Four-cluster analysis: a simple method to test phylogenetic hypotheses

A simple statistical test for comparing three alternative phylogenetic hypotheses for four monophyletic groups is presented. This test is based on the minimum-evolution principle, and it does not require any information regarding the branching order within each monophyletic group. It is computationally efficient and can be easily extended to five or more monophyletic groups.      

Reversible blockade of rodent whisking: Botulinum toxin as a tool for developmental studies

Studies of sensorimotor systems such as the whisking system of rodents have suggested that associations between body movements and their sensory consequences during development may make an important contribution to the functional organization of the system. In the present study we have explored the possible utility of Botulinum toxin for developmental studies of whisking. Botox selectively blocks whisking-generated afference leaving other sources of whisker afference intact. We describe appropriate modes of injection, define dosage levels, and assess the effects of prolonged whisking paralysis during development upon the basic motor competency of the adult rat. Our findings indicate that: (a) Botulinum toxin may be used to block whisking behavior in adult and developing rats, (b) that the duration of the whisking paralysis produced by Botox treatment blockade is dose dependent in both developing and adult animals, (c) that the blockade is functionally reversible and (d) that Botox treatment during development does not impair either the kinematics or the rhythmic patterning of adult whisking behavior. Botox may be a useful tool for studying the role of experiential factors in the development of "active touch" in rodents.     

Reversible blockade of rodent whisking: Botulinum toxin as a tool for developmental studies

Studies of sensorimotor systems such as the whisking system of rodents have suggested that associations between body movements and their sensory consequences during development may make an important contribution to the functional organization of the system. In the present study we have explored the possible utility of Botulinum toxin for developmental studies of whisking. Botox selectively blocks whisking-generated afference leaving other sources of whisker afference intact. We describe appropriate modes of injection, define dosage levels, and assess the effects of prolonged whisking paralysis during development upon the basic motor competency of the adult rat. Our findings indicate that: (a) Botulinum toxin may be used to block whisking behavior in adult and developing rats, (b) that the duration of the whisking paralysis produced by Botox treatment blockade is dose dependent in both developing and adult animals, (c) that the blockade is functionally reversible and (d) that Botox treatment during development does not impair either the kinematics or the rhythmic patterning of adult whisking behavior. Botox may be a useful tool for studying the role of experiential factors in the development of "active touch" in rodents.     

Adaptation of mouse skeletal muscle to a novel functional overload test: changes in myosin heavy chains and SERCA and physiological consequences

We have used a new approach to study the effects of overload on skeletal muscle phenotype in mice. The method used avoids any traumatising contact with muscles and the inflammatory reaction that this may provoke. Blocks of lead embedded in silicone were inserted under the skin of the lower part of the back. After 1 month, a 17% hypertrophy was found to have occurred in the tonic soleus muscle, but no change was observed in the fast-twitch extensor digitorum longus (EDL) muscle. The main effects on the contractile properties of the soleus muscle were a decrease in the tetanic relaxation rate and a reduction in the maximal velocity of shortening. Immunohistological analysis of the soleus muscles revealed an increase in the proportion of fibres that express myosin heavy chain (MHC) 1, from 54.2% to 73.9%, with a reduction in the proportion of MHC2a-positive fibres, from 45.8% to 30.2%. These changes were accompanied by an increase in the proportion of fibres that express the slow type of sarcoplasmic reticulum calcium pump (SERCA2a), from 61.8% to 84.7%. In EDL muscles, overload induced only minor changes. Thus, this method of overload affected the soleus muscle in particular. The observed changes in the control of muscle contraction were significantly larger than the changes in typical myofibrillar properties that were observed. These results indicate that there is a temporal dissociation between the relative expression of MHCs and SERCAs.     

Fucose expression in skeletal muscle: a lectin histochemical study

Summary Binding sites for three fucose specific lectins, Aleuria aurantia agglutinin (AAA), Lotus tetragonolobus agglutinin (LTA) and Ulex europeus I agglutinin (UEA I), were investigated in sections from normal human and rat muscles, in muscle from patients with Duchenne muscular dystrophy (DMD) and in denervated and devascularized rat muscle. In normal human and rat muscle AAA detected fucosylated glycocompounds in the sarcoplasm, sarcolemma, interfibre connective tissue and vascular structures. In normal human muscle addition of fucose to the AAA incubation medium or treatment of the sections with formaldehyde followed by periodic oxidation before lectin incubation strongly inhibited the staining at all sites other than endothelial cells. In normal rat muscle the same staining procedures strongly inhibited the AAA binding at all sites other than the sarcolemma. Incubation with LTA resulted in a diffuse reaction around the vascular structures in rat muscle, while in human muscle a moderate, homogeneous staining was present in all muscle fibres. Treatment of the sections with formaldehyde and periodic acid before incubation with LTA resulted in strongly labelled muscle capillaries in both human and rat muscle. The only elements in the muscle tissues that were stained with UEA I were human endothelial cells. In denervated and devascularized rat muscle incubation with AAA revealed a novel fucose expression that appeared intracellularly in some necrotic fibres. The AAA-positive fucose residues in the sarcolemma of normal muscle fibres that were resistant to periodic acid oxidation could not be shown by AAA in denervated muscle. In DMD muscle a cryptic sarcolemmal fucose expression could be shown with AAA. It is suggested that both the sarcoplasm and sarcolemma of diseased muscle fibres show altered fucose expression.     

Computational fluid dynamics as a tool for testing functional and ecological hypotheses in fossil taxa

Computational fluid dynamics is a method for simulating fluid flows that has been widely used in engineering for decades, and which also has applications for studying function and ecology in fossil taxa. However, despite the possible benefits of this approach, computational fluid dynamics has been used only rarely in palaeontology to date. The theoretical basis underlying the technique is outlined and the main steps involved in carrying out computer simulations of fluid flows are detailed. I also describe previous studies that have applied the method to fossils and discuss their potential for informing future research directions in palaeontology. Computational fluid dynamics can enable large‐scale comparative analyses, as well as exacting tests of hypotheses related to the function and ecology of ancient organisms. In this way, it could transform our understanding of many extinct fossil groups.     

Rab8a as a mitochondrial receptor for lipid droplets in skeletal muscle

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Macrophages in developing mammalian skeletal muscle: evidence for muscle fibre death as a normal developmental event.

Macrophages in the diaphragm of fetal, growing and adult rats were investigated using electron microscopy. In addition, frozen sections of the diaphragm, muscles of the anterior abdominal wall and muscles of the calf were stained for acid phosphatase activity and examined with the light microscope. Macrophages were frequently observed in the muscles at 20 days of gestation and until the animals were 2 weeks old. They were less frequently found in the 4-week-old rats and very rarely found in the 8-week-old and adult rats. They were found in intimate contact with, sometimes apparently surrounding, certain muscle fibres which were very electron dense and considered to be degenerating. In addition, myofibrils were found as phagosomes within the macrophage cytoplasm. There was evidence to support the theory that macrophages develop from mesenchymal cells in embryonic tissue. Cells considered to be early macrophages and containing small lysosomes were found in the muscles at 16 and 18 days of gestation. In all other respects these cells resembled mesenchymal cells. It is proposed that cell death may be a normal developmental event in skeletal muscle, in which macrophages play an important role in the removal of the dead fibres.     

Problems and paradigms: Dystrophin as a mechanochemical transducer in skeletal muscle

This review is primarily concerned with two key issues in research on dystrophin: (1) how the protein interacts with the plasma membrane of skeletal muscle fibres and (2) how an absence of dystrophin gives rise to Duchenne muscular dystrophy. In relation to the first point, we suggest that the post-translational acylation of dystrophin may contribute to its interaction with the plasma membrane. Regarding the second point, it is generally considered that an absence of dystrophin makes the plasma membrane susceptible to damage by contraction/relaxation cycles. In this connection, we propose that the progressive nature of Duchenne dystrophy, and the phenotypic characteristics of mdx mice, are more consistent with dystrophin functioning as a mechanical transducer that transmits growth stimuli from the enlarging skeleton to the muscle. On the basis of this hypothesis, dystrophin-deficient muscles would be unable to grow at the same rate as the skeleton.     

The toxin binding inhibition test as a reliable in vitro alternative to the toxin neutralization test in mice for the estimation of tetanus antitoxin in human sera

A method for the screening of human sera for tetanus antibodies has been developed and evaluated. The toxin binding inhibition test (ToBI-test) is based on inhibition of the binding of tetanus toxin to an antitoxin-coated immunoassay microtitre plate by tetanus antibodies. Serum samples from 191 healthy adults with different vaccination histories have been titrated for tetanus antibodies by the toxin neutralization (TN) test in mice, by toxoid-ELISA and by the ToBI-test. In every respect, the ToBI-test proved to be the best in vitro alternative to the TN-test in mice. Comparisons showed a higher degree of correlation between the ToBI-test and the TN-test than between the toxoid-ELISA and the TN-test. Furthermore, no overestimation of antibody content was seen in titrating low titre sera by the ToBI-test. In contrast, several false positive results were seen when using the toxoid-ELISA. It is concluded that the ToBI-test is a reliable and precise alternative to the TN-test and can be performed under simple laboratory conditions in a short time.     

Use of a metallochromic indicator to study intracellular calcium movements in skeletal muscle

The transient increase in free myoplasmic calcium concentration due to depolarization of a skeletal muscle fibre is the net result of the release of calcium from the sarcoplasmic reticulum (SR) and its simultaneous removal by binding to various sites and by reuptake into the SR. We review here procedures recently developed in this laboratory for empirically characterizing the calcium removal processes in voltage-clamped fibres and for using such characterization to determine the time course of SR calcium release during a depolarizing pulse.     

Increased cAMP as a positive inotropic factor for mammalian skeletal muscle in vitro

To test the hypothesis that an increased cAMP concentration improves skeletal muscle force development, we stimulated mouse soleus and extensor digitorum longus (EDL) in the presence of isoproterenol (1 x 10(-5) mol.L-1), a beta-adrenergic agonist, or N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dcAMP) (1 x 10(-3) mol.L-1), a membrane-permeable cAMP analogue. Drugs used in the challenges were dissolved in Krebs-Henseleit bicarbonate buffer (Krebs) at 27 degrees C and gassed with 95% O2 - 5% CO2. Stimulation at 50 impulses.s-1 for 0.5 s produced an isometric tetanic contraction. Over 25 min of contractions at 0.6 contractions.min-1, developed force increased significantly with the addition of isoproterenol (soleus, 2.5% +/- 1.1%; EDL, 13.8% +/- 2.0%) or dcAMP (soleus, 2.3% +/- 0.5%; EDL, 10.9% +/- 1.9%) as compared with vehicle controls (cont) with Krebs added (soleus, 0.0% +/- 0.2%; EDL, -2.5% +/- 0.7%). To investigate the role of Ca2+ availability, we amplified or attenuated sarcolemmal L-type Ca2+ channels with Bay K 8644 (Bay K) (5.6 x 10(-6) mol.L-1) or diltiazem hydrochloride (dilt) (10(-4) mol.L-1), respectively. Ca2+ release from the sarcoplasmic reticulum was increased with caffeine (2 x 10(-3) mol.L-1) or decreased with dantrolene sodium (dant) (4.2 x 10(-7) mol.L-1). With Ca2+availability modified, dcAMP addition in soleus significantly increased force development above control (cont, 2.3% +/- 0.4%; Bay K, 4.0% +/- 1.0%; dilt, 52.3% +/- 3.6%; caffeine, 2.3% +/- 0.7%; dant, 6.0% +/- 2.0%; dilt + dant, 55.0% +/- 23.0%). In EDL, the addition of dcAMP also increased force development above control (cont, 13.7% +/- 1.9%; Bay K, 17.0% +/- 4.0%; dilt, 170.0% +/- 40.0%; caffeine, 23.0% +/- 4.0%; dant, 72.0% +/- 10.0%; dilt + dant, 54.0% +/- 14.0%). Thus, a positive inotropic effect of cAMP existed in both fast- and slow-twitch mammalian skeletal muscle with both normal and altered Ca2+ flux into the sarcoplasm.     

Expression in Escherichia coli and a functional study of a beta-troponin T 25 kDa fragment of rabbit skeletal muscle.

A 25 kDa fragment of beta-type troponin T (beta-TnT) was expressed in Escherichia coli, and its function as a component of the regulatory system for actomyosin ATPase was compared with that of the authentic counterpart, the full length alpha-TnT. The expressed species, designated as beta-TnT(N'-208), consists of 208 residues. It lacks the entire variable region at the amino-terminus and, near the carboxyl-terminus, a segment of 14 residues is changed from the alpha-type to the beta-type sequence. Functional tests indicated that the truncated beta-TnT was not distinguishable from the full length alpha-TnT, suggesting that neither deletion of the variable N-terminal region nor alteration of the type has a significant effect on the regulatory action of TnT.     

Myosin heavy chain mRNA transform to faster isoforms in immobilized skeletal muscle: a quantitative PCR study

Jänkälä, Heidi, Veli-Pekka Harjola, NielsErik Petersen, and Matti Härkönen. Myosin heavy chainmRNA transform to faster isoforms in immobilized skeletal muscle: aquantitative PCR study. J. Appl.Physiol. 82(3): 977-982, 1997.A quantitative polymerase chain reaction (PCR) method was used to measure the quantities of type I, IIa, IIx, and IIb myosin heavy chain (MHC) mRNAin total RNA preparations of the soleus, gastrocnemius, and plantarismuscles of normal and hindlimb-immobilized rats. Type IIx and even typeIIb MHC mRNA were demonstrated at extremely low levels in normalsoleus, 2.1 ± 0.4 × 10⁵and 5.0 ± 0.2 × 10⁵molecules of mRNA per microgram total RNA, respectively. Immobilization for 1 wk significantly altered the gene expression of MHC isoforms. Insoleus, both type IIx and IIb MHC genes became significantly upregulated, 24-fold (P < 0.005) and 2.6-fold (P < 0.05),respectively. In gastrocnemius, the level of type IIa MHC mRNAdecreased by 51% (P < 0.01) and thelevel of type IIx MHC mRNA increased by 140%(P < 0.05). In plantaris, the levelof type IIa MHC mRNA decreased by 58%(P < 0.005). In conclusion,immobilization changed the MHC mRNA profile in three different types ofskeletal muscle toward faster isoforms. The quantitative results permitreliable evaluation of changes in mRNA levels.

     

Distribution of antibodies to the troponin complex,troponin-C and troponin-I in chicken skeletal muscle as determined by a simplified method for immuno-electron microscopy

Antisera against the troponin complex, troponin-C and troponin-I have been utilized to locate these proteins in normal, adult chicken skeletal muscle and in filaments prepared from chicken acetone dried powder. The antisera had been previously characterized by immunochemical methods and were employed to ascertain the distribution of the proteins by a simple method for immuno-electron microscopy. Glycerinated chicken breast muscle was treated with the antisera, unreacted antibody was washed from the muscle, and a goat anti-rabbit γ-globulin was added to enhance the electron density of the antigen-antibody complexes. A periodic distribution of anti-troponin-C at a mean interval of 389 Å was observed along the thin filaments in the sectioned tissue. Anti-troponin-I was deposited every 399 Å (P < 0.01). Thin filaments were prepared from acetone dried powder and reacted with the antisera. The anti-troponin-C was located every 386 Å; anti-troponin-I, every 399 Å (P < 0.01). Our technique for immuno-electron microscopy is compared with that used by others, and the significance of the findings is discussed.