Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.     

The effects of aflatoxin B1 on immature germinating maize (Zea mays) embryos

Immature maize (Zea mays L.) embryos were treated with aflatoxin B₁ concentrations, ranging from 0.1 g ml^–1 to 25 g ml^–1. Below 5 g ml^–1 aflatoxin B₁, root and shoot elongation was not significantly inhibited. Ultrastructurally, root tip cells showed little deterioration, except a possible diffused clearing in mitochondria and plastids. As the toxin concentration was increased above 5 gml^–1, shoot, and particularly root elongation, was progressively inhibited. Associated with this, there was an apparent decrease in the ribosome population. Furthermore, membranes, particularly the vacuolar membrane, became abnormal and vacuolar distension occurred. At 20 and 25 g ml^–1, these effects were exacerbated, and mitochondria and plastid structure was disrupted. At these concentrations, there was evidence of a disruption in lipid metabolism. The results are discussed in the context of known aflatoxin effects on cellular control mechanisms and ultrastructure in animal systems.     

Selective isolation of bacteria with dipeptidyl aminopeptidase type I activity from the sheep rumen

Five-hundred-and-six fresh isolates of rumen bacteria were tested for their ability to hydrolyse the synthetic substrate for dipeptidyl aminopeptidase type I, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), using a gel overlay technique. Twelve positive isolates were small Gram-negative rods which resembled Bacteroides ruminicola in their biochemical and morphological properties. SDS-PAGE of whole cell extracts indicated that two were similar to B. ruminicola strain B14, six resembled B. ruminicola strain M384, and four were similar to B. ruminicola GA33. All hydrolysed GlyArg-MNA, Ala2 and Ala5, and showed no activity against Leu-MNA. Ala3 and Ala2, but no Ala4, was produced from Ala5. The different groups had different, distinctive activity profiles. The two remaining positive isolates were Lactobacillus spp. with an exceptionally high Leu-MNA activity. It was concluded that, although different strains may only be distantly related, B. ruminicola forms the most important group of bacteria in the rumen to possess a dipeptidyl aminopeptidase type I activity.     

Molecular cloning of a GTPase activating protein specific for the Krev-1 protein p21rap1

The rap1/Krev-1 gene encodes a ras-related protein that suppresses transformation by ras oncogenes. We have purified an 88 kd GTPase activating protein (GAP), specific for the rap1/Krev-1 gene product, from bovine brain. Based on partial amino acid sequences obtained from this protein, a 3.3 kb cDNA was isolated from a human brain library. Expression of the cDNA in insect Sf9 cells resulted in high level production of an 85-95 kd rap1GAP that specifically stimulated the GTPase activity of p21rap1. The complete deduced amino acid sequence is not homologous to any known protein sequences, including GAPs specific for p21ras. Northern and Western blotting analysis indicate that rap1GAP is not ubiquitously expressed and appears most abundant in fetal tissues and certain tumor cell lines, particularly the Wilms' kidney tumor, SK-NEP-1, and the melanoma, SK-MEL-3, cell lines.     

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.     

Differentiation among populations of Sedum wrightii (Crassulaceae) in response to limited water availability: water relations,CO2 assimilation,growth and survivorship

Summary Sedum wrightii is one of only a few species in the Crassulaceae for which there is evidence for a high degree of variability in the ratio of daytime to nighttime CO₂ assimilation. There are both environmental and genetic components to this variability. S. wrightii grows over a wide altitudinal gradient. The purpose of this study was to compare low, intermediate, and high altitude populations with respect to the degree of CAM expression and the capability to tolerate limited water availability. We utilized clonallyreplicated genotypes of plants from each population in common environment greenhouse experiments. Genetic differences among the populations were found in long-term water use efficiency, in 24 hour CO₂ exchange patterns, in biomass ¹³C values, in carbon allocation, and in water status and ultimately survival during prolonged drought. The differences among the populations appear to be closely related to differences in the native habitats. The low altitude, desert plants had the greatest ability to grow and survive under conditions of limited water availability and appear to have the greatest shift to nighttime CO₂ uptake during periods without water, while the high altitude plants had the poorest performance under these conditions and appear to shut down net carbon uptake when severely water limited.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

Sex ratio and intrasexual kin competition in mammals

Summary Asymmetries in both intrasexual competition and generation overlap occur in Antechinus (Dasyuridae; Marsupialia). We show that the range of interpopulation variation in the sex ratio of pouch young spans and exceeds the range of sex ratios at birth hitherto recorded from eutherians (Clutton-Brock and Albon 1983). Although postweaning dispersal and male mortality are similar among all Antechinus populations, interpopulation variance in female longevity leads to variable duration of mother/daughter interaction. As this duration increases, parental investment is increasingly biased toward males, supporting the view that local competition among female kin for resources may influence mammalian sex ratios.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

Calcium-induced reorganization of desmosomal components in cultured human keratinocytes

We used antibodies raised against individual desmosomal components to study calcium-induced desmosome formation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the level of calcium ions in the culture medium, there is little contact between adjacent cells. Raising the level of calcium ions rapidly induces desmosome formation, and stratification occurs within 24 h. We found that before addition of calcium the 115,000- and 100,000-mol-wt core glycoproteins were distributed over the entire cell surface, whereas the plaque proteins (205,000 and 230,000 mol wt), the 82,000- and 86,000-mol-wt proteins, and the 150,000-mol-wt glycoprotein were located throughout the cytoplasm. 15 min after increasing the calcium ion concentration, all of these molecules appeared at the cell margins. The intensity of peripheral staining increased over the next 2 h and during this time the distribution of keratin filaments changed from predominantly perinuclear to extend throughout the cytoplasm. Keratinocytes could be dissociated with EDTA for up to 2 h after exposure to calcium. After 3 h of exposure to calcium the cells were no longer susceptible to EDTA dissociation and staining for desmosomal plaque antigens persisted in regions of intercellular contact. Desmosomal staining in stratified cultures became greatly reduced within 24 h of lowering the calcium ion concentration again. We have preliminary evidence that stratification occurs by breakdown of desmosomes at lateral surfaces and reformation at surfaces of contact between basal and suprabasal cells, rather than by rearrangement of existing desmosomes. Involucrin-positive cells in the monolayer appeared to contain more 205,000- and 230,000-mol-wt proteins free in the cytoplasm than involucrin-negative cells.