Comparison of an isoelectric focusing technique and high-performance liquid chromatography for determination of fetal hemoglobin levels.

The authors previously have reported measurements of fetal hemoglobin in infants in blood samples taken at autopsy using an isoelectric focusing (IEF) procedure. The current study was undertaken to compare this methodology with a high-performance liquid chromatography (HPLC) procedure. The correlation coefficient between the IEF and HPLC procedures was 0.938. The HPLC method is technically easier and has fewer disadvantages than the IEF procedure and is recommended for the determination of fetal hemoglobin levels.

Extramedullary hematopoiesis in the liver in sudden infant death syndrome.

Liver extramedullary hematopoiesis was examined in 54 victims of sudden infant death syndrome and in 21 infants who died of other causes in an attempt to confirm Naeye's findings of increased extramedullary hematopoiesis in cases of sudden infant death syndrome. Our data showed greater extramedullary hematopoiesis in victims of sudden infant death syndrome (F = 23.52), supporting Naeye's hypothesis that victims of sudden infant death syndrome have suffered a subtle, chronic hypoxemic condition before death.

Elevated fetal hemoglobin levels in sudden infant death syndrome.

The cause of sudden infant death syndrome (SIDS) is unknown, although deficits in cardiopulmonary function and central respiratory control have been suggested as possible mechanisms of the disorder. In this study, we tested the hypothesis that SIDS is associated with a delay in the maturation of hematopoiesis. Prolonged elevation in the levels of fetal hemoglobin (hemoglobin F) in infants with SIDS could denote a compromised delivery of oxygen to sensitive tissue sites. Normally, hemoglobin F (alpha 2 gamma 2) is largely replaced by adult hemoglobin, hemoglobin A (alpha 2 beta 2), during the first six months after birth. Using an isoelectric-focusing procedure for measuring stable hemoglobin subunits, we quantitated the levels of hemoglobin F in blood samples from 59 patients with SIDS and 40 controls (32 living and 8 dead) matched for postconceptional age. The level of hemoglobin F in the population with SIDS was significantly higher than that in the controls in the age range tested (39 to 75 weeks); the mean (+/- SEM) proportion of hemoglobin F was 63.2 +/- 3.6 percent in the group with SIDS, as compared with 48.1 +/- 5.0 percent in the controls (P less than 0.025). The difference in hemoglobin F levels was most pronounced 50 weeks after conception: the proportion of hemoglobin F in the 37 patients with SIDS with a postconceptional age of more than 50 weeks was 47.4 +/- 3.6 percent, as compared with 18.8 +/- 3.1 percent in the 19 controls of that age (P less than 0.0005). We conclude that hemoglobin F is a useful postmortem marker for the population with SIDS that we studied and that it may have value as a prospective marker for some infants at risk for SIDS.

Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C.

Binding of Ca2+ to the troponin C (TnC) subunit of troponin is necessary for tension development in skeletal and cardiac muscles. Tension was measured in skinned fibers from rabbit skeletal muscle at various [Ca2+] before and after partial substitution of skeletal TnC with cardiac TnC. Following substitution, the tension-pCa relationship was altered in a manner consistent with the differences in the number of low-affinity Ca2+-binding sites on the two types of TnC and their affinities for Ca2+. The alterations in the tension-pCa relationship were for the most part reversed by reextraction of cardiac TnC and readdition of skeletal TnC into the fiber segments. These findings indicate that the type of TnC present plays an important role in determining the Ca2+ dependence of tension development in striated muscle.

Shortening velocity and myosin heavy chains of developing rabbit muscle fibers.

The regulation of vertebrate muscle contraction with respect to the role of the different subunits of myosin remains somewhat uncertain. One approach to gaining a better understanding of the molecular basis of contraction is to study developing muscle which undergoes changes in myosin isozyme composition and contractile properties during the normal course of maturation. The present study utilizes single fibers from psoas muscles of rabbits at several ages as a model system for fast-twitch muscle development. This approach eliminates the inherent problems of interpreting results from studies on whole muscles which usually contain heterogeneous fiber types with respect to contractile properties and isoenzyme composition. Maximum velocity of shortening and tension-generating ability of individual fibers were measured and the myosin heavy chain composition of the same fibers was examined using an ultrasensitive sodium dodecyl sulfate-polyacrylamide gel system. The results indicate that 1) with regard to contractile properties, there is a transitional period from slow to fast shortening velocities within the first postnatal month; 2) a strong, positive correlation exists between the speed of shortening and tension-generating ability of individual postnatal day 7 fibers, suggesting that as more myosin is incorporated in these developing fibers it is of the fast type; and 3) there is a wide variation in maximum velocity of shortening among postnatal day 7 psoas fibers which is also a time when a mixture of heavy chain isoforms characterizes the myosin composition of single muscle fibers.

The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers.

The activation of contraction in vertebrate skeletal muscle involves the binding of Ca2+ to low-affinity binding sites on the troponin C (TnC) subunit of the regulatory protein troponin. The present study is an investigation of possible cooperative interactions between adjacent functional groups, composed of seven actin monomers, one tropomyosin, and one troponin, along the same thin filament. Single skinned fibers were obtained from rabbit psoas muscles and were then placed in an experimental chamber containing relaxing solution maintained at 15 degrees C. Isometric tension was measured in solutions containing maximally and submaximally activating levels of free Ca2+ (a) in control fiber segments, (b) in the same segments after partial extraction of TnC, and finally (c) after recombination of TnC into the segments. The extraction was done at 11-13 degrees C in 20 mM Tris, 5 mM EDTA, pH 7.85 or 8.3, a procedure derived from that of Cox et al. (1981. Biochem. J. 195:205). Extraction of TnC was quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the control and experimental samples. Partial extraction of TnC resulted in reductions in tension during maximal Ca activation and in a shift of the relative tension-pCa (i.e., -log[Ca2+]) relationship to lower pCa's. The readdition of TnC to the extracted fiber segments resulted in a recovery of tension to near-control levels and in the return of the tension-pCa relation to its original position. On the basis of these findings, we conclude that the sensitivity to Ca2+ of a functional group within the thin filament may vary depending upon the state of activation of immediately adjacent groups.

Shortening velocity in single fibers from adult rabbit soleus muscles is correlated with myosin heavy chain composition.

Extensive variations exist in the heavy and light chain components of myosin in vertebrate striated muscles. In the present study, we have characterized a specific contractile property, velocity of shortening, and protein subunit composition of single fibers from adult rabbit soleus muscles. Maximum velocity of shortening (Vmax) was measured using the slack test method, and the myosin composition of these same fibers was determined using an ultrasensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. While most fibers were found to have velocities between 0.5 and 1.0 muscle length/s, several had velocities distributed between 1.33 and 2.99 muscle length/s. The fibers in the slower group had myosin subunits that were solely of the slow type; however, those in the faster group contained both fast and slow heavy chains and light chains. The velocity of shortening measured in fibers having both myosin types was highly correlated with the myosin heavy chain composition, with velocity increasing as the proportion of fast-type heavy chain increased. Variations in light chain composition, particularly fast and slow myosin light chain 1, appeared to occur independently of the variations in heavy chain composition, suggesting that some myosin molecules consist of mixtures of slow- and fast-type subunits.

Analytical isoelectric focusing using a high-voltage vertical slab polyacrylamide gel system.

A commercially supplied vertical slab electrophoresis system has been modified to permit electrofocusing of thin gels using electrical potentials of 3000 V and higher. Polyacrylamide gels (5.65% T, 2.65% C; 2.4-3.3% (w/v) ampholytes; 0.35 mm thick X 98-105 mm long X 140 mm wide) were run under native and denaturing conditions. Accurate temperature regulation and atmospheric control were obtained by casting the gel between two glass plates, and then completely submerging the gel in the lower tank buffer. As many as 18 samples were loaded into wells at the top of each gel. Protein standards and mouse ascites fluid were focused on gels in the native state using a broad-range blend of commercial ampholytes from pH 3.5 to 10. Narrow-range pH ampholyte blends were also used: pH 2.5 to 6 under denaturing conditions resolving bovine calmodulins; pH 4 to 6 under a native condition for human plasma proteins including immunoglobulin G, fibronectin, and fibrinogen; pH 4 to 6 under denaturing conditions for myosin light chains; pH 6 to 9 under native conditions for human hemoglobins; and pH 9 to 11 under denaturing conditions to separate 30 S ribosomal subunit proteins. High-voltage vertical slab electrofocusing provides a means for rapid resolution of multiple protein samples using stable pH gradients. The method is especially valuable in ranges near pH 2.5 and pH 10.5 in which difficulties have previously been encountered with regard to atmospheric control and temperature regulation using conventional focusing techniques.

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.

Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels.

A sodium dodecyl sulfate-discontinuous polyacrylamide gel electrophoresis system for separation and quantitation of low-molecular-weight (75 to 10K Da) proteins from single muscle fibers is described. Slab gels (0.75 mm thick) were stained using an improved silver-stain technique which does not require photographic fixers in order to achieve low-level background staining. The modified staining procedure uses continuous flow washing to minimize the handling of gels. The procedure has high sensitivity and gave a linear response between approximately 2 and 70 ng of protein per band. In addition, a convenient method for mounting slab gels for photography, scanning, and long-term storage has been developed.

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.