Effect of 17days of bed rest on peak isometric force and unloaded shortening velocity of human soleus fibers

The purpose of this study was to examine the effect of prolongedbed rest (BR) on the peak isometric force(P_o) and unloaded shorteningvelocity (V_o)of single Ca²⁺-activated musclefibers. Soleus muscle biopsies were obtained from eight adult malesbefore and after 17 days of 6° head-down BR. Chemicallypermeabilized single fiber segments were mounted between a forcetransducer and position motor, activated with saturating levels ofCa²⁺, and subjected to slacklength steps. V_owas determined by plotting the time for force redevelopment vs. theslack step distance. Gel electrophoresis revealed that 96% of the pre-and 87% of the post-BR fibers studied expressed only the slow type Imyosin heavy chain isoform. Fibers with diameter >100 µm made uponly 14% of this post-BR type I population compared with 33% of thepre-BR type I population. Consequently, the post-BR type I fibers(n = 147) were, on average, 5%smaller in diameter than the pre-BR type I fibers(n = 218) and produced 13% lessabsolute P_o. BR had no overalleffect on P_o per fibercross-sectional area(P_o/CSA), even though halfof the subjects displayed a decline of 9-12% inP_o/CSA after BR. Type Ifiber V_oincreased by an average of 34% with BR. Although the ratio of myosinlight chain 3 to myosin light chain 2 also rose with BR, there was nocorrelation between this ratio andV_o for either thepre- or post-BR fibers. In separate fibers obtained from the originalbiopsies, quantitative electron microscopy revealed a 20-24%decrease in thin filament density, with no change in thick filamentdensity. These results raise the possibility that alterations in thegeometric relationships between thin and thick filaments may be atleast partially responsible for the elevatedV_o of the post-BRtype I fibers.

     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Naturally Occurring DNA Transfer System Associated with Membrane Vesicles in Cellulolytic Ruminococcus spp. of Ruminal Origin

A genetic transformation system with similarities to those reported for gram-negative bacteria was found to be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates. The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb (Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion, suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell. Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71 was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen.