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Biomedical subjects

A Babu

Publications and source records attributed to A Babu.

At least 37 records · Page 2Linked to original sources
PubMed1996

Epidemiology of symptomatic human rotaviruses in Bangalore and Mysore, India, from 1988 to 1994 as determined by electropherotype, subgroup and serotype analysis.

Epidemiology of symptomatic rotaviruses from Bangalore and Mysore in Southern India was investigated. While serotype G3 predominated throughout the 7-year study period from 1988 to 1994 in Bangalore, serotype G1 was more predominant than serotype G3 in Mysore during 1993 and 1994. Serotype G2 strains were either not detected or infrequently observed in both the cities. However, several strains with subgroup I and 'short' RNA pattern that exhibited high reactivity with typing MAbs specific for serotype 2 as well as other serotypes were detected throughout the period. Among the nonserotypeable strains from both cities, several exhibited dual subgroup (SGI + II) or subgroup I specificity and 'long' RNA pattern indicating their probable animal origin. Notably, a gradual, yet highly significant reduction in rotavirus gastroenteritis, from 45.3% in 1988 to 1.8% during 1994, was observed in Bangalore in stark contrast to the consistently high (about 34%) incidence of asymptomatic infections among neonates by I321-like G10P11 type strains during the same period. Moreover, I321-like asymptomatic strains were not detected in children with diarrhea.

Antibodies, Monoclonal↗
PubMed1993

Critical minimum length of the central helix in troponin C for the Ca2+ switch in muscular contraction.

In the troponin C (TnC) dumbbell, the NH2- and COOH-terminal lobes are well delineated, but the role of the central helix and especially the function of its long length remain doubtful. To study this, we used a cDNA construct encoding rabbit fast-twitch muscle TnC, comprising multiple restriction sequences to facilitate mutagenesis (Babu, A., Su, H., Ryu, Y. & Gulati, J. (1992) J. Biol. Chem. 267, 15469-15474). Systematically, we have deleted 3-12 amino acid residues from the central helix and examined their effects in maximally activated skinned muscle fibers. Limiting the deletions to 7 amino acid residues manifested little change in maximal force development (Sheng, Z., Francois, J. M., Hitchcock, S. E. & Potter, J. D. (1991) J. Biol. Chem. 266, 5711-5715). However, with further deletions, we now find that contractility was inhibited pari passu; by 12 deletions, the inhibition was complete. The critical minimum length for the central helix is thereby estimated as 27 A. The Ca2+ binding capacity (4 mol of Ca2+/mol of protein) as well as the structural characteristics (alpha-helicity by CD measurements and the fluorescence emitted by Tyr-109) indicated a well preserved global conformation of the short mutant. However, surprisingly, two of these short mutants filled each TnC slot under highly specific superloading conditions: one short molecule was taken up in EGTA solution, and the second molecule was captured and retained with Ca2+. They also rescued the contractile switch, evidently in a bimolecular reaction. Another short variant (putative skeletal fast muscle TnC-I-II), in which the NH2-terminal Ca(2+)-binding sites were incapacitated, failed to respond to superloading, indicating that sites III and IV could not substitute for sites I and II. The results suggest that a critical role of the central helix linker in TnC is to keep the two lobes optimally apart, evidently in proximity of their respective target sites on troponin I in the fiber.

Amino Acid Sequence↗
PubMed1993

Identification of the regions conferring calmodulin-like properties to troponin C.

The structural and functional correlations between troponin C (TnC) and calmodulin (CaM) were investigated by mutagenizing a synthetic cDNA coding rabbit skeletal muscle TnC. Compared with TnC, calmodulin lacks the N-terminal alpha-helical arm (N-helix), and its central helix is shorter due to the absence of 88KGK90 residues. Deleting both regions concomitantly (delta Nt delta KGK) elicited CaM-like regulation as tested (i) by smooth muscle contractility (maximal tension = 80 +/- 5% Po of control) and (ii) by the activation of phosphodiesterase (Vmax = 75 +/- 2% of control). The Ca(2+)-binding capacity of the mutant and the effect of the mutant on maximally Ca(2+)-activated tension of skinned rabbit psoas muscle fibers were both conserved. Furthermore, in the linker region of the central helix, replacing the TnC-characteristic 85EDAKGK90 successive residues with CaM-specific DTD residues generated a highly effective CaM mimic (Vmax = 96 +/- 2%) whether or not the N-helix was also retained. Apparent KD values (i.e. concentrations for half-maximal response) for the successful mutants were similar to each other but about 200-fold higher than that for CaM. A part of the alpha-helical linker region in CaM may unfold and bend to promote multiplicity of target interaction using all four hands (Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638; Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) Science 257, 1251-1255). In contrast, our results suggest that the TnC central helix evolved to be less pliable by the combined influences of 85EDAKGK90 residues and the alpha-helical extension in N terminus, thereby keeping the N-terminal hands well separated from their C-terminal counterparts.

Amino Acid Sequence↗
PubMed1993

The mechanism of Ca(2+)-coordination in the EF-hand of TnC, by cassette mutagenesis.

Genetic engineering of TnC and skinned fiber physiology on rabbit psoas muscle are combined to study the mechanisms of Ca(2+)-binding in the EF-hand in TnC. Of the six coordinating positions (X,Y,Z,-Y,-X & -Z) for Ca(2+)-binding in the loop, the X position is invariably occupied by an aspartate, and the -Z position by a glutamate. X-ray analysis has indicated that both oxygen atoms of the beta-carboxylate in aspartate (in X) are extensively hydrogen bonded to other residues in the loop. When this aspartate in site II was replaced by a glutamate (gamma-carboxylate), Ca(2+)-binding was annihilated, and the mutant was unable to regulate force development in the fiber. Similarly, glutamate for aspartate exchange in the -Z position of site I also inactivated the site as well as its function in skinned fiber. Mutations in the Y position indicated that a glutamate was unacceptable in place of aspartate but that an asparagine was acceptable. The Ca(2+)-sensitivity with asparagine was also similar to that of the wild type. The study indicates a powerful approach for defining the physicochemical principles governing Ca-coordination and sensitivity in Ca-binding proteins. Furthermore, by comparison with findings on chemically synthesized peptides, the results show that behavior of the EF-hand in TnC is modified by quaternary structure of the molecule.

Amino Acid Sequence↗
PubMed1992

Functional delineation of the Ca(2+)-deficient EF-hand in cardiac muscle, with genetically engineered cardiac-skeletal chimeric troponin C.

Cardiac and fast skeletal isoforms of TnC each comprise four putative EF-hand (helix-loop-helix) motifs as potential Ca(2+)-binding sites (sites 1-4), except that site 1 in cardiac TnC is deficient in Ca2+ coordination. In skeletal TnC, the N-terminal sites 1 and 2 are both essential for the trigger mechanism of the contraction switch. However, the mechanism in cardiac muscle is unsettled; it is obscure whether the cardiac site 1 is functionally inert due to calcium deficiency and consequently site 2 is the lone trigger site, or whether sites 1 and 2 perform interactively despite the impairment. These possibilities were addressed by mutagenizing site 1 in skeletal TnC to mimic the cardiac response. In one mutant (STnC-1), two selected Ca(2+)-ligands were abolished. In another (C1/S chimera), 41 N-terminal residues from cardiac TnC were spliced to STnC. The Ca(2+)-binding capacities as well as skinned fiber responses were measured. The STnC-1 derivative failed to switch on contraction. In contrast, the chimeric construct expressed close to full contractile potential in myocardium (74 +/- 3% Po; Po = maximal tension) and also the manifest cardiac phenotype. By devising supplemental chimeric constructs, cardiac-type N-terminal overhang together with cardiac-type EF-hand for site 1 both were found essential for the phenotype. We conclude that cardiac TnC site 1 is actively engaged in the trigger mechanism and in fact dominates the phenotype despite the inability to chelate Ca2+. The N-terminal overhang also participates in this mechanism, which is a novel finding. The conclusion that a non-chelating site functions interactively with a proximal site in cardiac TnC may have wider significance, inasmuch as similar pairings of disparate EF-hands are of common occurrence.

Amino Acid Sequence↗
PubMed1992

Double mosaic aneuploidy: 45,X/47,XY,+8 in a male infant.

We report on a 13-month-old boy with abnormalities consistent with mosaic trisomy 8 syndrome and male genitalia with partial penoscrotal transposition without hypospadias, a retractile left testis in inguinal canal, and an absent right testis. A voiding cystourethrogram showed an outpouching close to the lower right side of the bladder (utriculum) and bilateral hydronephrosis secondary to vesicoureteral reflux. Peripheral blood karyotype was 45,X/47,XY,+8. The karyotype of cultured skin fibroblasts was 47,XY,+8 with no 45,X cells detected among 20 cells counted. Tissues removed during surgery documented a 45,X/47,XY,+8 complement in the left testicle and utriculum, but only a 45,X line among 20 cells counted from vas deferens tissue. A possible mechanism for the origin of this previously unreported mosaicism might be an abnormal zygote with a 47,XY,+8 complement with subsequent simultaneous loss of chromosome Y and 8 in a cell at a very early embryonic stage.

Aneuploidy↗
PubMed1992

Determination of residue specificity in the EF-hand of troponin C for Ca2+ coordination, by genetic engineering.

Utilizing protein engineering of troponin C (TnC), combined with the physiology of skinned fibers, the present study sought to delineate the mechanisms for metal ion coordination and sensitivity in the sites (EF-hands) that execute the Ca2+ switch for contraction. A total TnC-encoding gene comprising multiple target sequences for restriction enzymes was synthesized, furnishing a pliant molecular handle to manipulate sites I and II in the NH2 terminus of the protein. Of the six positions (X, Y, Z, -Y, -X, and -Z) essential for metal ion chelation in a typical EF-hand, invariably the X position has aspartate, and -Z position has glutamate. In the X position of site II, mutation of aspartate for either glutamate (gamma-carboxylate) or asparagine (same side chain length as aspartate) yielded functionally inactive proteins with concomitantly diminished Ca2+ binding capacity. Similarly, in -Z position (site I), neither aspartate nor glutamine were compatible in exchange for the conserved glutamate. In contrast, for the Y coordinate of site II, a preference for asparagine comparable to that for wild-type aspartate was detected, but glutamate was impermissible. Evidently, physicochemical and steric factors both are critical in governing the mechanism for metal ion chelation in TnC in a physiological milieu. Furthermore, the findings manifest that the quaternary structure of hydrated TnC restrains the EF-hands during on-off operation of the Ca2+ switch.

Amino Acid Sequence↗
PubMed1992

T-cell chronic lymphocytic leukemia. Unusual morphologic, phenotypic, and karyotypic features in association with light chain amyloidosis.

BACKGROUND: Lymphocytes that display a phenotype of mature B-cells, T-cells, natural killer (NK) cells, or a combination of T-cells and NK cells can be found in patients with lymphoproliferations that manifest as expansions of peripheral blood lymphocytes (PBL). If these PBL expansions exhibit clonality, they can be classified as chronic lymphocytic leukemia (CLL). METHODS/RESULTS: A patient who had two simultaneous, clonal lymphoproliferative disorders manifested as an unusual T-cell CLL in conjunction with systemic light chain amyloidosis is described. Gene rearrangement studies of the PBL of the patient showed clonal rearrangements of both the T-cell receptor beta (T beta) chain and the immunoglobulin genes. Additional immunologic and microscopic studies of the T-cells of the patient showed that they were large, agranular, CD4+ T-cells that also expressed the NK cell marker CD57. Cytogenetics disclosed an unusual karyotype in the PBL. CONCLUSIONS: The pathogenesis of this T-cell CLL and whether it truly represents a malignant disorder, as well as its relation to amyloidosis, is discussed.

Aged↗
PubMed1991

The role of troponin C in the length dependence of Ca(2+)-sensitive force of mammalian skeletal and cardiac muscles.

1. Skinned fibre preparations of right ventricular trabeculae, psoas and soleus muscles from hamster and rabbit were activated by Ca2+ and the length dependencies of their pCa (-log [Ca2+])-force relationships were compared. 2. Ca2+ sensitivity of the myocardium was higher at 2.2-2.4 microns than that at 1.7-1.9 microns. The length dependence was at least twofold greater in cardiac muscle than in fast skeletal fibres at identical temperatures and salt concentrations. Slow-twitch fibres gave a response similar to that in the myocardium. 3. The effect of the troponin C (TnC) phenotype on the length dependence of Ca2+ sensitivity was measured on both fast skeletal fibres and cardiac muscle with TnC exchange in situ. The length-induced increase in Ca2+ sensitivity was found to be greater in the presence of cardiac TnC than with fast skeletal TnC. Thus the results indicate that a certain domain of TnC is specialized in this length function, and that this domain is different in the two phenotypes. 4. The possibility that the enhanced length dependence of Ca2+ sensitivity after cardiac TnC reconstitution was attributable to reduced TnC binding was excluded when the length dependence of partially extracted fast fibres was reduced to one-half the normal value after a 50% deletion of the native TnC. 5. Two recombinant forms of cardiac TnC (kindly provided by Dr John Putkey, Houston, TX, USA) were used next, to investigate the roles of two specific domains in TnC in the control of length dependence of Ca2+ sensitivity and in the contraction-relaxation switching of cardiac muscle: 6. Using mutant CBM1 [corrected], in which site 1 was modified such as to bind the 4th Ca2+ ion, as in skeletal TnC, the length-induced Ca2+ sensitivity in cardiac muscle was suppressed. The effect was intermediate between cardiac and skeletal TnCs under the same conditions. The pSr (-log [Sr2+])-force relationship of cardiac muscle was also measured. In the presence of the mutant, skinned trabeculae manifest pSr-activation curves identical to those of fast fibres. This indicates that the metal ion binding properties of site 1 in TnC modulate the regulatory action of site 2. 7. Using mutant CBM2A, in which site 2 was inactivated, the activation of cardiac muscle by both Ca2+ and Sr2+ ions was completely blocked. This is the expected result, since both regulatory sites were now inactive, regulatory site 1 being normally inactive in cardiac muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Central helix role in the contraction-relaxation switching mechanisms of permeabilized skeletal and smooth muscles with genetic manipulation of calmodulin.

A prominent common feature of calmodulin and troponin structures is the unusually long central helix which separates the two lobes, each containing two Ca2(+)-binding sites. To study the role of certain highly conserved residues in the helix in the contraction-relaxation switching mechanism in muscle, we measured the Ca2(+)-activated force of permeabilized skeletal and smooth muscles with three genetically manipulated forms of calmodulin. Mutated calmodulin was made to substitute for troponin-C in vertebrate skeletal fiber. The mutants had 1-4 deletions in the conserved cluster (positions 81-84) in the solvent-exposed region of the central helix, which also substantially shortened the helix. The force of the maximally activated fiber was found to be diminished only with the mutant in which the entire cluster Ser-81 to Glu-84 (CaM delta 81-84) was deleted. All such deletions were found to be completely ineffective in blocking the Ca2(+)-switching process in smooth muscle strips. The results show for the first time that at least a part of the highly conserved four-residue cluster in the central helix is critical for the contraction mechanism of striated muscle. Further, the possibility is raised that the reduced length of the central helix may be a determining factor in the Ca2(+)-switching mechanism in fast-twitch muscle. These findings combined with the results on smooth muscle indicate diversity in the structure-function specifications for the central helix of calmodulin for different target proteins.

Animals↗
PubMed1990

Trifluoperazine inhibition of contraction in permeabilized skeletal, cardiac and smooth muscles.

To gain insights into the mechanism of the central helix of calmodulin and troponin-C in the Ca2(+)-regulation of force development in striated and smooth muscles, the present study was made of the TFP induced inhibition of contraction, and of the uptake of these proteins by skinned fibers. Calmodulin was four-fold more sensitive to TFP than TnC, but the inhibition was found to be identical for skeletal and cardiac muscles despite the differences in their troponin-C isoforms. Also, the results were comparable between fast-twitch fiber, when calmodulin was exchanged for troponin-C to act on TnI, and smooth muscle, where calmodulin acts on myosin light chain kinase. These findings indicate that the inhibition of force by TFP is entirely due to its binding to the hydrophobic sites in the central helix. The uptakes of troponin-C and calmodulin were also different, and this is explained by a TFP-independent domain in troponin-C that binds TnI.

Animals↗
PubMed1990

Anatomy of human genome by restriction endonucleases Alul, Ddel, Haelll, Hinfl, Mobol and Rsal, and their application in clinical cytogenetics.

The application of restriction endonucleases Alul, Ddel, Haelll, Hinfl, Mbol and Rsal in clinical cytogenetics is described. The extensive inherent heterogeneity of heterochromatin in the C-band regions revealed by these enzymes provides a valuable tool for describing the origin of trisomies and abnormal chromosomes in humans. The heteromorphic markers identified by these enzymes have tremendous potential in clinical cytogenetics. Unlike the CBG technique, slides can be stained immediately after preparation providing a rapid diagnosis. Characteristic bands induced by each enzyme in the human complement are discussed in detail. Comparative analysis of the present data, with those described earlier, revealed certain discrepancies including chromosomes 19 and 20 by Alul, chromosomes 4, 5, 8 and 22 by Mbol and chromosomes 12 and 20 by Rasl. These controversies are examined in the light of heteromorphisms, technical variables and chromosome identification.

Chromosome Banding↗
PubMed1989

Characterization of the Ca2+-switch in skeletal and cardiac muscles.

To determine the significance of the global structure of the regulatory proteins in the mechanism of the Ca2+-switch in cardiac and skeletal muscle contractions, the properties of a family of Ca2+-binding proteins with 4 or 3 EF-hand motifs have been studied with desensitized skinned fiber preparations. Proteins with 4 EF hands (such as troponins C - TnCs) are dumb-bell shaped, those with 3 EF hands (parvalbumin) being ellipsoidal. The number of active sites varied between four and two. We find that the ability to anchor in the fiber is limited to proteins with 4 EF hands and, at least, two active Ca2+-binding sites, one each in the N- and C-termini. The results suggest that the dumb-bell shaped global structure is critical for the switching action in muscular contraction, and a trigger site in the N-terminus and a structural site in the C-terminus need to be active in order to regulate contractility.

Animals↗
PubMed1989

Down-regulation of fast-twitch skeletal muscle fiber with cardiac troponin-C and recombinant mutants. Structure/function studies with site-directed mutagenesis.

Structure/function relationships in troponin C are studied with vertebrate fast-twitch fibers by exchanging the skeletal troponin C with two bacterially synthesized recombinant proteins designed by site-directed mutagenesis of cardiac troponin C. One mutant (CBM1) contained an additional active site, by deleting Val-28 and converting Leu-29, Gly-30, Ala-31 and Glu-32 to Asp, Ala, Asp and Gly, respectively, in the normally inactive trigger site 1 in the N-terminus. In another mutant (CBM2A), the normally active site 2 was inactivated by conversion of Asp-65 to Ala. The fibers were found to be down-regulated with recombinant cardiac troponin C (CTnC3), as with tissue-cardiac-troponin-C. With mutants, in one case (CBM1) the regulation was unmodified despite Ca2+ coordination by all sites. In contrast, regulation was found to be completely blocked with the mutant (CBM2A) where both trigger sites were inactive. The results provide the first indication that structural specification of the entire EF-hand motif of site 1, and not just Ca2+ coordination, is needed to operate fully the Ca2+ switch in fast-twitch fibers.

Animals↗
PubMed1989

Effect of acidosis on Ca2+ sensitivity of skinned cardiac muscle with troponin C exchange. Implications for myocardial ischemia.

By using a novel approach for the study of the effects of pH variation in skinned myocardium, the present experiments were aimed to provide new insights into the mechanism of ischemia. Ca2+ sensitivity is decreased by acid pH, but the effect is more than double in cardiac myofilaments than that in fast-twitch skeletal muscle fibers. With the technique of troponin C exchange in myocytes, we find here that the effect of pH is the same with cardiac or skeletal troponin C. These results rule out a direct H+-Ca2+ competition on the Ca2+-binding sites of troponin C as a significant mechanism of ischemia. The findings provide conclusive evidence in favor of the idea that acidosis modulates the protein-protein interactions in the regulatory complex in cardiac muscle.

Animals↗
PubMed1989

Hybrid acute leukemia in an HIV-antibody-positive patient.

Although the great majority of acute leukemias have been designated as being of lymphocytic or myelocytic origin, recent reports have described elements of both in some patients. We describe here the first case of hybrid acute leukemia in an HIV-antibody-positive patient as well as the first hybrid involving B-cell (Burkitt) acute lymphocytic leukemia and acute myelomonocytic leukemia proven by cytochemical, immunologic, and cytogenetic methods. This case illustrates the increasingly complex difficulties in the diagnosis and treatment of AIDS-related malignancies.

Acute Disease↗