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S Chacko

Publications and source records attributed to S Chacko.

At least 73 records · Page 4Linked to original sources
PubMed1994

Expression of stress proteins (HSP-70 and HSP-90) in the rabbit urinary bladder subjected to partial outlet obstruction.

Partial obstruction of the rabbit bladder outlet induces a rapid hypertrophy characterized by increased bladder mass, increased smooth muscle content, and increased collagen deposition. In addition, partial outlet obstruction induces decreased contractile responses to both field stimulation and postsynaptic receptor stimulation. Although the morphological and contractile responses to partial outlet obstruction have been well characterized, there is little information on the cellular and molecular mechanisms of these changes. In a previous study, we demonstrated that one of the earliest genes to be expressed following partial outlet obstruction in rabbits was the gene expressing stress protein-70 (HSP-70). In order to further define the genetic and molecular basis of these responses, the expression of stress gene products HSP-70 and HSP-90 in rabbit urinary bladder subjected to partial outlet obstruction has been quantitatively evaluated by Western blot coupled with laser densitometry using anti-HSP-70 and -90 monoclonal antibodies. The data show that stress gene products HSP-70 and HSP-90 are constitutively expressed in control rabbit bladder tissue and transiently increased following partial outlet obstruction. Increased content of HSP-70 was detected at 6 hr after obstruction and reached a maximum (2.7-fold over the control level) at 24 hr. Increased HSP-90 was also detected at 6 hr but reached a maximum (4.5-fold over the control level) at 12 hr. By 7 day post-obstruction, the content of these two proteins returned to the control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Overexpression, purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system.

Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5'-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3'-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 micrograms per 10(6) cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36-48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of Ca(2+)-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant.

Actins↗
PubMed1994

Regulation of actomyosin and contraction in smooth muscle.

Unlike striated muscle cells, smooth muscle cells do not have an organized sarcomeric structure. However, all smooth muscle cells contain the contractile proteins, myosin, actin, and tropomyosin. Polymorphism of the myosin heavy chain exists in smooth muscle cells. Two myosin heavy chain (MHC) isoforms, SM1 (204 kDa) and SM2 (200 kDa), are present in smooth muscle cells; however, their ratios vary in smooth muscles from different sources. The hypertrophy of the urinary bladder induced by partial outlet obstruction in rabbits is associated with an alteration of the SM1-to-SM2 ratio from 1:3 to 1:1. Both heavy chains react with polyclonal antibody against smooth muscle myosin; however, antibody prepared against a peptide from the C-terminal region of the SM2 heavy chain cross-reacts only with the SM2 heavy chain. Removal of the obstruction reverses the bladder to normal mass with a concomitant change in the SM1-to-SM2 ratio back to 1:3. The expression of the SM1 mRNA is increased in response to obstruction-induced hypertrophy, and it also returns to normal upon removal of the obstruction. Urinary bladder smooth muscle contains predominantly gamma-actin. Obstruction-induced hypertrophy of the bladder smooth muscle is associated with an increase in the gamma-actin at both protein and mRNA levels. The beta-non-muscle actin is decreased and the alpha-smooth muscle actin is unchanged in response to obstruction-induced bladder hypertrophy. Contraction of all smooth muscles involves similar mechanisms. This review describes our current understanding of the mechanisms regulating contraction of the smooth muscle of the urinary bladder.

Actomyosin↗
PubMed1993

Expression of constitutive heat shock protein-70 in normal (non-stressed) rabbit urinary bladder tissue.

The expression of constitutive HSP-70 in the urinary bladder was determined by SDS-PAGE and western blotting using a mouse monoclonal antibody against HSP-70. The western blot analysis showed that the mouse anti-HSP-70 cross-reacted with a 70 kDa protein present in the extracts of the urinary bladder muscle and mucosa. Densitometric scanning of the western blots allowed us to specifically quantitate the relative amounts of the HSP-70. The quantitation of the HSP-70 by combining immunoblotting and densitometry using a laser scanner is reproducible and this technique requires only a small amount of tissue. The amounts of HSP-70 can be estimated from a standard curve of nanogram(ng) of HSP-70 vs absorption from the immunoblots. The amounts of HSP-70 in the muscular and mucosal layers in the body of the urinary bladder are more than those in the base of the bladder. The presence of HSP-70 in the muscle and mucosal epithelium of the bladder was demonstrated by immunohistochemical analysis of freshly removed tissue from the base and the body of bladder from normal animals.

Animals↗
PubMed1993

The epidemiology of AIDS in the Vellore region, southern India.

OBJECTIVE: To describe the epidemiology of patients with AIDS in Vellore region, Southern India. DESIGN AND METHODS: Sixty-one patients with AIDS were diagnosed and treated between July 1987 and June 1992. Information on their demographic characteristics and probable modes of acquiring HIV infection was collected at interviews with them and their spouses. RESULTS: There was a progressive increase in the number of patients seen over the 5 years. The mean ages of the 51 men and the 10 women were 33 and 29 years, respectively. Of the 44 patients from our district (population, 5 million), 28 were from Vellore town and 10 from rural areas. Forty-seven (92%) men had frequently used prostitutes. Of the women, four were prostitutes, one had had multiple sex partners and five had not had extramarital sexual contact. One man and one woman had no other risk factor except blood transfusion. Thirty-one (51%) patients had died by August 1992. CONCLUSION: The AIDS epidemic in this region is in its early ascending phase, with a doubling time of approximately 1 year. Most men with AIDS were infected by heterosexual contact with prostitutes, while some women were prostitutes themselves. Together with the male-to-female ratio of 5:1, these results suggest that the male population at risk has sex with a much smaller population of female prostitutes, constituting the major chain of transmission. HIV infection is occurring in both urban and rural populations.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Characteristics of arterial myosin in experimental renal hypertension in the dog.

We compared myosin samples isolated from iliac-femoral arteries of control and renal (stenosis) hypertensive dogs to determine the effects of increased blood pressure on the characteristics of the myosin. The ratio of 204-kd (SM-1) to 200-kd (SM-2) myosin heavy chains was approximately 1:0.75 for myosin from the iliac-femoral artery of normotensive dogs. This was not altered significantly in response to hypertension. Both SM-1 and SM-2 myosin heavy chains cross-reacted with antibody against smooth muscle myosin on Western blot analysis. In addition to these heavy chains, purified myosin from both groups showed a very faint protein band slightly below the 200-kd myosin heavy chain on electrophoresis on a highly porous sodium dodecyl sulfate-polyacrylamide gel. This protein band cross-reacted with antibody against nonmuscle myosin but not with smooth muscle myosin antibody. The 20- and 17-kd light chains of myosin isolated from normotensive and hypertensive dogs gave similar results on isoelectric focusing. Peptide maps of tryptic digests of heavy chains revealed both quantitative and qualitative differences. The Ca(2+)-activated myosin ATPase activity measured in high salt (0.5 mol/L KCl) was similar for myosin from both groups, whereas the potassium (ethylenedinitrilo)tetraacetic acid-stimulated ATPase of myosin from hypertensive animals was higher than that from normotensive animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Diffuse x-ray scattering from tropomyosin crystals.

Diffuse scattering analyses are emerging as a technique to extract additional dynamic information from x-ray diffraction data. In fact, when examined carefully, most protein crystals show significant diffuse scattering in addition to the usual Bragg diffraction. This diffuse scattering contains information about the disorder in the crystal that cannot be obtained from the Bragg diffraction data. Diffraction from tropomyosin crystals shows characteristic diffuse scattering streaks that are directly related to motion of the molecules. The structure of tropomyosin to 15 A resolution shows that the limited molecular contacts between molecules allow large conformational fluctuations of up to 8 A amplitude. Models for the three-dimensional motion of tropomyosin have been tested by comparing their predicted diffuse scattering patterns with the experimental data. From the parameters of the successful simulations, we were able to determine the amplitudes, directions, and distances over which the atomic displacements are correlated.

Biophysical Phenomena↗
PubMed1992

Smooth muscle myosin isoform distribution and myosin ATPase in hypertrophied urinary bladder.

Hypertrophy of the urinary bladder was produced in rabbit by partial ligation of the urethra. Electrophoresis of the bladder smooth muscle myosin on highly porous (3.5-7% gradient) SDS-polyacrylamide gel revealed two heavy chain isoforms, SM-1 and SM-2 with approximate molecular weights of 204,000 and 200,000, respectively. The ratio of the SM-2 to SM-1 heavy chain is 3:1 for myosin isolated from normal bladder smooth muscle, and this ratio changes to about 1:1 in hypertrophied bladder. Despite a change in the ratio of SM-2 to SM-1, the myosin ATPase and the actin-activated ATPase activities are not altered in response to hypertrophy.

Animals↗
PubMed1991

The mechanism for the inhibition of actin-activated ATPase of smooth muscle heavy meromyosin by calponin.

Calponin, an actin-binding protein, inhibited the acto-heavy meromyosin (HMM) MgATPase and lowered the binding of HMM to actin. The amount of calponin bound to actin or tropomyosin-actin was the same when the ATPase was inhibited 80-90%. While the KATPase was diminished only less than 2-fold in the presence of calponin, the Vmax was decreased 6-fold and 2-fold with actin and tropomyosin-actin, respectively. A comparison of the kinetic constants for the ATP hydrolysis obtained in the presence of actin-calponin and tropomyosin-actin-calponin revealed that the tropomyosin augmented the Vmax 5-fold from the inhibited level, but there was no effect on the KATPase.

Actins↗
PubMed1991

Mechanism for the inhibition of acto-heavy meromyosin ATPase by the actin/calmodulin binding domain of caldesmon.

Caldesmon, an actin/calmodulin binding protein, inhibits acto-heavy meromyosin (HMM) ATPase, while it increases the binding of HMM to actin, presumably mediated through an interaction between the myosin subfragment 2 region of HMM and caldesmon, which is bound to actin. In order to study the mechanism for the inhibition of acto-HM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lacks the myosin binding portion. The 38-kDa fragment inhibits the actin-activated HMM ATPase to the same extent as does the intact caldesmon molecule. In the absence of tropomyosin, the 38-kDa fragment decreased the KATPase and Kbinding without any effect on the Vmax. However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3-fold decrease in the Vmax, in addition to lowering the KATPase and the Kbinding. The 38-kDa fragment-induced inhibition is partially reversed by calmodulin at a 10:1 molar ratio to caldesmon fragment; the reversal was more remarkable in 100 mM ionic strength at 37 degrees C than in 20 or 50 mM at 25 degrees C. Results from these experiments demonstrate that the 38-kDa domain of caldesmon fragment of myosin head to actin; however, when the actin filament contains bound tropomyosin, caldesmon fragment affects not only the binding of HMM to/actin but also the catalytic step in the ATPase cycle. The interaction between the 38-kDa domain of caldesmon and tropomyosin-actin is likely to play a role in the regulation of actomyosin ATPase and contraction in smooth muscle.

Actins↗
PubMed1991

Structural studies of tropomyosin by cryoelectron microscopy and x-ray diffraction.

A comparison has been made between cryoelectron microscope images and the x-ray structure of one projection of the Bailey tropomyosin crystal. The computed transforms of the electron micrographs extend to a resolution of approximately 18 A compared with the reflections from x-ray crystallography which extend to 15 A. After correction of the images for lattice distortions and the contrast transfer function, the structure factors were constrained to the plane group (pmg) symmetry of this projection. Amplitude and phase data for five images were compared with the corresponding view from the three-dimensional x-ray diffraction data (Phillips, G.N., Jr., J.P. Fillers, and C. Cohen. 1986. J. Mol. Biol. 192: 111-131). The average R factor between the electron microscopy and x-ray amplitudes was 15%, with an amplitude-weighted mean phase difference of 4.8 degrees. The density maps derived from cryoelectron microscopy contain structural features similar to those from x-ray diffraction: these include the width and run of the filaments and their woven appearance at the crossover regions. Preliminary images obtained from frozen-hydrated tropomyosin/troponin cocrystals suggest that this approach may provide structural details not readily obtainable from x-ray diffraction studies.

Animals↗
PubMed1990

Effects of phosphorylation, magnesium, and filament assembly on actin-activated ATPase of pig urinary bladder myosin.

The relationship between the light-chain phosphorylation and the actin-activated ATPase activity of pig urinary bladder myosin was either linear or nonlinear depending on the free Mg2+ concentration. Varying the free [Mg2+] in the presence of 50 mM ionic strength (I) had a biphasic effect on the actin-activated ATPase. In 100 mM I, the activity increased on raising the free [Mg2+]. The activity of the phosphorylated myosin was 3-23-fold higher than that of the unphosphorylated myosin at all concentrations of free Mg2+, pH, and temperature used in this study. The increase in the turbidity and sedimentability of both phosphorylated and unphosphorylated myosins on raising the free [Mg2+] was associated with a rise in the actin-activated ATPase activity. However, myosin light-chain phosphorylation still had a remarkable effect on the actin activation. The myosin polymers formed under these conditions were sedimented by centrifugation. Experiments performed with myosin polymers formed in mixtures of unphosphorylated and phosphorylated myosins showed that the presence of phosphorylated myosin in these mixtures had a slight effect on the sedimentation of the unphosphorylated myosin but it had no effect on the actin-activated ATP hydrolysis. Electron microscopy showed that the unphosphorylated myosin formed unorganized aggregates while phosphorylated myosin molecules assembled into bipolar filaments with tapered ends. These data show that although the unphosphorylated and phosphorylated myosins have the same level of sedimentability and turbidity, the filament assembly present only with the phosphorylated myosin can be associated with the maximal actin activation of Mg-ATPase.

Actins↗
PubMed1990

Cooperativity of actin-activated ATPase of gizzard heavy meromyosin in the presence of gizzard tropomyosin.

The mechanism for the potentiation of the actin-activated ATPase of smooth muscle myosin by tropomyosin is investigated using smooth muscle actin, tropomyosin, and heavy meromyosin. In the presence of tropomyosin, an increase in Vmax occurs with no effect on KATPase and Kbinding at 20 mM ionic strength. Utilizing N-ethylmaleimide-treated subfragment-1, which forms rigor complexes with actin in the presence of ATP but does not have ATPase activity, experiments were carried out to determine if the tropomyosin-actin complex exists in both the turned-off and turned-on forms as in the skeletal muscle system. At both 60 and 100 mM ionic strengths, the presence of rigor complexes on the smooth muscle actin filament containing bound tropomyosin causes a 2-3-fold increase in Vmax and about a 3-fold increase in KATPase, resulting in about a 4-fold increase in ATPase activity at moderate actin concentration. The increase in KATPase is correlated with an increase in Kbinding. The finding that rigor complexes increase Vmax and the binding constant for heavy meromyosin to tropomyosin-actin at an ionic strength close to physiological conditions indicates that the tropomyosin-actin complex can be turned on by rigor complexes in a cooperative manner. However, in contrast to the situation in the skeletal muscle system, the increase in KATPase is associated with a corresponding increase in Kbinding. Furthermore, there is only a 3-fold increase in KATPase in the smooth muscle system rather than a 10-fold increase as in the skeletal muscle system.

Actins↗
PubMed1989

Caldesmon inhibits the cooperative turning-on of the smooth muscle heavy meromyosin by tropomyosin-actin.

The 38-kDa chymotryptic fragment of caldesmon, which possesses the actin/calmodulin binding domain, was purified and utilized to study the mechanism for the inhibition of acto-myosin ATPase by caldesmon. The intact caldesmon inhibited the acto-HMM ATPase although it caused an increase in the binding of HMM to actin, presumably due to the interaction between the S-2 region of HMM and the caldesmon located on the actin filament. The 38-kDa fragment, which lacks the S-2 binding domain, inhibited both the acto-HMM ATPase and the HMM binding to actin. The ATPase and the HMM binding to actin decreased in parallel on increasing the 38-kDa fragment bound to actin. In the presence of tropomyosin, the ATPase activity fell more rapidly than did the HMM binding to actin. Binding of intact caldesmon or 38-kDa fragment to actin inhibited the cooperative turning-on of tropomyosin-actin by NEM.S-1, which forms rigor complexes in the presence of ATP. The absence of cooperative turning-on of the acto-HMM ATPase by rigor complexes in the presence of 38-kDa fragment was associated with an inhibition of the binding of HMM to tropomyosin-actin. Addition of NEM.S-1 to tropomyosin-actin-caldesmon caused a gradual decrease in the caldesmon-induced binding of HMM to actin. The calmodulin restored the caldesmon-induced binding of HMM to tropomyosin-actin, but it had only a slight effect on the acto-HMM ATPase. These data suggest that the cooperative turning-on of the smooth muscle tropomyosin-actin by rigor bonds is modulated by the interaction of caldesmon, tropomyosin, and calmodulin on the thin filament.

Actins↗
PubMed1989

Characteristics of the myosin and tropomyosin binding regions of the smooth muscle caldesmon.

Limited digestion of caldesmon by alpha-chymotrypsin generates mainly 110, 80, 60, 38, and 28 kDa fragments. Affinity chromatography of these fragments on columns immobilized with myosin, HMM, or tropomyosin showed that the bound fraction from these columns was similar and it contained 110, 80, 60 and 28 kDa fragments. These fragments did not bind to myosin filaments, acto-HMM, actin or tropomyosin-actin in the solution, and they had no effect on the actin-activated ATPase of HMM. In contrast, the flow-through fraction from these affinity columns inhibited the actin-activated ATPase. Binding studies revealed that the 38 kDa fragment and its break down products bound to actin and tropomyosin-actin, and they were released partially from actin by calmodulin with a concomitant increase in the ATPase activity. These results indicate that, unlike the actin binding domain, the myosin and tropomyosin binding domains require the caldesmon molecule to be intact in order to exert their effects on the protein-protein interaction.

Actins↗
PubMed1988

Interaction between caldesmon and tropomyosin in the presence and absence of smooth muscle actin.

Cysteine residues of caldesmon were labeled with the fluorescent reagent N-(1-pyrenyl)maleimide. The number of sulfhydryl (SH) groups in caldesmon was around 3.5 on the basis of reactivity to 5,5'-dithiobis(2-nitrobenzoate); 80% of the SH groups were labeled with pyrene. The fluorescence spectrum from pyrene-caldesmon showed the presence of excited monomer and dimer (excimer). As the ionic strength increased, excimer fluorescence decreased, disappearing at salt concentrations higher than around 50 mM. The labeling of caldesmon with pyrene did not affect its ability to inhibit actin activation of heavy meromyosin Mg-ATPase and the release of this inhibition in the presence of Ca2+-calmodulin. Tropomyosin induced a change in the fluorescence spectrum of pyrene-caldesmon, indicating a conformational change associated with the interaction between caldesmon and tropomyosin. The affinity of caldesmon to tropomyosin was dependent on ionic strength. The binding constant was 5 x 10(6) M-1 in low salt, and the affinity was 20-fold less at ionic strengths close to physiological conditions. In the presence of actin, the affinity of caldesmon to tropomyosin was increased 5-fold. The addition of tropomyosin also changed the fluorescence spectrum of pyrene-caldesmon bound to actin filaments. The change in the conformation of tropomyosin, caused by the interaction between caldesmon and tropomyosin, was studied with pyrene-labeled tropomyosin. Fluorescence change was evident when unlabeled caldesmon was added to pyrene-tropomyosin bound to actin. These data suggest that the interaction between caldesmon and tropomyosin on the actin filament is associated with conformational changes on these thin filament associated proteins. These conformational changes may modulate the ability of thin filament to interact with myosin heads.

Actins↗