Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

Summary N-acetylglucosaminidase (NAG), acid phosphatase (ACP) and alkaline phosphatase (AKP) were localised histochemically in fixed cells from the 37-day-old rat epididymis grown in static monolayer culture for 2–8 days. ACP and NAG were cytosolic enzymes found in perinuclear positions, whereas staining of AKP was consistent with a membranous position. These enzymes were also examined in frozen tissue sections of the epididymis, from rats of the equivalent age, where NAG had intense activity in both supra- and infra-nuclear cytoplasm and ACP was more active apically. For the first time AKP was localised along basolateral membranes of the epithelium and in the lumen of the mid-caput region. The monolayer in culture was of principal cells only and they maintained their polarity and ultrastructural characteristics, but the height of the cells was reduced compared to that obtained in situ.     

Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart

cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

The importance of worm identity and life history in determining the vertical distribution of tubificids (Oligochaeta) in a riverine mud

The vertical distribution of Tubifex tubifex, Limnodrilus hoffmeisteri and L. udekemianus, in a riverine mud was studied over a year. Distribution was dependent upon species identity, season and locality. The size structure of the population, and hence species life history, was of fundamental importance in determining temporal patterns of vertical distribution. A positive correlation between worm length and depth of penetration was clearly demonstrated for all three species. However, the mid-stream populations of the three species were closer to the mud surface during the summer and autumn than were the near-bank populations, although the size structures of the populations were not significantly different. Thus, another factor, possibly the distribution of food, also influenced the vertical distribution of the worms. Cocoon distribution was similar to that of the adult worms. Cocoons of the larger species, L. udekemianus, occurred deeper in the mud than those of T. tubifex. A comparison between the vertical distributions of tubificids in cores sectioned in situ, on the stream bank, and 30 mins later, showed there to be no significant change in distribution with time.     

Enhanced tumor susceptibility of immunocompetent mice infected with lymphocytic choriomeningitis virus

Summary Mice infected i.v. with high doses of lymphocytic choriomeningitis virus (LCMV; 10⁵–10⁶ plaqueforming units) 8–10 days prior to challenge with the methylcholanthrene-induced fibrosarcoma tumor cell line MC57G or the melanoma cell line B16 tumor cells showed an enhanced tumor susceptibility with respect to both growth kinetics of the tumor and the minimal dose necessary for tumor take. After transient initial growth, MC57G tumor cells were all rejected by uninfected C57BL/6 mice by day 14. Mice preinfected i.v. with LCMV 3 weeks before or at the time of tumor challenge, but not those infected 2 months before or 7 days after, showed increasing tumor growth, the tumor take being 100% for 10⁶, 50% for 10⁵ and 37% for 10⁴ MC57G tumor cells injected into the footpad compared with resistance to 10⁶ cells in normal mice. B16 melanoma cells also grew more rapidly in LCMV-preinfected mice and by day 40 tumors were established with about 100 times fewer cells, i.e. about 10³ compared with 3×10⁴–3×10⁵ for uninfected mice. Analysis of the growth of tumor cells in normal and in LCMV-carrier mice revealed that the latter mice were not more susceptible to LCMV-infected than to uninfected MC57G. Since LCMV-carrier mice fail to mount LCMV-specific T cell responses, these results suggest that anti-LCMV-specific T cells may be responsible for acquired immunodeficiency hampering immune surveillance against the tumors studied.Supported by grants from the Swiss National Science Foundation 3.259–0.87 and the Kanton of Zürich     

The time-dependent distribution of 125I-asialo-orosomucoid-horseradish peroxidase and 131I-immunoglobulin A among three endosomal subfractions isolated from rat liver.

Three discrete endosomal fractions showing a time-dependent uptake of radioactive ligand were partially purified from rat liver. The 3,3'-diaminobenzidine (DAB)-induced density-shift protocol of Courtoy, Quintart & Baudhuin [(1984) J. Cell Biol. 98, 870-876] was used to study the distribution among these three endosomal fractions of two ligands with different intracellular destinations. Rats received both 125I-asialo-orosomucoid-horseradish peroxidase (125I-ASOR-HRP) and 131I-dIgA simultaneously by intraportal injection. The liver was fractionated at various times after injection, the three ligand-containing endosomal fractions (A, B and C) were separated and each was subjected separately to the DAB-induced density-shift procedure in which only vesicles containing 125I-ASOR-HRP are increased in density. Information on whether 131I-dIgA was co-localized or segregated from 125I-ASOR-HRP was obtained. The two ligands in the A fraction were partly segregated and partly co-localized, and this distribution appeared to be relatively unchanged with time. The two ligands in the B fraction were co-localized at all times studied. We have tentatively identified the B fraction as a compartment in which vesicle fusion has occurred. The two ligands in the C fraction were also partly co-localized and partly segregated, but the 131I-dIgA became increasingly segregated with time. This represents the first report of the purification of an endosomal subfraction specifically involved in the accumulation of multiple ligands.     

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.     

Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.     

Distinct interactions between Ca2+/calmodulin and neurotransmitter stimulation of adenylate cyclase in striatum and hippocampus

1. Ca2+ and cAMP both act as intracellular second messengers of receptor activation. In neuronal tissue, Ca2+ acting via calmodulin can elevate cAMP levels. This regulation by Ca2+ provides a means whereby the elevation of intracellular [Ca2+] might modulate cAMP generation. 2. In the present studies, the impact of the Ca2+/calmodulin regulation on receptor-mediated stimulation of activity is compared in striatum and hippocampus--regions of differing sensitivity to Ca2+/camodulin. Ca2+/calmodulin stimulated striatal and hippocampal adenylate cyclase activity by 1.4- and 2.7-fold respectively, while dopamine and vasoactive intestinal peptide (VIP) stimulated the enzyme activity of these respective regions by 1.3- and 2-fold. 3. In the presence of Ca2+/calmodulin, the dopamine dose-response curve in the striatum was shifted upward, without alteration of the slope of the curve or of the maximal stimulation of activity elicited by dopamine. In the hippocampus, the ability of VIP to stimulate adenylate cyclase activity was reduced by the presence of calmodulin. 4. The dose dependence of these actions of calmodulin was examined. In the striatum, the stimulation of adenylate cyclase activity by 0.1 to 0.3 microM calmodulin obscured dopamine stimulation, while 1 to 10 microM was additive with the dopamine stimulation. In the hippocampus, all concentrations of calmodulin (0.1 to 10 microM) reduced VIP-mediated stimulation of enzyme activity. 5. These data suggest that the ratio of calmodulin-sensitive to calmodulin-insensitive adenylate cyclase activity varies in different rat brain regions and that, in those regions in which this ratio is low (e.g., rat striatum and most peripheral systems), calmodulin- and receptor-mediated activation of adenylate cyclase activity will be additive, while in those systems in which this ratio is high (e.g., most of the central nervous system), calmodulin will reduce receptor-mediated stimulation of enzyme activity.