The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols

A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.     

Phosphorylation of phospholamban at threonine-17 reduces cardiac adrenergic contractile responsiveness in chronic pressure overload-induced hypertrophy

Physiological hemodynamic stress, such as aerobic exercise, is intermittent and requires an increase in Ca2+ -dependent contractility through sympathetic nervous system activation. Pathological hemodynamic stress, such as hypertension, is persistent and requires sustained increases in cardiac function. Over time, this causes left ventricular hypertrophy (LVH)-reduced responsiveness to sympathetic stimulation. In this study, we examined the hypothesis that blunted in vivo adrenergic contractile responsiveness in pressure overload (PO)-induced cardiac hypertrophy is caused by abnormalities in the abundance and/or basal phosphorylation state of Ca2+ regulatory proteins. PO, induced by aortic constriction, caused concentric LVH or dilated LVH. Only animals with dilation exhibited a decrease in baseline left ventricle function [fractional area change (FAC); measured with echocardiography]. All PO animals had a reduced contractile response to adrenergic agonists (increase in FAC with 40 microg.kg(-1).min(-1) dobutamine, control 0.30 +/- 0.04, n = 5 vs. banded 0.10 +/- 0.03, n = 10; P < 0.01). PO animals had reduced phospholamban (PLB) protein abundance (P = 0.07, not significant) and increased PLB phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLB-Thr17, P < 0.05) but not at the protein kinase A-specific site (PLB-Ser16). PLB-Thr17 phosphorylation was inversely correlated with dobutamine-induced increases in contractility in PO animals (r2 = 0.81, P < 0.05). Continuous induction of Ca2+ transients in isolated ventricular myocytes for 24 h increased phosphorylation at PLB-Thr17 and diminished inotropic responsiveness and PLB-Ser16 phosphorylation after exposure to isoproterenol (P < 0.05). These data show that reduced adrenergic responsiveness in feline PO hypertrophy and failure involves increases in basal PLB-Thr17 phosphorylation, suggesting that activation of CaMKII in PO hypertrophy contributes to defective adrenergic reserve in compensated LVH and early heart failure.     

Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the "subcellular AQPs". In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72+/-0.03x10(-13) cm(3)/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

Assortment of phosphatidylinositol 3-kinase complexes--Atg14p directs association of complex I to the pre-autophagosomal structure in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.     

Energetics and volume changes of the intermediates in the photolysis of octopus rhodopsin at a physiological temperature

Enthalpy changes (Delta H) of the photointermediates that appear in the photolysis of octopus rhodopsin were measured at physiological temperatures by the laser-induced transient grating method. The enthalpy from the initial state, rhodopsin, to bathorhodopsin, lumirhodopsin, mesorhodopsin, transient acid metarhodopsin, and acid metarhodopsin were 146 +/- 15 kJ/mol, 122 +/- 17 kJ/mol, 38 +/- 8 kJ/mol, 12 +/- 5 kJ/mol, and 12 +/- 5 kJ/mol, respectively. These values, except for lumirhodopsin, are similar to those obtained for the cryogenically trapped intermediate species by direct calorimetric measurements. However, the Delta H of lumirhodopsin at physiological temperatures is quite different from that at low temperature. The reaction volume changes of these processes were determined by the pulsed laser-induced photoacoustic method along with the above Delta H values. Initially, in the transformation between rhodopsin and bathorhodopsin, a large volume expansion of +32 +/- 3 ml/mol was obtained. The volume changes of the subsequent reaction steps were rather small. These results are compared with the structural changes of the chromophore, peptide backbone, and water molecules within the membrane helixes reported previously.     

Exogenous cytosine deaminase gene expression in Bifidobacterium breve I-53-8w for tumor-targeting enzyme/prodrug therapy

Bifidobacteria are nonpathogenic, anaerobic domestic bacteria with health-promoting properties for the host. In our previous study, Bifidobacterium longum (B. longum) were found to be localized selectively and to proliferate within solid tumors after systemic application. Additionally, B. longum transformed by shuttle-plasmid including the cytosine deaminase (CD) gene expressed active CD, converted the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). We also demonstrated antitumor efficacy with a transformant of B. longum in rats. In this study, we found that Bifidobacterium breve (B. breve), the smallest species of human-derived bifidobacterium, expressed the exogenous transgene (CD), that CD enzymatic activity in the transformant of B. breve was much higher, and that the segregational stability of the plasmid was greater than that of B. longum. Thus, numerous transformants of B. breve were detected solely in the tumors after systemic administration. We consider the transformant of B. breve to be more beneficial in our enzyme/prodrug therapy.     

Platelet-rich plasma provides nucleus for mineralization in cultures of partially differentiated periodontal ligament cells

Summary Platelet-rich plasma (PRP) has been used to promote periodontal regeneration following the premise that constituent transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor-AB will stimulate cell proliferation at the site of application. In previous studies, we demonstrated that PRP mimics TGF-β1 to modulate proliferation in a cell type-specific manner, that fibrin clot formation by PRP upregulates type I collagen, and that an unidentified factor(s) in PRP increases alkaline phosphatase (ALP) activity in human periodontal ligament (PDL) cell cultures. We have now examined the effects of PRP on in vitro mineralization. Platelet-rich plasma and PDL cells were prepared from human adult volunteers or rats. After 20 d of continuous treatment with PRP in dexamethazone (Dex)-containing osteogenic medium, PRP time dependently promoted mineralization by rat PDL cells but failed to fully induce the osteoblastic phenotype. Furthermore, when human PDL cells were induced to increase ALP activity in osteogenic medium that lacked Dex, a condition that should delay (or suppress) osteoblastic differentiation, transmission electron microscopy revealed that mineralized spicules were initially deposited onto PRP-derived platelet aggregates. Taken together with our previous data, these findings suggest that PRP provides platelet aggregates as nuclei to initiate mineralization while stimulating PDL cell proliferation, differentiation, and collagen production. The combination of these effects may effectively mediate PRP's ability to promote regeneration of periodontal tissue, including skeletal tissue, at the site of injury.     

Nardilysin enhances ectodomain shedding of heparin-binding epidermal growth factor-like growth factor through activation of tumor necrosis factor-alpha-converting enzyme

Like other members of the epidermal growth factor family, heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a transmembrane protein that can be shed enzymatically to release a soluble growth factor. Ectodomain shedding is essential to the biological functions of HB-EGF and is strictly regulated. However, the mechanism that induces the shedding remains unclear. We have recently identified nardilysin (N-arginine dibasic convertase (NRDc)), a metalloendopeptidase of the M16 family, as a protein that specifically binds HB-EGF (Nishi, E., Prat, A., Hospital, V., Elenius, K., and Klagsbrun, M. (2001) EMBO J. 20, 3342-3350). Here, we show that NRDc enhances ectodomain shedding of HB-EGF. When expressed in cells, NRDc enhanced the shedding in cooperation with tumor necrosis factor-alpha-converting enzyme (TACE; ADAM17). NRDc formed a complex with TACE, a process promoted by phorbol esters, general activators of ectodomain shedding. NRDc enhanced TACE-induced HB-EGF cleavage in a peptide cleavage assay, indicating that the interaction with NRDc potentiates the catalytic activity of TACE. The metalloendopeptidase activity of NRDc was not required for the enhancement of HB-EGF shedding. Notably, a reduction in the expression of NRDc caused by RNA interference was accompanied by a decrease in ectodomain shedding of HB-EGF. These results indicate the essential role of NRDc in HB-EGF ectodomain shedding and reveal how the shedding is regulated by the modulation of sheddase activity.     

A novel approach and protocol for discovering extremely low-abundance proteins in serum

The proteomic analysis of serum (plasma) has been a major approach to determining biomarkers essential for early disease diagnoses and drug discoveries. The determination of these biomarkers, however, is analytically challenging since the dynamic concentration range of serum proteins/peptides is extremely wide (more than 10 orders of magnitude). Thus, the reduction in sample complexity prior to proteomic analyses is essential, particularly in analyzing low-abundance protein biomarkers. Here, we demonstrate a novel approach to the proteomic analyses of human serum that uses an originally developed serum protein separation device and a sequentially linked 3-D-LC-MS/MS system. Our hollow-fiber-membrane-based serum pretreatment device can efficiently deplete high-molecular weight proteins and concentrate low-molecular weight proteins/peptides automatically within 1 h. Four independent analyses of healthy human sera pretreated using this unique device, followed by the 3-D-LC-MS/MS successfully produced 12 000-13 000 MS/MS spectra and hit around 1800 proteins (>95% reliability) and 2300 proteins (>80% reliability). We believe that the unique serum pretreatment device and proteomic analysis protocol reported here could be a powerful tool for searching physiological biomarkers by its high throughput (3.7 days per one sample analysis) and high performance of finding low abundant proteins from serum or plasma samples.