Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

Receptor tyrosine phosphatase gamma (PTPRG, or RPTPγ) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98–99% reduction of RPTPγ gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTPγ C1060S, replaced the wild type. We showed that human wild type RPTPγ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPγ C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPγ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPγ for use in an assay for identifying inhibitors, “substrate trapping” mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPγs retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPγ did not. This suggested that wild type RPTPγ might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTPγ. Additional investigation of RPTPγ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.     

Cytological and mapping analysis of a novel male sterile type resulting from spontaneous floral organ homeotic conversion in marigold (<Emphasis Type="Italic">Tagetes erecta</Emphasis> L.)

A male sterile line was isolated in marigold (Tagetes erecta L.) and cytological analysis determined this to be a novel genic male sterility trait (Tems). Through the use of amplified fragment length polymorphisms (AFLPs) and bulked segregant analysis (BSA), tightly linked markers of Tems were identified with a view towards a map-based cloning strategy. It was found that spontaneous homeotic conversion of floral organs was the underlying cause of the male sterility in this marigold line. Thus, petals of male sterile plants resembled sepal-like structures and the stamens were partially converted to styles, although without the full characteristics or function of the true style organs. We have constructed a fine marker-based map for the Tems gene. This is intended to provide a tool for marker assisted selection (MAS) strategies in hybrid breeding and map-based cloning strategies for the male sterility locus. We discuss the significance of this spontaneously derived genic male sterility trait relating to the homeotic conversion of floral organs in marigold.     

Photobiomodulation by diffusing optical fiber on spinal cord: A feasibility study in piglet model

Previous studies on spinal cord injury (SCI) have confirmed that percutaneous photobiomodulation (PBM) therapy can ameliorate immunoinflammatory responses at sites of injury, accelerate nerve regeneration, suppress glial scar formation and promote the subsequent recovery of locomotor function. The current study was performed to evaluate a large‐animal model employing implanted optical fibers to accurately irradiate targeted spinal segments. The method's feasibility and irradiation parameters that do not cause phototoxic reaction were determined, and the methodology of irradiating the spinal cord with near‐infrared light was investigated in detail. A diffusing optical fiber was implanted above the T9 spinal cord of Bama miniature pigs and used to transfer near‐infrared light (810 nm) onto the spinal cord surface. After daily irradiation with 200, 300, 500 or 1000 mW for 14 days, both sides of the irradiated area of the spinal cord were assessed for temperature changes. The condition of the spinal cord and the position of optical fiber were investigated by magnetic resonance imaging (MRI), and different parameters indicating temperature increases or phototoxicity were measured on the normal spinal cord surface due to light irradiation (ie, heat shock responses, inflammatory reactions and neuronal apoptosis), and the animals' lower‐limb neurological function and gait were assessed during the irradiation process. The implanted device was stable inside the freely moving animals, and light energy could be directly projected onto the spinal cord surface. The screening of different irradiation parameters preliminary showed that direct irradiation onto the spinal cord surface at 200 and 300 mW did not significantly increase the temperature, stress responses, inflammatory reactions and neural apoptosis, whereas irradiation at 500 mW slightly increased these parameters, and irradiation at 1000 mW induced a significant temperature increase, heat shock, inflammation and apoptosis responses. HE staining of spinal cord tissue sections did not reveal any significant structural changes of the tissues compared to the control group, and the neurological function and gait of all irradiated animals were normal. In this study, we established an in‐vivo optical fiber implantation method, which might be safe and stable and could be used to directly project light energy onto the spinal cord surface. This study might provide a new perspective for clinical applications of PBM in acute SCI.     

Molecular architecture and assembly of the yeast kinetochore MIND complex

The MIND multiprotein complex is a conserved, essential component of eukaryotic kinetochores and is a constituent of the tripartite KMN network that directly attaches the kinetochore to the mitotic spindle. The primary microtubule-binding complex in this network, NDC80, has been extensively characterized, but very little is known about the structure or function of the MIND complex. In this study, we present biochemical, hydrodynamic, electron microscopy, and small-angle x-ray scattering data that provide insight into the overall architecture and assembly of the MIND complex and the physical relationship of the complex with other components of the KMN network. We propose a model for the overall structure of the complex and provide data on the interactions with NDC80, Spc105p, and thus the mitotic spindle.     

The disordering effect of hyoscyamine drugs on phospholipid membranes

The effect of hyoscyamine drugs on the fluidity of dipalmitoylphosphatidylcholine liposomes has been studied by differential scanning calorimetry (DSC), electron spin resonance spectroscopy (ESR), fluorescence polarization and freeze-fracture electron microscopic techniques. DSC results indicate that anisodamine, anisodine, atropine and scopolamine all increase the fluidity of dipalmitoylphosphatidylcholine liposomes but with different degrees of efficiency. The increasing of fluidity of dipalmitoylphosphatidylcholine liposomes by hyoscyamine drugs is in a dose-dependent way. Increase of the fluidity of phosphatidylcholine liposomes by anisodamine was also shown by the other three methods. The possible mechanism of hyoscyamine-membrane interaction is discussed.     

Foliage terpenoids of Chinese Cupressus species

Foliage of trees from five Chinese Cupressus species was analysed for volatile monoterpenoids, sesquiterpenoids and diterpenoids. Multivariate analysis of the terpenoid data indicated that C. gigantea is most distinct, but otherwise no obvious chemotaxonomic groupings were evident. Comparison of the Cupressus analytical data with that of specimens of five Chamaecyparis species indicated that Cupressus funebris should not be reclassified into Chamaecyparis.     

Comparing the effects of atamestane, toremifene and tamoxifen alone and in combination, on bone, serum lipids and uterus in ovariectomized rats

Complete estrogen blockade remains under investigation as a means to optimize anti-estrogen therapy in breast cancer thus both the efficacy and end-organ toxicities are of interest with combinations. We hypothesized that a steroidal aromatase inhibitor (AI) atamestane (ATA) alone, and in combination with the anti-estrogens tamoxifen (TAM) or toremifene (TOR) would have beneficial effects in ovariectomized (OVX) rats on key end-organ functions including bone and lipid metabolism and on the endometrium. Significant positive effects on bone were noted with ATA, TOR, TAM, ATA + TOR, or ATA + TAM. TOR, TAM, ATA + TOR, or ATA + TAM caused significant decreases in serum cholesterol and low-density lipoprotein cholesterol whereas ATA had no effect. Uterine weight and epithelium lining height were not increased by ATA but were by TOR and TAM. No significant differences were found in the key parameters outlined above between OVX rats given TOR and ATA + TOR, or TAM and ATA + TAM. Our data show that ATA in combination with TOR or TAM is equivalent to TOR or TAM alone in terms of end-organ effects within a range of clinically relevant doses. Further studies of combinations of AIs with anti-estrogens on end-organ function are merited.     

The gene for severe combined immunodeficiency disease in Athabascan-speaking Native Americans is located on chromosome 10p.

Severe combined immunodeficiency disease (SCID) consists of a group of heterogeneous genetic disorders. The most severe phenotype, T-B- SCID, is inherited as an autosomal recessive trait and is characterized by a profound deficiency of both T cell and B cell immunity. There is a uniquely high frequency of T-B- SCID among Athabascan-speaking Native Americans (A-SCID). To localize the A-SCID gene, we conducted a genomewide search, using linkage analysis of approximately 300 microsatellite markers in 14 affected Athabascan-speaking Native American families. We obtained conclusive evidence for linkage of the A-SCID locus to markers on chromosome 10p. The maximum pairwise LOD scores 4.53 and 4.60 were obtained from two adjacent markers, D10S191 and D10S1653, respectively, at a recombination fraction of straight theta=.00. Recombination events placed the gene in an interval of approximately 6.5 cM flanked by D10S1664 and D10S674. Multipoint analysis positioned the gene for the A-SCID phenotype between D10S191 and D10S1653, with a peak LOD score of 5.10 at D10S191. Strong linkage disequilibrium was found in five linked markers spanning approximately 6.5 cM in the candidate region, suggesting a founder effect with an ancestral mutation that occurred sometime before 1300 A.D.     

Effects of exogenous abscisic acid on leaf carbohydrate metabolism during cucumber seedling dehydration

Cucumber (Cucumis sativus L.) seeds were pretreated with exogenous abscisic acid (ABA) prior to germination. After germination, seedlings with three leaves were exposed to gradual dehydration. The effects of ABA on photosynthetic rate (Pn), daily water loss (WL) and water utilization efficiency (WUE) during dehydration were investigated, in addition to the variation of carbohydrates in leaves. ABA improved the Pn, WL and WUE of cucumber seedlings during dehydration. After rehydration, the seedlings pretreated with ABA showed a higher recovery in Pn, WL and WUE, as compared to those without an ABA pretreatment. Subsequent to dehydration, concentration of stachyose, raffinose, sucrose, glucose, and fructose increased in seedlings pretreated with ABA. Dehydration altered the proportions of the sugars in the total carbohydrates, and accelerated the accumulation of stachyose, raffinose and sucrose. After rehydration, carbohydrate concentrations of seedlings pretreated with ABA recovered to levels observed prior to dehydration. These results demonstrated that pretreatment of seeds with exogenous ABA enhanced carbohydrate tolerance to dehydration of cucumber seedlings.