, 2008). Two additional genes, contained www.selleckchem.com/products/PF-2341066.html in an

operon with amtB, have also been proposed to be under GlnR control: glnK (msmeg_2426; PII-type protein) and glnD (msmeg_2427; an adenylyl-transferase enzyme) (Amon et al., 2008). Therefore, glnA1, amtB and amt1 were selected for analysis in this study. Four other genes were also chosen for investigation owing to their proposed role in nitrogen metabolism in Mycobacteria (Amon et al., 2009). These were amtA (msmeg_4635), an ammonium transporter; nirB (msmeg_0427), a nitrite reductase enzyme; gltD (msmeg_3226), a glutamate synthase enzyme involved in glutamate synthesis during nitrogen limitation; and glnE (msmeg_4293), a bifunctional adenylyl-transferase thought to modulate GS enzymatic activity. Expression of glnR (msmeg_5784) itself was also included. Wild-type and mutant strains were grown in nitrogen-limiting or nitrogen-excess conditions for 13 h. Expression values for each gene analysed were compared with the housekeeping gene sigA (msmeg_2758) whose expression did not alter in the conditions tested (data not shown). Genes previously shown to be under GlnR control, glnA1, amtB and amt1, were all highly up-regulated in the wild type during nitrogen limitation when compared with their expression in nitrogen-excess conditions (Fig. 2). glnA1 expression was induced approximately 13-fold, amtB 153-fold and amt1 219-fold (Table 3). However, there was no induction of these genes in either GlnR mutant

grown under nitrogen limitation enough (Fig. 2 and Table 3). To account for the fact that the GlnR mutants deplete the external ammonium at a slower rate than the wild type (Fig. 1) and check details may not be stressed at 13 h, we repeated the qRT-PCR using samples taken at 19 h, when external nitrogen was no longer detectable. However, there was also no induction of gene expression in either mutant at this later time point (data not shown). Transcriptional control of other genes proposed to be involved in mycobacterial nitrogen metabolism, not shown previously to be under GlnR control, was subsequently investigated. Figure 3 shows that amtA, gltD and nirB were up-regulated in the wild-type strain in response

to nitrogen limitation at 13 h, compared with nitrogen excess, while glnE expression was down-regulated. During nitrogen limitation, amtA was induced approximately 337-fold, nirB 103-fold and gltD 8-fold; glnE was down-regulated 3-fold. Again, no significant change in the expression levels of these genes was observed in the GlnR mutants at either 13 h (Fig. 3 and Table 3) or 19 h (data not shown). To exclude the possibility that the GlnR_D48A mutation inhibited glnR expression, leading to the observed null phenotype of this strain, transcriptomic analysis of glnR was performed. No significant change in glnR expression was observed under nitrogen-limiting conditions for either the wild type or GlnR_D48A mutant (Table 3 and Fig. 2), confirming previous observations that M.

, 2008). Two additional genes, contained selleck compound in an

operon with amtB, have also been proposed to be under GlnR control: glnK (msmeg_2426; PII-type protein) and glnD (msmeg_2427; an adenylyl-transferase enzyme) (Amon et al., 2008). Therefore, glnA1, amtB and amt1 were selected for analysis in this study. Four other genes were also chosen for investigation owing to their proposed role in nitrogen metabolism in Mycobacteria (Amon et al., 2009). These were amtA (msmeg_4635), an ammonium transporter; nirB (msmeg_0427), a nitrite reductase enzyme; gltD (msmeg_3226), a glutamate synthase enzyme involved in glutamate synthesis during nitrogen limitation; and glnE (msmeg_4293), a bifunctional adenylyl-transferase thought to modulate GS enzymatic activity. Expression of glnR (msmeg_5784) itself was also included. Wild-type and mutant strains were grown in nitrogen-limiting or nitrogen-excess conditions for 13 h. Expression values for each gene analysed were compared with the housekeeping gene sigA (msmeg_2758) whose expression did not alter in the conditions tested (data not shown). Genes previously shown to be under GlnR control, glnA1, amtB and amt1, were all highly up-regulated in the wild type during nitrogen limitation when compared with their expression in nitrogen-excess conditions (Fig. 2). glnA1 expression was induced approximately 13-fold, amtB 153-fold and amt1 219-fold (Table 3). However, there was no induction of these genes in either GlnR mutant

grown under nitrogen limitation Chloroambucil (Fig. 2 and Table 3). To account for the fact that the GlnR mutants deplete the external ammonium at a slower rate than the wild type (Fig. 1) and PS-341 molecular weight may not be stressed at 13 h, we repeated the qRT-PCR using samples taken at 19 h, when external nitrogen was no longer detectable. However, there was also no induction of gene expression in either mutant at this later time point (data not shown). Transcriptional control of other genes proposed to be involved in mycobacterial nitrogen metabolism, not shown previously to be under GlnR control, was subsequently investigated. Figure 3 shows that amtA, gltD and nirB were up-regulated in the wild-type strain in response

to nitrogen limitation at 13 h, compared with nitrogen excess, while glnE expression was down-regulated. During nitrogen limitation, amtA was induced approximately 337-fold, nirB 103-fold and gltD 8-fold; glnE was down-regulated 3-fold. Again, no significant change in the expression levels of these genes was observed in the GlnR mutants at either 13 h (Fig. 3 and Table 3) or 19 h (data not shown). To exclude the possibility that the GlnR_D48A mutation inhibited glnR expression, leading to the observed null phenotype of this strain, transcriptomic analysis of glnR was performed. No significant change in glnR expression was observed under nitrogen-limiting conditions for either the wild type or GlnR_D48A mutant (Table 3 and Fig. 2), confirming previous observations that M.

subtilis 168, YH2M (MW) and the double

mutant 8R. As shown in Fig. 6a, the half-life of 168 and single-mutant MW was ≈1.5 min, whereas the half-life of 8R was calculated to be ≈3 min. This twofold increase of the half-life of the double-mutant must be due to a contribution of single-mutant WM at position +6, demonstrating that this mutation leads to the stabilization of the bmrA mRNA. Figure 6b shows the mRNA secondary structures predicted for the bmrA 5′ untranslated region. The transition at position +6 leads to a change of the predicted structure and a decrease in Gibbs free energy ΔG. According to http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi, the first stem–loop is stabilized. This is in accordance with previous observations on the mRNA-stabilizing function of 5′-terminal stem–loops (Hansen et al., 1994; Hambraeus et al., 2000). Because antibiotic resistance is most often only transiently advantageous to bacteria, an efficient way to escape the U0126 cost lethal action of drugs Stem Cell Compound Library ic50 is the regulation of resistance gene expression at the transcriptional or the translational level following mutations or the movement of mobile genetic elements (Depardieu et al., 2007). Piddock (2006) reported that chromosomally encoded efflux pumps may be overexpressed due to mutations in the local repressor, mutations

in global regulatory genes, promoter mutations or insertion sequences. In an induction experiment, we confirmed the finding of Steinfels et al. (2004) that bmrA is not inducible by any specific substrate. Furthermore, using EMSA and a radioactively labelled fragment of the bmrA upstream region, no specific binding protein acting as an activator or a repressor could be identified in crude protein extracts of the mutant or the wild-type strain (data not shown). Instead, we identified a mechanism of adaptation without fine-tuning, resulting in antibiotic resistance by constitutively upregulated expression of a specific protein. Such proteins may encompass ABC transporters, permeases, transcription

factors or sigma factors. For instance, Stirrett et al. (2008) reported the upregulated expression of several efflux pumps in Y. pestis by overexpression of the transcriptional regulator RobAYp from a multicopy plasmid. So far, spontaneous constitutively resistant mutants in Gram-positive bacteria revealing overexpression due to promoter mutations have only C1GALT1 been detected in a few cases (Piddock, 2006). For instance, the triclosan efflux pump of Pseudomonas aeroginosa was upregulated by a mutation in the −35 region of the promoter (Mima et al., 2007), while in M. smegmatis a G to T transversion in the −10 region of the promoter increased the copy number of the d-alanine racemase conferring resistance to d-cycloserine (Cáceres et al., 1997). Similar data were obtained by Ohki & Tateno (2004), who reported the increased expression of the bmr3 efflux transporter due to a +4 mutation that also resulted in the stabilization of the corresponding bmr3 mRNA.

There were no significant changes in these enzymes in the cells exposed to H2O2 (Fig. 3). Hence, these data point to the channeling of substrates towards the formation of KG and NADPH with the subsequent decrease in the synthesis of NADH. This strategy ensures that during oxidative stress, sufficient NADPH, a potent reductive fuel, and KG, a powerful scavenger of ROS, are available.

The decrease in the Kinase Inhibitor Library solubility dmso generation of NADH will further help decrease the oxidative burden as this moiety drives the production of ROS via the electron transport chain (ETC). Furthermore, it is critical that during oxidative stress, the effectors mediating ROS production be attenuated. Oxidative phosphorylation is a major generator of ROS (Ludwig et al., 2001; Murphy, 2009). Hence, it is quite conceivable that the complexes mediating this process are downgraded. These Fe-containing complexes are susceptible to H2O2 (Touati, 2000; Middaugh et al., 2005). Indeed, sharp reduction was observed in the activities of Complexes I, II, and IV (Fig. 4). The nature of Complexes I and IV was further confirmed by

the inclusion of rotenone CP-690550 nmr and KCN in the assay mixture. The former is a specific inhibitor for Complex I, while Complex IV is inhibited by KCN. The activity band was not detected in the control CFE in the presence of these inhibitors, respectively (data not included). This strategy of limiting the formation of NADH, coupled with decreased activities of the enzymes involved in its oxidation, provides an effective tool to mitigate H2O2 insult. Pseudomonas fluorescens appears to adopt this tactic in an effort to survive in the oxidative environment induced by H2O2. Numerous STK38 organisms do indeed resort to decreased oxidative phosphorylation and anaerobiosis with the goal of coping with a ROS challenge (Chen et al., 2003; Chenier et al., 2008). In eukaryotic systems, the promotion of the hypoxia-inducible factor (HIF-1α), an activator of anaerobic respiration, is favored (Mailloux et al., 2009a, b). As the catabolism of histidine was

providing glutamate, a moiety involved in the generation of the antioxidant KG, it was important to ascertain whether the enzymes involved in the formation and utilization of KG were modulated by H2O2. When control cells were exposed to H2O2 stress, the decrease in KGDH activity was coupled with the increase in GDH activity. However, when H2O2-stressed cells were introduced into control media, the reverse trend was observed i.e. the activity of KGDH was recovered while the activity of GDH was decreased. Western blot analyses revealed that the latter enzyme was more abundant in the H2O2-treated cells and was affected by this oxidative modulator (Fig. 5). Hence, it is clear that H2O2 was indeed controlling the status of KGDH, GDH, and ICDH and subsequently the levels of KG and NADPH.

Salmonella enterica serovar Typhimurium strain LT2 harbours four prophages, including Gifsy-1, Gifsy-2, Fels-1 and Fels-2 (McClelland et al., 2001; Brüssow et al., 2004). Both the Gifsy-3 and the SopE prophages, found in S. Typhimurium strains 14028 and SL1344, respectively, are absent in S. Typhimurium strain LT2 (Figueroa-Bossi et al., 2001; Brüssow et al., 2004; Thomson et al., 2004). Salmonella enterica serovar Typhimurium strains SL1344

and 14028 both contain Gifsy-1 and Gifsy-2, but not Fels-1 and Fels-2 (Figueroa-Bossi et al., Tyrosine Kinase Inhibitor Library 2001). Salmonella enterica serovar Typhi harbours seven distinct prophage-like elements, spanning >180 kb, that are generally conserved between strains (Fig. 2) (Thomson et al., 2004). The modular nature of prophage genomes makes a significant contribution to serovar variation

and comprises most of the variation in gene content among strains of the same serovar (Boyd et al., 2003; Vernikos et al., 2007). Salmonella has many virulence-associated genes found within clusters in its genome, which are known as SPIs (Mills et al., 1995). Virulence factors encoded by SPI genes tamper with host cellular mechanisms and are thought to dictate the host specificity of the different S. enterica serovars (Eswarappa et al., 2008). Many of the SPIs are found next to a tRNA gene (Supporting Information, Fig. S1) and their G+C content differs from the rest of the genome (Fig. 2). Hence, such genomic islands were most likely inserted into the DNA of Salmonella by horizontal transfer events, although Tolmetin this explanation remains uncertain (Amavisit Epacadostat mouse et al., 2003). Twenty-one SPIs are known to date in Salmonella (McClelland et al., 2001; Parkhill et al., 2001; Chiu et al., 2005; Shah et al., 2005; Vernikos & Parkhill, 2006; Fuentes et al., 2008; Blondel et al., 2009). The S. Typhimurium and S. Typhi genomes contain 11 common SPIs (SPIs-1 to 6, 9, 11, 12, 13 and 16) (Fig. 2). SPIs-8 and 10 were initially found in S. Typhi, and considered as absent in S. Typhimurium. However, at both locations in S. Typhimurium, there is a completely different set of genes. There is only one SPI specific to S. Typhimurium, SPI-14 (Shah et

al., 2005), and four SPIs are specific to S. Typhi (SPIs-7, 15, 17 and 18) (Fig. 2). SPIs-19, 20 and 21 are absent in both of these serovars and will not be discussed further (Blondel et al., 2009). Even if many of these islands are found in both serovars, differences emerge when comparing equivalent SPIs. In the following section, the genomic differences between S. Typhimurium and S. Typhi are described for each SPI using S. Typhimurium strain LT2 and S. Typhi strain CT18 as the genomic references. Amino acid alignments of SPIs between these strains were performed using the xbase software (Chaudhuri & Pallen, 2006) and can be seen in Fig. S1. SPI-1 is a 40 kb locus located at centisome 63 encoding a type three secretion system (T3SS) (Mills et al.

It has been shown that enhancing spinal inhibitory transmission alleviates hyperalgesia and allodynia. Therefore, the spinal neuronal network is a pivotal target

to counteract pain symptoms. Thus, any increase in spinal 3α5α-reduced steroid production enhancing GABAergic inhibition should reduce nociceptive message integration and the pain response. Previously, it has been this website shown that high levels of plasma glucocorticoids give rise to analgesia. However, to our knowledge, nothing has been reported regarding direct non-genomic modulation of neuronal spinal activity by peripheral CORT. In the present study, we used combined in vivo and in vitro electrophysiology approaches, associated with measurement of nociceptive mechanical sensitivity and plasma CORT level measurement, to assess the impact of circulating CORT on rat nociception. We showed that CORT plasma level elevation produced analgesia via a reduction in C-fiber-mediated spinal responses. In the spine, CORT is reduced to the neuroactive metabolite allotetrahydrodeoxycorticosterone, which specifically enhances lamina II GABAergic synaptic transmission. Selumetinib The main consequence is a reduction in lamina II network excitability,

reflecting a selective decrease in the processing of nociceptive inputs. The depressed neuronal activity at the spinal level then, Mephenoxalone in turn, leads to weaker nociceptive message transmission to supraspinal structures and hence to alleviation of pain.

“
“Disambiguation of a noisy visual scene with prior knowledge is an indispensable task of the visual system. To adequately adapt to a dynamically changing visual environment full of noisy visual scenes, the implementation of knowledge-mediated disambiguation in the brain is imperative and essential for proceeding as fast as possible under the limited capacity of visual image processing. However, the temporal profile of the disambiguation process has not yet been fully elucidated in the brain. The present study attempted to determine how quickly knowledge-mediated disambiguation began to proceed along visual areas after the onset of a two-tone ambiguous image using magnetoencephalography with high temporal resolution. Using the predictive coding framework, we focused on activity reduction for the two-tone ambiguous image as an index of the implementation of disambiguation. Source analysis revealed that a significant activity reduction was observed in the lateral occipital area at approximately 120 ms after the onset of the ambiguous image, but not in preceding activity (about 115 ms) in the cuneus when participants perceptually disambiguated the ambiguous image with prior knowledge.

Patients preferred shorter waiting times,[40] high satisfaction with pharmacy ratings, quality certificates and extended opening hours.[37] A study by Wellman and Vidican,[39] piloting the addition of medication management services in prescription benefit insurance models, demonstrated that respondents placed the greatest value on pharmacist provision of comprehensive medication management services. Several of the studies also indicated the existence of ‘status-quo’ bias (i.e. tendency to prefer their current service) among patients with respect to

pharmacy choices.[35-37] Four studies examined pharmacist preferences, of which only three elicited preferences with respect to pharmacy services,[41-43] while one was related to preferences for a specific new technology.[44] Grindrod et al.[42] investigated pharmacist preferences for specialised service provision and showed SB525334 manufacturer that pharmacists preferred to provide medication and disease-management services but did not have significant preferences for screening services. This was contrary to Scott et al.[43] who investigated pharmacist preferences for extended roles in primary care. Significant predictors of pharmacists’ job choices included having an extended pharmacy team, integration with primary and secondary care as well as higher income whereas provision of chronic disease

management and health promotion services was not preferred. Using the latent class model, one study[42] showed the existence of preference heterogeneity in pharmacists’ preferences MAPK Inhibitor Library nmr with pharmacists falling into three classes, indicating that groups of pharmacists may have different preferences with respect to specialised services provision. Pharmacist preferences were mainly investigated for process-related aspects such as duration of service, type or level of service provision, setting and integration with primary/secondary care and professional service/job-related aspects including job satisfaction, educational requirements and personal income. In the majority of the

studies eliciting new preferences for the delivery of specialised pharmacy services (medication or disease management), income was an important attribute, with pharmacists preferring higher incomes.[41-43] Only two studies investigated preferences of both patients and providers for haemophilia therapy.[45, 46] These were also the only studies that included health-outcome related attributes in addition to process-related outcomes. While patients and providers showed substantial consensus for some attributes (e.g. cost), preferences for other attributes were quite different. Patients were more focused on process-related attributes as compared to providers. The relatively few pharmacy DCE studies make it hard to make definitive conclusions about pharmacy services from both the provider and recipient viewpoints. However, a few findings may be highlighted.

The aim of this review is to compare the clinical features, pathophysiology and management of these conditions. Common clinical features due to a raised anion gap metabolic acidosis are seen. Reduced carbohydrate intake is a usual contributor to ketogenesis. Treatment

is primarily with intravenous glucose, insulin if there is insulin deficiency and potassium as needed. The value of using bedside monitors to measure β-hydroxybutyrate levels both for diagnosis and monitoring of response to treatment is emphasised. Early recognition of ketoacidosis and treatment with glucose rather than saline is important for optimum outcome. Copyright © 2013 John Wiley & Sons. Practical Diabetes 2013; 30(4): 167–171 “
“A previously fit and well 35-year-old man presented to the acute medical ABT-199 in vivo take with a three-week history mTOR inhibitor of general malaise; he complained of polyuria, polydipsia, weight loss, abdominal pain and vomiting. He was diagnosed with type 1 diabetes (capillary blood glucose 20.7mmol/L) but was not ketoacidotic (pH 7.43) and was commenced on intravenous fluids and an intravenous insulin infusion. After four hours of appropriate fluid resuscitation and normalisation of blood glucose, the patient had deteriorated becoming tachycardic and hypotensive. At this time the patient was reassessed and a short synacthen

test with a baseline ACTH was carried out. Hydrocortisone was administered to the patient immediately after the short synacthen test. Addison’s many disease was confirmed (cortisol 0 minutes: 315nmol/L, 30 minutes: 337nmol/L, ACTH 627ng/L). Further investigations during that admission also identified primary hypothyroidism (TSH 48.5mU/L, free T4 19.2pmol/L) but no other endocrinopathies. For the first time in the literature this is a case report of a young man presenting with type 1 diabetes,

Addison’s disease and primary hypothyroidism simultaneously. Copyright © 2011 John Wiley & Sons. Type II polyglandular autoimmune syndrome is a rare endocrine disorder with a frequency of 1.4–2.0/100 0001 and is more common in women than men.1 The condition is characterised by autoimmune adrenal failure, autoimmune thyroid disease and/or type 1 diabetes. Other autoimmune conditions associated with it are pernicious anaemia, vitiligo, hypergonadotrophic hypogonadism, chronic autoimmune hepatitis, coeliac disease, autoimmune diabetes insipidus and rarely lymphocytic hypophysitis, stiff-person syndrome and myasthenia gravis.1 Type 1 diabetes is the first presentation in 57–63% of cases; adrenal failure is the first presentation in 23–35% of cases and both present simultaneously in 8–10% of cases.2 Carpenter noticed the association of Schmidt’s syndrome (Addison’s disease and hypothyroidism) and diabetes mellitus in his case review in 1964:3 in this review, 20% of Schmidt’s syndrome cases also had diabetes mellitus and the triad is sometimes referred to as Carpenter’s syndrome.

, 2006).

Other studies have shown that ecological proximity may be linked to HGT. For example, a yeast wine strain (S. cerevisiae EC118) has gained 65 KB of genetic material from Zygosaccharomyces bailii (a major contaminant of wine fermentations; Novo et al., 2009). The genome of Mycosphaerella graminicola also displays evidence of whole chromosomal transfer (Goodwin et al., 2011). M. graminicola contains 21 chromosomes; eight of these are dispensable and originated from an unknown fungal source, which is most likely the result of a somatic fusion with another species that had eight or more chromosomes (Goodwin et al., 2011). Another process linked to HGT in fungal species is anastomosis. Filamentous fungi frequently fuse conidia and conidial germlings using a specialized hypha known as conidial anastomosis tubes; these allow interconnected germlings to act as a single coordinated individual (regulating CH5424802 supplier water, nutrients, signal molecules, nuclei and organelles; Read

et al., 2009) and also allow for genetic exchange (Roca et al., 2004). Although non-self-recognition systems have evolved in fungi (Glass & Kaneko, 2003), there is evidence to suggest NVP-LDE225 that interspecies anastomosis between fungal pathogens may have occurred (Friesen et al., 2006; Xie et al., 2008). As well as mechanisms that facilitate fungal HGT, there are also potential barriers that may oppose it. For example, fungal nuclei are membrane bound, and also differential intron processing and incompatible gene promoters may need to be overcome (Keeling & Palmer, 2008). Furthermore, fungal genetic material is stored in chromatin; while gene-silencing mechanisms such as repeat induced point mutation and methylation induced premeiotically systems have the potential to pseudogenize foreign genes with repetitive elements. The process Janus kinase (JAK) of meiotic silencing by unpaired DNA (Shiu et al., 2001) is yet another possible barrier to HGT; indeed, it has been proposed that (meiotic) sex has evolved in eukaryotes as a mechanism to

check the identity and limit the impact of foreign DNA (Glansdorff et al., 2009). Another possible barrier to HGT is an alternative genetic code. The human pathogen Candida albicans and close relatives translate the codon CTG as serine instead of leucine. Recent analyses of species from the CTG clade (Fitzpatrick et al., 2006) could only locate four incidences of bacterial to fungal HGT since the CTG codon reassignment approximately 170 million years ago (Fitzpatrick et al., 2008; Marcet-Houben & Gabaldon, 2010). Such low incidences of HGT over such a long time period support the hypothesis that genetic code alterations act as barriers to HGT. Comparative fungal genomic analyses have shown the importance that HGT plays in the evolution of fungi. For example, Hall and Dietrich have shown that S.

After electrophoresis, gel was stained with Coomassie Brilliant Blue R-250. For activity staining, zymographic analysis of the protease was performed using gelatin (0.1%) as the substrate as Raf inhibition described by Karbalaei-Heidari et al. (2009). Zymographic analysis for the amylase was performed on nondenaturing electrophoresis slab gels (10% polyacrylamide) prepared with 10% of sucrose, as described by Cadenas & Engel (1994). The amylase activity, with soluble starch as the substrate,

was determined using DNS (3,5-dinitrosalicylic acid) method (Miller, 1959). One unit (U) of amylase activity was defined as the amount of enzyme necessary to produce 1 μmol of reducing sugar per minute under the assay conditions. Protease activity was measured as described previously (Karbalaei-Heidari et al., 2009). One unit (U) of protease activity was defined as the amount of enzyme yielding 1 μmol of tyrosine per minute under the assay conditions. The effect of pH on enzyme activity was studied over a pH range of 4.0–12.0. The pH stability of the enzymes was determined by incubation with different buffer systems at 30 °C for 24 h. The following buffer systems (100 mM) were used: glycine-HCl buffer, pH 4.0; sodium acetate buffer, pH 5.0–6.0; potassium phosphate buffer, pH 7.0; Tris–HCl buffer, pH 8.0–8.5; glycine–NaOH buffer, pH 9.0–12.0. To investigate the effect of temperature, the assay

was conducted under different temperatures from 30 to 90 °C. The thermostability of the enzyme was determined by pre-incubating Enzalutamide solubility dmso the enzyme sample at various temperatures for 24 h, and residual activity was measured Florfenicol using the standard assay. The activity of the purified enzyme was measured in enzyme reaction mixture containing 0–20% NaCl. Salt stability of the enzyme was determined by incubating

the enzyme with different concentrations of NaCl for 24 h, and the remaining activity was determined under standard assay conditions. The effect of organic solvents with different log Pow values at 50% (v/v) concentration on the purified enzyme was determined by incubating the enzyme solution in different organic solvents at 30 °C. Residual activity was measured under the standard conditions. If residual activity was more than 50% after 10 days, half-life was taken as ‘> 10 days’. While activity was < 50% after 1 h, half-life was taken as ‘< 1 h’. Effects of different metal ions and chemical reagents [ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), phenylarsine oxide (PAO), diethyl pyrocarbonate (DEPC), β-mercaptoethanol, SDS, Triton X-100, and Tween-80] on the activity of purified enzymes were examined after they had been pre-incubated with them at 30 °C for 12 h, respectively, and the residual activity was determined under optimal assay conditions. Activity of the enzyme assayed in the absence of any additives was taken as 100%.