Eph/Ephrin membrane proteins: a mammalian expression vector pTIg-BOS-Fc allowing rapid protein purification

There is an urgent need for high purity, single chain, fully functional Eph/ephrin membrane proteins. This report outlines the pTIg-BOS-Fc vector and purification approach resulting in rapid increased production of fully functional single chain extracellular proteins that were isolated with high purity and used in structure-function analysis and pre-clinical studies.     

Multiple Shoot Regeneration from Young Shoots of Kenaf (Hibiscus cannabinus)

A method was developed to initiate multiple shoots from the young shoot of kenaf. Young shoots along with the cotyledons were excised from ten-day old aseptically germinated seeds and pre-cultured for two weeks in Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) or a combination of BA and kinetin. After two weeks in culture, elongated shoots were excised above the cotyledonary nodes and cultured on fresh medium of the same composition. Multiple shoots were initiated within eight weeks. The number of shoots varied among cultivars. The highest number of shoots (11/explant) occured in cultivar Tainung 2 (T2) cultured in MS medium supplemented with 8.8 μM BA. Concentrations of BA higher than 8.8 μM had a negative effect on the number of shoots. Furthermore, callus growth was initiated from which morphologically abnormal shoots were induced. Kinetin had a significant effect only on cultivar Everglades 41 (E41). Shoot elongation and rooting were obtained simultaneously in half strength MS basal medium with no plant growth regulators. About 98% of the rooted plants were grown to maturity under greenhouse conditions. This method was successful with all four genotypes tested. However, significant genotypic variations were observed among the genotypes.     

Generation and characterization of EphA1 receptor tyrosine kinase reporter knockout mice

Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock-in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in approximately 80% of homozygous adults. A second, distinct abnormality present in approximately 18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone-induced apoptotic processes.     

Localization and secretion of inhibins in the equine fetal ovaries

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.     

Microbial transformation of xanthohumol

Microbial transformation of xanthohumol using the culture broth of Pichia membranifaciens afforded three metabolites, (E)-2"-(2"'-hydroxyisopropyl)-dihydrofurano[2",3":4',3']-2', 4-dihydroxy-6'-methoxychalcone, (2S)-2"-(2"'-hydroxyisopropyl)-dihydrofurano[2",3":7,8]-4'-hydroxy-5-methoxyflavanone and (E)-2"-(2"'-hydroxyisopropyl)-dihydrofurano[2",3":2',3']-4'-hydroxy-5-methoxychalcone.     

Anthropogenic signature in the incidence and distribution of an emerging pathogen of poplars

The introduction and establishment of non-native plant pathogens into new areas can result in severe outbreaks. Septoria leaf spot and canker caused by Sphaerulina musiva is one of the most damaging poplar diseases in northeastern and north-central North America. Stem and branch cankers can be devastating on susceptible trees, leading to tree death and reduced biomass in commercial plantations. In the Pacific Northwest region of North America, the first report of the disease was made in 2006 in the Fraser Valley of British Columbia (BC), Canada. To investigate the incidence and distribution of S. musiva from its point of introduction into BC, five plantations of Populus trichocarpa (black cottonwood), 500 P. trichocarpa trees from natural populations, and 23 plantations of hybrid poplars were surveyed by using real-time PCR assays targeting S. musiva and its native sister species, S. populicola. Our survey suggests a strong anthropogenic signature to the emergence of the non-native S. musiva. Detection frequency of S. musiva was high in hybrid poplar plantations (116 trees infected, 54.2 % of the sampled trees), while detection of the native S. populicola was limited to 13.1 % (22 trees infected). By contrast, in natural stands of P. trichocarpa, less than 2 % of the trees were positive for S. musiva (7 trees) while ~75 % were positive for S. populicola (433 trees). All the S. musiva detections in natural stands of the native P. trichocarpa were from trees located in the vicinity (<2.5 km) of hybrid poplar plantations. Identification of the genotypes found in the hybrid poplar plantations revealed that they are in majority F1 progeny from P. trichocarpa × P. deltoides (T × D) (82 %) and P. nigra × P. maximowiczii (N × M) (7.8 %) crosses, which are generally susceptible (intermediate level of susceptibility between the two parental species) to the canker disease. Our results suggest that the emergence of S. musiva in BC is related to the planting of susceptible hybrid poplars. Even if the disease has not yet established itself in natural poplar populations outside of the Fraser Valley, infected plantations could act as a reservoir that could promote its spread into nearby native P. trichocarpa populations.     

Triterpenoid,coumarin and quinone constituents of eleven Diospyros species (Ebenaceae)

From the bark and/or timber extracts of Diospyros hirsuta, D. moonii, D. quaesita, D. spinescens, D. thwaitesii and D. walkeri, the following compounds have been isolated; lupeol, betulin, betulinic acid sitosterol, taraxerol, taraxerone, ursolic acid, oleanolic acid scopoletin, plumbagin, elliptinone, diospyrin and diosindigo A. TLC examination of the bark and timber extract of D. acuta, D. chaetocarpa, D. oblongifolia, D. oppositifolia and D. rheophytica is reported. Lupeol betulin, oleanolic acid and sitosterol have been isolated from the fruit of D. oblongfolia.     

Photosynthetic rates of citronella and lemongrass

Ten selections of citronella (Cymbopogon nardus [L.] Rendle) were grown at 32/27, 27/21, or 15/10 C day/night temperatures, and plants from three populations of lemongrass (Cymbopogon citratus [D.C.] Stapf from Japan or Sri Lanka and Cymbopogon flexuosus [D.C.] Stapf from India) were grown at 8- or 15-hour photoperiods. Net photosynthetic rates of mature leaves were measured in a controlled environment at 25 C and 260 microeinsteins per meter² per second. Rates declined with increasing leaf age, and from the tip to the base of the leaf blade. Rates for citronella leaves grown at 15/10 C were extremely low for all selections. Highest rates of net photosynthesis were recorded for four selections grown at 27/21 C and for two selections grown at 32/27 C. Lemongrass grown at 8-hour photoperiod had higher photosynthetic rates than that grown at 15-hour photoperiod.     

Carbon dioxide compensation values in citronella and lemongrass

Carbon dioxide compensation values of mature leaves from 10 selections of citronella (Cymbopogon nardus [L.] Rendle) grown at 32/27 or 27/21 C day/night temperatures and three strains of lemongrass (Cymbopogon citratus [D.C.] Stapf. and Cymbopogon flexuosus [D.C.] Stapf.) grown at 8- or 15-hour photoperiods were measured in a controlled environment at 25 C. All leaves had low compensation values but citronella varied from 1.3 to 9.7 μl/liter and lemongrass from 0.7 to 3.5 μl/liter. Lower growing temperature generally resulted in lower compensation values for citronella but there was no consistent photoperiod effect on lemongrass.     

Developmental aberrations of liver gene expression in bovine fetuses derived from somatic cell nuclear transplantation

Cloning by somatic cell nuclear transfer (NT) has been accomplished. However, the process itself is inefficient since most clones die before birth and survivors often display various anomalies. In an effort to determine global expression profiles of developmentally regulated liver genes in NT bovine fetuses, we employed a custom-made bovine liver complementary DNA (cDNA) microarray. The NT fetuses in early pregnancy were derived from cumulus cells as the nuclear donor cells. Normal fetuses were derived from in vitro fertilization (IVF) and artificial insemination (AI). Gene expression levels in NT, IVF, and AI fetal livers were obtained by comparing individual fetal liver samples with that of adult liver of nonpregnant cycling cows. Statistical analyses of the expression data showed widespread dysregulation of developmentally important genes in the three NT fetuses examined. It was found that the number of dysregulated genes was within a range of 3.5-7.7% of the tested genes in the NT fetal livers. The analyses revealed that one NT fetus was markedly different in liver gene expression profile from the other two NT fetal livers in which the expression profiles were highly correlated. Thus, our findings demonstrate that widespread dysregulation of liver genes occurs in the developing liver of NT bovine fetuses. It is possible that inappropriate genomic reprogramming after NT is a key factor associated with abnormal gene expressions in the livers of NT fetuses, whereas distinct expression patterns between the fellow cloned fetuses likely have resulted from variable epigenetic status of the donor nuclei.