Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression.

Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

Growth hormone secretagogue increases muscle strength during remobilization after canine hindlimb immobilization.

Twenty-two beagles were divided into two equal groups, and the right hindlimb of each animal was immobilized at 105 degrees of knee flexion by external fixation. After 10 weeks of fixation, the device was removed, allowing free mobility for the following 5 weeks. Each day throughout the 15 weeks, one group received a growth hormone secretagogue (treatment) at a dose of 5 mg/kg, and the other received a lactose placebo (control). At weeks 0, 10, and 15, strength as indicated by maximum isometric extension torque was measured in the right hindlimb, biopsies of the vastus lateralis muscle were taken, and the dogs were weighed. Weekly blood samples were analyzed for insulin-like growth factor-1, blood urea nitrogen, and creatine phosphokinase. Between weeks 0 and 10, tetanic torque declined by about 60% (p < 0.001) in both groups, with no significant difference between the groups (p > 0.7). Between weeks 10 and 15, tetanic torque in the treated group increased by 0.81 Nm; this was significantly greater than the increase of 0.25 Nm in the placebo group (p < 0.05). The diameters of slow (type-1) and fast (type-2) fibers measured from the vastus lateralis muscle followed the same trend. At all time points, fiber diameter correlated strongly with torque; this argues against nonmuscular causes such as nerve injury for strength loss. The mean levels of insulin-like growth factor-1 increased 100% by week 4 in the treated group and remained elevated by about 60% throughout the experiment. Levels of insulin-like growth factor-1 in the placebo group decreased 30% within week 1 and remained depressed throughout the experiment. Our interpretation of these data suggests that the growth hormone secretagogue elevated levels of serum insulin-like growth factor-1, which in turn increased the size and strength of the quadriceps muscle during remobilization. These data may ultimately have therapeutic application to humans during rehabilitation after prolonged inactivity.

Increased oxidative capacity does not protect skeletal muscle fibers from eccentric contraction-induced injury.

Isometric electrical stimulation was delivered to rabbit dorsiflexor muscles at 10 Hz for 1 s on and 1 s off over 30 min, 5 days/wk for 3 wk to induce an increase in muscle oxidative capacity. Stimulation-trained muscles as well as untrained muscles were then subjected to a 30-min eccentric exercise bout to test whether increased oxidative capacity provided a protective effect against muscle injury. Electrical stimulation results in significant training of both the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, with EDL citrate synthase (CS) activity increasing an average of 67% (P < 0.0001) and TA CS activity increasing by 27% (P < 0.05). For all parameters measured, the magnitude of change was much greater for EDL than for TA muscle. Dorsiflexor fatigability decreased significantly during the 3-wk training period (P < 0.0001), whereas the EDL TA individually showed strong decreasing trends in fatigability after training. TA and EDL capillary density measured histomorphometrically increased from 839 +/- 56 to 1,026 +/- 71 mm-2 (P = 0.07) and from 589 +/- 37 to 792 +/- 66 mm-2 (P < 0.05), respectively. TA and EDL capillary-to-fiber ratio increased from 1.32 +/- 0.10 to 1.55 +/- 0.16 (P > 0.2) and 1.08 +/- 0.07 to 1.36 +/- 0.14 (P > 0.1), respectively. Type 2A fiber type percentage increased after stimulation training by 68% (P < 0.0001) for the EDL and by 32% (P > 0.1) for the TA at the expense of type 2D fibers. Despite the large training effect for the EDL and the modest training effect for the TA, no differences were observed between stimulation-trained and untrained groups for maximum dorsiflexion torque (P > 0.3) or maximum tetanic tension (P > 0.3) after eccentric contraction-induced injury. Additionally, no significant correlation was observed between CS activity and maximum tetanic tension after eccentric contraction-induced injury for either muscle (P > 0.2). Thus we conclude that increasing muscle oxidative capacity by isometric electrical stimulation training did not protect muscle against eccentric contraction-induced injury.

Four novel myosin heavy chain transcripts define a molecular basis for muscle fibre types in Rana pipiens.

1. Differential expression of myosin heavy chain (MHC) isoforms dramatically affects mechanical and energetic properties of skeletal muscle fibre types. As many as five different fibre types, each with different mechanical properties, have been reported in frog hindlimb muscles. However, only two frog MHC isoforms have previously been detected by SDS-PAGE and only one adult hindlimb MHC isoform has been cloned. 2. In the present study, four different fibre types (type 1, type 2, type 3 and tonic) were initially identified in adult Rana pipiens anterior tibialis muscle based on myosin ATPase histochemistry, size and location. Each fibre type exhibited unique reactivity to a panel of MHC monoclonal antibodies. Single fibre analysis using SDS-PAGE revealed that MHCs from immunohistochemically defined type 1, type 2 and type 3 fibres ran as three distinct isoform bands, while MHC of tonic fibres co-migrated with type 1 MHC. The combined data from immunohistochemistry and SDS-PAGE suggests that Rana fibre types are composed of four different MHCs. 3. Four novel MHC cDNAs were cloned and expression of the corresponding transcripts was measured in single immuno-identified fibres using specific polymerase chain reaction (PCR) primer pairs. Each of the four transcripts was found to be primarily expressed in a different one of the four fibre types. 4. Coexpression of MHC isoforms was observed only between types 1/2 and types 2/3 at both the protein and mRNA level. 5. These data provide a molecular basis for differentiation between frog fibre types and permit future molecular studies of MHC structure/function and gene regulation in this classic physiological system. 6. Comparison of sequence homology among amphibian, avian and mammalian MHC families supports the concept of independent evolution of fast MHC genes within vertebrate classes subsequent to the amphibian/avian/mammalian radiation.