Myocardial cell vulnerability to exogenous phospholipase attack

Myocardial cell vulnerability to phospholipase C (PC-PLC) attack was investigated in three different preparations of rat myocardial cells: triacylglycerol (TG)-loaded, hypothermic/rewarmed and energy depleted myocytes. The attack by PC-PLC was evaluated as PC-PLC induced glycerol output due to the combined action of phospholipase C and intracellular lipases. PC-PLC induced glycerol output was significantly higher (p < 0.05) in all three myocyte preparations, compared to their respective controls. Cell morphology (% rod shaped myocytes) of TG-loaded or hypothermic/rewarmed myocytes was not different from their controls, whereas energy depleted myocytes almost exclusively were rounded up, due to hypercontraction of the myofilaments. Hypothermic/rewarmed and energy depleted myocytes showed a significantly higher release of lactate dehydrogenase (LDH), compared to their controls although the difference was much more pronounced in the latter. Finally, the cellular contents of ATP were maintained both in TG-loaded and hypothermic rewarmed myocytes, while energy depleted myocytes contained only about 25% of the normal ATP level. These results demonstrate that attack from exogenously added phospholipases can occur, not only in seriously damaged cardiac myocytes, but in myocytes with a more subtle damage as well. (Mol Cell Biochem 116: 47–52, 1992)     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

Immunocytochemical localization of atrial natriuretic peptide in the venae cavae and the pulmonary veins of the rat

Summary In the present investigation atrial natriuretic peptide (ANP) was localized in striated myocytes of the venae cavae and the pulmonary veins in the rat by the use of immunohistochemical and immunocytochemical staining techniques. ANP was stored in granules which appeared to be morphologically similar to the atrial specific granules (ASG) of the atria. In general, the amount of ASG in the great thoracic veins was less than observed in the atria, and the specific granules appeared to be more evenly distributed throughout the sarcoplasm. However, the presence of ANP-containing specific granules in the venae cavae and the pulmonary veins may suggest participation of these veins in the production and secretion of the hormone.     

The carbon assimilation pathways of Methylococcus capsulatus, Pseudomonas methanica and Methylosinus trichosporium (OB3B) during growth on methane

d-arabino-3-Hexulose 6-phosphate was prepared by condensation of formaldehyde with ribulose 5-phosphate in the presence of 3-hexulose phosphate synthase from methane-grown Methylococcus capsulatus. The 3-hexulose phosphate was unstable in solutions of pH greater than 3, giving a mixture of products in which, after dephosphorylation, allulose and fructose were detected. A complete conversion of d-ribulose 5-phosphate and formaldehyde into d-fructose 6-phosphate was demonstrated in the presence of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase (prepared from methane-grown M. capsulatus). d-Allulose 6-phosphate was prepared from d-allose by way of d-allose 6-phosphate. No evidence was found for its metabolism by extracts of M. capsulatus, thus eliminating it as an intermediate in the carbon assimilation process of this organism. A survey was made of the enzymes involved in the regeneration of pentose phosphate during C(1) assimilation via a modified pentose phosphate cycle. On the basis of the presence of the necessary enzymes, two alternative routes for cleavage of fructose 6-phosphate are suggested, one route involves fructose diphosphate aldolase and the other 6-phospho-2-keto-3-deoxygluconate aldolase. A detailed formulation of the complete ribulose monophosphate cycle of formaldehyde fixation is presented. The energy requirements for carbon assimilation by this cycle are compared with those for the serine pathway and the ribulose diphosphate cycle of carbon dioxide fixation. A cyclic scheme for oxidation of formaldehyde via 6-phosphogluconate is suggested.     

Relationships of the chromosomal species in the eurasian mole rats of theSpalax ehrenbergi group as determined by DNA-DNA hybridization,and an estimate of the spalacid-murid divergence time

Summary DNA-DNA hybridization was used to measure the average genomic divergence among the four chromosomal species of the Eurasian mole rats belonging to theSpalax ehrenbergi complex (Rodentia: Spalacidae). The percent nucleotide substitutions in the single-copy nuclear DNA among the species ranged from 0 to 5%, suggesting that speciation has occurred with minor genomic changes in these animals. The youngest chromosomal species appear to differ by 0.2–0.6% base pair mismatch, which is only between one and three base differences in a 500-bp fragment. The interspecific values of percent nucleotide differences permit the recognition of two well-separated speciation events in theS. ehrenbergi complex, the older (of Lower Pleistocene age) having isolated the chromosomal species 2n=54 before the divergence of the three other species.DNA-DNA hybridization was also used to compare the Spalacinae (Eurasian mole rats), Murinae (Old World rats and mice), and Arvicolinae (voles and lemmings). These data enabled us to estimate the time of divergence of the spalacids at ca. 19 million years ago. The dates of divergence among the other rodent lineages, as predicted by DNA hybridization results, agree well with paleontological data. These dates of divergence are obtained by the relation between geological time and single-copy nuclear DNA change, a relation that was calibrated by Catzeflis et al. (1987) through the use of fossil Arvicolinae and Murinae data.     

The polymerase-like core of brome mosaic virus 2a protein, lacking a region interacting with viral 1a protein in vitro, maintains activity and 1a selectivity in RNA replication.

Brome mosaic virus (BMV), a member of the alphavirus-like super-family of positive-strand RNA viruses, encodes two proteins required for viral RNA replication: 1a and 2a. 1a contains m7G methyltransferase- and helicase-like domains, while 2a contains a polymerase (pol)-like core flanked by N- and C-terminal extensions. Genetic studies show that BMV RNA replication requires 1a-2a compatibility implying direct or indirect 1a-2a interaction in vivo. In vitro, la interacts with the N-terminal 125-amino-acid segment of 2a preceding the pol-like core, and prior deletion studies suggested that this 2a segment was essential for RNA replication. We have now used protein fusions and deletions to explore possible parallels between noncovalent 1a-2a interaction and covalent fusion of similar protein domains in tobacco mosaic virus and to see whether the N-terminal 2a-1a interaction was the primary basis for 1a-2a compatibility in vivo. We found that 2a can function as part of a tobacco mosaic virus-like 1a-2a fusion and that a 2a segment (amino acids 162 to 697) comprising the pol-like core was sufficient to provide 2a functions in such a fusion. Unexpectedly, the unfused 2a core segment also supported RNA replication when it and wild-type la were expressed as separate proteins. Moreover, in gene reassortant experiments with the related cowpea chlorotic mottle virus, the unfused 2a core segment showed the same 1a compatibility requirements as did wild-type BMV 2a. Thus, the pol-like core of 2a must interact with la in a way that is selective and essential for RNA synthesis, and 1a-2a interactions are more complex than the single, previously mapped interaction of the N-terminal 2a segment with 1a.     

Microtubule-associated distribution of specific granules and secretion of atrial natriuretic factor in primary cultures of rat cardiomyocytes

A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent.     

Targeting of superantigens

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on MHC class II antigens. In order to induce T lymphocytes to reject a tumor, we substituted the specificity of SEA for MHC class II molecules with specificity for tumor cells by combining SEA with a MAb recognizing colon carcinomas. Chemical conjugates or recombinant fusion proteins of the MAb C215 and SEA retained excellent antigen binding properties whereas the binding to MHC class II was markedly reduced. The hybrid proteins directed SEA responsive T cells to tumors with specificity determined by the specificity of the MAb. Significant tumor cell killing was obtained at picomolar concentrations of the hybrid proteins and was the result of direct cell mediated by cytotoxicity as well as production of tumoricidal cytokines by T cells. Targeting of superantigens represents a novel approach to specific immunomodulation and deserves further study as a potential therapy for malignant disease.     

Crustacean plankton in Høylandet

Crustacean plankton was studied in 12 lakes in theHøylandet area in 1986–87. Basic lake characteristicsare elevation 134–415 m, surface area 9–530 ha, pH 5.9–7.1,conductivity (25 °C) 12–40 µS cm^-1 andSecchi depth 4–9 m. Number ofspecies present varied between 3 and 11. Populationnumbers between 4000 and 400 000 per m² andbiomasses were within the range 30 to1800 mg m^-2 dry weight. Cladocera dominated overCopepoda in lakes with allopatric brown trout (Salmo trutta L.), on the contrary to lakes also populatedby Arctic charr (Salvelinus alpinus (L.)). Thesevariations are caused by differences in elevation,lake morphometry, water quality, fish predation andthe general distribution of the species. The largestlakes at lowest elevation were richest in species. Theacid sensitive genus Daphnia was represented by 3species. The lakes Storgrønningen (530 ha) andRøyrtjønna (27 ha) were sampled monthly in theice-free seasons of 1986–89, and Storgrønningen moreintensively from June to November in 1987 and 1988. The same6 species of Cladocera and 5 of Copepoda were presentin both lakes. Their life cycles were traditional orknown from several other Scandinavian lakes. Meanseasonal biomasses were of the range600–750 mg m^-2. At the species level, there wereconsiderable variations between years inStorgrønningen and particularly in Røyrtjønna. Noeffects of human impacts on the crustacean planktonwere found. The Høylandet lakes are representative forScandinavian oligotrophic to almost ultra-oligotrophiclakes. Storgrønningen is well qualified as a referencesystem. The between year variations in Røyrtjønna areso extreme, that any human impact could only be traced at alevel causing the extinction of species.