She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast,Saccharomyces cerevisiae

She4p/Dim1p, a member of the UNC-45/CRO1/She4p (UCS) domain-containing protein family, is required for endocytosis, polarization of actin cytoskeleton, and polarization of ASH1 mRNA in Saccharomyces cerevisiae. We show herein that She4p/Dim1p is involved in endocytosis and actin polarization through interactions with the type I myosins Myo3p and Myo5p. Two-hybrid and biochemical experiments showed that She4p/Dim1p interacts with the motor domain of Myo3/5p through its UCS domain. She4p/Dim1p was required for Myo5p localization to cortical patch-like structures. Using random mutagenesis of the motor region of MYO5, we identified four independent dominant point mutations that suppress the temperature-sensitive growth phenotype of the she4/dim1 null mutant. All of the amino acid substitutions caused by these mutations, V164I, N168I, N209S, and K377M, could suppress the defects of endocytosis and actin polarization of the she4/dim1 mutant as well. She4p/Dim1p also showed two-hybrid interactions with the motor domain of a type II myosin Myo1p and type V myosins Myo2p and Myo4p, and was required for proper localization of Myo4p, which regulates polarization of ASH1 mRNA. Our results suggest that She4p/Dim1p is required for structural integrity or regulation of the motor domain of unconventional myosins.     

Changes in N-glycosylation of human stromal cells by telomerase expression

It was established that remarkable changes in the N-glycosylation are induced in immortalized cancer cells. Whether changes were induced in human stromal cells immortalized by transfection with the human telomerase catalytic subunit (hTert) cDNA was examined by lectin blot analysis. Morphological appearance and growth rate of the gene-transfected stromal cells were not changed significantly. However, lectin blot analysis of membrane glycoprotein samples showed that bindings of Ricinus communis agglutinin-I (RCA-I) and of leuko-agglutinating phytohemagglutinin to glycoprotein bands increase significantly in the gene-transfected cells. No lectin binding was observed when blotted filters were treated with diplococcal beta-1,4-galactosidase or N-glycanase prior to incubation with RCA-I. In contrast, no changes in Coomassie brilliant blue-staining and in binding of concanavalin A were obtained between the primary and gene-transfected stromal cells. These results indicate that the highly branched N-glycosylation with augmented galactosylation is induced in human stromal cells immortalized by the telomerase expression.     

Bcl-xL gene transfer protects the heart against ischemia/reperfusion injury

Ischemia and reperfusion (I/R) injury causes the progression of cardiac dysfunction. The prevention of cardiomyocyte-loss due to I/R injury is important for the treatment of heart failure. Therefore, we employed antiapoptotic Bcl-xL protein to prevent I/R injury in the heart and evaluated the cardioprotective effect of Bcl-xL transduction by adenoviral vector (Adv) after I/R injury. Adv with Bcl-xL gene was injected in the rat heart 4 days prior to I/R. The prevention of cardiac performance-loss and the reduction of cardiac apoptosis, after 30min ischemia and 30min reperfusion of global I/R, were demonstrated in the heart with adenoviral Bcl-xL transduction. Also, significant reductions of the infarct size and serum creatine kinase levels were observed in the heart transduced with Bcl-xL gene compared with control after 30min ischemia and 24h reperfusion of the left anterior coronary artery. Thus, Bcl-xL may serve as a potential therapeutic tool for cardioprotection.     

Hybrid respiration in the denitrifying mitochondria of Fusarium oxysporum

Induction of the mitochondrial nitrate-respiration (denitrification) system of the fungus Fusarium oxysporum requires the supply of low levels of oxygen (O(2)). Here we show that O(2) and nitrate (NO(3)(-)) respiration function simultaneously in the mitochondria of fungal cells incubated under hypoxic, denitrifying conditions in which both O(2) and NO(3)(-) act as the terminal electron acceptors. The NO(3)(-) and nitrite (NO(2)(-)) reductases involved in fungal denitrification share the mitochondrial respiratory chain with cytochrome oxidase. F. oxysporum cytochrome c(549) can serve as an electron donor for both NO(2)(-) reductase and cytochrome oxidase. We are the first to demonstrate hybrid respiration in respiring eukaryotic mitochondria.     

Over-expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants

Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.     

Determination of free N-acetylneuraminic acid in urine by high-performance liquid chromatography using 3-[(1-[[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent

A highly sensitive high-performance liquid chromatography (HPLC) method for the determination of urinary N-acetylneuraminic acid (NeuAc) using 3-[(1-[[4-(5,6-dimethoxy-1-oxoisoindolin-2-yl)-2-methoxyphenyl]sulfonyl]pyrrolidin-2-yl)carbonylamino]phenylboronic acid as a fluorescent labeling reagent was developed. The labeling reaction was carried out at 30 degrees C for 30 min in the presence of pyridine. The derivative was monitored at Ex 314 nm and Em 388 nm. The detection limit of NeuAc was about 48 fmol per injection. The relative standard deviations of within-day and between-day precisions were 2.6-3.3 and 1.7-3.3%, respectively. Urine diluted 10 times with distilled water was analyzed by employing the standard-addition method. The concentrations were 8-89 nmol/mg creatinine (30+/-28 nmol/mg creatinine, n=9).