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Biomedical subjects

P Krishna

Publications and source records attributed to P Krishna.

At least 19 recordsLinked to original sources
PubMed2001

Hsp90-binding immunophilins in plants: the protein movers.

Studies of cytoplasmic-nuclear trafficking of the glucocorticoid receptor in mammalian cells suggest that the hsp90/hsp70-based chaperone system and the hsp90-binding immunophilin FKBP52 are involved in targeted movement of the receptor along microtubule tracts. Over the past few years, plant cells have been found to possess a similar multiprotein chaperone machinery. Plant cells also contain high molecular weight FKBPs that bind to plant hsp90 via a conserved protein interaction involving tetratricopeptide repeat domains. The hsp90/hsp70-based machinery and the plant FKBPs might be used to target the trafficking of signalling proteins in plants.

Animals↗
PubMed2001

Post-tonsillectomy bleeding: a meta-analysis.

OBJECTIVE/HYPOTHESIS: To reconcile conflicting reports and opinions of the value of preoperative coagulation studies for patients undergoing tonsillectomy. STUDY DESIGN: Meta-analysis: Articles were identified by MEDLINE search, references from review articles, textbook chapter, and retrieved reports. Independent observers selected prospective trials of patients undergoing tonsillectomy or adenoidectomy and tonsillectomy. Retrospective studies meeting other inclusion and exclusion criteria were included for sensitivity analyses of results. METHODS: Data were abstracted from studies for an end point of bleeding with normal and abnormal coagulation tests. Four prospective studies met all inclusion and exclusion criteria. These four studies were used in the data synthesis. An additional eight retrospective studies met all other criteria and were used in the sensitivity analysis. RESULTS: Pooled analysis of 3384 patients revealed a rate of 3.3% (95% confidence interval [CI], 2.5%-4.1%) for post-tonsillectomy bleeding in patients with normal coagulation studies. A rate of 8.7% (95% CI, 1.5%-15.9%) was obtained for bleeding in patients with abnormal coagulation studies. No significant rate difference in post-tonsillectomy bleeding was demonstrated. CONCLUSION: There is no difference in the rate of post-tonsillectomy bleeding in patients with abnormal coagulation studies as compared with patients with normal coagulation studies obtained preoperatively.

Blood Coagulation Disorders↗
PubMed2001

The Hsp90 family of proteins in Arabidopsis thaliana.

The 90-kDa heat shock protein (Hsp90) is an essential molecular chaperone in eukaryotic cells, with key roles in the folding and activation of proteins involved in signal transduction and control of the cell cycle. A search for Hsp90 sequences in the Arabidopsis thaliana genome revealed that this family includes 7 members. The AtHsp90-1 through AtHsp90-4 proteins constitute the cytoplasmic subfamily, whereas the AtHsp90-5, AtHsp90-6, and AtHsp90-7 proteins are predicted to be within the plastidial, mitochondrial, and endoplasmic reticulum compartments, respectively. The deduced amino acid sequences of each of the cytoplasmic proteins contains the highly conserved C-terminal pentapeptide MEEVD. All of the AtHsp90 sequences include a conserved adenosine triphosphate-binding domain, whereas only the cytoplasmic and endoplasmic reticulum-resident sequences include an adjacent charged linker domain that is common in mammalian and yeast sequences. The occurrence of multiple AtHsp90 proteins in the cytoplasm and of family members in other subcellular compartments suggests a range of specific functions and target polypeptides.

Amino Acid Sequence↗
PubMed2000

A glucosinolate mutant of Arabidopsis is thermosensitive and defective in cytosolic Hsp90 expression after heat stress.

The TU8 mutant of Arabidopsis previously described to be deficient in glucosinolate metabolism and pathogen-induced auxin accumulation was found to be remarkably less tolerant upon exposure to elevated temperatures than wild-type plants. Although moderately increased temperature only affected shoot growth, exposure to severe heat stress led to a dramatic decay of mutant plants. By contrast, wild-type seedlings showed little or no damage under the same conditions. Analysis of different heat stress proteins (Hsps) in TU8 seedlings revealed that only expression of cytoplasmic Hsp90 was affected in these plants. Although Hsp90 was present under control conditions, its level declined in mutant plants at elevated temperatures. Northern-blot analysis indicated that the decrease in Hsp90 protein was accompanied with a reduction of hsp90 transcript levels. Transient expression of Hsp90 in mutant protoplasts increased their survival rate at higher temperatures to near equivalent that of wild-type protoplasts. These data suggest that the reduced level of Hsp90 in TU8 mutants may be the primary cause for the observed reduction in thermostability.

Arabidopsis↗
PubMed1999

Pediatric hemorrhagic thyroid nodule: a case report.

Hemorrhagic thyroid nodules are rare in the pediatric age group. They present as rapidly enlarging neck masses. Diagnostic modalities available are laboratory evaluation, ultrasound, radionuclide imaging, and fine needle aspiration. Depending on the pattern of growth of the lesion, one may observe or proceed with surgery. A rapidly enlarging thyroid mass raises the suspicion of malignancy, and hemorrhagic nodules, though rare, must be considered in the differential diagnosis.

Biopsy↗
PubMed1999

Mass spectral study of substituted allyl aryl and allyl alkyl selenides and some analogous sulfides

The electron impact (EI) mass spectra of allyl aryl selenides showed abundant molecular ions and many fragment ions containing the selenium atom. alpha-Cleavage is the dominant process in the fragmentation of selenides, and cleavage product ions are characteristic of the substituents. In the case of 3-methyl allyl and related aryl selenides, characteristic delta-hydrogen migration to the selenium atom is observed. A McLafferty-type rearrangement is found in benzyl allyl selenides and substituted alkyl allyl selenides. The charge on the rearrangement products preferably remains on the fragments containing the phenyl group. The [M - SeH](+), [M - CH(3)](+) and [M - C(2)H(4)](+) ions are found only in the EI mass spectrum of allyl phenyl selenide, and are attributed to a Claisen rearrangement in the source of the mass spectrometer. All structurally informative fragmentation processes are supported by collision induced dissociation spectra of molecular ions. The fragmentation patterns found in methane chemical ionization (CI) spectra of the selenides were significantly different from those observed in EI. The EI and CI mass spectra of analogous sulfides showed similar behaviour to that observed in the corresponding selenides. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗
PubMed1999

Treatment with 24-epibrassinolide, a brassinosteroid, increases the basic thermotolerance of Brassica napus and tomato seedlings.

Brassinosteroids are plant growth-promoting compounds that exhibit structural similarities to animal steroid hormones. Recent studies have indicated that brassinosteroids are essential for proper plant development. In addition to a role in development, several lines of evidence suggest that brassinosteroids exert anti-stress effects on plants. However, the mechanism by which they modulate plant stress responses is not understood. We show here that Brassica napus and tomato seedlings grown in the presence of 24-epibrassinolide (EBR) are significantly more tolerant to a lethal heat treatment than are control seedlings grown in the absence of the compound. Since a preconditioning treatment of seedlings was not required to observe this effect, we conclude that EBR treatment increases the basic thermotolerance of seedlings. An analysis of heat shock proteins (HSPs) in B. napus seedlings by western blot analysis indicated that the HSPs did not preferentially accumulate in EBR-treated seedlings at the control temperature. However, after heat stress, HSP accumulation was higher in EBR-treated than in untreated seedlings. The results of the present study provide the first direct evidence for EBR-induced expression of HSPs. The higher accumulation of HSPs in EBR-treated seedlings raises the possibility that HSPs contribute, at least in part, to thermotolerance in EBR-treated seedlings. A search for factors other than HSPs, which may directly or indirectly contribute to brassinosteroid-mediated increase in thermotolerance, is underway.

Adaptation, Physiological↗
PubMed1998

Brassica napus hsp90 can autophosphorylate and phosphorylate other protein substrates.

A Brassica napus cDNA encoding the 90 kDa heat shock protein, hsp90, was modified to add 6 histidines at the C-terminus and expressed in insect cells to prepare a recombinant histidine-tagged hsp90. The recombinant protein was purified over Ni2+-NTA agarose columns and its identity was confirmed by Western blotting, using a plant hsp90-specific antiserum. Incubation of purified hsp90 with [gamma-32P] ATP in the presence of Mn2+ resulted in its autophosphorylation on serine residues. The purified hsp90 could also phosphorylate other protein substrates such as histones and casein in the presence of Mn2+. Analysis of phosphorylated casein revealed that serine residues are phosphorylated by hsp90. This is the first demonstration that a cytosolic hsp90 homolog can phosphorylate other protein substrates.

Animals↗
PubMed1998

High-molecular-weight FK506-binding proteins are components of heat-shock protein 90 heterocomplexes in wheat germ lysate.

In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249-15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.

Amino Acid Sequence↗
PubMed1997

Folding of the glucocorticoid receptor by the heat shock protein (hsp) 90-based chaperone machinery. The role of p23 is to stabilize receptor.hsp90 heterocomplexes formed by hsp90.p60.hsp70.

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor.hsp90 heterocomplexes. We have reconstituted a minimal receptor.hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833-12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR.hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). In this work, we show that addition of p23 to native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR.hsp90 heterocomplexes prepared with the minimal (hsp90.p60.hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR.hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90.p60.hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR.hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90.p60.hsp70 forms a GR.hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR.hsp90 heterocomplex to inactivation and disassembly.

Adenosine Triphosphate↗
PubMed1997

Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts.

A polyclonal antibody, R2, was raised against a fusion protein consisting of a portion of plant hsp90 fused to the trpE protein of Escherichia coli. This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins. The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants. Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings. An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca. 680 kDa in cell extracts of spinach cotyledons and leaves, B. napus seedlings and wheat germ. Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer. When 35S-labelled B. napus cytosolic extracts were immunoprecipitated with the R2 antiserum, hsp90 and two additional proteins with approximate molecular masses of 49 and 45 kDa were detected in the immunoprecipitates. These results are consistent with the idea that hsp90:protein heterocomplexes exist in plant cells.

Antibody Formation↗
PubMed1996

Binding of immunophilins to the 90 kDa heat shock protein (hsp90) via a tetratricopeptide repeat domain is a conserved protein interaction in plants.

In animal cell lysates, multiprotein complexes containing hsp90, hsp70, p60, p23, and several immunophilins can assemble steroid receptors and oncogenic protein kinases, such as v-Src and v-Raf, into heterocomplexes that contain hsp90 and either immunophilins or, in the case of protein kinases, p50. The complexes with hsp90 are required for the proper functioning of these signal transduction systems. Wheat germ lysate contains a similar protein folding activity that forms functional steroid receptor complexes with hsp90, but not all the components of this system have been identified. The plant chaperone system has conserved interactions with animal chaperones in that wheat hsp70 functions in the rabbit reticulocyte lysate heterocomplex assembly system and human p23 functions in the wheat germ lysate. Here, we ask if wheat germ lysate also contains immunophilins of the FK506-binding class (FKBPs) that bind to the hsp90 component of the chaperone complex via tetratricopeptide repeat (TPR) domains. To demonstrate the plant heterocomplex, we add purified mammalian p23, preadsorbed with the JJ3 antibody to protein A-Sepharose, to wheat germ lysate and allow ATP-dependent formation of an animal p23. plant hsp90 complex. The complex is then washed and incubated with the radiolabeled immunosuppressant drug [3H]FK506, which binds in a specific manner to a coimmunoadsorbed plant FKBP. Binding of the plant FKBP to plant hsp90 is prevented by adding to wheat germ lysate a purified fragment containing the TPR domains of human cyclophilin-40. Geldanamycin, a benzoquinone ansamycin that binds to animal hsp90s and prevents their chaperone activity, binds in a temperature-dependent manner to wheat hsp90 to block formation of the p23.hsp90.FKBP heterocomplex. These data show that immunophilin binding to hsp90 via TPR domains is conserved in the plant kingdom as well as in the animal kingdom and that geldanamycin will be an important tool for the study of hsp90-mediated protein chaperoning in plant cells.

Adenosine Triphosphate↗
PubMed1996

Animal and plant cell lysates share a conserved chaperone system that assembles the glucocorticoid receptor into a functional heterocomplex with hsp90.

The hormone-binding domain of the glucocorticoid receptor must be bound to heat shock protein (hsp) 90 for it to have a high-affinity steroid-binding conformation. Cell-free assembly of a glucocorticoid receptor-hsp90 heterocomplex is brought about in reticulocyte lysate by a preformed protein-folding complex containing hsp90, hsp70, and other proteins [Hutchison, K.A., Dittmar, K. D., & Pratt, W.B. (1994) J. Biol. Chem. 269, 27894-27899]. In this "foldosome" system, hsp70 is required for assembly of the receptor-hsp90 complex and concomitant activation of steroid-binding activity [Hutchison, K.A., Dittmar, K.D., Czar, M.J., & Pratt, W.B. (1994) J. Biol. Chem. 269, 22157-22161]. All previous experiments involving cell-free assembly of both receptor-hsp90 and protein kinase-hsp90 heterocomplexes have been carried out with the protein-folding system in rabbit reticulocyte lysate. In this work, we show that concentrated lysates of receptor-free mouse (L cells) and insect (Sf9) cells and also a plant (wheat germ) lysate fold the immunopurified glucocorticoid receptor into a functional (i.e., steroid binding) heterocomplex with hsp90. Receptor heterocomplex formation in animal lysates and in the plant lysate are not identical in that the dynamics of complex assembly are different, but both systems produce a functional complex that binds steroid. Also, in contrast to animal and insect complexes, receptor-plant hsp90 complexes are not stabilized by molybdate. When added to the other lysate, purified plant and animal hsp90s show partial complementarity, in that a receptor-hsp90 complex is formed but the receptor is not converted to the steroid-binding conformation. When added to rabbit reticulocyte lysate that has been depleted of endogenous hsp70, purified wheat germ and mouse hsp70's are equally active in promoting both assembly of receptor-hsp90 heterocomplexes and conversion of receptor to the steroid-binding conformation. Thus, hsp70 from the plant kingdom has conserved the ability to interact functionally with chaperone proteins of the animal kingdom to cooperate in protein folding as evidenced by formation of a functional receptor-hsp90 heterocomplex.

Animals↗
PubMed1995

The 23-kDa acidic protein in reticulocyte lysate is the weakly bound component of the hsp foldosome that is required for assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90.

The heat shock proteins hsp90 and hsp70 have been immunopurified from rabbit reticulocyte lysate in a multiprotein complex that acts as a self-sufficient protein folding machine. This immunopurified "foldosome" directs the assembly of the glucocorticoid receptor-hsp90 complex and refolds the receptor to the steroid binding state (Hutchison, K.A., Dittmar, K.D., and Pratt, W.B. (1994) J. Biol. Chem. 269, 27894-27899). Extensive washing of the immunoadsorbed foldosome eliminates a weakly bound component required for receptor heterocomplex assembly and folding. This protein factor is contained in a Centricon C-100 filtrate of lysate which reconstitutes the receptor activating activity of the washed foldosome. This hsp90-associated protein folding system is present in both animal and plant cells, and the Centricon C-100 fraction of rabbit reticulocyte lysate potentiates receptor folding directed by wheat germ lysate. We have used this ability to stimulate wheat germ lysate-directing folding of the glucocorticoid receptor as a rapid assay for the factor. We demonstrate that the activity segregates with the 23-kDa acidic protein component of the hsp90 foldosome when rabbit reticulocyte lysate is fractionated by ammonium sulfate precipitation and ion exchange chromatography. Immunoadsorption of the Centricon C-100 filtrate with a monoclonal antibody against p23 eliminates its ability to stimulate the wheat germ heterocomplex assembly/receptor folding system, and the activity is replaced by purified, bacterially expressed p23. Immunodepletion of p23 also eliminates the ability of the Centricon C-100 filtrate to reconstitute receptor activating activity of the washed foldosome and addition of purified, bacterially expressed p23 restores its activity, confirming that p23 is the weakly bound component of the foldosome complex required for refolding of the receptor to the steroid binding conformation.

Animals↗
PubMed1993

Interactions of anti-DNA antibodies with Z-DNA.

Systemic lupus erythematosus (SLE) sera, two classes of serum lipoproteins, and IgG antibodies from SLE and normal sera were tested for their reactivity with a Z-DNA polymer, Br-poly (dG-dC). In all cases preferential binding to Z-DNA over B-DNA was observed. This interaction, for the most part, could be inhibited by the negatively charged phospholipid, cardiolipin, which suggests that most of the anti-Z-DNA activity associated with sera arises from relatively non-specific ionic interactions between proteins and polyanionic molecules. An assay has been described that can eliminate proteins cross-reactive with negatively charged phospholipids.

Antibodies, Antinuclear↗
PubMed1991

Interaction of recA protein with left-handed Z-DNA.

The ability of recA protein to interact with a Z-DNA polymer, Br-poly(dG-dC), or M13 bacteriophage single-stranded DNA was investigated. RecA protein binds more avidly to Z-DNA than to single-stranded DNA in the absence of a nucleotide cofactor. This binding pattern changes in the presence of adenosine 5'-(gamma-thio)triphosphate (ATP[S]), however, such that the binding to Z-DNA decreases while binding to single-stranded DNA increases roughly 2-fold. When present together, the two forms of DNA compete with each other in the presence of ATP[S]. Experiments involving recA protein binding to recombinant plasmids showed neither a preferential binding of recA protein to the plasmid containing Z-DNA nor a similar effect of ATP[S] to that observed with the Z-DNA polymer. In contrast, maximal binding was obtained with a plasmid (linear or supercoiled) containing a polypurine.polypyrimidine insert, thus suggesting that recA protein displays sequence preferences in its interaction with DNA. The results of the present study provide no evidence that recA protein specifically interacts with or stabilizes the Z-DNA insert of a recombinant plasmid in the left-handed conformation.

Adenosine Triphosphate↗
PubMed1990

Are many Z-DNA binding proteins actually phospholipid-binding proteins?

We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In all cases, this Z-DNA binding was strongly blocked by competition with acidic phospholipids, such as cardiolipin. Our results raise the question whether many of the Z-DNA binding proteins previously isolated are actually phospholipid-binding proteins.

Carrier Proteins↗