The effect of lake restoration measures on the physical,chemical and phytoplankton variables of Lake Bled

Monthly changes of physical, chemical and biological variables due a combination of artificial inflow of clean water, removal of hypolimnetic water, and diversion of sewage were studied in Lake Bled from December 1980 to December 1982.During the winter period 1981/82 the species composition of the phytoplankton changed. New species replaced those observed in previous years. We conclude that the combined effect of these three lake restoration measures was responsible for the sudden disappearance ofOscillatoria rubescens D.C. A marked decrease in some nutrients and an increase in temperature and oxygen concentration also occurred.     

Identification, purification, and characterization of truncated forms of the human nerve growth factor receptor

We report the presence of truncated forms of the nerve growth factor receptor (NGFRt) in the conditioned medium of the human melanoma cell line A875 and in human urine and amniotic fluid. Radioiodinated nerve growth factor (125I-NGF) specifically bound to NGFRt was chemically cross-linked. After immunoprecipitation, labeled receptor species were visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NGFRts were purified from human adult male urine or a mixture of human amniotic fluid and infant urine by using a combination of either ion exchange chromatography (adult) or ammonium sulfate precipitation (infant) and immunoaffinity chromatography. Typical yields were about 1 microgram/liter of adult urine and 75 micrograms/liter of amniotic fluid/infant urine. The purified proteins, with molecular masses of 45, 40, and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%), were confirmed to be NGFRts by amino-terminal sequencing and were designated NGFRt-1, NGFRt-2, and NGFRt-3, respectively. The isoelectric points of these three species ranged from 3.3 to 3.95 and displayed intraspecies heterogeneity; subsequently, amino acid residues covalently modified with sialic acid-containing carbohydrates were documented. The binding affinities of these species for nerve growth factor were comparable to that of the low affinity cell surface receptor. The potential to isolate milligram quantities of human NGFRts allows for model studies of the physicochemical structure of the intact receptor and the generation of polyclonal antibodies to study the biological functions of the NGF receptor.     

Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells.

Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections. Although most research with P30 focuses on its targeting to and gating of plasmodesmata, the mechanisms of P30 intracellular movement to plasmodesmata have not been defined. To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 coding sequences under a constitutive promoter and for infection with tobacco mosaic virus particles. In both systems, P30 appears as filaments that colocalize primarily with microtubules. To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin. This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-viral RNA complex through the cytoplasm to and possibly through plasmodesmata.     

Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2

The majority of phospholipase A2 activity in myocardium is calcium-independent and selective for hydrolysis of plasmalogen substrate (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303; Hazen, S. L., Stuppy, R. J., and Gross, R. W. (1990) J. Biol. Chem. 265, 10622-10630). Accordingly, identification of an inhibitor which selectively targets calcium-independent phospholipases A2 would facilitate elucidation of the biologic significance of this class of intracellular phospholipases. We now report that the haloenol lactone, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Compound 1), is a potent, irreversible, mechanism-based inhibitor of myocardial calcium-independent phospholipase A2 which is greater than 1000-fold specific for inhibition of myocardial calcium-independent phospholipase A2 in comparisons with multiple calcium-dependent phospholipases A2. Mechanism-based inhibition of myocardial cytosolic calcium-independent phospholipase A2 by Compound 1 was established by demonstrating: 1) time-dependent irreversible inactivation; 2) covalent binding of [3H]Compound 1 to the purified phospholipase A2; 3) ablation of covalent binding of [3H]Compound 1 after chemical inactivation of phospholipase A2 enzymic activity; 4) identical inhibition of myocardial phospholipase A2 by Compound 1 in the absence or presence of nucleophilic scavengers; 5) Compound 1 is a substrate for myocardial calcium-independent phospholipase A2 resulting in the generation of the electrophilic alpha-bromomethyl ketone; 6) phospholipase A2 inhibition requires the in situ generation of the reactive electrophile (i.e. neither the alpha-bromomethyl ketone nor the diproteoenol lactone analog are inhibitory); and 7) concomitant attenuation of the inhibitory potency and the extent of covalent adduct formation in the presence of saturating substrate. Collectively, these results demonstrate that the haloenol lactone, Compound 1, is a substrate for, covalently binds to, and irreversibly inhibits canine myocardial cytosolic calcium-independent phospholipase A2.     

Intracellular delivery of trehalose renders mesenchymal stromal cells viable and immunomodulatory competent after cryopreservation

Cytotechnology - Trehalose is a nontoxic disaccharide and a promising cryoprotection agent for medically applicable cells. In this study, the efficiency of combining trehalose with reversible...     

Glutathione S-transferase polymorphisms influence the level of oxidative DNA damage and antioxidant protection in humans

The fem-3 gene of Caenorhabditis elegans was employed to determine the mutation frequency as well as the nature of mutations induced by low earth orbit space radiation ambient to Space Shuttle flight STS-76. Recovered mutations were compared to those induced by accelerated iron ions generated by the AGS synchrotron accelerator at Brookhaven National Laboratory. For logistical reasons, dauer larvae were prepared at TCU, transported to either Kennedy Space Center or Brookhaven National Laboratory, flown in space or irradiated, returned to TCU and screened for mutants. A total of 25 fem-3 mutants were recovered after the shuttle flight and yielded a mutation frequency of 2.1x10(-5), roughly 3.3-fold higher than the spontaneous rate of 6.3x10(-6). Four of the mutations were homozygous inviable, suggesting that they were large deletions encompassing fem-3 as well as neighboring, essential genes. Southern blot analyses revealed that one of the 25 contained a polymorphism in fem-3, further evidence that space radiation can induce deletions. While no polymorphisms were detected among the iron ion-induced mutations, three of the 15 mutants were homozygous inviable, which is in keeping with previous observations that high LET iron particles generate deficiencies. These data provide evidence, albeit indirect, that an important mutagenic component of ambient space radiation is high LET charged particles such as iron ions.     

Effect of C677T methylenetetrahydrofolate reductase gene polymorphism on plasma homocysteine levels in ethnic groups

The objective of this study was to examine plasma homocysteine levels and C677T methylenetetrahydrofolate reductase (MTHFR) gene polymorphism in two ethnic groups from Slovakia. The samples consisted of general Slovak-Romany population (68 men and 81 women) from Southwestern Slovakia and the Slovak-Caucasians (174 men and 177 women) who participated in the CINDI project. The homocysteine levels were examined by HPLC, the analysis of MTHFR genotypes was done by PCR. The Slovak-Romany men (12.0+/-5.6 (S.D.) micromol/l) and women (9.2+/-2.6 microol/l) have significantly lower plasma homocysteine levels (p<0.024 and p<0.00001) when compared to Caucasians (13.3+/-5.1 micromol/l in men and 11.3+/-4.3 micromol/l in women). The genetic equilibrium is assumed for the gene frequencies of the MTHFR polymorphism in both samples. The distribution of MTHFR genotypes did not differ between the two populations (TT 13 vs. 10.6 %; CT 46.6 vs. 41.7 %; CC 40.4 vs. 47.7 %, chí(2)2 = 2.315, df=2, ns). The effect of MTHFR genotypes on homocysteine levels was not confirmed in the Slovak-Romanies and TT homozygosity significantly increased plasma homocysteine levels only in Slovak-Caucasians (11.5+/-4.4 micromol/l, ns; vs. 14.8+/-4.8 micromol/l, p 0.002, respectively). To our knowledge, this is the first epidemiological study in the Romany population examining distribution of the MTHFR genotypes and their effect on homocysteine levels. Further studies are needed to establish the variety of cardiovascular risk factors among Romanies in order to evaluate the significance of particular factors.     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.     

Pentylenetetrazol-induced seizures and kindling: changes in free fatty acids,superoxide dismutase,and glutathione peroxidase activity

The whole brain free fatty acid (FFA) level, as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were determined in the frontal cortex, cerebellum, hippocampus, and pons-medulla region of the single pentylenetetrazol (PZT)-treated and PZT-kindled Hannover-Wistar rats. PZT administration in the convulsive dose caused significant increase of the brain FFA content. Decreased SOD activity was detected in the frontal cortex of PZT-kindled rats, whereas decreased GPX activity was found in the frontal cortex and cerebellum of all treated rats, as well as in the hippocampus and pons-medulla of PZT-kindled rats. Kindling caused distinctive change of antioxidative defense in the frontal cortex, hippocampus, and pons-medulla region.