, 2007; Pandhal et al., 2007). iTRAQ was chosen as this technique has a clear advantage over more conventional proteomic methods through conferring reproducibility and statistical confidence to the measurements SB431542 order of protein abundance

within a cell at a fixed point in time (Ross et al., 2004; Gan et al., 2007). For a complete description of the Materials and methods refer to the Supporting Information Appendix S1; in brief, however, P. marinus strain MED4 was grown in biological triplicate under two separate conditions: P-deplete and P-replete PCR-S11 media. The cells were harvested at the same time in the late exponential phase (after 10 days), and proteins were extracted (Meijer & Wijffels, 1998). Approximately 100 μg of protein from each replicate was then reduced, alkylated, digested and labelled with 8-plex iTRAQ reagents according to the manufacturer’s (Applied Biosystems, Framingham, MA) protocol. The replicates were then pooled before primary strong cation exchange (SCX)

fractionation (Pandhal et al., 2007). Mass spectrometeric analysis of the SCX fractions was performed using both a HCTUltra ESI TRAP MS/MS (Bruker Daltonics GmbH, Bremen, Germany) and a QStar XL Hybrid ESI Quadrupole time-of-flight tandem mass spectrometer, ESI-qQ-TOF-MS/MS (Applied Biosystems; MDS-Sciex, Concord, Ontario, Canada), coupled with an online capillary liquid chromatography system (Ultimate 3000, Dionex/LC Packings, the Netherlands) (Pandhal et al., 2007). Preliminary data analysis, peptide identification and quantification were carried out using the phenyx [Geneva Bioinformatics (GeneBio), Geneva, Switzerland] software. Ninety-eight proteins Selleckchem Antidiabetic Compound Library were identified by ≥1 peptides [coefficient of variation (CV)=1.07] and eight false positives were identified [false positive rate (FPR)=0.016]. However, for accurate determination of protein identification, ≥2 peptides are required. With this restriction, 68 proteins were identified (CV=1.05), with three false positives (FPR=0.05), with quantification only possible for 62 of the identified proteins. For a full list

of identified proteins, see Supporting Information, Table S1. This figure, while lower than other iTRAQ experimental Edoxaban data of other cyanobacteria, such as Synechocystis sp. PCC6803 (Gan et al., 2007) and Nostoc sp. (Ow et al., 2009a), shows a broad coverage across the chromosome for MED4 (Fig. 1). It is also similar to the only other iTRAQ shotgun proteomic experiment conducted on MED4, where 70 proteins were identified by ≥2 peptides (Pandhal et al., 2007). Also, there was a significant bias towards identification of particular proteins within the results, where 75% of the peptides identified only mapped to 19% of the identified proteins (Table S1). This strongly suggests that the cell’s proteome, particularly under P-stressed conditions, is dominated by a small number of these particular proteins.

β-Galactosidase activity due to the core 16S/23S rRNA gene promoter in Sulfolobus was 1.7–3-fold lower in the stationary phase than in exponential growth (Fig. 3). The pattern of β-galactosidase activity did not change significantly when normalized for

the absolute copy number of the lacS gene by qPCR, indicating that the increase in activity in exponential growth Selleck Target Selective Inhibitor Library was due to regulation of the 16S/23S rRNA gene promoter, not gene dosage (Fig. 3b). The 42-bp 16S/23S rRNA gene core promoter is the smallest reported regulated promoter for Sulfolobus. These findings are consistent with evidence of upregulation of rRNA transcription during exponential growth in E. coli and Saccharomyces cerevisiae (yeast) (Nomura, 1999) and with microarray data from halophilic archaea showing that ribosomal protein gene transcription is higher during exponential growth than in the stationary phase (Lange et al., 2007). Moreover, rRNA in crude preparations from Natronococcus occultus decreases in the stationary phase (Nercessian

& Conde, 2006). The mechanism for core rRNA promoter regulation in S. solfataricus is obscure. The decrease in β-galactosidase activity observed during the stationary phase may be due to growth rate-dependent transcriptional regulation or stringent control in response to decreasing nutrient availability and/or charged tRNAs. The latter has been AZD4547 datasheet shown to decrease total stable RNA accumulation in Sulfolobus (Cellini et al., 2004). As in E. coli and yeast, it is likely that there are multiple mechanisms contributing to regulation of the Sulfolobus 16S/23S rRNA gene operon. There is considerable

evidence that archaeal transcriptional regulators interact with core promoters, either binding between or overlapping the TATA box and the transcriptional start site (Peng et al., 2011). In vivo and in vitro analyses have determined several regulatory regions and the start site of the 16S/23S rRNA gene in S. shibatae Dolutegravir manufacturer (Hudepohl et al., 1990; Reiter et al., 1990; Hain et al., 1992; Qureshi et al., 1997). The core promoter sequences necessary for transcription initiation in vitro are between −38 and −2 bases relative to the transcription start, identical to those used here in vivo. This region encompasses the proximal promoter element (PPE) (an AT-rich sequence −11 to −2 conserved in Sulfolobus stable RNA promoters), the TATA box, and several bases upstream thereof (Reiter et al., 1990), later identified as a transcription factor B (TFB) recognition element (BRE) (Qureshi & Jackson, 1998). A weak positive regulatory region between −354 and −190 and a negative regulatory region between −93 and −38 were also found (Reiter et al., 1990).

CD4 cell count (cells/μL) HBV requiring treatment* HBV not requiring treatment HCV with immediate plan to start HCV treatment* HCV with no immediate plan to start HCV treatment *See BHIVA

guidelines for the management of hepatitis viruses in adults infected with HIV 2013 [31] for indications to treat hepatitis B and C We recommend patients with HIV and hepatitis B virus coinfection who have a CD4 cell count <500 cells/μL are treated with fully suppressive ART inclusive of anti-HBV active antivirals (1B). We recommend patients with HIV and HBV coinfection who have a CD4 cell count ≥500 cells/μL and who have an HBV-DNA ≥2000 IU/mL and/or evidence of more than minimal fibrosis (Metavir ≥F2) are treated with fully suppressive ART inclusive

of anti-HBV active antivirals (1C). Proportion of patients with a CD4 cell count ≥500 cells/μL and an HBV DNA ≥2000 IU/mL BAY 73-4506 and/or evidence of more than minimal TSA HDAC fibrosis commencing ART inclusive of anti-HBV antivirals. Rationale. Because of the negative effect of immune depletion on HBV disease progression, the availability of single drugs with high level dual hepatitis B and HIV antiviral activity, and the increased risk of liver-related deaths in patients with CD4 cell counts ≥500 cells/μL, coinfected patients with active HBV disease (HBV viral load ≥2000 IU/mL or Metavir F2 or above) and those with CD4 cell counts below 500 cells/μL should start ART inclusive of anti-HBV active antivirals [2]. Patients Venetoclax research buy with CD4 cell counts ≥500 cells/μL and HBV DNA of <2000 IU/mL, minimal or no evidence of liver inflammation or fibrosis, and a repeatedly normal ALT should be given the option to commence treatment or defer and be monitored not less than 6-monthly with HBV DNA and ALT and at least yearly for evidence of fibrosis.

For more information on the indications to start treatment for hepatitis B infection please refer to the BHIVA guidelines for the management of hepatitis viruses in adults infected with HIV 2013 [31]. We recommend TDF/FTC as part of a fully suppressive ART combination should be given to all patients where HBV treatment is deemed necessary (1C). We recommend neither 3TC nor FTC be used as the sole active drug against HBV in ART due to the rapid emergence of HBV resistant to these agents (1B). We recommend 3TC/FTC may be omitted from the ART regimen and tenofovir be given as the sole anti-HBV active agent if there is clinical or genotypic evidence of 3TC/FTC-resistant HBV or HIV (1D). Proportion of patients with a CD4 cell count <500 cells/μL receiving TDF/FTC or TDF/3TC as part of a fully suppressive combination ART regimen. Proportion of patients receiving 3TC or FTC as the sole active drug against HBV in ART. TDF, FTC and 3TC are agents that have good antiviral activity against both HIV and hepatitis B.

Given the results of a previous study showing that TA systems are ubiquitous in VRE (Moritz & Hergenrother, 2007), and the current survey showing that TA systems are also highly prevalent and transcribed in MRSA and PA, it appears that these problematic bacterial pathogens would indeed be susceptible to TA-based antibacterial strategies. Specifically, activation of MazEFSa should be considered for MRSA, and activation of RelEPa or HigBAPa appear to be attractive strategies against PA. This work was supported

Galunisertib in vivo by National Institutes of Health Grant 2R01-GM068385. J.J.W and E.M.H. were partially supported by a National Institutes of Health Cell and Molecular Biology Training grant T32 GM007283. E.M.D. was partially supported by the Center for Nano-CEMMS (NSF DMI-0328162) at the University of Illinois. We thank the bacterial laboratories at Carle Foundation Hospital (Urbana, IL),

Memorial Medical Center (Springfield, IL), and Delnor Community Hospital (Geneva, IL) for the MRSA isolates. We also thank Cubist Pharmaceuticals Inc. (Lexington, MA) and Carle Foundation Hospital (Urbana, IL) for the PA isolates. J.J.W. and E.M.H. contributed equally to this work. Fig. S1. Alignment of mazEFSa sequences. Fig. S2. Alignment of parDEPa sequences. Fig. S3. Alignment of relBEPa sequences. Fig. S4. Alignment of higBAPa sequences. Fig. S5. Alignment of mazEFSa upstream (a) and downstream (b) flanking sequences. Fig. S6. Alignment of parDEPa upstream (a) and downstream (b) flanking sequences. Fig. S7. Alignment of relBEPa anti-PD-1 antibody upstream (a) and downstream (b) flanking sequences. Fig. S8. Alignment of higBAPa upstream (a) and downstream (b) flanking sequences. Table S1. Presence of TA Systems in MRSA and Pseudomonas aeruginosa. Table S2. Flanking regions of TA Systems in MRSA Cytidine deaminase and Pseudomonas aeruginosa. 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. “
“Phytophthora ramorum,Phytophthora alni, and Phytophthora kernoviae present significant threats to biosecurity. As zoosporic oomycetes, these plant pathogens may spread through natural waterways and irrigation systems. However, survival of these pathogens in aquatic systems in response to water quality is not well understood. In this study, we investigated their zoospore survival at pH 3–11 in a 10% Hoagland’s solution over a 14-day period. The results showed that all three pathogens were most stable at pH 7, although the populations declined overnight irrespective of pH. Extended survival of these species depended on the tolerance of pH of their germinants. Germinants of P. alni ssp. alni and P.

Conversely, a lack of comparator data for ZDV monotherapy and potential toxicities arising from ZDV use PD-0332991 order may limit the relevance of these data. Of note, further to peripheral toxicities, which are well described with ZDV use, biomarker data suggest there may also be CNS toxicities associated with the use of ZDV-containing regimens [18]. In summary, we recommend patients with NC impairment start standard combination ART regimens and the choice should be determined, as with other patients, by different factors, including baseline

VL, side effect profile, tolerability, DDIs and patient preference. Novel ARV strategies, including protease-inhibitor monotherapy continue to be assessed in clinical trials as cost-beneficial treatment regimens with the potential for reduced long-term toxicities. Concerns have been raised regarding the cerebral effects of PI monotherapy [19], with such concerns based on the hypotheses that PI monotherapy comprises only one effective ARV agent that may not adequately suppress ongoing HIV replication in sanctuary sites such as the CNS, and on pharmacokinetic modelling that suggests that not all PIs have optimal penetration across the blood–brain barrier [13]. Furthermore, isolated cases describing the evolution of CNS disease in previously stable HIV-positive subjects SP600125 solubility dmso receiving PI monotherapy have been reported [20]. One study was specifically

designed to assess the cerebral effects of LPV/r monotherapy [21]; however, it was terminated early due to a lack of efficacy in the plasma compartment. Although cases of CNS disease were reported within this study, such results must be interpreted with caution as virological endpoints in the plasma compartment were not met and therefore

such cases may be driven by poor ARV efficacy per se, rather than distinct CNS disease itself [22]. In the MONET study assessing DRV/r vs. standard therapy, no differences in patient-reported cognitive function are observed between the study treatment arms over 3 years of therapy MycoClean Mycoplasma Removal Kit [23]. Although reassuring, these data represent changes in patient-reported observations rather than observations from formal neuropsychological testing. Interestingly, in a small substudy within MONET, improvements in detailed neuropsychological testing and improvements in cerebral biomarkers measured via imaging techniques, were reported in both treatment arms [24]. In the ongoing UK PIVOT study, detailed neuropsychological testing is being assessed prospectively in subjects on PI monotherapy vs. standard therapy, the results of which will be of great interest to this field. Given the above theoretical concerns regarding the CNS activity of PI monotherapy, and for the majority of HIV-positive subjects it may be possible to select other ARV regimens, we suggest this approach is currently avoided in neurologically symptomatic subjects.

[29] Recent evidence also suggests that 6TGN measurement is beneficial in this disease. In a prospective study of 70 patients with autoimmune hepatitis, patients underwent AZA dose escalation to 2.0 mg/kg/day and steroid withdrawal. For patients who remained in remission (alanine aminotransferase

[ALT] < 33 IU/mL), median 6TGN levels were 237 versus 177 for those who relapsed (P = 0.025).There was no correlation between dose and 6TGN levels. Patients in remission with higher 6TGN levels tended to be on lower dosages of AZA (1.7 mg/kg) compared with relapsers (2.0 mg/kg) (P = 0.08).[30] Further studies are required to establish the therapeutic window for 6TGN levels Fulvestrant in vivo in autoimmune hepatitis. There is a paucity of publications investigating the measurement

of thiopurine metabolites in rheumatological diseases. In a cohort of 23 patients with various systemic connective tissue diseases, no correlation was seen between AZA dose and 6TGN levels.[31] Thirteen patients with SLE had higher levels of 6TGN than 13 patients with other systemic rheumatological conditions despite similar AZA dose (2 mg/kg/day vs. 2 mg/kg/day, P = NS).[32] Another study of 17 SLE patients found no correlation between 6TGN levels and disease activity indices, perhaps because median 6TGN levels were < 160.[33] It is difficult to draw firm conclusions from these studies, in view of the small numbers of patients and the inclusion of heterogeneous rheumatological diseases, as there may be variation in thiopurine metabolism, efficacy and therapeutic 6TGN thresholds selleck chemical for different disease entities. In 2009, an open-label dose escalation study of AZA to 3.5 mg/kg or 6TGN within the therapeutic range (235–400) in 50 patients with SLE was published. There was no difference in 6TGN levels between Mannose-binding protein-associated serine protease responders (average 6TGN = 159) and non-responders (average 6TGN = 202), but there was a correlation between 6TGN and AZA dose (Pearson correlation coefficient = 0.39, P < 0.0001). Only 38% of

responders and 40% of non-responders achieved a 6TGN level above 235. The authors comment that given the small sample size, they could not establish a therapeutic dose or metabolic threshold.[34] In conclusion, while the literature does not refute the utility of thiopurine metabolites, the lack of high quality data prevents endorsement for routine measurement of thiopurine metabolites in patients with rheumatologic diseases. Further prospective, well designed studies are required to elucidate a therapeutic window for 6TGN levels in rheumatological conditions. A well-known side effect of thiopurine therapy is myelosuppression, in particular leucopenia. Myelotoxicity tends to occur later than other thiopurine side effects. In patients with normal TPMT activity, myelotoxicity can occur as early as 3 months after the commencement of therapy,[2] but can be as late as 18 months.

Four focus groups (with Androgen Receptor Antagonist library 31 participants) were held in 2012 with Australian hospital pharmacists who work with children. Written notes and audio recordings were used to produce verbatim transcriptions and extract themes. There was consensus across groups that formal recognition of advanced pharmacy practice was valuable to the profession and to individuals. Elements should include a strong grounding in clinical practice, commitment to education, research and service improvement outside the department and institution. A framework for career development should be used to describe the levels of practice leading to advanced practice. Assessment should involve multiple

separate criteria, and incorporate direct observation, peer review and a PLX4032 professional portfolio. Postgraduate qualifications are desirable but not considered essential. Different knowledge and skills are required in paediatrics; however, the definition of

advanced practice remains the same. Recognition of advanced practice is valuable for the profession and for individuals. Multiple methods of assessment should be used. Specialty areas such as paediatrics can be defined and assessed similar to other specialties, with acknowledgement of the specific paediatric knowledge and skills required. “
“N. Umarua, S. Dhillona, K. Andrzeja, P. Sivab, C. Janetb aUniversity of Hertfordshire, Hertfordshire, UK, bLuton and Dunstable Hospital NHS Foundation, Bedfordshire, UK To examine Medicines Related Hospital Admissions (MRHA) in older patients 65 years and over. A mixed method study based on triangulation of data

collected from a review of patients’ hospital admission notes, interviews with patients prior to discharge and a review of respective patients’ health records held at the GP surgery. Factors contributing to a MRHA included non-adherence, very limited discussions with healthcare professionals, patient-related inability to self-manage healthcare and lack of caution when initiating additional medication therapy. Previous studies have attributed the causes of MRHAs to communication failures, knowledge gaps and problems in the medication use process. Older patients OSBPL9 are at a greater risk of experiencing MRPs resulting in hospital admissions of which an estimated two thirds are preventable. Concerns regarding unnecessary admission to hospital have been raised due to patient safety issues and use of limited resources within a healthcare system desperate to streamline its activities. Concerns arising due to MRHAs have largely been reported from the perspective of healthcare professionals. This study aimed to examine and incorporate the views of older patients admitted to hospital due to a MRP.

The eyes open/closed paradigm elicited alpha-band modulations in both lighting conditions, manifested in occipital and frontal electrodes. Fig. 2A depicts an example of alpha-band (8–12 Hz) modulations for a single subject, while the averaged results for all subjects can be seen in Fig. 2B. Alpha amplitude

was significantly larger when derived from occipital electrodes compared with frontal electrodes in both dark and light conditions, during eyes open as well as eyes closed (all paired t-tests, P < 0.005). Additionally, occipital alpha amplitude was larger during the eyes-open condition in the dark compared find more to light (paired t-test, P < 0.05). In accordance PD-166866 in vitro with these results, an anova

conducted on alpha amplitude in all conditions (location, lighting and eye state) revealed a significant main effect for both eye state and location (P < 0.0001) but not for light (P < 0.88). Furthermore, a significant interaction was found for eyes × location (P < 0.002) but not for eyes × lighting (P < 0.23). The EEG classifier revealed a significant contribution of the alpha rhythm to eye state classification across subjects in both lighting conditions [P < 0.05, false discovery rate (FDR)-corrected; see Figs. 3A and B]. The weight of the alpha rhythm was 0.27 in the light condition and 0.2 in the complete darkness condition, indicating the amount of variability explained by the alpha rhythm between eyes open/closed in both lighting conditions. The chosen electrode for each subject was mostly located in the occipital regions under the light condition (in 79% of all subjects)

and in frontal regions under complete darkness (in 64% of all subjects). In accordance, highest classification rates were achieved in occipital electrodes under the light condition and in frontal electrodes under the complete darkness condition (for a distribution of classification rates across the scalp see Figs. 3C and D). Furthermore, the classifier revealed two more EEG bands as significantly contributing to eye state inference Fenbendazole – a wide beta (24–33 Hz) contribution during the light condition and a contribution of theta (4–7 Hz) during complete darkness. Accordingly, we found a significant correlation (average r = 0.46, P < 0.0002) between the alpha and theta timecourses in the dark condition in 79% of all subjects. This finding suggests a link between alpha and theta modulations mostly evident in the complete darkness condition, which could be related to internal mental context, as discussed later, or reflect changes in subjects’ vigilance state. Nonetheless, theta-related fMRI activation did not reach high statistical significance and therefore is not further discussed in the current paper, which focuses on the alpha band.

Currently, the treatment of choice

for T. vaginalis infections is metronidazole. The increase in metronidazole-resistant parasites and undesirable side effects of this drug make the search for alternative chemotherapeutic approaches a priority for the management of trichomoniasis. Here, the antiproliferative and ultrastructural effects of sterol biosynthesis inhibitors against T. vaginalis were RG7204 mouse investigated. It was found that 22,26-azasterol (5 μM) and 24(R,S),25-epiminolanosterol (10 μM), known inhibitors of Δ24(25)-sterol methyltransferase, exhibited antiproliferative effects on T. vaginalis trophozoites cultured in vitro. Morphological analyses showed that azasterols induced changes in the ultrastructure of T. vaginalis. The most significant alterations

were (1) membrane blebbing and disruption, (2) wrinkled cells and (3) the formation of cell clusters. In addition, autophagic vacuoles, Golgi duplication arrest, an abnormal Golgi enlargement and damaged hydrogenosomes were also observed. Nonspecific cytotoxicity assays using the cultured mammalian cell lines Madin–Darby canine kidney cells showed no effect of the azasterols on the viability and proliferation AZD1208 cell line of these cells at a concentration that significantly inhibited the proliferation of T. vaginalis, indicating a selective antiparasitic action. Taken together, these results suggest that azasterols could be important compounds in the development of novel chemotherapeutic approaches against T. vaginalis. Trichomonas vaginalis is a parasitic protozoan that is the aetiological agent of trichomoniasis, the most common nonviral sexually transmitted disease worldwide.

According to WHO (2001), 173 million new cases occur annually. This disease has been associated with serious health problems for female patients, particularly during pregnancy, and it has been implicated as a risk factor for cervical cancer. Infected individuals are also predisposed to a higher transmission rate of HIV (Petrin et al., 1998). In addition, a recent study showed a relationship between trichomoniasis and prostate cancer (Sutcliffe et al., 2009). Currently, the compound of choice for the treatment of T. Arachidonate 15-lipoxygenase vaginalis infections is metronidazole, which has been effectively used since the 1960s (Durel et al., 1960). An increase in metronidazole-resistant trichomoniasis has been observed (Lossick et al., 1986); moreover, a clinical resistance to metronidazole has been reported since 1962 (Robinson, 1962; Dunne et al., 2003; Goldman et al., 2009). Furthermore, undesirable side effects (nausea and hypersensitivity reactions) are common in patients undergoing treatment with this drug (Kurohara et al., 1991; Smilack et al., 1991). Undoubtedly, the development of safer and more potent chemotherapeutic agents is a top priority for the management of this worldwide health problem.

This enzyme possesses a number of conserved residues, which include H204, F213, Y236, L263, T265, C266 and R275 that are commonly present among different classes of sortases from various bacteria. These conserved residues are located primarily in domains D2 and D3 (Dramsi et al., 2005). For example, H204 and F213 are located in domain D2, Y236 is positioned between domains D2 and D3, and L263, T265, C266 and R275 are found in Domain selleck inhibitor D3. Thus, the roles of these conserved

residues may provide valuable information for developing potent and selective inhibitors for both this particular sortase and other sortases. Herein, we report the identification of the transcription starting site of the srtC1 determined by rapid amplification of cDNA ends (RACE) method and several conserved residues essential for its

catalytic function revealed by site-directed mutagenesis. Bacterial strains and plasmids used in this study are listed in Table 1. The Escherichia coli strains used for subcloning and plasmid isolation were grown in Luria–Bertani medium (Difco Laboratories, Detroit, MI) at 37 °C in the presence of the appropriate selective substances. Actinomyces oris T14V and its mutants were grown in Todd–Hewitt broth (THB) (Difco Laboratories), or as otherwise indicated, at 37 °C without agitation. When needed, kanamycin and selleck screening library trimethoprim were included in growth media at concentrations of 50 and 100 μg mL−1, respectively. Total RNA from exponentially growing wild-type A. oris cells was extracted using the RNeasy

Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Residual DNA in the total RNA samples was removed by DNase I treatment. Total RNA was concentrated by ethanol precipitation, resuspended in a small volume of RNase-free water and stored at −80 °C. To determine the transcription start site(s) of A. oris srtC1, 5′RACE-PCR experiments were carried out using SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA) with 3 μg of total RNA. The sequences of oligo primers used are shown in Table 2. Briefly, the first strand of cDNA synthesis was carried out at 42 °C for Thiamet G 1.5 h using a gene-specific primer: primer 1 for fimQ, primer 3 for fimP and primer 5 for srtC1. RACE-PCR was performed using the above cDNA as the template and using SMART PCR primer UPM and gene-specific primers: primer 2 for fimQ, primer 4 for fimP and primer 6 for srtC1. The amplified PCR products were further cloned into Zero Blunt TOPO vector (Invitrogen, Carlsbad, CA) and transformed into E. coli competent cells. Plasmid DNAs were isolated with QIAprep Spin Miniprep Kit (Qiagen). Cloned fragments were sequenced in both directions (ACGT Inc., Wheeling, IL) using an ABI automated sequencer and Dye Terminator Cycle Sequencing Kit, and the transcription start site was determined.