The relative changes in isometric force and work during fatigue and recovery in isolated toad sartorius muscle

Toad sartorius muscle was subjected to sinusoidal varying length changes at 2 Hz to measure work. Both isometric tetanic force and work per cycle were measured before, during, and after a 3-min fatigue. Both isometric tetanic force and positive work, the work done by the muscle during the shortening part of the cycle, rapidly decreased in parallel in the first 40 s of fatigue. Thereafter, force continued to decrease, but at a slower rate, to about 10% of prefatigue values, whereas positive work levelled off at about 30% of prefatigue values. Negative work, the work done on the muscle during the lengthening part of the cycle, increased during fatigue to the extent that net work became negative. This was due to a prolonged relaxation, which resulted in active force still being generated while the muscle was being stretched. Work and force recovered at about the same rate. Isometric force measurements alone do not give any clear indication that net work will be negative under a particular set of experimental conditions.     

Effect of cycle frequency and excursion amplitude on work done by rat diaphragm muscle

Strips of isolated rat diaphragm muscle were attached to a servomotor-transducer apparatus, and the muscle length was cycled in a sinusoidal fashion about the length at which maximum isometric twitch force was developed, Lo. The amplitude of the length displacement (excursion amplitude) and rate of cycling were varied between 3 and 13% Lo and 1-4 Hz respectively. The muscle was tetanically stimulated (100 Hz, supramaximal voltage, stimulus duration (duty cycle) 20% of the length cycle period) during the shortening stage of the imposed length cycle at the phase that yielded maximum net positive work. The force and displacement of the muscle were recorded. Work per cycle was calculated from the area of the loop formed by plotting force against length for one full stretch-shorten cycle. Work per cycle decreased, but power increased, as cycle frequency was increased from 1 to 4 Hz. Maximum work done per cycle was about 12.8 J/kg at a cycle frequency of 1 Hz. Maximum mean power developed was about 27 W/kg and occurred at a cycle frequency of 4 Hz. Work and power were maximum at an excursion amplitude of 13% of Lo (i.e., Lo +/- 6.5%). Measured work and power output are considerably less than values estimated from length-tension and force-velocity curves.     

Clinical and Molecular Characterization of Patients with Distal 11q Deletions

Jacobsen syndrome is caused by segmental aneusomy for the distal end of the long arm of chromosome 11. Typical features include mild to moderate psychomotor retardation, trigonocephaly, facial dysmorphism, cardiac defects, and thrombocytopenia, though none of these features are invariably present. To define the critical regions responsible for these abnormalities, we studied 17 individuals with de novo terminal deletions of 11q. The patients were characterized in a loss-of-heterozygosity analysis using polymorphic dinucleotide repeats. The breakpoints in the complete two-generation families were localized with an average resolution of 3.9 cM. Eight patients with the largest deletions extending from 11q23.3 to 11qter have breakpoints, between D11S924 and D11S1341. This cytogenetic region accounts for the majority of 11q⁻ patients and may be related to the FRA11B fragile site in 11q23.3. One patient with a small terminal deletion distal to D11S1351 had facial dysmorphism, cardiac defects, and thrombocytopenia, suggesting that the genes responsible for these features may lie distal to D11S1351. Twelve of 15 patients with deletion breakpoints as far distal as D11S1345 had trigonocephaly, while patients with deletions distal to D11S912 did not, suggesting that, if hemizygosity for a single gene is responsible for this dysmorphic feature, the gene may lie distal to D11S1345 and proximal to D11S912.     

Molecular localization of the inhibitory arachidonic acid binding site to the pore of hIK1

We previously demonstrated that the endogenously expressed human intermediate conductance, Ca(2+)-activated K(+) channel (hIK1) was inhibited by arachidonic acid (AA) (Devor, D. C., and Frizzell, R. A. (1998) Am. J. Physiol. 274, C138-C148). Here we demonstrate, using the excised, inside-out patch-clamp technique, that hIK1, heterologously expressed in HEK293 cells, is inhibited 82 +/- 2% (n = 16) with 3 microm AA, being half-maximally inhibited (IC(50)) at 1.4 +/- 0.7 microm. In contrast, AA does not inhibit the Ca(2+)-dependent, small conductance K(+) channel, rSK2, another member of the KCNN gene family. Therefore, we utilized chimeric hIK1/rSK2 channels to define the AA binding domain on hIK1 to the S5-Pore-S6 region of the channel. Subsequent site-directed mutagenesis revealed that mutation of Thr(250) to Ser (T250S) resulted in a channel with limited sensitivity to block by AA (8 +/- 2%, n = 8), demonstrating that Thr(250) is a key molecular determinant for the inhibition of hIK1 by AA. Likewise, when Val(275) in S6 was mutated to Ala (V275A) AA inhibited only 43 +/- 11% (n = 9) of current flow. The double mutation T250S/V275A eliminated the AA sensitivity of hIK1. Introducing the complimentary single amino acid substitutions into rSK2 (S359T and A384V) conferred partial AA sensitivity to rSK2, 21 +/- 3% and 31 +/- 3%, respectively. Further, introducing the double mutation S359T/A384V into rSK2 resulted in a 63 +/- 8% (n = 9) inhibition by AA, thereby demonstrating the ability to introduce this inhibitory AA binding site into another member of the KCNN gene family. These results demonstrate that AA interacts with the pore-lining amino acids, Thr(250) and Val(275) in hIK1, conferring inhibition of hIK1 by AA and that AA and clotrimazole share similar, if not identical, molecular sites of interaction.     

Peroxisomal proteomics, a new tool for risk assessment of peroxisome proliferating pollutants in the marine environment

In an attempt to improve the detection of peroxisome proliferation as a biomarker in environmental pollution assessment, we have applied a novel approach based on peroxisomal proteomics. Peroxisomal proteins from digestive glands of mussels Mytilus galloprovincialis were analyzed using 2-DE and MS. We have generated a reference 2-DE map from samples obtained in a well-studied reference area and compared this with peroxisomal proteomes from other sequenced genomes. In addition, by comparing 2-DE maps from control samples with samples obtained in a polluted area, we have characterized the peroxisome proliferation expression pattern associated with exposure to a polluted environment. Over 100 spots were reproducibly resolved per 2-DE map; 55 differentially expressed spots were quantitatively detected and analyzed, and 14 of these showed an increase in protein expression of more than fourfold. Epoxide hydrolase, peroxisomal antioxidant enzyme, and sarcosine oxidase (SOX) have been identified by ESI MS/MS, and acyl-CoA oxidase, multifunctional protein, and Cu,Zn-superoxide dismutase were immunolocalized by Western blotting. Our results indicate that a peroxisomal protein pattern associated to marine pollutant exposure can be generated, and this approach may have a greater potential as biomarker than traditional, single-protein markers.     

Contractile abilities of normal and "mini" triceps surae muscles from mice (Mus domesticus) selectively bred for high voluntary wheel running.

As reported previously, artificial selection of house mice caused a 2.7-fold increase in voluntary wheel running of four replicate selected lines compared with four random-bred control lines. Two of the selected lines developed a high incidence of a small-muscle phenotype ("mini muscles") in the plantar flexor group of the hindlimb, which apparently results from a simple Mendelian recessive allele. At generations 36-38, we measured wheel running and key contractile characteristics of soleus and medial gastrocnemius muscles from normal and mini muscles in mice from these selected lines. Mice with mini muscles ran faster and a greater distance per day than normal individuals but not longer. As expected, in mini-muscle mice the medial and lateral gastrocnemius muscles were approximately 54 and 45% the mass of normal muscles, respectively, but the plantaris muscles were not different in mass and soleus muscles were actually 30% larger. In spite of the increased mass, contractile characteristics of the soleus were unchanged in any notable way between mini and normal mice. However, medial gastrocnemius muscles in mini mice were changed markedly toward a slower phenotype, having slower twitches; demonstrated a more curved force-velocity relationship; produced about half the mass-specific isotonic power, 20-50% of the mass-specific cyclic work and power (only 10-25% the absolute power if the loss in mass is considered); and fatigued at about half the rate of normal muscles. These changes would promote increased, aerobically supported running activity but may compromise activities that require high power, such as sprinting.     

The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1

The wheat pathogen Stagonospora nodorum produces multiple necrotrophic effectors (also called host-selective toxins) that promote disease by interacting with corresponding host sensitivity gene products. SnTox1 was the first necrotrophic effector identified in S. nodorum, and was shown to induce necrosis on wheat lines carrying Snn1. Here, we report the molecular cloning and validation of SnTox1 as well as the preliminary characterization of the mechanism underlying the SnTox1-Snn1 interaction which leads to susceptibility. SnTox1 was identified using bioinformatics tools and verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid protein with the first 17 amino acids predicted as a signal peptide, and strikingly, the mature protein contains 16 cysteine residues, a common feature for some avirulence effectors. The transformation of SnTox1 into an avirulent S. nodorum isolate was sufficient to make the strain pathogenic. Additionally, the deletion of SnTox1 in virulent isolates rendered the SnTox1 mutated strains avirulent on the Snn1 differential wheat line. SnTox1 was present in 85% of a global collection of S. nodorum isolates. We identified a total of 11 protein isoforms and found evidence for strong diversifying selection operating on SnTox1. The SnTox1-Snn1 interaction results in an oxidative burst, DNA laddering, and pathogenesis related (PR) gene expression, all hallmarks of a defense response. In the absence of light, the development of SnTox1-induced necrosis and disease symptoms were completely blocked. By comparing the infection processes of a GFP-tagged avirulent isolate and the same isolate transformed with SnTox1, we conclude that SnTox1 may play a critical role during fungal penetration. This research further demonstrates that necrotrophic fungal pathogens utilize small effector proteins to exploit plant resistance pathways for their colonization, which provides important insights into the molecular basis of the wheat-S. nodorum interaction, an emerging model for necrotrophic pathosystems.     

A first genome assembly of the barley fungal pathogen <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis>

Background  

Pyrenophora teres f. teres is a necrotrophic fungal pathogen and the cause of one of barley's most important diseases, net form of net blotch. Here we report the first genome assembly for this species based solely on short Solexa sequencing reads of isolate 0-1. The assembly was validated by comparison to BAC sequences, ESTs, orthologous genes and by PCR, and complemented by cytogenetic karyotyping and the first genome-wide genetic map for P. teres f. teres.