Core substructure in Mastigocladus laminosus phycobilisomes

A native high molecular complex (M_r 850000) containing about 50% of the allphycocyanin of the phycobilisome but lacking allophycocyanin B was separated from isolated phycobilisomes by gel electrophoresis. It was designated AP_CM since the large linker polypeptide L_CM was exclusively localized in this complex. The complex exhibited a ?196°C fluorescence emission maximum at 673 nm (671 nm at 25°C). In addition, a core complex (designated AP_C, M_r≥1000000) consisting of both AP_CM and AP 680 was isolated by combined gel filtration and linear gradient centrifugation. At 25°C this complex showed dual emission peaks at 670 and 680 nm demonstrating functional independence of the terminal emitters. A complex similar to AP_CM can be isolated from phycobilisomes of Anabaena variabilis. This is evidence that AP_CM is the constitutive center of the tricylindrical core of hemidiscoidal cyanobacterial phycobilisomes. Two models summarizing the structural and functional consequences of the results are presented in the discussion.     

Artificial dimers of native actin: Preparation and properties in biological functions

With the aid of tartryl-bis--aminocaprylazide artificial dimers were produced from F actin from rabbit striated muscle. These derivatives will not polymerize by themselves but are able to copolymerize fully with native G actin. By modification of a single side chain per dimer, this copolymerization was completely inhibited. The dimers are able to activate subfragment I ATPase of myosin and bind to DNase I with inactivation of the enzyme in the same manner as native G actin. Within the dimer, one ADP is immobilized and will exchange against ATP extremely slowly. The dimers do not bind to the mushroom toxin phalloidin.     

The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.     

A re-evaluation of the cytology of cat Pacinian corpuscles

Summary The ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristic concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.     

Gender difference in the relationship of performance in the handgrip and standing long jump tests to lean limb volume in young adults

Groups of young, adult males and females performed the handgrip and standing long jump tests. Their total forearm and leg volumes were calculated from a series of circumference and length measurements, and the lean volumes (bone + muscle) calculated by taking the skinfold thickness into consideration. In the handgrip, the mean female performance was 298 N compared with 496 N for the males. In the standing long jump, mean performance expressed as distance x body mass was 87.3 kg.m for females compared with 137.7 kg.m for males. These superior performances of males could simply reflect their greater muscle mass, as the mean lean volumes of female and male limbs respectively were 0.54 l and 0.89 l for forearms, and 11.82 l and 14.82 l for the two legs. However, when the performances of males and females were grouped by lean limb volume, it was found that while in both tests there were linear relationships, males and females did not share a common line. In both tests the male relationship was at a higher level than the female; therefore, for a given lean volume, the male performance was significantly superior to that of the female. The gender difference found in this study has not been seen in other studies in which the performance of skeletal muscle has been related to the cross-sectional area of the active muscles and the possible reasons for the differences are considered.     

Effects of training at simulated altitude on performance and muscle metabolic capacity in competitive road cyclists

Differences between the effects of training at sea level and at simulated altitude on performance and muscle structural and biochemical properties were investigated in 8 competitive cyclists who trained for 3-4 weeks, 4-5 sessions/week, each session consisting of cycling for 60-90 min continuously and 45-60 min intermittently. Four subjects, the altitude group (AG), trained in a hypobaric chamber (574 torr = 2300 m above sea level), and the other four at sea level (SLG). Before and after training work capacity was tested both at simulated altitude (574 torr) and at sea level, by an incremental cycle ergometer test until exhaustion. Work capacity was expressed as total amount of work performed. Venous blood samples were taken during the tests. Leg muscle biopsies were taken at rest before and after the training period. AG exhibited an increase of 33% in both sea level and altitude performance, while SLG increased 22% at sea level and 14% at altitude. Blood lactate concentration at a given submaximal load at altitude was significantly more reduced by training in AG than SLG. Muscle phosphofructokinase (PFK) activity decreased with training in AG but increased in SLG. All AG subjects showed increases in capillary density. In conclusion, work capacity at altitude was increased more by training at altitude than at sea level. Work capacity at sea level was at least as much improved by altitude as by sea level training. The improved work capacity by training at altitude was paralleled by decreased exercise blood lactate concentration, increased capillarization and decreased glycolytic capacity in leg muscle.     

The role of phospholipase A2 in calcium-induced damage in cardiac and skeletal muscle

Summary This study compares the action of inhibitors of the eicosanoid cascade on calcium-induced myofilament damage in cardiac muscle of the perfused frog heart and incubated frog ventricle slices, and in skeletal muscle of incubated mammalian diaphragm and isolated and saponin-skinned amphibian pectoris cutaneous muscle. Mepacrine (10^-5M) and indomethacin (3×10^-6M) protected completely against myofilament damage induced by entry of calcium in the calcium-paradox in frog heart. However, inhibition of phospholipase A₂ (PLA₂) (with chlorpromazine, 2×10^-4M, or mepacrine, 10^-5M, 5x10^-5M), of cyclo-oxygenase enzymes (with indomethacin, 3x10^-6M to 10^-5M or BW755C, 3.8x10^-4M), or of lipoxygenase enzymes (with BW755C, 3.8x10^-4M or nordihydroguaiaretic acid, 2x10^-6M or 5x10^-6M) all failed in intact cardiac or skeletal muscle cells to prevent the myofilament damage that is rapidly triggered by 10^-2M caffeine, 6x10^-6M ruthenium red, 10^-4M DNP or 5 g ml^-1 A23187. These agents also failed completely to protect against myofilament damage in saponin-skinned amphibian skeletal muscle when [Ca]_i was raised to 8x10^-6M. Thus, inhibition of PLA₂ does not protect the myofilament apparatus against calcium released intracellularly, and it is suggested that mepacrine and indomethacin can block entry of calcium in the calcium-paradox in the amphibian heart. Chlorpromazine (2x10^-4M) and mepacrine (10^-3M) at zero [Ca] caused severe myofilament damage in skinned muscle, possibly due to an effect on membranes. Since inhibitors of PLA₂ and of lipoxygenases prevent efflux of creatine kinase and sarcolemma damage in mammalian skeletal muscle, it is evident that experimentally-induced rises in [Ca]_i (by caffeine or A23187) can trigger two separate pathways: (i) PLA₂ and the arachidonic acid cascade which culminate in membrane damage, and (ii) a different, Ca-activated system that causes rapid damage of myofilaments.     

Comparison of incremental and steady state tests of endurance training

To compare the results obtained by incremental or constant work load exercises in the evaluation of endurance conditioning, a 20-week training programme was performed by 9 healthy human subjects on the bicycle ergometer for 1 h a day, 4 days a week, at 70-80% VO2max. Before and at the end of the training programme, (1) the blood lactate response to a progressive incremental exercise (18 W increments every 2nd min until exhaustion) was used to determine the aerobic and anaerobic thresholds (AeT and AnT respectively). On a different day, (2) blood lactate concentrations were measured during two sessions of constant work load exercises of 20 min duration corresponding to the relative intensities of AeT (1st session) and AnT (2nd session) levels obtained before training. A muscle biopsy was obtained from vastus lateralis at the end of these sessions to determine muscle lactate. AeT and AnT, when expressed as % VO2max, increased with training by 17% (p less than 0.01) and 9% (p less than 0.05) respectively. Constant workload exercise performed at AeT intensity was linked before training (60% VO2max) to a blood lactate steady state (4.8 +/- 1.4 mmol.l-1) whereas, after training, AeT intensity (73% VO2max) led to a blood lactate accumulation of up to 6.6 +/- 1.7 mmol.l-1 without significant modification of muscle lactate (7.6 +/- 3.1 and 8.2 +/- 2.8 mmol.kg-1 wet weight respectively). It is concluded that increase in AeT with training may reflect transient changes linked to lower early blood lactate accumulation during incremental exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal relationship between nerve-stump-length-dependent changes in the autophosphorylation of a cyclic AMP-dependent protein kinase and the acetylcholine receptor content in skeletal muscle

The acetylcholine receptor (AChR) content and the autorphosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) were evaluated in rat soleus muscles at 24, 30 and 66 hr after surgical denervation by cutting the nerve at a short distance (short-nerve-stump) and at a long distance (long-nerve-stump) from the muscle. AChR content was based on the specific binding of [¹²⁵I]alpha-bungarotoxin (BUTX); changes in the autophosphorylation of R-II were based upon the predominant in vitro³²P-phosphorylation of a 56-Kd soluble protein in cytosolic fractions of solei. The AChR content and the³²P-autophosphorylation of R-II were increased in samples from short-nerve-stump solei, but not from long-nerve-stump solei, after a denervation-time of 30 hr. This nerve-stump-length dependency indicates that the two denervation effects are not related to the immediate halt of impulse-evoked muscle contractility. Furthermore, the results show that alterations in the³²P-autophosphorylation of R-II occurred before, as well as whenever, increases in the AChR content were found. Speculatively, this temporal relationship may be significant with respect to the potential role of R-II in gene expression.Abbreviations ACh acetylcholine - AChR acetylcholine receptor(s) - BUTX alpha-bungarotoxin - Kd kilodalton - PAGE polyacrylamide gel electrophoresis - R-II regulatory subunit of cyclic AMP-dependent protein kinase type II - SDS sodium dodecyl sulfate     

Structural and functional domains of cellobiohydrolase I from trichoderma reesei

Limited proteolysis (papain) of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei led to the seperation of two functional domains: a core protein (55 kDa) containing the active site, and a C-terminal glycopeptide (10 kDa) implicated in binding to the insoluble matrix (cellulose). The quaternary structures of the intact CBH I and its core in solution are now compared by small angle X-ray scattering (SAXS) measurements. The molecular parameters derived for the core (Rg=2.09 nm, D_max=6.5 nm) and for the intact enzyme (Rg=4.27 nm, D_max=18 nm) indicate very different shapes. The resulting models show a tadpole-like structure for the intact enzyme where the isotropic part coincides with the core protein and the flexible tail part should be identified with the C-terminal glycopeptide. Thus in this enzyme, functional differentiation is reflected in structural peculiarities.Abbreviations SAXS small angle X-ray scattering - SDS-PAGE SDS-polyacrylamide gel electrophoresis - IEF-PAG polyacrylamide gel isoelectric focusing; cellobiohydrolase (CBH, 1,4--glucan cellobio hydrolase (E.C.3.2.1.91)) - D_max maximum diameter - Rg radius of gyration