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Physiological effects accompanying the removal of myosin LC2 from skinned skeletal muscle fibers.

The possible role of the LC2 light chain of myosin in the contraction of vertebrate striated muscle has long been a subject of interest. This problem has been addressed in the present study in which the mechanical effects of partial removal of LC2 from skinned fibers of rabbit psoas muscle have been investigated. Each fiber was divided into three segments, thus allowing determinations of the LC2 content of the fiber 1) prior to extraction of the LC2 subunit, i.e. control, 2) following extraction of LC2, and 3) following readdition of LC2 to the fiber. Measurements of isometric tension and the maximum velocity of shortening were made in these fiber segments at each of the above stages of the extraction protocol. LC2 was partially removed from the fiber segments by treatment with a solution containing 20 mM EDTA, 50 mM KCl, 5 mM phosphate buffer, pH 7.0, for 120 min at 30 degrees C, a procedure modified from Chantler and Szent-Gyorgyi (Chantler, P. D., and Szent-Gyorgyi, A. G. (1980) J. Mol. Biol. 138, 473-492). LC2 content was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoretic techniques. The results indicate that removal of about one-third of the total LC2 within a fiber segment reduced Vmax by nearly 50%, with very little effect upon isometric tension. Readdition of LC2 to these fiber segments resulted in recovery of Vmax to near control values. These findings suggest that LC2 may modulate the kinetics of interaction of myosin with actin in mammalian skeletal muscle.

Animals↗

Pneumococcal psoas abscess: report of a case and review of the world literature.

A 39-year-old man was admitted to the hospital with fever and right hip pain. CT scan of the pelvis showed a psoas muscle abscess. Pus, which was surgically evacuated, grew Streptococcus pneumoniae. The patient fully recovered after 30 days of intravenous antibiotics. Review of the literature revealed only nine similar cases of primary pneumococcal psoas abscess. Three cases were complicated with pneumococcal meningitis. In four cases there was a history of trauma to the involved area. Pneumococcal psoas abscess is a rare entity that carries a good prognosis provided early surgical drainage is performed and parenteral antibiotic therapy is administered to the patient.

Adult↗

Evidence for cross-bridge attachment in relaxed muscle at low ionic strength.

In vitro at low ionic strength (mu = 0.02 M) and 5 degrees C, myosin subfragment-1 shows significant binding to regulated actin in the presence of ATP, independent of the concentration of free Ca2+. Under the same conditions, single skinned rabbit psoas muscle fibers develop force only in the presence of Ca2+ and are relaxed in its absence. However, the stiffness, measured with very rapid stretches (0.5% of muscle length in 0.1 ms), is high even when the fibers are relaxed. This "rapid stiffness" of the resting muscle is sensitive to ionic strength, becoming small at normal ionic strength (mu = 0.17 M). At low ionic strength, the rapid stiffness is approximately proportional to the overlap between the actin and myosin filaments. At zero overlap (sarcomere length = 3.8 microns), the stiffness is less than 20% of the value measured at full overlap. This remaining 20% is relatively insensitive to ionic strength, like the passive resting tension, and it may in fact be due to the structures responsible for the resting tension. Thus, both in vitro binding and the effect of overlap on rapid stiffness measurements in fibers suggest that cross-bridges are attached to actin in relaxed muscle at low ionic strength.

Actins↗

Myosin heads have a broad orientational distribution during isometric muscle contraction: time-resolved EPR studies using caged ATP.

To study the orientation of spin-labeled myosin heads in the first few seconds after the production of saturating ATP, we have used a laser flash to photolyze caged ATP during EPR data acquisition. Rabbit psoas muscle fibers were labeled with maleimide spin label, modifying 60% of the myosin heads without impairing muscle fiber biochemical and physiological activity (ATPase and force). The muscle bundles were incubated for 30 min with 5 mM caged ATP prior to the UV flash. The flash, from an excimer laser, liberated 2-3 mM ATP, generating maximum force in the presence of Ca2+ and relaxing fully in the absence of Ca2+. Control experiments, using fibers decorated with labeled myosin subfragment, showed that the flash liberates sufficient ATP to saturate myosin active sites in all regions of the muscle bundles. To increase the time resolution, and to minimize the time of the contraction, we followed in time the intensity at a single spectral position (P2), which is associated with the high degree of orientational order in rigor. ATP liberation produced a rapid decrease of P2 with liberation of ATP, indicating a large decrease in orientational order in both relaxation and contraction. This transient was absent when caged AMP was used, ruling out nonspecific effects of the UV flash and subsequent photochemistry. The steady-state level of P2 during contraction was almost as low as that reached in relaxation, although the duration of the steady state was much more brief in contraction. Upon depletion of ATP in contraction, the P2 intensity reverted to the original rigor level, accompanied by development of rigor tension. The steady-state results obtained in the brief contractions induced by caged ATP are quantitatively consistent with those obtained in longer contractions by continuously perfusing fibers with ATP. In isometric contraction, most (88% +/- 4%) of the heads are in a population characterized by a high degree of axial disorder, comparable to that observed for all heads in relaxation. Since the stiffness of these fibers in contraction is 80% of the stiffness in rigor, it is likely that most of the heads in this highly disoriented population are attached to actin in contraction and that most actin-attached heads in contraction are in this disoriented population.

Adenosine Triphosphate↗

[A case of psoas abscess associated in the elderly].

A case of psoas abscess associated with diabetes mellitus in the elderly is reported. A 81-year-old male who had been followed for cerebral thrombosis, diabetes mellitus and basal cell carcinoma was admitted to our hospital because of high fever. Leukocytosis, a positive CRP test and pyuria were seen. Proteus mirabilis and Escherichia coli were detected by urine and blood culture, respectively. He was treated with antibiotic therapy for urinary tract infection and sepsis. After starting treatment, a low grade fever continued. On the twenty first hospital day he developed pyrexia again, and a large abscess was demonstrated in the right psoas muscle by pelvic couputed tomography. The abscess was drained and a specimen from it yielded E. coli on culture. Treatment with antibiotics and drainage resulted in symptomatic improvement. In Japan, 82 cases of psoas abscess have been reported from 1990 to 1994. Four cases of these reports were above eighty years old. The experience with this case indicates the necessity of adequate care in cases of elderly diabetes complicated by psoas abscess.

Aged↗

Saturation transfer electron paramagnetic resonance of spin-labeled muscle fibers. Dependence of myosin head rotational motion on sarcomere length.

We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 microns (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 degrees), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread greater than 90 degrees) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.

Animals↗

Inositol trisphosphate has no direct effect on the contractile apparatus of skinned cardiac muscles.

Alpha-adrenoceptor stimulation produces a positive inotropic effect in heart muscle via mechanisms that are not well understood. The purpose of our study was to test the hypothesis that the increase in inositol 1,4,5 trisphosphate [Ins(1,4,5)P3] concentration that accompanies alpha stimulation contributes to the inotropic effect by increasing the calcium sensitivity of the contractile proteins, an effect which Ins(1,4,5)P3 has been shown to have in skeletal muscle. We determined the calcium sensitivity of the contractile apparatus of small, chemically skinned bundles from papillary muscles of rabbit, rat and dog hearts. These preparations were chosen because they exhibit a range of sensitivity to alpha agonists. In addition, we measured the calcium sensitivity of chemically skinned, single fibers from rabbit psoas muscle. All preparations were skinned with Triton X-100, a non-ionic detergent that disrupts the sarcolemmal, sarcoplasmic reticular, and mitochondrial membranes. In all cardiac preparations, we found that 38 microM Ins(1,4,5)P3 had no effect on either the calcium sensitivity or maximum calcium-activated force. Thus, there was no correlation between inotropic response to alpha stimulation and myocardial response to Ins(1,4,5)P3. On the other hand, the maximum calcium-activated force of skinned skeletal muscle was slightly increased by Ins(1,4,5)P3. Moreover, Ins(1,4,5)P3 significantly increased the sensitivity of these fibers to calcium.

Animals↗

[Translumbar radical nephrectomy of renal cell carcinoma].

We invented a new surgical approach to the kidney through the flank to perform a radical nephrectomy for renal cell carcinoma. With the patient in usual lateral decubitus position keeping the dorsum vertical to the operating table, a skin incision is made over the XII rib from the posterior axillary line to the lateral edge of the rectus muscle. After the tip of XII rib is resected by about 5 cm, the retroperitoneal space is entered. Blunt dissection of the posterior aspect of Gerota's fascia from fasciae of the quadratus lumborum and psoas muscle is easily carried out with a liver retractor or intestinal spatula. The pulsating renal artery can be identified through Gerota's fascia when the renal hilus is exposed. Following ligation and division of the artery, renal vein is exposed. On the left side, adrenal, gonadal and occasionally lumbar veins are also ligated and severed in addition to the renal vein. Then, the kidney in Gerota's fascia is removed en bloc with perinephric adipose tissue and adrenal gland. Of 21 patients with renal cell carcinoma seen during 1 year and 3 months from June 1987, 11 underwent this operation, and other 10 patients transperitoneal radical nephrectomy because of the possible tumor extension into the renal vein, inferior vena cava or adjacent organ, the severe spinal deformity or metastases and the necessity of additional surgical procedures for concomitant benign intraperitoneal diseases. The blood loss was smaller and operating time was shorter significantly in the translumbar group than the evaluable transperitoneal group. None of those in the translumbar group received blood transfusion. As complications, pneumothorax due to pleural injury during operation and postoperative incisional hernia occurred each in one patient, but no other serious one was found. From the above results, this approach seems to be one of choices for the surgical treatment of renal cell carcinoma, as long as the tumor is not likely to extend to adjacent organs, ipsilateral nodes or the inferior vena cava.

Adult↗

Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers.

We have measured the orientation of a region of the myosin head, close to the junction with the rod, during active force generation. Paramagnetic probes were attached specifically to a reactive cysteine (Cys 125) of purified myosin light chain 2 (LC2) and exchanged into myosin heads in glycerinated rabbit psoas muscle. Electron paramagnetic resonance spectroscopy was used to monitor the orientation of the probes. Previous work has shown that the LC2 bound spin probes are significantly ordered in rigor and muscle in the presence of adenosine diphosphate (ADP). In contrast, there is a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, all of the LC2 bound spin probes (98 +/- 1.6%) show an angular distribution similar to that of relaxed muscle. These findings contrast with results obtained from probes attached to Cys 707 on the cross-bridge, located close to the actin binding site, where, during active force generation, a proportion of the spin probes were ordered as in rigor, whereas the remaining probes were disordered as in relaxation. To test the hypothesis that this ordered component is due to modification of Cys 707, we measured the spectra obtained from probes attached to LC2 in fibers modified at Cys 707. The modification of Cys 707 did not produce an ordered component in these spectra. The absence of an ordered component at the LC2 site limits the populations of some states in active fibers. An actin/myosin/ADP state is thought to be the major force-producing state. Our present results show that the populations of states with ordered probes on LC2 are < 2% in active fibers; thus, the major force-producing state is different from the one obtained by addition of ADP to rigor fibers.

Adenosine Triphosphate↗

X-ray diffraction studies on the structural changes of rigor muscles induced by binding of phosphate analogs in the presence of MgADP.

To clarify the structure of the ATP hydrolysis intermediates (ADP.Pi bound state) formed by actomyosin crossbridges, the effects of various phosphate analogs in the presence of MgADP on the structures of the thin and thick filaments in glycerinated rabbit psoas muscle fibers in the rigor state have been investigated by X-ray diffraction with a short exposure time using synchrotron radiation. When MgADP and phosphate analogs such as metallofluorides (BeFx = 3,4 and AlF4) and vanadate (VO4(Vi)) were added to rigor fibers in the presence of the ATP-depletion backup system, the intensities of the actin-based layer lines were markedly weakened. The greatest effect (approximately 50% decrease in intensity) was observed in the presence of BeFx among the analogs examined. The intensity distribution of the 5.9 nm actin-based layer line shifted towards that observed in the Ca(2+)-activated fibers, while the first actin layer line at approximately 1/36.7 nm-1 retained a rigor-like profile with an intensity weakened by approximately 50%. The intensity of the equatorial 10 reflection increased while that of the 11 reflection changed little, resulting in only a small increase (approximately 1.7 fold) in the intensity ratio of the 10 to the 11 reflection. No resting-like pattern appeared upon the addition of MgADP and BeFx. These results indicate that a substantial fraction (approximately 40%) of the myosin heads dissociate from actin but the detached heads remain in the vicinity of the actin filaments when MgADP and BeFx bind. The states produced by binding phosphate analogs to a rigor muscle differ from the resting-like state produced by adding them to a contracting muscle (Takemori et al., J. Biochem. (Tokyo) 117 (1995) 603-608). Our conclusion put forward to explain the data is that one of the two heads of a crossbridge is detached and the other retains a rigor-like attachment.

Adenosine Diphosphate↗

Primary ilio-psoas abscess extending to the thigh in a neonate: US, CT and MR findings.

Psoas abscess in children, and especially in neonates, is an uncommon condition which is difficult to diagnose clinically. The US, CT and MR findings of a psoas abscess in a neonate, which extended to the thigh, are reported. Imaging was helpful in revealing the abnormality, in demonstrating its extension and in determining its nature. The child was treated conservatively and the abscess resolved, leaving atrophy of the psoas muscle.

Humans↗

A spin label that binds to myosin heads in muscle fibers with its principal axis parallel to the fiber axis.

We have used an indane-dione spin label (2-[-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methenyl]in dane-1,3-dione), designated InVSL, to study the orientation of myosin heads in bundles of chemically skinned rabbit psoas muscle fibers, with electron paramagnetic resonance (EPR) spectroscopy. After reversible preblocking with 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB), we were able to attach most of the spin label covalently and rigidly to either Cys 707 (SH1) or Cys 697 (SH2) on myosin heads. EPR spectra of labeled fibers contained substantial contributions from both oriented and disordered populations of spin labels. Similar spectra were obtained from fibers decorated with InVSL-labeled myosin heads (subfragment 1), indicating that virtually all the spin labels in labeled fibers are on the myosin head. We specifically labeled SH2 with InVSL after reversible preblocking of the SH1 sites with 1-fluoro-2,4-dinitrobenzene (FDNB), resulting in a spectrum that indicated only disordered spin labels. Therefore, the oriented and disordered populations correspond to labels on SH1 and SH2, respectively. The spectrum of SH2-bound labels was subtracted to produce a spectrum corresponding to SH1-bound labels, which was used for further analysis. For this corrected spectrum, the angle between the fiber axis and the principal axis of the spin label was fitted well by a Gaussian distribution centered at theta o = 11 +/- 1 degree, with a full width at half-maximum of delta theta = 15 +/- 2 degrees. The unique orientation of InVSL, with its principal axis almost parallel to the fiber axis, makes it complementary to spin labels previously studied in this system. This label can provide unambiguous information about axial rotations of myosin heads, since any axial rotation of the head must be reflected in the same axial rotation of the principal axis of the probe, thus changing the hyperfine splitting. Therefore, InVSL-labeled fibers have ideal properties needed for further exploration myosin head orientation and rotational motion in muscle.

Animals↗

Association between muscle trauma and heterotopic ossification in spinal cord injured patients: reflections on their causal relationship and the diagnostic value of ultrasonography.

Paraplegic patients presenting with a subacute limitation of hip joint mobility were subjected to serial sonographic examinations. In four patients the initial sonographic study disclosed discontinuity with fluid collection in the psoas muscle, which was diagnostic of a traumatic muscle rupture. All four patients subsequently developed sonographic and radiographic evidence of heterotopic ossification. Our findings confirm that ultrasonography is an easy and inexpensive screening method for the early diagnosis of heterotopic ossification. The sonographic results obtained in these four paraplegic patients are indicative of a possible traumatic origin of heterotopic ossification around the hip.

Adult↗

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers.

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.

Animals↗

High-resolution field emission scanning electron microscope imaging of internal cell structures after Epon extraction from sections: a new approach to correlative ultrastructural and immunocytochemical studies.

The availability of high-resolution field emission scanning electron microscopes (FESEM) and the recent development of a less destructive method for extracting Epon from sections motivated us to investigate these techniques for the study of internal cell structures. We chose the nuclear pore complex (NPC) and insect striated muscle as test objects. Chemically fixed or rapidly cryoimmobilized samples were embedded in Epon 812. The Epon was extracted from 200- to 300-nm-thick sections with a modified potassium methoxide-crown ether complex. The samples were viewed with high-resolution FESEM at low voltages. In tangential sections of isolated nuclear envelopes from Xenopus oocytes the cytoplasmic and intranuclear components ("fishtraps") of NPCs appeared identical to what has been described from whole mounts. In cross sections, fishtraps are seen in side view, which is possible only with this technique. In longitudinal and cross sections of insect flight muscle the classical arrangement of myofilaments and cross-bridges is well preserved. This method now makes it possible to image internal cell structures from any desired angle by high-resolution FESEM. Immunolabeling studies on the rabbit psoas muscle demonstrated that antigenicity of alpha-actinin was retained in Epon-extracted sections. Immunogold labeling with antibodies against alpha-actinin conjugated to 3-nm gold beads was intense, highly specific, and restricted to the Z lines. This method can overcome the penetration problem of immunogold labeling, since any cell component can be positioned at the surface of the section. Obviously this approach can become a powerful new tool for many areas of structural cell biology.

Actinin↗

Psoas abscess as a complication of pyogenic sacroiliitis: report of a case.

A psoas abscess is, either primary or secondary, a rare entity for a general surgeon. Images by ultrasonography and computed tomography (CT) can help a general surgeon to make an accurate diagnosis when encountering the patient complaining of unilateral lower abdominal deep pain with fever. A case of pyogenic abscess of the psoas muscle as a result of sacroiliitis in a 22-year-old man is reported herein. The abdominal CT and magnetic resonance imaging scans demonstrated a large multilocular abscess extending along the iliopsoas muscle, and erosion and a widening of the left sacroiliac joint. The abscess was drained with an open surgical approach and the patient responded well to antibiotic therapy. Aggressive surgical and medical treatment is necessary in patients with psoas abscess to prevent complications.

Adult↗

[A case of hepatocellular carcinoma complicated with psoas abscess].

Psoas abscess is relatively rare and often difficult to make early diagnosis. We treated a patient suffering from hepatocellular carcinoma due to hepatitis C virus infection who was admitted to our hospital complaining of right inguinodynia and a high fever. Positive CRP test were seen. Staphylococcus aureus was detected from blood culture and he was treated for sepsis with antibiotic therapy. After starting treatment, his inguinodynia continued and abscesses were demonstrated in the right psoas muscle by pelvic computed tomography (CT). The abscesses were drained and a specimen yielded S. aureus on culture. After drainage, the symptoms improved and the abscesses disappeared on pelvic CT. Pelvic CT can be successfully used to diagnose psoas abscess and to monitor the efficiency of the treatment.

Aged↗

An investigation into the use of MR imaging to determine the functional cross sectional area of lumbar paraspinal muscles.

The purpose of this study was to investigate the use of magnetic resonance (MR) imaging and image processing software to determine the functional cross-sectional area (FCSA) (the area of muscle isolated from fat) of the lumbar paraspinal muscles. The measurement of the morphology of the lumbar paraspinal muscles has become the focus of several recent investigations into the aetiology of low back pain. However, the reliability and validity of determining the FCSA of the lumbar paraspinal muscles using MR imaging are yet to be reported. T2 axial MR scans at the L1-S1 spinal levels of six subjects were obtained using identical MR systems and scanning parameters. Lean paraspinal muscle, vertebral body bone and intermuscular fat were manually segmented using image analysis software to assign a grey scale range to the MR signal intensity emitted by each tissue type. The resultant grey scale range for muscle was used to determine FCSA measurements for each of the paraspinal muscles, psoas, quadratus lumborum, erector spinae and lumbar multifidus on each scan slice. As various biological, instrument and measurement factors can affect MR signal intensity, a sensitivity analysis was conducted to determine the error associated in calculating FCSA for paraspinal muscle using a discrete grey scale range. Cross-sectional area and FCSA measurements were repeated three times and reliability indices for the FCSA measurements were obtained, showing excellent reliability, intra class correlation coefficient (mean=0.97, range 0.90-0.99) and %SEM (mean=2.6%, range 0.7-4.8%). In addition, the error associated with miscalculation of the grey scale range for the MR signal intensity of muscle was calculated and found to be low with an error of 20 grey scale units at the upper end of the muscle's grey scale range resulting in a very small error in the measured muscle FCSA. The method presented in this paper has a variety of practical applications in areas such as evidence-based rehabilitation, biomechanical modelling and the determination of segmental inertial parameters.

Adult↗