Quantitative Assay for CASF (Ca2+-activated sarcoplasmic factor) Activity,and Effect of CASF Treatment on ATPase Activities of Rabbit Myofibrils

The ability of CASF (Ca²⁺-activated sarcoplasmic factor), a proteolytic enzyme that has recently been isolated from muscle and that removes Z-disks from myofibrils, to remove soluble material from myofibrils and to alter the Mg²⁺-modified ATPase activity of myofibrils was studied. A new assay involving determination of soluble material released from myofibrils was developed to measure CASF activity quantitatively. Optimum pH and optimum Ca²⁺ concentration for CASF activity as determined by this new assay were 7.0 and 1 mm, respectively. Proteolytic activity of CASF on myofibrils was prevented completely by excess EDTA. CASF treatment of myofibrils at CASF to myofibril ratios of 1: 20 by weight for 30 min caused a 20~25% increase in Mg²⁺-modified ATPase activity. CASF treatment for 360 min under these same conditions caused a decrease in Mg²⁺-modified ATPase activity at the highest ionic strengths used in this study (46.7 and 66.7 mm KCI). The increase in Mg²⁺-modified ATPase activity may originate from CASF degradation of troponin, whereas the decrease in Mg²⁺- modified ATPase activity may be due to CASF destruction or release of α-actinin from myofibrils. Digestion of myofibrils by CASF causes in the myofibrils (degradation of Z-lines, increase of ATPase activity) that are very similar to the changes caused by postmortem storage.     

Correlating Function and Imaging Measures of the Medial Longitudinal Fasciculus

Objective

To test the validity of diffusion tensor imaging (DTI) measures of tissue injury by examining such measures in a white matter structure with well-defined function, the medial longitudinal fasciculus (MLF). Injury to the MLF underlies internuclear ophthalmoparesis (INO).

Methods

40 MS patients with chronic INO and 15 healthy controls were examined under an IRB-approved protocol. Tissue integrity of the MLF was characterized by DTI parameters: longitudinal diffusivity (LD), transverse diffusivity (TD), mean diffusivity (MD) and fractional anisotropy (FA). Severity of INO was quantified by infrared oculography to measure versional disconjugacy index (VDI).

Results

LD was significantly lower in patients than in controls in the medulla-pons region of the MLF (p < 0.03). FA was also lower in patients in the same region (p < 0.0004). LD of the medulla-pons region correlated with VDI (R = -0.28, p < 0.05) as did FA in the midbrain section (R = 0.31, p < 0.02).

Conclusions

This study demonstrates that DTI measures of brain tissue injury can detect injury to a functionally relevant white matter pathway, and that such measures correlate with clinically accepted evaluation indices for INO. The results validate DTI as a useful imaging measure of tissue integrity.     

The conversion of vitamin K epoxide to vitamin K quinone and vitamin K quinone to vitamin K hydroquinone uses the same active site cysteines

Vitamin K epoxide (or oxido) reductase (VKOR) is the target of warfarin and provides vitamin K hydroquinone for the carboxylation of select glutamic acid residues of the vitamin K-dependent proteins which are important for coagulation, signaling, and bone metabolism. It has been known for at least 20 years that cysteines are required for VKOR function. To investigate their importance, we mutated each of the seven cysteines in VKOR. In addition, we made VKOR with both C43 and C51 mutated to alanine (C43A/C51A), as well as a VKOR with residues C43-C51 deleted. Each mutated enzyme was purified and characterized. We report here that C132 and C135 of the CXXC motif are essential for both the conversion of vitamin K epoxide to vitamin K and the conversion of vitamin K to vitamin K hydroquinone. Surprisingly, conserved cysteines, 43 and 51, appear not to be important for either reaction. For the in vitro reaction driven by dithiothreitol, the 43-51 deletion mutation retained 85% and C43A/C51A 112% of the wild-type activity. The facile purification of the nine different mutations reported here illustrates the ease and reproducibility of VKOR purification by the method reported in our recent publication [Chu, P.-H., Huang, T.-Y., Williams, J., and Stafford, D. W. (2006) Proc. Natl. Acad. Sci. U S A. 103, 19308-19313].     

Two-dimensional crystallization of human vitamin K-dependent gamma-glutamyl carboxylase

Planar-tubular two-dimensional (2D) crystals of human vitamin K-dependent gamma-glutamyl carboxylase grow in the presence of dimyristoyl phosphatidylcholine (DMPC). Surprisingly, these crystals form below the phase transition temperature of DMPC and at the unusually low molar lipid-to-protein (LPR) ratio of 1, while 2D crystals are conventionally grown above the phase transition temperature of the reconstituting lipid and significantly higher LPRs. The crystals are up to 0.75 microm in the shorter dimension of the planar tubes and at least 1 microm in length. Due to the planar-tubular nature of the crystals, two lattices are present. These are rotated by nearly 90 degrees in respect to each other. The ordered arrays exhibit p12(1) plane group symmetry with unit cell dimensions of a=83.7 A, b=76.6 A, gamma=91 degrees. Projection maps calculated from images of negatively stained and electron cryo-microscopy samples reveal the human vitamin K-dependent gamma-glutamyl carboxylase to be a monomer.     

A computational study on the influence of catheter-delivered intravascular probes on blood flow in a coronary artery model

Catheter-delivered intravascular probes are widely used in clinical practice to measure coronary arterial velocity and pressure, but the artefactual effect of the probe on the variables being measured is not well characterised. A coronary artery was simulated with a 180 degrees curved tube 3mm in diameter and the effect of catheters of different diameters was modelled numerically under pulsatile flow conditions. The presence of a catheter increased pressure by 1.3-4.3 mmHg depending on its diameter, and reduced velocity-pressure phase-lag. For an ultrasound sample volume 5mm downstream from the probe tip, the underestimation in velocity measurement attributed to catheter blockage is approximately 15-21% for an average inlet velocity of 0.1m/s. The velocity measurement error is lower at higher mean flow velocity. Accuracy of clinical velocity measurements could be improved by moving the sample volume farther downstream from the probe tip, because the centrifugal pressure gradient intrinsic to the curvature promotes re-development of flow.     

An effect of trypsin on the actin-myosin interaction

Three different lines of evidence were obtained to show that trypsin modifies the actin-myosin interaction: (I) At trypsin to actomyosin or myosin ratios between 1 to 300 and 1 to 500, 30 min of trypsin treatment causes an 8-fold increase in the Ca²⁺-modified ITPase activity of actomyosin but has no effect on the Ca²⁺-modified ITPase activity of myosin alone. At these same trypsin to actomyosin ratios, the Mg²⁺ + Ca²⁺-modified ATPase activity increases by 10–30% during the first 1–2 min of trypsin digestion, and then decreases rapidly to less than 20% of its original activity after 60 min of digestion. Trypsin has no effect on the Mg²⁺ + Ca²⁺-modified ATPase activity of pure myosin. (2) The rate of turbidity response of reconstituted actomyosin suspensions is first increased and then decreased by trypsin treatment. At trypsin to actomyosin ratios of 1 to 3000, rate of turbidity response is maximal after 5 min of trypsin digestion and then decreases; after 60 min, the turbidity response is much slower than the response of the control actomyosin. (3) Supercontracted sarcomeres, shortened to less than 50% of their initial length, are lengthened to 70% of their initial length by 4 min of trypsin treatment. Myosin B from such lengthened sarcomeres has less than 35% of its myosin converted to light meromyosin and heavy meromyosin.

These results show that trypsin modifies the actin-myosin interaction in at least two ways: (1) a very rapid initial modification that increases the Mg²⁺ + Ca²⁺-modified ATPase activity and the rate of turbidity increase, and (2) a slower modification that decreases the Mg²⁺ + Ca²⁺-modified ATPase activity and rate of turbidity response, and that lengthens contracted sarcomeres. Tryptic modification is not due to cleavage of myosin to light and heavy meromyosin. Since tryptic modification occurs more rapidly than conversion of myosin to light and heavy meromyosin, all heavy meromyosin preparations will be modified.     

Effects of copper on branchial ionoregulation in the rainbow trout,Salmo gairdneri Richardson

Summary Hard- and softwater acclimated adult rainbow trout were statically exposed to copper (12.5, 25, 50, 100, and 200 ppb) for two, 12 h periods at neutral and pH 5.0. Unidirectional Na⁺, and Cl⁻, and net Na⁺, Cl⁻, K⁺, and ammonia fluxes were monitored as a measure of branchial ionoregulatory disturbance. Copper concentrations as low as 12.5 ppb led to measurable ion losses. Net Na⁺, Cl⁻, and K⁺ losses were concentration dependent and unaffected by prior acclimation to either hard- or softwater at both neutral pH and pH 5. From 12.5 to 50 ppb net NaCl losses arose primarily as a result of the inhibition ofJ _in, and at higher concentrations,J _out was also stimulated. In softwater,J _in was more resistant to inhibition than in hardwater. However, in hardwater,J _out recovered to normal levels during the second 12 h period, but no such recovery was found in softwater. Plasma NaCl was inversely correlated with [copper], while plasma glucose and ammonia increased with [copper]. At pH 5.0 and [copper] from 12.5 to 50 ppb, H⁺ contributed significantly to the total ion loss, while at 100 and 200 ppb, ion losses were no greater at pH 5.0 than at neutral pH. In no case were the effects of copper and H⁺ strictly additive.     

A Genetic and Biochemical Analysis of the Temperature Sensitive, Normal-Winged Alleles of the Rudimentary Locus of DROSOPHILA MELANOGASTER

The genetic and biochemical characteristics of a particular class of mutants at the rudimentary locus are described. The mutants are pyrimidine auxotrophs, like classical rudimentary alleles, but they are unique in that they do not alter the size or shape of the wing (Falk and Nash 1974b). Aspartate transcarbamylase and dihydroorotase activities have been measured in seven different normal-winged mutants, and the results indicate that these strains are enzymologically "leaky" mutants. Previous studies have shown that three genetic functions (corresponding to the first three enzymes of pyrimidine synthesis) are associated with the rudimentary locus. Four of the seven mutants appear to affect all three of these functions. Each of the four is temperature sensitive, and a biochemical analysis of the temperature sensitivity of one of these mutants, (r)pyr1-3, suggests that a process affecting the synthesis or assembly of these enzymes is altered at high temperatures.     

Functional diversity within the rudimentary locus of Drosophila melanogaster

Summary The rudimentary locus of Drosophila melanogaster is shown to be at least bifunctional. Mutants in different regions of the locus have either no CPSase or no ATCase activity; some mutants lack both activities. The results are discussed in correlation with the complementation and genetic map of the locus.Supported by National Research Council of Canada, grant A-1764 and National Cancer Institute of Canada, grant 6051 to D. T. Suzuki.Researcher, Centre National de la Recherche Scientifique, France and recipient of European Molecular Biology Organization Fellowship.