Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis

Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo‐spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel‐specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia‐inducible factor (HIF)‐1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo. Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.     

Melanic pigmentation and light preference within and between two Drosophila species

Environmental adaptation and species divergence often involve suites of co‐evolving traits. Pigmentation in insects presents a variable, adaptive, and well‐characterized class of phenotypes for which correlations with multiple other traits have been demonstrated. In Drosophila, the pigmentation genes ebony and tan have pleiotropic effects on flies'' response to light, creating the potential for correlated evolution of pigmentation and vision. Here, we investigate differences in light preference within and between two sister species, Drosophila americana and D. novamexicana, which differ in pigmentation in part because of evolution at ebony and tan and occupy environments that differ in many variables including solar radiation. We hypothesized that lighter pigmentation would be correlated with a greater preference for environmental light and tested this hypothesis using a habitat choice experiment. In a first set of experiments, using males of D. novamexicana line N14 and D. americana line A00, the light‐bodied D. novamexicana was found slightly but significantly more often than D. americana in the light habitat. A second experiment, which included additional lines and females as well as males, failed to find any significant difference between D. novamexicana‐N14 and D. americana‐A00. Additionally, the other dark line of D. americana (A04) was found in the light habitat more often than the light‐bodied D. novamexicana‐N14, in contrast to our predictions. However, the lightest line of D. americana, A01, was found substantially and significantly more often in the light habitat than the two darker lines of D. americana, thus providing partial support for our hypothesis. Finally, across all four lines, females were found more often in the light habitat than their more darkly pigmented male counterparts. Additional replication is needed to corroborate these findings and evaluate conflicting results, with the consistent effect of sex within and between species providing an especially intriguing avenue for further research.     

Lumican is a major proteoglycan component of the bone matrix.

MC3T3-E1 mouse calvaria cells are a clonal population of committed osteoprogenitors that in the presence of appropriate supplements form a mineralized bone matrix. The development of the MC3T3-E1 cells can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Recently, using the cDNA microarray technology we found lumican to be abundantly expressed during the mineralization and differentiation stages of the MC3T3-E1 development and not during the proliferation stage. Lumican has been shown to play essential roles in regulating collagen fibril formation in different extracellular matrices but its expression in the developing bone matrix remains elusive. By examining the expression profile of this gene during the different stages of MC3T3-E1 development, utilizing the 'real-time' PCR technology, we observed that the expression of lumican increases as the osteoblast culture differentiates and matures, suggesting that lumican may be involved in regulating collagen fibrillogenesis in bone matrices. Using immunostaining, we observed that during the early embryonic development of mouse (E11 to E13), lumican is mainly expressed in the cartilaginous matrices. However, in the older embryos (E14 to E16), the expression of lumican is more prominent in the developing bone matrices. Our data suggest that lumican is a significant proteoglycan component of bone matrix, which is secreted by differentiating and mature osteoblasts only and therefore it can be used as a marker to distinguish proliferating pre-osteoblasts from the differentiating osteoblasts.     

Enhancement of Phloem Exudation from Fraxinus uhdei Wenz. (Evergreen Ash) using Ethylenediaminetetraacetic Acid

Ethylenediaminetetraacetic acid (EDTA) enhanced the exudation of ¹⁴C-labeled assimilates from excised leaflets and whole plant specimens of Fraxinus uhdei Wenz. A 2 millimolar EDTA concentration was found to be most effective in promoting exudation from excised leaflets, while 10 millimolar EDTA was most effective in whole plants experiments. Exudation rate reached a maximum after 24 hours in both experiments. The continuous presence of EDTA throughout the treatment period was required for maximum exudation from excised leaflets. Stachyose, raffinose, verbascose, and sucrose were the principal compounds found to occur in exudate samples. These compounds are typically transported in sieve elements of various Fraxinus species suggesting the exudate was of phloem origin. Electron microscope studies of petiolule sieve plate pores from excised leaflets showed substantially less callose appearing after treatment with EDTA than after H₂O treatment. It is suggested that EDTA enhances phloem exudation by inhibiting or reducing callose formation in sieve plate pores. The exudation enhancement technique described for whole plant specimens is suggested as a useful means of collecting phloem sap and studying translocation in woody plants.     

The isolation and biochemical characterization of three subfractions of myelin from central nervous tissue of the adult rat

Abstract— By techniques of isosmotic density gradient ultracentrifugation three subfractions of myelin were isolated from homogenates of whole rat brain at densities of 1.054 g/ml (myelin I), 1.060 g/ml (myelin II) and 1.066 g/ml (myelin III). The stability of these fractions was demonstrated by the zonal centrifuge profile analysis of recycled fractions. Examination of the three myelin subfractions by techniques of electron microscopy and thin layer chromatography detected no obvious morphological or chemical differences. However, analysis for protein, cholesterol, phospholipids and cerebrosides did reveal differences among myelin I, myelin II and myelin III. Myelin I contained relatively more cholesterol than II or III. Myelin III contained relatively more phospholipids than I or II. The cerebroside-to-protein ratios were the same in all three fractions. Quantitative differences in fatty acid composition (as detected by gas-liquid chromatography) were also observed.     

Development of a serum-free medium using computer-assisted factorial design and analysis

Stepwise screening of media supplements using factorial design and analysis was employed in the development of serum-free medium for a recombinant Chinese hamster ovary cell line. The effects (growth and target protein production) of different combinations were measured at two time points to ensure adequate response. The results were analysed by a computer program specialized in factorial analysis. The formulation deduced from the previous experiment was used as the new basal medium for the next screening. Certain significant nutrients were studied again in a more advanced formulation in order to analyse the potential synergistic effects with new media components. Compared to cells grown in serum-containing medium, cells adapted to the final formulation of the serum-free medium had a comparable growth rate but a four fold increase in the active protein production.Abbreviations ANOVA Analysis of variance - BSA bovine serum albumin - CHO Chinese hamster ovary - FBS fetal bovine serum - MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - PBS phosphate buffered saline - SFM serum-free medium     

Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures: Comparison of different substrata and tissue culture media formulations

Summary Long-term monolayer cultures of adult rat hepatocytes were tested for their ability to glucuronize phenol red and to maintain initial levels of cell proteins, glucose consumption, and lactic acid production. Lactate dehydrogenase leakage served as an index of culture status because a high value indicates cell death. Three tissue culture (TC) media formulations were the main variables introduced to determine ideal conditions for cell survival in vitro. Investigations of long-term cultures were preceded by studies of hepatocyte attachment to polystyrene surfaces. This attachment was influenced by the amount of substrate deposited and the number of cells seeded, but not by the uniformity of the substrate coating. A statistical analysis of our data revealed that in the absence of fetal bovine serum (FBS), air dried collagen (ADC) and Biomatrix (BMX) were superior to saline precipitated collagen and fibronectin as attachment substrates. In the presence of 10% FBS, all of the substrates performed equally. Chee's Medium (CEM) proved to be the best for preserving cell proteins over a time course of 28 d and Williams' E medium also performed adequately up to 14 d. The glucuronization of phenol red was at 50% of initial values at Day 7 in CEM-ADC hepatocytes in contrast to 30% for cells in Williams' E medium and 5% for cells grown in Waymouth's. At 14 d glucuronization was still present at 40% of original values in CEM-ADC cells but had ceased in the other two media. When BMX was used, none of the TC media supported glucuronization levels comparable to ADC cells. This research was supported in part by grant 1R01-AM-26520 from the National Institute of Arthritis, Diabetes and Digestive Kidney Diseases, NIH, Bethesda, Maryland.     

Functional properties of ficoll and their influence on anther culture responses of wheat

The effects of ficoll in liquid culture media have been contradictory in previous reports. The objective of this study was to determine the functional properties of ficoll in potato 4 (P4) liquid induction medium and their influence on anther culture responses of wheat. Ficoll addition significantly (p0.01) reduced callus production from the anthers of spring wheat cv. Pavon 76. The reduction was directly related to the concentration of ficoll added within the range of 50 to 200 g l^-1 medium. Although the addition of ficoll significantly (p0.01) increased the percentage of regenerable calli and the ratio of green vs. albino plants, the final yield of green plants per 100 anthers was significantly lower. Consistent results also were obtained with four other spring wheat genotypes (Chris, Butte 86, WA 6916, and Edwall). Ficoll concentration affected the density, viscosity, and osmolality of the liquid media. The higher medium density caused by ficoll addition increased the percentage of floating calli, as well as the percentage of regenerable calli and the ratio of green vs. albino plants. However, the increased medium viscosity by ficoll addition significantly (p0.01) reduced callus production. Ficoll addition also increased medium osmolality, which affected callus production by interacting with the sugar concentration of the induction media. Using response functions, the estimated maltose concentration for maximum callus production was 105 g l^-1 for the standard P4 media, compared with 68 g l^-1 for the ficoll-containing P4 media. These results clearly demonstrate that ficoll addition to the liquid P4 induction medium containing high sucrose concentration (90 g l^-1) is deleterious to the maximum production of green plants from wheat anther culture.