Osmotic stress-induced alterations in rice (Oryza sativa L.) and recovery on stress release

In laboratory experiments, rice plants cv Kranti were stressed osmotically using polyethylene glycol 6000 and mannitol, whereas, in pots, plants were drought stressed by withholding water supply. Both osmotic and drought stress influenced different aspects of nitrogen metabolism, resulting in a decline in the activities of aspartate aminotransferase, alanine amino-transferase and an increase in protease activity accompanied by increased free proline and alterations in other amino acid content. An influence on -amylase activity, total free sugars and starch contents was also observed, reflecting the impact of water stress on interconversion between starch and simpler sugars. Effects of polyethylene glycol 6000 as an osmotic agent were more consistent than those of mannitol during short-term (18 h) stress imposition, probably because of slight absorption of mannitol. Since the recovery for most of the parameters was substantial on release of stress, one can infer that the duration and magnitude of stress applied in the present experiments did not cause major permanent alterations in the rice cultivar Kranti. The significant basic information gathered in such experiments, particularly on recovery potential, can be utilised for varietal screening.     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

Elevated CO₂ (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO₂ uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO₂ also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO₂ treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m^-2 s^-1 for both elevated and ambient CO₂ Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m^-2 s^-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m^-2 s^-1. Hence, no significant differences between elevated and ambient CO₂ treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO₂. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO₂, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO₂ on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO₂. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.     

Promotion of murine antitumour activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α

Summary The effect of prothymosin (ProT) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10⁵ syngeneic leukaemic L1210 cells developed ascites within 8–12 days and died 10–14 days later. Treatment with ProT consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%–60% of the animals up to 70 days. The most effective treatment schedule of ProT was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor (TNF) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF and tumoricidal activity peaked 7 days after the last injection of ProT and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT and a wide range of thymosin 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF and exhibited negligible tumoricidal activity. Our data demonstrate that ProT has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProT-activated PE cells. These cells were demonstrated to produce high levels of TNF in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.Supported by a CEC grant to Dr. M. Papamichail     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

Induction of Somatic Embryogenesis and Plant Regeneration of ‘Ataulfo’ Mango (Mangifera indica)

Nucellar explants from the immature fruit of Ataulfo mango, 3–4 cm long, were used. The process had a very low efficiency (<10%); however, Ataulfo was more embryogenic than other cultivars such as Tommy Atkins or Haden in our hands. Overall, the process from nucellus to plantlet stage took 5 months. Twelve regenerated plants were obtained and are currently growing in the greenhouse. They appear to be normal and free of abnormalities. Some biochemical and molecular tests are currently being carried out.     

Formation of nucleoside 5′-phosphoramidates under potentially prebiological conditions

Summary Adenosine 5-phosphoramidates form when solutions containing adenosine 5-polyphosphates p_nA (n 3) or P₁, P₂-diadenosine 5-diphosphate and amines are allowed to dry out. Mg ions catalyze these reactions. We have studied systems containing ammonia, imidazole, glycine, ethylenediamine and histamine. The yields of adenosine 5-phosphoramidates range from 10–50 % based on the nucleotide. The prebiotic significance of the reactions is discussed.Abbreviations Im imidazole - hist histamine - gly glycine - en ethylenediamine - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - P_n (n = 1, 2 ) linear polyphosphate containing n phosphate residues - p_nA adenosine 5-polyphosphate containing n phosphate residues - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - AppA P₁, P₂-diadenosine 5-diphosphate - gly-pA adenylyl-(5N)-glycine - ImpA adenosine 5-phosphorimidazolide - NH₂-pA adenosine 5-phosphoramidate - en-pA adenylyl-(5N)-ethylenediamine - hist (NH) - pA adenosine 5-phospho-[2-(4-imidazolyl)-ethylamide] - hist(Im)-pA adenosine 5-phospho-[4-(2-aminoethyl)-imidazolide] - enP_1,2 phosphoramidates of ethylenediamine derived from H₃PO₄ and H₄P₂O₇     

Enhanced killing capacity of human Kupffer cells after activation with human granulocyte/macrophage-colony-stimulating factor and interferon γ

In this study we investigated the effect of the cytokines human granulocyte/macrophage-colony-stimulating Factor (hGM-CSF) and interferon (IFN) on human Kupffer-cell-mediated cytotoxicity against the SW948 coloncarcinoma cell line. Kupffer cells were isolated from small liver wedge biopsies, taken from 14 patient who had had abdominal surgery for colon carcinoma or partial hepatectomy. The cells were incubated with hGM-CSF (100 ng/ml), or with IFN (100 U/ml) or with their combination and the perecentage cytotoxicity was determined using a recently described modified assay. Additional experiments were performed with tumour-necrosis-factor-(TNF)-sensitive U937 cells as target. The TNF secretion of Kupffer cells was measured and we evaluated the effect of TNF on colon tumour targets. We performed human-Kupffer cell-mediated cytotoxicity blocking experiments with anti-TNF and used paraformaldehydefixed Kupffer cells to demonstrate lysis of TNF-sensitive WEHI-164 cells and of SW948 cells. The overall cytotoxicity against SW948 caused by unactivated Kupffer cells (n=14), and by Kupffer cells activated with hGM-CSF (n=14), IFN (n=6) or their combination (n=6) was respectively: 19.5±2.6%, 25.3±2.9% 41±9.4% and 45.6±8% at E/T=1 and 28.2±2.9%, 35.6±3.2%, 55.6±9.7% and 62.8% at E/T=5. All differences were statistically significant (P<0.05). No growth-promoting activity by hGM-CSF on the SW948 tumour cells was observed. U937 cells were highly susceptible to Kupffer-cell-mediated cytotoxicity. The TNF secretion by human Kupffer cells increased in parallel to their cytotoxicity after incubation with these cytokines. Soluble TNF had only a slight anti-proliferative effect on SW948 cells, while specific anti-TNF blocked Kupffer cell cytotoxicity by up to 80%. Finally, paraformaldehyde-fixed Kupffer cells were able to lyse WEHI-164 and SW948 cells. This indicates that expression of cell-associated TNF is the main cytolytic mechanism of human-Kupffer-cell-mediated cytotoxicity. The implications for the use of hGM-CSF and IFN in vivo are discussed.     

Restriction fragment analysis of ‘null’ forms at the Gli-1 loci of bread and durum wheats

Summary Wheat accessions lacking some of the - and -gliadin components encoded by the Gli-1 loci on the short arm of chromosome 1D in bread wheat and chromosome 1A in durum wheat were studied by two-dimensional polyacrylamide gel electrophoresis and restriction fragment analysis. Digested genomic DNAs of normal and null forms were probed with a cDNA clone related to -/-gliadins and with a genomic clone encoding an LMW subunit of glutenin. The hybridisation patterns with the -/-gliadin probe were similar to those of cvs Chinese Spring and Langdon used as standards for bread and durum wheats, respectively, but several restriction fragments located on the 1D chromosome of bread wheat and the 1A chromosome of durum wheat were absent in the null forms. In addition, specific LMW glutenin fragments encoded by the same chromosomes were also absent in the null forms, suggesting that simultaneous deletions of blocks of genes for both -/-gliadins and LMW glutenins had occurred. Comparisons of the protein and RFLP patterns enabled some proteins to be mapped to specific restriction fragments.     

Localization of 5′-nucleotidase in bovine brain myelin fraction and myelin subfractions

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) and 5-nucleotidase (5N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5N activity maxima suggest that myelin may be a predominant locus of 5N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5N activity were studied, and the results also suggest the intrinsic nature of 5N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5N activity, but not for the solubilization of this enzyme.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Field experiments on the optical orientation of pelagic rotifers

Avoidance of shore by pelagic rotifers is considered to be the result of an optical orientation. Field experiments show that the spatial light distribution in the shore region determines the preferred direction of migration. The behaviour of Eudiaptomus gracilis was tested in comparison to that of rotifers.This publication is dedicated to Pater Dr. Josef Donner on the occasion of his 70th birthday     

Purification, properties and possible gene assignment of an α1,3-fucosyltransferase expressed in human liver

1,3-Fucosyltransferase solubilized from human liver has been purified 40 000-fold to apparent homogeneity by a multistage process involving cation exchange chromatography on CM-Sephadex, hydrophobic interaction chromatography on Phenyl Sepharose, affinity chromatography on GDP-hexanolamine Sepharose and HPLC gel exclusion chromatography. The final step gave a major protein peak that co-chromatographed with 1,3-fucosyltransferase activity and had a specific activity of 5–6 µmol min^–1 mg^–1 and anM _r 44 000 deduced from SDS-PAGE and HPLC analysis. The purified enzyme readily utilized Gal1-4GlcNAc, NeuAc2-3Gal1-4GlcNAc and Fuc1-2Gal1-4GlcNAc, with a preference for sialylated and fucosylated Type 2 acceptors. Fuc1-2Gal1-4Glc and the Type 1 compound Gal1-3GlcNAc were very poor acceptors and no incorporation was observed with NeuAc2-6Gal1-4GlcNAc. A polyclonal antibody raised against the liver preparation reacted with the homologous enzyme and also with the blood group Lewis gene-associated 1,3/1,4-fucosyltransferase purified from the human A431 epidermoid carcinoma cell line. No cross reactivity was found with 1,3-fucosyltransferase(s) isolated from myeloid cells. Examination by Northern blot analysis of mRNA from normal liver and from the HepG2 cell line, together with a comparison of the specificity pattern of the purified enzyme with that reported for the enzyme expressed in mammalian cells transfected with theFuc-TVI cDNA, suggests a provisional identification ofFuc-TVI as the major 1,3-fucosyltransferase gene expressed in human liver.Died June, 1991     

Onychomycosis caused by Scopulariopsis brumptii

Scopulariopsis brumptii was isolated from nail lesions in left hand of a 42 year-old-farmer. The direct microscopic examination of the nail samples revealed light brown, septate, branched fungal hyphae along with thick-walled spherical cells. The histopathological examination showed involvement of internal phase of the nail plate. Amongst the antimycotics tested against S. brumptii In vitro oxiconazole was found to be the most active with MIC value of 10 g/ml^–1. This report documents the first instance of onychomycosis caused by S. brumptii.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Chromosome and isoenzyme studies on cells derived from protoplast fusion of Nicotiana glauca with glycine max-Nicotiana glauca cell hybrids

Summary The somatic hybrids of Glycine max (L)Merr.-Nicotiana glauca Grah. exhibited a preferential loss of N. glauca chromosomes. When protoplasts from such hybrid cells were back fused twice to N. glauca protoplasts, a considerable increase in stability of the N. glauca chromosomes was observed. Gel electrophoresis studies of aspartate aminotransferase showed that the chromosome(s) responsible for this enzyme was stabilized in the back fused hybrid cell lines. The data suggest that the back fusion technique described in this study might aid in stabilizing somatic hybrids.NRCC No. 18040     

Tnf-α and IL-1α inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: Evidence for primary impairment of mitochondrial function

Cytokines such as tumor necrosis factor (TNF) and Interleukin-1 (IL1) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNF and IL1. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNF (1, 10, 100, 1000 I.U./ml) and IL1 (0.1, 1, 10, 100 I.U./ ml).TNF- and IL1- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNF and IL1 and a subsequent down regulation of PDH activity.     

Isolation and partial sequence of goat spleen prothymosin α

Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT Prothymosin - T₁ Thymosin ₁ - MLR Mixed Lymphocyte Response - HPLC High Performance Liquid Chromatography - RIA Radioimmunoassay - B Aspartic acid or Asparagine - Z Glutamic acid or Glutamine     

Classification ofPythium ‘group F’ based on mycelial protein and isozyme patterns

Taxonomic characteristics were compared among 10 isolates ofPythium group F in tems of the electrophoretic patterns of their mycelial proteins and isozymes. These isolates were obtained from water of three ponds in different seasons and have an identical morphology of zoosporangia. Attempts to cross the isolates with each other themselves and with other isolates from the same group failed.Pythium group F is the most dominant of the pythia in the aquatic ecosystem and is difficult to identify because of the lack of sexual reproductive structures. Isozyme analysis proved useful in this respect. Comparisons of banding patterns of total soluble proteins and isozymes revealed five subgroups inPythium group F. Two isolates obtained from water of different ponds in different seasons showed the same protein and isozyme patterns. Our findings indicate that the use of total soluble protein and isozyme patterns for determining the variation withinPythium group F could become a valuable adjunct to the morphological and physiological criteria.     

Mutagen sensitivity and mutability in lentil

Summary Seeds of two cultivars, each of macrosperma and microsperma varietal groups of lentil were mutagenised with gamma-rays and NMU to determine their mutagen sensitivity and mutability. The increasing doeses of gamma-rays and NMU decreased germination, root and shoot length, pollen fertility and plant survival, but increased the occurrence of leaf spots. The root system was found to be more sensitive to both mutagens than the shoot. The macrosperma varietal group was more sensitive to both the mutagens than microsperma group. In microsperma group, variety Pusa-1 was more sensitive to both the mutagens than L-259, while in the macrosperma group L-1491 showed more sensitivity to the mutagens than L-1492. Radio-sensitivity corresponded positively with chemosensitivity in both varietal groups. There was a positive relationship between radio- and chemo-sensitivity of the genotypes and their mutability. The results also revealed the existence of a parallelism between radiomutability and chemo-mutability. Due to saturation in the mutational events and vigour of both diplontic and haplontic selection in the biological material, the mutation frequency either decreased or remained constant at higher doses of the mutagens.     

Expression of α-amylase in response to wounding in mung bean

The activity of -amylase (EC 3.2.1.1) in mung bean (Vigna radiata (L.) Wilczek) cotyledons increased markedly in response to wounding. The changes in enzyme activity were in parallel with those in enzyme content. The level of -amylase mRNA also notably increased in wounded cotyledons and attained its maximum level during the period between 1 and 2 d after wounding. The level of mRNA for phenylalanine ammonia-lyase, which is one of the well-characterized stress-inducible proteins, also increased after wounding, but the increase in mRNA level was faster than that of -amylase mRNA. On the other hand, the content of mRNA for actin, a housekeeping protein, was almost the same in wounded and unwounded cotyledons. The increase in -amylase mRNA level in wounded cotyledons was severely inhibited by -amanitin and cordycepin. -Amylase expression in the first leaves of mung-bean seedlings was also induced by wounding.Abbreviations PAL phenylalanine ammonia-lyase - SSC standard saline citrate We greatly acknowledge Prof. Richard Meagher, Department of Genetics, University of Georgia, Athens, USA for the gift of soybean actin gene clone. We also thank Mr. Kaoru Ishiwata for technical assistance.