The Control Region of Maternally and Paternally Inherited Mitochondrial Genomes of Three Species of the Sea Mussel Genus Mytilus

Species of the mussel genus Mytilus possess maternally and paternally transmitted mitochondrial genomes. In the interbreeding taxa Mytilus edulis and M. galloprovincialis, several genomes of both types have been fully sequenced. The genome consists of the coding part (which, in addition to protein and RNA genes, contains several small noncoding sequences) and the main control region (CR), which in turn consists of three distinct parts: the first variable (VD1), the conserved (CD), and the second variable (VD2) domain. The maternal and paternal genomes are very similar in gene content and organization, even though they differ by >20% in primary sequence. They differ even more at VD1 and VD2, yet they are remarkably similar at CD. The complete sequence of a genome from the closely related species M. trossulus was previously reported and found to consist of a maternal-like coding part and a paternal-like and a maternal-like CR. From this and from the fact that it was extracted from a male individual, it was inferred that this is a genome that switched from maternal to paternal transmission. Here we provide clear evidence that this genome is the maternal genome of M. trossulus. We have found that in this genome the tRNA^Gln in the coding region is apparently defective and that an intact copy of this tRNA occurs in the CR, that one of the two conserved domains is missing essential motifs, and that one of the two first variable domains has a high rate of divergence. These features may explain the large size and mosaic structure of the CR of the maternal genome of M. trossulus. We have also obtained CR sequences of the maternal and paternal genomes of M. californianus, a more distantly related species. We compare the control regions from all three species, focusing on the divergence among genomes of different species origin and among genomes of different transmission routes.     

Hydrolysis of 2-arachidonoylglycerol in Tetrahymena thermophila. Identification and partial characterization of a Monoacylglycerol Lipase-like enzyme

Tetrahymena thermophila is a model organism for molecular and cellular biology. Previous studies from our group showed that Tetrahymena contains major components of the endocannabinoid system, such as various endocannabinoids and FAAH. In mammalian cells the endocannabinoid 2-arachidonoylglycerol is inactivated mainly by MAGL. In this study we showed that 2-arachidonoylglycerol and 2-oleoylglycerol are hydrolyzed by the combined actions of MAGL and FAAH. MAGL-like activity was examined in the presence of FAAH specific inhibitors, URB597 or AM374 and showed optimum pH of 8-9, apparent K(M) of 14.1μM and V(max) of 5.8nmol/min×mg. The enzyme was present in membrane bound and cytosolic isoforms; molecular mass was determined at ～45 and ～40kDa. MAGL and FAAH could also inactivate endogenous signaling lipids, which might play an important role in Tetrahymena as suggested in mammals. Tetrahymena could be used as a model system for testing drugs targeting enzymes of the endocannabinoid system.     

The effects of a twenty-four-week aquatic training program on muscular strength performance in healthy elderly women

The purpose of the present study was to determine the effectiveness of a 24-week aquatic training (AT) program, which included both aerobic and resistance components, on muscle strength (isometric and dynamic), flexibility, and functional mobility in healthy women over 60 years of age. Twenty-two subjects were assigned randomly to either an AT (n = 12) or a control (C, n = 10) group. Volunteers participated in a supervised shallow-water exercise program for 60 minutes a day, 3 days a week; the exercise program consisted of a 10-minute warm-up and stretching, 25 minutes of endurance-type exercise (dancing) at 80% of heart rate (HR)(max), 20 minutes of upper- and lower-body resistance exercises with specialized water-resistance equipment, and a 5-minute cool down. Maximal isometric torque of knee extensors (KEXT) and knee flexors (KFLEX) were evaluated by a Cybex Norm dynamometer, grip strength (HGR) was evaluated using a Jamar hydraulic dynamometer, and dynamic strength was evaluated via the 3 repetition maximum (3RM) test for chest press, knee extension, lat pull down, and leg press. Jumping performance was evaluated using the squat jump (SJ), functional mobility with the timed up-and-go (TUG) test, and trunk flexion with the sit-and-reach test. Body composition was measured using the bioelectrical impedance method. The AT induced significant improvements in KEXT (10.5%) and KFLEX (13.4%) peak torque, HGR strength (13%), 3RM (25.7-29.4%), SJ (24.6%), sit-and-reach (11.6%), and TUG (19.8%) performance. The AT group demonstrated a significant increase in lean body mass (3.4%). No significant changes in these variables were observed in the C group. The results indicate that AT, with both aerobic and resistance components, is an alternative training method for improving neuromuscular and functional fitness performance in healthy elderly women.     

Comparison of methods for the concentration of Cryptosporidium oocysts and Giardia cysts from raw waters

This study compared the recovery of Cryptosporidium oocysts and Giardia cysts ((oo)cysts) from raw waters using 4 different concentration-elution methods: flatbed membranes, FiltaMax foam, Envirochek HV capsules, and Hemoflow ultrafilters. The recovery efficiency of the combined immunomagnetic separation and staining steps was also determined. Analysis of variance of arcsine-transformed data demonstrated that recovery of Cryptosporidium oocysts by 2 of the methods was statistically equivalent (flatbed filtration 26.7% and Hemoflow 28.3%), with FiltaMax and Envirochek HV recoveries significantly lower (18.9% and 18.4%). Recovery of Giardia cysts was significantly higher using flatbed membrane filtration (42.2%) compared with the other 3 methods (Envirochek HV 29.3%, FiltaMax 29.0%, and Hemoflow 20.9%). All methods were generally acceptable and are suitable for laboratory use; 2 of the methods are also suitable for field use (FiltaMax and Envirochek HV). In conclusion, with recoveries generally being statistically equivalent or similar, practical considerations become important in determining which filters to use for particular circumstances. The results indicate that while low-turbidity or "finished" waters can be processed with consistently high recovery efficiencies, recoveries from raw water samples differ significantly with variations in raw water quality. The use of an internal control with each raw water sample is therefore highly recommended.     

An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml(-1)) followed by achromopeptidase (60 U ml(-1)) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.     

Synthesis of pyrimidinopyridine-triazene conjugates targeted to abl tyrosine kinase

The synthesis and abl tyrosine kinase inhibitory activities of alkyltriazenes conjugated to phenylaminopyrimidines are described. Significant abl inhibitory activities were observed only when a benzamido spacer was inserted between the 1,2,3-triazene chain and the 2-phenyaminopyridopyrimidine moiety.     

Induction and inhibition of Friend erythroleukemia cell differentiation by pyrimidine analogs: analysis of the requirement for intracellular accumulation and incorporation into DNA.

Alkyldeoxyuridines which differ from thymidine by a C5 substitution of straight or branched alkyl chains of two to six carbon atoms have been tested for their ability to be taken up, phosphorylated, and incorporated into DNA. Analysis of the uptake of 5-ethyl-2'-deoxyuridine and 5-propyl-2'-deoxyuridine (n-PrdU)--similar to both thymidine and 5-bromo-2'-deoxyuridine--indicates that transport is dependent upon a functional cellular thymidine kinase. All of the aforementioned pyrimidines with the exception of n-PrdU are phosphorylated to the triphosphate and incorporated into DNA. The homologs 5-iso-propyl-2'-deoxyuridine (iso-PrdU) and 5-hexyl-2'-deoxyuridine are neither transported into the cell, phosphorylated, nor incorporated into DNA. These analogs were tested (i) for their ability to induce in the absence of dimethyl sulfoxide and (ii) to determine whether they enhance or inhibit dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. Inhibition of erythroid differentiation appears to require the incorporation of thymidine analogs into DNA, and thus only 5-ethyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine were effective in inhibiting dimethyl sulfoxide-induced differentiation. The observation that iso-PrdU, and to a lesser extent n-PrdU and 5-propyldeoxyuridine monophosphate, induce differentiation under conditions in which they are not detectable intracellularly is strong evidence that this class of inducer acts at the cell membrane.     

Endogenous Interleukin 6 Conveys Resistance to cis-Diamminedichloroplatinum-Mediated Apoptosis of the K562 Human Leukemic Cell Line

Cisplatin is an effective chemotherapeutic agent that elicits its antineoplastic activity by binding to DNA and disrupting template functions. IL-6 is a cytokine which has been shown to play a central role in host immunological defense mechanisms. Although K562 leukemic cells have been shown to secrete IL-6, little is known of whether there exists a correlation between the expression of IL-6 and the resistance of these cells to anticancer chemotherapeutic agents. To determine the contribution of IL-6 to the regulation of cisplatin-induced apoptosis in K562 cells, we examined whether treatment of K562 cells and cisplatin-resistant K562 subclones with anti-IL-6 mAb enhances their sensitivity to cisplatin. The results show that cis-diamminedichloroplatinum (CDDP) resistance was overcome by treatment with nontoxic doses of CDDP in combination with anti-IL-6 mAb. When we tested if the synergistic effect of anti-IL-6 and cisplatin could restore the ability of K562 mutant cells to undergo apoptosis, we found the typical DNA laddering in these cells, even in the presence of a nontoxic dose of the drug. Treatment of cells with anti-IL-6 reduced the levels of glutathione. The current studies show that anti-IL-6 mAb sensitized CDDP-resistant K562 cells to CDDP by induction of apoptotic death and the reduction of glutathione levels might be implicated in the enhanced cytotoxicity observed.