Biomedical subjects

A Poddar

Publications and source records attributed to A Poddar.

16 recordsLinked to original sources

PubMed2001

Chaperone-like activity of tubulin. binding and reactivation of unfolded substrate enzymes.

The eukaryotic cytoskeletal protein tubulin is a heterodimer of two subunits, alpha and beta, and is a building block unit of microtubules. In a previous communication we demonstrated that tubulin possesses chaperone-like activities by preventing the stress-induced aggregation of various proteins (Guha, S., Manna, T. K., Das, K. P., and Bhattacharyya, B. (1998) J. Biol. Chem. 273, 30077-30080). As an extension of this observation, we explored whether tubulin, like other known chaperones, also protected biological activity of proteins against thermal stress or increased the yields of active proteins during refolding from a denatured state. We show here that tubulin not only prevents the thermal aggregation of alcohol dehydrogenase and malic dehydrogenase but also protects them from loss of activity. We also show that tubulin prevents the aggregation of substrates during their refolding from a denatured state and forms a stable complex with denatured substrate. The activity of malic dehydrogenase, alpha-glucosidase, and lactate dehydrogenase during their refolding from urea or guanidium hydrochloride denatured states increased significantly in presence of tubulin compared with that without tubulin. These results suggest that tubulin, in addition to its role in mitosis, cell motility, and other cellular events, might be implicated in protein folding and protection from stress.

Alcohol Dehydrogenase↗

PubMed2001

Role of the carboxy-termini of tubulin on its chaperone-like activity.

Mutational analysis and the enzymatic digestion of many chaperones indicate the importance of both hydrophobic and hydrophilic residues for their unique property. Thus, the chaperone activity of alpha-crystallin is lost due to the substitution of hydrophobic residues or upon enzymatic digestion of the negatively charged residues. Tubulin, an eukaryotic cytoskeletal protein, exhibits chaperone-like activity as demonstrated by prevention of DTT-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase, betagamma-crystallin, and other proteins. We have shown that the tubulin lost its chaperone-like activity upon digestion of its negatively charged C-termini. In this article, the role of the C-terminus of individual subunits has been investigated. We observe that the digestion of C-terminus of beta-subunit with subtilisin causes loss of chaperone-like activity of tubulin. The contribution of C-terminus of alpha-subunit is difficult to establish directly as subtilisin cleaves C-terminus of beta-subunit first. This has been ascertained indirectly using a 14-residue peptide P2 having the sequence corresponding to a conserved region of MHC class I molecules and that binds tightly to the C-terminus of alpha-subunit. We have shown that the binding of P2 peptide to alphabeta-tubulin causes complete loss of its chaperone-like activity. NMR and gel-electrophoresis studies indicate that the P2 peptide has a significant higher binding affinity for the C-terminus of alpha-subunit compared to that of beta-subunit. Thus, we conclude that both the C-termini are necessary for the chaperone-like activity of tubulin. Implications for the chaperone functions in vivo have been discussed.

Amino Acid Sequence↗

PubMed2001

The IML3/MCM19 gene of Saccharomyces cerevisiae is required for a kinetochore-related process during chromosome segregation.

The mcm19 mutation in budding yeast affects minichromosome maintenance. In this work we have shown that this mutation leads to defects in the segregation of minichromosomes and chromosomes. The mutant cells show defective kinetochore function as judged by three criteria-- relaxation of the transcriptional block normally associated with a CEN box, stable maintenance of a dicentric plasmid in mutant cells, and mild sensitivity to the antimicrotubule drug benomyl. The MCM19 gene has been cloned and found to be the same as IML3, which codes for the ORF YBR107C. Deletion of the gene was not lethal, nor did it confer any growth defects on the mutant cells. However, the mcm19 null mutation conferred growth defects in the presence of a mutation in the TUB1 gene coding for alpha-tubulin. Two-hybrid experiments showed an interaction between Im13p/Mcm19p and the kinetochore protein Ch14, indicating that the Im13/Mcm19 protein has a role in kinetochore function.

Benomyl↗

PubMed2001

The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain.

We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed1999

MCM21 and MCM22, two novel genes of the yeast Saccharomyces cerevisiae are required for chromosome transmission.

The minichromosome maintenance genes, MCM21 and MCM22, have been cloned and are shown to code for the ORFs YDR318W and YJR135C respectively. Mutations in these genes caused a decrease in the stability of the minichromosome. This decrease in stability was associated with an increase in the copy number of the minichromosome in cells carrying it. Small circular dicentric plasmids were maintained relatively stably and structurally intact in the mutants compared with the wild-type strain. In the latter, such plasmids were mitotically unstable and, upon recovery, showed frequent rearrangements of their DNA. A centromere offered less obstruction to transcription in mutant cells than in the wild type, showing that both these mutants had a more relaxed kinetochore assembly. The mutant strains showed elevated rates of chromosome loss but not those of recombination. Both the mutations caused the cells to display a higher sensitivity towards the anti-mitotic drug benomyl. All these observations suggest that MCM21 and MCM22 are important for chromosome segregation with a potential role in kinetochore function. These genes are non-essential, as their deletions from chromosomes did not cause loss of cell viability. However, exponentially growing mutant cells carrying the deletion of the MCM21 gene had a significant population of large-budded cells with a single nucleus at the neck. Furthermore, the DNA content of these cells showed a shift towards 2N, suggesting a temporary pause of cells in G2 or in an early phase of mitosis. The mcm21 and mcm22 mutations do not show synthetic lethality or any further enhancement of growth defects, implying that they could be carrying out non-overlapping functions in chromosome segregation.

Cell Cycle Proteins↗

PubMed1997

Receptor induction regulates the synergistic effects of substance P with IL-1 and platelet-derived growth factor on the proliferation of bone marrow fibroblasts.

The neuropeptide substance P (SP) stimulates CFU in bone marrow (BM) cultures. Although the methylcellulose matrix used in these assays does not provide an appropriate substratum to support adherent-dependent cells, we have observed that cultures containing optimal SP (10(-8)-10(-10) M) develop confluent areas of reticular/fibroblastoid-like cells with CFUs predominantly localized within their vicinity. Characterization (cytochemical and immunofluorescence) of the reticular/fibroblastoid-like cells indicated that they were fibroblasts, the major constituent of the BM stroma. Hemopoietic effects by SP are mediated by the stroma that expresses SP receptors. We studied the effects of SP (10(-7)-10(-11) M) with suboptimal platelet-derived growth factor-BB (PDGF-BB; 5 ng/ml) and IL-1alpha (2 ng/ml), two fibrogenic cytokines, and also hemopoietic regulators. SP by itself and in synergy with either cytokine induced fibroblast proliferation. At optimum SP, IL-1alpha induced 1.6 times the proliferation of PDGF-BB (87 +/- 7 vs 55 +/- 5; n = 12; p < 0.05). The effects of SP were blunted by a specific neurokinin-1 antagonist. Scatchard analysis indicated that SP binds to BM fibroblasts with an approximate Kd of 5 nM. SP induced steady state mRNA for IL-1 receptor IL-1RI and PDGF-BB (PDGF-AR, PDGF-BR) receptors by 7.5-, 6.2-, and 10.5-fold, respectively. Their up-regulation may be partly responsible for the synergistic effects of SP and their ligands. Induction (3-fold) of neurokinin-1 mRNA by IL-1alpha compared with no induction by PDGF-BB may explain the preferred synergism between SP and IL-1alpha. This study indicates that induction of SP, IL-1alpha, and PDGF-BB receptors is important to their synergistic effects on BM fibroblast proliferation. These results bring new insights into stroma-mediated hemopoietic regulation.

Animals↗

PubMed1997

The mcm17 mutation of yeast shows a size-dependent segregational defect of a mini-chromosome.

Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not stably maintain mini-chromosomes. Out of these, the ARS-specific class has been more extensively studied and is found to lose chromosomes and mini-chromosomes due to a defect in the initiation of DNA replication at yeast ARSs. In the present study we have identified a number of mcm mutants which show size-dependent loss of mini-chromosomes. When the size of the mini-chromosome was increased, from about 15 kb to about 60 kb, there was a dramatic increase in its mitotic stability in these mutants, but not in the ARS-specific class of mutants. One mutant, mcm17, belonging to the size-dependent class was further characterized. In this mutant, cells carried mini-chromosomes in significantly elevated copy numbers, suggesting a defect in segregation. This defect was largely suppressed in the 60-kb mini-chromosome. A non-centromeric plasmid, the TRP1ARS1 circle, was not affected in its maintenance. This mutant also displayed enhanced chromosome-III loss during mitosis over the wild-type strain, without elevating mitotic recombination. Cloning and sequencing of MCM17 has shown it to be the same as CHL4, a gene required for chromosome stability. This gene is non-essential for growth, as its disruption or deletion from the chromosome did not affect the growth-rate of cells at 23 degrees C or 37 degrees C. This work suggests that centromere-directed segregation of a chromosome in yeast is strongly influenced by its length.

Cell Cycle Proteins↗

PubMed1997

Hematopoietic regulation mediated by interactions among the neurokinins and cytokines.

This review summarizes the current data regarding the mechanisms by which two mammalian neurokinins (tachykinins), substance P (SP) and neurokinin-A (NK-A) are involved in hematopoiesis. SP and NK-A are derived from the preprotachykinin-I (PPT-I) gene which can be induced by cytokines and neurotrophic factors. In the bone marrow (BM), nerve fibers and stroma are potential sources for the PPT-I gene products. SP and NK-A interact with either of three cloned receptors, neurokinin-1 (NK-1), NK-2 or NK-3, although SP and NK-A exhibit binding preferences for NK-1 and NK-2 respectively. Through specific receptors, SP and NK-A exert dichotomous hematopoietic effects mediated mostly by the BM stroma. SP enhances the proliferation of primitive BM stem cells and progenitors and these effects correlate with the induction of stimulatory hematopoietic growth factors. NK-A appears to be protective to stem cells through the induction of TGF-beta. Proliferation of myeloid progenitors is inhibited by NK-A, effects which correlate with the induction of two suppressive factors, TGF-beta and MIP-1alpha. Stimulation of NK-2 leads to partial blunting of the enhanced stimulatory effects mediated by NK-1. Furthermore, stimulatory hematopoietic cytokines upregulate NK-1 expression and downregulate the constitutively expressed NK-2 in BM stroma. Together, the experimental evidence suggests that NK-A-NK-2 interactions could be a feedback to hematopoietic stimulation. Expression of NK-1 and NK-2 in CD34+ cell lines and also, the presence of SP binding sites on primary CD34+ cells suggest that the neurokinins could be interacting directly with BM progenitors and stem cells. In BM stroma, cytokines and neurokinins regulate the expression of each other and also, their respective receptors. In summary, the current literature pertaining to hematopoietic regulation indicates the involvement of a complex network that includes, but not exclusive of the cytokines and neurokinins. The current models that pertain to stem cell proliferation and differentiation should therefore add neuropeptides to the list of hematopoietic modulators.

Animals↗

PubMed1995

Predatory aggression induced by hypothalamic stimulation: modulation by midbrain periaqueductal gray (PAG).

Adequate electrical stimulation of extreme lateral hypothalamic regions of healthy, non-aggressive male cats was employed to produce aggression on live but anaesthetized rats. Stimulus response (S-R) curves based on scoring systems for both somatic and affective display components of behaviour were used to assess how manipulation of midbrain PAG by electrocoagulative lesions or drug microinjections affected the sensitivity of attack producing hypothalamic loci. Anodal lesions of dorsal PAG and adjoining tectum increased the excitability of hypothalamic loci producing predatory attack. Microinjection of 250 ng of delta-alanine-methionine enkephalin (DAME) in dPAG completely suppressed the somatomotor components of attack behaviour and markedly inhibited the affective display components. Administration of naloxone, an opioid antagonist (1 microgram) at the same sites facilitated the hypothalamically induced attack behaviour and annulled the inhibitory effect of DAME. These findings indicate the involvement of midbrain enkephalinergic mechanisms in the modulation of predatory attack behaviour elaborated by hypothalamic stimulation.

Aggression↗

PubMed1993