Evidence that guanine-nucleotide binding regulatory proteins couple cell-surface receptors to the breakdown of inositol-containing lipids during T-lymphocyte mitogenesis

We have studied the role of guanine-nucleotide binding regulatory proteins (G proteins) in the stimulation of inositol lipid breakdown during mitogenic activation of normal human T lymphocytes. The effect of the mitogen phytohemagglutinin (PHA) was compared with the action of two G-protein activators, fluoroaluminate (AlF4-) and guanosine-5'-O-thiotriphosphate (GTP gamma S). PHA and AlF4- stimulated the breakdown of inositol lipids via both the phospholipase A and C pathways when added to intact lymphocytes. PHA, AlF4- and GTP gamma S also triggered both these pathways when added to permeable lymphocytes. The magnitude of the response obtained with AlF4- and GTP gamma S was about four-fold less than with PHA. This difference was attributable to increases in cAMP elicited by AlF4- and GTP gamma S which inhibited the phospholipase pathways. AlF4-, GTP gamma S, and PHA all stimulated the phosphorylation of a 42 kDa protein on tyrosine residues. We propose a model for the early steps following mitogen binding, including sequential activation of a G protein, phospholipase C, protein kinase C and a tyrosine protein kinase. A parallel pathway involving G protein mediated activation of phospholipase A is also implicated.     

Phytohemagglutinin treatment of T lymphocytes stimulates rapid increases in activity of both particulate and cytosolic protein kinase C

We have measured the activity of protein kinase C in particulate and cytosolic fractions prepared from lymphocytes following stimulation with phytohemagglutinin. Activity in the particulate fraction increased approximately three-fold within 5 min, and declined to nearly zero between 20 and 60 min. Cytosolic activity increased in a biphasic manner, with an initial increase at 5 min, a decline at 10 min, and a further increase by 20 min, which was sustained for at least 60 min. By contrast, 12-O-tetradecanoylphorbol-13-acetate caused a rapid translocation of protein kinase C from cytosol to the particulate fraction which was sustained for at least 1 h. The results suggest that agents, such as phytohemagglutinin, which both generate diacylglycerol and mobilize intracellular Ca2+ stores, result in changes in subcellular distribution and activity of protein kinase C which are different from those elicited by 12-O-tetradecanoylphorbol-13-acetate.     

Comparative study of methods for the quantification of fungal spores at the leaf surfaces ofPopulus xeuramericana

Summary The removal of fungal spores, urediniospores ofMelampsora medusae and conidia ofPestalozzia sp., from the leaf surfaces ofPopulus xeuramericana (Dode) Guinier cv. I-488 was assessed using three cultural techniques conventionally employed in phylloplane studies. The method of removal and the original density of spore deposition, but not the interaction of these factors, were significant determinants of variability in spore removal. Irrespective of the original density of deposition, the leaf print method was the most, and the leaf washing technique the least, efficient means of spore removal from the leaf surface. Factors which could contribute to this difference in efficiency are discussed.     

Alternative metabolic fates of thymine nucleotides in human cells.

Three types of experiments have been used to study the metabolism of thymine nucleotides by human cells. (1) Cells were labelled continuously with [3H]thymidine and the incorporation of label into DNA compared with the specific radioactivities of pools of individual thymine nucleotides separated by chromatography on polyethylene-imine-cellulose. (2) Cellular thymine nucleotides were labelled with [3H]thymidine at 13 degrees C, followed by incubation at 37 degrees C in unlabelled medium. Incorporation of label into DNA and loss of label from the nucleotide pools were monitored during the 'chase' period at 37 degrees C. (3) The experiments described in (2) above were repeated in the presence of the DNA-synthesis inhibitor cytosine arabinoside, in order to demonstrate more clearly and to quantify degradative pathways for thymine nucleotides. In phytohaemagglutinin-stimulated lymphocytes and in bone-marrow cells, only a proportion (25-60%) of labelled thymine nucleotide was incorporated into DNA, the rest being rapidly degraded and lost from the cell. In contrast, an established cell line (HPB-ALL) from a patient with acute lymphoblastic leukaemia of thymic origin incorporated 100% of its exogenously labelled thymine nucleotides into DNA. These results indicated that alternative metabolic routes are open to thymine nucleotides in human cells. In lymphocytes from patients with megaloblastic anaemia and in normal lymphocytes treated with methotrexate, the utilization of labelled thymine nucleotides for DNA synthesis was more efficient than in controls. These results offer an explanation for the observation of a normal pool of thymidine triphosphate in the cells of patients with untreated megaloblastic anaemia even though the amount of this compound available for DNA synthesis appears to be decreased.     

Urea hydrolysis in some tea soils

The influence of application of hexachlorocyclohexane (HCH=gamma BHC), to a submerged tropical field soil at rates equivalent to recommended field rates (1–2.5kg a.i./ha) and twice this level, upon the rhizosphere soil nitrogenase, nitrogen fixers, and soil redox potential (Eh) was investigated. The rhizosphere soil from HCH-treated field exhibited significantly higher nitrogenase activity than that from untreated fields. HCH retarded the drop in redox potential of the field soil upto 80 days after transplantation under submerged conditions. Populations of nitrogen-fixingAzospirillum sp. and Azotobacter, to a greater extent, and anaerobic organisms, to a lesser extent, were stimulated in HCH-treated soils. Results indicate the stimulation of heterotrophic nitrogen-fixing bacteria by HCH in submerged paddy soils.     

Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants

A wide-ranging examination of plastid (pt)DNA sequence homologies within higher plant nuclear genomes (promiscuous DNA) was undertaken. Digestion with methylation-sensitive restriction enzymes and Southern analysis was used to distinguish plastid and nuclear DNA in order to assess the extent of variability of promiscuous sequences within and between plant species. Some species, such as Gossypium hirsutum (cotton), Nicotiana tabacum (tobacco), and Chenopodium quinoa, showed homogenity of these sequences, while intraspecific sequence variation was observed among different cultivars of Pisum sativum (pea), Hordeum vulgare (barley), and Triticum aestivum (wheat). Hypervariability of plastid sequence homologies was identified in the nuclear genomes of Spinacea oleracea (spinach) and Beta vulgaris (beet), in which individual plants were shown to possess a unique spectrum of nuclear sequences with ptDNA homology. This hypervariability apparently extended to somatic variation in B. vulgaris. No sequences with ptDNA homology were identified by this method in the nuclear genome of Arabidopsis thaliana.      

Incidence and levels of fumonisin contamination in Colombian corn and corn products

A survey was conducted to determine the levels of fumonisins B1 and B2 in corn and corn-based products available in Colombia for human and animal consumption. A total of 120 samples were analyzed by acetonitrile-water extraction, cleanup with a strong-anion-exchange column, and liquid chromatography with o-phthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. The samples of corn and corn-based products for animal intake were taken at different feed manufacturing plants, whereas the samples used for human foods where purchased from local retail stores. The number of positive samples for fumonisin B1 was 20.0% higher in corn and corn-based products for animal intake (75.0%) than in corn and corn-based products for human consumption (55.0%). The levels of fumonisin B1 were also higher in corn and corn-based products for animal intake (mean = 694 μg/kg; range = 32–2964 μg/kg), than in corn and corn-based products for human intake (mean = 218 μg/kg; range = 24–2170 μg/ kg). The incidence and levels of fumonisin B2 were lower than those for fumonisin B1. Corn and corn-based products for animal consumption had an incidence of fumonisin B2 of 58.3%, with a mean value of 283 μg/kg, and a range of 44–987 μg/kg. The incidence of fumonisin B2 in corn-based products for human intake was 35.0%, with a mean value of 118 μg/kg and a range of 21–833 μg/kg. The highest incidence and levels of fumonisins were found in samples of hominy feed, with concentrations ranging from 86 to 2964 μg/kg fumonisin B1 and 57 to 987 μg/kg fumonisin B2.     

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.