Chemical approaches to penicillin allergy—IV. Binding of carrier receptor protein with penicillin and its analogues

The availability of electrophoretically homogeneous rabbit penicillin carrier receptor protein (CRP) by affinity chromatography afforded an idealin vitro system to calculate the thermodynamic parameters of binding of penicillin and analogues with CRP as well as competitive binding of such analogues with CRP in presence of14C-penicillin G. The kinetics of association of CRP with 7-deoxy penicillin which does not bind covalently with CRP have been studied through equilibrium dialysis with14C-7-deoxybenzyl penicillin and found to be K=2·79×106M−1.−ΔG=8·106 k cal/mole as well as fluorescence quenching studies with exciter λ 280 K=3·573×106M−1,−ΔG=8·239 k cal/mole. The fluorescence quenching studies have been extended to CRP-benzyl penicillin and CRP-6-aminopenicillanic acid (6APA) systems also. The fluorescence data with benzyl penicillin indicate two conformational changes in CRP—a fast change corresponding to the non-covalent binding to CRP with 7-deoxy penicillin and a slower change due to covalent bond formation. With 6-APA the first change is not observed but the conformational change corresponding to covalent binding is only seen. Competitive binding studies indicate that the order of binding of CRP with the analogues of penicillin is as follows: methicillin > 6APA > carbenicillin >o-nitrobenzyl penicillin > cloxacillin ≈ benzyl penicillin ≈ 6-phenyl acetamido penicillanyl alcohol ≈ 7 phenyl acetamido desacetoxy cephalosporanic acid ≈p-amino benzyl penicillin ≈p-nitro benzyl penicillin > ticarcillin >o-amino benzyl penicillin > amoxycillin > 7-deoxy benzyl penicillin > ampicillin.From these data it has been possible to delineate partially the topology of the penicillin binding cleft of the CRP as well as some of the functional groups in the cleft responsible for the binding process.

Diabetic cardiomyopathy and post-infarct ventricular remodelling : Effects of levosimendan in a rodent model of type II diabetes

Type 2 diabetes is a risk factor for the development of cardiovascular disease. Recently, the term diabetic cardiomyopathy has been proposed to describe the changes in the heart that occur in response to chronic hyperglycemia and insulin resistance. Ventricular remodelling in diabetic cardiomyopathy includes left ventricular hypertrophy, increased interstitial fibrosis, apoptosis and diastolic dysfunction. Mechanisms behind these changes are increased oxidative stress and renin-angiotensin system activation. The diabetic Goto-Kakizaki rat is a non-obese model of type 2 diabetes that exhibits defective insulin signalling. Recently two interconnected stress response pathways have been discovered that link insulin signalling, longevity, apoptosis and cardiomyocyte hypertrophy. The insulin-receptor PI3K/Ak pathway inhibits proapoptotic FOXO3a in response to insulin signalling and the nuclear Sirt1 deacetylase inhibits proapoptotic p53 and modulates FOXO3a in favour of survival and growth. --- Levosimendan is a calcium sensitizing agent used for the management of acute decompensated heart failure. Levosimendan acts as a positive inotrope by sensitizing cardiac troponin C to calcium and exerts vasodilation by opening mitochondrial and sarcolemmal ATP-sensitive potassium channels. Levosimendan has been described to have beneficial effects in ventricular remodelling after myocardial infarction. The aims of the study were to characterize whether diabetic cardiomyopathy associates with cardiac dysfunction, cardiomyocyte apoptosis, hypertrophy and fibrosis in spontaneously diabetic Goto-Kakizaki (GK) rats, which were used to model type 2 diabetes. Protein expression and activation of the Akt FOXO3a and Sirt1 p53 pathways were examined in the development of ventricular remodelling in GK rats with and without myocardial infarction (MI). The third and fourth studies examined the effects of levosimendan on ventricular remodelling and gene expression in post-MI GK rats. The results demonstrated that diabetic GK rats develop both modest hypertension and features similar to diabetic cardiomyopathy including cardiac dysfunction, LV hypertrophy and fibrosis and increased apoptotic signalling. MI induced a sustained increase in cardiomyocyte apoptosis in GK rats together with aggravated LV hypertrophy and fibrosis. The GK rat myocardium exhibited decreased Akt- FOXO3a phosphorylation and increased nuclear translocation of FOXO3a and overproduction of the Sirt1 protein. Treatment with levosimendan decreased cardiomyocyte apoptosis, senescence and LV hypertrophy and altered the gene expression profile in GK rat myocardium. The findings indicate that impaired cardioprotection via Akt FOXO3a and p38 MAPK is associated with increased apoptosis, whereas Sirt1 functions in counteracting apoptosis and the development of LV hypertrophy in the GK rat myocardium. Overall, levosimendan treatment protects against post-MI ventricular remodelling and alters the gene expression profile in the GK rat myocardium.

Interaction of rat testis protein, TP, with nucleosome core particle

Circular dichroism studies have revealed that addition of testis specific protein, TP in vitro, to rat testes nucleosome core particle resulted in a decrease in the compaction of the core particle DNA. This was also corroborated by thermal denaturation analysis. Addition of TP to nucleosome core particle resulted in the conversion of a biphasic transition towards a single phase. However, at the same time there was a 20% reduction in the overall hyperchromicity of core particle DNA at core particle to TP molar ratios of 1:2 and 1:3. These observations along with our earlier report, showing the DNA melting properties of TP, suggest that TP may play an important role in the disassembly process of nucleosome core particle during spermiogenesis.

Isolation Of A Cytokinin-Binding Protein From Cucumis-Sativus Cotyledons

A cytokinin-binding protein which exists as monomer and dimer was isolated from cucumber (Cucumis sativus L. var Guntur) cotyledons by affinity chromatography on AH-Sepharose-pi6 Ap /s> column. The protein bound to [3H]-N6-(Δ2-isopentenyl) adenosine trialcohol. On Sephadex G-50 chromatography it gave 2 peaks corresponding to molecular weight 4000 and 8000 daltons. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis, it gave only one band with an apparent molecular weight of 4000 daltons.

Identification of TGFβ signaling, p53, and actin stress fibers as targets of LKB1 tumor suppressor activity

Tumorigenesis is a consequence of inactivating mutations of tumor suppressor genes and activating mutations of proto-oncogenes. Most of the mutations compromise cell autonomous and non-autonomous restrains on cell proliferation by modulating kinase signal transduction pathways. LKB1 is a tumor suppressor kinase whose sporadic mutations are frequently found in non-small cell lung cancer and cervical cancer. Germ-line mutations in the LKB1 gene lead to Peutz-Jeghers syndrome with an increased risk of cancer and development of benign gastrointestinal hamartomatous polyps consisting of hyperproliferative epithelia and prominent stromal stalk composed of smooth muscle cell lineage cells. The tumor suppressive function of LKB1 is possibly mediated by 14 identified LKB1 substrate kinases, whose activation is dependent on the LKB1 kinase complex. The aim of my thesis was to identify cell signaling pathways crucial for tumor suppression by LKB1. Re-introduction of LKB1 expression in the melanoma cell line G361 induces cell cycle arrest. Here we demonstrated that restoring the cytoplasmic LKB1 was sufficient to induce the cell cycle arrest in a tumor suppressor p53 dependent manner. To address the role of LKB1 in gastrointestinal tumor suppression, Lkb1 was deleted specifically in SMC lineage in vivo, which was sufficient to cause Peutz-Jeghers syndrome type polyposis. Studies on primary myofibroblasts lacking Lkb1 suggest that the regulation of TGFβ signaling, actin stress fibers and smooth muscle cell lineage differentiation are candidate mechanisms for tumor suppression by LKB1 in the gastrointestinal stroma. Further studies with LKB1 substrate kinase NUAK2 in HeLa cells indicate that NUAK2 is part of a positive feedback loop by which NUAK2 expression promotes actin stress fiber formation and, reciprocally the induction of actin stress fibers promote NUAK2 expression. Findings in this thesis suggest that p53 and TGFβ signaling pathways are potential mediators of tumor suppression by LKB1. An indication of NUAK2 in the promotion of actin stress fibers suggests that NUAK2 is one possible mediator of LKB1 dependent TGFβ signaling and smooth muscle cell lineage differentiation.

Vitamin A and thyroxine carrier proteins in chicken plasma Steady-state control of the plasma level of free retinol-binding protein and free thyroxine

1. 1. The binding parameters of prealbumin-2 with retinol-binding protein and thyroxine (T4) revealed the existence of distinct and multiple sites for both retinol-binding protein and T4. 2. 2. From the analysis of binding parameters of retinol-binding protein with prealbumin-2 it is clear that under steady-state conditions about 99% of the holo-retinol-binding protein remains bound to prealbumin-2. 3. 3. Equilibrium dialysis studies on binding properties of thyroid hormones with prealbumin-2 revealed that it has a single high affinity site and three low affinity sites. 4. 4. The occurrence of three carrier proteins for thyroid hormones, thyroxine-binding globulin, prealbumin-2 and albumin has been demonstrated. However, the chicken thyroxine-binding globulin differs from human thyroxine-binding globulin by being relatively less acidic and occuring at a two-fold lower concentration. But the thyroid hormone binding parameters are comparable. 5. 5. Highly sensitive methods were developed for determination of T4 binding capacities of the various proteins and plasma level of total T4 by fractionation of carrier proteins and further quantitatively employing in electrophoresis and equilibrium dialysis. 6. 6. The thyroxine-binding proteins were found to be two types, one (viz., thyroxine-binding globulin) of great affinity but of low binding capacity, which mainly acts as reservoir of T4, and another (viz.,prealbumin-2) of low affinity but of high binding capacity, which can participate predominantly in the control of the free T4 pool.

High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (ICFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K-D similar to 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access In recent years, IGFBPs have been implicated in a variety of cancers However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Eschericha coli Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E coli and first structural characterization of a full-length IGFBP (C) 2010 Elsevier Inc. All rights reserved

Purification of goat serum retinol-binding protein and preparation of its antibodies

The method for the purification of goat serum retinol-binding protein consists of DEAE-cellulose chromatography of the serum followed by preparative polyacrylamide disc gel electrophoresis. After electrophoresis, the retinol-binding protein containing zone is identified by the specific fluorescence of retinol. For raising the antibodies, the portion of the gel containing retinol binding protein is homogenized and injected intradermally and intramuscularly to rabbits. The availability of this simple method for the isolation of retinol-binding protein and production of its antibodies enables the development of a radioimmunoassay for this protein.

A simple model for protein thermal denaturation

Whether proteins denature in all-or-none fashion or in a continuous fashion is as yet an unresolved problem. The all-or-none process implies that while the process of denaturation is going on, only two kinds of protein molecules can exist. One is completely unchanged and the other is altered. The altered protein molecules are indistinguishable. Underlying the 'continuum' models is the assumption that all the chains in a protein globule undergo similar changes so that it is enough to consider a single chain.

Functional linkages can reveal protein complexes for structure determination

The study of molecular machines, and protein complexes in general, is a growth area of biology. Is there a computational method for inferring which combinations of proteins in an organism are likely to form a crystallizable complex? We use the Protein Data Bank (PDB) to assess the usefulness of inferred functional protein linkages for this task. We find that of 242 nonredundant prokaryotic protein complexes (complexes excluding structural variants of the same protein) from organisms that are shared between the current PDB and the Prolinks functional linkage database, 44% (107/242) contain proteins that are linked at high-confidence by one or more methods of computed functional linkages. This suggests that computing functional linkages will be useful in defining protein complexes for structural studies. We offer a database of such inferred linkages corresponding to likely protein complexes for some 629,952 pairs of proteins in 154 prokaryotes and archea.

Interaction of water molecules in non-identical protein structures

The interaction of the protein atoms with the surrounding water oxygen atoms has been computed for 392 protein chains from 369 protein structures belonging to 90% non-homologous high resolution (<= 1.5 angstrom) protein Structures with a crystallographic R-factor <= 20%. The percentage composition of the polar atoms is found to be 36.3%. An average of 82.55% of water oxygen atoms are found to be in the primary hydration shell and 15.12% in the secondary hydration shell. The average Percentage of interactions of water oxygen atoms with the polar atoms of the main chain and side chain are 54% and 46%. respectively. The interaction of the acidic residues, aspartate and glutamate, with the water oxygen atoms is more when compared to that of the other residues.

Genome-wide analysis and experimentation of plant serine/threonine/tyrosine-specific protein kinases

Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.

mRNA-binding protein HuR in breast carcinogenesis

Breast cancer is the most common cancer among women. Although its prognosis has improved nowadays, methods to predict the progression of the disease or to treat it are not comprehensive. This thesis work was initiated to elucidate in breast carcinogenesis the role of HuR, a ubiquitously expressed mRNA-binding protein that regulates gene expression posttranscriptionally. HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm, and this nucleocytoplasmic translocation is important for its function as a RNA-stabilizing and translational regulator. HuR has been associated with diverse cellular processes, for example carcinogenesis. The specific aims of my thesis work were to study the prognostic value of HuR in breast cancer and to clarify the mechanisms by which HuR contributes to breast carcinogenesis. My ultimate goal is, by better understanding the role of HuR in breast carcinogenesis, to aid in the discovery of novel targets for cancer therapies. HuR expression and localization was studied in paraffin-embedded preinvasive (atypical ductal hyperplasia, ADH, and ductal carcinoma in situ, DCIS) specimens as well in sporadic and familial breast cancer specimens. Our results show that cytoplasmic HuR expression was already elevated in ADH and remained elevated in DCIS as well as in cancer specimens. Clinicopathological analysis showed that cytoplasmic HuR expression associated with the more aggressive form of the disease in DCIS, and in cancer specimens it proved an independent marker for poor prognosis. Importantly, cytoplasmic HuR expression was significantly associated with poor outcome in the subgroups of small (2 cm) and axillary lymph node-negative breast cancers. HuR proved to be the first mRNA stability protein the expression of which is associated in breast cancer with poor outcome. To explore the mechanisms of HuR in breast carcinogenesis, lentiviral constructs were developed to inhibit and to overexpress the HuR expression in a breast epithelial cell line (184B5Me). Our results suggest that HuR mediates breast carcinogenesis by participating in processes important in cell transformation, in programmed cell death, and in cell invasion. Global gene expression analysis shows that HuR regulates genes participating in diverse cellular processes, and affects several pathways important in cancer development. In addition, we identified two novel target transcripts (connective tissue growth factor, CTGF, and Ras oncogene family member 31, RAB31) for HuR. In conclusion, because cytoplasmic HuR expression in breast cancer can predict the outcome of the disease it could serve in clinics as a prognostic marker. HuR accumulates in the cytoplasm even at its non-invasive stage (ADH and DCIS) of the carcinogenic process and supports functions essential in cell alteration. These data suggest that HuR contributes to carcinogenesis of the breast epithelium.