The biophysics of leaf growth in salt-stressed barley. A study at the cell level

Biophysical parameters potentially involved in growth regulation were studied at the single-cell level in the third leaf of barley (Hordeum vulgare) after exposure to various degrees of NaCl stress for 3 to 5 d. Gradients of elongation growth were measured, and turgor pressure, osmolality, and water potentials (psi) were determined (pressure probe and picoliter osmometry) in epidermal cells of the elongation zone and the mature blade. Cells in the elongation zone adjusted to decreasing external psi through increases in cell osmolality that were accomplished by increased solute loads and reduced water contents. Cell turgor changed only slightly. In contrast, decreases in turgor also contributed significantly to psi adjustment in the mature blade. Solute deposition rates in the elongation zone increased at moderate stress levels as compared with control conditions, but decreased again at more severe NaCl exposure. Growth-associated psi gradients between expanding epidermal cells and the xylem were significant under control and moderate stress conditions (75 mM NaCl) but seemed negligible at severe stress (120 mM NaCl). We conclude that leaf cell elongation in NaCl-treated barley is probably limited by the rate at which solutes can be taken up to generate turgor, particularly at high NaCl levels.     

Regulating role of various amino acids in development of apoptosis in organotypic culture of the nervous and lymphoid tissue

The effect of aminoacids L-arginin, L-lysine, L-asparagin was investigated in organotypic tissue culture of brain cortex and spleen of 1-day old rats. The aminoacids in concentrations 0.05 and 0.1 ng/ml are active, inducing a less intensive growth zone as compared to the control, excluding the effect of asparagin on lymphoid tissue. Method of fluorescent staining shows a negative correlation between growth zone size and apoptotic cell number. The nerve and lymphoid tissue express apoptosis in response to aminoacids.     

Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.     

Apoplasmic barriers and oxygen transport properties of hypodermal cell walls in roots from four amazonian tree species

The formation of suberized and lignified barriers in the exodermis is suggested to be part of a suite of adaptations to flooded or waterlogged conditions, adjusting transport of solutes and gases in and out of roots. In this study, the composition of apoplasmic barriers in hypodermal cell walls and oxygen profiles in roots and the surrounding medium of four Amazon tree species that are subjected to long-term flooding at their habitat was analyzed. In hypodermal cell walls of the deciduous tree Crateva benthami, suberization is very weak and dominated by monoacids, 2-hydroxy acids, and omega-hydroxycarboxylic acids. This species does not show any morphological adaptations to flooding and overcomes the aquatic period in a dormant state. Hypodermal cells of Tabernaemontana juruana, a tree which is able to maintain its leaf system during the aquatic phase, are characterized by extensively suberized walls, incrusted mainly by the unsaturated C(18) omega-hydroxycarboxylic acid and the alpha,omega-dicarboxylic acid analogon, known as typical suberin markers. Two other evergreen species, Laetia corymbulosa and Salix martiana, contained 3- to 4-fold less aliphatic suberin in the exodermis, but more than 85% of the aromatic moiety of suberin are composed of para-hydroxybenzoic acid, suggesting a function of suberin in pathogen defense. No major differences in the lignin content among the species were observed. Determination of oxygen distribution in the roots and rhizosphere of the four species revealed that radial loss of oxygen can be effectively restricted by the formation of suberized barriers but not by lignification of exodermal cell walls.     

Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment

We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-alpha-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-alpha-D-mannosyl-D-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-alpha-D-mannosyl-D-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-alpha-D-mannosyl-D-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.     

Biosynthesis of fungal melanins and their importance for human pathogenic fungi

For more than 40 years fungi have been known to produce pigments known as melanins. Predominantly these have been dihydroxyphenylalanine (DOPA)-melanin and dihydroxynaphthalene (DHN)-melanin. The biochemical and genetical analysis of the biosynthesis pathways have led to the identification of the genes and corresponding enzymes of the pathways. Only recently have both these types of melanin been linked to virulence in some human pathogenic and phytopathogenic fungi. The absence of melanin in human pathogenic and phytopathogenic fungi often leads to a decrease in virulence. In phytopathogenic fungi such as Magnaporthe grisea and Colletotrichum lagenarium, besides other possible functions in pathogenicity, DHN-melanin plays an essential role in generating turgor for plant appressoria to penetrate plant leaves. While the function of melanin in human pathogenic fungi such as Cryptococcus neoformans, Wangiella dermatitidis, Sporothrix schenckii, and Aspergillus fumigatus is less well defined, its role in protecting fungal cells has clearly been shown. Specifically, the ability of both DOPA- and DHN-melanins to quench free radicals is thought to be an important factor in virulence. In addition, in several fungi the production of fungal virulence factors, such as melanin, has been linked to a cAMP-dependent signaling pathway. Many of the components involved in the signaling pathway have been identified.     

Zinc serum level in human immunodeficiency virus-infected patients in relation to immunological status

In human immunodeficiency virus (HIV) infection, serum level of zinc, an important micronutrient for immune function, is frequently diminished. The aim of this study was to determine the zinc status in relation to immunological parameters and disease stage in 79 HIV-1 seropositive patients. The median serum level of zinc was within normal limits (12.5 micromol/L) but in 23% of patients, zinc deficiency was seen. Decreased serum zinc was associated with a low CD4 cell count, high viral load, and increased neopterin and IgA levels. According to current treatment recommendations, the majority of patients received antiretroviral triple therapy. Zinc levels in treated and untreated patients were comparable. Referring to disease stage (CDC classification, 1993), the mean zinc level was highest in stage C and lowest in stage A. In conclusion, even under antiretroviral triple therapy, zinc deficiency is still of great importance in HIV infection, and zinc substitution in zinc deficient individuals should be taken into account to optimize therapeutical success.     

LOCATION AND EXPRESSION OF FRUSTULINS IN THE PENNATE DIATOMS CYLINDROTHECA FUSIFORMIS, NAVICULA PELLICULOSA, AND NAVICULA SALINARUM (BACILLARIOPHYCEAE)

A detailed immunocytochemical and biochemical study of the location and expression of frustulins, a family of proteins associated with the frustules of diatoms, has been performed for Cylindrotheca fusiformis Reimann et Lewin, Navicula pelliculosa (Brébisson et Kützing) Hilse, and Navicula salinarum (Grunow) Husted. Immunocytochemistry revealed that frustulins, which share homologous epitopes but are different in size, were predominantly located in the organic casing. Based on timed immunolocalization experiments and Western blotting analysis of cell extracts obtained sequentially after repleting silicate to Si-synchronized cells, the continuous presence of the frustulins in the mature and parental organic casing of the examined species was observed. The frustulins of N. pelliculosa appeared as proteins similar to those of C. fusiformis, sharing identical epitopes. The extractions, however, yielded a markedly lower abundance of frustulins in N. pelliculosa. Peak concentrations of extracted frustulins appeared to be expressed just ahead of the silicification process in C. fusiformis, whereas the level of expression in N. pelliculosa increased along with maturation of the new valves. For N. salinarum, the presence of the frustulins could not be confirmed properly by Western blotting, most probably because of the small sample volumes, inefficient extraction, and a lower amount of homologous frustulins in the casing of this species. It is concluded that the frustulins of these species are not associated with the silicalemma of the newly formed silica deposition vesicles and therefore do not seem to be involved in the silicification process itself. Overall, the results imply a structural role of the frustulins in the casing of diatoms rather than a regulation of the silicification process.     

A new Georissa (Gastropoda: Neritopsina: Hydrocenidae) from a limestone cave in Malaysian Borneo

In a previous paper, we have reported on a new, troglobiticspecies of Georissa and its possible parapatric origin fromGeorissa saulae Benthem-Jutting, 1966, which occurs outsidethe cave system of Batu Sanaron, a limestone outcrop in Sabah,Malaysian Borneo. These analyses were based on genetic and morphometricdata. Here, we formally describe the new species adding anatomicaldata derived from dissections and histological serial sectionsas well as fine structural data of shell, operculum and radula,and compare it with its stem species. The two species differin shell and radular morphology as well as genital characters.Since we found anatomical differences between very closely relatedspecies, we assume that dissections would be of general usefor the taxonomy of Georissa and the remaining nominal genus-grouptaxa of the poorly known Hydrocenidae.