The ΔacfB fragment was digested with BamHI and EcoRI and the ΔtcpI∷Cm fragment was digested with KpnI, and then each was ligated into pKAS32 (Skorupski & Taylor, 1996), which was digested appropriately to generate pKEK870 and pKEK1117, respectively. The expression plasmid containing acfB was created by PCR amplification using

the oligonucleotides acfBMet and acfBXbaI. The PCR fragment was digested with XbaI and ligated into pBAD24 (Guzman et al., 1995) that had been digested with NcoI, treated with Klenow fragment to fill in the 5′ overhand, and then digested with XbaI, to form pKEK149. The expression plasmid containing tcpI was created by PCR amplification with oligonucleotides tcpI F BamHI and tcpI R EcoRI, followed by digestion with BamHI and EcoRI, and ligation into pWSK30 (Wang & Kushner, 1991) digested similarly to form pKEK1306. Table 1 contains a list of the bacterial strains Selleck Alectinib used in this study. Escherichia coli strain DH5α (Hanahan, 1983) was used for all cloning experiments, while the E. coli strain WM3046 (a gift from William Metcalf, University of Illinois) was used to transfer plasmids to V. cholerae by conjugation. The ΔacfB, ΔtcpI∷Cm, and ΔcheY-3 V. cholerae strains KKV2089, KKV2060, and KKV2090 were constructed as described previously (Skorupski & Taylor, 1996) by mating pKEK870, pKEK1117, and pSB27, respectively, into

V. cholerae strain O395. The ΔacfB, ΔtcpI∷Cm strain KKV2061 was constructed by CPT1ts transduction (Hava & Camilli, 2001) of ΔtcpI∷Cm

into strain KKV2089. The correct construction Selleck BI 6727 of all strains was verified by PCR and sequencing. CT in the culture supernatants was measured using a GM1-enzyme-linked immunosorbent assay with rabbit polyclonal antiserum against the purified B subunit of CT (Svennerholm & Holmgren, 1978). TCP was measured by CTXφ-Kan phage transduction (Waldor & Mekalanos, 1996). The in AMP deaminase vivo colonization assays were performed as described by Gardel & Mekalanos (1996) using 5–6-day-old CD-1 suckling mice. The inocula consisted of ∼105 CFU for both wild-type and mutant strains, and intestines were harvested 22 h postinoculation. For strains carrying pKEK149, inocula also contained 0.1% arabinose. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the University of Texas, San Antonio. Within the VPI lie the acfB (VC0825) and tcpI (VC0840) genes, which are predicted to encode putative MCPs (Everiss et al., 1994; Harkey et al., 1994) (Fig. 1). The tcpI gene is in a single gene operon divergently transcribed from the regulatory genes tcpPH, while the acfB gene lies within an operon downstream of toxT and tcpJ. Both AcfB and TcpI have been demonstrated to be positively regulated by ToxT (Peterson & Mekalanos, 1988; DiRita et al.

The ΔacfB fragment was digested with BamHI and EcoRI and the ΔtcpI∷Cm fragment was digested with KpnI, and then each was ligated into pKAS32 (Skorupski & Taylor, 1996), which was digested appropriately to generate pKEK870 and pKEK1117, respectively. The expression plasmid containing acfB was created by PCR amplification using

the oligonucleotides acfBMet and acfBXbaI. The PCR fragment was digested with XbaI and ligated into pBAD24 (Guzman et al., 1995) that had been digested with NcoI, treated with Klenow fragment to fill in the 5′ overhand, and then digested with XbaI, to form pKEK149. The expression plasmid containing tcpI was created by PCR amplification with oligonucleotides tcpI F BamHI and tcpI R EcoRI, followed by digestion with BamHI and EcoRI, and ligation into pWSK30 (Wang & Kushner, 1991) digested similarly to form pKEK1306. Table 1 contains a list of the bacterial strains buy Docetaxel used in this study. Escherichia coli strain DH5α (Hanahan, 1983) was used for all cloning experiments, while the E. coli strain WM3046 (a gift from William Metcalf, University of Illinois) was used to transfer plasmids to V. cholerae by conjugation. The ΔacfB, ΔtcpI∷Cm, and ΔcheY-3 V. cholerae strains KKV2089, KKV2060, and KKV2090 were constructed as described previously (Skorupski & Taylor, 1996) by mating pKEK870, pKEK1117, and pSB27, respectively, into

V. cholerae strain O395. The ΔacfB, ΔtcpI∷Cm strain KKV2061 was constructed by CPT1ts transduction (Hava & Camilli, 2001) of ΔtcpI∷Cm

into strain KKV2089. The correct construction 17-AAG of all strains was verified by PCR and sequencing. CT in the culture supernatants was measured using a GM1-enzyme-linked immunosorbent assay with rabbit polyclonal antiserum against the purified B subunit of CT (Svennerholm & Holmgren, 1978). TCP was measured by CTXφ-Kan phage transduction (Waldor & Mekalanos, 1996). The in Urease vivo colonization assays were performed as described by Gardel & Mekalanos (1996) using 5–6-day-old CD-1 suckling mice. The inocula consisted of ∼105 CFU for both wild-type and mutant strains, and intestines were harvested 22 h postinoculation. For strains carrying pKEK149, inocula also contained 0.1% arabinose. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of the University of Texas, San Antonio. Within the VPI lie the acfB (VC0825) and tcpI (VC0840) genes, which are predicted to encode putative MCPs (Everiss et al., 1994; Harkey et al., 1994) (Fig. 1). The tcpI gene is in a single gene operon divergently transcribed from the regulatory genes tcpPH, while the acfB gene lies within an operon downstream of toxT and tcpJ. Both AcfB and TcpI have been demonstrated to be positively regulated by ToxT (Peterson & Mekalanos, 1988; DiRita et al.

digitatum have been limited. Mitochondria are generally accepted as having a common origin and play an important role in phylogenetic studies. Many programmes,

such as the Fungal Mitochondrial Genome Project (Paquin et al., 1997), have significantly increased the data on fungal mitochondria and more than 80 complete fungal mitochondrial genomes are available in NCBI (www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=4751&opt=organelle). Information acquired from mitochondria, e.g. gene content and arrangement, exon–intron structure, as well as molecular phylogeny based on single or concatenated mitochondrial protein sequences, has largely increased our knowledge of Penicillium and its closely related Aspergillus species (Woo et al., Caspase inhibitor 2003; Juhasz et al., 2004, 2008). However, phytopathogenic Penicillium species have not been well described.

To reveal the mechanisms of molecular plant–pathogen interactions, whole genome sequencing of P. digitatum has been initiated in our laboratory. Here we reveal the mitochondrial genome and use comparative analysis to further confirm the species’ evolutionary degree, and to explore polymophism in Penicillium mitochondrial genomes. Penicillium digitatum strain Pd01 was isolated from green mould diseased citrus fruit collected in Zhejiang province, China, in 2000. It was maintained on potato selleck screening library dextrose agar medium at 4 °C. The mycelium of Pd01 was cultured in potato dextrose broth in a rotary shaker at 150 g at 25 °C for 4 days. Fresh harvested Pd01 mycelia (100 mg) were homogenized in a mortar precooled with liquid nitrogen.

The subsequent powder was transferred to a 2-mL Eppendorf tube, then incubated at 65 °C for 1 h after adding 0.8 mL however CTAB buffer (1% CTAB, 1 M NaCl 100 mM Tris, 20 mM EDTA; 1% polyvinyl polypyrolidone and 1% β-mercaptoethanol). Thereafter, 0.8 mL chloroform/isoamyl alcohol (24 : 1, v/v) was added to the tube. After being vortexed for 10 min, the mixture was centrifuged at 14 000 g for 10 min. The aqueous phase was transferred to a new tube, and extracted with a mixture of equal volume of phenol/chloroform for 1 min, and centrifuged at 4000 g for 10 min. The extraction was repeated twice. The supernatant was transferred to a new tube containing isopropanol (two-thirds the volume of supernatant), then gently mixed at room temperature for 10 min, and centrifuged at 14 000 g for 10 min. After pouring off and being dried in air, the obtained pellet was suspended in 0.3 mL TE buffer (50 mM Tris-HCl, 10 mM EDTA, pH 8.0), then stored at −20 °C for 1 h after adding 0.2 mL 5 M NaCl and 1 mL frozen ethanol. The obtained DNA was precipitated by centrifugation at 14 000 g for 15 min and washed twice with 75% ethanol, then re-suspended in 50 μL TE buffer. The DNA was qualified and quantified by agar electrophoresis and spectrophotometrics, as described by Sambrook & Russell (2001).

From 1995 to 1999, HIV-2 infection was more frequently found in female patients (64; 67.4%). Portugal was the country of birth of 54.7% of individuals. Cases attributed to transfusions declined to 10.5%, while those attributed to heterosexual intercourse increased INNO-406 clinical trial to 65.3%. Three cases of vertical transmission were diagnosed, while for 17 patients (17.9%) the mode of transmission was not specified. During this period, 63.2% (60) of the diagnoses were made in hospitals located in the south of the country. From January 2000 to December 2004, 127 additional patients were identified. Most

cases were still among female patients (84; 66.1%). The major differences from the previous periods were the patients’ country of origin and residence area, with the majority (77; 60.6%) coming from West African countries and being diagnosed in Lisbon (100; 78.7%). Heterosexual intercourse remained the primary mode of HIV-2 acquisition (75; 59.1%) while blood transfusions almost

disappeared as a cause of infection (6; 4.7%). In 31.5% of cases the route of transmission was not specified. Most patients had no AIDS-defining illness at diagnosis (80; 63.0%), although the stage at diagnosis was not possible to ascertain for 20 patients (15.7%). In the last three years of the study period (2005–2007), 73 additional patients were diagnosed with HIV-2 infection: 39 women and 34 men. The average age CP-868596 cost at diagnosis was

higher than in the previous periods (43.0 years for women and 48.7 years for men). West African origin was reported for 64.4% of patients (47), while 23.3% (17) were Portuguese. More than 80% of the diagnoses were made at one of the participant hospitals located in Lisbon. Most patients were Interleukin-2 receptor infected heterosexually (39; 53.4%) and only 4.1% through blood transfusions. No case of vertical transmission was documented. However, the mode of transmission was not specified for 30 patients (41.1%). This sample of 442 HIV-2-infected patients is the largest sample of HIV-2-infected patients ever described. The sample represents 37% of all HIV-2 (mono)infections notified in Portugal as of the end of 2007 and includes patients from hospitals that cover a wide geographical area. The proportion of cases identified over each time period resembles the pattern observed for notified cases and the sample is representative of the transmission dynamics of HIV-2 in the country (Table 2). From 1985 to 2007, HIV-2-infected patients included in the sample presented distinct characteristics according to the period of diagnosis. Until 2000, the majority of HIV-2-infected patients were Portuguese-born men living in the north of the country, but from 2000 to 2007 most patients diagnosed with HIV-2 infection had a West African origin, were predominantly female and were living in the capital, further south.

In general, children who have been fully vaccinated before there is evidence of immunocompromisation should be tested for vaccine antibody levels LDK378 in vitro when primary vaccination and booster doses have been completed, i.e. at around 4–6 years of age. Those who were vaccinated when they had any degree of immunosuppression should have specific immunity re-checked after approximately 5 years (at age 9–11 years) and again 5 years later, before transfer to adult care (at age 14–16 years). Accepted

cut-off protective titres for vaccine-preventable diseases [40, 106-113] are suggested (Table 3), acknowledging that the evidence base is limited in some areas. Assays that meaningfully reflect the level of individual protection are not routinely available for all vaccines and, when available, defined levels of protection may not be relevant to HIV-positive individuals. > 10 IU/L protective > 100 IU/L optimal A further challenge is how to vaccinate those children whose vaccine status is unknown or incomplete, including

children from other countries. Unless a reliable vaccine history is available, individuals should be assumed to be unimmunized and a full course of immunization should BTK inhibitor mouse be planned. In the absence of vaccination details, serology provides partial guidance on their immunization status but prevents assessment of the durability of seroprotection or the capacity for anamnestic responses. The following guidance addresses catch-up immunization priories. HBV: measure serology

and offer a complete series to susceptible children (three doses), ideally using combined HAV and HBV vaccine. Figure 1 is an algorithm for immunizing HIV-infected children with uncertain or incomplete immunization Cediranib (AZD2171) status; this schedule is based on the routine vaccine schedule and formulations currently available in the UK and on guidance provided by the UK Health Protection Agency. The schedule can be modified according to local schedules and availability. It should be noted (as discussed in section 5) that the use of PPV23 is controversial and is not included in this guidance. PCV should be considered for previously unimmunized children over 5 years of age, ensuring that 2 doses are given at least 2 months apart. Even after normalization of the CD4 cell count on HAART, vaccine responsiveness may be inadequate because of pre-existing and irreversible immune impairment, given that responsiveness to vaccination is related to the nadir CD4 cell count for some vaccines [114]. Moreover, impaired B-cell memory responses persist despite effective HAART [115, 116]. A suboptimal response to primary vaccinations and a requirement for additional reinforcing doses of vaccine should be anticipated and, if the patient was severely immunocompromised when primary vaccination courses were administered, then complete revaccination after immune recovery on HAART should be the standard of care.

2a and c), while for the GST fusion proteins, FpdNK + GST and PddNK + GST, it was around 50 kDa, because of the GST part (Fig. 2b and d). Kinetic parameters were determined for purified recombinant PdTK1, PddNK + GST, FpTK1, and FpdNK + GST (Table 1).

The activity was measured at 37 °C, except for FpTK1 and PdTK1, which were tested at 21 °C. All enzyme reactions followed classical Michaelis–Menten kinetics. The FpTK1 specifically phosphorylated dT and dU, having dT as the preferred substrate, because the Km for dT was 2.2 μM, which is ~ 64 times lower signaling pathway than for dU. The apparent maximal velocity (Vm-app) was 5.78 U mg−1 for dT and 4.64 U mg−1 for dU (Table 1, Fig. S2a and b). Similarly, also PdTK1 preferred dT as substrate over dU, with Km for dT being 32 μM, which is ~ 27 times lower than for dU. The Vm-app for dT was 3.43 and 2.11 U mg−1 for dU (Table 1, Fig. S2e and f). The catalytic efficiency (Vm-app/Km) was manyfold higher for FpTK1 than for PdTK1, and the activity with dT was higher for both enzymes. This indicates that FpTK1 has higher specificity toward dT and dU (Table 1). We could not detect any significant phosphorylation of dA, dC, or dG by either FpTK1 or PdTK1. The FpdNK was able to phosphorylate both dA

and dC, but had dA as the preferred substrate, with the Km for dA being 4.5 times lower than for dC. The catalytic efficiency (Vm-app/Km) was manyfold higher for dA than for dC, indicating that dA was the preferred substrate (Table 1, Fig. S2c and d). Also PddNK preferred dA as substrate over dC, the catalytic efficiency being almost 6-fold higher for dA than for dC (Table 1, Fig. S2g and h). In short, both GSI-IX non-TK1 kinases were much more Rapamycin in vivo specific

for dA than for dC, and none of them was able to phosphorylate dG. Initially, attempts to measure the substrate specificity of pure recombinant PdTK1 and FpTK1 at 37 °C failed; therefore, we decided to determine PdTK1 phosphorylating activity as a function of temperature, by measuring the activity at 500 μM 3H-dT and 2.5 mM ATP, at different temperatures, and with prolonged sampling times. It turned out that the activity of PdTK1 increased with temperature, up to 21 °C, where the highest activity was detected (7.77 ± 1.56 U mg−1); thereafter, the activity decreased with increasing temperature (Fig. 3). Therefore, 21 °C was used for further investigation of PdTK1 and FpTK1. Upon pre-incubating the enzyme at 0 °C for one hour, the obtained activity at 21 °C was considerably lower (0.71 ± 0.04 U mg−1), and after pre-incubation at 37 °C for one hour, the enzyme was irreversibly denatured, because we could not detect any activity at all. One of the approaches to estimate the aquatic bacteria biomass production is the incorporation of 3H-dT into newly synthesized DNA, and it is based on the assumption that all actively growing bacteria can incorporate external dT into DNA (Furhman & Azam, 1982).

2a and c), while for the GST fusion proteins, FpdNK + GST and PddNK + GST, it was around 50 kDa, because of the GST part (Fig. 2b and d). Kinetic parameters were determined for purified recombinant PdTK1, PddNK + GST, FpTK1, and FpdNK + GST (Table 1).

The activity was measured at 37 °C, except for FpTK1 and PdTK1, which were tested at 21 °C. All enzyme reactions followed classical Michaelis–Menten kinetics. The FpTK1 specifically phosphorylated dT and dU, having dT as the preferred substrate, because the Km for dT was 2.2 μM, which is ~ 64 times lower PTC124 than for dU. The apparent maximal velocity (Vm-app) was 5.78 U mg−1 for dT and 4.64 U mg−1 for dU (Table 1, Fig. S2a and b). Similarly, also PdTK1 preferred dT as substrate over dU, with Km for dT being 32 μM, which is ~ 27 times lower than for dU. The Vm-app for dT was 3.43 and 2.11 U mg−1 for dU (Table 1, Fig. S2e and f). The catalytic efficiency (Vm-app/Km) was manyfold higher for FpTK1 than for PdTK1, and the activity with dT was higher for both enzymes. This indicates that FpTK1 has higher specificity toward dT and dU (Table 1). We could not detect any significant phosphorylation of dA, dC, or dG by either FpTK1 or PdTK1. The FpdNK was able to phosphorylate both dA

and dC, but had dA as the preferred substrate, with the Km for dA being 4.5 times lower than for dC. The catalytic efficiency (Vm-app/Km) was manyfold higher for dA than for dC, indicating that dA was the preferred substrate (Table 1, Fig. S2c and d). Also PddNK preferred dA as substrate over dC, the catalytic efficiency being almost 6-fold higher for dA than for dC (Table 1, Fig. S2g and h). In short, both click here non-TK1 kinases were much more second specific

for dA than for dC, and none of them was able to phosphorylate dG. Initially, attempts to measure the substrate specificity of pure recombinant PdTK1 and FpTK1 at 37 °C failed; therefore, we decided to determine PdTK1 phosphorylating activity as a function of temperature, by measuring the activity at 500 μM 3H-dT and 2.5 mM ATP, at different temperatures, and with prolonged sampling times. It turned out that the activity of PdTK1 increased with temperature, up to 21 °C, where the highest activity was detected (7.77 ± 1.56 U mg−1); thereafter, the activity decreased with increasing temperature (Fig. 3). Therefore, 21 °C was used for further investigation of PdTK1 and FpTK1. Upon pre-incubating the enzyme at 0 °C for one hour, the obtained activity at 21 °C was considerably lower (0.71 ± 0.04 U mg−1), and after pre-incubation at 37 °C for one hour, the enzyme was irreversibly denatured, because we could not detect any activity at all. One of the approaches to estimate the aquatic bacteria biomass production is the incorporation of 3H-dT into newly synthesized DNA, and it is based on the assumption that all actively growing bacteria can incorporate external dT into DNA (Furhman & Azam, 1982).

Conclusions.  Orthodontic treatment carries a higher risk of mucosal lesions and implies greater awareness of better oral hygiene as shown by the results of this study. Oral hygiene instructions and early treatment of oral lesions are important considerations in better patient’s motivation, treatment planning, and successful outcome. “
“International Journal of Paediatric Dentistry 2013; 23: 131–137 Aim.  To estimate the prevalence, intensity and associated factors of dental pain in 7- and 8-year-old schoolchildren in a Southern Brazilian city. Design.  A cross-sectional study was carried out involving a representative sample (n = 401) of schoolchildren of Tubarão, Brazil. The data were

obtained through oral examinations, following WHO criteria. Dental pain was analysed using a specific questionnaire developed to measure Selleck 17-AAG it. Prevalence and intensity of spontaneous pain and pain caused by cold and hot food and liquids were analysed. Association studies were carried out using chi-square test followed by nonconditional multiple logistic regression analysis to test for independence of association between outcomes and explanatory variables. Sirolimus solubility dmso Results.  The prevalence of spontaneous dental pain and dental pain caused by cold and hot food and liquids was 31.7 and 28.1%, respectively. Females and schoolchildren who had visited the dentist at least once showed statistically higher prevalence of spontaneous pain and pain caused by cold and

hot food and liquids. Eight-year-old schoolchildren

and those presenting cavities in the primary dentition also showed higher prevalence of spontaneous dental pain. Conclusions.  The prevalence and intensity of dental pain were considered high. The prevalence showed to be associated with female gender, higher age, the presence of cavities in the primary dentition and dental visit. “
“International Journal of Paediatric Dentistry 2011; 22: 17–26 Background.  Pain following the extraction of the primary canine in children with palatally displaced canines (PDC) as an interceptive treatment has not been investigated. Galactosylceramidase Aims.  To describe pain, discomfort, dental anxiety, and use of analgesics following the extraction of primary canines in children with PDC. Design.  Forty-four children, aged 10–13 with PDC, were included. Pain intensity, discomfort, and analgesic consumption were rated the first evening and 1 week after the extraction of the primary canine. Dental anxiety was assessed pre-extraction, using the dental anxiety scale (DAS). A matched reference group also completed the DAS. Results.  No significant differences were found between the study and the reference group regarding the pre-extraction assessments. Post-extraction pain and discomfort was low. The experience of the injection was graded worse than the extraction, and more pain was rated at the evening post-extraction than during the extraction. Analgesics were used only the first evening.

HRQL assessment has become one of the most widely used subjective health evaluations in chronic illness. Life experiences of HIV-infected people are as heterogeneous as the population affected. HRQL assessment in these patients provides valuable information about the effects of ART, disease progression and prognosis, and the factors that influence prognosis; results that clinical analysis is unable to provide. It must be taken into account that the evaluation of HRQL by the patient does OSI744 not necessarily coincide with the severity of the illness as defined by the patient’s doctor. HRQL provides valuable information for health care managers

and authorities, as it allows evaluation of the efficiency, effectiveness and cost–benefit ratio of health care programmes, and for pharmaceutical companies that gather data on effectiveness, clinical benefit, satisfaction with treatment and treatment adherence [9–11]. The literature shows the importance of factors most closely related to HRQL in HIV-infected people. These factors are psychological aspects and sociodemographic characteristics, clinical indicators unrelated to the infection and the individual illness [6,12–15]. HRQL in the HIV-infected population has not previously been investigated

in our region, and so the aim of this study was to determine the impact of various sociodemographic, clinical and psychological factors on HRQL in an HIV-infected population receiving care at the HIV clinic of a tertiary Spanish Osimertinib hospital, Arachidonate 15-lipoxygenase and to identify variables that allow us to establish a predictive model to evaluate HRQL in this population and these patients’ overall perception of their health status. A cross-sectional study

was conducted in HIV-infected patients under follow-up at the Río Hortega University Hospital in Valladolid (Spain). The target population comprised individuals with HIV infection who agreed to participate in the study in the period March 2007 to April 2008. Exclusion criteria were: (a) recent diagnosis with HIV infection (less than 6 months ago); (b) age <16 years; (c) the patient not being frequently seen by our specialists; (d) refusal to participate in the study; (e) a physical or mental condition that made interviewing the patient problematic. Nine persons refused to participate in the study (six men and three women) and did not sign the medical consent form; these patients were not a homogeneous group in terms of sociodemographic, epidemiological or clinical characteristics. Following consultation with the Investigation Department, a total of 150 out-patients were consecutively selected after they had signed the medical consent form according to the principles of the Declaration of Helsinki.

Fig.

S2. Nucleotide sequences of tclipG (GenBank accession no. AB237774). Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Bacteria often have multiple copies of ribosomal RNA (rrn) genes in their genomes. The presence of multiple rrn operons suggests an advantage to the organism, perhaps Selleck Lumacaftor through adjustable control of protein expression in response to altered environmental conditions. In the work described here, the strengths of the seven rRNA promoters of Pseudomonas sp. UW4 were individually assessed by separately cloning each promoter region into an expression vector and monitoring the activity of the reporter protein, the Escherichia coli lacZ gene product. The lacZ expression was the highest for the rrnE promoter under all growth conditions, with the various promoters demonstrating a range of strengths. These findings indicate that these promoters are not functionally identical. This observation suggests that the differential expression of rrn operons under various physiological conditions and growth stages allows better regulation

of rRNA, conferring an advantage to P. sp. UW4 through a more fine-tuned control of protein expression in a wide range of environmental situations. “
“Nitrogenase produces hydrogen as a normal byproduct of the reduction of dinitrogen to ammonia. The Nif2 nitrogenase in Anabaena variabilis is an alternative Mo-nitrogenase and is expressed in vegetative cells grown with fructose Selleckchem Vorinostat under strictly anaerobic conditions. We report here that the V75I substitution in the α-subunit of Nif2 showed greatly impaired acetylene reduction and reduced levels of 15N2 fixation but had similar hydrogen production rates as the wild-type enzyme under argon. Another mutant containing a substitution in the α-subunit, V76I, would result in a decrease in the size of the putative gas channel of nitrogenase and, thus, was hypothesized to affect substrate selectivity of nitrogenase.

However, this substitution Calpain had no effect on the enzyme selectivity, suggesting that access by gases to the active site through this putative gas channel is not limited by the increased size of the amino acid side chain in the α-subunit, V76I substitution. Hydrogen produced from photosynthetic microorganisms such as cyanobacteria is an attractive biofuel because it is made from water using sunlight as the energy source. In filamentous cyanobacteria, the primary enzyme used to produce H2 is nitrogenase, which reduces H+ to H2 as part of the mechanism of reduction of N2 to ammonia (Tamagnini et al., 2007). Hydrogen production by nitrogenase is not dependent upon the reduction of N2; in an argon atmosphere, nitrogenase produces only H2 (Benemann & Weare, 1974; Barney et al., 2004).