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P Malik

Publications and source records attributed to P Malik.

At least 19 recordsLinked to original sources
PubMed2000

Perfusion chromatography for very rapid purification of class I and II MHC proteins.

Major histocompatibility complex (MHC) proteins are surface glycoproteins that are strongly associated with either self or foreign peptides. Their interaction with the T-cell receptor on the T-cells initiates an immune response and help in discriminating between self and non-self, respectively. We describe here a novel means of rapidly purifying human MHC molecules on either small scale or large scale from the cell lysate of lymphoblastoid B cell line and from insect cell culture supernatants by using affinity perfusion chromatography. As representative cases HLA-B2705, a class I MHC molecule, and HLA-DR1, a class II MHC molecule were purified from EBV-transformed human lymphoblastoid B cells, LG2. Soluble HLA-DR1 was also purified from the cell culture supernatant of insect cells. The peptides eluted from the purified HLA-B2705 were pool sequenced and found to have the same motif as has previously been published. This new method provides a very rapid means of purifying MHC protein molecules, applicable to both large scale and small scale purification, which in turn greatly enhances the accuracy of further analysis of the associated peptides through mass spectrometry.

Animals↗
PubMed2000

Ocean tides.

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Canada↗
PubMed1999

Constitutive HOXA5 expression inhibits erythropoiesis and increases myelopoiesis from human hematopoietic progenitors.

The role of the homeobox gene HOXA5 in normal human hematopoiesis was studied by constitutively expressing the HOXA5 cDNA in CD34(+) and CD34(+)CD38(-) cells from bone marrow and cord blood. By using retroviral vectors that contained both HOXA5 and a cell surface marker gene, pure populations of progenitors that expressed the transgene were obtained for analysis of differentiation patterns. Based on both immunophenotypic and morphological analysis of cultures from transduced CD34(+) cells, HOXA5 expression caused a significant shift toward myeloid differentiation and away from erythroid differentiation in comparison to CD34(+) cells transduced with Control vectors (P =.001, n = 15 for immunophenotypic analysis; and P <.0001, n = 19 for morphological analysis). Transduction of more primitive progenitors (CD34(+)CD38(-) cells) resulted in a significantly greater effect on differentiation than did transduction of the largely committed CD34(+) population (P =.006 for difference between HOXA5 effect on CD34(+) v CD34(+)CD38(-) cells). Erythroid progenitors (burst-forming unit-erythroid [BFU-E]) were significantly decreased in frequency among progenitors transduced with the HOXA5 vector (P =.016, n = 7), with no reduction in total CFU numbers. Clonal analysis of single cells transduced with HOXA5 or control vectors (cultured in erythroid culture conditions) showed that HOXA5 expression prevented erythroid differentiation and produced clones with a preponderance of undifferentiated blasts. These studies show that constitutive expression of HOXA5 inhibits human erythropoiesis and promotes myelopoiesis. The reciprocal inhibition of erythropoiesis and promotion of myelopoiesis in the absence of any demonstrable effect on proliferation suggests that HOXA5 diverts differentiation at a mulitpotent progenitor stage away from the erythroid toward the myeloid pathway.

Cells, Cultured↗
PubMed1999

Origins of heart rate variability. Inducibility and prevalence of a discrete, tachycardic event.

BACKGROUND: We propose that heart period sequences are linearly organized, like sentences, and that there is a lexicon of recurrent, similarly shaped transient structures like words. Each word (or lexon) has a characteristic physiological basis. One potential lexon is the transient, reversible tachycardia that is induced by exercise initiation under laboratory conditions. We hypothesized that this lexon was inducible and observable on ambulatory ECGs of most or all subjects, was morphologically similar in both induced and detected bursts, and shared a plausible origin in both circumstances. METHODS AND RESULTS: Ten healthy subjects (mean age, 36 years) underwent a protocol in which subjects rolled themselves from supine to lateral decubitus positions and back. Transient tachycardias ("bursts") were seen in 36 of 40 rollovers. Bursts were characterized by an initial monoexponential heart period decay (K=0.39+/-0.23 s-1), a maximum heart period decrease of 277+/-109 ms after 10.8+/-4.5 seconds, and a subsequent return to baseline 23.3+/-10.8 seconds after roll initiation. The roll-induced bursts were detected with 97% sensitivity and 99% specificity with a search algorithm that incorporated morphological parameters. In 24-hour ambulatory ECGs of 10 healthy subjects (mean age, 38 years; range, 17 to 69 years), 117+/-59 bursts were detected. Induced and detected bursts were similar in most morphological parameters. Finally, many bursts occurred at night, when rolling over also occurs. CONCLUSIONS: Bursts are inducible, transient tachycardias that occur clinically and constitute a lexon with an understandable physiology.

Adolescent↗
PubMed1999

Human CD16 as a lysis receptor mediating direct natural killer cell cytotoxicity.

In addition to their role in peptide antigen presentation, class I MHC proteins also play a critical role in inhibiting natural killer (NK) cytotoxicity through interaction with NK inhibitory receptors. Thus, NK cells are cytotoxic to virus-infected and tumor cells that have lost class I MHC protein expression. However, the nature of the receptors involved in the triggering of lysis of target cells is poorly understood. CD16 (Fcgamma receptor III) has been described as a receptor expressed on NK cells that facilitates antibody-dependent cellular cytotoxicity (ADCC) by binding to the Fc portion of various antibodies. However, we show here that CD16 has a broader function and is directly involved in the lysis of some virus-infected cells and tumor cells, independent of antibody binding. The presence of a putative CD16 ligand on appropriate target cells has also been demonstrated by the use of a CD16-Ig fusion protein.

Antibodies, Monoclonal↗
PubMed1999

Recognition of HIV-derived B and T cell epitopes displayed on filamentous phages.

The amino acid sequence of HIV reverse transcriptase (RT) from residue 248 to residue 262 was expressed on the surface of filamentous phage fd, fused to the major coat protein gVIIIp. The chimeric phage was used to assess the ability of anti-RT (248-262) human T cell lines and clones to become activated by the phage-displayed peptide. The RT peptide displayed on phage was recognized by the T-cells and induced production of Abs. However, not all T cells raised against the synthetic RT (248-262) peptide could respond. Lack of recognition did not depend on differences in the ability of different APCs to present the phage, but was apparently determined by the TCR specificity. The results presented here may be relevant to the design of recombinant protein-based subunit vaccines.

Animals↗
PubMed1999

Effects of temperature and Y21M mutation on conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd.

Solid-state NMR spectroscopy was used to analyze the conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd. Both one and two-dimensional solid-state NMR spectra of magnetically aligned samples of fd bacteriophage reveal that an increase in temperature and a single site substitution (Tyr21 to Met, Y21M) reduce the conformational heterogeneity observed throughout wild-type pVIII. The NMR results are consistent with previous studies indicating that conformational flexibility in the hinge-bend segment that links the amphipathic and hydrophobic helices in the membrane-bound form of the protein plays an essential role during phage assembly, which involves a major change in the tertiary, but not secondary, structure of the coat protein.

Capsid↗
PubMed1999

Interaction of the globular domains of pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli.

Gene 3 protein (pIII), a minor coat protein at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus of Escherichia coli cells that display an F-pilus. Infection is thought to start with the adsorption of the D2 domain of pIII to the tip of the pilus, retraction of the pilus, and penetration of the E. coli cell membrane mediated by an interaction between the D1 domain of pIII and the Tol protein complex in the membrane. A subgene encoding the pIII-D1D2 di-domain was created, and the subgene was successfully overexpressed in E. coli cells. Domains D1 and D2 were separated after limited proteolysis of a modified pIII-D1D2 (designated pIII-D1D2.trp) into which two tryptic cleavage sites were introduced at appropriate points. The purified pIII-D1D2 di-domain and pIII-D2 domain were able to bind to the F-pilus, competing with the wild-type pIII and delaying infection by the intact filamentous phage. The pIII-D1 domain was unable to bind to the F-pilus by this criterion. This provides conclusive evidence that the pIII-D2 domain is responsible for the adsorption to the tip of the F-pilus and can achieve this in the absence of domain D1, opening the way to identifying the molecular basis of the interaction of pIII-D2 with the pilus.

Binding Sites↗
PubMed1999

Biotinylation of class I MHC molecules abrogates recognition by W6/32 antibody.

W6/32 is one of the most common monoclonal antibodies (mAb) used to characterize human class I major histocompatibility complex (MHC) molecules. It recognizes a conformational epitope on the intact MHC molecule containing both beta2-microglobulin (beta2-m) and the heavy chain. Labelling proteins by biotinylation is a very useful technique of for their detection, purification and analysis. A common method for biotinylating proteins is through the use of N-hydroxysuccinimide (NHS) biotin or Sulfo-NHS-biotin where the free amino groups on the protein are used for coupling the biotin moiety. However, W6/32 was unable to effectively immunoprecipitate biotinylated human class I MHC molecules including the human non-classical HLA-G molecule. FACScan analysis confirmed that biotinylating human class I MHC and HLA-G molecules prevents the recognition of these molecule by W6/32. In contrast, the recognition by another conformation-dependent monoclonal antibody, ME1, specific to HLA-B27 molecules, remained totally unaffected.

Antibodies, Monoclonal↗
PubMed1999

Molecular characterization of an Indian isolate of Japanese encephalitis virus that shows an extended lag phase during growth.

The biological properties of an Indian isolate (GP78) of Japanese encephalitis virus (JEV) were characterized in tissue-cultured cells and mice and these were compared with the JaOArS982 strain from Japan. The GP78 strain had a markedly extended lag phase during its growth in porcine stable kidney (PS) cells. There were no obvious defects in the penetration of GP78 into PS cells. However, viral RNA and protein synthesis were significantly delayed in GP78-infected PS cells. Fusion-from-within assays carried out in C6/36 cells indicated that GP78 was less fusogenic than the JaOArS982 strain of JEV. Moreover, maximum fusion in GP78-infected cells occurred at pH 5.5, whereas JaOArS982-infected cells showed maximum fusion at pH 6.0. These results suggested that there may be a lesion in the virus-cell fusion process. The GP78 strain also showed delayed growth in brains of 1-week-old BALB/c mice. Although JEV GP78 was as virulent as the JaOArS982 strain in these mice, the appearance of clinical symptoms of JEV infection was delayed by a day in mice infected with the GP78 strain and these animals showed an increased average survival time. Comparison of the nucleotide sequences of the GP78 and the JaOArS982 strains of JEV identified a number of amino acid substitutions in structural proteins. Of these, a Thr --> Met substitution at residue 76 of the envelope protein is predicted to be causally associated with the altered biology of the GP78 strain during growth.

Amino Acid Sequence↗
PubMed1999

Complete nucleotide sequence of an Indian strain of Japanese encephalitis virus: sequence comparison with other strains and phylogenetic analysis.

The RNA genome of an Indian strain of Japanese encephalitis virus (JEV), GP78, was reverse transcribed and the cDNA fragments were cloned in bacterial plasmids. Nucleotide sequencing of the cDNA clones covering the entire genome of the virus established that the GP78 genome was 10,976 nucleotides long. An open reading frame of 10,296 bases, capable of coding for a 3,432 amino acid polyprotein, was flanked by 95- and 585-base long 5'- and 3'-non-coding regions, respectively. When compared with the nucleotide sequence of the JaOArS982 strain, the JEV GP78 genome had a number of nucleotide substitutions that were scattered throughout the genome except for the 5'-noncoding region, the sequence of which was fully conserved. Comparison of the complete genome sequences of different JEV isolates showed a 1.3-4.1% nucleotide sequence divergence among them, which resulted in 0.6-1.8% amino acid sequence divergence. Analysis based on the complete genome sequences of different JEV isolates showed that the GP78 isolate from India was phylogenetically closer to the Chinese SA14 isolate.

Adult↗
PubMed1999

Hidden treasure.

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Canada↗