Our collections of L. menziesii are the first reported from the Neotropics and their morphological features match those of Polyporus menziesii as described by Ryvarden and Johansen (1980) and our personal observations

(isotype – K). The third species here VS-4718 mentioned as ‘Leiotrametes sp.’ from French Guiana does not match any species known to us nor described in the literature. Nevertheless hymenial surface of this species could evoke the temperate Daedalea quercina RepSox supplier (L.: Fr.) Fr., a phylogenetically unrelated species producing a brown rot (also showing other morphological discrepancies). Since Daedalea quercina was mentioned by Patouillard (in Duss 1903) after a collection by Duss in Guadeloupe and taking into account its unlikely occurrence in the Carribean (see Courtecuisse and Welti 2011) it is possible that Duss’s material represents this still undescribed Leiotrametes sp. The main characteristic separating Leiotrametes from Trametes and Pycnoporus is the glabrous upper surface, the lack of black line under the pileipellis and of parietal crystals (red in Pycnoporus, colorless in T. cingulata and blue in T. versicolor). Another interesting character is the brown resinous substance filling

the lumen of the skeletal hyphae in the pileipellis, particularly those concentrated in the narrow grayish concentric zones (Fig. 4e). They were also found in KU-57788 concentration some species of Trametes: T. gibbosa and T. villosa. A comparable resinous content also appears in T. cingulata and T. ljubarskyi

but differs by its conspicuous accumulation in uppermost level inducing Gemcitabine cost cellular walls rupture (Fig. 4g) and so generating a glossy and brown, surface. ‘Lenzites’ warnieri, of still unsolved phylogenetic position, also showed similar resinous hyphae; nevertheless, they appear less abundant in the upper surface level and did not show resinous accumulation at the surface (Fig. 4e). ‘Trametes’ cingulata and ‘Trametes’ ljubarskyi The position of Trametes cingulata and T. ljubarskyi has already been shown to be ambiguous according to our study. However the Bayesian analyses on ITS + RPB2 (Fig. 1) and to a lesser degree on 28S rLSU, suggest a sister-clade relationship between both species and Pycnoporus. As a support to this hypothesis we detected crystals darkening in 5% KOH under the upper surface of T. cingulata. Furthermore, the orange-brown, dry basidiomes of this species, as well as its tendancy to turn blackish with 5% KOH 5%, at a lower degree the characteristic of Pycnoporus species (red basidiomes and KOH reaction). So far a close relationship between Trametes ljubarskyi and T.

Materials and methods Patients and tissue samples A total of 100 patients undergoing LT for HCC and the follow-up data about the patients in this study were obtained from Liver Transplantation Surgery, Shanghai First People’s Hospital, Shanghai, China, from 2002 to 2007. All the patients were followed until December 2010. The median recurrence-free period was 12 months for patients with HCC recurrence and 64 months for patients without HCC recurrence. All of these 100 patients fulfilled the Up-To-Seven transplantation criteria Alpelisib datasheet for HCC [14] and none of them had macro-vascular invasion. HCC samples were from the FFPE tissue blocks and the normal liver tissues were from the

liver hemangioma resection. The clinicopathological features of patients were summarized in Table 1. Pre-LT serum AFP level stratification was according to the previous study [15]. All patients provided informed consent according to the protocols approved by the Institutional Review Boards of Shanghai First People’s Hospital. Table 1 Clinical characteristics of the 100 HCC patients according to high- or low miR-20a expression level Parameter N Patients with low miR-20a expression Patients with high miR-20a expression P-value Age 100 57.820 ± 7.330 53.64 ± 8.341 0.212† Sex           Male 84 44 40 0.585‡   Female 16 6 10   Underlying

liver disease           HBV 95 47 48 1.000§   others 5 3 2   Gemcitabine research buy Cirrhosis           Yes 95 47 48 1.000 §   No 5 3 2   Tumor stage           I + II 66 32 34 0.673‡   III 34 18 16   Histologic grade           Differentiated 88 41 47 0.065§   Undifferentiated 12 9 3   Milan criteria           In 55 24 31 0.159‡   Out 45 26 19   Tumor size (cm)           ≤5 Tolmetin 60 24 36 0.014‡   >5 40 26 14   Multinodular           Yes 43 25 18 0.034 ‡   No 57 25 32   Micro-vascular invasion           Yes 22 16 6 0.016 ‡   No 78 34 44   pre-LT serum AFP level           ≤400

(ng/ml) 63 30 33 0.534 ‡   >400 (ng/ml) 37 20 17     Overall survival 42/100 11/50 31/50 –   HCC recurrence 58/100 37/50 21/50 – NOTE: AFP, alpha-fetoprotein. †Unpaired student t test; ‡chi-square test; §Fisher’s exact test. Cell culture and transfection All the cell lines used in this study were KU55933 purchased from the cell bank of the Chinese Academy of Sciences and grown in DMEM (GIBCO, Grand Island, NY), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO), 2 mM glutamine, 100 U of penicillin/ml and 100 μg of streptomycin/ml (Cambrex, Verviers, Belgium). All cells were incubated at 37°C in a humidified chamber supplemented with 5% CO2. Control negative oligonucleotide, and double-stranded RNAs that mimic endogenous precursor miR-20a were purchased from Ambion (Ambion, Austin, TX) were transfected into cells using Oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction.

Antimicrob Agents Chemother 2004, 48 (6) : 2153–2158.PubMedCrossRef 16. Scott NW, JQEZ5 price Harwood CR: Studies on the influence of the Tozasertib ic50 cyclic AMP system on major outer membrane proteins of Escherichia coli K12. FEMS Microbiol Lett 1980, 9: 95–98.CrossRef 17. Huang L, Tsui P, Freundlich M: Positive and negative control of ompB transcription in Escherichia coli by cyclic AMP and the cyclic AMP receptor protein. J Bacteriol 1992, 174 (3) : 664–670.PubMed 18. Zhou D, Yang R: Molecular Darwinian

evolution of virulence in Yersinia pestis. Infect Immun 2009, 77 (6) : 2242–2250.PubMedCrossRef 19. Brzostek K, Raczkowska A: The YompC protein of Yersinia enterocolitica: molecular and physiological characterization. Folia Microbiol (Praha) 2007, 52 (1) : 73–80.CrossRef 20. Delihas N: Annotation and evolutionary relationships of a small regulatory RNA gene micF and its target ompF in Yersinia species. BMC Microbiol 2003, 3 (1) : 13.PubMedCrossRef 21. Brzostek K, Hrebenda J, Benz R, Boos W: The OmpC protein of Yersinia enterocolitica: purification and properties. Res Microbiol 1989, 140 (9) : 599–614.PubMedCrossRef 22. Zhou

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Discussion In this study, a novel RCC species was found growing in the anaerobic fungal subcultures. Many studies have shown that a large group of RCC inhabited the rumen of a variety of ruminant species on various diets [1, 2, 4–11]. Thus, the RCC species grown in the anaerobic fungal cultures in the Cilengitide in vitro present study just represented a small group of the

total RCC. It has been proposed that the RCC in the rumen and its relatives in other environments could constitute the seventh order of the methanogens (Methanoplasmatales) [17]. Methanogens within this new methanogenic order distantly related to the Thermoplasmatales, have been shown to be present in various environments, including marine habitats, soil, and also the intestinal tracts of termites and mammals, suggesting their ubiquitous in various environments. The whole order was proposed to form three big clusters, Ca. M. alvus EX 527 supplier Cluster, M. luminyensis Cluster and Lake Pavin Cluster [15]. The novel RCC species in the present study was grouped in the Ca. M. alvus Cluster. The present study reported the first account for RCC species grown in the fungal cultures from the goat rumen. Nevertheless this single species may not represent the whole RCC community in the rumen. Therefore, further research is needed to uncover this community and its features in the rumen. Interestingly, this novel species could survive

in the long-term transferred fungal subcultures (even in the 62nd subcultures). Thus, there must be an underlying mechanism supporting the growth of this novel RCC species in the fungal subcultures. A similar phenomenon for protozoa was

reported by Irbis and Ushida [20]. When testing a single protozoa cell for the 16S rRNA gene sequences Janus kinase (JAK) of archaea, they found that the cultured rumen protozoa Isotricha intestinalis and Ophryoscolex purkynjei from goats carried Thermoplasma sp. related sequences (GenBank: AB189868, 99% similarity to LGM-AF04). Recent studies showed that methanogens belonging to this group [8, 14–17] could strictly use hydrogen to reduce methanol and methylamines to methane. It is well known that both anaerobic fungi and protozoa could produce hydrogen, which is one of the substrates for methanogens [19, 21]. This may make it possible for anaerobic fungi to provide RCC species with hydrogen. Methanol and methylamines could be derived from the microbial degradation of pectin, betaine, and choline from diets in the rumen [22]. Ametaj et al. [23] demonstrated that there were methanol and methylamines in the rumen fluid of lactating dairy cows fed graded amounts of barley grain. In this study, the medium for the mTOR inhibitor co-culture of anaerobic fungi and methanogens contained rice straw and clarified rumen fluid. Anaerobic fungi could degrade the pectin of rice straw by pectinolytic enzymes [24, 25], accompanying the release of methanol.

Similarly with previous reported [11, 12], most genes involved in ergosterol biosynthesis were repressed for both strains in this study. It is possible that the regulatory functions of the biosynthesis may not be significantly affected at transcriptional levels under the conditions of this study. The PDR gene group is a new set of genes examined for ethanol tolerance in this study. Many PDR genes function as transporters of ATP-binding cassette proteins and are encoded for plasma membrane proteins that mediate membrane translocation of ions and a wide range of substrates. It impacts lipid and cell wall compositions and major facilitator

superfamily proteins for cell detoxifications [60]. We previously found that PDR genes and regulatory elements played significant roles for tolerance and in situ detoxification of lignocellulose-derived inhibitors [61]. Selleck AZD6244 Since plasma membrane and cell walls are major targets of ethanol damages, we anticipated the involvement of these genes for reconditioning and remodeling membrane

and cell walls in response to ethanol challenges. The significantly enriched background of transcriptional Selleckchem CB-839 abundance and continuously increased expressions of several genes in this group for the ethanol tolerant yeast observed in this study support our hypothesis (Table 3). The expressions of PDR genes are mainly controlled by transcription factor Pdr1p and Pdr3p [62]. As demonstrated in our study, many genes share the common transcription protein binding motif of Pdr1p/Pdr3p. Expressions of PDR1 in the tolerant Y-50316 Cyclin-dependent kinase 3 was not significantly induced but constantly expressed at all time

points compared with the parental strain. It needs to be pointed out that unless it is repressed, PDR1 does not have to be greatly induced to allow potential Pdr1p functions as a regulator [32, 60]. We consider the ability of its expression under the stress is a tolerance response and suggest Pdr1p as a potential regulator involving the ethanol tolerance of Y-50316. As discussed above, genes able to express or recover to express normally under the stress are SHP099 important to maintain gene interactions and cell functions. On the other hand, transcription factor genes MSN4, MSN2, YAP1 and HSF1 of the tolerant strains were highly abundance under the ethanol stress. Since many ethanol tolerance candidate genes sharing protein binding motifs of Msn4p/Msn2p, Yap1p and Hsf1p, these transcription factors are likely a core set of regulators for interactive expressions of ethanol tolerance. An HSF1-deletion mutant showed repressed expressions for its target genes usually induced by ethanol [63]. It has been demonstrated that Msn2p and Msn4p induces gene expression via a stress response element and triggers transcriptional response of the downstream genes [64, 65]. Condition-specific roles in gene expression regulation by these transcription factors were also suggested [66].

J Bone Miner Res 16:1846–1853CrossRefPubMed”
“Background Telomeric DNA is GDC-0941 mouse protected and maintained at the ends of chromosomes by the action of the enzyme telomerase. Whilst the shortening of DNA telomeres during

repeated cell division is a natural part of the cellular ageing mechanism, one of the hallmarks of cancer is the expression of telomerase by cancer cells which allows them to maintain telomeric length and adopt immortal characteristics [1, 2]. Telomerase requires a single-stranded DNA primer as BIBW2992 manufacturer substrate for the addition of telomeric repeats (TTAGGG) [3], his terminal telomere G-rich single stranded tract, also called G-overhang, can fold into four-stranded G-quadruplex (G4) structures consisting of G-tetrads coordinated around a monovalent cation [4, 5]. G4 stabilization, deny access of telomerase to its substrate, representing a valid

tool for telomerase targeted approach in cancer https://www.selleckchem.com/products/lxh254.html therapy [6]. Nevertheless, for direct telomerase inhibition, a time-dependent response is observed, related to the basal length of the telomeres, due to the slow attrition of telomeres experienced after each cell division, thus limiting the efficacy of agents designed to inhibit telomerase alone [7–9]. The extremely rapid and potent cytotoxic effect triggered by G4 ligands interacting with telomeric DNA sequences (‘Telomere Targeting Agents’: TTAs) is explained by a dual mechanism of action. On one hand the inhibition of telomerase, Methamphetamine and, on the other hand, disruption of the shelterin complex, a nulcleo-protein complex which stabilises

and protects the ends of chromosomes from being recognized as double-strand breaks [7, 8]. The presence of G4 structures has been recently showed in non telomeric regions, as already hypothesized on the base of predictive studies [10]. In particular, G4 forming regions were already found in the promoter of several cancer related genes (c-myc, bcl2, hif1, hTERT), and for some of those genes, a transcriptional inhibitory function was attributed to these structures. Consequently, G4 targeting molecules could have additional extra-telomeric features, which could improve their potential as anti-cancer agents. The pentacyclic quinoacridinium salt RHPS4 (1: Figure  1) has many of the attributes of an ideal TTA. We have shown previously by NMR studies that the agent stacks above and below the G3 core of a (TTAGGGT)4 parallel-stranded quadruplex; [11] it binds with high efficiency to the h-Tel DNA sequence as measured by surface plasmon resonance [11], circular dichroism and ESI-MS [12, 13]; it is an active inhibitor of telomerase (IC50 0.33 μM) as revealed in a Trap assay [14] and disrupts the shelterin complex of the telomere with the liberation of the POT-1 protein [15].

01) (Figure 5B). Statins have been found to decrease the up regulation of adhesion molecules on endothelial cells in several models of inflammation [20–22]. Because we observed a dose-dependent

reduction in neutrophil influx, yet only mice on the HSD had lower production of the neutrophil chemoattractant KC, we went on to assess whether statins were reducing neutrophil infiltration by modulating the upregulation of adhesion molecules. In agreement with the observed reduction in neutrophils influx, mice receiving statins had a strong dose-dependent reduction in the protein levels of ICAM-1 present www.selleckchem.com/products/VX-680(MK-0457).html in the lungs prior to infection with S. pneumoniae (Control versus LSD, P = 0.04; Control versus HSD, P = 0.004) (Figure 6A). Whereas at 24 hpi, only mice on the HSD continued to have decreased protein levels of ICAM-1 in the lungs (P = 0.02) (Figure 6B). Taken together these findings suggest that statins exert a dose-dependent effect to reduce neutrophil infiltration during pneumococcal pneumonia by reducing neutrophil chemotaxis and transcytosis without suppressing pro-inflammatory mediators required to enhance antibacterial defense mechanism. Selleckchem Birinapant Figure 6 Statins decrease ICAM-1 protein expression prior to and following infection with S. pneumoniae. Lungs from mice on Control,

Low, and High statin diet (n = 6/group) were examined for protein expression of ICAM-1 prior to and 24 h following intratracheally infection with 1 X 105 cfu by see more western blot analysis of whole lung protein lysates (n = 3/group for uninfected and n = 6/group for infected mice). Mice receiving statins had significantly less ICAM-1 protein levels present in the lungs both A) prior to and B) following infection. Data are presented as the mean ± SEM. Statistics were determined by a two-tailed student’s t-test. P < 0.05 was considered significant in comparison

to Control fed mice. Surivival of infected mice on statins Finally, we sought to directly test if prophylactic statin therapy improved disease outcomes in a clinically relevant infection model. Since individuals with CAP receive antimicrobials, we the tested for survival of mice in a model where beginning at 48 hpi mice received ampicillin at 12 h intervals. Despite the protective effects observed above, mice on LSD or HSD had equivalent survival over time as controls (Figure 7). Thus, the overall protective effects of statins were modest and may not necessarily impact disease outcomes in humans. Figure 7 Survival of simvastatin fed mice following infection with S. pneumoniae . Kaplan–Meier plot demonstrating percent survival of challenged mice. Mice on Control (n = 19), Low (n = 19) or High (n = 20) diet were challenged intratracheally with 1 X 105 cfu. After 48 h ampicillin (80 mg/kg) was administered every twelve hours. Significance was determined by Log-Rank test.

Med Mycol 2005,43(Suppl 1):S267–270.PubMedCrossRef 5. Marr KA, Carter RA, Boeckh M, Martin P, Corey L: Invasive aspergillosis in allogeneic eFT508 in vivo stem cell transplant recipients: changes in epidemiology and risk factors. Blood 2002,100(13):4358–4366.PubMedCrossRef 6. Garcia-Vidal C, Upton A, Kirby KA, Marr KA: Epidemiology of invasive mold infections in allogeneic stem cell transplant recipients: biological risk factors for infection

according to time after transplantation. Clin Infect Dis 2008,47(8):1041–1050.PubMedCrossRef 7. Lionakis MS, Kontoyiannis DP: Glucocorticoids and invasive fungal infections. Lancet 2003,362(9398):1828–1838.PubMedCrossRef 8. Philippe B, Ibrahim-Granet O, Prevost MC, Gougerot-Pocidalo MA, Sanchez Perez M, Meeren A, Latge JP: Killing of Aspergillus fumigatus by alveolar macrophages is mediated by reactive oxidant intermediates. Infect Immun 2003,71(6):3034–3042.PubMedCrossRef 9. Ibrahim-Granet O, Philippe B, Boleti H, Boisvieux-Ulrich E, Grenet D, Stern M, Latge JP: Phagocytosis and intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages. Infect Immun 2003,71(2):891–903.PubMedCrossRef 10. Romani L, Montagnoli C, Bozza S, Perruccio K, Spreca A, Allavena P, Verbeek S, check details Calderone RA, Bistoni F, Puccetti P: The exploitation of distinct recognition receptors in dendritic cells determines the full range of host immune

relationships with Candida albicans. Int Immunol 2004,16(1):149–161.PubMedCrossRef 11. Bellocchio S, Moretti S, Perruccio K, Fallarino buy Niraparib F, Bozza S, Montagnoli C, Mosci P, Lipford GB, Pitzurra L, Romani L: TLRs govern neutrophil activity in aspergillosis. J Immunol 2004,173(12):7406–7415.PubMed 12. Hohl TM, Van Epps HL, Rivera A, Morgan LA, Chen PL, Feldmesser M, Pamer EG: Aspergillus fumigatus Triggers Inflammatory Responses by Stage-Specific beta-Glucan Display. PLoS Pathog 2005,1(3):e30.PubMedCrossRef 13. Steele C, Rapaka RR, Metz A, Pop SM, Williams DL, Gordon S, Kolls JK, Brown GD: The beta-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus. PLoS Pathog 2005,1(4):e42.PubMedCrossRef

14. Gersuk GM, Underhill DM, Zhu L, Marr KA: Dectin-1 and TLRs Ribonucleotide reductase permit macrophages to distinguish between different Aspergillus fumigatus cellular states. J Immunol 2006,176(6):3717–3724.PubMed 15. Chignard M, Balloy V, Sallenave JM, Si-Tahar M: Role of Toll-like receptors in lung innate defense against invasive aspergillosis. Distinct impact in immunocompetent and immunocompromized hosts. Clin Immunol 2007,124(3):238–243.PubMedCrossRef 16. Brock M, Jouvion G, Droin-Bergere S, Dussurget O, Nicola MA, Ibrahim-Granet O: Bioluminescent Aspergillus fumigatus, a new tool for drug efficiency testing and in vivo monitoring of invasive aspergillosis. Appl Environ Microbiol 2008,74(22):7023–7035.PubMedCrossRef 17.

VacA s/i/d/m region subtyping was accomplished by three single PCR amplification assays. The signal-sequence (SS) region was amplified using primer M13-SeqS.se and SeqS.as; the intermediate and deletion region (IR and DR) using primer M13-SeqVac.se and SeqVac.as; the midregion (MR) using primer M13-SeqM.se and VAG-R (Figure  2; Table  2), respectively Amplification conditions used were identical in all assays as described previously [45]. Prior to sequencing, amplicons were analysed

by automated capillary gel electrophoresis using a QIAxcel system and a QIAxcel DNA High Resolution kit (Qiagen, Hilden, Germany). cagA EPIYA motif and vacA s/i/d/m-region sequence analysis M13-tagged cagA EPIYA and vacA TSA HDAC datasheet amplicons were sequenced using M13 uni (−21) sequencing primer and a customer sequencing service (Eurofins MWG Operon, Ebersberg, Germany). The obtained GS-4997 in vitro cagA and vacA sequences were aligned and compared

with catalogued H. pylori 26695 [GenBank:AE000511, H. pylori J99 [GenBank:AE001439], H. pylori P12 [GeneBank:CP001217], H. pylori G27 [GenBank:CP001173], and H. pylori Shi470 [GeneBank:CP001072] sequences using the CLC DNA Workbench version 5.5 [55]. Sequences were retrieved from the NCBI nucleotide database [56]. CagE and cag-PAI (empty-site) amplicon sizes were analysed by capillary gel electrophoresis only. Statistical analysis Selleckchem A-1210477 Binary logistic regression analysis of data was performed using Minitab 15 software. Statistical significance was assumed at P < 0.05. All statistical analyses presented here were significant according to Hosmer-Lemeshow

(HL) goodness-of-fit test, with HL p values >0.05. In the logistic regression analysis and the GLM analysis, a 95% confidence interval including 1.0 was next regarded as non-significant. Odds ratios with 95% confidence intervals (CI) were calculated to explore possible associations of individual genotypes to peptic ulcer or gastric atrophy. Age and sex were included as covariates. With regard to atrophy, data from duodenal biopsies were not included in the statistical analysis. Acknowledgements The study was supported by grants from the Research Council in the South-East of Sweden (FORSS, the ALF program, the committee for medical R&D, and the Molecular Biology program at Clinical Microbiology, Laboratory Medicine Centre-DC, University Hospital, Linköping, Sweden. We are grateful to Statistician Olle Eriksson, PhD, for statistical calculations and advice. Electronic supplementary material Additional file 1: Results from cagA and vacA genotyping, including clinical data. (PDF 18 KB) References 1. Marshall BJ, Warren JR: Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984,1(8390):1311–1315.PubMedCrossRef 2. Cover TL, Blaser MJ: Helicobacter pylori and gastroduodenal disease. Annu Rev Med 1992, 43:135–145.PubMedCrossRef 3.

Arg136 is further positioned in AlrSP by a hydrogen bond to Ser309. Sequences of alanine racemases that contain a lysine in position 129 almost always have an accompanying serine or cysteine residue in the equivalent of position 309 [36]. Recently, the AlrBA structure was found to contain an aspargine residue bound to a chloride ion at the equivalent position of Lys129, which appears to play the same role as the carbamylated Lys of positioning the active site arginine [36]. An alignment of alanine racemase sequences by Couñago et al. revealed that the presence of an aspargine residue can occur at the equivalent position

of Lys129 in AlrSP and is likely to be indicative of an internal chloride within the active site in the place of a carbamylated lysine. Notably this change from Lys to Ser appears to always be accompanied by a threonine at the equivalent position CFTRinh-172 concentration of Ser309, even though the threonine does not directly

interact with the chloride ion. The environments on either side of the pyridine ring of PLP are quite different, as reported previously for AlrGS [29, 33]. The side of the PLP that faces the dimer interface is polar in character, with many hydrophilic amino acid residues (including carbamylated Lys129, Arg136, His165 and Arg218), several water molecules and the hydrogen-bond network. The nonpolar side of PLP, in contact with the α/β barrel, contains several hydrophobic residues Isotretinoin (Val38, Leu83, Leu85 and Phe163), no charged residues and no water molecules. learn more As observed in several other alanine racemase structures [[29, 32, 34, 36]], we identified extra density in the active site of AlrSP adjacent to the PLP cofactor (Figure 4C). The position of this density corresponds to that of the acetate modeled in AlrGS. In other structures, this VX-680 mw location has been reported to contain propionate, alanine phosphonate, and a putative substrate molecule in DadXPA [[28–30, 38]]. Water molecules in the same location are found in the AlrMT and AlrSL structures. After unsuccessfully attempting to model a

variety of small molecules into the extra density, including acetate, we left this region of the model empty. Active site entryway The entryway to the active site in AlrSP comprises the α/β barrel domain of one monomer and residues from the C-terminal domain of the other monomer, and is about 13 Å from the active site C4″” atom of PLP. The entryway has a funnel-like shape, with its widest end towards the outside of the enzyme, narrowing as it approaches the PLP. The highly conserved residues comprising the entryway are distributed in layers beginning at the PLP site (Figures 6A and 6B): charged near the entrance, and mainly hydrophobic near the active site [33, 34]. Mutagenesis has shown that these hydrophobic residues have an important role in controlling the substrate specificity of alanine racemase [51].