Gene transfer, expression and inheritance of pRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (Linnaeus)

A recombinant plasmid containing the Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter linked to rainbow trout (Salmo gairdneri) growth hormone (GH) cDNA was microinjected into fertilized carp eggs. Genomic DNA extracted from pectoral fin of individual presumptive transgenic fish was analyzed by dot blot and Southern blot hybridization, using the RSV-LTR and/or the GH cDNA sequences as probes. Out of 365 presumptive transgenic fish analyzed, 20 individuals were found to contain pRSV-rtGH-cDNA sequence in the genomic DNA. Expression of the trout GH polypeptide was detected by immunobinding assay in the red blood cells of nine transgenic fish tested. The level of expression, however, varied among the transgenics and could not be correlated with exogenous DNA copy number. Although there was considerable variation in the sizes of the transgenic fish, those microinjected during the one-cell stage were (P less than 0.05) 22% larger, on the average, than their sibling controls. A randomly selected fraction of the progeny derived from crosses between transgenic males and non-transgenic females inherited the foreign DNA. These transgenic progeny grew faster (P less than 0.05) than their non-transgenic siblings.     

Light-modulation of nitrate reductase activity in leaves and roots of maize

The nuclear DNA content in ray cells from the 1-year-old vascular cambium of white ash ( Fraxinus americana L.) trees was determined at intervals during the annual cycle of cambial activity and dormancy by using Feulgen microspectrophotometry. By 10 September, these cells had entered dormancy in G₁ with a normal DNA distribution and a minimal average DNA content of 2.65 pg. The average amount of DNA increased to 3.51 pg by 30 November, remained at this elevated value until at least 30 March, when the cambium was still dormant, then declined to the minimum level on 1 May and 10 June, when the cells were mitotically active. The springtime decline appeared to occur both before and during cell division. Between 1 May and 10 June, the prophase (4C) and telophase (2C) DNA contents decreased significantly. The amount of nuclear DNA measured by microspectrophotometry was verified by using flow cytometry and image analysis. The results support the view that there is an annual oscillation in the nuclear genome size of shoot meristematic cells in tree species native to the northern temperate zone.     

Double labelling study of the spreading depression effect on the incorporation of labelled leucine into the proteins of the brain mitochondrial subfractions

T opical application of potassium chloride solutions (above 0·25 M) as well as some other agents onto the surface of the brain cortex provokes typical changes in the basic performance of the brain. Serious impairment of learning as well as depression of spontaneous activity, depolarization of the cortical cells, disappearance of the evoked responses, changes in unit activity and alteration of energy metabolism are among the detectable features of this phenomenon called cortical spreading depression (CSD) by its discoverer LEÃ (1944). For reviews on CSD see M arshall (1959); B urést (1962); O chs (1962). The inhibitory effect of CSD on the incorporation of the labelled amino acids into the proteins of the rat cerebral cortex has been reported (R uščák 1964; B ennett and E delman , 1969; K řivánek , 1969, 1970) and the distribution of the effect among the crude subcellular fractions was studied by K řivánek (1970). Possible involvement of macromolecules, particularly proteins in memory formation and storage has been claimed. It was tempting to see whether the proteins of some mitochondrial subfractions namely nerve endings particles and pure mitochondria, could reveal a differential susceptibility to the potassium action which, in vivo , induces such profound changes in the brain cortex function.     

Interactions of small molecules with phospholipid bilayers. Binding to egg phosphatidylcholine of some uncharged molecules (2-acetylaminofluorene, 4-dimethylaminoazobenzene, oestrone and testosterone) that bind to ligandin and aminoazo-dye-binding protein A

1. To assess the possible involvement of ligandin and aminoazo-dye-binding protein A in intracellular transport it is necessary to know how their ligands, most of which are molecules with hydrophobic moieties, interact with cellular membranes. To obtain such information we have examined the interactions of 2-acetylaminofluorene, 4-dimethylaminoazobenzene, oestrone and testosterone with aqueous dispersions of egg phosphatidylcholine and egg phosphatidylcholine/cholesterol (1:1, molar ratio) by equilibrium dialysis and spectrophotometry. 2. At 25 degrees C and pH7.4, the partition coefficients for binding to phosphatidylcholine [expressed as (mol of ligand bound/mol of phosphatidylcholine)/unbound ligand concentration] were: for 2-acetylaminofluorene, 5.0x10(3) litre.mol(-1); for 4-dimethylaminoazobenzene, 2.1x10(4) litre.mol(-1); for oestrone, 3.1x10(3) litre.mol(-1); and for testosterone, 4.2x10(2) litre.mol(-1). In the ranges studied these values were independent of concentration. The results for the two steroids confirm those of Heap, Symons & Watkins [(1970) Biochim. Biophys. Acta218, 482-495]. 3. The introduction of cholesterol into the lipid bilayers caused large decreases in the partition coefficients of oestrone and testosterone, but had relatively little effect on the binding of 2-acetylaminofluorene and 4-dimethylaminoazobenzene. 4. By assuming that the interactions with egg phosphatidylcholine bilayers resemble those with the phospholipid components of mammalian intracellular membranes the phosphatidylcholine partition coefficients, together with data for binding to the intracellular proteins ligandin and aminoazo-dye-binding protein A, enable the subcellular distributions of the four compounds to be estimated. For the rat hepatocyte up to 98, 99, 89 and 58% of the total 2-acetylaminofluorene, 4-dimethylaminoazobenzene, oestrone and testosterone respectively may be membrane-bound.     

PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles

The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.     

Discovery of potent and selective PPARα/δ dual antagonists and initial biological studies

We previously published on the design and synthesis of novel, potent and selective PPARα antagonists suitable for either i.p. or oral in vivo administration for the potential treatment of cancer. Described herein is SAR for a subsequent program, where we set out to identify selective and potent PPARα/δ dual antagonist molecules. Emerging literature indicates that both PPARα and PPARδ antagonism may be helpful in curbing the proliferation of certain types of cancer. This dual antagonism could also be used to study PPARs in other settings. After testing for selective and dual potency, off-target counter screening, metabolic stability, oral bioavailability and associated toxicity, compound 11, the first reported PPARα/δ dual antagonist was chosen for more advanced preclinical evaluation.     

Expression and activity of cyclooxygenase isoforms in skeletal muscles and myocardium of humans and rodents.

Conflicting data have been reported on cyclooxygenase (COX)-1 and COX-2 expression and activity in striated muscles, including skeletal muscles and myocardium, in particular it is still unclear whether muscle cells are able to produce prostaglandins (PGs). We characterized the expression and enzymatic activity of COX-1 and COX-2 in the skeletal muscles and in the myocardium of mice, rats and humans. By RT-PCR, COX-1 and COX-2 mRNAs were observed in homogenates of mouse and rat hearts, and in different types of skeletal muscles from all different species. By Western blotting, COX-1 and -2 proteins were detected in skeletal muscles and hearts from rodents, as well as in skeletal muscles from humans. Immunoperoxidase stains showed that COX-1 and -2 were diffusely expressed in the myocytes of different muscles and in the myocardiocytes from all different species. In the presence of arachidonic acid, which is the COX enzymatic substrate, isolated skeletal muscle and heart samples from rodents released predominantly PGE(2). The biosynthesis of PGE(2) was reduced between 50 and 80% (P < 0.05 vs. vehicle) in the presence of either COX-1- or COX-2-selective blockers, demonstrating that both isoforms are enzymatically active. Exogenous PGE(2) added to isolated skeletal muscle preparations from rodents did not affect contraction, whereas it significantly fastened relaxation of a slow type muscle, such as soleus. In conclusion, COX-1 and COX-2 are expressed and enzymatically active in myocytes of skeletal muscles and hearts of rodents and humans. PGE(2) appears to be the main product of COX activity in striated muscles.     

Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (Rhamdia quelen)

Silver catfish (Rhamdia quelen; Teleostei) were exposed to commercial formulation Roundup, a glyphosate herbicide: 0 (control), 0.2 or 0.4 mg/L for 96 h. Fish exposed to glyphosate showed an increase in hepatic glycogen, but a reduction in muscle glycogen at both concentrations tested. Glucose decreased in liver and increased in muscle of fish at both herbicide concentrations. Glyphosate exposure increased lactate levels in liver and white muscle at both concentrations. Protein levels increased in liver and decreased in white muscle while levels of ammonia in both tissues increased in fish at both glyphosate concentrations. Specific AChE activity was reduced in brain after treatments, no changes were observed in muscle tissue. Catalase activity in liver did not change during of exposure. Fish exposed to glyphosate demonstrated increased TBARS production in muscle tissue at both concentrations tested. For both glyphosate concentrations tested brain showed a reduction of TBARS after 96 h of exposure. The present results showed that in 96 h, glyphosate changed AChE activity, metabolic parameters and TBARS production. The parameters measured can be used as herbicide toxicity indicators considering environmentally relevant concentration.     

Effects of Triclosan on Mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possible modes of action on non target organisms

Pharmaceuticals and Personal Care Products (PPCPs) are a class of emerging environmental pollutants with the potential of affecting various aquatic organisms through unexpected modes of action. Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) (TCS), is a common antibacterial agent that is found in significant amounts in the aquatic environment. In this work, the possible effects and modes of action of TCS were investigated in the marine bivalve Mytilus galloprovincialis Lam. In mussel immune cells, the hemocytes, in vitro short-term exposure to TCS in the low microM range reduced lysosomal membrane stability (LMS) and induced extracellular release of lysosomal hydrolytic enzymes. The effects on LMS were mediated by activation of ERK MAPKs (Extracellularly Regulated Mitogen Activated Protein Kinases) and PKC (protein kinase C) alpha and betaII isoforms, as demonstrated by both specific kinase inhibitors and Western blotting with specific anti-phospho-antibodies. The effects of TCS were confirmed in vivo, in the hemocytes of mussels injected with different concentrations of TCS (corresponding to 0.29, 2.9 and 29 ng/g dry weight) and sampled at 24 h post-injection. The possible in vivo effects of TCS were also evaluated on the activity of different enzymes in the digestive gland, the tissue mainly involved in accumulation and metabolism of organic contaminants in mussels. Significant increases were observed in the activity of the glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase), as well as of GST (GSH transferase) and GSR (GSSG reductase), whereas a decrease in catalase activity was observed. The results demonstrate that in mussels TCS can act on kinase-mediated cell signalling, lysosomal membranes and redox balance in different systems/organs. Although further studies are needed in order to evaluate possible consequences of environmental exposure to TCS on mussel health, the results represent the first data on the possible modes of action of this widespread antibacterial in aquatic invertebrates.     

Determination of pindolol enantiomers in amniotic fluid and breast milk by high-performance liquid chromatography: applications to pharmacokinetics in pregnant and lactating women

A method for the determination of pindolol enantiomers in amniotic fluid and breast milk was developed, validated, and applied to the investigation of six pregnant women treated with rac-pindolol (10 mg/12 h). Biological samples were extracted with tert-methyl-butyl ether, and the pindolol enantiomers were resolved on a Chiralpak AD column. Amniotic fluid/plasma and milk/plasma concentrations ratios ranged from 0.4 to 4.5 and from 0.6 to 3.7, respectively, for (+)-R-pindolol and from 0.5 to 3.5 and from 1.1 to 2.8, respectively, for (-)-S-pindolol. Preliminary data suggest that amniotic fluid and breast milk are routes of fetal exposure to pindolol enantiomers.