Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

Triplex formation by a psoralen-conjugated oligodeoxyribonucleotide containing the base analog 8-oxo-adenine.

Oligodeoxyribonucleotides containing thymidine and 8-oxo-2'-deoxyadenosine can form pyr.pur.pyr type triplexes with double-stranded DNA. Unlike triplexes whose third strands contain thymidine and deoxycytidine, the stability of these triplexes is independent of pH. We have prepared d-ps-TAAATAAATTTTTAT-L [I(A)], where A is 8-oxo-2'-deoxyadenosine, ps is 4'-hydroxymethyl-4,5',8- trimethylpsoralen and L is a 6-amino-2-(hydroxymethyl)hexyl linker. The oligomer is designed to interact with a homopurine sequence in the promoter region of the human gene coding for the 92 kDa form of collagenase type IV. Oligomer I(A) and oligomer I(C), which contains 2'-deoxycytidine in place of 8-oxo-2'-deoxycytidine, both form stable triplexes at pH 6.2, but only I(A) forms a stable triplex with a model duplex DNA target at pH 7.5, as determined by UV melting experiments. Triplex formation is stabilized by the presence of the psoralen group. Upon irradiation both I(A) and I(C) form photoadducts with the DNA target at pH 6.2, but only I(A) forms a photoadduct at pH 7.5. In these photoreactions oligomer I(A) appears to selectively form a photoadduct with a C in the purine-rich strand of the duplex target. Although a T residue is present in the pyrimidine-rich strand of the target at the duplex/triplex junction, essentially no adduct formation takes place with this strand, nor is interstrand cross-linking observed. The extent of photoadduct formation decreases with increasing temperature, behavior which is consistent with the UV melting curve of the triplex. A tetramethylrhodamine derivative of I(A) was prepared and found to cross-link less extensively than I(A) itself. Oligomer I(A) is completely resistant to hydrolysis when incubated for 24h in the presence of 10% fetal bovine serum at 37 degree C, although it is hydrolyzed by S1 nuclease. The properties of oligomer I(A) suggest that 8-oxo- containing oligomers may find utility as antigene oligonucleotide reagents.     

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

Selective reversal of H-2 linked genetic unresponsiveness to lysozymes

Genes outside of the mouse major histocompatibility complex (H-2) were found to be capable of specifically reversing the previously described nonresponsiveness to hen egg-white lysozyme (HEL) owing to H-2 ^b immune response (Ir) genes. C3H.SW, BALB.B, and C57L, all of the H-2 ^b haplotype, showed responsiveness to HEL, but not to human lysozyme (H UL). Mapping of the reversing gene(s) was attempted by testing H-2 ^b recombinant inbred (RI) strains of mice carrying C3H, BALB, and C57L non-H-2 ^b genes. Analysis of the strain distribution pattern of responsiveness with both CXB and BXH RI strains was consistent with the location of the responsible site within the H-3 region on chromosome 2. The anti-HEL proliferative responsiveness in two H-3 congenic strains of mice, B10.C(28NX) ^SN and B10.C-H-3 ^{cH-3 a} , that have BALB/c genes within the H-3 region confirmed the mapping, as well as localized the reversing gene(s) near the Ir-2 gene. The data are discussed with regard to the site of expression of the reversing gene(s) and its mechanism of action.Abbreviations used in this paper MHC major histocompatibility complex - HEL hen egg-white lysozyme - Ir immune response gene - HUL human lysozyme - SDP strain distribution pattern - PFC plaque-forming cells; ₂ m, ₂-microglobulin - CFA complete Freund's adjuvant - PT-LN parathymic lymph nodes - RI recombinant inbred mice     

Extracts of black and brown rice powders improve hepatic lipid accumulation via the activation of PPARα in obese and diabetic model mice

Rice powder extract (RPE) from black and brown rice (Oryza sativa L. indica) improves hepatic lipid accumulation in obese and diabetic model mice via peroxisomal fatty acid oxidation. RPE showed PPARα agonistic activity which did not differ between black and brown RPE despite a higher anthocyanin content in black RPE.     

Regulation of ecto-5'-nucleotidase (CD73) in cultured cortical astrocytes by different inflammatory factors

Ecto-5'-nucleotidase (e-5NT) is a cell-surface located, rate-limiting enzyme in the extracellular metabolism of ATP, catalyzing the final step of the conversion of AMP to adenosine. Since this enzyme shifts the balance from pro-inflammatory ATP to anti-inflammatory adenosine, it is considered to be an important regulator of inflammation. Although up-regulation of e-5NT was repeatedly reported in several in vivo models of brain injury, the regulation of its expression and function remains largely unknown. We have studied effects of several pro-inflammatory factors, namely, bacterial endotoxin lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), glutamate (Glu) and hydrogen peroxide (H(2)O(2)) on e-5NT (i) activity, (ii) mRNA expression and (iii) membrane protein abundance in primary cultured cortical astrocytes. We are clearly able to demonstrate a stimulus-specific regulation of the e-5NT pathway. IFN-γ, LPS, Glu and H(2)O(2) decrease, while TNF-α increases e-5NT activity. The analysis of e-5NT gene expression and e-5NT membrane protein levels revealed that tested factors regulate e-5NT at different levels and by employing different mechanisms. In summary, we provide evidence that e-5NT activity is tightly regulated in a stimulus-specific manner.     

Transoceanic ships as vectors for nonindigenous freshwater bryozoans

Aim The transport of organisms in ships’ ballast tanks is a dominant vector for aquatic invasions worldwide. Until recently, efforts to manage this vector have overlooked the potential transport of invertebrate resting stages in the residual waters and sediments within emptied ballast tanks, i.e. NOBOB (‘No Ballast On Board’) tanks. The resting stages (statoblasts) of freshwater bryozoans are often buoyant and locally abundant and thus can be taken up easily during ballasting operations. They are also resistant to extreme environmental conditions and can generate new colonies after being dormant for decades; as such, they would likely remain viable propagules after lengthy transport in ship ballast tanks. This study quantified the occurrence of freshwater bryozoan statoblasts in ballast tank sediments of transoceanic ships. Location North American Great Lakes. Methods We quantified the frequency of occurrence, abundance and diversity of bryozoans (as statoblasts) in residual sediment samples taken from 51 NOBOB tanks of 33 transoceanic ships visiting the Great Lakes from 2000 to 2002. Results Our study identified 11 species, comprising nearly 12% of the total number of freshwater bryozoans known worldwide. These include two exotic species unrecorded in the Great Lakes (Fredericella sultana and Lophopus crystallinus), an exotic species already established in the region (Lophopodella carteri) and three cosmopolitan species (Plumatella casmiana, P. fungosa and P. repens). Our estimates suggest that a ship with NOBOB tanks may carry up to 10⁶ statoblasts. Main conclusions The discovery of species unrecorded in the Great Lakes and the potentially large numbers of statoblasts being transported in ship ballast tanks indicate a significant risk of new species introductions. Furthermore, the presence of cosmopolitan species and an exotic species already established in the Great Lakes suggests the strong possibility of cryptic invasions via the introduction of exotic genotypes.     

Preparation of chromosome suspensions from cells of a solid experimental tumour for measurement by flow cytometry

A method is described by which metaphase chromosomes are isolated from cells of a transplantable rat sarcoma. The chromosomes are derived from cells in a suspension prepared by trypsinisation of tumours from rats that have been treated with vindesine 24 h before excision in order to accumulate cells in mitosis. Histograms obtained for the chromosomes of the solid tumour are compared with flow karyotypes of cells cultured for 20 h or for several generations in vitro.     

Mechanism of ascorbic acid oxidation by cytochrome b(561)

The 1 equiv reaction between ascorbic acid and cytochrome b(561) is a good model for redox reactions between metalloproteins (electron carriers) and specific organic substrates (hydrogen-atom carriers). Diethyl pyrocarbonate inhibits the reaction of cytochrome b(561) with ascorbate by modifying a histidine residue in the ascorbate-binding site. Ferri/ferrocyanide can mediate reduction of DEPC-treated cytochrome b(561) by ascorbic acid, indicating that DEPC-inhibited cytochrome b(561) cannot accept electrons from a hydrogen-atom donor like ascorbate but can still accept electrons from an electron donor like ferrocyanide. Ascorbic acid reduces cytochrome b(561) with a K(m) of 1.0 +/- 0.2 mM and a V(max) of 4.1 +/- 0.8 s(-1) at pH 7.0. V(max)/K(m) decreases at low pH but is approximately constant at pH >7. The rate constant for oxidation of cytochrome b(561) by semidehydroascorbate decreases at high pH but is approximately constant at pH <7. This suggests that the active site must be unprotonated to react with ascorbate and protonated to react with semidehydroascorbate. Molecular modeling calculations show that hydrogen bonding between the 2-hydroxyl of ascorbate and imidazole stabilizes the ascorbate radical relative to the monoanion. These results are consistent with the following mechanism for ascorbate oxidation. (1) The ascorbate monoanion binds to an unprotonated site (histidine) on cytochrome b(561). (2) This complex donates an electron to reduce the heme. (3) The semidehydroascorbate anion dissociates from the cytochrome, leaving a proton associated with the binding site. (4) The binding site is deprotonated to complete the cycle. In this mechanism, an essential role of the cytochrome is to bind the ascorbate monoanion, which does not react by outer-sphere electron transfer in solution, and complex it in such a way that the complex acts as an electron donor. Thermodynamic considerations show that no steps in this process involve large changes in free energy, so the mechanism is reversible and capable of fulfilling the cytochrome's function of equilibrating ascorbate and semidehydroascorbate.