Seedling ecology of two miombo woodland trees

The development of seedlings of two miombo trees, Brachystegia spiciformis Benth. and Julbernardia paniculata (Benth.) Troupin, was studied during two growing seasons (December 1989–April 1991) at a Zambian grassland site. Seed germination rates under laboratory and field conditions were not significantly different although germination in the field was delayed by 1–2 weeks due to insufficient rainfall. After one year of storage J. paniculata seed germination had declined from 67% to 17% while germination of B. spiciformis seeds remained at about 83%.Leaf production was confined to the rainy season. Leaf fall occurred during the dry season and in J. paniculata this was followed by shoot die-back during the hot dry period (August–November). Two-thirds of B. spiciformis seedlings experienced shoot die-back but shoot die-back did not necessarily result in seedling mortality. Seedling deaths occurred during the germination period (6–10 weeks after planting) and in the hot dry period (40–50 weeks after planting) during September–November. Survivorship of B. spiciformis seedlings was 74% at the end of the second growing season while this was 46% for J. paniculata.Shoot growth was negligible during the second growing season. In fact mean maximum leaf area of B. spiciformis decreased significantly from 19.7 cm² (SD=5.7) per plant at the end of the first growing season to 13.3 cm² (SD=5.8) at the end of the second growing season (t=3.31, P<0.01). However, root biomass of B. spiciformis seedlings increased 2.8 times during the second growing season.These results suggest that shoot die-back in seedlings of miombo trees is caused by drought and that the slow shoot growth is the result of allocating most of the biomass to root growth during seedling development.     

Transformation of CW-15 Mutant Cells of Chlamydomonas reinhardtii Dang. with pCTVHyg Plasmid

A pCTVHyg plasmid was constructed in a unicellular green alga Chlamydomonas reinhardtii Dang. by using the hygromycin phosphotransferase gene (hpt) as a selectable marker and the Escherichia coli transposon Tn5 under the early SV40 viral gene promoter. CW-15 mutant cells devoid of cell walls were transformed by electroporation in an electric field of 1 kV/cm and a pulse duration of 2 ms. A suspension density of 10⁶ cell/ml and the mid-logarithmic growth phase were the optimum conditions for transformation, producing up to 10³ hygromycin-resistant (Hyg^R) clones per 10⁶ Hyg^R recipient cells. Exogenous DNA integrated in the nuclear genome of C. reinhardtii was steadily inherited in subsequent generations within at least a 8-month period; however, the Hyg^R trait manifestation was not stable. The comparative analysis of frequencies in codon usage in hpt and in the nuclear genes of C. reinhardtii significantly excluded the possibility that the bias in codon usage was the primary factor affecting foreign gene expression. The advantages of using theCW-15 mutant and the described selection system are discussed in the context of heterologous transformation of C. reinhardtii.     

Flow cytometric analysis of porcine preadipocytes.

In this report, conditions have been established for utilizing monoclonal antibodies and fluorescence activated flow cytometry in studying antigen expression by primary porcine stromal-vascular cells cultured under various conditions. Single cells were isolated from cultures maintained in DME/F12 medium containing 10% fetal bovine serum, 2% pig serum, and containing 2% pig serum and 10 nM dexamethasone supplemented with growth hormone (GH), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta). Flow cytometric analyses revealed that the proportion of cells expressing detectable levels of the AD-1 cells surface antigen was greater in cultures supplemented with 2% pig serum and 10 nM dexamethasone than in other media. In cultures, GH, TNF-alpha and TGF-beta each inhibited lipid deposition, whereas TNF-alpha and TGF-beta, but not GH, inhibited AD-1 antigen expression. Inhibition of lipid deposition as well as antigen expression by TNF-alpha and TGF-beta was reversible, but inhibition of cluster formation by GH was not reversed upon removal from cultures. In summary, differential effects of factors on surface antigen expression by preadipocytes are detectable by flow cytometry. Flow cytometric analysis using monoclonal antibodies produced against key developmentally regulated cell surface antigens is potentially a powerful analytical approach to the study of adipocyte development.     

Albumin stabilizes 14,15-leukotriene A4

14,15-Leukotriene A4 is a pivotal biosynthetic intermediate in 15-lipoxygenase initiated leukotriene biosynthesis. This compound hydrolyzes instantaneously in phosphate buffer at pH 7.4. However, addition of human or bovine albumin to otherwise identical buffer solutions increases its stability. Intact 14,15-leukotriene A4 then decomposes by first-order kinetics with rate constants inversely proportional to the albumin concentration. Stabilization of 14,15-leukotriene A4 under certain conditions may influence its proportionate transformation by enzymatic vs non-enzymatic processes.     

The effect of growth state on the activity of the protein kinase isoenzymes an increase in type II cyclic AMP-dependent protein kinase is correlated with growth arrest in G1 phase

Native polyacrylamide gels have been used to resolve protein kinase isoenzymes from cultured cells and the protein kinases have been identified by carrying out phosphorylation reactions in the gel. Following electrophoresis, the gels were incubated with histome and [γ-³²P]ATP. The gels were then thoroughly washed and dried down, and the protein kinases were located by autoradiography. Protein kinase activity as measured in the gel system was a linear function of cytosol protein concentration up to about 100 μg per channel and incorporation of ³²P into histone was time dependent. Three bands of protein kinase activity were resolved in cytosol samples from baby hamster kidney (BHK) fibroblasts. The band with the lowest relative mobility utilized histone IIA or casein equally well as substrate protein whereas bands 2 and 3 demonstrated a clear preference for histone. Bands 2 and 3 displayed a relative mobility in electrophoresis that was identical to that observed for cyclic AMP-dependent protein kinases I and II from rat liver. Treatment of cyctosol samples with cyclic AMP prior to electrophoresis resulted in the disappearance of cyclic AMP-dependent protein kinases from the gel profile. This method was employed to identify bands 2 and 3 as cyclic AMP-dependent protein kinases. The protein kinases in growth-arrested cells were compared with proliferating cells. We have observed a 3.5-fold increase in the activity of Type II protein kinase as the cells arrest growth in G₁ phase of the cell cycle. This increase in Type II is correlated with the increase in cells blocked in G₁ and a decrease in II Type activity appears to be an early event in permitting cells to leave G₁ and resume growth.     

GRF-induced feeding: Evidence for protein selectivity and opiate involvement

Growth hormone-releasing factor (GRF) is a hypothalamic peptide named for its ability to induce release of growth hormone from the anterior pituitary. GRF also acts as a neurotransmitter in the suprachiasmatic nucleus/medial preoptic area (SCN/MPOA) to stimulate food intake. The purpose of this series of experiments was to explore the nature of GRF-induced feeding, with a particular emphasis on macronutrient selectivity, and to examine the role of opiate activity in the paraventricular nucleus of the hypothalamus (PVN). Chow intake stimulated by GRF microinjection (1 pmol/0.5 μl) into the SCN/MPOA was blocked by injection of methyl-naltrexone (3 μg/0.5 μl) into the PVN. In animals habituated to macronutrient diets (Teklad, WI), GRF preferentially stimulated intake of protein at 2 and 4 h postinjection, whereas it had no effect on carbohydrate intake. Further, this effect was blocked by injection of naloxone (40 nmol/0.5 μl) into the PVN. Microinjection of morphine (0, 1, 10, and 17 μg/0.5 μl) into the PVN also specifically stimulated protein intake at 2 and 4 h postinjection. These results suggest that feeding derived from GRF actions in the SCN/MPOA is macronutrient selective, and is dependent on PVN opiate activity for expression.     

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak^−/− MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak^−/− MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak^−/− MEF cells (Ahnak^−/−-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak^−/−-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak^−/− MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.