Chemical approaches of penicillin allergy—III Isolation of the penicillin-free carrier receptor protein (CRP) on a polymeric 7-deoxy penicillin analogue template and its role in penicillin immunogeneses in rabbit and man

Specific penicillin-carrier receptor proteins (CRP) have been isolated from the sera of penicillin allergic rabbits and human subjects in the unconjugated native state in electrophoretically homogeneous form by employing a synthetic polymeric affinity template containing the 7-deoxy analogue of penicillin G. The synthesis of the 7-deoxy analogue has been described. In this affinity system the antipenicillin-antibody is desorbed by 0·9M thiourea and the CRP in 8M urea. The CRP after incubation with penicillin is converted into the full-fledged antigen. Studies on the origin of CRP and the nature of antibody as well as comparative studies on the properties of the rabbit antibody and those of antibodies elicited by a BSA-BPO conjugate are reported.

Differential regulation of MRN (Mre11-Rad50-Nbs1) complex subunits and telomerase activity in cancer cells

Several lines of evidence suggest that cancer progression is associated with up-regulation or reactivation of telomerase and the underlying mechanism remains an active area of research. The heterotrimeric MRN complex, consisting of Mre11, Rad50 and Nbs1, which is required for the repair of double-strand breaks, plays a key role in telomere length maintenance. In this study, we show significant differences in the levels of expression of MRN complex subunits among various cancer cells and somatic cells. Notably, siRNA-mediated depletion of any of the subunits of MRN complex led to complete ablation of other subunits of the complex. Treatment of leukemia and prostate cancer cells with etoposide lead to increased expression of MRN complex subunits, with concomitant decrease in the levels of telomerase activity, compared to breast cancer cells. These studies raise the possibility of developing anti-cancer drugs targeting MRN complex subunits to sensitize a subset of cancer cells to radio- and/or chemotherapy. (C) 2010 Elsevier Inc. All rights reserved.

Enhanced photodynamic effect of cobalt(III) dipyridophenazine complex on thyrotropin receptor expressing HEK293 cells

Ternary cobalt(III) complexes CoL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3) are synthesized, characterized from X-ray crystallographic, analytical and spectral techniques, and their utility in photodynamic therapy (PDT) of thyroid diseases caused by TSH receptor dysfunction is probed. The complexes display a visible spectral band within the PDT spectral window at similar to 690 nm. Photodynamic potential was estimated through DNA cleavage activity of the dpq and dppz complexes in UV-A light of 365 nm and red light of 676 nm. The reactions proceed via the hydroxyl radical pathway. The complexes retain their DNA photocleavage activity in red light under anaerobic conditions, a situation normally prevails in hypoxic tumor core. Investigation into the photocytotoxic potential of these complexes showed that the dppz complex 3 is approximately 4-fold more active in the HEK293 cells expressing human thyrotropin receptor (HEK293-hTSHR) than in the parental cell line and has an insignificant effect on an unrelated human cervical carcinoma cell line (HeLa). Photoexcitation of complex 3 in HEK293-hTSHR cells leads to damage hTSHR as evidenced from the decrease in cAMP formation both in absence and presence of hTSH and decrease in the TSHR immunofluorescence with a concomitant cytoplasmic translocation of the membrane protein, cadherin. The involvement of hTSHR is evidenced from the ability of complex 3 to bind to the extracellular domain of hTSHR (hTSHR-ECD) with a K-d value of 81 nM and from the photocleavage of hTSHR-ECD.

Gonadal cell surface receptor for plasma retinol-binding protein A method for its radioassay and studies on its level during spermatogenesis

A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps: direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme.The binding of retinol-binding protein to the receptor is saturable and reverible. The interaction shows a Kd value of 2.1 · 10−10 M. The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testoterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifically induced by testoterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome.

Coated pits,endosome CURLing and intracellular receptor--ligand trafficking

In mediating endocytosis of extracellular macromolecules; the major mechanism in which cells ingest nutrients, degrade hormones and maintain the protein and lipid compositions of their organelle membrane, the cell surface receptors encounter 'coated pits', migrate continuously from one organelle to another, deliver the 'cargo' and often recycle back to the cell surface. This article is an attempt to give an account of the recent advances in our understanding of the molecular events involved in the 'round trip itinerary' of cell surface receptors.

Development of an LH receptor assay capable of measuring serum LH/CG in a wide variety of species

The development of a radioreceptor assay (RRA) that can measure serum LH in a variety of species and CG in sera and urine of pregnant women and monkeys is reported. Using sheep luteal membrane as the receptor source and I-125-labelled hLH/hCG as the tracer, dose-response (displacement) curves were obtained using hLH or hCG as standard. The addition of LH-free serum (200 mul per tube) had no affect on the standard displacement curve. The assay is simple, requires less than 90 min to complete and provides reproducible results. The sensitivity of the assay was 0.6 ng hLH per tube and the intra- and interassay variations were 9.6 and 9.8, respectively. Sera obtained from male and female bonnet monkeys (Macaca radiata) and monkey pituitary extract showed parallelism to the standard curve. The concentrations of LH measured correlated with the physiological status of the animals. Sera of rats, rabbits, hamsters, guinea-pigs, sheep and humans showed parallelism to the hLH standard curve indicating the viability of the RRA to measure serum LH of different species. Since the receptors recognize LH and CG, detection of pregnancy in monkeys and women was possible using this assay. The sensitivity of the assay for hCG was 8.7 miu per tube. This RRA could be a convenient alternative to the Leydig cell bioassay for obtaining the LH bioactivity profile of sera and biological fluids.

Calcium/Calmodulin Dependent Protein Kinase II Bound to NMDA Receptor 2B Subunit Exhibits Increased ATP Affinity and Attenuated Dephosphorylation

Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATP gamma S, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr(286)-alpha-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr(286) autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.

A novel method for mapping assembled epitopes in batches: Identification of three epitopes at the receptor binding region of human chorionic gonadotropin

Identification of conformation-specific epitopes of hCG beta has been done using a simple batch method, Chemically or enzymatically-modified hCG beta has been prepared in a batch and the effect of modifications on the integrity of different epitope regions has been investigated in a quantitative manner using monoclonal antibodies (MAbs) immobilized on plastic tubes from culture supernatants. Based on the extent of damage done to different regions by different modifications, three conformation-specific epitopes of hCG beta have been identified. The method has been shown to have important advantages over the existing methods on many considerations, Using this approach, these epitopes have been shown to be at/near the receptor-binding region.

Progesterone receptor expression in the human placenta

The presence of progesterone receptors (PR) in the human placenta has been demonstrated using the reverse transcriptase-polymerase chain reaction technique. It was observed that the amount of PR in the human placenta is less during late gestation. Electrophoretic mobility shift assays with nuclear extract isolated from the first trimester and term placenta revealed three complexes when incubated with [P-32]dCTP-labelled progesterone response element, and, in competition with unlabelled progesterone response element, the formation of all three complexes was inhibited. When supershift analysis of these complexes was carried out using antibodies which cross-react with both the A and B types of the PR or only with the B type receptor, only the A-form of PR was detected in the human placenta.

Structure and activity cyclase activated of OK-GC: A kidney receptor-guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4�75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4-75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13 peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.

Expression of the receptor guanylyl cyclase C and its ligands in reproductive tissues of the rat: A potential role for a novel signaling pathway in the epididymis

Guanylyl cyclase C (GC-C) is a membrane-associated form of guanylyl cyclase and serves as the receptor for the heat-stable enterotoxin (ST) peptide and endogenous ligands guanylin, uroguanylin, and lymphoguanylin. The major site of expression of GC-C is the intestinal epithelial cell, although GC-C is also expressed in extraintestinal tissue such as the kidney, airway epithelium, perinatal liver, stomach, brain, and adrenal glands. Binding of ligands to GC-C leads to accumulation of intracellular cGMP, the activation of protein kinases G and A, and phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates salt and water secretion. We examined the expression of GC-C and its ligands in various tissues of the reproductive tract of the rat. Using reverse transcriptase and the polymerase chain reaction, we demonstrated the presence of GC-C, uroguanylin, and guanylin mRNA in both male and female reproductive organs. Western blot analysis using a monoclonal antibody to GC-C revealed the presence of differentially glycosylated forms of GC-C in the caput and cauda epididymis. Exogenous addition of uroguanylin to minced epididymal tissue resulted in cGMP accumulation, suggesting an autocrine or endocrine activation of GC-C in this tissue. Immunohistochemical analyses demonstrated expression of GC-C in the tubular epithelial cells of both the caput epididymis and cauda epididymis. Our results suggest that the GC-C signaling pathway could converge on CFTR in the epididymis and perhaps control fluid and ion balance for optimal sperm maturation and storage in this tissue.