Disturbance of sphingolipid biosynthesis abrogates the signaling of Mss4, phosphatidylinositol-4-phosphate 5-kinase, in yeast

The functional relationships between phosphoinositides and sphingolipids have not been well characterized to date. ISP-1/myriocin is a potent inhibitor of sphingolipid biosynthesis and induces severe growth defects in eukaryotic cells because of the sphingolipid deprivation. We characterized a novel multicopy suppressor gene of ISP-1-mediated cell death in yeast, MSS4. MSS4 encodes a phosphatidylinositol-4-phosphate 5-kinase that synthesizes phosphatidylinositol (4,5)-bisphosphate (PI4,5P(2)). We demonstrate here that ISP-1 treatment of yeast causes defects both in the activity and subcellular localization of Mss4. The effect of the Mss4 defect on the downstream signaling was examined, because interaction between the Mss4 product, PI4,5P(2), and the pleckstrin-homology domain of Rom2 mediates recruitment of Rom2 to the membrane, which is the crucial step for subsequent Rho1/2 activation. Indeed, failure of Rom2 recruitment was observed in ISP-1-treated cells as well as in csg2-deleted cells, which have reduced mannosylated inositolphosphorylceramide. These data suggested that proper sphingolipids are required for the signaling pathway involving Mss4.     

Spectral differentiation of blue opsins between phylogenetically close but ecologically distant goldfish and zebrafish

Zebrafish and goldfish are both diurnal freshwater fish species belonging to the same family, Cyprinidae, but their visual ecological surroundings considerably differ. Zebrafish are surface swimmers in conditions of broad and shortwave-dominated background spectra and goldfish are generalized swimmers whose light environment extends to a depth of elevated short wavelength absorbance with turbidity. The peak absorption spectrum (lambdamax) of the zebrafish blue (SWS2) visual pigment is consistently shifted to short wavelength (416 nm) compared with that of the goldfish SWS2 (443 nm). Among the amino acid differences between the two pigments, only one (alanine in zebrafish and serine in goldfish at residue 94) was previously known to cause a difference in absorption spectrum (14-nm lambdamax shift in newt SWS2). In this study, we reconstructed the ancestral SWS2 pigment of the two species by applying likelihood-based Bayesian statistics and performing site-directed mutagenesis. The reconstituted ancestral photopigment had a lambdamax of 430 nm, indicating that zebrafish and goldfish achieved short wavelength (-14 nm) and long wavelength (+13 nm) spectral shifts, respectively, from the ancestor. Unexpectedly, the S94A mutation resulted in only a -3-nm spectral shift when introduced into the goldfish SWS2 pigment. Nearly half of the long wavelength shift toward the goldfish pigment was achieved instead by T116L (6 nm). The S295C mutation toward zebrafish SWS2 contributed to creating a ridge of absorbance around 400 nm and broadening its spectral sensitivity in the short wavelength direction. These results indicate that the evolutionary engineering approach is very effective in deciphering the process of functional divergence of visual pigments.     

Transgenic studies of alpha(1)-adrenergic receptor subtype function

Mice with altered alpha(1)-adrenergic receptor (AR) genes have become important tools in elucidating the subtype-specific functions of the three alpha(1)-AR subtypes because of the lack of sufficiently subtype-selective pharmacological agents. Mice with a deletion (knockout, KO) or an overexpression (transgenic, TG) of the alpha(1A)-, alpha(1B)-, or alpha(1D)-AR subtypes have been generated. The alpha(1)-ARs are the principal mediators of the hypertensive response to alpha(1)-agonists in the cardiovascular system. Studies with these mice indicate that alpha(1A)-AR and alpha(1B)-AR subtypes play an important role in cardiac development and/or function as well as in blood pressure (BP) response to alpha(1)-agonists via vasoconstriction. The alpha(1B)- and alpha(1D)-subtypes also appear to be involved in central nervous system (CNS) processes such as nociceptive responses, modulation of memory consolidation and working memory. The ability to study subtype-specific functions in different mouse strains by altering the same alpha(1)-AR in different ways strengthens the conclusions drawn from these studies. Although these genetic approaches have limitations, they have significantly increased our understanding of the functions of alpha(1)-AR subtypes.     

Apoptosis of CTLL-2 cells induced by an immunosuppressant, ISP-I, is caspase-3-like protease-independent

In our previous study, the sphingosine-like immunosuppressant ISP-1 was shown to induce apoptosis in the mouse cytotoxic T cell line CTLL-2. In this study, we characterized the ISP-1-induced apoptotic pathway. Although caspase-3-like protease activity increases concomitantly with ISP-1-induced apoptosis in CTLL-2 cells, the apoptosis is not inhibited by caspase-3-like protease inhibitors, i.e. DEVD-cho and z-DEVD-fmk. In contrast, sphingosine-induced apoptosis in CTLL-2 cells is caspase-3-like protease-dependent. A caspase inhibitor with broad specificity, z-VAD-fmk, protects cells from apoptosis induced by ISP-1, indicating that ISP-1-induced apoptosis is dependent on caspase(s) other than caspase-3. Overexpression of Bcl-2 or Bcl-xL suppresses the apoptosis induced by ISP-1, although sphingosine-induced apoptosis is not efficiently inhibited by Bcl-2. Finally, ISP-1-induced mitochondrial depolarization, which is thought to be a checkpoint dividing the apoptotic pathway into upstream and downstream stages, is not inhibited by DEVD-cho, but is inhibited by z-VAD-fmk. These data suggest that a pathway dependent on caspase(s) other than caspase-3 is involved upstream of mitochondrial depolarization in ISP-1-induced apoptosis.     

Analysis of the Genetic Diversity in the Vigna minima Complex and Related Species in East Asia

Vigna subgenus Ceratotropis (V. angularis, V. minima, V. nakashimae, V. reflexo-pilosa, V. riukiuensis and V. umbellata) was investigated using AFLP methodology. Species in the V. minima complex, V. minima, V. nakashimae and V. riukiuensis, had greater intra-specific polymorphism than the other species analyzed. Vigna species from East Asia analysed could be clearly distinguished. The relationship between species was consistent using different analyses and showed V. riukiuensis and V. nakashimae are closely related. The phenetic distance between species (1-Jaccard's similarity coefficient) ranged from 0.279 between V. riukiuensis and V. nakashimae to 0.688 between V. reflexo-pilosa and V. minima. Genetic distance based on inferred nucleotide diversity (π) ranged from 0.012 between V. riukiuensis and V. nakashimae and 0.053 between both V. riukiuensis and V. nakashimae and V. reflexo-pilosa. The diversity (Ha) within species, based on Shannon's information index of phenotypic diversity, was lowest in V. reflexo-pilosa (0.006) and highest in V. minima (0.095). Korean and Japanese populations of V. nakashimae had distinct polymorphism. Vigna riukiuensis populations from Miyako island were genetically different from populations of other islands of southern Okinawa. Received 21 April 2000/ Accepted in revised form 5 September 2000     

Out of the Orient: Post‐Tethyan transoceanic and trans‐Arabian routes fostered the spread of Baorini skippers in the Afrotropics

The origin of taxa presenting a disjunct distribution between Africa and Asia has puzzled biogeographers for more than a century. This biogeographic pattern has been hypothesized to be the result of transoceanic long‐distance dispersal, Oligocene dispersal through forested corridors, Miocene dispersal through the Arabian Peninsula or passive dispersal on the rifting Indian plate. However, it has often been difficult to pinpoint the mechanisms at play. We investigate biotic exchange between the Afrotropics and the Oriental region during the Cenozoic, a period in which geological changes altered landmass connectivity. We use Baorini skippers (Lepidoptera, Hesperiidae) as a model, a widespread clade of butterflies in the Old World tropics with a disjunct distribution between the Afrotropics and the Oriental region. We use anchored phylogenomics to infer a robust evolutionary tree for Baorini skippers and estimate divergence times and ancestral ranges to test biogeographic hypotheses. Our phylogenomic tree recovers strongly supported relationships for Baorini skippers and clarifies the systematics of the tribe. Dating analyses suggest that these butterflies originated in the Oriental region, Greater Sunda Islands, and the Philippines in the early Miocene c. 23 Ma. Baorini skippers dispersed from the Oriental region towards Africa at least five times in the past 20 Ma. These butterflies colonized the Afrotropics primarily through trans‐Arabian geodispersal after the closure of the Tethyan seaway in the mid‐Miocene. Range expansion from the Oriental region towards the African continent probably occurred via the Gomphotherium land bridge through the Arabian Peninsula. Alternative scenarios invoking long‐distance dispersal and vicariance are not supported. The Miocene climate change and biome shift from forested areas to grasslands possibly facilitated geodispersal in this clade of butterflies.     

Cinnamtannin B-1 Promotes Migration of Mesenchymal Stem Cells and Accelerates Wound Healing in Mice

Substances that enhance the migration of mesenchymal stem cells to damaged sites have the potential to improve the effectiveness of tissue repair. We previously found that ethanol extracts of Mallotus philippinensis bark promoted migration of mesenchymal stem cells and improved wound healing in a mouse model. We also demonstrated that bark extracts contain cinnamtannin B-1, a flavonoid with in vitro migratory activity against mesenchymal stem cells. However, the in vivo effects of cinnamtannin B-1 on the migration of mesenchymal stem cells and underlying mechanism of this action remain unknown. Therefore, we examined the effects of cinnamtannin B-1 on in vivo migration of mesenchymal stem cells and wound healing in mice. In addition, we characterized cinnamtannin B-1-induced migration of mesenchymal stem cells pharmacologically and structurally. The mobilization of endogenous mesenchymal stem cells into the blood circulation was enhanced in cinnamtannin B-1-treated mice as shown by flow cytometric analysis of peripheral blood cells. Whole animal imaging analysis using luciferase-expressing mesenchymal stem cells as a tracer revealed that cinnamtannin B-1 increased the homing of mesenchymal stem cells to wounds and accelerated healing in a diabetic mouse model. Additionally, the cinnamtannin B-1-induced migration of mesenchymal stem cells was pharmacologically susceptible to inhibitors of phosphatidylinositol 3-kinase, phospholipase C, lipoxygenase, and purines. Furthermore, biflavonoids with similar structural features to cinnamtannin B-1 also augmented the migration of mesenchymal stem cells by similar pharmacological mechanisms. These results demonstrate that cinnamtannin B-1 promoted mesenchymal stem cell migration in vivo and improved wound healing in mice. Furthermore, the results reveal that cinnamtannin B-1-induced migration of mesenchymal stem cells may be mediated by specific signaling pathways, and the flavonoid skeleton may be relevant to its effects on mesenchymal stem cell migration.     

S100A10 protein expression is associated with oxaliplatin sensitivity in human colorectal cancer cells

Background

C-reactive protein (CRP) is a predictor of cardiovascular risk. It circulates as a pentameric protein in plasma. Recently, a potential dissociation mechanism from the disc-shaped pentameric CRP (pCRP) into single monomers (monomeric or mCRP) has been described. It has been shown that mCRP has strong pro-inflammatory effects on monocytes. To further define the role of mCRP in determining monocyte phenotype, the effects of CRP isoforms on THP-1 protein expression profiles were determined. The hypothesis to be tested was that mCRP induces specific changes in the protein expression profile of THP-1 cells that differ from that of pCRP.

Methods

Protein cell lysates from control and mCRP, pCRP or LPS-treated THP-1 cells were displayed using 2-dimensional SDS PAGE and compared. Differentially expressed proteins were identified by MALDI-TOF MS and confirmed by Western blotting.

Results

mCRP significantly up-regulates ubiquitin-activating enzyme E1, a member of the ubiquitin-proteasome system in THP-1 monocytes. Furthermore, HSP 70, alpha-actinin-4 (ACTN4) and alpha-enolase/enolase 1 were upregulated. The proteomic profile of LPS and pCRP treated monocytes differ significantly from that of mCRP.

Conclusion

The data obtained in this study support the hypothesis that isoform-specific effects of CRP may differentially regulate the phenotype of monocytes.