Comparison of vector space model methodologies to reconcile cross-species neuroanatomical concepts.

Generating informational thesauri that classify, cross-reference, and retrieve diverse and highly detailed neuroscientific information requires identifying related neuroanatomical terms and acronyms within and between species (Gorin et al., 2001) Manual construction of such informational thesauri is laborious, and we describe implementing and evaluating a neuroanatomical term and acronym reconciliation (NTAR) system to assist domain experts with this task. NTAR is composed of two modules. The neuroanatomical term extraction (NTE) module employs a hidden Markov model (HMM) in conjunction with lexical rules to extract neuroanatomical terms (NT) and acronyms (NA) from textual material. The output of the NTE is formatted into collections of term- or acronym-indexed documents composed of sentences and word phrases extracted from textual material. The second information retrieval (IR) module utilizes a vector space model (VSM) and includes a novel, automated relevance feedback algorithm. The IR module retrieves statistically related neuroanatomical terms and acronyms in response to queried neuroanatomical terms and acronyms. Neuroanatomical terms and acronyms retrieval obtained from term-based inquiries were compared with (1) term retrieval obtained by including automated relevance feedback and with (2) term retrieval using "document-to-document" comparisons (context-based VSM). The retrieval of synonymous and similar primate and macaque thalamic terms and acronyms in response to a query list of human thalamic terminology by these three IR approaches was compared against a previously published, manually constructed concordance table of homologous cross-species terms and acronyms. Term-based VSM with automated relevance feedback retrieved 70% and 80% of these primate and macaque terms and acronyms, respectively, listed in the concordance table. Automated feedback algorithm correctly identified 87% of the macaque terms and acronyms that were independently selected by a domain expert as being appropriate for manual relevance feedback. Context-based VSM correctly retrieved 97% and 98% of the primate and macaque terms and acronyms listed in the term homology table. These results indicate that the NTAR system could assist neuroscientists with thesauri creation for closely related, highly detailed neuroanatomical domains.

Proteomics in early detection of cancer.

Early detection is critical in cancer control and prevention. Biomarkers help in this process by providing valuable information about a the status of a cell at any given point in time. As a cell transforms from nondiseased to neoplastic, distinct changes occur that could be potentially detected through the identification of the appropriate biomarkers. Biomarker research has benefited from advances in technology such as proteomics. We discuss here ongoing research in this field, focusing on proteomic technologies. The advances in two-dimensional electrophoresis and mass spectrometry are discussed in light of their contribution to biomarker research. Chip-based techniques, such as surface-enhanced laser desorption, and ionization and emerging methods, such as tissue and antibody arrays, are also discussed. The development of bioinformatic tools that have and are being developed in parallel to proteomics is also addressed. This report brings into focus the efforts of the Early Detection Research Network at the National Cancer Institute in harnessing scientific expertise from leading institutions to identify and validate biomarkers for early detection and risk assessment.

Genetic mapping and functional studies of a natural inhibitor of the insulin receptor tyrosine kinase: the mouse ortholog of human alpha2-HS glycoprotein.

Fetuin/alpha2-HS glycoprotein (alpha2-HSG) homologs have been identified in several species including rat, sheep, pig, rabbit, guinea pig, cattle, mouse and human. Multiple physiological roles for these homologs have been suggested, including ability to bind to hydroxyapatite crystals and to specifically inhibit the tyrosine kinase (TK) activity of the insulin receptor (IR). In this study we report the identification, cloning, and characterization of the mouse Ahsg gene and its function as an IR-TK inhibitor. Genomic clones derived from a mouse Svj 129 genomic library were sequenced in order to characterize the intron-exon organization of the mouse Ahsg gene, including an 875 bp subclone containing 154 bp upstream from the transcription start site, the first exon, and part of the first intron. A second genomic subclone harboring a 3.45 kb Bgl II fragment contained exons 2, 3 and 4 in addition to two adjacent elements within the first intron-a repetitive element of the B1 family (92 bp) and a 271 bp tract of (T,C)n*(A,G)n. We have mapped mouse Ahsg at 16 cM adjacent to the Diacylglycerol kinase 3 (Dagk3) gene on chromosome 16 by genotyping interspecific backcross panels between C57BL/6J and Mus spretus. The position is syntenic with human chromosome 3q27, where the human AHSG gene resides. Using recombinant mouse alpha2-HSG expressed from a recombinant baculovirus, we demonstrate that mouse alpha2-HSG inhibits insulin-stimulated IR autophosphorylation and IR-TKA in vitro. In addition, mouse alpha2-HSG (25 microg/ml) completely abolishes insulin-induced DNA synthesis in H-35 rat hepatoma cells. Based on the sequence data and functional analysis, we conclude that the mouse Ahsg gene is the true ortholog of the human AHSG gene.

Trends in biomarker research for cancer detection.

A key challenge in cancer control and prevention is detection of the disease as early as possible, enabling effective interventions and therapies to contribute to reduction in mortality and morbidity. Biomarkers are important as molecular signposts of the physiological state of a cell at a specific time. Active genes, their respective protein products, and other organic chemicals made by the cell create these signposts. As a normal cell progresses through the complex process of transformation to a cancerous state, biomarkers could prove vital for the identification of early cancer and people at risk of developing cancer. We discuss current research into the genetic and molecular signatures of cells, including microsatellite instability, hypermethylation and single-nucleotide polymorphisms. The use of genomic and proteomic high-throughput technology platforms to facilitate detection of early cancer by means of biomarkers, and issues on the analysis, validation, and predictive value of biomarkers based on these technologies are also discussed. We report on recent advances in identifying sources of biomarkers that can be accessed by noninvasive techniques, such as buccal-cell isolates, as well as traditional sources such as serum or plasma. We also focus on the work of the Early Detection Research Network at the National Cancer Institute, harnessing expertise from leading national and international institutions, to identify and validate biomarkers for the detection of precancerous and cancerous cells in assessing risk of cancer. The network also has a role in linking discovery to process development, resulting in early detection tests and clinical assessment.

Alpha2-HSG, a specific inhibitor of insulin receptor autophosphorylation, interacts with the insulin receptor.

Human fetuin, [alpha2-Heremans Schmid Glycoprotein (alpha2-HSG)], is a natural inhibitor of insulin receptor tyrosine kinase activity (IR-TKA). Previously, we have demonstrated that alpha2-HSG inhibits the mitogenic pathway without affecting the metabolic arm of insulin signal transduction. In this study, we demonstrate the time-course and specificity of inhibition, its interaction with IR and probable physiological role. In intact rat1 fibroblasts overexpressing the human insulin receptor (HIRc B), incubation of recombinant human alpha2-HSGbac (1.8 microM) inhibited insulin-induced IR autophosphorylation by over 80%. This inhibitory effect of alpha2-HSGbac on insulin-induced IR autophosphorylation was blunted by half in 60 min. Interestingly, alpha2-HSGbac at similar concentrations (0.9 or 1.8 microM), had no effect on EGF- or IGF-I-induced cognate receptor autophosphorylation. Anti-alpha2-HSG immunoprecipitates of alpha2-HSGbac-treated HIRc B cell lysates demonstrated the presence of IR. Our data suggest that alpha2-HSGbac preferentially interacts with the activated IR. To further characterize the site(s) of interaction, the effect of alpha2-HSGbac on trypsin-treated IR autophosphorylation was studied. Trypsin-treatment of intact HIRc B cells results in proteolysis of the IR alpha-chain and constitutive activation of IR-TKA. We demonstrate that alpha2-HSGbac (0.1 microM) completely inhibited trypsin-activated IR autophosphorylation and TKA in vitro indicating that this effect was not mediated by its interaction with the proximal 576 amino acid residues of the IR alpha-subunit. The physiological relevance of these observations was explored by characterizing the effects of alpha2-HSG injection in rats. Alpha2-HSGbac (2 microM), acutely injected through the portal vein of normal rats, inhibited insulin-stimulated IR autophosphorylation and IRS-1 phosphorylation in liver and hindlimb muscle. Taken together our results suggest that alpha2-HSG, by interacting with IR, specifically inhibits insulin-stimulated IR autophosphorylation and may play a physiological role in the regulation of insulin signaling.

Prevalence of osteoporosis in women: variation with skeletal site of measurement of bone mineral density.

OBJECTIVE: To determine retrospectively the prevalence of osteoporosis in a referral population of female patients and to compare the sensitivity for diagnosing osteoporosis by dual-energy x-ray absorptiometry (DXA) measurements of bone mineral density (BMD) at multiple skeletal sites. METHODS: We studied the data from 625 consecutive women (mean age, 57.3 +/- 13.9 years), who had been referred to our center for lumbar spine (anteroposterior [AP] and lateral region) and hip (femoral neck [FN], Ward's triangle [WT], trochanter, intertrochanteric region, and total hip) BMD measurements with use of DXA (Hologic QDR-2000) between June 1994 and July 1998. RESULTS: Osteoporosis (based on the World Health Organization definition--T-score of -2.5 or lower for BMD) was diagnosed by DXA at the following sites: AP spine in 21.7%, lateral spine in 43.2%, FN in 33.6%, WT in 49.1%, trochanter in 26.1%, intertrochanteric region in 25.9%, and total hip in 28.4% of study patients. Significant site differences were found in the prevalence of osteoporosis between the lateral and AP spine (P < 0.001), as well as between WT and the FN, trochanter, intertrochanteric region, and total hip (P < 0.001). In a subgroup of 71 women, forearm (ultradistal radius and radius 1/3 region) BMD results indicated low sensitivity for diagnosing osteoporosis, similar to that seen at the AP spine, trochanter, and intertrochanteric region. Not surprisingly, the prevalence of osteoporosis increased with advancing age (15.5% in patients younger than 50 years, in comparison with 59.6% in those older than 69 years of age). The frequency of misclassification of patients (osteoporosis at one site and normal BMD at another) with use of the seven measurement sites was 16.6% (104 of the 625 patients). CONCLUSIONS: For diagnosis of osteoporosis, DXA BMD measurements are significantly more sensitive at the lateral spine than at the AP spine, as well as at WT than at the FN, trochanter, intertrochanteric region, and total hip sites.

Alpha2-Heremans Schmid glycoprotein inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in rat adipose cells.

Alpha2-Heremans Schmid glycoprotein (alpha2-HSG) is a member of the fetuin family of serum proteins whose biological functions are not completely understood. There is a consensus that alpha2-HSG plays a role in the regulation of tissue mineralization. However, one aspect of alpha2-HSG function that remains controversial is its ability to inhibit the insulin receptor tyrosine kinase and the biological actions of insulin. Interestingly, some studies suggest that alpha2-HSG differentially inhibits mitogenic, but not metabolic, actions of insulin. However, these previous studies were not carried out in bona fide insulin target cells. Therefore, in the present study we investigate the effects of alpha2-HSG in the physiologically relevant rat adipose cell. We studied insulin-stimulated translocation of the insulin-responsive glucose transporter GLUT4 in transfected rat adipose cells overexpressing human alpha2-HSG. In addition, we measured insulin-stimulated glucose transport in adipose cells cultured with conditioned medium from the transfected cells as well as in freshly isolated adipose cells treated with purified human alpha2-HSG. Compared with control cells, we were unable to demonstrate any significant effect of alpha2-HSG on insulin-stimulated translocation of GLUT4 or glucose transport. In contrast, we did demonstrate that overexpression of alpha2-HSG in adipose cells inhibits both basal and insulin-stimulated phosphorylation of Elk-1 (a transcription factor phosphorylated and activated by mitogen-activated protein kinase and other related upstream kinases). Interestingly, we did not observe any major effects of alpha2-HSG to inhibit insulin-stimulated phosphorylation of the insulin receptor, insulin receptor substrate-1, -2, or -3, in either transfected or freshly isolated adipose cells. We conclude that alpha2-HSG inhibits insulin-stimulated Elk-1 phosphorylation, but not glucose transport, in adipose cells by a mechanism that may involve effector molecules downstream of insulin receptor substrate proteins.

Calcium and protein kinase C mediate high-glucose-induced inhibition of inducible nitric oxide synthase in vascular smooth muscle cells.

Abnormal vascular smooth muscle (VSMC) proliferation is a key feature in diabetes-associated atherosclerotic disease. Since nitric oxide inhibits VSMC tone, migration, adhesion, and proliferation, we examined the effects of high glucose on IL-1beta-induced NO release from VSMCs in culture. Confluent smooth muscle cells, preincubated with either 5 mmol/L (mM) or 20 mmol/L (mM) glucose for 48 hours, were stimulated with IL-1beta. Nitrite was measured in the culture medium after 24 hours. IL-1beta-induced a 15-fold increase in NO production in normal glucose medium. Glucose (10 to 30 mmol/L (mM)) significantly reduced the response to IL-1beta. High glucose (20 mmol/L (mM)) inhibited IL-1beta-evoked NO production by approximately 50%. IL-1beta-stimulated [3H] citrulline-forming activity of the nitric oxide synthase (NOS) was also significantly lower in high-glucose-exposed cells, and this was reflected in diminished cellular levels of NOS protein. To assess the role of protein kinase C (PKC), membrane PKC activity was measured, and glucose (20 mmol/L (mM)) significantly increased it. Immunoblotting of the membranes revealed a glucose-induced increase in the PKC betaII isoform. 1,2-Dioctanoyl-glycerol, a PKC activator, mimicked the high-glucose effect on IL-1beta-induced NO release, while staurosporine, a PKC inhibitor, reversed it. The role of calcium in the glucose-mediated inhibition of cytokine-induced NO release was determined by treatment with BAPTA, an intracellular chelator of calcium. BAPTA partially reversed the inhibitory effects of glucose. Increasing intracellular calcium by A23187, an ionophore or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, significantly decreased IL-1beta-induced NO release and NOS expression. These results indicate that glucose-induced inhibition of IL-1beta-stimulated NO release and NOS expression may be mediated by PKC activation and increased intracellular calcium.

Bovine fetuin is an inhibitor of insulin receptor tyrosine kinase.

Fetuin has been identified earlier as the bovine homolog of the human plasma protein, alpha2-Heremans Schmid glycoprotein (alpha2-HSG). Although bovine fetuin shares over 70% amino acid sequence similarity with alpha2-HSG and rat fetuin, no common function(s) have been identified. We report that immunoaffinity purified bovine fetuin acts as an inhibitor of insulin receptor tyrosine kinase activity (IR-TKA) with half-maximal inhibition at 1.5 microM. In vitro, bovine fetuin (1.5 microM) blocked insulin-induced autophosphorylation of the human IR completely and the half-maximal inhibitory effect was observed at 0.5 microM. Incubation of HIRcB cells (rat1 fibroblasts transfected with wild-type human insulin receptor cDNA) with bovine fetuin (1.5 microM) inhibited insulin-induced tyrosine phosphorylation of the IR beta-subunit by 40%. In addition, bovine fetuin (2 microM) completely blocked insulin-stimulated DNA synthesis in H-35 rat hepatoma cells. Our results, together with earlier reports on rat fetuin and human alpha2-HSG, indicate a common IR-TK inhibitory function for fetuin homologs.

Recombinant human alpha 2-HS glycoprotein inhibits insulin-stimulated mitogenic pathway without affecting metabolic signalling in Chinese hamster ovary cells overexpressing the human insulin receptor.

Insulin acts on its target tissues by specific interaction with the cell surface insulin receptor (IR). The IR possesses an intrinsic tyrosine kinase (TK) activity which is stimulated by insulin binding. This TK activity is required for many aspects of insulin signalling. We had earlier reported that human plasma alpha 2-HS glycoprotein (alpha 2-HSG) inhibits insulin-stimulated mitogenesis at the level of IR-TK (Mol Endo 7: 1445-1455, 1993). In the present study, using recombinant alpha 2-HSG, which possesses 50-100 times the specific activity of plasma alpha 2-HSG, we have further investigated the molecular basis of this effect. We examined the insulin-stimulated Ras signalling pathway in Chinese Hamster Ovary cells overexpressing the human IR. alpha 2-HSG inhibits insulin-induced tyrosine phosphorylation of IRS-1 and the subsequent association of GRB2, as well as Sos, with IRS-1. This inhibition results in reduced guanine nucleotide exchange in p21ras. alpha 2-HSG also inhibits the stimulation of Raf phosphorylation, in response to insulin, leading to inhibition of MEK activity. In a parallel pathway, alpha 2-HSG also inhibits insulin-induced tyrosine phosphorylation of Shc. However, alpha 2-HSG does not affect any of the metabolic actions of insulin rested in these cells. These results suggest that, while insulin's mitogenic effects can be abolished by inhibition of insulin-induced IR-TK, propagation of signals for metabolic activities might utilize alternate of rescue mechanisms.

Effects of metformin on tyrosine kinase activity, glucose transport, and intracellular calcium in rat vascular smooth muscle.

Metformin enhances peripheral insulin action and reduces blood pressure in hypertensive rats. Our group has previously reported that insulin and insulin-like growth factor I (IGF-1) attenuate both agonist-induced vascular smooth muscle cell (VSMC) contraction and associated increases in cytosolic free calcium ([Ca]i). Thus, changes in insulin actions may explain in part metformin's vascular effects. However, metformin's mechanism of action at the vasculature had not been elucidated. Therefore, the purpose of this study was to determine whether metformin evokes alterations in VSMC insulin and IGF-I receptors, glucose transport, and/or [Ca]i. We quantitated hormone binding and tyrosine kinase (TK) activity in partially purified insulin and IGF-I receptors prepared from metformin-treated (100 microM) and control rat aortic VSMC in culture. Glucose transport was assessed by 2-deoxyglucose uptake. Metformin exposure for 24 h 1) increased basal TK activity (metformin, 3.49 +/- 0.39; control, 1.77 +/- 0.39 pmol 32P incorporated/mg protein; P < 0.01) without changes in insulin-or IGF-I stimulated TK activity, 2) increased 2-deoxyglucose transport in a dose-dependent manner, 3) decreased thrombin-induced elevation in [Ca]i (metformin, 10.3%; control, 35.3% over basal; P < 0.05), These insulin/IGF-I-like effects of metformin may help explain some of its vascular actions.

Baculoviral expression of a natural inhibitor of the human insulin receptor tyrosine kinase.

We had earlier reported that the human serum alpha 2-HS glycoprotein (1) is a physiological and specific inhibitor of the human insulin receptor tyrosine kinase (IR-TK). We have now expressed this human protein in the baculoviral expression system using the Sf-9 and High Five insect cells. The protein was optimally expressed at 72 h post infection. alpha 2-HSGbac completely inhibited the insulin-stimulated autophosphorylation and TK activity of partially purified IR preparations. It also abolished insulin-induced DNA synthesis in the H-35 rat hepatoma cell line. The effective concentration of the baculoviral derived alpha 2-HSG necessary for inhibiting IR-TK activity was significantly lower than that of the protein purified from human plasma.

Inhibitors of the insulin receptor tyrosine kinase.

Insulin is a polypeptide hormone consisting of 51 amino acids. Insulin promotes a variety of anabolic enzymatic pathways and inhibits many catabolic enzymatic pathways involved in energy storage, as well as in synthesis of structural tissue proteins. In addition, insulin serves as a growth factor, modulating mitogenesis, growth and differentiation. Insulin mediates all of its effects by initially binding and activating its specific cell-surface receptor. Conformational changes induced by insulin binding lead to activation of intrinsic receptor tyrosine kinase. Thus, the study of tyrosine kinase inhibitors, whether synthetically produced or purified from microorganisms or humans, has led to elucidation of molecular details of physiological insulin signaling.

Serum alpha 2-HS-glycoprotein is an inhibitor of the human insulin receptor at the tyrosine kinase level.

The insulin-dependent tyrosine kinase activity (TKA) of the insulin receptor (IR) plays an essential role in insulin signaling. Thus, dysregulation of IR-TKA might be an important element in the states of insulin resistance. A phosphorylated rat hepatic glycoprotein (pp63) acting as an inhibitor of IR-TK has been described. In search of the human homolog of pp63, we isolated a cDNA clone from a human liver lambda gt11 cDNA library. DNA sequence analysis reveals identity with the mRNA product of a human gene AHSG encoding a serum protein, alpha 2-Heremans Scmid-glycoprotein (alpha 2HSG), with heretofore unknown physiological function. Northern blot analysis demonstrates a 1.8-kilobase mRNA in human liver and HepG2 hepatoma cells. alpha 2HSG, purified from human serum, specifically inhibits insulin-stimulated IR autophosphorylation in vitro and in vivo as well as exogenous substrate tyrosine phosphorylation. alpha 2HSG also inhibits both insulin-induced tyrosine phosphorylation of IRS-1 and the association of IRS-1 with the p85 subunit of phosphatidylinositol-3 kinase in H-35 hepatoma cells. alpha 2HSG inhibits insulin-dependent mitogenesis, but does not affect insulin-stimulated induction of the metabolic enzyme tyrosine aminotransferase. alpha 2HSG does not compete with insulin for binding to IR. Finally, the action of alpha 2HSG is specific toward the IR-TK; its effect does not extend to insulin-like growth factor-I-stimulated TKA. Our results allow us to assign a biochemical function for human alpha 2HSG, namely regulation of insulin action at the IR-TK level.

p56lck stably associates with a 115 kDa substrate.

Using antibodies directed against p56lck, we have identified a 115 kDa protein (p115) that is specifically immunoprecipitated with p56lck from whole cell lysates. The p56lck/p115 complex is stable in the presence of nonionic detergents. p115 becomes phosphorylated on tyrosine residues in p56lck immune-complex kinase assays. Treatment of whole cells with 12-O-tetradecanoyl phorbol-13-acetate decreases the subsequent tyrosine phosphorylation of p115 in immune-complex kinase assays.

Partial purification and characterization of the lck protein-tyrosine kinase from bovine thymus.

Soluble extracts prepared from bovine thymus contain an angiotensin-I-phosphorylating activity that is activated several-fold by high concentrations of NaCl. Fractionation of this protein-tyrosine kinase activity by chromatography on DEAE-cellulose yields a major diffuse peak of activity. The enzymes responsible for this activity are found at much higher levels in extracts from bovine thymus than from bovine spleen. The peak of activity from the DEAE-cellulose column can be further separated into two major peaks by chromatography on heparin-agarose. The second peak to elute from the heparin-agarose column was previously purified through several chromatographic steps to yield a 40 kDa protein-tyrosine kinase (p40). We have now partially purified the early-eluting peak of kinase activity by chromatography on columns of butyl-agarose, protamine-agarose and Sephacryl S200. The enzyme was identified following covalent modification with 5'-fluorosulphonylbenzoyladenosine (FSBA) by reactivity with anti-FSBA antibodies. This procedure labelled a series of 52-56 kDa proteins. These proteins were also recognized by polyclonal anti-peptide antibodies raised against the C-terminal 33 amino acids of p56lck, a major T lymphocyte protein-tyrosine kinase. Peptide maps of the partially purified enzyme were identical to maps generated from p56lck obtained from LSTRA cells. These data suggest that bovine thymus p56lck is responsible for the activity found in the early-eluting peak from heparin-agarose. Antibodies raised against a peptide corresponding to amino acids 39-64 of p56lck, a sequence found near the N-terminus, recognized the slower-migrating, but not the faster-migrating, form of the enzyme, indicating that a fraction of the protein had been proteolysed near the N-terminus during purification. The partially purified bovine enzyme exhibited a restricted substrate specificity in vitro and did not readily phosphorylate human erythrocyte band 3, casein or histone, but was able to phosphorylate acid-treated enolase. The dilute enzyme present in fractions eluting from chromatography columns was unable to catalyse an autophosphorylation reaction. Autophosphorylation could be detected in more concentrated enzyme samples and was readily observed in immune-complex assays. The phosphorylation of angiotensin I by bovine thymus p56lck was weakly activated by polyionic compounds such as heparin and polylysine, and was strongly activated by high concentrations of NaCl.