Evidence for regulation of ER/Golgi SNARE complex formation by hsc70 chaperones

SNARE proteins control intracellular membrane fusion through formation of membrane-bridging helix bundles of amphipathic SNARE motifs. Repetitive cycles of membrane fusion likely involve repetitive folding/unfolding of the SNARE motif helical structure. Despite these conformational demands, little is known about conformational regulation of SNAREs by other proteins. Here we demonstrate that hsc70 chaperones stimulate in vitro SNARE complex formation among the ER/Golgi SNAREs syntaxin 5, membrin, rbetl and sec22b, under conditions in which assembly is normally inhibited. Thus, molecular chaperones can render the SNARE motif more competent for assembly. Partially purified hsc70 fractions from brain cytosol had higher specific activities than fully purified hsc70, suggesting the involvement of unidentified cofactors. Using chemical crosslinking of cells followed by immunoprecipitation, we found that hsc70 was associated with ER/Golgi SNAREs in vivo. Consistent with a modulatory role for hsc70 in transport, we found that excess hsc70 specifically inhibited ER-to-Golgi transport in permeabilized cells.     

Role of <Emphasis Type="Italic">Leishmania (Leishmania) chagasi</Emphasis> amastigote cysteine protease in intracellular parasite survival: studies by gene disruption and antisense mRNA inhibition

Background  

The parasitic protozoa belonging to Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L.) chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L.) chagasi within the U937 macrophage cells.     

Genomic organization and functional expression of differentially regulated cysteine protease genes of Leishmania donovani complex

For the first time, we report the genomic organization and characterization of Cathepsin L-like cysteine protease gene cluster from the members of Leishmania donovani complex. The cysteine protease gene cluster of Leishmania chagasi has five copies of tandemly arranged genes. The first gene (Ldccys1A) is identical to Ldccys1 cDNA and is predominantly expressed in promastigotes. The last gene (Ldccys1E) is identical to Ldccys1A with a 13 amino acids deletion in the mature domain, including one of the active site histidine residues and a truncated carboxyl terminal extension. It has a diverged 3' untranslated region and is also constitutively expressed in the parasite. Results from rapid amplification of cDNA ends (RACE) suggest that there are three different types of 3' untranslated regions, one of them is identical to that of Ldccys1A whereas another to Ldccys1E. The third one is also identical to Ldccys1A, but has a 154 nucleotides deletion near the polyA region and this gene is constitutively expressed. Gene organization and expression in L. donovani cluster is similar to that of L. chagasi. However, the last gene (Lddcys1F) is different from Ldccys1E as it lacks 13 amino acid deletions. Also, L. donovani possesses an additional copy of the gene (Lddcys1E), which is located away from the cluster. Furthermore, for the first time we have expressed full-length cysteine protease genes in an insect expression system. Ldccys1A and Ldccys1F cleaved gelatin whereas Ldccys1E was found to be inactive in gelatin assays.     

Pseudomembranous Type of Oral Candidiasis is Associated with Decreased Salivary Flow Rate and Secretory Immunoglobulin A Levels

Saliva plays an important role in maintaining microbial homeostasis in the oral cavity, while salivary gland hypofunction predisposes the oral mucosa to pathologic alteration and increases the risk for oral candidiasis. This study sought to determine the salivary flow rate (SFR) and secretory immunoglobulin A (SIgA) levels in HIV-positive and HIV-negative individuals and evaluate their relationship with the determinants of oral candidiasis. Sixty HIV-positive (30 with and 30 without oral candidiasis) and 30 healthy HIV-negative individuals were enrolled. Cotton pellet was weighed pre- and post-saliva collection for the assessment of SFR, while SIgA levels were estimated by commercial ELISA (Diametra, Italy) kit. The mean ± SD, SFR and SIgA levels in HIV-positive individuals with candidiasis, without candidiasis and HIV-negative controls were 0.396 ± 0.290, 0.546 ± 0.355 and 0.534 ± 0.214 ml/min and 115.891 ± 37.621, 136.024 ± 51.075 and 149.418 ± 31.765 µg/ml, respectively. A positive correlation between low CD4 counts (indicator of immunodeficiency) and SIgA was observed in HIV-positive individuals with candidiasis (r = 0.373, p = 0.045). We also report here for the first time the significant decrease in SFR and SIgA levels in individuals presenting with pseudomembranous type of oral candidiasis and Candida albicans infection.     

A Comparative Study of Caffeine Degradation by Four Different Fungi

The objective of the present study is to investigate the caffeine-degrading abilities of different fungi and to apply this knowledge to environmental remediation and industrial decaffeination process. Chrysosporium keratinophilum, Gliocladium roseum, Fusarium solani, and Aspergillus restrictus were isolated from the coffee pulp obtained from a coffee estate. Pure cultures of fungi were isolated on standard conventional potato dextrose broth (PDB) medium and authenticated. Pure cultures were subjected to a caffeine tolerance study at different concentrations of caffeine (1–8 g/L) in potato dextrose agar (PDA) and minimal media. On PDA, Fusarium solani could tolerate caffeine concentration up to 8 g/L, whereas Chrysosporium keratinophilum, Gliocladium roseum, and Aspergillus restrictus could tolerate up to 6 g/L. On minimal agar medium containing different concentrations of caffeine (1–8 g/L), Fusarium solani tolerated up to 8 g/L and the other fungi up to 2 g/L. A time-bound caffeine degradation study was undertaken at 1 g/L concentration of caffeine and glucose in nitrogen-containing and nitrogen-free liquid minimal media by subjecting the four fungi to shake flask culture at 120 rpm and 30°C. Degradation of caffeine up to 7 days at 24-h intervals was analyzed by high-performance liquid chromatography (HPLC). Gliocladium roseum followed by Aspergillus restrictus showed maximum degradation of caffeine at 0.47 and 0.3 mg/ml, respectively, by 96 h in nitrogen-containing minimal medium, whereas Fusarium solani showed maximum degradation of caffeine by 48 h (0.35 mg/ml) and Chrysosporium keratinophilum by 72 h (0.29 g/ml). In nitrogen-free minimal medium, Chrysosporium keratinophilum showed maximum degradation of caffeine at 72 h (0.45 mg/ml), followed by Gliocladium roseum, Fusarium solani (0.3 mg/ml), and Aspergillus restrictus (0.25 mg/ml) at 96 h. Overall, Chrysosporium keratinophilum showed a comparatively higher rate of caffeine degradation in minimal medium with or without a nitrogen source as compared with the other three fungi, indicating that nitrogen affects caffeine metabolism.     

Identification and characterization of a 66–68-kDa protein as a methotrexate-binding protein in murine leukemia L1210 cells

We previously observed an unidentified, tyrosine-phosphorylated, membrane-associated, 66–68-kDa protein which was present in the L1210 murine leukemia cells but not present, at least in the tyrosine-phosphorylated form, in cisplatin–methotrexate (CDDP–MTX) cross-resistant L1210/DDP cells. We purified and characterized this 66–68-kDa protein by affinity chromatography purification using its two identified properties, tyrosine phosphorylation and MTX-binding, and yielded a single band of 66–68 kDa. The purified protein was subjected to trypsin digestion and the isolated peptide fragments were sequenced and yielded two partial peptide sequences: VEIIANDQ and VTNAVVTVPAYFNDSQRQA. The two peptide sequences were used to search for the mouse genome at the national center for biotechnology information (NCBI) database for Open Reading Frame Sequence (ORFs) containing these peptides using the TBLASTN function. A single gene was identified containing both sequences, the HSPa8 gene, which codes for the heat shock family protein, HSC70. We further demonstrated that HSC70 is a MTX-binding protein using a binding assay with MTX-agarose beads followed by Western blotting. The HSC70 also existed in various cancer cell lines and showed binding to MTX. Additionally, the HSC70 protein, cloned from the L1210 murine leukemia cells, was expressed and purified from E. coli cells using a polyhistidine-tag purification system and it also showed the binding properties with MTX. DnaK, the HSC70 homologue in E. coli, also binds to MTX. By using the purified truncated HSC70 domains, we identified the adenosine triphosphatase (ATPase) domain of HSC70 that can bind to MTX. Thus, we have tentatively characterized a new, novel property of HSC70 as a MTX-binding protein.     

Biometric ratio in estimating widths of maxillary anterior teeth derived after correlating anthropometric measurements with dental measurements

doi: 10.1111/j.1741‐2358.2012.00648.x Biometric ratio in estimating widths of maxillary anterior teeth derived after correlating anthropometric measurements with dental measurements Objective: To correlate dental measurements i.e. combined mesiodistal width of six maxillary anterior teeth with facial measurements i.e. inner canthal distance, interpupillary distance and intercommissural width and acquire a biometric ratio to serve as a preliminary guide in selection of the maxillary anterior teeth. Background: In the absence of pre‐extraction records, the resultant denture can lead to patient dissatisfaction towards the aesthetic appeal of their dentures. The maxillary anterior teeth play a pivotal role in denture aesthetics. Various techniques and biometric ratios have been described in literature for selection of the maxillary anteriors. This study derives a biometric ratio for the same, obtained after correlating anthropometric measurements with dental measurements. Materials and methods: Two standardized digital photographs of the face were generated; one, when the facial muscles were relaxed and the other, when the subject was smiling; thereby, revealing the maxillary anterior teeth upto the canine tip. Inner canthal distance, interpupillary distance, intercommissural distance, distance between the tips of the maxillary canines and distance between the distal surfaces of the canines were measured. On the cast, the distance between tips of maxillary canines and distance between distal surfaces of maxillary canines were noted. The data was analysed using Spearman’s rank correlation coefficient. Results: A high correlation was found between the intercommissural measurement with distance between the tips of the canines on the photograph and between the tips of the canines on the cast with the interpupillary distance, giving a biometric ratio of 1:1.35 and 1:1.41 respectively. The least correlation was between the inner canthal distance and the tips of the canines measured on the photograph. Conclusions: Extra oral anthropometric measurements of the interpupillary distances and the intercommissural distances with the help of standardised photographs can help us determine the combined widths of the anterior teeth accurately, thus aiding their selection in the absence of pre‐extraction records.     

Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His(6) tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8-11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40180 ± 240 Da (without Mg(2+) ions) or 41290 ± 330 Da (with Mg(2+) ions). These values, close to the sequence-derived mass of 41711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~3.22 S for activated ERK2 with or without 10 mM MgCl(2). The frictional coefficient ratio (f/f(0)) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ~8.3 × 10(-7) cm(2) s(-1) calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~57 kDa with the cytoplasmic scaffold protein PEA-15. Thus, ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the nonphysiological His(6) tag shows substantial dimerization under the same ionic conditions.