Cloning and Characterization of the Xyn11A Gene from <Emphasis Type="Italic">Lentinula edodes</Emphasis>

Hemicellulose represents a rich source of biomass that can be converted into useful chemical feedstocks. One of the main components of hemicellulose is xylan, a polymer of xylose residues. Xylanase enzymes that hydrolyze xylan are therefore of great commercial interest. We have cloned a gene (xyn11A) that encodes a 283-amino acid xylanase enzyme from the fungus Lentinula edodes. The enzyme has a pI of 4.6 and belongs to the highly conserved glycosyl hydrolase family 11. The xylanase gene was cloned into a Pichia pastoris expression vector that secretes active enzyme into both solid and liquid media. The optimal reaction conditions were at pH 4.5 and 50°C. The enzyme had a K_m of 1.5 mg/ml and a V_max of 2.1 mmol/min/mg. Xyn11A produced primarily xylobiose, xylotriose, and xylotetraose from a birchwood xylan substrate. This is the first report on the cloning of a hemicellulase gene from L. edodes.     

Reprogramming mediated by stem cell fusion

Advances in mammalian cloning prove that somatic nuclei can be reprogrammed to a state of totipotency by transfer into oocytes. An alternative approach to reprogram the somatic genome involves the creation of hybrids between somatic cells and other cells that contain reprogramming activities. Potential fusion partners with reprogramming activities include embryonic stem cells, embryonic germ cells, embryonal carcinoma cells, and even differentiated cells. Recent advances in fusion-mediated reprogramming are discussed from the standpoints of the developmental potency of hybrid cells, genetic and epigenetic correlates of reprogramming, and other aspects involved in the reprogramming process. In addition, the utility of fusion-mediated reprogramming for future cell-based therapies is discussed.     

Proteomics analysis of growth hormone isoforms in the human pituitary

In order to elucidate the roles of human growth hormone (hGH) in the normal (control) pituitary and in adenomas, the hGH isoforms in the human pituitary were analyzed with two-dimensional gel electrophoresis, immobilized metal affinity column (Ga(+3)) chromatography, mass spectrometry (MS), and bioinformatics. Twenty-four hGH-containing proteins, with significantly different expression proportions of their isoforms were found. The proportions of isoforms were as follows: isoform 1 (87.5%) > isoform 2 (8.1%) > isoform 3 (3.3%) > isoform 4 (1.1%). Deamidation of asparagine to aspartate was identified with matrix-assisted laser desorption/ionization-time of flight MS. Tandem mass spectrometry data demonstrated that hGH is a phosphoprotein (spot 6); phosphorylation was found at Ser-77 in the tryptic peptide (68)YSFLQNPQTSLCFSESIPTPSNR(90), at Ser-176 in the tryptic peptide (172)FDTNSHNDDALLK(184), and at Ser-132 in the peptide (126)SLVYGASDSNVYDLLK(141). The phosphorylation sites at Ser-77 and Ser-176 were consistent with computer-program predictions (NetPhos). These results provide novel clues for further studies of the functions, and mechanisms of action, of hGH in the human pituitary and in growth hormone-related diseases.     

Determination of peptide substrate specificity for mu-calpain by a peptide library-based approach: the importance of primed side interactions

Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca(2+) signaling. Because of their involvement in pathological conditions such as post-ischemic injury and Alzheimer and Parkinson disease, calpains form a class of pharmacologically significant targets for inhibition. We have determined the sequence preference for the hydrolysis of peptide substrates of the ubiquitous mu-calpain isoform by a peptide library-based approach using the proteolytic core of mu-calpain (muI-II). The approach, first described by Turk et al. (Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C. (2001) Nat. Biotechnol. 19, 661-667), involved the digestion of an N-terminally acetylated degenerate peptide library in conjunction with Edman sequencing to determine the specificity for residues found at primed positions. The cleavage consensus for these positions was then used to design a second, partially degenerate library, to determine specificity at unprimed positions. We have improved upon the original methodology by using a degenerate peptide dendrimer for determination of specificity at unprimed positions. By using this modified approach, the complete cleavage specificity profile for muI-II was determined for all positions flanking the cleaved peptide. A previously known preference of calpains for hydrophobic amino acids at unprimed positions was confirmed. In addition, a novel residue specificity for primed positions was revealed to highlight the importance of these sites for substrate recognition. The optimal primed site motif (MER) was shown to be capable of directing cleavage to a specific peptide bond. Accordingly, we designed a fluorescent resonance energy transfer-based substrate with optimal cleavage motifs on the primed and non-primed sides (PLFAER). The mu-calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of alpha-spectrin or the proteolytic sequence consensus compiled from substrate alignments.     

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.     

Angiotensin-converting enzyme inhibitor captopril prevents volume overload cardiomyopathy in experimental chronic aortic valve regurgitation

The efficacy of angiotensin-converting enzyme inhibitors (ACEIs) in the treatment of chronic aortic regurgitation (AR) is not well established and remains controversial. The mechanisms by which ACEIs may protect against left-ventricular (LV) volume overload are not well understood, and clinical trials performed until now have yielded conflicting results. This study was therefore performed to assess the effectiveness of two different doses of the ACEI captopril in a rat model of chronic AR. We compared the effects of a 6-month low-dose (LD) (25 mg/kg) or higher dose (HD) (75 mg/kg) treatment with captopril on LV function and hypertrophy in Wistar rats with severe AR. Untreated animals developed LV eccentric hypertrophy and systolic dysfunction. LD treatment did not prevent hypertrophy and provided modest protection against systolic dysfunction. HD treatment preserved LV systolic function and dimensions and tended to slow hypertrophy. The cardiac index remained high and similar among all AR groups, treated or not. Tissue renin-angiotensin system (RAS) analysis revealed that ACE activity was increased in the LVs of AR animals and that only HD treatment significantly decreased angiotensin II receptor mRNA levels. Fibronectin expression was increased in the LV or AR animals, but HD treatment almost completely reversed this increase. The ACE inhibitor captopril was effective at high doses in this model of severe AR. These effects might be related to the modulation of tissue RAS and the control of fibrosis.     

Assembly of the MexAB-OprM multidrug efflux system of Pseudomonas aeruginosa: identification and characterization of mutations in mexA compromising MexA multimerization and interaction with MexB

The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of P. aeruginosa is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization). In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of Escherichia coli, AcrA, that is implicated in binding to its RND component, AcrB (C. A. Elkins and H. Nikaido, J. Bacteriol. 185:5349-5356, 2003). Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important for and define the MexA interaction with its RND component, MexB. Still, all but one of the MexA mutations studied compromised MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this.     

Hypertriglyceridemia in lecithin-cholesterol acyltransferase-deficient mice is associated with hepatic overproduction of triglycerides, increased lipogenesis, and improved glucose tolerance

Lecithin-cholesterol acyltransferase deficiency is frequently associated with hypertriglyceridemia (HTG) in animal models and humans. We investigated the mechanism of HTG in the ldlr-/- x lcat-/- (double knockout (dko)) mice using the ldlr-/- x lcat+/+ (knock-out (ko)) littermates as control. Mean fasting triglyceride (TG) levels in the dko mice were elevated 1.75-fold compared with their controls (p < 0.002). Both the very low density lipoprotein and the low density lipoprotein/intermediate density lipoprotein fractions separated by fast protein liquid chromatography were TG-enriched in the dko mice. In vitro lipolysis assay revealed that the dko mouse very low density lipoprotein (d < 1.019 g/ml) fraction separated by ultracentrifugation was a more efficient substrate for lipolysis by exogenous bovine lipoprotein lipase. Post-heparin lipoprotein lipase activity was reduced by 61% in the dko mice. Hepatic TG production rate, determined after intravenous Triton WR1339 injection, was increased 8-fold in the dko mice. Hepatic mRNA levels of sterol regulatory element binding protein-1 (srebp-1) and its target genes acetyl-CoA carboxylase-1 (acc-1), fatty acid synthase (fas), and stearoyl-CoA desaturase-1 (scd-1) were significantly elevated in the dko mice compared with the ko control. The hepatic mRNA levels of LXRalpha (lxralpha) and its target genes including angiopoietin-like protein 3 (angptl-3) in the dko mice were unchanged. Fasting glucose and insulin levels were reduced by 31 and 42%, respectively in the dko mice, in conjunction with a 49% reduction in hepatic pepck-1 mRNA (p = 0.014). Both the HTG and the improved fasting glucose phenotype seen in the dko mice are at least in part attributable to an up-regulation of the hepatic srebp-1c gene.     

Phosphorylation of actopaxin regulates cell spreading and migration

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.