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Biomedical subjects

P K Mehta

Publications and source records attributed to P K Mehta.

At least 19 recordsLinked to original sources
PubMed2001

Molecular emergence of acute myeloid leukemia during treatment for acute lymphoblastic leukemia.

Therapy-related acute myeloid leukemias (t-AML) with translocations of the MLL gene are associated with the use of topoisomerase II inhibitors. We established the emergence of the malignant clone in a child who developed t-AML with a t(11;19) (q23;p13.3) during treatment for acute lymphoblastic leukemia (ALL). The MLL-ENL and the reciprocal ENL-MLL genomic fusions and their chimeric transcripts were characterized from samples collected at the time of t-AML diagnosis. We used PCR with patient-specific genomic primers to establish the emergence of the MLL-ENL fusion in serially obtained DNA samples. The MLL-ENL fusion was not detectable in bone marrow at the time of ALL diagnosis or after 2 months of chemotherapy (frequency <8.3 x 10(-7) cells(-1)). The genomic fusion was first detected in bone marrow after 6 months of treatment at a frequency of one in 4,000 mononuclear bone marrow cells; the frequency was one in 70 cells after 20 months of therapy. At the first detection of MLL-ENL, the only topoisomerase II inhibitors the patient had received were one dose of daunorubicin and two doses of etoposide. The MLL-ENL fusion was not detectable in blood at the time of ALL diagnosis or after 0.7, 2, 8, 10, and 12 months of therapy but was detectable in blood at 16 months (one in 2.3 x 10(4) cells). Recombinogenic Alu sequences bracketed the breakpoints in both fusions. These data indicate that the malignant clone was not present before therapy, arose early during chemotherapy, and was able to proliferate even during exposure to antileukemic therapy.

Adolescent↗
PubMed2001

From cofactor to enzymes. The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes.

The pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes that act on amino acid substrates have multiple evolutionary origins. Thus, the common mechanistic features of B(6) enzymes are not accidental historical traits but reflect evolutionary or chemical necessities. The B(6) enzymes belong to four independent evolutionary lineages of paralogous proteins, of which the alpha family (with aspartate aminotransferase as the prototype enzyme) is by far the largest and most diverse. The considerably smaller beta family (tryptophan synthase beta as the prototype enzyme) is structurally and functionally more homogenous. Both the D-alanine aminotransferase family and the alanine racemase family consist of only a few enzymes. The primordial pyridoxal-5'-phosphate-dependent protein catalysts apparently first diverged into reaction-specific protoenzymes, which then diverged further by specializing for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages, providing examples of convergent enzyme evolution. The functional specialization of most B(6) enzymes seems to have already occurred in the universal ancestor cell before the divergence of eukaryotes, archebacteria, and eubacteria 1500 million years ago. Pyridoxal-5'-phosphate must have emerged very early in biological evolution; conceivably, metal ions and organic cofactors were the first biological catalysts. To simulate particular steps of molecular evolution, both the substrate and reaction specificity of existent B(6) enzymes were changed by substitution of active-site residues, and monoclonal pyridoxal-5'-phosphate-dependent catalytic antibodies were produced with selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis.

Amino Acid Isomerases↗
PubMed2001

Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13;q13) of acute megakaryoblastic leukemia.

t(1;22) is the principal translocation of acute megakaryoblastic leukemias. Here we show this chromosomal rearrangement to result in the fusion of two novel genes, RNA-binding motif protein-15 (RBM15), an RNA recognition motif-encoding gene with homology to Drosophila spen, and Megakaryoblastic Leukemia-1 (MKL1), a gene encoding an SAP (SAF-A/B, Acinus and PIAS) DNA-binding domain.

Chromosomes, Human, Pair 1↗
PubMed2000

Rates of evolution of pyridoxal-5'-phosphate-dependent enzymes.

The pyridoxal-5'-phosphate-dependent enzymes (B(6) enzymes), that operate in the metabolism of amino acids, are of multiple evolutionary origin. To estimate their rates of evolution, a total of 180 sequences of 21 B(6) enzymes from distantly related eukaryotic species were compared. The enzymes belong to all four evolutionarily independent families of B(6) enzymes with different folds, i.e., the large alpha family, the beta family, the d-alanine aminotransferase family, and the alanine racemase family. Their unit evolutionary periods, i.e., the time for a 1% sequence difference to accumulate between branches, ranged from 4.6 to 45.1 million years. Both, fastest changing serine pyruvate aminotransferase and most slowly changing glutamate decarboxylase are members of the alpha family. The evolutionary rates of the few enzymes belonging to the other three families were interspersed among those of the alpha family members. Enzymes that catalyze the same reaction, e.g., transamination or amino acid decarboxylation, with different substrates show widely varying rates. The absence of correlations of the rate of evolution with either protein fold or type of catalyzed reaction suggests that individual functional constraints have determined the differential rates of evolution of B(6) enzymes.

Animals↗
PubMed2000

The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes.

The pyridoxal-5-phosphate-dependent enzymes (B6 enzymes) that act on amino acid substrates are of multiple evolutionary origin. The numerous common mechanistic features of B6 enzymes thus are not historical traits passed on from a common ancestor enzyme but rather reflect evolutionary or chemical necessities. Family profile analysis of amino acid sequences supported by comparison of the available three-dimensional (3-D) crystal structures indicates that the B6 enzymes known to date belong to four independent evolutionary lineages of homologous (or more precisely paralogous) proteins, of which the alpha family is by far the largest. The alpha family (with aspartate aminotransferase as the prototype enzyme) includes enzymes that catalyze, with several exceptions, transformations of amino acids in which the covalency changes are limited to the same carbon atom that carries the amino group forming the imine linkage with the coenzyme (i.e., Calpha in most cases). Enzymes of the beta family (tryptophan synthase beta as the prototype enzyme) mainly catalyze replacement and elimination reactions at Cbeta. The D-alanine aminotransferase family and the alanine racemase family are the two other independent lineages, both with relatively few member enzymes. The primordial pyridoxal-5-phosphate-dependent enzymes apparently were regio-specific catalysts that first diverged into reaction-specific enzymes and then specialized for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages. Comparison of sequences from eukaryotic, archebacterial, and eubacterial species indicates that the functional specialization of most B6 enzymes has occurred already in the universal ancestor cell. The cofactor pyridoxal-5-phosphate must have emerged very early in biological evolution; conceivably, organic cofactors and metal ions were the first biological catalysts. In attempts to stimulate particular steps of molecular evolution, oligonucleotide-directed mutagenesis of active-site residues and directed molecular evolution have been applied to change both the substrate and reaction specificity of existent B6 enzymes. Pyridoxal-5-phosphate-dependent catalytic antibodies were elicited with a screening protocol that applied functional selection criteria as they might have been operative in the evolution of protein-assisted pyridoxal catalysis.

Alanine Racemase↗
PubMed1999

Recognizing very distant sequence relationships among proteins by family profile analysis.

Family profile analysis (FPA), described in this paper, compares all available homologous amino acid sequences of a target family with the profile of a probe family while conventional sequence profile analysis (Gribskov M, Lüthy R, Eisenberg D. Meth Enzymol 1990;183:146-159) considers only a single target sequence in comparison with the probe family. The increased input of sequence information in FPA expands the range for sequence-based recognition of structural relationships. In the FPA algorithm, Zscores of each of the target sequences, obtained from a probe profile search over all known amino acid sequences, are averaged and then compared with the scores for sequences of 100 reference families in the same probe family search. The resulting F-Zscore of the target family, expressed in "effective standard deviations" of the mean Zscores of the reference families, with value above a threshold of 3.5 indicates a statistically significant evolutionary relationship between the target and probe families. The sensitivity of FPA to sequence information was tested with several protein families where distant relationships have been verified from known tertiary protein architectures, which included vitamin B6-dependent enzymes, (beta/alpha)8-barrel proteins, beta-trefoil proteins, and globins. In comparison to other methods, FPA proved to be significantly more sensitive, finding numerous new homologies. The FPA technique is not only useful to test a suspected relationship between probe and target families but also identifies possible target families in profile searches over all known primary structures.

Algorithms↗
PubMed1998

Bacterial selenocysteine synthase--structural and functional properties.

Selenocysteine synthase from Escherichia coli is a pyridoxal-5'-phosphate-containing enzyme which catalyses the conversion of seryl-tRNA(Sec) into selenocysteyl-tRNA(Sec). Analysis of amino acid sequences indicated that selenocysteine synthase belongs to the alpha/gamma superfamily of pyridoxal-5'-phosphate-dependent enzymes. To identify the lysine residue carrying the prosthetic group, the genes coding for the selenocysteine synthases from Moorella thermoacetica and Desulfomicrobium baculatum were cloned and sequenced and their derived amino acid sequences were aligned with those from E. coli and Haemophilus influenzae. Three lysine residues were found to be conserved; they were mutated into asparagine and one of them, Lys295, was found to be essential for activity. Proteolytic fragmentation of the E. coli enzyme reduced with borohydride, and mass-spectrometric and sequence analysis of the chromophoric peptide proved that Lys295 was modified. Kinetic analysis of the enzyme showed that thiophosphate served as a substrate leading to cysteyl-tRNA(Sec) synthesis, albeit with a 330-fold lower catalytic efficiency. Selenide and, to a much lesser degree, sulfide could also be used by the enzyme but only at much higher concentrations. These data together with the finding that selenophosphate synthetase is highly specific for selenide indicate that the phosphate moiety of selenophosphate provides selenocysteine synthase with the discrimination specificity against sulfur.

Amino Acid Sequence↗
PubMed1996

Comparison of in vitro models for the study of Mycobacterium tuberculosis invasion and intracellular replication.

We recently evaluated several tissue culture model systems for the study of invasion and intracellular multiplication of Mycobacterium tuberculosis. These model systems include a human alveolar pneumocyte epithelial cell line, a murine macrophage cell line (J774), and fresh human peripheral blood-derived macrophages. Our data indicated that the initial level of association of M. tuberculosis with human alveolar pneumocyte cells (2%) was less than that observed with fresh human peripheral blood macrophages (9%) or J774 murine macrophages (13%) within 6 h of the addition of the bacteria. M. tuberculosis replicated in association with the pneumocyte cells by more than 55-fold by day 7 postinfection. In contrast, total bacteria] growth in the J774 cells and human macrophages was considerably less, with increases of only fourfold and threefold, respectively, over the same 7-day period. Amikacin, an aminoglycoside antimicrobial agent, was added to inhibit the growth of extracellular bacteria after the initial 6-h infection period. Decreases in viable counts were observed in all three cell cultures within the first 3 days after infection. However, unlike the case with either macrophage culture, intracellular bacterial CFU obtained from the infected pneumocytes increased by fourfold by day 7 after the addition of amikacin. These data indicate that M. tuberculosis infects and multiplies intracellularly in human lung epithelial cells and that these cells may be an alternative in vitro model for the study of intracellular multiplication of M. tuberculosis in the human lung.

Animals↗
PubMed1995

A simple and fast approach to prediction of protein secondary structure from multiply aligned sequences with accuracy above 70%.

To improve secondary structure predictions in protein sequences, the information residing in multiple sequence alignments of substituted but structurally related proteins is exploited. A database comprised of 70 protein families and a total of 2,500 sequences, some of which were aligned by tertiary structural superpositions, was used to calculate residue exchange weight matrices within alpha-helical, beta-strand, and coil substructures, respectively. Secondary structure predictions were made based on the observed residue substitutions in local regions of the multiple alignments and the largest possible associated exchange weights in each of the three matrix types. Comparison of the observed and predicted secondary structure on a per-residue basis yielded a mean accuracy of 72.2%. Individual alpha-helix, beta-strand, and coil states were respectively predicted at 66.7, and 75.8% correctness, representing a well-balanced three-state prediction. The accuracy level, verified by cross-validation through jack-knife tests on all protein families, dropped, on average, to only 70.9%, indicating the rigor of the prediction procedure. On the basis of robustness, conceptual clarity, accuracy, and executable efficiency, the method has considerable advantage, especially with its sole reliance on amino acid substitutions within structurally related proteins.

Algorithms↗
PubMed1995

False positive modified Widal test in acute malaria.

The present study reports results of modified Widal test carried out on 96 cases of acute malaria and 25 healthy controls. Out of the 96 patients studied, 30 had complicated P.falciparum (CPF), 36 had uncomplicated P.falciparum (UPF) and 30 had P.vivax (pv) malaria. Modified widal test was done to know the Widal titers due to Igm type of anti-salmonella antibodies, which is considered very specific for diagnosis of recent enteric fever. This test was positive in 14.58% and 10.41% of malaria patients for salmonella 'o' and 'H' titers respectively. On four weeks follow up most of the positive test become negative. Non-specific polyclonal B lymphocyte stimulation due to malaria was postulated to be responsible for this phenomenon.

Acute Disease↗
PubMed1994

Evolutionary relationships among pyridoxal-5'-phosphate-dependent enzymes. Regio-specific alpha, beta and gamma families.

Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio-specificity. In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the imine linkage with the coenzyme. This family was thus named alpha family. It comprises glycine hydroxymethyltransferase, glycine C-acetyltransferase, 5-aminolevulinate synthase, 8-amino-7-oxononanoate synthase, all aminotransferases (with the possible exception of subgroup III), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol-lyase which, however, catalyze beta-elimination reactions. The beta family includes L- and D-serine dehydratase, threonine dehydratase, the beta subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze beta-replacement or beta-elimination reactions. The gamma family incorporates O-succinylhomoserine (thiol-lyase, O-acetylhomoserine (thiol)-lyase, and cystathionine gamma-lyase, which catalyze gamma-replacement or gamma-elimination reactions, as well as cystathionine beta-lyase. The alpha and gamma family might be distantly related with one another, but are clearly not homologous with the beta family. Apparently, the primordial pyridoxal-5'-phosphate-dependent enzymes were regio-specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal-5'-phosphate-dependent enzymes seem to be unrelated with the alpha, beta or gamma family by the criterion of profile analysis:alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B6 enzymes.

Amino Acid Sequence↗
PubMed1994

Homology of 1-aminocyclopropane-1-carboxylate synthase, 8-amino-7-oxononanoate synthase, 2-amino-6-caprolactam racemase, 2,2-dialkylglycine decarboxylase, glutamate-1-semialdehyde 2,1-aminomutase and isopenicillin-N-epimerase with aminotransferases.

Profile analysis showed the title enzymes to be homologous with the aminotransferases. 1-Aminocyclopropane-1-carboxylate synthase is closely related to subgroup I of aminotransferases which includes aspartate, alanine, histidinol-phosphate, tyrosine and phenylalanine aminotransferase. 2,2-Dialkylglycine decarboxylase, glutamate-1-semialdehyde 2,1-aminomutase and 2-amino-6-caprolactam racemase are most similar to subgroup II which comprises aminotransferases with omega-amino acids as substrates. 8-Amino-7-oxononanoate synthase is closely related to both subgroup I and II, and isopenicillin-N-epimerase to subgroup IV with serine and phosphoserine aminotransferase. Aminotransferases and the title enzymes belong to a regio-specific family of evolutionarily related pyridoxal-5'-phosphate-dependent enzymes.

Acyltransferases↗
PubMed1993

Aminotransferases: demonstration of homology and division into evolutionary subgroups.

A total of 150 amino acid sequences of vitamin B6-dependent enzymes are known to date, the largest contingent being furnished by the aminotransferases with 51 sequences of 14 different enzymes. All aminotransferase sequences were aligned by using algorithms for sequence comparison, hydropathy patterns and secondary structure predictions. The aminotransferases could be divided into four subgroups on the basis of their mutual structural relatedness. Subgroup I comprises aspartate, alanine, tyrosine, histidinol-phosphate, and phenylalanine aminotransferases; subgroup II acetylornithine, ornithine, omega-amino acid, 4-aminobutyrate and diaminopelargonate aminotransferases; subgroup III D-alanine and branched-chain amino acid aminotransferases, and subgroup IV serine and phosphoserine aminotransferases. (N-1) Profile analysis, a more stringent application of profile analysis [Gribskov, M., McLachlan, A. D. and Eisenberg, D. (1987) Proc. Natl Acad. Sci. USA 84, 4355-4358], established the homology among the enzymes of each subgroup as well as among all subgroups except subgroup III. However, similarity of active-site segments and the hydropathy patterns around invariant residues suggest that subgroup III, though most distantly related, might also be homologous with the other aminotransferases. On the basis of the comprehensive alignment, a new numbering of amino acid residues applicable to aminotransferases (AT) in general is proposed. In the multiply aligned sequences, only four out of a total of about 400 amino acid residues proved invariant in all 51 sequences, i.e. Gly(314AT)197, Asp/Glu(340AT)222, Lys(385AT)258 and Arg(562AT)386, the number not in parentheses corresponding to the structure of porcine cytosolic aspartate aminotransferase. Apparently, the aminotransferases constitute a group of homologous proteins which diverged into subgroups and, with some exceptions, into substrate-specific individual enzymes already in the universal ancestor cell.

Animals↗
PubMed1993

Homology of pyridoxal-5'-phosphate-dependent aminotransferases with the cobC (cobalamin synthesis), nifS (nitrogen fixation), pabC (p-aminobenzoate synthesis) and malY (abolishing endogenous induction of the maltose system) gene products.

Bacterial deletion mutants have indicated that the gene products of cobC, nifS, pabC and malY participate in important metabolic pathways, i.e. cobalamin synthesis, nitrogen fixation, synthesis of p-aminobenzoate and the regulation of the maltose system, respectively. However, the proteins themselves and their specific functions have not yet been identified. In the course of our studies on the evolutionary relationships among aminotransferases, we have found that the above gene products are homologous to aminotransferases. Profile analysis [Gribskov, M., Lüthy, R. & Eisenberg, D. (1990) Methods Enzymol. 183, 146-159] based on the amino acid sequences of certain subgroups of aminotransferases as probes attributed significant Z scores in the range 5-20 SD to the deduced amino acid sequences of the above gene products as included in the protein data base. Reciprocal profile analyses confirmed the homologies. All known aminotransferases are pyridoxal-5'-phosphate-dependent enzymes and catalyze the reversible transfer of amino groups from amino acids to oxo acids. The sequence homologies suggest that the above gene products are aminotransferases or other closely related pyridoxal-5'-phosphate-dependent enzymes probably catalyzing transformations of amino acids involving cleavage of a bond at C alpha.

4-Aminobenzoic Acid↗
PubMed1990

Immunogenicity of mannophosphoinositides of mycobacteria: effect of cord factor (trehalose-6,6'-dimycolate).

Attempts were made to enhance the immunogenicity of mannophosphoinositides of mycobacteria against experimental tuberculosis. It was found that immunization of mice with a combination of mannosides and cord factor, coupled to methylated bovine serum albumin (MBSA) could not further alter the protective efficacy of mannoside-MBSA complexes against challenge with a LD50 dose of M. tuberculosis H37Rv as seen by survival-to-mortality rate, root-specific lung mass, lung density and bacterial counts recovered from the infected organs.

Animals↗
PubMed1990

Evolutionary and biosynthetic aspects of aspartate aminotransferase isoenzymes and other aminotransferases.

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase are homologous proteins. Both are encoded by nuclear DNA and synthesized on free polysomes. The organization of their genes is very similar, five out of a total of eight introns are located at the same nucleotide position. A variant consensus sequence was observed at the 3' splice site of introns of genes of imported mitochondrial proteins which may reflect the existence of splicing factors specific for the genes of this particular group of nuclear-encoded proteins. To date the amino acid sequences of 22 aminotransferases are known. A rigorous analysis yielded clear evidence that aspartate, tyrosine, and histidinol-phosphate aminotransferases are homologous proteins despite their low degree of sequence identity. The evolutionary relationship among the vitamin B6-dependent enzymes in general appears less clear. Conceivably, their common structural and mechanistic features are dictated by the chemical properties of pyridoxal 5'-phosphate rather than being due to a common ancestor of their protein moieties. In agreement with this notion, the ubiquitous active-site lysine residue that forms a Schiff base with the coenzyme can be replaced in the case of aspartate aminotransferase by a histidine residue without complete loss of catalytic competence.

Animals↗
PubMed1989

Evolutionary relationships among aminotransferases. Tyrosine aminotransferase, histidinol-phosphate aminotransferase, and aspartate aminotransferase are homologous proteins.

A data base was compiled containing the amino acid sequences of 12 aspartate aminotransferases and 11 other aminotransferases. A comparison of these sequences by a standard alignment method confirmed the previously reported homology of all aspartate aminotransferases and Escherichia coli tyrosine aminotransferase. However, no significant similarity between these proteins and any of the other aminotransferases was detected. A more rigorous analysis, focusing on short sequence segments rather than the total polypeptide chain, revealed that rat tyrosine aminotransferase and Saccharomyces cerevisiae and Escherichia coli histidinol-phosphate aminotransferase share several homologous sequence segments with aspartate aminotransferases. For comparison of the complete sequences, a multiple sequence editor was developed to display the whole set of amino acid sequences in parallel on a single work-sheet. The editor allows gaps in individual sequences or a set of sequences to be introduced and thus facilitates their parallel analysis and alignment. Several clusters of invariant residues at corresponding positions in the amino acid sequences became evident, clearly establishing that the cytosolic and the mitochondrial isoenzyme of vertebrate aspartate aminotransferase, E. coli aspartate aminotransferase, rat and E. coli tyrosine aminotransferase, and S. cerevisiae and E. coli histidinol-phosphate aminotransferase are homologous proteins. Only 12 amino acid residues out of a total of about 400 proved to be invariant in all sequences compared; they are either involved in the binding of pyridoxal 5'-phosphate and the substrate, or appear to be essential for the conformation of the enzymes.

Amino Acid Sequence↗