Mg-ATPase Activity Research Articles

Oxidative stress and Na,K-ATPase activity differential regulation in brainstem and forebrain of Wistar Audiogenic rats may lead to increased seizure susceptibility

The Wistar Audiogenic Rat (WAR) is a well-characterized seizure-prone, inbred rodent strain that, when acutely stimulated with high-intensity sounds, develops brainstem-dependent tonic-clonic seizures that can evolve to limbic-like, myoclonic (forebrain) seizures when the acoustic stimuli are presented chronically (audiogenic kindling). In order to investigate possible mechanisms underlying WAR susceptibility to seizures, we evaluated Na,K-ATPase activity, Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and oxidative stress markers in whole forebrain and whole brainstem samples of naïve WAR, as compared to samples from control Wistar rats. We also evaluated the expression levels of α1 and α3 isoforms of Na,K-ATPase in forebrain samples. We observed increased Na,K-ATPase activity in forebrain samples and increased oxidative stress markers (lipid peroxidation, glutathione peroxidase and superoxide dismutase) in brainstem samples of WAR. The Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and expression levels of α1 and α3 isoforms of Na,K-ATPase were unaltered. In view of previous data showing that the membrane potentials from naïve WAR’s neurons are less negative than that from neurons from Wistar rats, we suggest that Na,K-ATPase increased activity might be involved in a compensatory mechanism necessary to maintain WAR’s brains normal activity. Additionally, ongoing oxidative stress in the brainstem could bring Na,K-ATPase activity back to normal levels, which may explain why WAR’s present increased susceptibility to seizures triggered by high-intensity sound stimulation.

Increasing zinc levels in phytase-supplemented diets improves the performance and nutrient utilization of broiler chickens

Three hundred and thirty-six day-old Ross-308 male broiler chicks were used in a 35-day trial to investigate the effect of different concentrations of dietary Zn and phytase on broiler performance and energy utilization. Twelve day-old birds were used for the initial slaughter group to provide baseline body compositional data, while the remaining 324 birds were randomly distributed to six experimental diets. The treatments consisted of a 3 x 2 factorial arrangement with three levels of Zn (low, mid, and high; 30, 40, and 50 mg/kg, respectively) and two levels (0, 500 FTU/kg) of microbial phytase. Each dietary treatment was fed to 6 cages (9 birds/cage). Low Zn diet significantly decreased feed intake and body weight gain at days 1-24. Phytase supplementation improved body weight gain at d 24, irrespective of Zn level. The digestibility of P was improved in birds fed high-Zn diet with phytase supplementation, and the reverse was the case for Fe and Zn digestibility. High dietary Zn increased the Zn and Fe deposition in liver. The activity of AP, Ca-ATPase and Mg-ATPase in the jejunum was high in the phytase supplemented mid-level Zn diet. Phytase supplemented to the mid and high level Zn diets significantly improved most energy utilization parameters. This result indicate that the Zn concentrations used in this study were not inhibitory to phytase activity and broiler performance. Therefore, it can be concluded that dietary zinc level in phytase-supplemented diets could be increased up to 50 mg/kg without any negative effect on phytase-mediated broiler response.Keywords: Bone quality, net energy, nutrient utilization

Increased iron level in phytase-supplemented diets reduces performance and nutrient utilisation in broiler chickens

ABSTRACT1. The effect of different levels of dietary iron on phytase activity and its subsequent effect on broiler performance were investigated in a 3 × 2 factorial arrangement. A total of 360 day-old Ross 308 male broiler chicks were distributed to 6 experimental diets, formulated with three levels of Fe (60, 80 and 100 mg/kg) and two levels of phytase (0 and 500 FTU/kg).2. Phytase supplemented to mid-Fe diets increased feed consumption more than the non-supplemented diet at d 24. From hatch to d 35, Fe × phytase interaction significantly influenced the feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR). The high-Fe diet supplemented with phytase significantly reduced FI and BWG of broilers than those supplemented with low- or mid-Fe diets. The overall FCR was significantly better in birds fed on the mid-Fe diets with phytase supplementation.3. A significant improvement in ileal digestibility of N, P, Mg and Fe was observed in birds feed diets containing 60 mg Fe/kg, with significant interaction between Fe and phytase.4. Phytase improved the bone breaking strength when supplemented to low- or mid-Fe diets, compared to the non-supplemented diets. There was a significant Fe × phytase interaction effect. Tibia Fe content was higher in birds fed on phytase-free diets with high Fe but the reverse was the case when phytase was added and their interaction was significant. High dietary Fe significantly increased the accumulation of Fe in liver.5. Phytase improved Ca-Mg-ATPase, Ca-ATPase and Mg-ATPase activities in jejunum when supplemented to the diet containing 80 mg Fe/kg.6. This study indicates that high (100 mg/kg) dietary Fe inhibited phytase efficacy and subsequently reduced the overall performance and nutrient utilisation of broilers.

The mechano-sensitivity of cardiac ATP-sensitive potassium channels is mediated by intrinsic MgATPase activity

Cardiac ATP-sensitive K+ (KATP) channel activity plays an important cardio-protective role in regulating excitability in response to metabolic stress. Evidence suggests that these channels are also mechano-sensitive and therefore may couple KATP channel activity to increased cardiac workloads. However, the molecular mechanism that couples membrane stretch to channel activity is not currently known. We hypothesized that membrane stretch may alter the intrinsic MgATPase activity of the cardiac KATP channel resulting in increased channel activation. The inside-out patch-clamp technique was used to record single-channel and macroscopic recombinant KATP channel activity in response to membrane stretch elicited by negative pipette pressure. We found that stretch activation requires the presence of the SUR subunit and that inhibition of MgATPase activity with either the non-hydrolysable ATP analog AMP-PNP or the ATPase inhibitor BeFx significantly reduced the stimulatory effect of stretch. We employed a point mutagenic approach to determine that a single residue (K1337) in the hairpin loop proximal to the major MgATPase catalytic site in the SUR2A subunit is responsible for the difference in mechano-sensitivity between SUR2A and SUR1 containing KATP channels. Moreover, using a double cysteine mutant substitution in the hairpin loop region revealed the importance of a key residue-residue interaction in this region that transduces membrane mechanical forces into KATP channel stimulation via increases in channel MgATPase activity. With respect to KATP channel pharmacology, glibenclamide, but not glicalizide or repaglinide, was able to completely inhibit KATP channel mechano-sensitivity. In summary, our results provide a highly plausible molecular mechanism by which mechanical membrane forces are rapidly converted in changes in KATP channel activity that have implications for our understanding of cardiac KATP channels in physiological or pathophysiological settings that involve increased workload.

Evidence for the Essential Role of Myosin Subfragment-2 in Muscle Contraction: Functional Communication between Myosin Head and Subfragment-2

In 1971, Harrington et al. put forward a hypothesis, in which helix-coil transition in the hinge region of myosin subfragment-2 (S-2) contributes to muscle contraction. The helix-coil transition hypothesis has been, however, ignored by muscle investigators over many years. In 1992, we worked with him to examine the effect of polyclonal antibody to myosin subfragment-2 (anti-S-2 antibody), and found that the antibody eliminated Ca2+-activated isometric force generation of skinned vertebrate muscle fibers without affecting MgATPase activity. Further studies using the same antibody indicated functional communication between myosin head and myosin S-2, including regulation of binding strength between myosin head and actin filament during Ca2+-activated contraction in vertebrate muscle fibers. These findings indicate that the swinging lever arm hypothesis, in which muscle contraction results from active rotation of myosin head converter domain, is incomplete because it ignores of the role of myosin S-2. Much more experimental work is necessary to reach full understanding of muscle contraction mechanism at the molecular level.

Acute exposure to progesterone attenuates cardiac contraction by modifying myofilament calcium sensitivity in the female mouse heart.

We investigated myocardial effects of acute application of progesterone. In females, but not males, progesterone attenuates and slows cardiomyocyte contraction with no effect on calcium transients. Progesterone also reduces myofilament calcium sensitivity in female hearts. This may adversely affect heart function, especially when serum progesterone levels are high in pregnancy.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/acute-progesterone-modifies-cardiac-contraction/.

Crystal structure of yeast V1-ATPase in the autoinhibited state.

Vacuolar ATPases (V-ATPases) are essential proton pumps that acidify the lumen of subcellular organelles in all eukaryotic cells and the extracellular space in some tissues. V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, wherein the soluble catalytic sector, V1, is released from the membrane and its MgATPase activity silenced. The crystal structure of yeast V1 presented here shows that activity silencing involves a large conformational change of subunit H, with its C-terminal domain rotating ~150° from a position near the membrane in holo V-ATPase to a position at the bottom of V1 near an open catalytic site. Together with biochemical data, the structure supports a mechanistic model wherein subunit H inhibits ATPase activity by stabilizing an open catalytic site that results in tight binding of inhibitory ADP at another site.

Effects of Elevated Temperature on the Shell Density of the Large Benthic Foraminifera Amphistegina lobifera.

This study investigated the effects of elevated temperature on shell density and Mg-ATPase activity of Amphistegina lobifera. This species is abundant in shallow reef habitats, and can be vulnerable to daily physicochemical fluctuations. To assess potential responses and acclimation mechanisms of A.lobifera to changing temperature conditions, we performed a blocked-design experiment exposing specimens collected from different reef sites (inshore and offshore) to three temperature treatments (Control: 24°C, +2°C: 26°C and +5°C: 29°C) for 30days. The final size and shell density of inshore reef foraminifera were unaffected by elevated temperature, and the enzyme activity in these individuals showed that they were able to acclimate to new temperature conditions. In contrast, offshore A.lobifera were more sensitive to changes in temperature, and heat stress caused growth impairment and inhibited Mg-ATPase activity. However, newly added chambers were not affected. These results suggested that Mg-ATPase plays an important role in regulating intracellular Mg2+ ions, but has little influence in the onset of calcification in A.lobifera. Moreover, it suggests that even though A.lobifera can regulate intracellular functions, local habitat seems to play a crucial role in determining how foraminifera respond to environmental changes.

Non-Straub type actin from molluscan catch muscle

We have developed a method of obtaining natural actin from smooth muscles of the bivalves on the example of the Сrenomytilus grayanus catch muscle. The muscles were previously rigorized to prevent a loss of thin filaments during homogenization and washings. Thin filaments were isolated with a low ionic strength solution in the presence of ATP and sodium pyrophosphate. Surface proteins of thin filaments-tropomyosin, troponin, calponin and some minor actin-binding proteins-were dissociated from actin filaments by increasing the ionic strength to 0.6M KCL. Natural fibrillar actin obtained in that way depolymerizes easily in low ionic strength solutions commonly used for the extraction of Straub-type actin from acetone powder. Purification of natural actin was carried out by the polymerization-depolymerization cycle. The content of inactivated actin remaining in the supernatant is much less than at a similar purification of Straub-type actin. A comparative investigation was performed between the natural mussel actin and the Straub-type rabbit skeletal actin in terms of the key properties of actin: polymerization, activation of Mg-ATPase activity of myosin, and the electron-microscopic structure of actin polymers.

Evidence against the Swinging Lever Arm Mechanism in Muscle Contraction Based on the Effect of Antibodies to Myosin Head

During muscle contraction, myosin heads extending from myosin filaments first attach to actin filaments, perform power stroke producing force and motion in muscle, and then detach from actin filaments. A myosin head (or myosin subfragment-1, S-1) consists of catalytic and lever arm domains (CAD and LD), which are connected via converter domain (COD). It is widely believed that the myosin head power stroke is caused by swinging lever arm mechanism, which assumes active rotation of the LD around the COD, caused by structural changes in and around the COD. The lever arm mechanism is, however, constructed from nucleotide-dependent structural changes of crystals of truncated myosin head, consisting only of the CAD and the COD, and therefore overlooks possible role of the LD and myosin subfragment-2 (S-2), connecting myosin heads to myosin filament backbone. In this article, we present evidence against the lever arm hypothesis based on the following results: (1) antibody to reactive lysine residue (Lys83) located in the COD (anti-RLR antibody) has no effect on Ca2+-activated muscle fiber contraction; (2) using the gas environmental chamber attached to electron microscope, we record ATP-induced power stroke of myosin heads, position-marked with anti-RLR antibody; (3) antibodies to myosin head LD (anti-LD antibody) and to myosin S-2 (anti-S-2 antibody) inhibit Ca2+-activated contraction without changing MgATPase activity. The absence of inhibitory effect of anti-RLR antibody, attaching to RLR in the COD, makes the lever arm mechanism unlikely, since attachment of bulky antibody (IgG) to RLR in the COD is expected to inhibit structural changes in and around the COD. Meanwhile, inhibitory effect of anti-LD and anti-S-2 antibodies indicate essential role of the LD and the S-2 in muscle contraction.

A Failure to Communicate: MYOSIN RESIDUES INVOLVED IN HYPERTROPHIC CARDIOMYOPATHY AFFECT INTER-DOMAIN INTERACTION

Our molecular modeling studies suggest a charge-dependent interaction between residues Glu-497 in the relay domain and Arg-712 in the converter domain of human β-cardiac myosin. To test the significance of this putative interaction, we generated transgenic Drosophila expressing indirect flight muscle myosin with charge reversal mutations in the relay (E496R) or converter (R713E). Each mutation yielded dramatic reductions in myosin Ca-ATPase activity (~80%) as well as in basal (~67%) and actin-activated (~84%) Mg-ATPase activity. E496R myosin-induced in vitro actin-sliding velocity was reduced by 71% and R713E myosin permitted no actin motility. Indirect flight muscles of late pupae from each mutant displayed disrupted myofibril assembly, with adults having severely abnormal myofibrils and no flight ability. To understand the molecular basis of these defects, we constructed a putative compensatory mutant that expresses myosin with both E496R and R713E. Intriguingly, ATPase values were restored to ~73% of wild-type and actin-sliding velocity increased to 40%. The double mutation suppresses myofibril assembly defects in pupal indirect flight muscles and dramatically reduces myofibril disruption in young adults. Although sarcomere organization is not sustained in older flies and flight ability is not restored in homozygotes, young heterozygotes fly well. Our results indicate that this charge-dependent interaction between the myosin relay and converter domains is essential to the mechanochemical cycle and sarcomere assembly. Furthermore, the same inter-domain interaction is disrupted when modeling human β-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease.

Molecular determinants of ATP-sensitive potassium channel MgATPase activity: diabetes risk variants and diazoxide sensitivity.

ATP-sensitive K(+) (KATP) channels play an important role in insulin secretion. KATP channels possess intrinsic MgATPase activity that is important in regulating channel activity in response to metabolic changes, although the precise structural determinants are not clearly understood. Furthermore, the sulfonylurea receptor 1 (SUR1) S1369A diabetes risk variant increases MgATPase activity, but the molecular mechanisms remain to be determined. Therefore, we hypothesized that residue-residue interactions between 1369 and 1372, predicted from in silico modelling, influence MgATPase activity, as well as sensitivity to the clinically used drug diazoxide that is known to increase MgATPase activity. We employed a point mutagenic approach with patch-clamp and direct biochemical assays to determine interaction between residues 1369 and 1372. Mutations in residues 1369 and 1372 predicted to decrease the residue interaction elicited a significant increase in MgATPase activity, whereas mutations predicted to possess similar residue interactions to wild-type (WT) channels elicited no alterations in MgATPase activity. In contrast, mutations that were predicted to increase residue interactions resulted in significant decreases in MgATPase activity. We also determined that a single S1369K substitution in SUR1 caused MgATPase activity and diazoxide pharmacological profiles to resemble those of channels containing the SUR2A subunit isoform. Our results provide evidence, at the single residue level, for a molecular mechanism that may underlie the association of the S1369A variant with type2 diabetes. We also show a single amino acid difference can account for the markedly different diazoxide sensitivities between channels containing either the SUR1 or SUR2A subunit isoforms.

Role of Negative Charge on C2 Insert of Nonmuscle Myosin II‐C: Intermittent Structural Oscillation and Aggregation in Neuritogenesis

Previous studies reported that inclusion of 41 aa (33 aa in human) insert in loop 2 of mouse nonmuscle myosin (NM) II‐C heavy chain makes the molecule to become independent of its regulatory light chain (RLC) phosphorylation in MgATPase activity and in vitro motility, and to localize in the neurites as puncta form. However, the molecular mechanism behind these unique properties among myosin II family is not known. Here, we show that deletion of the N‐terminal 12 aa, but not the C‐terminal 8 aa or the middle 21 aa of C2 insert, diminishes the fluorescence oscillation property of GFP tagged‐NM II‐C2 molecule in the neurites of neuro‐2a cells as determined by fluorescence lifetime imaging microscopy analysis. Also, we demonstrate that inclusion of 41 aa insert increases the population of extended monomers whereas deletion of N‐terminal 12 aa, middle 21 aa or C‐terminal 8 aa of 41 aa insert increases the folded autoinhibited NM II‐C2 monomers as revealed by fluorescence correlation spectroscopy (FCS) analysis in neuronal cells. Furthermore, FCS analysis reveals that NM II‐Δ12C2‐GFP significantly diffuses very slowly compared with NM II‐Δ8C2‐ or NM II‐Δ21C2‐GFP in the neurites, suggesting that radius (rH) of NM II‐Δ12 C2‐GFP is higher than that of wildtype or mutants Δ21 and Δ8C2‐GFP. These studies suggest that N‐terminal 12 aa (contributor of net charge of C2) plays a significant role in determining the oscillation and aggregation properties of the molecule.Research Support: Department of Science and Technology, India

Ocean acidification induces biochemical and morphological changes in the calcification process of large benthic foraminifera.

Large benthic foraminifera are significant contributors to sediment formation on coral reefs, yet they are vulnerable to ocean acidification. Here, we assessed the biochemical and morphological impacts of acidification on the calcification of Amphistegina lessonii and Marginopora vertebralis exposed to different pH conditions. We measured growth rates (surface area and buoyant weight) and Ca-ATPase and Mg-ATPase activities and calculated shell density using micro-computer tomography images. In A. lessonii, we detected a significant decrease in buoyant weight, a reduction in the density of inner skeletal chambers, and an increase of Ca-ATPase and Mg-ATPase activities at pH 7.6 when compared with ambient conditions of pH 8.1. By contrast, M. vertebralis showed an inhibition in Mg-ATPase activity under lowered pH, with growth rate and skeletal density remaining constant. While M. vertebralis is considered to be more sensitive than A. lessonii owing to its high-Mg-calcite skeleton, it appears to be less affected by changes in pH, based on the parameters assessed in this study. We suggest difference in biochemical pathways of calcification as the main factor influencing response to changes in pH levels, and that A. lessonii and M. vertebralis have the ability to regulate biochemical functions to cope with short-term increases in acidity.

Molecular mechanism of Mg-ATPase activity.

Mg-ATPase is very important in living organisms. To better understand the molecular mechanism of Mg-ATPase activity, we applied the method of kinetic analysis of multi-sited enzyme systems; this is a suitable approach used for kinetic investigation of multi-sited enzyme systems. The study of Mg-ATPase has demonstrated: (1) It is a multi-sited enzyme system whose functional unit is minimum a dimmer; (2) Its substrate is MgATP, while free ATP and Mg(2+) appear to be the enzyme modifiers with a dual effect; (3) The enzyme system for MgATP has at least three sites: i.e., the essential activator, full inhibitor, and partial effect modifiers sites; (4) Mg-ATPase carries out Mg(2+) transport through the 1Mg(2+):1ATP stochiometry. Based on the results of these analyses, the kinetic scheme for Mg-ATPase has been developed.

The R146N and R249Q Myosin Mutations Disrupt Motor Function and Myofibrillar Structure and cause Cardiomyopathy in Drosophila

Numerous myosin-based myopathies have been identified without a detailed understanding of their molecular basis. To pursue mechanistic investigations, we generated transgenic Drosophila expressing the R146N or R249Q myosin mutations that cause hypertrophic cardiomyopathy in humans. Our studies show severely compromised flight ability in homozygotes, with an age-dependent worsening of muscle function and myofibrillar disarray in the indirect flight muscle ultrastructure. The R146N mutation caused an increase in basal Ca-ATPase and Mg-ATPase activities, with a significant decrease in actin motility. Semi-automated optical heartbeat analysis performed using high-speed movies of semi-intact mutant heterozygous hearts indicated restrictive cardiac physiology and diastolic dysfunction. Based upon molecular modeling, we predict 1) the R146N mutation alters ionic interaction of the N-terminal motor domain with E774 of the lever arm, resulting in failure of the lever arm to cock prior to the power-stroke; and 2) the R249Q mutation disrupts electrostatic interaction with D262 of the central β-sheet domain, interrupting communication between the ATP-binding and actin-binding sites. Thus, the models imply that these conserved charge interactions are critical for myosin function. To test these predictions, lines expressing R146E, E774R, R249D and D262R mutations, as well as putative suppressor lines R146E+E774R and R249D+D262R, will be studied using an integrative biochemical, biophysical and physiological approach. Transgenic Drosophila expressing either R146E or R249D myosin revealed more severe phenotypes relative to cardiomyopathy mutations. Both homozygous mutants displayed flightless phenotypes and highly compromised myofibrillar ultrastructure, with the R146E mutants showing increased basal Ca-ATPase and Mg-ATPase activities, and no actin motility. Heterozygotes present with severe restricted cardiac physiology and diastolic dysfunction. Overall our studies indicate that the Drosophila is a valuable tool to test significant myosin interactions during health and disease. Supported by NIH R01GM32443 to SIB.

The Functional Consequences of Hypertrophic Cardiomyopathy Troponin C Mutations in the Regulation of Slow Skeletal Muscle Contraction: The Protective Role of Slow Skeletal Troponin I

Mutations in cardiac troponin C (cTnC) have been linked to hypertrophic cardiomyopathy (HCM) in humans. The cTnC subunit of the troponin complex is expressed in both slow skeletal and cardiac muscle. The cTnC HCM mutations A8V, C84Y and D145E increase the Ca2+-sensitivity of contraction in cardiac muscle; however, their functional consequences in slow skeletal muscle regulation are unknown. Here we investigated the Ca2+-sensitivity of TnC-extracted rabbit soleus skinned fibers reconstituted with either WT or HCM mutant cTnC. Surprisingly, the slow skeletal skinned fibers extinguished the Ca2+-sensitization typical of cardiac fibers for A8V (pCa50 = 6.01) and D145E (pCa50 = 6.05), while for C84Y (pCa50 = 6.33) the Ca2+-sensitization was maintained compared to WT (pCa50 = 6.00). To determine whether slow skeletal TnI (ssTnI) alone was responsible for the protective effect of incorporating the A8V and D145E mutants into the slow skeletal myofilaments, cardiac skinned fibers were reconstituted with a hybrid cardiac troponin complex containing cTnT, ssTnI and cTnC-WT or -HCM mutants. The presence of ssTnI in cardiac fibers partially protected against Ca2+-sensitization by the cTnC mutants in cardiac skinned fibers, with the exception of C84Y. MgATPase activity of slow or cardiac myofibrils replaced with exogenous HCM cTnC mutants were also affected. The absence of increased Ca2+-sensitivity of contraction arising from the A8V and D145E in the soleus muscle suggests that the aberrant effects of cardiomyopathic cTnCs may be tissue-specific, and ssTnI is an important partner that partially protects the slow muscle from a deleterious effect of the HCM cTnC mutants. Supported by NIH HL103840 (JRP).

Definite differences between in vitro actin-myosin sliding and muscle contraction as revealed using antibodies to myosin head.

Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.

Regulation of Nonmuscle Myosin II by Tropomyosin Isoforms

Tropomyosin (Tm) regulates actin myosin interactions in eukaryotic cells ranging from yeast to mammalian muscle and nonmuscle cells. Tropomyosin is a α-helical coiled-coil actin binding protein that associates end-to-end to form continuous strands along both sides of the actin filament. Tropomyosins can inhibit or activate actomyosin MgATPase activity and motility depending on the myosin and Tm isoforms. In this study, we have attempted to determine whether activation or inhibition is specified by the Tm or the myosin isoform. We carried out in vitro motility assays with four myosin isoforms (skeletal muscle myosin, and nonmuscle IIA, IIB and IIC HMMs) in the presence of five Tm isoforms (skeletal muscle αTm, and nonmuscle isoforms, Tm2, Tm5a, Tm5NM1 and Tm4) and skeletal muscle actin. With skeletal muscle myosin, actin-Tm filament velocities were inhibited by αTm (∼60%) but activated by Tm5a and Tm5NM1 (30-60%) relative to actin alone, whereas Tm2 and Tm4 had little or no effect. In the case of nonmuscle IIA and IIC HMMs, all nonmuscle Tms activated filament velocities, whereas αTm had no effect, relative to actin alone. None of the Tm isoforms affected filament velocities with nonmuscle IIB HMM. Therefore, the primary determinant of the effect of Tm on actin filament velocities on myosin is the myosin isoform. The actin-activated MgATPase activities of IIA, IIB and IIC HMMs were measured to determine the mechanism of activation by a nonmuscle Tm, Tm5NM1. Tm5NM1 increased the Vmax of both IIA (26%), and IIC (19%), with a much smaller effect on IIB (12%) compared to actin alone thus supporting the motility data. Tm5NM1 also decreased the KATPase of IIA and IIB. Therefore, Tm5NM1 activates the MgATPase activity of IIA and IIB by increasing the Vmax and decreasing KATPase. Supported by NIH.

Effect of Some New Aza – Uracil Derivatives in Treatment of Schistosoma mansoni Infection

Praziquantel is now the drug of choice for treatment of schistosomiasis, its use in the control ofschistosomiasis at a population level faces some problems, and thus a search for new drugs withantischistosomal activity is urgently needed. In the present study we worked on newlysynthesized 1, 2, 4-triazine derivative compounds. The study was carried out on 190 femalemice for the assessment of the antischistosomal activity of 1,2,4-triazine compounds, withspecial reference to histopathological examination and some liver tissues biochemicalparameters. The results showed four compounds 3, 6, 7 and 10 caused suppressive effect on thedevelopment of granuloma reaction as compared with Schistosoma infected only. Compound 10had the highest effect in reducing worm burden lesser sever enzymatic dysfunction. Somecompounds caused a marked improvement in almost enzymatic activity particularly 5`-nucleotidase, G6P, Na-K ATPase and Mg-ATPase activities. ALT and AST activities decreasedthan the normal. Also, some compounds showed improvement in catalase and GST enzymeactivities indicating their antioxidant effect .Some compounds as 9 and 10 showed antischistosomalactivity; also compounds 3, 6 and 10 had anti-inflammatory activity.