperative complications (29.1 vs 21.2%; P < 0.0001), S-SSI (9.0 vs 5.9%; P = 0.003) and DVT (1.2 vs 0.3%; P = 0.001). The LC group had a shorter LOS (8.7 vs 6.4 days; P < 0.0001), while mortality was comparable between the two groups (4.0 vs 4.1%; P = 0.578). CONCLUSION: LC is associated with a lower incidence of S-SSI and DVT than OC. Previously suggested advantages for laparoscopy, such as shorter length of stay and overall rate of complications, were observed even after controlling for differences in comorbidity.

Is colectomy for fulminant Clostridium difficile colitis life saving? A systematic review.

AIM: It is unclear whether colectomy for fulminant Clostridium difficile colitis (FCDC) leads to a improvement in survival compared with continued medical therapy for this moribund population. METHOD: Selected studies from 1994-2010 were identified through a comprehensive search theme applied to MEDLINE (OvidSP and PubMed), EMBASE and by hand searching. Data regarding mortality rates between medically and surgically treated patients were extracted. Risk of bias was assessed using a Newcastle-Ottawa Scale score. A meta-analysis of the odds ratios for mortality between surgical and medical treatment for FCDC was conducted using the Mantel-Haenszel method and fixed-effects modelling. RESULTS: Five hundred and ten patients with FCDC were identified in six studies. The pooled adjusted odds ratio of mortality comparing surgery with medical therapy was 0.70 (0.49-0.99), suggesting that surgery provided a survival benefit. CONCLUSION: Emergent colectomy for patients with FCDC provides a survival advantage compared with continuing antibiotics. Though there is selection bias of patients having surgery, the results of this systematic review suggest that colectomy has a therapeutic role in treating severe forms of C. difficile colitis.

Impact of Clostridium difficile colitis following closure of a diverting loop ileostomy: results of a matched cohort study.

AIM: Previous reports describing Clostridium difficile colitis (CDC) developing after the closure of a loop ileostomy suggest it is severe. In this study the incidence of CDC following ileostomy closure and its effect on the postoperative outcome have been studied. METHOD: Patients undergoing closure of loop ileostomy from 2004 to 2008 were analysed using the Nationwide Inpatient Sample. Patients who developed postoperative CDC (n = 217) were matched 10:1 to a propensity-score-matched cohort of patients without CDC (n = 13 245). Linear and logistic regression were used to examine the effect of CDC on hospital cost (US dollars), length of stay and mortality rates. Population resampling was performed using nearest neighbour bootstrapping to confirm the validity of the results. RESULTS: The incidence of CDC following ileostomy closure was 16 per 1000 patients. The mean length of stay was 11.5 days longer among CDC patients (P < 0.0001), with a greater cost of hospitalization of US$21 240 (P < 0.0001). There was no difference in mortality between the cohorts. CONCLUSION: CDC following ileostomy closure is an uncommon, costly and morbid complication. Patients undergoing stoma closure are at high risk for an adverse outcome if they have CDC. Should it develop they should be aggressively treated.

The direct cellular target of topically applied pimecrolimus may not be infiltrating lymphocytes.

BACKGROUND: Topically applied calcineurin inhibitors have been shown to be effective in the treatment of atopic dermatitis. When systemically administered, these agents cause immunosuppression via inhibition of calcineurin in lymphocytes. As topical agents, the mechanism of action is poorly defined. OBJECTIVES: To test the hypothesis that skin-infiltrating lymphocytes are directly targeted when calcineurin inhibitors are applied to the skin. METHODS: Ten patients with atopic dermatitis were treated with 1% pimecrolimus cream twice daily to target lesions. Skin biopsies were performed before and 48 h after beginning therapy. We assessed the cellular localization of NFAT1 and NFAT2 as a surrogate measure of intracellular calcineurin activity (e.g. increasing cytoplasmic localization with increasing calcineurin inhibition). RESULTS: All patients showed a clinical response, at both 48 h and 2 weeks. As previously described, NFAT2 localized to the follicular keratinocytes, and its activation was partially inhibited by topical pimecrolimus. NFAT1 was found to be expressed by follicular and interfollicular keratinocytes, and its mostly nuclear localization was not affected by topical pimecrolimus therapy. Both NFAT1 and NFAT2 were found in the infiltrating lymphocytes. However, using both manual counting as well as an automated method to assess nuclear intensity of NFAT staining, we found that the proportion of infiltrating leucocytes with nuclear ('activated') NFAT did not change following therapy with pimecrolimus. CONCLUSIONS: Our results suggest that topical pimecrolimus does not act primarily by inhibiting the calcineurin/NFAT axis in lymphocytes but may instead act by other mechanisms, possibly by decreasing NFAT2 activity in follicular keratinocytes.

The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin. A possible mechanism for regulating cadherin turnover.

Cadherins are single pass transmembrane proteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion by linking the cytoskeletons of adjacent cells. In adherens junctions, the cytoplasmic domain of cadherins bind to beta-catenin, which in turn binds to the actin-associated protein alpha-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with beta-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and (1)H NMR measurements indicate that murine E-cadherin cytoplasmic domain is unstructured. Upon binding to beta-catenin, the domain becomes resistant to proteolysis, suggesting that it structures upon binding. Cadherin-beta-catenin complex stability is modestly dependent on ionic strength, indicating that, contrary to previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also reveals the presence of PEST sequences, motifs associated with ubiquitin/proteosome degradation, that overlap the previously identified beta-catenin-binding site. It is proposed that binding of cadherins to beta-catenin prevents recognition of degradation signals that are exposed in the unstructured cadherin cytoplasmic domain, favoring a cell surface population of catenin-bound cadherins capable of participating in cell adhesion.

Differential regulation of endogenous cadherin expression in Madin-Darby canine kidney cells by cell-cell adhesion and activation of beta -catenin signaling.

Cadherins mediate cell-cell adhesion, but little is known about how their expression is regulated. In Madin-Darby canine kidney (MDCK) cells, the cadherin-associated cytoplasmic proteins alpha- and beta-catenin form high molecular weight protein complexes with two glycoproteins (Stewart, D. B., and Nelson, W. J. (1997) J. Biol. Chem. 272, 29652-29662), one of which is E-cadherin and the other we show here is the type II cadherin, cadherin-6 (K-cadherin). In low density, motile MDCK cells, the steady-state level of cadherin-6 is low, but protein is synthesized. However, following cell-cell adhesion, cadherin-6 becomes stabilized and accumulates by >50-fold at cell-cell contacts while the E-cadherin level increases only 5-fold during the same period. To investigate a role of beta-catenin in regulation of cadherin expression in MDCK cells, we examined the effects of expressing signaling-active beta-catenin mutants (DeltaGSK, DeltaN90, and DeltaN131). In these cells, while levels of E-cadherin, alpha- and beta-catenin are similar to those in control cells, levels of cadherin-6 are significantly reduced due to rapid degradation of newly synthesized protein. Additionally, these cells appeared more motile and less cohesive, as expression of DeltaGSK-beta-catenin delayed the establishment of tight confluent cell monolayers compared with control cells. These results indicate that the level of cadherin-6, but not that of E-cadherin, is strictly regulated post-translationally in response to Wnt signaling, and that E-cadherin and cadherin-6 may contribute different properties to cell-cell adhesion and the epithelial phenotype.

T cell factor-activated transcription is not sufficient to induce anchorage-independent growth of epithelial cells expressing mutant beta-catenin.

N-terminal mutations in beta-catenin that inhibit beta-catenin degradation are found in primary tumors and cancer cell lines, and increased beta-catenin/T cell factor (TCF)-activated transcription in these cells has been correlated with cancer formation. However, the role of mutant beta-catenin in cell transformation is poorly understood. Here, we compare the ability of different N-terminal mutations of beta-catenin (DeltaN131, DeltaN90, DeltaGSK) to induce TCF-activated transcription and anchorage-independent growth in Madin-Darby canine kidney epithelial cells. Expression of DeltaN90 or DeltaGSK beta-catenin increased TCF-activated transcription but did not induce significant anchorage-independent cell growth. In contrast, deletion of the alpha-catenin-binding site in DeltaN131 beta-catenin reduced TCF-activated transcription, compared with that induced by DeltaN90 or DeltaGSK beta-catenin, but significantly enhanced anchorage-independent cell growth.

Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells.

The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.

Networking antioxidants in the isolated rat heart are selectively depleted by ischemia-reperfusion.

Although cardiac endogenous antioxidants have been reported to be oxidized and decreased by ischemia-reperfusion, little is known whether the changes in these antioxidants are correlated with each other in a systematic relationship. In this study, isolated rat hearts were subjected to various periods of ischemia-reperfusion using the Langendorff method, and the content and/or redox status of tissue antioxidants were analyzed. Significant losses in the tissue hydrophilic antioxidants, ascorbate, and glutathione were observed. These losses were dependent on the duration of the reperfusion period (between 0-40 min) but not of ischemia (20-60 min). Marked increases of dehydroascorbate and glutathione disulfide, the oxidized forms of ascorbate and glutathione, respectively, were found during reperfusion, but these changes were not observed during ischemia. These findings indicate that the tissue hydrophilic antioxidants are easily oxidized and may be the first line of antioxidant defenses during reperfusion. Lipophilic antioxidants, like ubiquinol 9 and vitamin E, were not decreased during ischemia-reperfusion using regular buffer; however, if oxidative stress was induced by addition of H2O2 to the buffer solution during reperfusion after 20 min of ischemia, decreases in both the hydrophilic and hydrophobic antioxidants were noticeable. With 100 microM H2O2, the tissue antioxidant decreases were ubiquinol 9 (39%), vitamin E (3%), glutathione (44%) and ascorbate (58%). Only with 500 microM H2O2 treatment were marked decreases in tissue vitamin E (65%) observed; this was associated with almost complete depletion of tissue ubiquinol 9 (95%). These results suggest that prior to the consumption of vitamin E, other antioxidants are depleted and that vitamin E may serve as the ultimate antioxidant, protecting the integrity of cellular membranes. Thus, in this work, cardiac antioxidants were demonstrated to change in a systematically organized relationship under ischemia-reperfusion. This graded utilization of antioxidants supports the redox based antioxidant network concept, found to be present in other biological systems.

Identification of four distinct pools of catenins in mammalian cells and transformation-dependent changes in catenin distributions among these pools.

Catenins are cytoplasmic proteins that were initially identified in a complex with cadherins, a superfamily of transmembrane glycoproteins important for cell adhesion in normal and disease states. We have used gel filtration to identify four complexes of catenins in extracts from normal and transformed epithelial cells. In normal Madin-Darby canine kidney epithelial cells, a significant fraction of alpha- and beta-catenin and plakoglobin co-elute with cadherin in a high molecular weight complex (complex I). A portion of alpha-catenin and the remainder of beta-catenin and plakoglobin co-elute in a high molecular weight complex that does not contain cadherin (complex II). The remainder of alpha-catenin elutes in a low molecular weight fraction (complex III). In extracts from two colon carcinoma cell lines, HCT116 and SW480, beta-catenin elutes in an additional low molecular weight pool (complex IV) not present in Madin-Darby canine kidney cell extracts. In two subclones derived from SW480 cells, SW-E8 and SW-R2, beta-catenin is distributed evenly between high and low molecular weight pools in SW-E8 cells, whereas it elutes primarily in the low molecular weight pool (complex IV) in SW-R2 cells. These changes in beta-catenin elution profiles correlate with an increase in transformed phenotype and decreased cell-cell adhesion in the SW-R2 cells.

Role of ascorbate in protection by nitecapone against cardiac ischemia-reperfusion injury.

The antioxidant properties of nitecapone, a catechol derivative and an inhibitor of catechol-O-methyltransferase, were reported recently. In the present study, the influence of nitecapone on isolated rat heart ischemia-reperfusion injury was investigated to elucidate its cardioprotective role. Nitecapone, administered in the perfusion buffer from the beginning of the pre-ischemic phase, significantly improved recovery of cardiac mechanical function, suppressed enzyme leakage in the coronary effluent, and minimized loss of ascorbate, compared with the control group. In rats fed a diet containing 4% ascorbate, myocardial ascorbate content in ascorbate-fed rats after ischemia-reperfusion was higher than that in control rats fed a normal diet without ischemia. However, supplemented rats did not show any beneficial effects on cardiac mechanical recovery or enzyme leakage, suggesting that maintenance of tissue ascorbate level is not the cause, but the result of the protective effects of nitecapone against cardiac ischemia-reperfusion injury. The iron-chelating effect of nitecapone was also tested. It was confirmed, using electron spin resonance, that 50 microM nitecapone chelates the same concentration of iron released from the heart into the coronary effluent. Hence, the iron-chelating ability of nitecapone may be responsible, at least in part, for its cardioprotective effects in ischemia-reperfusion injury.

Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex.

The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types. Although proteins of this complex have been identified, little is known about their interactions. Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that beta-catenin is the linker protein between E-cadherin and alpha-catenin and that E-cadherin does not bind directly to alpha-catenin. We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of alpha-catenin are independent binding sites for beta-catenin. In addition to beta-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin. However, unlike beta-catenin, p120 does not bind alpha-catenin in vitro, although a complex of p120 and endogenous alpha-catenin could be immunoprecipitated from cell extracts. In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and alpha-catenin revealed two catenin pools in cell lysates: an approximately 1000- to approximately 2000-kDa complex bound to E-cadherin and an approximately 220-kDa pool that did not contain E-cadherin. Only beta-catenin in the approximately 220-kDa pool bound exogenous E-cadherin. Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.

Interactions between drought and elevated CO2 on growth and gas exchange of seedlings of three deciduous tree species.

Interactions between elevated atmospheric CO2 and drought on growth and gas exchange of American sycamore (Platanus occidentalis L.), sweetgum (Liquidambar styraciflua L.) and sugar maple (Acer saccharum Marsh.) were investigated using 1-yr-old seedlings, planted in 8 1 pots and grown in four open-top chambers, containing either ambient air or ambient air enriched with 300 µmol mol-1 CO2 . Two soil moisture regimes were included within each chamber: a 'well-watered' treatment with plants watered daily and a 'drought' treatment in which plants were subjected to a series of drought cycles. Duration and depth of the drought cycles were determined by soil matric potential. Mean soil water potential at rewatering for the water-stressed seedlings under ambient CO2 for sugar maple, sweetgum and sycamore was -0.5, -0.7 and -1.8 MPa, respectively, compared with > -0.1 MPa for all well-watered plants. Elevated CO2 increased relative growth rate of well-watered sugar maple by 181%, resulting in a 4.3-fold increase in total plant dry weight after 81 d, compared with 1.4 and 1.6-fold increases for sweetgum and sycamore, respectively, after 69 d. Although elevated CO2 increased net CO2 assimilation rate of sugar maple by 115%, there was a 10-fold increase in leaf area which contributed to the growth response. Although drought did not eliminate a growth response of sugar maple to elevated CO2 it greatly reduced the elevated CO2 -induced enhancement of relative growth rate. In contrast, relative growth rates of sweetgum and sycamore were not significantly increased by elevated CO2 . Drought, under elevated CO2 , reduced leaf area of all three species to a greater extent than it reduced net CO2 assimilation rate. The response ranged from no effect in sugar maple to a 40 % reduction in sycamore, with sweetgum exhibiting an intermediate response. Results indicate that drought may alter the growth response, gas exchange and water relations of tree species growing in an elevated CO2 atmosphere. Under high nutrient and water availability, sugar maple may benefit the most (of the three species studied) from a CO2 - enriched atmosphere, but productivity gains will be limited if frequent drought is prevalent.

Long-run social and economic responses of fertility in the United States.

PIP: The classic linear relationship of fertility rates, either in terms of completed family size or birth timing, to income fails to take adequately into consideration social variables, such as marriage rate, female unemployment rate, urban vs rural residence, divorce rate, and contraceptive sales levels. Even when only income is considered, its effect on fertility is not linear because the opportunity cost of children is high at both ends of a marriage -- during the 1st 2 years because income is low and during the last 6 years because income is high enough for other values, such as leisure, to compete with children. The relation between income and fertility is therefore parabolic. If one analyzes US census data from 1900 to 1977, the threshold level of permanent income at which the influence of income becomes negative is $2599. This value of permanent income occurs between 1958 and 1959, and the US birth rate began to decline in 1957. Income also affects fertility indirectly because it affects marriage rates and the timing of births. Divorce rates also affect fertility in 2 ways. They limit the completed family size of any 1 family, and they tend to have a conservative effect on risk-taking behavior, such as having children. Thus, increasing marriage rates have a positive effect on fertility; increasing divorce rate, a negative one. Female employment increases the opportunity cost of having a child, since a child may be considered as 1 kind of durable good, which competes for the woman's time. Migration from rural to urban areas also effects a decline in fertility. War affects fertility in at least 3 ways. It influences the number of men away, the number of men returning at the same time, and the amount of stress on a military family whether there is a war or not. What these various relationships indicate is that it would be premature to assume that as income increases fertility rates will necessarily decline. If the behavior of divorce and marriage rates remains stable and the rate of growth in permanent per capita income remains at 2.5%-3.0% per year, given the present age structure of the population, marriage rates, and thus fertility rates, will increase.^ieng

Recruitment of naphthalene dissimilatory enzymes for the oxidation of 1,4-dichloronaphthalene to 3,6-dichlorosalicylate, a precursor for the herbicide dicamba.

Pseudomonas putida expresses plasmid-encoded enzymes and regulatory proteins for the dissimilation of naphthalene through salicylate and the alpha-keto acid pathway. A strain of P. putida (NAH:Tn5/G67) defective in salicylate hydroxylase (nahG) was assessed for its ability to oxidize 1,4-dichloronaphthalene. Washed cell suspensions were shown to accumulate 3,6-dichlorosalicylate, which, after further chemical treatment, yields the herbicide dicamba (3,6-dichloro-2-methoxybenzoate). However, the rate of dichlorosalicylate formation from dichloronaphthalene was less than 1% of the rate of salicylate formation from unsubstituted naphthalene.

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.