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Synthesis of a spin-labeled photoaffinity ATP analogue, and its use to specifically photolabel myosin cross-bridges in skeletal muscle fibers.

A spin-labeled photoaffinity ATP analogue 3'(2')-O-[4-[4-oxo-(4-amido-2,2,6,6-tetramethyl-piperidino-1-oxyl)]-benz oyl]benzoyl adenosine 5'-triphosphate (SL-Bz2ATP) was synthesized and used to photolabel myosin in muscle fibers. Previous work has shown that 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP) photolabeled Ser-324 of the 50 kDa tryptic fragment of skeletal S1 heavy chain. In this work, [alpha-32P]SL-Bz2ATP was hydrolyzed and trapped as the diphosphate analogue with Co2+ and orthovanadate at the active site of myosin in rabbit psoas muscle fibers. After UV irradiation, the myosin heavy chain was the only protein band found to be significantly photolabeled as assayed by gel electrophoresis and radioactivity counting. The labeling was localized after brief trypsin digestion by SDS-PAGE to be on the 50 kDa tryptic fragment of the S1 heavy chain. Ca. 35% of the myosin in fibers was covalently photolabeled. The fibers photolabeled with SL-Bz2ATP had the same active tension and maximum shortening velocity as the control fibers. The resulting spin label on myosin was too mobile to report the orientation of the heads in fibers. Nonetheless, this is the first work to show the feasibility of utilizing active site binding and photoaffinity labeling to place covalent spectroscopic probes at the myosin active site in fibers with high specificity and yield without affecting mechanical function.

Adenosine Triphosphate↗

Ca2+ binding to troponin C in skinned skeletal muscle fibers assessed with caged Ca2+ and a Ca2+ fluorophore. Invariance of Ca2+ binding as a function of sarcomere length.

Ca2+ sensitivity of tension varies with sarcomere length in both skeletal and cardiac muscles. One possible explanation for this effect is that the Ca2+ affinity of the regulatory protein troponin C decreases when sarcomere length is reduced. To examine length dependence of Ca2+ binding to troponin C in skeletal muscle, we developed a protocol to simultaneously monitor changes in sarcomere length, tension, and Ca2+ concentration following flash photolysis of caged Ca2+. In this protocol, [Ca2+] was rapidly increased by flash photolysis of caged Ca2+, and changes in [Ca2+] due to photolysis and the subsequent binding to troponin C were assessed using a Ca2+ fluorophore. Small bundles of fibers from rabbit skinned psoas muscles were loaded with Ca2+ fluorophore (Fluo-3) and caged Ca2+ (dimethoxynitrophenamine or o-nitrophenyl-EGTA). The bundles were then transferred to silicone oil, where [Ca2+]free, tension, and sarcomere length were monitored before and after photolysis of caged Ca2+. Upon photolysis of caged Ca2+, fluorescence increased and then decayed to a new steady-state level within approximately 1 s, while tension increased to a new steady-state level within approximately 1.5 s. After extracting troponin C, fibers did not generate tension following the flash, but steady-state post-flash fluorescence was significantly greater than when troponin C was present. The difference in [Ca2+]free represents the amount of Ca2+ bound to troponin C. In fibers that were troponin C-replete, Ca2+ binding to troponin C did not differ at short (approximately 1.97 microm) and long (approximately 2.51 microm) sarcomere length, yet tension was approximately 50% greater at the long sarcomere length. These results show that the affinity of troponin C for Ca2+ is not altered by changes in sarcomere length, indicating that length-dependent changes in Ca2+ sensitivity of tension in skeletal muscle are not related to length-dependent changes in Ca2+ binding affinity of troponin C.

Aniline Compounds↗

Muscle fiber typing in routinely processed skeletal muscle with monoclonal antibodies.

Muscle fiber typing is conventionally performed using mATPase enzyme histochemistry on cryostat sections. After pre-incubation of sections at pH 4.3, 4.6 and 10.3, based on the pattern of enzyme reactivity, the fibers can be classified in types I, II (subtypes A, AB and B) and the intermediate C (I and II) fibers. We have attempted to perform fiber typing of human psoas muscle by immunohistochemistry, using monoclonal antibodies R11D10 (specific for cardiac and type I skeletal myosin) and MY-32 (specific for fast muscle fibers) on cryostat as well as on paraffin sections. Staining of consecutive cryostat sections showed that type I fibers are R11D10 reactive whereas type II fibers are MY-32 reactive. Subtyping of type II fibers could not be performed by immunohistochemistry. Quantitative analysis of type I and II fibers showed that enzyme histochemical and immunohistochemical analysis are in close agreement.

Antibodies, Monoclonal↗

Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres.

In muscle fibres labelled with iodoacetamidotetramethylrhodamine at Cys707 of the myosin heavy chain, the probes have been reported to change orientation when the fibre is activated, relaxed or put into rigor. In order to test whether these motions are indications of the cross-bridge power stroke, we monitored tension and linear dichroism of the probes in single glycerol-extracted fibres of rabbit psoas muscle during mechanical transients initiated by laser pulse photolysis of caged ATP and caged ADP. In rigor dichroism is negative, indicating average probe absorption dipole moments oriented more than 54.7 degrees away from the fibre axis. During activation from rigor induced by photoliberation of ATP from caged ATP in the presence of calcium, the dichroism reversed sign promptly (half-time 12.5 ms for 500 microM-ATP) upon release of ATP, but then changed only slightly during tension development 20 to 100 milliseconds later. During the onset of rigor following transfer of the fibre from an ATP-containing relaxing solution to a rigor medium lacking ATP, force generation preceded the change in dichroism. The dichroism change occurred slowly (half-time 47 s), because binding of ADP to sites within the muscle fibre limited its rate of diffusion out of the fibre. When ADP was introduced or removed, the dichroism transient was similar in time course and magnitude to that obtained after the introduction or removal of ATP. Neither adding nor removing ADP produced substantial changes in force. These results demonstrate that orientation of the rhodamine probes on the myosin head reflects mainly structural changes linked to nucleotide binding and release, rather than rotation of the cross-bridge during force generation.

Actomyosin↗

Obturator internus muscle abscess in children.

The authors describe four cases of obturator internus muscle (OIM) abscess in children, including their clinical presentations and treatment. This was a retrospective chart review. Children and adolescents younger than 18 years discharged between July 1, 1985, and September 30, 1998, from Brenner Children's Hospital with the diagnosis of muscle abscess or pelvic abscess were identified. A total of 56 patients were identified with the diagnosis of muscle abscess or pelvic abscess. OIM abscess was defined by radiologic findings of an inflammatory process with fluid collection in the OIM, along with the clinical findings suggestive of an OIM abscess. Four of the patients met the definition of OIM muscle abscess. The common presenting features were fever, limp, and hip pain. Computed tomography or magnetic resonance imaging was diagnostic in all four patients, and Staphylococcus aureus was the causative agent in each. All the patients recovered, one after surgical drainage and the other three after antimicrobial therapy alone or with needle aspiration. The presentation of OIM pyomyositis is similar to that of psoas muscle pyomyositis and other infectious processes of the pelvis and hip. The S. aureus is the most common etiologic agent but not the only one reported. Most patients can be managed without open surgical drainage, but needle aspirations may be helpful both therapeutically and diagnostically.

Abscess↗

Load-dependent mechanical efficiency of individual myosin heads in skeletal muscle fibers activated by laser flash photolysis of caged calcium in the presence of a limited amount of ATP.

Although a contracting muscle regulates its energy output depending on the load imposed on it ("Fenn effect"), the mechanism underlying the load-dependent energy output remains obscure. To explore the possibility that the mechanical efficiency, with which chemical energy derived from ATP hydrolysis is converted into mechanical work, of individual myosin heads changes in a load-dependent manner, we examined the auxotonic shortening of glycerinated rabbit psoas muscle fibers, containing ATP molecules almost equal in number to the myosin heads, following laser flash photolysis of caged calcium. Immediately before laser flash activation, almost all of the myosin heads in the fiber are in the state, M.ADP.Pi, and can undergo only one ATP hydrolysis cycle after activation. When the fibers were activated to shorten under various auxotonic loads, the length, force and power output changes were found to be scaled according to the auxotonic load. Both the power and energy outputs were maximal under a moderate auxotonic load. The amount of M.ADP.Pi utilized at a time after activation was estimated from the amount of isometric force developed after interruption of fiber shortening. This amount was minimal in the isometric condition, and increased nearly in proportion to the distance of fiber shortening. These results are taken as evidence that the efficiency of chemo-mechanical energy conversion in individual myosin heads changes in a load-dependent manner.

Adenosine Triphosphate↗

Evidence for the load-dependent mechanical efficiency of individual myosin heads in skeletal muscle fibers activated by laser flash photolysis of caged calcium in the presence of a limited amount of ATP.

Although a contracting muscle regulates its energy output depending on the load imposed on it ("Fenn effect"), the mechanism underlying the load-dependent energy output remains obscure. To explore the possibility that the mechanical efficiency, with which chemical energy derived from ATP hydrolysis is converted into mechanical work, of individual myosin heads changes in a load-dependent manner, we examined the auxotonic shortening of glycerinated rabbit psoas muscle fibers, containing ATP molecules almost equal in number to the myosin heads, after laser-flash photolysis of caged calcium. Immediately before laser-flash activation, almost all of the myosin heads in the fiber are in the state M.ADP.Pi, and can undergo only one ATP hydrolysis cycle after activation. When the fibers were activated to shorten under various auxotonic loads, the length, force, and power output changes were found to be scaled according to the auxotonic load. Both the power and energy outputs were maximal under a moderate auxotonic load. The amount of M.ADP.Pi utilized at a time after activation was estimated from the amount of isometric force developed after interruption of fiber shortening. This amount was minimal in the isometric condition and increased nearly in proportion to the distance of fiber shortening. These results are taken as evidence that the efficiency of chemomechanical energy conversion in individual myosin heads changes in a load-dependent manner.

Acetates↗

Muscle infections caused by Salmonella species: case report and review.

We describe a patient with salmonella pyomyositis and review 30 other cases reported during the past 4 decades. Men outnumbered women by 2.9 to 1, and the median age of the patients was 51 years. Approximately one-half the cases were caused by Salmonella enteritidis. Infected vascular aneurysms were observed in seven patients. Prior salmonella infections and local trauma or lesions were common. Diverse underlying conditions, mainly diabetes and human immunodeficiency virus infection, were present in 81% of the patients, and the psoas muscle was involved in 55% of the cases. One-third of the patients died, and relapses were common after a median time of 5 weeks (range, 4.5-27 weeks) in those who survived. Most patients had anemia, and pathogens were recovered from blood samples from two-thirds of the patients. Salmonella should be considered as a causative agent of muscle infections in the appropriate clinical setting, particularly in patients with underlying diseases or preexisting vascular aneurysms.

Aged↗

Infected pseudocyst in tropical pancreatitis presenting as psoas abscess.

Infected pseudocyst as a consequence of tropical pancreatitis presenting as psoas abscess is unusual. We report a 40-year-old man who presented with pain in the right lumbar region. CT and MRI of the abdomen revealed pancreatic pseudocysts with abscess formation in the psoas muscle and evidence of chronic calcific pancreatitis. He was managed by percutaneous drainage of the abscess along with antibiotics and other supportive measures.

Adult↗

[Hematoma of the iliac psoas secondary to anticoagulant treatment. Urological implications].

We describe the urological manifestations arising from hematoma of the left iliac psoas that developed in the course of anticoagulant therapy in a 54-year-old patient with a mitral prosthesis. The onset of the clinical picture was acute and mimicked left reno-ureteral colic. Ultrasound and CT were decisive in making the diagnosis, revealing a left retroperitoneal mass in the anterolateral aspect of the left psoas muscle. Intravenous urography evidenced the urological repercussions of the disease process consisting of medialization of the left ureter, ureterohydronephrosis, and anterior and lateral displacement of the kidney. Although this condition is considered by most to be a surgical emergency, we opted for conservative treatment because the patient had no hemodynamic instability, severe obstructive uropathy or signs or crural paralysis. The literature on the urological repercussions of psoas hematomas due to anticoagulant therapy is briefly reviewed.

Acenocoumarol↗

[Ureterocystoneostomy using the psoas hitch technic--outside variant].

This paper presents the "outside" variant of the standard psoas hitch procedure. Two basic principles of the procedure are: lateralisation and fixation of the bladder at the psoas muscle; the long and straight reimplantation of the ureter on the posterior, lateral or anterolateral bladder wall using the extravesical approach. The main indications were the extensive changes of the ureter in case of neurogenic bladder dysfunction and the reduced bladder capacity with the changes of the mucous membrane. The procedure was also applied in those cases where the standard procedure would have been indicated. The advantages and disadvantages of the procedure in relation to the standard psoas hitch procedure are discussed. During 1982 to 1985 this procedure was applied in 67 cases. The age ranged from 2 months to 14 years. With 9 patients the operation was performed bilaterally. The time interval between the operations was from 3 to 6 months. Tailoring of the ureter was undertaken in 3 cases. The complications were: One ureter stenosis that was solved by means of the same technique, and three parahiatal diverticula formations without clinical manifestations.

Adolescent↗

Mechanical characterization of skeletal muscle myofibrils.

A new instrument, based on a technique described previously, is presented for studying mechanics of micron-scale preparations of two to three myofibrils or single myofibrils from muscle. Forces in the nanonewton to micronewton range are measurable with 0.5-ms time resolution. Programmed quick (200-microseconds) steps or ramp length changes are applied to contracting myofibrils to test their mechanical properties. Individual striations can be monitored during force production and shortening. The active isometric force, force-velocity relationship, and force transients after rapid length steps were obtained from bundles of two to three myofibrils from rabbit psoas muscle. Contrary to some earlier reports on myofibrillar mechanics, these properties are generally similar to expectations from studies on intact and skinned muscle fibers. Our experiments provide strong evidence that the mechanical properties of a fiber result from a simple summation of the myofibrillar force and shortening of independently contracting sarcomeres.

Animals↗

Percutaneous drainage of large tuberculous iliopsoas abscess via a subinguinal approach: a report of two cases.

Two patients who had large tuberculous abscesses that were successfully treated with percutaneous drainage alone are reported. A new approach, called the "subinguinal approach," was used. This new technique avoids the bowel loops and pelvic organs (which can be limitations when using the anterior abdominal approach) by inserting the catheter through the subinguinal portion of the psoas muscle into the abscess. Compared to the posterior approach, this technique is more comfortable for the patient (who prefers a supine position) and is not limited by the iliac bone. The technique, clinical course, and outcome are described.

Adult↗

Ratio of total sulfur amino acids to lysine for finishing pigs.

We conducted two experiments to determine the optimum ratio of total sulfur amino acids (TSAA) to Lys for late finishing pigs. In Exp. 1, 50 barrows and 50 gilts were allotted to treatments with three replicates of three or four pigs per replicate in a randomized complete block (RCB) design within a split-plot arrangement of treatments. Sex was the whole plot and TSAA:Lys ratio was the subplot. Average initial and final BW were 77 and 111 kg. Barrows and gilts were fed diets formulated to contain .55 and .65% Lys, respectively. The ratios of TSAA:Lys were .50, .55, .60, .65, and .70. Diets met or exceeded an ideal amino acid pattern for all indispensable amino acids (except TSAA), and all diets were isonitrogenous and equal in electrolyte balance. In Exp. 2, 60 gilts were allotted to five treatments with four replicates of three gilts each in a RCB design. Average initial and final BW were 74 and 110 kg. Gilts were fed diets formulated to contain .65% Lys. The ratios of TSAA:Lys were .35, .425, .50, .575, and .65. In Exp. 1, there were no TSAA:Lys ratio effects (P > .10) for ADG, final BW, percentage muscle, longissimus muscle area, carcass length, percentage fat-free lean (PFFLEAN), lean gain per day (LGD), total fat (TOFAT), percentage TOFAT (PTOFAT), fat gain per day (FGD), lean:fat, retained energy in TOFAT as ether extractable lipid (RE-F), retained energy (RE), or serum urea N (SUN). Feed intake (ADFI) was greater (quadratic, P < .05) for pigs fed .70 TSAA:Lys than for pigs fed any other treatment. Hot carcass weight, psoas muscle weight, 10th rib fat thickness, dressing percentage, fat-free lean (FFLEAN), and retained energy in FFLEAN as protein (RE-P) responded inconsistently to TSAA:Lys ratio, resulting in cubic (P < .09) effects. In Exp. 2, ADFI (linear, P < .08), TOFAT (linear, P < .05), PTOFAT (linear, P < .07), FGD (linear, P < .05), RE-F (linear, P < .05), RE (linear, P < .05), and SUN (linear, P < .02; quadratic, P < .01) decreased as TSAA:Lys ratio increased. Also, gain:feed (GF) (linear, P < .01; quadratic, P < .04), PFFLEAN (linear, P < .04), and lean:fat (linear, P < .04) increased as TSAA:Lys ratio increased. One-slope, broken-line regression models estimated required ratios of TSAA:Lys of .44 (SUN), .40 (ADG), .47 (ADFI), .45 (GF), .45 (FFLEAN), .44 (LGD), .65 (TOFAT), .65 (FGD), .44 (RE-P), .65 (RE-F), .65 (RE), and .57 (lean:fat). Thus, for growth and muscling traits of late finishing pigs, the optimum ratio of TSAA:Lys is less than the current proposed ratio (.65), but to minimize fat accretion, the ratio is .65.

Amino Acids, Sulfur↗

Ultrasound guidance for the psoas compartment block: an imaging study.

UNLABELLED: We conducted this study to develop an ultrasound-guided approach to the psoas compartment and to assess its feasibility and accuracy by means of computed tomography (CT). Two examiners performed ultrasound-guided approaches at three levels (L2-3, L3-4, and L4-5) on 10 embalmed cadavers, which were seated prone. After each needle had been advanced into the psoas compartment under ultrasound guidance, the positions of their tips were computed by using two coordinates (A and B). Subsequently, axial transverse CT scans were made to verify the ultrasound measurements by using the same coordinates. In total, 48 approaches were performed (Examiner 1, n = 20; Examiner 2, n = 28). CT revealed that 47 of 48 ultrasound-guided approaches were performed exactly. In 1 of 48 approaches (L3-4), the tip of the needle was located posterior to the psoas muscle. The median differences between ultrasound and CT coordinates were 0.3 plus minus 0.3 cm for A and 0.2 plus minus 0.3 for B. Kendall's coefficient of concordance was 0.9 (P < 0.001) between ultrasound and CT measurements for both coordinates. These results indicate that ultrasound enables exact needle placement, as proved by CT. We conclude that ultrasound guidance might be a useful adjunct to increase the safety and efficacy of the psoas compartment block at these levels. IMPLICATIONS: We developed an ultrasound-guided approach to the psoas compartment at the levels L2-3, L3-4, and L4-5. Feasibility and accuracy were tested on embalmed cadavers and verified by means of computed tomography. Ultrasound guidance proved to be feasible and accurate for the performance of psoas compartment blocks.

Aged↗

The challenge of diagnosing psoas abscess.

Diagnosing psoas abscess in patients with low back pain might be hampered by their atypical symptoms. We describe a case in which a 39-year-old retired provost marshal presented to our emergency facility with acute onset of low back pain following an episode of lumbar strain during a flight. His initial computed tomography of lumbar spine and abdominal sonography were inconclusive. Two weeks later, he was admitted to our rehabilitation unit for similar symptoms. One day later, leukocytosis and elevated c-reactive protein following rising body temperature were found. Fever of unknown origin (FUO) was impressed after series of laboratory studies. However, an inflammatory gallium scan performed 2 weeks later due to persistent fever revealed active infection in the L4 vertebra and soft tissue of the L5 paraspinal region. MRI was then performed and demonstrated abnormal enhancement at the bodies of the L2 and L3 vertebrae, prevertebral soft tissue and bilateral psoas muscles. A CT-guided percutaneous aspiration of 10 cc yellowish pus was attained, which yielded no pathogens. His symptoms subsided 1 week after being given appropriate antibiotics. This case showed the importance of nuclear scanning and MRI in patients with low back pain and FUO. It also suggests that repeated imaging study and/or other modality should be considered when the initial imaging result is not compatible with the clinical presentation of psoas abscess, which was initially missed by image studies.

Adult↗

Alteration of cross-bridge kinetics by myosin light chain phosphorylation in rabbit skeletal muscle: implications for regulation of actin-myosin interaction.

Myosin light chain phosphorylation in permeable skeletal muscle fibers increases isometric force and the rate of force production at submaximal levels of calcium activation; myosin light chain phosphorylation may underlie the increased rate and extent of force production associated with isometric twitch potentiation in intact fibers. To understand the mechanism by which myosin light chain phosphorylation manifests these effects, we have measured isometric force, isometric stiffness, rate of isometric force redevelopment after isotonic shortening, and isometric ATPase activity in permeabilized rabbit psoas muscle fibers. These measurements were made in the presence and absence of myosin light chain phosphorylation over a range of calcium concentrations that caused various levels of activation. The results were analyzed with a two-state cross-bridge cycle model as suggested by Brenner [Brenner, B. (1988) Proc. Natl. Acad. Sci. USA 85, 3265-3269]. The results indicate that myosin light chain phosphorylation exerts its effect on force generation and the isometric rate of force redevelopment in striated muscle through a single mechanism, namely, by increasing the rate constant describing the transition from non-force-generating cross-bridges to force-generating states (fapp). gapp, the reverse rate constant, is unaffected by phosphorylation as are the number of cycling cross-bridges. Since both calcium and myosin light chain phosphorylation increase fapp, the possibility is considered that modulation of fapp may represent a general mechanism for regulating force in actin-myosin systems.

Actins↗

[The role of echography in osteolytic tubercular abscesses].

Tubercular abscesses are relatively common complications of tubercular spondylodiscitis. Fifty-one patients with suspected abscesses were selected from a group of 97 patients with tubercular spondylodiscitis and submitted to US. In 10 cases CT was performed before US and detected 7 abscesses, all of them confirmed by US. In the extant 41 cases, CT followed US; in 13 cases only US poorly visualized ilio-psoas muscles. As for the group of 23 patients who underwent both CT and US, if the former method is assumed as the reference gold standard, overall US sensitivity is 97% (1 false negative) and its specificity is 100%. In all cases where US findings were accurate and specific enough, CT was not performed; the patients were followed every seventh month and no abscesses found. US showed abscesses in the iliac fossa in 20 cases, along the psoas fascia in 6 and in the thighs in 3 cases. Two cases of gluteal localization were observed, together with 1 Grynfelt's triangle abscess, 1 Petit's triangle and 1 Scarpa's triangle abscesses; finally, 1 abscess was found in the knee. The most common appearance of tubercular abscesses is a hypoechoic and inhomogeneous pattern; sometimes caseum makes the abscess solid and hyperechoic. Calcifications were unusual in our series. All patients were submitted to percutaneous drainage under US guidance. The results proved US to allow the early and unquestionable diagnosis of tubercular abscesses and to confirm clinical suspicion. Moreover, US is also useful to guide percutaneous drainage and to follow the patients after drainage. As for CT, it remains the method of choice to depict vertebral involvement, but, in our series, it exhibited no significant advantages over US in the study of abscessual lumbar collections.

Abscess↗