Histochemical and contractile responses of rat medial gastrocnemius to 2 weeks of complete disuse.

We studied the histochemical and in situ contractile changes in a rat ankle extensor, medial gastrocnemius, in which activation of muscle fibres by motoneurones was blocked for 14 days, using the sodium channel blocker tetrodotoxin applied to the sciatic nerve. Muscles were atrophied and showed slower twitch responses, greater fusion at subtetanic frequencies of stimulation, and higher twitch/tetanic ratios. Tetanic force/mm2 of fibre area and fatiguability were unchanged. Type II fibres were more atrophied and showed greater decreases in mitochondrial succinate dehydrogenase activity than type I fibres. The contractile changes resulting from complete disuse do not occur in models in which weight-bearing alone has been removed (space flight, hindlimb suspension), suggesting that the residual motoneurone activity reported in models of weightlessness is sufficient to prevent these responses. Similarly, the finding of a greater type II fibre susceptibility to complete disuse, which differs from the pattern seen in models of weightlessness, suggest that this residual motoneurone activity in the latter influences atrophic responses in a manner that is variable among motor unit types, to produce the reported preferential type I atrophy characteristic of removal of weight-bearing.

Rostrocaudal pattern of fiber-type changes in an overloaded rat ankle extensor.

Our aim was to quantify the overload-induced hypertrophy and conversion of fiber types (type II to I) occurring in the medial head of the gastrocnemius muscle (MG). Overload of MG was induced by a bilateral tenotomy/retraction of synergists, followed by 12-18 wk of regular treadmill locomotion (2 h of walking/running per day on 3 of 4 days). We counted all type I fibers and determined type I and II mean fiber areas in eight equidistant sections taken along the length of control and overloaded MG. Increase in muscle weights (31%), as well as in total muscle cross-sectional areas (37%) and fiber areas (type I, 57%; type II, 34%), attested to a significant hypertrophic response in overloaded MG. An increase in type I fiber composition of MG from 7.0 to 11.5% occurred as a result of overload, with the greatest and only statistically significant changes (approximately 70-100%) being found in sections taken from the most rostral 45% of the muscle length. Results of analysis of sections taken from the largest muscle girth showed that it significantly underestimated the extent of fiber conversion that occurred throughout the muscle as a whole. These data obtained on the MG, which possesses a compartmentalization of fiber types, support the notion that all fiber types respond to this model with a similar degree of hypertrophy. Also, they emphasize the complex nature of the adaptive changes that occur in these types of muscles as a result of overload.

Plasmid from photosynthetic bacterium Ectothiorhodospira Sp. carries a transposable streptomycin resistance gene.

Centrifugation through a cesium chloride density gradient and agarose gel electrophoresis of the DNA from the purple non-sulfur photosynthetic bacterium Ectothiorhodospira sp. resolved a single extrachromosomal element, plasmid pDG1. Its size was estimated to be 13.2 kilobases by restriction endonuclease mapping. Plasmid pDG1 and two restriction fragments thereof were cloned in Escherichia coli C600 with plasmid pBR327 as a vector to form mixed plasmids pDGBR1, pDGBR2, and pDGBR3. The resistance to streptomycin and mercury found in Ectothiorhodospira sp. was transferred to E. coli C600 after transformation with pDGBR1 but not with pDGBR2 and pDGBR3. The replication origin of pDG1 was estimated to be within a 2-kilobase restriction fragment of pDG1 by monitoring its replication in E. coli HB101, using a kanamycin resistance reporter gene. High stringency molecular hybridization with 32P-labeled pDG1 identified specific fragments of genomic DNA, suggesting the integration of some plasmid sequences. In accordance with the hypothesis that this integration is due to a transposon, we tested the transfer of streptomycin resistance from pDG1 into plasmid pVK100 used as a target. For this test, we regrouped in the same cells of E. coli HB101, pDGBR1 and mobilizable plasmid pVK100 (tetr,kmr). We used the conjugation capacity of the pVK100/pRK2013 system to rescue the target plasmid pVK100 into nalidixic acid-resistant E. coli DH1. The transfer frequency of streptomycin resistance into pVK100 was 10(-5), compatible with a transposition event. In line with the existence of a transposon on pDG1, heteroduplex mapping indicated the presence of inverted repeats approximately 7.5 kb from one another.

The structural genes coding for the L and M subunits of Rhodospirillum rubrum photoreaction center.

In Rhodospirillum rubrum, pufL, and pufM, the structural genes coding for the photoreaction center L and M polypeptides, are comprised respectively of 831 and 921 nucleotides. They are separated by a stretch of 12 nucleotides between the TAA stop codon of pufL and the first base of the ATG initiation codon of pufM. The predicted amino acid sequence of the L and M polypeptides, respectively, contain 275 and 305 residues with corresponding molecular weights of 30,473 and 33,978. Their sequences are highly homologous to those of Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis. As can be deduced from the crystallographic structure of other photoreaction centers, the regions of greatest similarity are the binding sites of the cofactors involved in the photochemical reaction rather than the protein secondary structure. L and M contain, at conserved positions of their sequences, three main clusters of positively charged residues on the cytoplasmic side of the membrane. This arrangement may be involved in protein orientation during membrane assembly. Evolutionary distance of pufL and pufM, as assessed by substitution frequency analysis, confirms the closeness of the two Rhodobacter species, the other two species being equidistant from one another. Interspecies evolutionary distance is greater for pufL than for pufM.

Molecular cloning and sequence of the B880 holochrome gene from Rhodospirillum rubrum.

Restriction fragments of genomic Rhodospirillum rubrum DNA were selected according to size by electrophoresis followed by hybridization with [32P]mRNA encoding the two B880 holochrome polypeptides. The fragments were cloned into Escherchia coli C600 with plasmid pBR327 as a vector. The clones were selected by colony hybridization with 32P-holochrome-mRNA and counterselected by hybridization with Rs. rubrum ribosomal RNA, a minor contaminant of the mRNA preparation. Chimeric plasmid pRR22 was shown to contain the B880 genes by hybrid selection of B880 holochrome-mRNA. We report a restriction map of its 2.2-kilobase insert and the sequence of a 430 base pair fragment thereof. Genes alpha and beta are nearly contiguous, indicating that they are transcribed as a single operon. The predicted amino acid sequences coincide with the sequences of the alpha and beta polypeptides established in other laboratories, except for additional C-terminal tails of 10 and 13 amino acid residues, respectively. We suggest that these tail sequences may serve, during membrane assembly, to give these intrinsic membrane proteins their peculiar orientation with their C-terminus facing the periplasm and their N terminus facing the cytoplasm. Intraspecific sequence homology between the alpha and beta genes of R. rubrum is low, showing no evolutionary relatedness. This is in contrast to the high interspecific homology between the corresponding sequences of Rs. rubrum and Rhodopseudomonas capsulata B880 genes.