Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

Prolactin-Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C-Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL-induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL-induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine, or polymyxin B blocked the PRL-induced increase in cell number with IC₅₀ values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α-phorbol 12,13-didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U-shaped dose-response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose-response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL-induced increase in cell number. Finally, TPA treatment decreased cell-surface binding of ¹²⁵I-PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.     

trans complementation of cap-independent translation directed by poliovirus 5' noncoding region deletion mutants: evidence for RNA-RNA interactions.

Poliovirus (PV) RNA is translated by a cap-independent mechanism involving the internal entry of ribosomes onto the 5' noncoding region (NCR). Using the vaccinia virus-T7 RNA polymerase transient expression system, we showed previously that deletion of certain individual predicted secondary structures within the PV 5' NCR rendered the element defective in directing internal initiation when assayed alone. However, these defective 5' NCRs were functional when coexpressed within cells with full-length PV cDNA (N. Percy, G. J. Belsham, J. K. Brangwyn, M. Sullivan, D. M. Stone, and J. W. Almond, J. Virol. 66:1695-1701, 1992). We have extended the study to demonstrate that when these predicted secondary structures are deleted in combination, the enhanced activity in the presence of the full-length PV cDNA is still observed. Indeed, a poliovirus 5' NCR devoid of all predicted secondary structures is capable of initiating protein synthesis under these conditions. Surprisingly, we also found that this enhancement of activity requires neither any PV protein nor the inhibition of cap-dependent translation. The results indicate that the defective PV 5' NCR elements can be complemented in trans by functional 5' NCRs in a highly sequence specific manner.     

Mitotic regulation of protein phosphatases by the fission yeast sds22 protein

BACKGROUND: Cell cycle progression requires the activity of protein kinases and phosphatases at critical points in the cell cycle in all eukaryotes. We have previously reported that the dis2(+) and sds2(+) genes of fission yeast encode redundant catalytic subunits of a type 1-like protein phosphatase. The sds22(+) gene was shown to be essential for cell viability and to interact genetically with dis2(+) and sds21(+). RESULTS: Here we show by immunoprecipitation that the sds22 protein physically interacts with the dis2 and sds21 proteins, and that sds22-associated phosphatase activity has altered substrate specificity, The loss of sds22 function by a temperature sensitive mutation leads to cell cycle arrest at mid-mitosis, at which point cdc2-dependent histone Hl kinase activity is high while sds22-dependent H1 phosphatase activity is low. To examine the unusual properties of sds22 protein structure, we analyzed a collection of sds22 deletion and point mutants by a variety of functional criteria. CONCLUSION: We propose that sds22 is a regulatory subunit of the dis2/sds21 phosphatase catalytic subunits and that sds22-bound phosphatase carries a key phosphatase activity essential for the progression from metaphase to anaphase. Mutational analysis indicates that dis2/sds21 interacts with the central repetitive domain of sds22, while the C-terminal and central regions of sds22 may be involved in subcellular targeting and the N-terminus is important for stability.     

IDENTIFICATION OF CYTOKININS IN SARGASSUM MUTICUM (PHAEOPHYTA) AND PORPHYRA PERFORATA (RHODOPHYTA)1

Combined gas chromatography-mass spectrometry (GCMS) was used to identify and quantify specific cytokinins from Porphyra perforate J. Ag. and Sargassum muticum (Yendo) Fensh. The level of isopentenyladenosine was estimated to be 0.6 μ·kg^?1 fresh weight in Porphyra and 0.9 μ·kg^?1 fresh weight in Sargassum. The level of cis-zeatin riboside was estimated to be 0.2 μ·kg^?1 fresh weight in Sargassum. This is the first definitive identification of a cytokinin from a red alga, and the second report from a brown alga.     

North Sea cod and climate change – modelling the effects of temperature on population dynamics

In order to examine the likely impacts of climate change on fish stocks, it is necessary to couple the output from large‐scale climate models to fisheries population simulations. Using projections of future North Sea surface temperatures for the period 2000–2050 from the Hadley General Circulation Model, we estimate the likely effects of climate change on the North Sea cod population. Output from the model suggests that increasing temperatures will lead to an increased rate of decline in the North Sea cod population compared with simulations that ignore environmental change. Although the simulation developed here is relatively simplistic, we demonstrate that inclusion of environmental factors in population models can markedly alter one's perception of how the population will behave. The development of simulations incorporating environment effects will become increasingly important as the impacts of climate change on the marine ecosystem become more pronounced.     

The molecular chaperone alpha-crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress.

Alpha-crystallin, a molecular chaperone and lens structural protein protects soluble enzymes against heat-induced aggregation and inactivation by a variety of molecules. In this study we investigated the chaperone function of alpha-crystallin in a more physiological system in which alpha-crystallin was incorporated into red cell 'ghosts'. Its ability to protect the intrinsic membrane protein Na/K-ATPase from external stresses was studied. Red cell ghosts were created by lysing the red cells and removing cytoplasmic contents by size-exclusion chromatography. The resulting ghost cells retain Na/K-ATPase activity. alpha-Crystallin was incorporated in the cells on resealing and the activity of Na/K-ATPase assessed by ouabain-sensitive 86Rb uptake. Incubation with fructose, hydrogen peroxide and methylglyoxal (compounds that have been implicated in diabetes and cataract formation) were used to test inactivation of the Na/K pump. Intracellular alpha-crystallin protected against the decrease in ouabain sensitive 86Rb uptake, and therefore against inactivation induced by all external modifiers, in a dose-dependent manner.     

Intraspecific molecular variation inHieracium sect.Alpina (Asteraceae), an apomictic group

Intraspecific variation of four agamospecies ofHieracium sect.Alpina was studied using RAPD and isozyme techniques. No variation in either multiprimer RAPD or multi-enzyme phenotypes was observed withinH. holosericeum, suggesting that this widespread species consists of only a single genotype. A low level of within-population isozyme variation was seen inH. tenuifrons andH. calenduliflorum, the origin of which appears to be consistent with somatic mutation. Most isozyme and all RAPD variation in these two species was partitioned between populations. A strong correlation with geography suggests that its cause may be due to polytopic (-polyphyletic?) origin or perhaps to mutation and dispersal. The most variable species wasH. alpinum, in which isozyme variation occurred mostly within populations rather than between them, suggesting occasional sexual events or that the parents ofH. alpinum were heterozygous. RAPD variation in this species, in contrast, was partitioned between Scottish and Swiss populations, suggesting the existence of geographical races.