Coupled motions in the SH2 and kinase domains of Csk control Src phosphorylation

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.     

Superior molasses assimilation, stress tolerance, and trehalose accumulation of baker's yeast isolated from dried sweet potatoes (hoshi-imo)

Yeast strains were isolated from dried sweet potatoes (hoshi-imo), a traditional preserved food in Japan. Dough fermentation ability, freeze tolerance, and growth rates in molasses, which are important characteristics of commercial baker's yeast, were compared between these yeast strains and a commercial yeast derivative that had typical characteristics of commercial strains. Classification tests including pulse-field gel electrophoresis and fermentation/assimilation ability of sugars showed that almost the stains isolated belonged to Saccharomyces cerevisiae. One strain, ONY1, accumulated intracellular trehalose at a higher level than commercial strain T128. Correlated with intracellular trehalose contents, the fermentation ability of high-sugar dough containing ONY1 was higher. ONY1 also showed higher freeze tolerance in both low-sugar and high-sugar doughs. The growth rate of ONY1 was significantly higher under batch and fed-batch cultivation conditions using either molasses or synthetic medium than that of strain T128. These results suggest that ONY1 has potential commercial use as baker's yeast for frozen dough and high-sugar dough.     

SSA/Ro52 autoantigen interacts with Dcp2 to enhance its decapping activity

We identified human decapping enzyme 2 (hDCP2) as a binding protein with Ro52, being colocalized in processing bodies (p-bodies). We also showed that the N-terminus and C-terminus of Ro52 bound to hDCP2. Moreover, Ro52 enhanced decapping activity of hDCP2 in a dose-dependent manner. Our data support the novel notion of the association between Ro52 with hDCP2 protein in cytoplasmic p-bodies, playing a role in mRNA metabolism in response to cellular stimulation.     

Molecular genetic studies on sleep-wake regulation, with special emphasis on the prostaglandin D(2) system.

To elucidate the exact role of the PGD(2) system in sleep-wake regulation in vivo, the sleep behavior of knockout mice, generated in the author's and other laboratories, was examined for lipocalin-type PGD synthase (L-PGDS), PGD receptor, adenosine A(2A) receptor, and histamine H(1) receptor; transgenic mice overexpressing the human L-PGDS gene, generated in the author's laboratory, were also examined. The circadian profiles of sleep patterns of wild-type and the genetically manipulated mice were essentially identical, indicating the possibility that the deficiency of one system may be effectively compensated by some other systems during development. Available evidence indicated that the PGD(2) system is involved in the homeostatic regulation of non-rapid eye movement sleep and that the arousal effect of orexin A is mediated by the histamine H(1) receptor system.     

Characterization of the cells in the repair tissue of full-thickness articular cartilage defects

 It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue, cells of which are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair cells biochemically, full-thickness defects were created in rabbit knee joints and the repair tissues taken at 3, 6, and 12 weeks after surgery. The repair cells were cultured and examined biochemically to investigate the effects of four exogenous growth factors with regard to the metabolism of type II collagen and proteoglycans. A significant increase of carboxy-terminal type II procollagen peptide production was observed in the conditional medium of the repair cells, especially taken at 6 weeks after surgery, in the presence of each growth factor. Glycosaminoglycan content was also increased and proteoglycan synthesis stimulated. The repair cells taken at the early stage of the repair process could originally have more activity of type II collagen synthesis, and the growth factors used could enhance the differentiation of the repair cells in vitro. Accepted: 3 November 1997     

Coordinated accumulation of the chloroplastic and cytosolic pyruvate,Pi dikinases with enhanced expression of CAM in Kalanchoë blossfeldiana

Recent studies reveal that the intracellular localization of pyruvate,Pi dikinase (PPDK, EC 2.7.9.1) in mesophyll cells of malic enzyme (ME)-dependent Crassulacean acid metabolism (CAM) plants varies among species, occurring not only in the chloroplasts but also in the cytosol in some species. The facultative CAM plant Kalanchoë blossfeldiana accumulates PPDK in both compartments of the mesophyll cells. In this study, the patterns of accumulation of the chloroplastic and cytosolic PPDKs were investigated for K. blossfeldiana plants with different CAM activities by immunogold labeling and electron microscopy. Greater CAM activity was found in plants grown under drought conditions with short days than under well-watered conditions with long days, and in lower leaves than in higher leaves. There was a trend that plants and leaves with greater CAM activity show denser labeling for PPDK in both the cytosol and chloroplasts. However, the ratio of the density of PPDK labeling in the cytosol to that in the chloroplasts was almost constant (2.4–3.0). Higher labeling for phosphoenolpyruvate carboxylase (EC 4.1.1.31) in the cytosol was also correlated with higher CAM activity but there was almost no difference in the density of labeling for ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in the chloroplasts. These results indicate that the increase in accumulation of cytosolic PPDK is closely associated with the increase of chloroplastic PPDK during enhanced CAM expression. This suggests that both PPDKs are involved in CAM function.     

DDB accumulates at DNA damage sites immediately after UV irradiation and directly stimulates nucleotide excision repair.

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC.HR23B, TFIIH, XPF.ERCC1 and XPG, up to 17-fold for CPDs and approximately 2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors.     

Simulation analysis of excitation conduction in the heart: Propagation of excitation in different tissues

The normal excitation and conduction in the heart are maintained by the coordination between the dynamics of ionic conductance of each cell and the electrical coupling between cells. To examine functional roles of these two factors, we proposed a spatially-discrete model of conduction of excitation in which the individual cells were assumed isopotential. This approximation was reasoned by comparing the apparent space constant with the measured junctional resistance between myocardial cells. We used the four reconstruction models previously reported for five kinds of myocardial cells. Coupling coefficients between adjacent cells were determined quantitatively from the apparent space constants. We first investigated to what extent the pacemaker activity of the sinoatrial node depends on the number and the coupling coefficient of its cells, by using a one-dimensional model system composed of the sinoatrial node cells and the atrial cells. Extensive computer simulation revealed the following two conditions for the pacemaker activity of the sinoatrial node. The number of the sinoatrial node cells and their coupling coefficients must be large enough to provide the atrium with the sufficient electric current flow. The number of the sinoatrial node cells must be large so that the period of the compound system is close to the intrinsic period of the sinoatrial node cell. In this simulation the same sinoatrial node cells produced action potentials of different shapes depending on where they were located in the sinoatrial node. Therefore it seems premature to classify the myocardial cells only from their waveforms obtained by electrical recordings in the compound tissue. Second, we investigated the very slow conduction in the atrioventricular node compared to, for example, the ventricle. This was assumed to be due to the inherent property of the membrane dynamics of the atrioventricular node cell, or to the small value of the coupling coefficient (weak intercellular coupling), or to the electrical load imposed on the atrioventricular node by the Purkinje fibers, because the relatively small atrioventricular node must provide the Purkinje fibers with sufficient electric current flow. Relative contributions of these three factors to the slow conduction were evaluated using the model system composed of only the atrioventricular cells or that composed of the atrioventricular and Purkinje cells. We found that the weak coupling has the strongest effect. In the model system composed of the atrioventricular cells, the propagation failure was not observed even for very small values of the coupling coefficient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of AQP6 in the Mercury-Sensitive Osmotic Lysis of Rat Parotid Secretory Granules

In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73–80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²⁺, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²⁺ (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5–2.0 μM Hg²⁺, concentrations that activate AQP6. The Hg lysis was completely blocked by β-mercaptoethanol which disrupts Hg²⁺-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO₃ ^? > Br^? > I^? > Cl^? and was facilitated by acidic pH. The anion selectivity for NO₃ ^? and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²⁺-sensitive anion channel in rat parotid secretory granules.