Characterization of the calcium sensitivity of differentiation in SCC-13 human squamous carcinoma cells

Summary The sensitivity to calcium of the human squamous carcinoma cell line, SCC-13, was demonstrated and characterized. Cultures grown to confluence in the presence of 0.2 to 2 mM calcium had approximately 10-fold higher levels of particulate transglutaminase activity and envelope competence than those grown in low calcium (0.025 to 0.05 mM) medium. Raising the calcium from 0.025 to 1.8 mM induced expression of this enzyme and of competence over the course of a week. Conversely, for cultures grown to confluence in 1.8 mM calcium, subsequent reduction of calcium to 0.025 mM resulted in a substantial decline in transglutaminase over a similar time period. Immunoprecipitable transglutaminase was clearly identifiable in cultures grown in 1.8 mM calcium-containing medium but not in those grown in low calcium medium or in the presence of retinoic acid, suggestive of regulation at the level of mRNA accumulation or translation rather than posttranslational modification. This research was supported by Public Health Service grant AR 27130 from the National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, and National Research Service postdoctoral fellowship ES 05336 from the National Institute of Environmental Health Sciences, Research Triangle Park, NC.     

Simultaneous determination of ascorbic acid and dehydroascorbic acid in cultures of C3H/10T1/2 cells

Summary A reproducible method is described for the separation and quantification of ascorbic acid and dehydroascorbic acid by ion-pairing reverse-phase high performance liquid chromatography and detection by absorbance at 232 nm. Lowest detectable concentrations with a linear response of detection were 5 nmol for ascorbic acid and 50 nmol for dehydroascorbic acid. This method was applied to the analysis of C3H/10T1/2 cells and culture medium after influx or efflux experiments and single or multiple treatments with ascorbic acid. Subsequent measurement of the radioactivity in the eluted fractions increased the detectability of both ascorbic acid and dehydroascorbic acid to 10 to 20 pmol. This research was supported by grant CA 09320 and CA 31574 from the National Cancer Institute, Bethesda, MD, and grant BC441 from The American Cancer Society.     

Environmentally directed mutations in the dehalogenase system of Pseudomonas putida strain PP3

Favourable mutations involving the two dehalogenases (DehI and DehII) of Pseudomonas putida PP3 and derivative strains containing the cloned gene for DehI (dehI) occurred in response to specific environmental conditions, namely: starvation conditions; the presence of dehalogenase substrates (halogenated alkanoic acids — HAAs) which were toxic to P. putida; and/or the presence of a potential growth substrate. Fluctuation tests showed that these mutations were environmentally directed by the presence of HAAs. the mutations were associated with complex DNA rearrangements involving the movement of dehI located on a transposon DEH. Some mutations resulted in switching off the expression of either one or both of the dehalogenases, events which were effective in protecting P. putida from toxic compounds in its growth environment. Other mutations partially restored P. putida's dehalogenating capability under conditions where toxic substrates were absent. Restoration of the capability to untilize HAAs was favoured when normal growth substrates were present in the environment.     

A family with RP3 type of X-linked retinitis pigmentosa: an association with ciliary abnormalities

Summary The results of linkage analysis in a family with X-linked retinitis pigmentosa (XLRP) are presented. Probe M27B (DXS255), localised to Xp11.22, was only loosely linked to XLRP, whereas pHOC3 (OTC), in the more distal Xp21.1 region, was tightly linked. In this family, the conditional probability of an RP3 locus (in Xp21.1–p11.4) was found to be 0.978 compared with 0.021 for an RP2 locus (in Xp11.4–p11.2). Risk assessment showed that 2 out of 4 at risk females showing no clinical abnormality have a high probability of being genetic carriers of XLRP. Some affected males have recurrent respiratory infections as a result of a condition indistinguishable from the immotile cilia syndrome; indeed, there is an association between XLRP and susceptibility to respiratory infections in the majority of affected males. The possibility that previously observed ciliary abnormalities in XLRP patients might be associated specifically with an RP3 locus abnormality is discussed.     

A nomenclature for the genes encoding the chlorophylla/b-binding proteins of higher plants

We propose a nomenclature for the genes encoding the chlorophylla/b-binding proteins of the light-harvesting complexes of photosystem I and II. The genes encoding LHC I and LHC II polypeptides are namedLhca1 throughLhca4 andLhcb1 throughLhcb6, respectively. The proposal follows the general format recommended by the Commision on Plant Gene Nomenclature. We also present a table for the conversion of old gene names to the new nomenclature.     

Metabolism of oxygen radicals in peroxisomes and cellular implications.

Peroxisomes are subcellular respiratory organelles which contain catalase and H2O2-producing flavin oxidases as basic enzymatic constituents. These organelles have an essentially oxidative type of metabolism and have the potential to carry out different important metabolic pathways. In recent years the presence of different types of superoxide dismutase (SOD) have been demonstrated in peroxisomes from several plant species, and more recently the occurrence of SOD has been extended to peroxisomes from human and transformed yeast cells. A copper,zinc-containing SOD from plant peroxisomes has been purified and partially characterized. The production of hydroxyl and superoxide radicals has been studied in peroxisomes. There are two sites of O2- production in peroxisomes: (1) in the matrix, the generating system being xanthine oxidase; and (2) in peroxisomal membranes, dependent on reduced nicotinamide adenine dinucleotide (NADH), and the electron transport components of the peroxisomal membrane are possibly responsible. The generation of oxygen radicals in peroxisomes could have important effects on cellular metabolism. Diverse cellular implications of oxyradical metabolism in peroxisomes are discussed in relation to phenomena such as cell injury, peroxisomal genetic diseases, peroxisome proliferation and oxidative stress, metal and salt stress, catabolism of nucleic acids, senescence, and plant pathogenic processes.     

Carotenoids and abscisic acid (ABA) biosynthesis in higher plants

Recent research has revealed that abscisic acid (ABA), synthesised in response to water stress, is an apo-carotenoid. Two potential carotenoid precursors, 9'- cis -neoxanthin and 9- cis -violaxanthin, have been identified in light-grown and etiolated leaves, and in roots of a variety of species. Experiments utilizing etiolated Phaseolus vulgaris leaves and deuterium oxide strongly suggest that 9'- cis -neoxanthin, synthesised from all- trans -violaxanthin, is the immediate pre-cleavage precursor of ABA. The cleavage of 9'- cis -neoxanthin, performed by an inducible and specific dioxygenase, is likely to be the rate-limiting step in ABA biosynthesis. Any apocarotenoids formed as by-products of cleavage are probably rapidly degraded by lipoxygenase or related enzymes. After cleavage xanthoxin is converted via ABA-aldehyde to ABA by constitutive enzymes in the cytosol.     

Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants

A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons.