Determination of retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection

A method for separating and detecting retinoids by reversed-phase capillary liquid chromatography with amperometric electrochemical detection is described. Packed columns with an inner diameter of 180 μm were employed for the separation using a C₁₈ stationary phase and a mobile phase containing acetonitrile-water-methanol (65:32.5:2.5, v/v/v) with 1% tetrabutylammonium perchlorate and 0.174 M acetate buffered at pH 5. The detection cell consisted of a carbon fiber barrel electrode held at 0.9 V versus an Ag/AgCl reference. Injection volumes of 2 μl produced detection limits of 2.73, 0.472, 0.428, and 0.267 fmol (or 410, 64.1, 60.9, and 38.2 pg ml⁻¹) for 13-cis-retinoic acid, all-trans-retinoic acid, retinaldehyde, and retinol, respectively. This represents an improvement in detection limits of at least three orders of magnitude for similar analyses using liquid chromatography and UV absorbance detection. The detector signal was linear over two orders of magnitude of analyte concentration. Retinoid concentrations in bovine serum were determined and found to be in good agreement with previously reported values.     

Human papilloma virus E6/E7 genes can expand the lifespan of human corneal fibroblasts

Summary Human corneal fibroblasts were infected with a retroviral delivery vector containing the E6 and E7 genes from human Papilloma virus type 16 in order to produce cell lines that have an expanded lifespan in culture. Morphologically, some of the trasfected corneal fibroblast lines appeared to have the normal spindle-shape morphology of diploid fibroblasts, whereas other lines appeared to have a more elongated morphology. All the cell lines were anchorage-dependent. Cells that had a normal morphology grew at a rate similar to normal diploid human corneal fibroblasts and had a population doubling time of 48 h. All E6/E7 expressing cell lines, regardless of morphology, produce types I, III, and V collagen, at levels similar to those observed in the parent corneal diploid fibroblast. These corneal fibroblast lines will be a usefulin vitro system to study collagen expression and fibril formation, as well as normal stroma development. These results also demonstrate that the use of E6/E7 genes to expand a cell’s lifespan can be a powerful tool because it does not appear to alter either the growth rate of the cell or collagen expression.     

Human immunodeficiency virus type 1 envelope protein does not stimulate either prostaglandin formation or the expression of prostaglandin H synthase in THP-1 human monocytes/macrophages.

Prostaglandin E2 is observed at elevated levels during human immunodeficiency virus (HIV) infection and thus may contribute to the HIV-dependent immunosuppression. The mechanisms responsible for this increase are not understood. Evidence indicates that the viral envelope proteins perturb membrane signaling mediated by the CD4 receptor, suggesting that the free envelope protein and/or the intact virus may be responsible for the increase in prostaglandin E2 levels. In this study, we have used THP-1 human monocytes and THP-1 cells differentiated by 12-O-tetradecanoylphorbol-13-acetate treatment into macrophages to determine if the HIV envelope protein, gp120, or an anti-CD4 receptor antibody stimulates prostaglandin formation by interacting with the CD4 receptor. Incubation of THP-1 cells with OKT4A antibody greatly stimulated the CD4-p56lck receptor complex as estimated by enhanced p56lck autophosphorylation, while the gp120 gave small but significant responses. Monocytic THP-1 cells poorly metabolized arachidonic acid to prostaglandin E2 and thromboxane B2 as measured by high-pressure liquid chromatography analysis. Western blot (immunoblot) and Northern (RNA) blot analyses revealed that unstimulated monocytes expressed little prostaglandin H synthase 1 and 2 (PGHS-1 and -2). Incubation of the monocytes with lipopolysaccharide, OKT4A, or gp120 did not increase the formation of prostaglandins. The expression of PGHS-1 or PGHS-2 was also not increased. Differentiation of the monocytes to macrophages by 12-O-tetradecanoylphorbol-13-acetate treatment resulted in increased expression of PGHS-1 and increased formation of prostaglandins compared with that for the monocytes. Lipopolysaccharide stimulation of the macrophages increased the formation of prostaglandins and increased the expression of PGHS-2 in the macrophages. However, OKT4A or gp120 preparation, at concentrations that stimulated p56lck autophosphorylation, did not enhance the formation of prostaglandins or the expression of PGHS-1 or PGHS-2. OKT4A and gp120 also did not stimulate the release of arachidonic acid, indicating that phospholipase A2 was not activated by the CD4 receptor in either the THP-1 monocytes or macrophages. These results indicate that activation of the CD4-p56lck receptor signal transduction pathway by the HIV envelope protein does not increase prostaglandin formation.     

Spatial variation in infection by digenetic trematodes in a population of freshwater snails (Potamopyrgus antipodarum)

Larval digenetic trematodes commonly castrate their first intermediate hosts, and should therefore impose strong selection on the timing and mode of host reproduction. Here we examine spatial variation in infection by trematodes in the freshwater snail Potamopyrgus antipodarum. Snails were collected at 11 different sites at Lake Alexandrina on the South Island of New Zealand from transects that ran perpendicular to the shore and across several different habitat types (from 0 to 8 m deep). Logistic regression was used to analyze the relationships between the frequency of trematode infection and snail size, habitat type, and transect location. On average, the probability of infection increased 3.3 times with each 1 mm increase in shell length. Prevalence of infection by the most common species of trematode, Microphallus sp., was highest in the shallow-water habitats where its final hosts (waterflow) spend most of their time. Prevalence of infection by another parasite, Telogaster ophistorchis (final host: eels) increased with depth, but because Microphallus was much more common, total infection by all trematodes decreased with depth. The effects of transect location were minor for Telogaster, but there was significant variation in Microphallus prevalence among transects, especially in the shore-bank habitat. Taken together, these results suggest that the risk of infection is spatially variable, but generally higher in shallow-water habitats, which may explain the greater frequency of sexual individuals as well as earlier reproduction among individuals near shore.     

Role of arginine-43 and arginine-69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites

The Hin recombinase mediates the site-specific inversion of a segment of the Salmonella chromosome between two flanking 26 bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4 bp added to the centre of the site, unlike wild-type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA-binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.     

Molecular evidence that diploid Stylosanthes humilis and diploid Stylosanthes hamata are progenitors of allotetraploid Stylosanthes hamata cv. Verano.

Restriction fragment length polymorphism analysis, using peroxidase, O-methyltransferase, phenylalanine ammonia-lyase, and coniferyl alcohol dehydrogenase cDNAs isolated from Stylosanthes humilis, as probes, provided molecular evidence for the genetic origin of the naturally occuring allotetraploid genotype Stylosanthes hamata cv. Verano (2n = 4x = 40). Hybridization patterns strongly suggest that the likely progenitors of S. hamata cv. Verano were a diploid S. humilis (2n = 2x = 20) and a diploid S. hamata (2n = 2x = 20) species.     

The effect of GABA on lumbar terminations of rubrospinal neurons in the cat spinal cord

Although GABA and piperidine-4-sulphonic acid depolarize I a afferent terminations in the cat spinal cord by activation of bicuculline-sensitive GABA receptors, no evidence was obtained for a bicuculline-sensitive alteration by either gabamimetic of the electrical threshold of rubrospinal terminations in the spinal intermediate nucleus. The terminal axonal arborizations in the spinal cord of neurons in the red nucleus thus do not have GABA receptors similar to those on the cell bodies. The results are discussed in relation to the depolarizing action of GABA on some central neurons, and on neurons with peripheral cell bodies, and to probable differences in the intracellular chloride content of neurons having peripheral or central cell bodies, and thus of different embryological origin. A presynaptic depolarizing inhibitory process mediated by GABA appears to be confined to the terminals of primary afferent fibres in the mammalian central nervous system.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

Abscission-inducing properties of methyl jasmonate, ABA, and ABA-methyl ester and their interactions with ethephon, AgNO3, and malformin

The biological activities of methyl jasmonate, ABA, methyl abscisate, and malformin were compared in a variety ofVigna radiata abscission tests. Although each compound diminished or completely negated the antiethylene properties of Ag⁺, differences in potency were observed. ABA and ABA-Me stimulated leaf abscission in the dark, potentiated abscission with low concentrations of ethephon, and interacted synergistically with malformin, whereas methyl jasmonate was inactive in each of these tests. Methyl jasmonate was most active in potentiating leaf abscission induced by high ethephon concentrations and stimulated petiole abscission, whether applied proximally or distally, from debladed explants. In two tests, negation of Ag⁺ activity and interaction with malformin, ABA concentrations as low as 0.1 M were biologically active and indicated that ABA can be a highly active abscission-inducing compound. Based on differences in biological activity, it was concluded that the modes of action of methyl jasmonate, ABA, and malformin were different.Journal Paper No. 9811 of the Purdue Agricultural Experiment Station.