The effect of chromium picolinate on muscular strength and body composition in women athletes.

Fifteen women softball athletes were randomly divided into 2 groups, the chromium treatment group (n = 8) and the placebo control group (n = 7) to examine the effect of chromium, in the form of chromium picolinate (CrPic) supplementation, on muscular strength, body composition (body weight, percent body fat, and lean body mass), and urinary excretion. The CrPic supplementation consisted of a 500 ug dosage taken once per day. All participants trained 3 times per week with 2-3 sets of 8-12 repetitions at 80% of 1 repetition maximum (1RM) using variable resistance machines and free weights. No significant (p < 0.05) differences in muscular strength or body composition were found after 6 weeks of resistance training. In addition, chromium excretion (microg per 24 every hours) was examined and increased significantly with the treatment group after the 6-week period.

Formation of proteasome-PA700 complexes directly correlates with activation of peptidase activity.

The proteolytic activity of the eukaryotic 20S proteasome is stimulated by a multisubunit activator, PA700, which forms both 1:1 and 2:1 complexes with the proteasome. Formation of the complexes is enhanced by an additional protein assembly called modulator, which also stimulates the enzymatic activity of the proteasome only in the presence of PA700. Here we show that the binding of PA700 to the proteasome is cooperative, as is the activation of the proteasome's intrinsic peptidase activity. Modulator increases the extent of complex formation and peptidase activation, while preserving the cooperative kinetics. Furthermore, the increase in activity is not linear with the number of PA700 assemblies bound to the proteasome, but rather with the number of proteasome-PA700 complexes, regardless of the PA700:proteasome stoichiometry. Hence the stimulation of peptidase activity is fully (or almost fully) effected by the binding of a single PA700 to the 20S proteasome. The stimulation of peptidase by modulator is explained entirely by the increased number of proteasome-PA700 complexes formed in its presence, rather than by any substantial direct stimulation of catalysis. These observations are consistent with a model in which PA700, either alone or assisted by modulator, promotes conformational changes in the proteasome that activate the catalytic sites and/or facilitate access of peptide substrates to these sites.

Structural and functional effects of PA700 and modulator protein on proteasomes.

Control and targeting of the proteolytic activity of the major intracellular protease, the proteasome, is accomplished by various regulatory protein complexes that may form higher-order assemblies with the proteasome. An activator of proteolytic activity, PA700, has been shown to have an ATP-dependent stimulatory effect on the peptidase activities of the proteasome, and another protein factor, the modulator, further enhances the effect of PA700. Here we show that the addition of PA700 endows the proteasome with the ability to cleave ubiquitinated proteins, a property associated with the previously isolated 26 S form of the proteasome. The modulator further stimulates this specific activity, without having any such effect on the proteasome alone. Using electron microscopy, we show that addition of PA700 causes the appearance of protein "caps" at one or both ends of proteasomes, forming structures that are indistinguishable from 26 S proteasomes. Quantitation of the numbers of uncapped, singly capped and doubly capped complexes indicates cooperativity in the association of PA700 with the two ends of the proteasome. Addition of modulator protein makes no further structural modification that is detectable by electron microscopy, but does cause an increase in the number of capped complexes visible at subsaturating concentrations of PA700. Hence PA700 converts the proteasome both functionally and structurally to the 26 S form, and the modulator promotes this transformation, apparently without stable association with the resulting complex.

Expression, purification, mass spectrometry, crystallization and multiwavelength anomalous diffraction of selenomethionyl PvuII DNA methyltransferase (cytosine-N4-specific).

The type II DNA-methyltransferase (cytosine N4-specific) M.PvuII was overexpressed in Escherichia coli, starting from the internal translation initiator at Met14. Selenomethionine was efficiently incorporated into this short form of M.PvuII by a strain prototrophic for methionine. Both native and selenomethionyl M.PvuII were purified to apparent homogeneity by a two-column chromatography procedure. The yield of purified protein was approximately 1.8 mg/g bacterial paste. Mass spectrometry analysis of selenomethionyl M.PvuII revealed three major forms that probably differ in the degree of selenomethionine incorporation and the extent of selenomethionine oxidation. Amino acid sequencing and mass spectrometry analysis of selenomethionine-containing peptides suggests that Met30, Met51, and Met261 were only partially replaced by selenomethionine. Furthermore, amino acid 261 may be preferentially oxidized in both native and selenomethionyl form. Selenomethionyl and native M.PvuII were crystallized separately as binary complexes of the methyl donor S-adenosyl-L-methionine in the monoclinic space group P2(1). Two complexes were present per asymmetric unit. Six out of nine selenium positions (per molecule), including the three that were found to be partially substituted, were identified crystallographically.

The PvuII DNA (cytosine-N4)-methyltransferase comprises two trypsin-defined domains, each of which binds a molecule of S-adenosyl-L-methionine.

Earlier studies have shown that PvuII methyltransferase is monomeric and transfers a methyl group from S-adenosyl-l-methionine (AdoMet) to cytosine, generating N4-methylcytosine in duplex 5'-CAGCTG-3' DNA. This study examines the interactions between PvuII methyltransferase and AdoMet. Trypsin preferentially cleaved the protein into two large fragments, with initial cleavages after Arg183 and Lys186. UV-mediated photochemical labeling with [3H-CH3]AdoMet, followed by trypsin digestion, revealed that both large fragments of the protein were labeled. Rapid gel filtration confirmed that each molecule of the intact enzyme bound two molecules of AdoMet (net Kd = 9.3 microM). When PvuII methyltransferase was preincubated with a range of [3H-CH3]AdoMet concentrations, bursts of product formation resulted upon DNA addition. These data indicate that PvuII methyltransferase is catalytically competent with one and with two bound molecules of AdoMet. These results, together with those from earlier studies, suggest possible roles for the second molecule of AdoMet.

Gene pvuIIW: a possible modulator of PvuII endonuclease subunit association.

The PvuII restriction-modification system has been found to contain three genes which code for a DNA methyltransferase (MTase), a restriction endonuclease (ENase) and a small protein required for expression of the ENase-encoding gene. In addition, there is a small open reading frame (ORF) within and opposite to the MTase-encoding gene. The region containing this ORF is transcribed, and the ORF has an excellent Shine-Dalgarno sequence with an ATA start codon. A closely related ORF is present in the SmaI system. The 28-amino-acid (aa) predicted peptide from the PvuII ORF resembles a region of the PvuII ENase at the dimer interface. We have cloned this ORF, giving it an ATG start codon and putting it under the control of an inducible promoter: induction leads to a slight but significant decrease in restriction of bacteriophage lambda. We also have obtained the 28-aa synthetic peptide, and are exploring the possibility that it modulates ENase subunit association. While this peptide has no detectable effect on dimeric PvuII ENase, it inhibits renaturation of urea-denatured ENase in a concentration-dependent manner. The ORF may represent an additional safeguard during establishment of the PvuII restriction-modification system in a new host cell, helping to delay the appearance of active ENase dimers, while the MTase accumulates and protects the host chromosome.

Determination of anaerobic threshold by ventilatory frequency.

Detection of anaerobic threshold (AT) requires either invasive techniques or expensive gas analyzers and somewhat complicated procedures. The purpose of this study was to determine if ventilatory frequency (f) could be used to detect AT. Thirteen (seven females) healthy, non-smoking, physically active adults (21-44 years) volunteered to perform progressive cycle exercise. A protocol of either 22.5- or 45-W increments every 2 min was used according to the subject's weight and fitness to assure steady state. Expiratory gas was measured using a computerized breath-by-breath system. Mean values of oxygen uptake (VO2, 1.min-1), ventilation (VE, 1.min-1), and f(br.min-1) were calculated each min. Peak VO2 ranged from 24.8 to 58.9 ml.kg-1.min with a group mean (+/- SD) of 45.1 +/- 11.6 ml.kg-1.min. Mean (+/- SD) VO2 at AT, as determined by disproportionate increase of VE, was 2.11 +/- 0.57 1.min-1. Mean (+/- SD) VO2 at the point of disproportionate increase of f was 2.09 +/- 0.58 1.min-1. A significant (P less than 0.05) correlation (r = 0.834) was found between the point of disproportionate increase in f and that of VE for individual data. A Student's t test indicated there was no significant difference in mean VO2 at AT. It was concluded that AT could be detected by f in healthy, physically active adults, thus providing a simplified and less expensive alternative method. This finding may have implications with regard to establishing and monitoring exercise intensity.

General characteristics, molecular and genetic analysis of two new UV-sensitive mutants of Chlamydomonas reinhardtii.

Two new UV-sensitive mutants of Chlamydomonas, UVS10 and UVS11, were isolated. Both behave as single nuclear mutations. UVS10 was mapped to linkage group I. UVS11 is a separate, unlinked mutation but has not yet been located to a specific linkage group. Both mutants are proficient in the excision of pyrimidine dimers from nuclear DNA. The survival of UV-irradiated UVS11 is increased when plated in the presence of 1.5 mM caffeine, similar to wild-type. Caffeine has no effect on the survival of UV-irradiated UVS10. UV-irradiated UVS11 frequently divides at least once before dying, in contrast to UVS10 or wild-type. UVS11 also exhibits a much increased frequency of mutation to streptomycin resistance after UV irradiation.

Toxoplasma modifies macrophage phagosomes by secretion of a vesicular network rich in surface proteins.

Modification of macrophage phagosomes begins shortly after formation as Toxoplasma cells secrete membranous vesicles that form a reticulate network within the vacuole. The Toxoplasma-modified compartments then resist normal endocytic processing and digestion. We have used the pronounced Ca++-dependent stability of the intraphagosomal membrane (IPM) network to purify and characterize the structural proteins of this assembly. In addition to the structural matrix, Toxoplasma secretes a discrete set of soluble proteins, including a newly described 22-kD calcium-binding protein. The IPM network adheres to intact Toxoplasma cells after host cell lysis in the presence of 1 mM Ca++; however, the network readily disperses in calcium-free buffer and was purified as vesicles that sedimented at 100,000 g. Purified IPM vesicles were specifically recognized by immune sera from mice with chronic Toxoplasma infection and consisted primarily of a 30-kD protein when analyzed by SDS PAGE. IPM network proteins share a major antigenic component located on the surface of extracellular Toxoplasma cells as shown by immunoperoxidase electron microscopy using a polyclonal antibody prepared against the IPM vesicles. Moreover, in Toxoplasma-infected macrophages, anti-IMP antibody confirmed that the extensive IPM array contains proteins also found on the Toxoplasma cell surface. Our results indicate the IMP network represents a unique structural modification of the phagosome comprised in part of Toxoplasma surface proteins.

Defective temporal and spatial control of flagellar assembly in a mutant of Chlamydomonas reinhardtii with variable flagellar number.

Wild-type Chlamydomonas reinhardtii carry two flagella per cell that are used for both motility and mating. We describe a mutant, vfl-1, in which the biflagellate state is disrupted such that the number of flagella per cell ranges from 0 to as many as 10. vfl-1 cells possess the novel ability to assemble new flagella throughout the G1 portion of the cell cycle, resulting in an average increase of about 0.05 flagella per cell per hour. Such uncoupling of the flagellar assembly cycle from the cell cycle is not observed in other mutants with abnormal flagellar number. Rather than being located in an exclusively apical position characteristic of the wild type, vfl-1 flagella can be at virtually any location on the cell surface. vfl-1 cells display abnormally wide variations in cell size, probably owing to extremely unequal cell divisions. Various ultrastructural abnormalities in the flagellar apparatus are also present, including missing or defective striated fibers and reduced numbers of rootlet microtubules. The pleiotropic defects observed in vfl-1 result from a recessive Mendelian mutation mapped to Chromosome VIII.

Temperature-Sensitive, Assembly-Defective Flagella Mutants of CHLAMYDOMONAS REINHARDTII.

We describe an efficient selection procedure for the isolation of mutants of Chlamydomonas reinhardtii with temperature sensitive flagella defects, with final yields of up to 11% of the population being mutant. Several mutants, all showing an inability to maintain flagellar integrity at the restrictive temperature, are described. We have examined flagellar stability and reassembly at various temperatures in the mutants. Mapping data are provided for these, as well as for some previously described mutants.

Chloroplast Gene Transmission in Chlamydomonas somatic fusion products : Effects of pretreatment with isolated flagella.

In sexual crosses between the two mating types of Chlamydomonas reinhardtii the progeny normally only receive chloroplast genes from the mt (+) parent. This pattern of inheritance is not seen following the rare mating events leading to the formation of vegetative diploids, or when the cells are fused using polyethylene glycol (PEG). Vegetative diploids usually carry chloroplast genes from both parents, and most of those that are uniparental carry the maternal parent's genes. In a PEG fusion the products are evenly divided among those uniparental for each of the parental chloroplast markers and those that are biparental. However, fusion in a sexual cross is usually preceded by the flagellaragglutination of the two mating types. When cells undergoing PEG fusion have been pretreated with isolated gametic flagella the transmission pattern in changed. If two mt (+) cells were fused, and one of them had been pretreated with isolated mt (-) flagella, then the majority of the progeny carried chloroplast genes only from the treated parent. This suggests that the signal for the induction of the events leading to uniparental maternal inheritance is the agglutination between mt (+) and mt (-) flagella that precedes cell fusion, and that the subsequent fate of chloroplast genes is controlled primarily by the mt (+) parent.

Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants.

Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally (pf-18, pf-19, and pf-15) or nearly totally (pf-20) the central microtubular pair complex in isolated axonemal preparations. Analysis of 35S-labeled axonemal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes. Analyses of axonemal proteins labeled by cellular growth on 32P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated. Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta serum. None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella. The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules. Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.

Human tecg changes during prolonged hyperbaric exposures breathing N2-O2 mixtures.

In an effort to determine whether hyperbaric exposures while breathing N2-O2 mixtures have an effect on cardiac depolarization and repolarization, electrocardiograms of 10 divers participating in four N2-O2 saturation dives were analyzed. In all cases, a decline in heart rate was observed upon compression to saturation depth (20-30%); a slow adaptation and return of heart rate toward normal was observed in those dives where the depth and environmental parameters remained constant. twhenever excursion dives were performed, the heart rate responded by decreasing on deeper excursions and increasing on upward excursions. Hyperbaric bradycardia disappeared after 8 days at pressure during the saturation dives at 50 and 60 feet seawater gauge (fswg), but was still present at this time at 200 fswg. The magnitude of the hyperbaric bradycardia produced by excursion dives following saturation at depth was influenced by the state of adaptation of heart rate. Decompression was uniformly accompanied by a rapid increase in heart rate resulting in a significant elevation in the postdive period. Alterations in myocardial repolarization as evidenced by Q-T interval, ST, and T wave changes were observed. Development of slight right ventricular conduction delay compatible with right ventricular strain was noted in four of the divers during the two deepest dives to 100 and 198 fswg. During the latter dive, progressive decrease in P wave amplitudes and eventual loss of P waves resulting in an apparent nodal rhythm was observed in one diver. Multiple premature ventricular contractions occurred in another diver. These observations, along with the reports by other authors, suggest that the different variables associated with the hyperbaric environment--gas density, pressure, inert gas--have a definite effect on the pacemaker activity of the heart and myocardial depolarization and repolarization.