In the BBH performed to identify common genes exclusive of pathogenic A1155463 bacteria, 851 clusters were obtained (Figure 2B). From these, 24 clusters involved in pathogenicity, protein secretion, and integration-recombination processes were selected, based on the best studied plant pathogen, Rhizobium tumefaciens C58 [27–29] in addition to clusters involved in biological nitrogen fixation.
R. tumefaciens was considered as the reference organism for pathogenesis because the symbionts in this study interact with plants and because in the animal pathogens of the Rhizobiales order, virulence-associated type IV secretion proteins homologous to R. tumefaciens Sepantronium price were identified [30–32]. Of the 24 clusters obtained, 11 of these clusters were analyzed in this study. The remaining 13 are related to protein secretion and integration-recombination (Figure 2B) (Table A2b of supplementary ICG-001 in vitro material in database). In the BBH performed with lower stringency for nitrogen-fixing
bacteria and bacteria involved in bioremediation, 41 extra clusters of interest were selected Fossariinae (Figure 2A, and Table A2a of supplementary material in database); however, they did not include all bacteria
used in the comparison. Among these clusters, two clusters were related to FixQ protein and two to NifS. Both FixQ and NifS clusters were composed by a separate group of bacteria. However, for each of these proteins, the clusters obtained were grouped in the analysis. Of the 41 clusters, 39 were analyzed. For pathogenic bacteria, of the clusters obtained in the analysis with lower stringency, 25 were obtained and 24 were selected for analysis (Figure 2B, and additional file 2) (in addition Table A2 of supplementary material in database). In the BBHs performed in this study, except for clusters related to protein secretion and integration-recombination, 96 clusters were selected. Of these, 81 are common or exclusive to nitrogen-fixing bacteria, bacteria involved in bioremediation, and pathogenic bacteria BBHs. Of these, 63 were of interest for analysis (except the clusters related to other evolutive mechanisms and those repeats for the same protein, which were considered as one) (Figure 2).
Enzymes such as trypsin-like serine proteases, which may cleave at the many Arg residues present in the sequence of Bac7(1-35), might have this effect. However, these results clearly indicate that the peptide should be quite stable in blood and its degradation occurs only after several hours, suggesting that the decreased activity of Bac7(1-35) Veliparib nmr is only in part due to its degradation. In vivo toxicity As a first step to evaluate the therapeutic potential of Bac7(1-35), its in vivo toxicity was determined in Balb/c and CBA/Ca mice after Ro 61-8048 research buy injection via i.p. of increasing single peptide doses. No
apparent toxic effect was observed when the peptide was administered i.p. up to 75 mg/kg, but the mice receiving the highest peptide dose (150 mg/kg) died 3 days post injection. This result confirms that Bac7(1-35) is much less toxic than other cathelicidin-derived peptides such as those belonging to the α-helical group  and, in this respect, it behaves similarly to insect proline-rich AMPs. For example, pyrrhocoricin protected mice against E. click here coli infection, and showed no toxicity up to the maximal applied dose i.v. of 50 mg/kg. Drosocin is completely devoid of toxicity to healthy animals when used via i.v. at 100 mg/kg . On the contrary, lytic peptides such as BMAP-27 and -28 are toxic via i.p. already at 10-15 mg/kg . In vivo Bac7(1-35)
activity in a mouse model of typhoid fever The potential of Bac7(1-35) to protect mice from a bacterial challenge was tested by a mouse model of Salmonella infection. Infected mice develop a systemic disease characterized by rapid bacterial multiplication in the liver and spleen that resembles typhoid fever caused by Salmonella serovar Typhi in humans . Cell-mediated immunity and macrophage activity play a key role in defence against murine salmonellosis,
and it has been shown that these immune responses are lacking in Balb/c mice [22, 23] so that also the antibiotic ciprofloxacin failed to prevent fatal S. typhimurium disease in this mouse strain . For this reason, we preferred to use CBA/Ca mice that show a lower susceptibility to Salmonella infection  to study the antimicrobial PRKD3 properties of Bac7(1-35). Nevertheless, an acute infection may be induced by i.p. injection of less than a hundred of CFU/mouse. Male CBA/Ca mice were infected via i.p. with a lethal dose of S. typhimurium ATCC 14028 (1 × 102 CFU/mouse), followed by i.p. injection of peptide at 30 mg/kg. The number of survivors was monitored for 60 days and compared to that of control mice that only received the lethal bacterial challenge. The survival curves of untreated and peptide-treated mice are significantly different (p = 0.01); the mean survival time of control mice was 10 days, while the treatment of infected mice with Bac7(1-35) increased the mean survival time to 24.5 days. It is worth noting that 36% of the infected mice treated with Bac7(1-35) were completely cured with respect to 0% survival for untreated animals (p = 0.
Fifty micrograms of cellular proteins were separated by 8% polyacrylamide-SDS inconsecutive gel electrophoresis. The separated proteins were electrophoretically transferred to polyvinylidene difluoride membrane. Membranes were blocked with a 5% skim milk in Tris-buffered saline (TBS) containing
0.1% Tween 20 at room temperature for 1 h and then incubated with mouse anti-human monoclonal HIF-1α (Abcam, USA) at a 1:500 dilusion and P-glycoprotein (P-Gp) antibody (Abcam, USA) at a 1:200 dilusion overnight at 4°C, followed by goat anti-mouse IgG for 1 h at room temperature. Signals were detected with enhanced chemiluminescence (ECL plus, Amersham, USA). Microtubule protein (Tubulin, Abcam, selleck kinase inhibitor USA) at a 1:1000 dilution was used as internal control to observe the BIBF 1120 changes of HIF-1α and MDR-1 bands. Immunocytochemistry analysis of HIF-1α expression Cells grew on coverslips in 6-well culture dishes to approach 70% confluence; they were then treated with BSO and NAC as above description, following 4 h hypoxic treatment. After the medium was completely removed by suction, the cells were rinsed briefly
with phosphate buffer saline (PBS). Then, 4% Formaldehyde was used to fix the cells on coverslips for GSK2245840 10 min at room temperature, and then methanol fixed the cells for 10 min at -20°C. To utilize 0.5% TritonX-100 enhanced permeabilizations of the cells for 10 min at room temperature. The coverclips were pre-incubated with 3% hydrogen peroxide (H2O2)-methyl alcohol mix solution for 10 min to block endogenous peroxidase activity, followed by incubation for 30 min with block solution at room temperature. Cells were incubated with primary antibody, a mouse anti-human monoclonal HIF-1α antibody, at a 1:1300 dilution overnight at 4°C. Then cells were incubated with biotinylated secondary antibody, followed by a routine immunoperoxidase processing.
After washed twice with PBS, these coverslips were developed using diaminobenzidine (-)-p-Bromotetramisole Oxalate (DAB) as a chromogen, rinsed, gradient dehydrated by alcohol, and then mounted on slides. The coverslips without primary antibody treatment was regarded as the negative control. H-score values were used as a semi-quantitative evaluation for immunocytochemistry . Statistical analysis Data were reported as the means ± SEM of three separate experiments. Statistical significance was measured by independent sample t test and analysis of variance. A value of p < 0.05 was considered as statistically significant. Results Selection of sublethal concentration of BSO In order to select the appropriate concentration of BSO for the study, a 12 h dose-response study was conducted by exposing cells to different concentrations of BSO. Cell viability was measured by the MTT assay. The results showed that there was not significant decrease in viability over a 12 h exposure to BSO concentration ranging from 12.5 to 200 μM (Figure 1). In subsequent studies, the concentrations of BSO used were set at 50, 100, 200 μM.
In contrast to the effect of COX-2 on angiogenesis, the
effects on lymphangiogenesis and lymphatic metastasis remain poorly understood. Recently, some studies have found that COX-2 expression is highly correlated with lymph node metastasis [20, 21]. Several lines of experimental evidence have shown that COX-2 might stimulate VEGFR-3 to promote lymphangiogenesis by up-regulating VEGF-C in breast and lung cancer cells [22, 23]. selleck products However, the role of COX-2 in lymphangiogenesis of gastric carcinoma remains unclear. Using immunohistochemistry, our study aimed to detect the expression of COX-2 and VEGF-C protein and the levels of lymphatic vessel density Combretastatin A4 in vitro (LVD) in human gastric cancer and analyze their correlations with clinicopathological characteristics and prognosis. Methods Patients and specimens Fifty-six patients with
histologically proven gastric adenocarcinoma and who underwent radical gastrectomy JNJ-26481585 order at West China Hospital, Sichuan University, China between January 2001 and October 2002, were included in the present investigation. In this investigation, paracancerous normal mucosal tissues from 25 patients were collected as a control. Patients undergoing neoadjuvant chemotherapy and/or radiotherapy were excluded. TNM staging was carried out according to the American Joint Committee on Cancer (AJCC) classification, and historical grading was performed according to WHO criteria. Paraffin-embedded, formalin-fixed surgical specimens were prepared and collected for immunohistochemical staining. Immunohistochemical staining Specimens were immunostained with the standard labeled streptavidin-biotin protocol. Briefly, after deparaffinization and antigen retrieval, 4-μm tissue sections were incubated with COX-2 antibodies (monoclonal rabbit anti-human, 1:100, Goldenbridge Biotechnology Co, Ltd, Beijing, China) and VEGF-C antibodies (polyclonal rabbit anti-human, 1:100, Goldenbridge Biotechnology Co., Ltd) at 37°C for 1 h then at 4°C overnight. The sections were then incubated with biotinylated goat anti-rabbit immunoglobulin G (1:200, Zymed Laboratories Inc, USA) and subsequently incubated with horseradish
labeled streptavidin Alanine-glyoxylate transaminase (1:200, Zymed Laboratories Inc). 3,3′-Diaminobenzidine was used as a chromogen and hematoxylin as a counterstain. For the staining of lymphatic vessels, a rabbit anti-human D2-40 polyclonal antibody (rabbit polyclonal, Dako Denmark A/S Co., Denmark) was used. The procedure for immunohistochemical staining of D2-40 is similar to that of the COX-2 staining at a dilution of 1:100. Evaluation of immunohistochemical staining The immunohistochemical score (IHS) based on the German immunoreactive score was used for COX-2 and VEGF-C immunohistochemical evaluation . The IHS is calculated by combining the quantity score (percentage of positive stained cells) with the staining intensity score. The quantity score ranges from 0 to 4, i.e.
The transition towards smaller cell
size is controlled EVP4593 clinical trial What kind of disturbance of cell size homeostasis is induced by depletion of YgjD? We considered two possibilities. First, it is possible that the control that couples cell division to cell size is lost, so that cells divide in an uncontrolled way, irrespective of their size. Second, it is conceivable that cell division remains coupled to cell size, but the target size that a cell needs to reach before initiating division decreases over time. If the decrease in cell size is the result of a controlled transition towards smaller cells, one would expect that, during the transition, the cell elongation rate and the timing of cell division would still be linked, but that this link would change quantitatively
over time. In fact this is what we observed when we analyzed each generation of cells during the depletion process separately (inserts Figure 3a and 3b). Within a given generation the time interval between divisions and the rate by which a cell elongated was negatively correlated: cells that grew faster than the average of their generation tended to initiate division more quickly; cells that grew more slowly initiated division later. This suggests that cell growth Dorsomorphin manufacturer and the timing of cell division are still linked within each generation in the depletion process, but that this link changes quantitatively over successive generations. This analysis has, however, an important limitation: cells within a given generation check details are not independent from each other. Some of these cells are more closely related, because they derive from the same mother or grandmother. This can lead to spurious correlations
between traits; in our case, this effect could lead to artificial correlations between cell elongation rates and interdivision intervals. This problem of relatedness in lineage trees is known from phylogenetic studies, where it is referred to as phylogenetic dependence . In the https://www.selleckchem.com/products/blu-285.html context of phylogenetic studies, these dependencies can be resolved by analyzing differences between independent pairs of species, rather than calculating correlations on the basis of the whole phylogenetic lineage . We used a variation of this approach to get an unbiased view on the relationship between cell growth and the timing of cell division: for each generation, we analyzed pairs of cells emerging from the same cell division, and calculated the difference in growth rates and in the time to division for each pair. We refer to two cells emerging from the same division as ‘sisters’ (thereby ignoring that these two cells have cell poles of different ages, [22, 23]). The differences for all sister pairs represent independent data points, and we can use them to calculate the correlation between cell growth and time to division in an unbiased way.
De Boer et al.  demonstrated in vivo genetic exchange Adriamycin in vivo between C. jejuni strains coinfecting chickens. Phase variation via slip-strand mutagenesis in homopolymeric tracts has been demonstrated in a motility-related gene , a capsular synthesis gene , and a lipo-oligosaccharide (LOS) synthesis gene . In the latter case, phase variation results in switching the genes encoding the LOS structure between forms mimicking GM1 or GM2 gangliosides found in neural tissue; it is thought that the reaction with neural tissue of autoimmune antibodies directed against LOS molecules that mimic neural gangliosides
underlies the development of Guillain-Barré and Miller Fisher syndromes. Prendergast et al. demonstrated in vivo phase variation in the LOS genes in experimentally AZD3965 infected human volunteers . Evolutionary changes in pathogenicity of pathogens (i. e., increase or decrease in virulence) are thought to be the result of trade-offs between host mortality and probability of transmission to a new host, although immunopathology resulting from damage caused by the immune response may modulate the selective process [31–33]. Both host and pathogen genetic factors may be important
in the evolutionary process . Serial passage experiments that explore virulence evolution have usually resulted in increased pathogen-induced damage to the host [35, 36]. A few serial passage experiments have been conducted with C. jejuni. Fernández et al.  showed that serial intraperitoneal passage in mice of ten click here C. jejuni strains that could not invade HEp2 cells in culture restored and then enhanced this ability, but pathogenicity of the passaged strains in intestinal infections of mice was not determined. Chickens are commensally colonized by C. jejuni and
are an important reservoir for human infection. Ringoir and Korolik  showed that serial passage of four C. jejuni strains in chickens reduced the minimum infectious dose required for colonization. Jones et al.  showed for that passage of a poorly motile variant of C. jejuni 11168 in chickens increased the ability of this strain to colonize and persist in chickens; this change was accompanied by an increase in motility. Development of a murine model of C. jejuni infection in which C57BL/6 IL-10+/+ mice are colonized by C. jejuni 11168 while C57BL/6 IL-10-/- mice are both colonized and experience enteritis allowed us to explore the relationship between genetic variation in C. jejuni and disease expression in a model in which host genetic factors are close to identical and host environmental factors can be either standardized or varied in a controlled way . Our first hypothesis was that C. jejuni strains from humans, chickens, and cattle vary in their ability to colonize and cause enteritis in C57BL/6 IL-10-/- mice. Our second hypothesis was that serial passage of C.
Shake flask cultures were all performed in MSS medium containing heptakis(2,6-O-dimethyl)β-cyclodextrin [23, 24]. At 36 h, the production of PT was about doubled in strain Bp-WWD (3.77
± 0.53 μg/mL), compared with Bp-WWC (2.61 ± 0.16 μg/mL) and wild-type selleck chemicals llc Tohama (2.2 μg/mL) (Table 1), demonstrating that the level of PT expression was a function of the number of copies of the structural gene cluster. FHA in all three recombinant strains was about the same (Table 1). The production of PRN in shake flask cultures of Bp-WWC, Bp-WWD and Bp-WWE in MSS medium was analyzed by densitometry analysis of Western blot results. PRN amount in the clarified culture supernatants and extract of the separated cells at 60°C was assayed. The amount of PRN in cell extract of Bp-WWC and Bp-WWD was similar (2.48 ± 0.10 and 2.31 ± 0.17 μg/mL, respectively). A two-fold Selleck OSI-027 increase was found in Bp-WWE (4.18 ± 1.02 μg/mL), again showing a good correlation of the level of prn expression to the gene copy number. In all three
recombinant strains, the fraction of PRN found in the supernatant fraction in these flask cultures was small or negligible (less than 0.1 μg/mL, data not shown). Table 1 PT, FHA and PRN production by strains Bp-WWC and Bp-WWD and Bp-WWE Strain PT (μg/mL) FHA (μg/mL) PRN (μg/mL)** Tohama wt 2.2 ND* ND* Bp-WWC 2.61 ± 0.16 17.75 ± 3.30 2.48 ± 0.10 Bp-WWD 3.77 ± 0.53 14.33 ± 0.50 2.31 ± 0.17 Bp-WWE 4.49 ± 0.83 17.08 ± 2.21 4.18 ± 1.02 *ND = Not determined **The amount in cell extract The values were the mean of 3 independent Celastrol experiments with standard Pifithrin-�� chemical structure deviation except the data for PT of Tohama WT was obtained from two independent experiments Assessment of PT inactivation PT was purified from culture supernatants using a modification of the process published by Ozcengiz  where the initial ammonium sulphate precipitation was replaced by ligand exchange chromatography [26, 27]. The toxicity of the PT toxin from wild type B. pertussis and Bp-WWC (genetically inactivated PT) was analysed and compared by the Chinese hamster ovary (CHO) cell clustering assay
. This assay has a much higher sensitivity than other functional assays reported for PT. The native toxin purified from strain B. pertussis Tohama demonstrated a clustering endpoint at 2.6 pg per well. The genetically-inactivated PT did not promote clustering at the highest concentrations of 0.8-1.6 μg per sample obtained in this test (Figure 6). This assay can, therefore, detect toxicity reduction by a factor of 5 × 105 to 1 × 106, despite limitations imposed by the low solubility of PT. This result demonstrated that PT toxin purified from Bp-WWC was successfully inactivated by insertion of five nucleotide replacements resulting in two amino acid replacements in the PT subunit S1. Figure 6 CHO-cell clustering test.
DppV, a member of the dipeptidyl-peptidase family in A. fumigatus, is identical to one of the principal antigens used in the diagnosis of IA. Moreover, DppV can generate protection responses, and improve the survival rate of Aspergillus-infected mice . DppV can also bind with collagen or other human proteins and degrade them, which can damage the host. Recombinant DppV has shown a great potential in the serodiagnosis of IA in immunocompromised and immunocompetent patients . NAD-dependent malate dehydrogenase, a key enzyme in glycometabolism that catalyze the reversible conversion
between malate and oxaloacetate, was reported recently as an allergen of A. fumigatus and A. versicolor . Malate dehydrogenase was also shown to be a Paracoccidioides YM155 solubility dmso brasileinsis immunogenic protein  as well as a Candida albicans immunogen . Aspartyl aminopeptidase, an enzyme that specifically degrades only amino-terminal acidic amino acids from peptides, was recently reported as an antigen of A. fumigatus  . TR of A. fumigatus has been described as an extracellular antigenic protein by two recent studies [30, 31]. In one former
study, the secreted fraction of two geographically different strains (190/96 and DAYA) of A. fumigatus were used to identify new immunogenic selleck chemicals molecules reacting with pooled ABPA patient sera (IgG and IgE). TR was only detected on 2DE immunoblots of the secreted proteome of the DAYA strain probed with the IgE antibody fraction from pooled ABPA check details PtdIns(3,4)P2 patients sera . This result suggested that TR might not be a good biomarker for ABPA. In another study, the immunosecretome of A. fumigatus was detected using pooled patient sera (total n = 22 patients [ABPA, n = 11; aspergilloma, n = 5; IA, n = 6]). The immunoreactive intensity of TR was lower than most other proteins . A possible explanation is that the anti-TR antibody titers were not high in pooled sera because most cases included in the study were not IA. Although
investigators in other laboratories recently noted the antigenic nature of TR [30, 31], no study has found shown diagnostic value for TR in non-neutropenic patients with IA. We showed that TR (spot no. 2A-2 M) had the strongest immunoreactivity with patient sera. TR, a component of the gliotoxin biosynthetic cluster, provides self protection to A. fumigatus against gliotoxin [37, 38]. This protein has been described as an extracellular protein of A. fumigatus by Singh and Kumar [30, 31]. However, Schrettl et al. showed that GliT is preferentially localized in the cytoplasm and nuclei by a GFP-GliT construct . To predict whether or not GliT is actively secreted into the culture supernatant, we used two bioinformatic tools (SignalP and WoLF PSORT) to analyze its localization. Our results support the findings of Singh and Kumar [30, 31].
Similarly, anti-insect CP673451 price activity of crude ethanolic extracts from Streptomyces sp. in terms of larval mortality had been reported by Rishikesh et al. . The isolate showed a marked insecticidal activity against Sitophilus oryzae in a dose dependent manner with 100% mortality at concentration of 24 mg/ml. Later, Arasu et al.  documented 68.41% and 60.02% larvicidal activities by polyketide metabolite from Streptomyces sp. AP-123 against H. armigera and S. litura, respectively at 1000 ppm. Azadirachtin showed a more toxic effect towards S. litura
as compared to the crude extract of S. hydrogenans as 100% mortality was noticed at higher concentrations. Table 1 Influence of ethyl acetate extract of S. hydrogenans on and azadirachtin on various developmental parameters of S.litura Treatments Concentrations (μg/ml) Larval period (in days) (Mean ± S.E.) Pupal period (in days) (Mean ± S.E.) Total developmental period (in days) (Mean ± S.E.) AZD5582 cell line Streptomyces ethyl acetate extract 400 17.30 ± 0.19ab 10.36 ± 0.40ab 27.66 ± 0.40 800 19.97 ± 2.15ab 8.03 ± 0.76b 28.00 ± 0.93 1600 22.00 ± 2.11b – - f- value 3.30* 5.83** 0.62N.S R2 0.99 0.82 0.57 Azadirachtin 400 16.66 ± 0.33c 7.00 ± 0.36c – 800 – - – 1600 – - – f- value – - – R2 – - – Mean ± SE followed by different letters (superscript) with in a column are significantly different. Tukey’s test P ≤ 0.05, N.S = Non Significant, R2 = Coefficient of determination, *Significant
at 5% level, **Significant at 1% level. Table 2 Regression equation, lower as well PLK inhibitor as upper 95% confidence limits for LC 50 and LC 90 Regression equation 95% Confidence limit LC 50 LC 90 Lower Upper (μg/ml)
(μg/ml) Streptomyces ethyl acetate extract 1164.962a 1562.021a 1337.384 2070.516 Y = 6.751X-16.107 1729.403b 2989.165b 32.516c 363.252c 260.121 560.390 Azadirachtin Y = 3.866X-9.344 427.265d 1142.37d aLower and upper 95% confidence limits for LC50 for Streptomyces ethyl acetate extract, bLower and upper 95% confidence limits for LC90 Streptomyces ethyl acetate extract, cLower and upper 95% confidence limits for LC50 for azadirachtin, dLower and upper 95% Tolmetin confidence limits for LC90 for azadirachtin. Prepupal mortality (66.66%) was also higher at the highest concentration (P ≤ 0.01) (Table 3). Diet supplemented with extract of S. hydrogenans induced 48–100% pupal mortality. As compared to control, significantly higher mortality of more than 50% was recorded at highest concentrations (P ≤ 0.01) (Table 3). Similarly, dose dependent (125–1000 ppm) pupal mortality (18–62%) was reported by Arasu et al.  and documented that prolonged larval–pupal durations were directly proportional to the increase in pupicidal activities. The adverse effect of solvent extract was also observed on emergence and performance of adults emerged from treated larvae. Adult emergence was significantly lower when larvae were reared on diet amended with extract (P ≤ 0.
smegmatis (which was taken as a reference point to calculate fold change for all the strains) (Figure 4B).
In MSP1 glnA1 expression in low and high nitrogen conditions was up-regulated ~ 42 and ~ 15 fold respectively. The glnA1 expression in MSFP in high nitrogen was ~ 6 fold less than expression in low nitrogen while the same was only ~ 3 fold in MSP1. In case of MSP2, the expression of glnA1 gene was comparable in both low and high nitrogen conditions. In case of M. bovis, the expression of glnA1 was also ~ 36 fold up-regulated in low nitrogen conditions as compared to ~ 6.2 fold in high nitrogen conditions. Hence it was observed that in the strains, MSFP and M. bovis, where both the promoters P1 and P2 were present upstream to glnA1, the difference in the gene expression
levels PRN1371 mw in low and high nitrogen conditions were significantly higher as compared to the difference in expression levels in GSK126 mw strains having single promoter. It was concluded that deletion of any one of the two promoters decreased the stringent regulation of glnA1 gene at the transcriptional level. GS specific activity and expression in response to nitrogen limitation and excess Response selleckchem to nitrogen availability for GS enzyme was studied by measuring cellular GS activity by γ-glutamyl transferase assay . Exponential phase culture of MSFP, MSP1, MSP2, wild type M. smegmatis and M. bovis was harvested and cell pellet of 10 ml culture was further used for determining intracellular GS activity. Upon exposure to the nitrogen limiting conditions, the cellular GS activity in M. bovis, MSFP, MSP1 and MSP2 was 9.16, 12, 4.4 and 5 times higher than the high nitrogen condition respectively. Intracellular GS activity for all strains grown in high nitrogen condition was much less as compared to the activity in low nitrogen conditions (Figure 5B). Intracellular GS specific activity in MSP2 strain was 1 U/mg in low nitrogen and 0.2 U/mg in high nitrogen condition which was much less as compared to GS activity in MSFP and
MSP1 strain. The GS activity in extracellular fraction followed the same trend in all strains (Figure 5B). Western blotting of the intracellular protein fraction was done by using anti-GS antibodies (Figure 5A). Fluorometholone Acetate It was observed that in all strains the GS expression was higher in low nitrogen condition than high nitrogen condition. Although it was observed from western blotting result that the amount of GS in low nitrogen condition of MSP2 was very less but the activity of the enzyme was relatively higher than the activity of the enzyme in high nitrogen conditions of all the strains. This is in accordance with earlier findings that in high nitrogen conditions GlnE protein adenylylates the GS protein at a conserved tyrosine residue and hence, the enzyme becomes inactive.