Time-based gene expression programme following diaphragm injury in a rat model.

It was hypothesised that diaphragm injury activates a time-based programme of gene expression in muscle repair. Gene expression of different substances, such as proteases (calpain 94 (p94)), transcription factors (myogenin and cFos), growth factors (both basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF)-II), and structural proteins (myosin heavy chain (MHC) and titin), was quantified by RT-PCR in rat diaphragms exposed to caffeine-induced injury. Injured and noninjured (control) rat hemidiaphragms were excised at different time points (1-240 h). In injured hemidiaphragms, in comparison with control muscles, p94 expression levels peaked at 1 h post-injury (PI), cFos mRNA levels began to rise, after an initial dip, and peaked at 96 h PI, while myogenin mRNA levels started to increase as early as 12 h PI, IGF-II mRNA levels initially decreased until 48 h PI and increased thereafter, peaking at 72 h PI, bFGF mRNA levels rose to a maximum at 96 h PI, and MHC and titin mRNA levels were significantly elevated at 72 h PI. Caffeine-induced diaphragm injury is followed by a time-based expression programme of different genes tailored to meet muscle repair needs.

Morphological and functional recovery from diaphragm injury: an in vivo rat diaphragm injury model.

Our objective was to develop an in vivo model to study the timing and mechanisms underlying diaphragm injury and repair. Diaphragm injury was induced in anesthetized rats by the application of a 100 mM caffeine solution for a 10-min period to the right abdominal diaphragm surface. Diaphragms were removed 1, 4, 6, 12, 24, 48, 72, and 96 h and 10 days after the injury, with contractile function being assessed in strips in vitro by force-frequency curves. The extent of caffeine-induced membrane injury was indicated by the percentage of fibers with a fluorescent cytoplasm revealed by inward leakage of the procion orange dye. One hour after caffeine exposure, 32.9 +/- 3.1 (SE) % of fibers showed membrane injury that resulted in 70% loss of muscle force. Within 72-96 h, the percentage of fluorescent cells decreased to control values. Muscle force, however, was still reduced by 30%. Complete muscle strength recovery was observed 10 days after the injury. Whereas diaphragmatic fiber repair occurred within 4 days after injury induction, force recovery took up to 10 days. We suggest that the caffeine-damaged rat diaphragm is a useful model to study the timing and mechanisms of muscle injury and repair.

Induction of the ATP-sensitive potassium (uK(ATP)-1) channel by endotoxemia.

The effect of sepsis on the ubiquitously expressed ATP-sensitive potassium (uK(ATP)-1) channel expression was measured in Sprague-Dawley rat diaphragms. Rats were treated with either 0.5 ml saline or 20 mg/Kg E. coli lipopolysaccharides and sacrificed at 3, 6, 12, 24, or 48 h later. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that channel mRNA expression was increased at 3 h and continued to rise up to 48 h. Western blotting analysis showed a approximately 9-fold increase in channel protein expression 24 h after sepsis. Our results demonstrate that sepsis upregulates the uK(ATP)-1 channel.

Biología del daño y reparación muscular / The biology of muscle damage and repair

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Expression of myosin heavy-chain isoforms in the respiratory muscles following inspiratory resistive breathing.

We investigated the effect of inspiratory resistive breathing (IRB) on the expression of the genes encoding fast and slow isoforms of myosin heavy chain (MyHC) in respiratory muscles. Eleven mongrel dogs were studied for baseline MyHC messenger RNA (mRNA) expression, seven of which were also used to study the effects of IRB. For this latter objective, awake and spontaneously breathing animals were subjected to 2 h of IRB (80 cm H(2)O/L/s) per day for four consecutive days. mRNA expression was assessed in the diaphragm, external intercostal muscle, and a limb muscle, using both slot- blot and in situ hybridizations with isoform-specific probes. A current semiquantitative scoring method (from 0 to 4) was used to quantify the in situ mRNA expression levels, and slot-blot data were analyzed with densitometry. Prior to IRB, slow- and fast-MyHC mRNA expression was moderate, similar, and homogeneous throughout the different regions of the diaphragm, with scores of 1.50 +/- 0.54 (mean +/- SD) for slow and 2.13 +/- 0.35 for fast mRNAs in the costal region of the diaphragm, and of 1.81 +/- 0.37 for slow and 2. 13 +/- 0.64 for fast mRNAs in the crural region of the diaphragm. Although expression of fast-MyHC mRNA remained unchanged after IRB, the relative expression of the mRNA for the slow isoform increased in costal (+30%), crural (+12%), and external intercostal (+27%) muscles. MyHC mRNA expression did not change in limb muscles. We conclude that breathing with a moderate inspiratory resistance for a short period induces the expression of slow MyHC in respiratory muscles.

Effects of reducing reagents and temperature on conversion of nitrite and nitrate to nitric oxide and detection of NO by chemiluminescence.

To measure the concentration of nitrites and nitrates by chemiluminescence, we examined the efficiency of five reducing agents [V(III), Mo(VI) + Fe(II), NaI, Ti(III), and Cr(III)] to reduce nitrite (NO2-) and (or) nitrate (NO3-) to nitric oxide (NO). The effect of each reducing agent on the conversion of different amounts of NO2- and (or) NO3- (100-500 pmol, representing concentrations of 0.4 to 2 mu molar) to NO was determined at 20 degrees C for NO2- and at 80 degrees C for NO3-. The effect of temperature from 20 to 90 degrees C on the conversion of a fixed amount of NO2- or NO3- (400 pmol or 1.6 mu molar) to NO was also determined. These five reducing agents are similarly efficient for the conversion of NO2- to NO at 20 degrees C. V(III) and Mo(VI) + Fe(II) can completely reduce NO3- to NO at 80 degrees C. NaI and Cr(III) were unable to convert NO3- to NO. Increased temperature facilitated the conversion of NO3- to NO, rather than that of NO2- to NO. We evaluated the recovery of NO2- and NO3- from plasmas of pig and of dog. Recovery from plasma of both animals was reproducible and near quantitative.

A prespore-specific gene of Dictyostelium discoideum encodes the small subunit of ribonucleotide reductase.

We have isolated the gene. rnrB, that encodes the ribonucleotide reductase small subunit of Dictyostelium discoideum. The deduced amino acid sequence of rnrB exhibits about 60% sequence identity with its homologues in other eukaryotes. As demonstrated by RNA blot analysis the rnrB transcript is detected in growing cells and decreases dramatically at the onset of development. The rnrB transcript reappears after the cells have formed multicellular aggregates. To further examine the pattern of expression, we have fused the rnrB promoter and part of its coding sequence to lacZ. The transgenic strain bearing such a reporter construct expresses the fusion gene with a biphasic profile, which is indistinguishable from that of the endogenous rnrB. The multicellular aggregates of Dictyostelium are differentiated along the anterior-posterior axis. Cells in the anterior give rise to the stalk of the fruiting body while cells in the posterior are precursors of spores. Results from histochemical staining show that beta-galactosidase activity is detected exclusively in the posterior two-thirds of the aggregates. These data suggest that rnrB is expressed in prespore cells during postaggregative development and in vegetative cells.

Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of Escherichia coli K12.

The hemG gene of Escherichia coli K12 is involved in the activity of protoporphyrinogen oxidase, the enzyme responsible for the conversion of protoporphyrinogen IX into protoporphyrin IX during heme and chlorophyll biosynthesis. The gene is located at min 87 on the genetic map of E. coli K12. The hemG gene was isolated by a mini-Mu in vivo cloning procedure. As expected, the hemG gene is able to restore normal growth to the hemG mutant, and the transformed cells display strong protoporphyrinogen oxidase activity. Sequencing of the hemG gene allowed us to identify an open reading frame of 546 nucleotides (181 amino acids), within the minimal fragment able to complement the mutant. The presumed molecular mass of the HemG protein is 21,202 Da, in agreement with values found by SDS-PAGE, in a DNA-directed coupled transcription-translation system. The identity of the first 18 amino acids at the amino-terminal end of the protein was confirmed by microsequencing. To our knowledge, this is the first cloning of a gene involved in the protoporphyrinogen oxidase activity of E. coli.

Defective DNA injection by alkylated and nonalkylated bacteriophage T7.

DNA injection by alkylated and nonalkylated bacteriophage T7 has been analyzed by a physical method which involved Southern hybridization to identify noninjected regions of DNA. Treatment of phage with methyl methanesulfonate reduced the amount of DNA injected into wild-type Escherichia coli cells. This reduction was correlated with a decreased injection of DNA segments located on the right-hand third of the T7 genome. An essentially identical injection defect was observed when alkylated phage infected E. coli mutant cells unable to repair 3-methyladenine. Furthermore, untreated phage particles were discovered to be naturally injection-defective. Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome, while other injected no DNA at all. In the presence of rifampicin, untreated phages injected only segments from the left end of the genome. These results provide direct physical evidence that T7 DNA injection is strictly unidirectional, starting from the left end of the T7 genome. The injection defect quantified here for alkylated phage is probably partially, if not totally, responsible for phage inactivation, when that inactivation is measured in wild-type E. coli cells. Since alkylated phage injected the same DNA sequences into both wild-type and repair-deficient cells, we conclude that DNA injection is independent of the host-cell's capacity for repair of 3-methyladenine residues.

Mechanism of inhibition of bacteriophage T7 DNA synthesis in Escherichia coli B cells infected by alkylated bacteriophage T7.

Quantitative analysis of DNA replication, in E. coli B cells infected by methyl methanesulfonate-treated bacteriophage T7, showed that production of phage DNA was delayed and decreased. The cause of the delay appeared to be a delay in host-DNA breakdown, the process which provides nucleotides for phage-DNA synthesis. In addition, reutilisation of host-derived nucleotides was impaired. These observations can be accounted for by a model in which methyl groups on phage DNA slow down DNA injection and also reduce the replicational template activity of the DNA once it has entered the cell. Repair of alkylated phage DNA may be required not only for replication but also for normal injection of DNA.