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Biomedical subjects

M Sundaram

Publications and source records attributed to M Sundaram.

At least 19 recordsLinked to original sources
PubMed1999

A PP2A regulatory subunit positively regulates Ras-mediated signaling during Caenorhabditis elegans vulval induction.

We describe evidence that a regulatory B subunit of protein phosphatase 2A (PP2A) positively regulates an RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although reduction of sur-6 PP2A-B function causes few vulval induction defects in an otherwise wild-type background, sur-6 PP2A-B mutations suppress the Multivulva phenotype of an activated ras mutation and enhance the Vulvaless phenotype of mutations in lin-45 raf, sur-8, or mpk-1. Double mutant analysis suggests that sur-6 PP2A-B acts downstream or in parallel to ras, but likely upstream of raf, and functions with ksr-1 in a common pathway to positively regulate Ras signaling.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Functional production and reconstitution of the human equilibrative nucleoside transporter (hENT1) in Saccharomyces cerevisiae. Interaction of inhibitors of nucleoside transport with recombinant hENT1 and a glycosylation-defective derivative (hENT1/N48Q).

We have produced recombinant human equilibrative nucleoside transporter (hENT1) in the yeast Saccharomyces cerevisiae and have compared the binding of inhibitors of equilibrative nucleoside transport with the wild-type transporter and a N-glycosylation-defective mutant transporter. Equilibrium binding of 3H-labelled nitrobenzylmercaptopurine ribonucleoside {6-[(4-nitrobenzyl)thio]-9-beta-d-ribofuranosyl purine; NBMPR} to hENT1-producing yeast revealed a single class of high-affinity sites that were shown to be in membrane fractions by (1) equilibrium binding (means+/-S.D.) of [3H]NBMPR to intact yeast (Kd 1.2+/-0.2 nM; Bmax 5.0+/-0.5 pmol/mg of protein) and membranes (Kd 0.7+/-0.2 nM; Bmax 6.5+/-1 pmol/mg of protein), and (2) reconstitution of hENT1-mediated [3H]thymidine transport into proteoliposomes that was potently inhibited by NBMPR. Dilazep and dipyridamole inhibited NBMPR binding to hENT1 with IC50 values of 130+/-10 and 380+/-20 nM respectively. The role of N-linked glycosylation in the interaction of NBMPR with hENT1 was examined by the quantification of binding of [3H]NBMPR to yeast producing either wild-type hENT1 or a glycosylation-defective mutant (hENT1/N48Q) in which Asn-48 was converted into Gln. The Kd for binding of NBMPR to hENT1/N48Q was 10. 5+/-1.6 nM, indicating that the replacement of an Asn residue with Gln decreased the affinity of hENT1 for NBMPR. The decreased affinity of hENT1/N48Q for NBMPR was due to an increased rate of dissociation (koff) and a decreased rate of association (kon) of specifically bound [3H]NBMPR because the values for hENT1-producing and hENT1/N48Q-producing yeast were respectively 0.14+/-0.02 and 0. 36+/-0.05 min-1 for koff, and (1.2+/-0.1)x10(8) and (0.40+/-0. 04)x10(8) M-1.min-1 for kon. These results indicated that the conservative conversion of an Asn residue into Gln at position 48 of hENT1 and/or the loss of N-linked glycosylation capability altered the binding characteristics of the transporter for NBMPR, dilazep and dipyridamole.

Base Sequence↗
PubMed1999

Spruce budworm (Choristoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning.

We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.

Amino Acid Sequence↗
PubMed1999

Clinical significance of erosive azotemic osteodystrophy: a prospective masked study.

OBJECTIVE: To determine the relationship between joint symptoms and radiographically identifiable erosions in patients on maintenance hemodialysis for 4 or more years. PATIENTS AND DESIGN: A prospective study was carried out on 21 patients, who underwent rheumatological evaluation and radiographic surveys of all clinically examined joints. The radiologist was masked to the clinical information and the clinicians were masked to the radiographic findings. Cuprophane dialyzers were used on all patients. The statistical analysis was performed by unpaired t-test and Fisher's exact test. RESULTS AND CONCLUSIONS: Ten men and 11 women comprised the 21 patients, of whom 10 had joint symptoms and clinical signs whereas 11 did not. Age, gender, and duration of hemodialysis did not differ significantly between the symptomatic and asymptomatic group. Of the 21 patients, 10 had radiological evidence of erosions and 11 did not. The average age of patients with erosions was 64.9 years; this was significantly different from the age of the group without erosions, which was 54.1 years. The group with radiographic evidence of erosions had been on dialysis for an average of 9.6 years, while those without erosions had received dialysis for an average of 6.4 years. Of the 11 patients without radiographic evidence of erosions, three were symptomatic. Of the 10 patients with erosions, seven had musculoskeletal symptoms, but only in four was there concordance between radiological findings and symptoms of the corresponding joints. The positive predictive value of radiographic erosions in predicting clinically significant disease was 40%. There was poor correlation between the presence of radiographic erosions and clinical signs and symptoms of joint disease.

Adult↗
PubMed1999

Magnetic resonance imaging for solitary lesions of bone: when, why, how useful?

The radiologist and orthopedic surgeon have a wide array of imaging modalities to choose from in diagnosing and staging solitary tumors of bone. MR imaging is the examination of choice for staging solitary tumors of bone. The radiograph, however, remains the most reliable imaging modality for predicting tumor histology. Radiographic interpretation in most instances ought to be the most influential factor in determining whether further imaging is required. This article reviews the strengths and weaknesses of the various imaging modalities and attempts to present a logical, cohesive, reproducible approach to the diagnosis and evaluation of solitary tumors of bone.

Angiography↗
PubMed1999

EEG in epilepsy: current perspectives.

The electroencephalogram (EEG) plays an important diagnostic role in epilepsy and provides supporting evidence of a seizure disorder as well as assisting with classification of seizures and epilepsy syndromes. Emerging evidence suggests that the EEG may also provide useful prognostic information regarding seizure recurrence after a single unprovoked attack and following antiepileptic drug withdrawal. Continuous EEG video telemetry monitoring has an established role in the diagnosis of non-epileptic pseudo-seizures and in localizing the seizure focus for epilepsy surgery. Newer tools such as EEG mapping and magneto-encephalogram, although still investigational, appear potentially useful for defining the seizure focus in epilepsy. This review examines the traditional concepts of clinical EEG in the light of newly available data.

Electroencephalography↗
PubMed1999

Kinase suppressor of Ras forms a multiprotein signaling complex and modulates MEK localization.

Genetic screens for modifiers of activated Ras phenotypes have identified a novel protein, kinase suppressor of Ras (KSR), which shares significant sequence homology with Raf family protein kinases. Studies using Drosophila melanogaster and Caenorhabditis elegans predict that KSR positively regulates Ras signaling; however, the function of mammalian KSR is not well understood. We show here that two predicted kinase-dead mutants of KSR retain the ability to complement ksr-1 loss-of-function alleles in C. elegans, suggesting that KSR may have physiological, kinase-independent functions. Furthermore, we observe that murine KSR forms a multimolecular signaling complex in human embryonic kidney 293T cells composed of HSP90, HSP70, HSP68, p50(CDC37), MEK1, MEK2, 14-3-3, and several other, unidentified proteins. Treatment of cells with geldanamycin, an inhibitor of HSP90, decreases the half-life of KSR, suggesting that HSPs may serve to stabilize KSR. Both nematode and mammalian KSRs are capable of binding to MEKs, and three-point mutants of KSR, corresponding to C. elegans loss-of-function alleles, are specifically compromised in MEK binding. KSR did not alter MEK activity or activation. However, KSR-MEK binding shifts the apparent molecular mass of MEK from 44 to >700 kDa, and this results in the appearance of MEK in membrane-associated fractions. Together, these results suggest that KSR may act as a scaffolding protein for the Ras-mitogen-activated protein kinase pathway.

14-3-3 Proteins↗
PubMed1999

Primary leiomyosarcoma of bone.

OBJECTIVE: This study describes radiographic and MR imaging features of primary leiomyosarcoma of bone. SUBJECTS AND METHODS: Twelve patients (five men and seven women, 39-79 years old) who were treated at two oncology centers for primary leiomyosarcoma of bone involving the femurs, tibia, ilium, and inferior pubic ramus were studied. None of the patients had preexisting disease or disease elsewhere at the time of diagnosis. Pathologic diagnosis was obtained in all patients. RESULTS: Radiographs of all patients showed a matrix that was exclusively osteolytic. In long bones (seven patients), the tumor had an average length of 11 cm (range, 7-17 cm) and revealed an elongated configuration. In the pelvis (five patients), the average length of the tumor was 10 cm (range, 4-15 cm). MR imaging confirmed an intramedullary lesion in all patients, with extension into the soft tissues in eight patients and no identifiable soft-tissue mass in the remaining four patients. Four of the five pelvic tumors had a prominent soft-tissue mass, whereas only four of the seven long bone lesions revealed a soft-tissue mass that was, in all instances, small. The tumor was hypointense on T1-weighted images and showed heterogeneous signal intensity on T2-weighted conventional and fast spin-echo sequences. We saw low signal intensity (short T2) in eight patients and homogeneous hyperintense signal intensity in one patient. In the remaining three patients, T2-weighted spin-echo sequences obtained with fat saturation showed high signal intensity (long T2) in the tumors. CONCLUSION: Primary leiomyosarcoma of bone is a rare tumor that on radiography reveals no matrix and on MR imaging reveals areas of T2 shortening in relation to fat on conventional and fast spin-echo sequences.

Adult↗
PubMed1998

Chimeric constructs between human and rat equilibrative nucleoside transporters (hENT1 and rENT1) reveal hENT1 structural domains interacting with coronary vasoactive drugs.

We have recently isolated cDNAs from human placenta and rat jejunum encoding the prototypic human and rat equilibrative nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporters hENT1 and rENT1. The two proteins (456 and 457 residues, Mr 50,000) are 78% identical in amino acid sequence and contain 11 potential transmembrane segments (TMs) with a large putative extracellular loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. When expressed in Xenopus oocytes, recombinant hENT1 and rENT1 transport both purine and pyrimidine nucleosides, including adenosine, and are inhibited by nanomolar concentrations of NBMPR. hENT1 is also potently inhibited by coronary vasodilator drugs (dipyridamole, dilazep, and draflazine), whereas rENT1 is insensitive to inhibition by these compounds (dipyridamole IC50 values 190 nM (hENT1) and >/=10 microM (rENT1) at 10 microM uridine). In the present study, we have generated reciprocal chimeras between hENT1 and rENT1, using splice sites at residues 99 (end of TM 2) and 231 (end of TM 6), to identify structural domains of hENT1 responsible for transport inhibition by vasoactive compounds. Transplanting the amino-terminal half of hENT1 into rENT1 converted rENT1 into a dipyridamole/dilazep-sensitive transporter, whereas the amino-terminal half of rENT1 rendered hENT1 dipyridamole/dilazep-insensitive. Domain swaps within the amino-terminal halves of hENT1 and rENT1 identified residues 100-231 (incorporating TMs 3-6) of hENT1 as the major site of vasodilator interaction. Since these drugs function as competitive inhibitors of nucleoside transport and NBMPR binding, TMs 3-6 are likely to form part of the substrate-binding site.

Affinity Labels↗
PubMed1998

Expression, characterization and engineered specificity of rat epididymal retinoic acid-binding protein.

Epididymal retinoic acid-binding protein (ERABP) is the major androgen-dependent protein present in the lumen of the epididymis and is thought to be involved in sperm maturation. It displays a high degree of three-dimensional structural similarity to serum retinol-binding protein (RBP). Although both proteins interact with retinoids, RBP exhibits a broad specificity, binding retinol, retinoic acid and retinaldehyde with roughly equal affinities, whereas ERABP is specific for all-trans- and 9-cis-retinoic acids. Consistent with this, the binding pockets of the two proteins are different: in RBP it is predominantly hydrophobic, whereas that for ERABP is amphipathic, with a network of charged residues at the open end of the binding pocket. In order to investigate the roles of these charged residues, Arg-80 and Glu-63 have been mutated to isoleucine. The resultant double mutant, Glu-63-->Ile/Arg-80-->Ile, as well as the wild-type protein, were subsequently expressed in Escherichia coli as fusion proteins, with the streptavidin recognition sequence (Strep) tagged to their C-termini. The expressed proteins were purified in a single step by streptavidin-affinity chromatography and their ligand-binding properties were examined using fluorimetric titrations. Whereas the wild-type ERABP binds only retinoic acid, the double mutant is capable of binding retinol, retinoic acid and retinaldehyde with similar affinities. These observations provide experimental support for the proposition that the charged residues near the open end of the binding pocket are responsible for restricting the specificity of ERABP for retinoic acid. These studies demonstrate that changes in specificity can be engineered into lipocalins.

Animals↗
PubMed1998

The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor.

The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated. The experimental system consisted of the [3H]retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes. [3H]Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin. Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP. The effects are specific: other binding proteins, including beta-lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP. The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism. Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP.

Humans↗
PubMed1998

Basis for selective action of a synthetic molting hormone agonist, RH-5992 on lepidopteran insects.

The non-steroidal ecdysone agonist, RH-5992, induces a precocious incomplete molt in lepidopteran insects but is refractory to insects of other orders. We used two lepidopteran cell lines, FPMI-CF-203 (CF-203) and IPRI-MD-66 (MD-66) and two dipteran cell lines, DM-2 and Kc, to investigate the lepidopteran specificity of RH-5992. The mRNAs for hormone receptor 3 homologues cloned from Drosophila (DHR3) and Choristoneura (CHR3) are directly induced by 20-hydroxyecdysone (20E) and serve as suitable markers for studying ecdysone action. Dose response experiments showed that 10(-7) M 20E induced CHR3 mRNA in CF-203 cell and DHR3 mRNA in DM-2 cells. Concentrations of RH-5992 as low as 10(-10) M induced CHR3 mRNA in CF-203 cells, whereas concentrations as high as 10(-6) M induced only very low levels of DHR3 mRNA in DM-2 cells. Studies using 14C-RH-5992 revealed that lepidopteran cell lines (CF-203 and MD-66) retained more of this compound within the cells than the dipteran cell lines (DM-2 and Kc). The clearance of RH-5992 from DM-2 cells was temperature dependent and was blocked by 10(-5) M ouabain, an inhibitor of Na+, K(+)-ATPase suggesting that the efflux was due to active transport.

Animals↗