Mesenchymal stem cells: therapeutic outlook for stroke

Adult bone marrow-derived mesenchymal stem cells (MSCs) display a spectrum of functional properties. Transplantation of these cells improves clinical outcome in models of cerebral ischemia and spinal cord injury via mechanisms that may include replacement of damaged cells, neuroprotective effects, induction of axonal sprouting, and neovascularization. Therapeutic effects have been reported in animal models of stroke after intravenous delivery of MSCs, including those derived from adult human bone marrow. Initial clinical studies on intravenously delivered MSCs have now been completed in human subjects with stroke. Here, we review the reparative and protective properties of transplanted MSCs in stroke models, describe initial human studies on intravenous MSC delivery in stroke, and provide a perspective on prospects for future progress with MSCs.     

Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB

Background and Aims: Fluoroquinolone‐containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87and 91 of GyrA among fluoroquinolone‐resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Materials & Methods: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Results: Norfloxacin‐resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin‐resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Conclusion: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.     

Genome-wide analysis and expression profiling of half-size ABC protein subgroup G in rice in response to abiotic stress and phytohormone treatments

The roles of the proteins encoded by half-size adenosine triphosphate-binding cassette transporter subgroup G (ABCG) genes in abiotic stress responses are starting to be established in the dicot model Arabidopsis thaliana. In the monocot model rice, the functions of most half-size ABCG proteins in abiotic stress responses are unknown. Rcn1/OsABCG5 is an essential transporter for growth and development under abiotic stress, but its molecular function remains largely unclear. Here, we present a comprehensive overview of all 30 half-size ABCG genes in rice, including their gene structures, phylogeny, chromosome locations, and conserved motifs. Phylogenetic analysis revealed that the half-size OsABCG proteins were divided to four classes. All seven rice intronless genes, including Rcn1/OsABCG5, were in Class III, like the 12 intronless ABCG genes of Arabidopsis. The EST and FL-cDNA databases provided expression information for 25 OsABCG genes. Semi-quantitative and quantitative RT-PCR analyses demonstrated that seven OsABCG genes were up-regulated in seedlings, shoots or roots following treatments with abiotic stresses (6, 17, 42?°C, NaCl, or mannitol) and abscisic acid. Another 15 OsABCG genes were up-regulated under at least one of the abiotic stress conditions and other phytohormones besides abscisic acid. Hierarchical clustering analysis of gene expression profiles showed that expression of the OsABCG genes could be classified into four clusters. The Rcn1/OsABCG5 cluster was up-regulated by abscisic acid and included OsABCG2, 3, 13, and 27. The present study will provide a useful reference for further functional analysis of the ABCGs in monocots.     

Association of the physical and chemical properties and the cytotoxicity of metal oxide nanoparticles: metal ion release, adsorption ability and specific surface area

Association of cellular influences and physical and chemical properties were examined for 24 kinds of industrial metal oxide nanoparticles: ZnO, CuO, NiO, Sb(2)O(3), CoO, MoO(3), Y(2)O(3), MgO, Gd(2)O(3), SnO(2), WO(3), ZrO(2), Fe(2)O(3), TiO(2), CeO(2), Al(2)O(3), Bi(2)O(3), La(2)O(3), ITO, and cobalt blue pigments. We prepared a stable medium dispersion for each nanoparticle and examined the influence on cell viability and oxidative stress together with physical and chemical characterizations. ZnO, CuO, NiO, MgO, and WO(3) showed a large amount of metal ion release in the culture medium. The cellular influences of these soluble nanoparticles were larger than insoluble nanoparticles. TiO(2), SnO(2), and CeO(2) nanoparticles showed strong protein adsorption ability; however, cellular influences of these nanoparticles were small. The primary particle size and the specific surface area seemed unrelated to cellular influences. Cellular influences of metal oxide nanoparticles depended on the kind and concentrations of released metals in the solution. For insoluble nanoparticles, the adsorption property was involved in cellular influences. The primary particle size and specific surface area of metal oxide nanoparticles did not affect directly cellular influences. In conclusion the most important cytotoxic factor of metal oxide nanoparticles was metal ion release.     

Direct Labeling of Polyphosphate at the Ultrastructural Level in Saccharomyces cerevisiae by Using the Affinity of the Polyphosphate Binding Domain of Escherichia coli Exopolyphosphatase

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affinity for short-chain polyP and nucleic acids. To directly demonstrate polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze-substitution, specimens were labeled with PPBD containing an epitope tag and then the epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse immunoglobulin G antibody conjugated with Alexa 488 for laser confocal microscopy or with colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-peptone-dextrose medium (10 mM phosphate) for 10 h, polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of polyP at the electron microscopic level for the first time and enabled the visualization of polyP localization with much higher specificity and resolution than with other conventional methods.     

Preliminary study on female choice of mates versus sites in a wrasse, Cirrhilabrus temminckii

Female choice of mates versus sites was studied in a wrasse, Cirrhilabrus temminckii. Males had territories within a restricted area on a rocky slope at which females visited and pair-spawned pelagic gametes. Females visited several males or territories before spawning, suggesting the opportunity of female choice. Of the four characteristics of territorial males examined—body size, ratio of pelvic fin length to body size, courtship, frequency and territory depth—only territory depth was significantly correlated with daily mating success of males. The former three male characters were not related to territory depth. These results suggest that female C. temminckii chooses deep sites rather than specific mates in mating.     

Up-regulation of hyaluronan synthase genes in cultured human epidermal keratinocytes by UVB irradiation

Hyaluronan controls keratinocyte proliferation and regeneration. We examined effect of UV on the expression of hyaluronan synthases (HASs) and hyaluronidases in cultured normal human newborn foreskin epidermal keratinocytes, NHEK(F). HAS3 mRNA was expressed predominantly and HAS2 mRNA expressed in lesser amounts and both were up-regulated after a single irradiation with moderate UVB but hyaluronidases was unchanged. Increased accumulation of hyaluronan in the culture medium mirrored the UVB-induced increase in the mRNA levels of HAS3 and HAS2. Unexpectedly, hyaluronan derived from UVB-irradiated and non-irradiated cells had identical size distribution. Increased expression of KGF and IL-1β was detected just prior to the increase of HAS3 and HAS2 mRNAs after UVB irradiation. Antibody-neutralization study revealed that KGF and/or IL-1β were at least involved in the up-regulation of HAS3 and HAS2 expressions. UVB-irradiated cells may enhance hyaluronan production to maintain homeostasis through up-regulation of HAS3 and HAS2 genes via cytokine response mechanism.     

Expression of POTE protein in human testis detected by novel monoclonal antibodies

The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2γC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2γC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.     

Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers

The cell permeability of hesperetin and hesperidin, anti-allergic compounds from citrus fruits, was measured using Caco-2 monolayers. In the presence of a proton gradient, hesperetin permeated cells in the apical-to-basolateral direction at the rate (Jap-->bl) of 10.43+/-0.78 nmol/min/mg protein, which was more than 400-fold higher than that of hesperidin (0.023+/-0.008 nmol/min/mg protein). The transepithelial flux of hesperidin, both in the presence or absence of a proton gradient, was nearly the same and was inversely correlated with the transepithelial electrical resistance (TER), indicating that the transport of hesperidin was mainly via paracellular diffusion. In contrast, the transepithelial flux of hesperetin was almost constant irrespective of the TER. Apically loaded NaN3 or carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the Jap-->bl of hesperetin, in the presence of proton gradient, by one-half. In the absence of a proton gradient, both Jap-->bl and Jbl-->ap of hesperetin were almost the same (5.75+/-0.40 and 5.16+/-0.73 nmol/min/mg protein). Jbl-->ap of hesperetin in the presence of a proton gradient was lower than Jbl-->ap in the absence of a proton gradient. Furthermore, Jbl-->ap in the presence of a proton gradient remarkably increased upon addition of NaN3 specifically to the apical side. These results indicate that hesperetin is absorbed by transcellular transport, which occurs mainly via proton-coupled active transport, and passive diffusion. Thus, hesperetin is efficiently absorbed from the intestine, whereas hesperidin is poorly transported via the paracellular pathway and its transport is highly dependent on conversion to hesperetin via the hydrolytic action of microflora. We have given novel insight to the absorption characteristics of hesperetin, that is proton-coupled and energy-dependent polarized transport.