bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular response to double‐stranded RNA
Authors: Haines Dale S. , Strauss Kenneth I. , Gillespie David H.
Published: May 2005

The study of double‐stranded RNA (dsRNA) encompasses a variety of fields. Basic research in this area has contributed to a greater mechanistic understanding of gene induction, tumor cell growth arrest, the establishment of antiviral states, and immunomodulation. Because of the possible clinical value of these molecules, physicians are now exploring the use of synthetic dsRNA to treat patients with cancer, HIV‐1 disease, and immune dysfunction. Continued studies of the mechanisms of action of dsRNA are likely to suggest an even wider scope of clinical applications.
Leukemia inhibitory factor: A biological perspective
Authors: Hilton Douglas J. , Gough Nicholas M.
Published: May 2005

The notion that a single hormone may exert a broad range of effects has become well established. As such, leukemia inhibitory factor (LIF) is a prime example. LIF was initially described, purified, and genetically cloned on the basis of its ability to induce the differentiation and suppress the clonogenicity of the monocytic leukemia cell line, M1. Subsequently, it has become apparent that in vitro LIF inhibits the differentiation of pluripotential ES cells, stimulates the synthesis of hepatic acute‐phase proteins, induces a switch in neurotransmitter phenotype from adrenergic to cholinergic, suppresses adipocyte lipoprotein lipase activity, and results in an increase in bone resorption. Moreover, elevation of LIF levels in vivo has a number of patho‐physiological consequences, many of which parallel those effects observed in vitro. The challenge that lies ahead is to determine whether other sites of LIF action exist and to define more clearly the physiological role LIF plays in vivo.

A major mechanism of cell‐cell communication is by the production and secretion of polypeptide hormones by one cell type, which act either systemically or locally, via interaction with specific receptors on the surface of responsive cells. Recently, it has become apparent that hormones initially described and named, on the basis of a specific action, in many cases exert a spectrum of effects on a broad range of cell types. Moreover, the effects exerted are often mimicked closely by other hormones. Hormones that act in a pleiotropic manner are, for example, transforming growth factor‐β (TGF‐β), the various fibroblast growth factors (FGFs), interleukin‐6 (IL‐6), and leukemia inhibitory factor (LIF). This review will focus on the various biological effects ascribed to LIF.
Novel immunomodulators with pronounced in vivo effects caused by stimulation of cytokine release
Authors: Rasmussen L.‐T. , Seljelid R.
Published: May 2005

Beta‐1,3‐D‐polyglucose derivatives protect mice against otherwise lethal bacterial infections. This protective effect has been considered to be mediated through mononuclear phagocytes. By using radioactive labelling, we localized the β‐1,3‐D‐polyglucose derivatized microbeads (GDM) during the period following injection. The GDM was recovered mainly in the milky spots of the omentum. In animals treated with GDM, the total white cell number was significantly increased in peritoneal fluid of mice before and after challenge with E. coli. Bacterial counts in peritoneal fluid of GDM treated animals declined to zero after 24 h. In untreated animals there was a slight increase in bacterial counts until the animals died after about 12 h. Mouse peritoneal macrophages stimulated with GDM released significant amounts of IL‐1 and PGE2. There was no significant release of TNF. Levels of IL‐1 and PGE2 in peritoneal fluid increased significantly during the first 48 h after treatment with GDM. There was no increase of levels of TNF. After challenge with E. coli, the levels of IL‐1, TNF, and PGE2 were significantly lower compared with control animals. In untreated animals the levels of IL‐1 and TNF remained elevated until the animals died after about 12 h. These studies demonstrate that the raised levels of arachidonic acid metabolites after pretreatment with GDM or AG seems to inhibit the otherwise lethal elevation of IL‐1 and TNF in body fluids which is seen in untreated animals.
Annexin VI is associated with calcium‐sequestering organelles
Authors: Hazarika Parul , Kaetzel Marcia A. , Sheldon Adrian , Karin Norman J. , Fleischer Sidney , Nelson T. E. , Dedman John R.
Published: May 2005

Annexin VI is a member of a Ca2+‐dependent, phospholipid‐binding protein family. Although functions for this annexin have been proposed from in vitro studies, most remain controversial. Díaz‐Muñoz et al. (J Biol Chem 265:15894, 1990) demonstrated that annexin VI modified, in a Ca2+‐dependent manner, the gating behavior of the sarcoplasmic reticulum Ca2+‐release channel, reconstituted into artificial bilayers, by increasing both the open probability and the mean open time. This effect was specific to the trans chamber, which represents the luminal side of the sarcoplasmic reticulum. In agreement with those findings, we show herein that annexin VI produced no effect on Ca2+‐uptake or ‐release by intact heavy sarcoplasmic reticulum vesicles (analogous to the cis chamber). We also used monospecific antibodies to evaluate the subcellular localization of annexin VI by immunofluorescent microscopy. Studies in rat skeletal muscle suggest that annexin VI is present surrounding individual myofibrils. Double immunolocalization studies with cultured muscle cells (chick myotubes) using anti‐annexin VI and anti‐SR Ca2+‐ATPase antibodies demonstrated superimposable staining patterns. In non‐muscle tissue (normal rat kidney (NRK) cells), a punctate, perinuclear anti‐annexin VI staining pattern was observed. Collectively, these data suggest that annexin VI may play a regulatory role in the Ca2+‐release/uptake cycle in the sarcoplasmic reticulum as well as in non‐muscle organelles, a key process in stimulus‐response systems.
Bacterial resistances to mercury and copper
Authors: Morby Andrew P. , Parkhill Julian , Lee Barry T. O. , Brown Nigel L. , Rouch Duncan A. , Camakaris James , Williams Tracey
Published: May 2005

Heavy metals are toxic to living organisms. Some have no known beneficial biological function, while others have essential roles in physiological reactions. Mechanisms which deal with heavy metal stress must protect against the deleterious effects of heavy metals, yet avoid depleting the cell of a heavy metal which is also an essential nutrient. We describe the mechanims of resistance in Escherichia coli to two different heavy metals, mercury and copper. Resistance of E. coli to mercury is reasonably well understood and is known to occur by transport of mercuric ions into the cytoplasmic compartment of the bacterial cell and subsequent reductive detoxification of mercuric ions. Recent mutational analysis has started to uncover the mechanistic detail of the mercuric ion transport processes, and has shown the essential nature of cysteine residues in transport of Hg(II). Resistance to copper is much less well understood, but is known to involve the increased export of copper from the bacterial cell and modification of the copper; the details of the process are still being elucidated.

Expression of both metal resistance determinants is regulated by the corresponding cation. In each case the response enables the maintenance of cellular homeostasis for the metal. The conclusions drawn allow us to make testable predictions about the regulation of expression of resistance to other heavy metals.
Constitutive production of lymphocyte activating factors by normal tissues in the adult rat
Authors: Granholm Tina , Söder Olof
Published: May 2005

Lymphocyte activating factors (LAFs), e.g., interleukin‐1 (IL‐1) and IL‐1‐like factors, have previously been demonstrated outside the immune system in the skin, thymus epithelium, and the human and rat testis. We have studied the presence of LAFs in normal tissues of the adult rat, utilizing a highly IL‐1 sensitive murine thymocyte proliferation assay. We have demonstrated high amounts of LAF activity in the tongue, esophagus, proventricular part of the stomach, and the liver. Some activity was also demonstrated in the duodenum, placenta, spleen, Peyer's patches, glandular stomach, and jejunum, but no bioactivity was present in other gastrointestinal, endocrine, lymphoid, or haematopoietic tissues. We were also unable to detect any LAF activity in the reproductive organs (except for the testis), urinary tract, skeletal and muscular tissues, brain, eyes, salivary glands, or lung. In the esophagus the activity was mainly localized to the mucosa. The LAF activity in the skin was partly inhibited by treatment with a mixture of antibodies against human IL‐1α and IL‐1β Dose response curves and gel filtration on a Sephacryl S‐200 column suggested the presence of a high molecular weight (90,000–100,000 Da) LAF inhibitory factor in the liver. In all positive tissues, the demonstrated LAFs had a molecular weight of 15,000–25,000 Da, as determined by Sephacryl S‐200 gel filtration. Of the positive tissues, the skin, tongue, esophagus, and the proventricular part of the stomach all contain stratified squamous epithelium. It is tempting to suggest that the detected LAFs have a similar function in these barrier tissues, e.g., to serve as host defence factors, or, alternatively or additionally, as tissue growth factors.
Poly(ADP‐ribosylation) of atypical CS histone variants is required for the progression of S phase in early embryos of sea urchins
Authors: Imschenetzky M. , Montecino M. , Puchi M.
Published: May 2005

The patterns of poly(ADP‐ribosylation) in vivo of CS (cleavage stage) histone variants were compared in sea urchin zygotes at the entrance and the exit of S1 and S2 in the initial developmental cell cycles. This post‐translational modification was detected by Western immunoblots with rabbit sera anti‐poly(ADP‐ribose) that was principally reactive against ADP‐ribose polymers and slightly against ADP‐ribose oligomers. The effect of 3 aminobenzamide (3‐ABA), an inhibitor of the poly(ADP‐ribose) synthetase, on S phase progression was determined in vivo by measuring the incorporation of 3H thymidine into DNA. The results obtained indicate that the CS histone variants are poly(ADP‐ribosylated) in a cell cycle dependent manner. A significantly positive reaction of several CS variants with sera anti‐poly(ADP‐ribose) was found at the entrance into S phase, which decreases after its completion. The incubation of zygotes in 3‐ABA inhibited the poly(ADP‐ribosylation) of CS variants and prevented both the progression of the first S phase and the first cleavage division. These observations suggest that the poly(ADP‐ribosylation) of atypical CS histone variants is relevant for initiation of sea urchin development and is required for embryonic DNA replication.
Prostatic epithelial cells in culture: Phosphorylation of protein tyrosyl residues and tyrosine protein kinase activity
Authors: Bourassa C. , Nguyen L. T. , Durocher Y. , Roberts K. D. , Chevalier S.
Published: May 2005

The ability of dividing canine prostatic epithelial cells in primary monolayers to phosphorylate protein tyrosyl residues was evaluated by metabolic studies performed through incorporation of [32P]‐phosphate into alkali‐resistant phosphoproteins and by the assay of their tyrosine protein kinase activity. The presence of sodium orthovanadate during cell incubation with [32P]‐phosphate greatly enhanced the relative labelling intensity of a 44 kDa alkali‐resistant phosphoprotein and the total cellular content of phosphotyrosine in proteins; in this respect, growth factors such as epidermal growth factor, insulin, and insulin‐like growth factor I, and the steroids dihydrotestosterone and estradiol were inactive. When the cells were solubilized, sodium orthovanadate stimulated their tyrosine protein kinase activity and inhibited their phosphotyrosine phosphatase activity. To characterize the tyrosine protein kinase of these cultured cells, conditions for optimal activity were established using the substrate poly [Glu80Na, Tyr20]. The subcellular localization of the enzyme was determined upon cell fractionation: 88% of the kinase activity was associated with the particulate fraction and 30% of this activity was partially solubilized with 0.5% Triton X–100; this solubilization was improved to 83% in the presence of 0.25 M KCl. The enzyme directly solubilized from prostatic cells with Triton X–100 (38% of activity) mainly catalyzed the alkali‐resistant phosphorylation of pp63, pp59, and pp44, which contained phosphotyrosine. These proteins were also phosphorylated by the major peak of kinase activity which was eluted at an apparent molecular weight of 300–350 kDa upon gel filtration. On a cell basis, the kinase activity was four‐ to eleven‐fold higher in the dividing epithelial cells in culture compared to quiescent secretory and non‐secretory epithelial cells, freshly isolated from dog prostates. It is proposed that this tyrosine protein kinase is implicated in the regulation of the proliferation of prostatic epithelial cells.
Adenosine deaminase deficiency due to heterozygous abnormality consisting of a deletion of exon 7 and the absence of enzyme mRNA
Authors: Kashii Saburo , Ito Kazuhiko , Monden Sumie , Sasai Yoshiki , Tsuchida Kunihiro , Fujita Masahiro , Kawamoto Hiroshi , Norioka Mihoko , Okuma Minoru
Published: May 2005

An adenosine deaminase (ADA;EC 3.5.4.4)‐deficient B lymphoblastoid cell line BADO5 derived from a Japanese patient with severe combined immunodeficiency disease and two B lymphoblastoid cell lines, BAMO5 from his mother and BAFO5 from his father, were characterized. To identify mutations affecting ADA activity, we prepared cDNAs to ADA mRNAs of the BADO5 cell line for nucleotide sequencing. Sequence analysis of one of the BADO5 ADA cDNA clones revealed deletion of exon 7, and one point mutation of base 629 from G to A that did not affect the amino acid sequence. All clones of the BADO5 cell line so far examined showed the absence of exon 7 by Southern blotting analysis. Ribonuclease protection assay with an RNA probe spanning from exon 5 to exon 11 showed that the BADO5 ADA mRNA had a deletion of exon 7, the BAMO5 mRNA had normal length, and the BAFO5 mRNA had two species with a deletion of exon 7 and with normal length. Consequently, the patient's ADA genes resulted from one allele of the BAMO5 ADA gene that did not produce a detectable mRNA, and the other allele of the BAFO5 ADA gene producing an aberrant mRNA without exon 7.
Studies on the role of protein kinases in the TNF‐mediated enhancement of murine tumor cell‐endothelial cell interactions
Authors: Bereta Joanna , Bereta Michal , Cohen Stanley , Cohen Marion C.
Published: May 2005

We have previously demonstrated that the exposure of mouse microvascular endothelium (MME) to tumor necrosis factor‐α (TNF) led to the increased binding of mouse mastocytoma cells (P815) to endothelial monolayers (Bereta et al., in press). In the current study we examined the possible involvement of protein kinases in TNF signal transduction in the endothelial cells. PKA does not appear to play a role in the potentiation of binding by TNF. We found that the TNF‐generated signal is inhibited by H‐7 and sangivamycin, but not by staurosporine. TNF did not cause translocation of PKC to the cell membrane and its effect could not be completely mimicked by PMA nor by PMA in the presence of calcium‐raising agents. Thus, we concluded that the “classical” PKC pathway is not completely responsible for TNF signalling in this system. We also found that staurosporine itself strongly enhanced adhesion of tumor cells to endothelium, utilizing a mechanism distinct from that of TNF. Although the data provide evidence for the role of kinases in the effect of TNF on binding of tumor cells to MME, this role appears to be a complex one.
Dynamic chromatin: The regulatory domain organization of eukaryotic gene loci
Authors: Bonifer Constanze , Hecht Andreas , Saueressig Harald , Winter Diana M. , Sippel Albrecht E.
Published: May 2005

It is hypothesized that nuclear DNA is organized in topologically constrained loop domains defining basic units of higher order chromatin structure. Our studies are performed in order to investigate the functional relevance of this structural subdivision of eukaryotic chromatin for the control of gene expression. We used the chicken lysozyme gene locus as a model to examine the relation between chromatin structure and gene function. Several structural features of the lysozyme locus are known: the extension of the region of general DNAasel sensitivity of the active gene, the location of DNA‐sequences with high affinity for the nuclear matrix in vitro, and the position of DNAasel hypersensitive chromatin sites (DHSs). The pattern of DHSs changes depending on the transcriptional status of the gene. Functional studies demonstrated that DHSs mark the position of cis‐acting regulatory elements. Additionally, we discovered a novel cis‐activity of the border regions of the DNAasel sensitive domain (A‐elements). By eliminating the position effect on gene expression usually observed when genes are randomly integrated into the genome after transfection, A‐elements possibly serve as punctuation marks for a regulatory chromatin domain. Experiments using transgenic mice confirmed that the complete structurally defined lysozyme gene domain behaves as an independent regulatory unit, expressing the gene in a tissue specific and position independent manner. These expression features were lost in transgenic mice carrying a construct, in which the A‐elements as well as an upstream enhancer region were deleted, indicating the lack of a locus activation function on this construct. Experiments are designed in order to uncover possible hierarchical relationships between the different cis‐acting regulatory elements for stepwise gene activation during cell differentiation. We are aiming at the definition of the basic structural and functional requirements for position independent and high level gene expression. The result of these experiments will have important consequences for random gene transfer with predictable and reproducible expression of transgenes.
The nuclear matrix: A heuristic model for investigating genomic organization and function in the cell nucleus
Authors: Berezney Ronald
Published: May 2005

Despite significant advances in deciphering the molecular events underlying genomic function, our understanding of these intergrated processes inside the functioning cell nucleus has, until recently, met with only very limited success. A major conundrum has been the “layers of complexity” characteristic of all cell structure and function. To understand how the cell nucleus functions, we must also understand how the cell nucleus is put together and functions as a whole. The value of this neo‐holistic approach is demonstrated by the enormous progress made in recent years in identifying a wide variety of nuclear functions associated with the nuclear matrix. In this article we summarize basic properties of in situ nuclear structure, isolated nuclear matrix systems, nuclear matrix‐associated functions, and DNA replication in particular. Emphasis is placed on identifying current problems and directions of research in this field and illustrating the intrinsic heuristic value of this global approach to genomic organization and function.
DNA and RNA within the nucleus: How much sequence‐specific spatial organization?
Authors: Carter Kenneth C. , Lawrence Jeanne Bentley
Published: May 2005

Spatial organization of various nuclear components is often proposed as means by which nuclei more efficiently carry out their various tasks. Such functional compartmentalization may involve a sequence‐specific packaging and placement of DNA and RNA. Here we review recent insights, allowed primarily by advances in fluorescent in situ hybridization methodology, into the organization of nucleic acids within individual nuclei.
Influence of dexamethasone on the vitamin D–mediated regulation of osteocalcin gene expression
Authors: Schepmoes Grace , Breen Ellen , Owen Thomas A. , Aronow Michael A. , Stein Gary S. , Lian Jane B.
Published: May 2005

The influence of dexamethasone on expression of the osteocalcin gene which encodes the most abundant non‐collagenous and only reported bone‐specific protein was examined in ROS 17/2.8 osteosarcoma cells which express a broad spectrum of genes related to bone formation. Consistent with previous reports, quantitation of cellular osteocalcin mRNA levels by Northern blot analysis, osteocalcin gene transcription by activity of the osteocalcin gene promoter fused to a chloramphenicol acetyl‐transferase (CAT) mRNA coding sequence following transfection into ROS 17/2.8 cells, and osteocalcin biosynthesis by radioimmunoassay indicate that dexamethasone in a concentration range of 10‐6to 10‐9M only modestly modifies basal levels of osteocalcin gene expression. However, dexamethasone significantly inhibits these parameters of the vitamin D–induced upregulation of osteocalcin gene expression in both proliferating and in confluent ROS 17/2.8 cells. In this study, we observed that the extent to which abrogation of the vitamin D response occurs is dependent on basal levels of osteocalcin gene expression as reflected by a complete inhibition of the vitamin D–induced upregulation in a ROS 17/2.8K subline with low basal expression and only a partial reduction of the vitamin D stimulation in a ROS 17/2.8C subline with eightfold higher levels of basal expression. This effect of glucocorticoid appears to be at the transcriptional and post‐transcriptional levels as demonstrated by a parallel decline in the cellular representation of osteocalcin mRNA, osteocalcin gene promoter activity, and osteocalcin biosynthesis. The complexity of the glucocorticoid effect on vitamin D–mediated transcriptional properties of the osteocalcin gene is indicated by persistence of sequence‐specific protein‐DNA interactions at two principal osteocalcin gene promoter regulatory elements, the osteocalcin (CCAAT) box which modulates basal level of transcription, and the vitamin D responsive element, where vitamin D–mediated enhancement of osteocalcin gene transcription is controlled.
Effect of monensin on ricin and fluid phase transport in polarized MDCK cells
Authors: Melby Elisabeth Løkken , Prydz Kristian , Olsnes Sjur , Sandvig Kirsten
Published: May 2005

The effect of monensin on endocytosis, transcytosis, recycling and transport to the Golgi apparatus in filter‐grown Madin‐Darby canine kidney (MDCK) cells was investigated using 125I‐labeled ricin as a marker for membrane transport, and horseradish peroxidase (HRP) as a marker for fluid phase transport. Monensin (10 μM) stimulated transcytosis of both markers about 3‐fold in the basolateral to apical direction. Transcytosis of HRP in the opposite direction, apical to basolateral, was reduced to approximately 50% of the control by monensin, whereas that of ricin was slightly increased. Recycling of markers endocytosed from the apical surface was reduced in the presence of monensin and there was an increased accumulation of both ricin and HRP in the cells. Transport of ricin to the Golgi apparatus increased to the same extent as the increase in intracellular accumulation. No change in recycling or accumulation was observed with monensin when the markers were added basolaterally, but transport of ricin to the Golgi apparatus increased almost 3‐fold. Our results indicate that basolateral to apical transcytosis is increased in the absence of low endosomal pH, and they suggest that apical to basolateral transcytosis of a membrane‐bound marker (ricin) is affected by monensin differently from that of a fluid phase marker (HRP).
Engineering T lymphocyte antigen specificity
Authors: Bolhuis Reinder L. H. , Sturm Els , Gratama Jan Willem , Braakman Eric
Published: May 2005

Targeting of immune cells by bispecific antibodies has proven to be a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely, the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction, are the major topics of this review.
Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages
Authors: Landmann R. , Ludwig C. , Obrist R. , Obrecht J. P.
Published: May 2005

The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor‐alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL‐1, rIL‐2, rIL‐3, rIL‐4, rIL‐5, rIL‐6, rTNFa, rGM‐CSF, rM‐CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu‐M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL‐2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti‐IFNg antibody. rIFNa and rIL‐2 were ineffective in purified monocytes or macrophages. rIL‐4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose‐dependently inhibited by rIL‐4 and this inhibition is probably due to an IL‐4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL‐4, suggest that the LPS‐LBP receptor is involved in the feedback response of IFNg and IL‐4 to LPS stimulation.
Protein‐Tyrosine phosphatases and the regulation of insulin action
Authors: Goldstein Barry J.
Published: May 2005

Protein‐tyrosine phosphatases (PTPases) play an important role in the regulation of insulin action by dephosphorylating the active (autophosphorylated) form of the insulin receptor and attenuating its tyrosine kinase activity. PTPases can also modulate post‐receptor signalling by catalyzing the dephosphorylation of cellular substrates of the insulin receptor kinase. Dramatic advances have recently been made in our understanding of PTPases as an extensive family of transmembrane and intracellular proteins that are involved in a number of pathways of cellular signal transduction. Identification of the PTPase(s) which act on various components of the insulin action cascade will not only enhance our understanding of insulin signalling but will also clarify the potential involvement of PTPases in the pathophysiology of insulin‐resistant disease states. This brief review provides a summary of reversible tyrosine phosphorlyation events in insulin action and available data on candidate PTPases in liver and skeletal muscle that may be involved in the regulation of insulin action.
Insulin/IGF‐1 hybrid receptors: Implications for the dominant‐negative phenotype in syndromes of insulin resistance
Authors: Frattali Anne L. , Treadway Judith L. , Pessin Jeffrey E.
Published: May 2005

Classical insulin and IGF‐1 receptors are α2β2 heterotetrameric complexes synthesized from two identical αβ half‐receptor precursors [1,2]. Recent data strongly suggests, however, that nonidentical αβ half‐receptor precursors can assemble to generate hybrid holoreceptor species both in vivo and in vitro [3–6,41]. This review focuses primarily on two types of hybrid receptors. The first type is an insulin/IGF‐1 hybrid receptor generated by the association of an αβ insulin half‐receptor with an αβ IGF‐1 half‐receptor. The second type is one formed from a wildtype (kinase‐active) insulin or IGF‐1 αβ half‐receptor and a mutant (kinase‐inactive) insulin αβ half‐receptor. Although the functional properties of insulin/IGF‐1 hybrid receptors have not yet been completely defined, wildtype/mutant hybrid receptors are essentially substrate kinase inactive [6]. These data indicate that the mutant αβ half‐receptor exerts a transdominant inhibition upon the wildtype αβ half‐receptor within the α2β2 holoreceptor complex. This defect in substrate kinase activity may contribute to the molecular defect underlying some syndromes of severe insulin resistance and diabetes. Heterozygous individuals expressing both wildtype and mutant tyrosine kinase‐defective insulin receptor precursors demonstrate varying degrees of insulin resistance and diabetes [7–11]. In addition, cell lines which express both endogenous wildtype and transfected kinase‐defective insulin receptors display markedly decreased insulin and IGF‐1 sensitivity and responsiveness [12–14]. Formation of hybrid receptors which results in premature termination of insulin signal transduction may be one mechanism underlying the observation that kinase‐inactive receptors inhibit the function of native receptors.
Acute and chronic signals controlling glucose transport in skeletal muscle
Authors: Klip Amira , Marette André
Published: May 2005

Glucose transport into muscle cells occurs through facilitated diffusion mediated primarily by the GLUT1 and GLUT4 glucose transporters. These transporter proteins are controlled by acute and chronic exposure to insulin, glucose, muscle contraction, and hypoxia. We propose that acute responses occur through recruitment of pre‐formed glucose transporters from an intracellular storage site to the plasma membrane. In contrast, chronic control is achieved by changes in transporter biosynthesis and protein stability. Using subcellular fractionation of rat skeletal muscle, recruitment of GLUT4 glucose transporters to the plasma membrane is demonstrated by acute exposure to insulin in vivo. The intracellular pool appears to arise from a unique organelle depleted of transverse tubule, plasma membrane, or sarcoplasmic reticulum markers. In diabetic rats, GLUT4 content in the plasma membranes and in the intracellular pool is reduced, and incomplete insulin‐dependent GLUT4 recruitment is observed, possibly through a defective incorporation of transporters to the plasma membrane. The lower content of GLUT4 transporters in the muscle plasma membranes is reversed by restoration of normoglycemia with phlorizin treatment.

In some muscle cells in culture, GLUT1 is the only transporter expressed yet they respond to insulin, suggesting that this transporter can also be regulated by acute mechanisms. In the L6 muscle cell line, GLUT1 transporter content diminishes during myogenesis and GLUT4 appears after cell fusion, reaching a molar ratio of about 1:1 in the plasma membrane. Prolonged exposure to high glucose diminishes the amount of GLUT1 protein in the plasma membrane by both endocytosis and reduced biosynthesis, and lowers GLUT4 protein content in the absence of changes in GLUT4 mRNA possibly through increased protein degradation. These studies suggest that the relative contribution of each transporter to transport activity, and the mechanisms by which glucose exerts control of the glucose transporters, will be key subjects of future investigations.
Acetyl‐CoA carboxylase in reuber hepatoma cells: Variation in enzyme activity, insulin regulation, and cellular lipid content
Authors: Bianchi Antonella , Evans Joseph L. , Nordlund Ann‐Charlotte , Watts Thomas D. , Witters Lee A.
Published: May 2005

Ruber hapatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl‐CoA carboxylase (ACC), the rate‐limiting enzyme of faty acid biosynthesis. During investigations in deifferent clonal lines of these of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal liners, Fao and H356A‐1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (M, 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorscence‐activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhobition of ACC by the fatty acid analog, 5‐(tetradecyloxy)‐2‐furoic and (TOFA). Thsese results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting colonal lines of Ruber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role (s) in overla hepatic lipid metabolism.
mRNA association with the cytoskeletal framework likely represents a physiological binding event
Authors: Biegel Diane , Pachter Joel S.
Published: May 2005

A multitude of studies has indicated that the vast majority of mRNA and polyribosomes is associated with the detergent‐resistant cytoskeletal faramework (CSK). However, the nature and purpose of this association remain unclear. To begin unraveling the factors which may medicate this phenomenon, we examined the extent of association of four mRNAs (ubulin, vimentin, actin, and histone mRNA) with the CSKs NIH T3 cells over a wide range of salt concentrations. Results indicate that the vast majority ( > 90%) of each of these mRNAs remains associated with the CSK after detergent extration of cells in low ionic strength buffer (25 mM NaCI). This association is manifest under conditions that cause the complete depolymerization of microtubules but that leave microfilaments and intermediate filaments intact. Even after extensive washing in buffer of approximately physiological ionic strength (150 mM NaCI), 75–85% of these mRNAs still remain associated with the CSK. However, at least 50% of each of these mRNAs can be eluted from the CSK by washing with buffer containing 250 mM NaCI. Not all the mRNAs, though, display the same elution profile. This suggests that different binding sites and/or different binding affinities may exist for different mRNAs. Surprisingly, close to 50% of the polyribosome population remains bound to the CSK despite washing in as much as 1.0 M NaCI. These adherent polyribosomes appear to be of the same size as those that are eluted, alllaying the possibility that they are retained by the CSK nor physically trapped within the meshwork of cytoskeletal filaments. Instead, mRNA appears to be bound to the CSK via interactions that are, at least partially, ionic in nature and strong enough to exist intracellulary. Moreover, the binding sites for these mRNAs on the CSK are not associated with microtubules, but may be associated with wither the microflament or intermedicate filament network.
Identification of a core motif that is recognized by three members of the HMG class of transcriptional regulators: IRE‐ABP, SRY, and TCF‐1α
Authors: Alexander‐Bridges M. , Ercolani Louis , Kong X. F. , Nasrin Nargis
Published: May 2005

Insulin induces glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) gene transcription in part by regulating one or more proteins that bind a cis‐acting element, IRE‐A. We have recently cloned a protein, IRE‐ABP, that binds the IRE‐A element. IRE‐ABP is a member of the HMG class of transcriptional regulators and is 67% identical within its HMG box domain to the candidate gene for the testis‐determining factor, SRY. IRE‐ABP and SRY share binding specificity for the IRE‐A motif. This sequence is also highly conserved with a core motif, 5′‐Py‐ctttg(a/t)‐3′, contained in T‐cell specific genes that have high affinity for TCF‐1α, another member of the HMG class of transcriptional regulators. Thus, diverse members of the HMG family interact with similar nucleotide sequences to regulate expression of genes that initiate and maintain the differentiated phenotype. We have found this core motif in the upstream region of many genes that are positively and negatively regulated by insulin. These observations suggest that IRE‐ABP or a related family member may coordinate the expression of these genes. The HMG family of proteins has diverse functions ranging from the regulation of differentiation and mating type in yeast to the regulation of tissue‐and species‐specific gene expression in mammals. Insulin regulates GAPDH gene transcription in a tissue‐specfic manner. We propose that members of the IRE‐ABP family play an important role in controlling tissue specificity of the insulin response.
Insulin/IGF‐I receptor hybrids: A mechanism for increasing receptor diversity
Authors: Moxham Cary P. , Jacobs Steven
Published: May 2005

Insulin and IGF‐l receptors are homologous disulfide linked α2β2 tetramers. These tetramers are formed biosynthetically when proreceptors containing α and β subunits in a single uninterrupted linear peptide from disulfide linked homodimers and are subsequently proteolytically cleaved at the α‐β junctions. Cells expressing both receptors also express hybrid receptors that contain one insulin receptor α and β subunit, and one IGF‐l receptor α and β subunit. These presumably from by the association of mixed proreceptors. Hybrid receptors greatly expand the possible repertoire of cellular responses to hormonal stimulation. Although not yet examined in detail, both the hormone binding and the signaling properties of the hybrid receptor appear to be different from that of either insulin or IGF‐l receptor. Regulatory mechanisms that involve either insulin or IGF‐l receptor, at the level of expression or subsequently, could alter the expression or function of the hybrid receptor or the other receptor. Similarly, pathology in one receptor could affect both the hybrid and other receptor, or perhaps be partially compensated for by a hybrid receptor. The magnitude of these effects could vary greatly in different tissues depending upon the relative level of expression of the different receptor forms. These postulated responses might explain some of the complex heterogeneity and linkage of these receptors that have been observed previously.
Heterotrimeric configuration is essential to the adhesive function of laminin
Authors: Lissitzky J. C. , Cantau P. , Martin P. M.
Published: May 2005

Mouse PFHR9 laminin, B1B2‐heterodimers, and free B1‐chains were separated from one another by gel filtration on superose 6. The cell attachment promoting activity of these species was measured after immunoprecipitation with monoclonal anti‐laminin antibodies coupled to Sepharose 6MB beads. These antibodies, Which did not react with the laminin E8 fragment, were directed against epitopes in the NH2‐terminus of the laminin B1‐chain and in the central region of laminin. After incubation with purified EHS laminin, the immunosorbents revealed efficient adhesion substrates for a rat rhabdomyosarcoma cell line which attached preferentially to the laminin E8 fragment. Although both were immunoprecipitated efficiently, B1B2‐heterodimers and B1‐chains, unlike PFHR9 laminin, did not support the attachment of RMS cells. On a molar basis B1B2‐heterodimers were 24 times less efficient than PFHR9 laminin or EHS laminin in supporting cell attachment. These data suggest that heterotrimeric configuration is essential to the adhesive function of the laminin E8 fragment.
The role of ion channels in insulin secretion
Authors: Boyd A. E.
Published: May 2005

Ion channels in beta cells regulate electrical and secretory activity in response to metabolic, pharmacologic, or neural signals by controlling the permeability to K+ and Ca2+. The ATP‐sensitive K+ channels act as a switch that responds to fuel secretagogues or sulfonylureas to initiate depolarization. This depolarization opens voltage‐dependent calcium channels (VDCC) to increase the amplitude of free cytosolic Ca2+ levels ([Ca2+]i), which triggers exocytosis. Acetyl choline and vasopressin (VP) both potentiate the acute effects of glucose on insulin secretion by generating inositol 1,4,5‐trisphosphate to release intracellular Ca2+; VP also potentiates sustained insulin secretion by effects on depolarization. In contrast, inhibitors of insulin secretion decrease [Ca2+]i by either hyperpolarizing the beta cell or by receptor‐mediated, G‐protein‐coupled effects to decrease VDCC activity. Repolarization is initiated by voltage‐ and Ca2+‐activated K+ channels. A human insulinoma voltage‐dependent K+ channel cDNA was recently cloned and two types of alpha1 subunits of the VDCC have been identified in insulin‐secreting cell lines. Determining how ion channels regulate insulin secretion in normal and diabetic beta cells should provide pathophysiologic insight into the beta cell signal transduction defect characteristic of non‐insulin dependent diabetes (NIDDM).
Monoclonal antibody to the human insulin receptor, but not insulin, stimulates S6 kinase via human insulin receptors mutated at three major tyrosine autophosphorylation sites
Authors: Sung Chin K.
Published: May 2005

Studies were carried out to examine the role of the major insulin receptor tyrosine autophosphorylation sites in stimulation of S6 kinase activity. For these studies, we employed HTC rat hepatoma cells transfected with and expressing human insulin receptors. In cells transfected with and expressing a large number of normal human insulin receptors (HTC‐IR cells), the sensitivity of cells to insulin to stimulate S6 kinase was increased tenfold when compared to untransfected wild type HTC cells (HTC‐WT cells). However, in cells transfected with and expressing a large number of mutated human insulin receptors where the tyrosines at three major autophosphorylation sites (1158, 1162, and 1163) were mutated to phenylalanines (HTC‐F3 cells), there was no change in insulin sensitivity when compared to HTC‐WT cells. We next studied the effect of a human‐specific monoclonal antbody to the human insulin receptor, MA‐5, on S6 kinase activation. In HTC‐WT cells, MA‐5 did not interact with endogenous rat insulin receptors and thus did not stimulate S6 kinase. In HTC‐IR cells expressing normal human insulin receptors, MA‐5 stimulated S6 kinase. Interestingly, MA‐5, unlike insulin, was also able to stimulate S6 kinase in HTC‐F3 cells expressing mutated receptors. In order to further understand the signaling mechanisms by MA‐5 and insulin, two potential intermediate protein kinases were investigate. Neither insulin nor MA‐5 appears to activate either microtubule‐associated protein 2 (MAP‐2) kinase or protein kinase C in these cells.

These studies suggest therefore that: 1) insulin and MA‐5 may signal S6 kinase activation by independent mechanisms that do not employ either MAP‐2 kinase or protein kinase C; and 2) under certain circumstances, S6 kinase appears to be activated by mechanisms that are independent of insulin receptor tyrosine autophosphorylation.
Transcriptional control of vitamin D‐regulated proteins
Authors: Lian Jane B. , Stein Gary S.
Published: May 2005

Vitamin D is a physiological regulator of gene transcription associated with control of a broad spectrum of biological processes that include but is not restricted to growth, differentiation and calcium‐mediated homeostatic control. Transcriptional regulation is mediated by sequence‐specific interactions of a 1,25(OH)2D3‐vitamin D receptor‐accessory factor complex with vitamin D responsive elements (VDRE) residing in the promoters of hormone responsive genes. Functioning primarily as a transcription enhancer, activity at the VDRE is controlled by diverse and integrated cellular signalling pathways acting synergistically and/or antagonistically with a series of basal regulatory elements and other hormone regulated sequences that are components of modularly organized vitamin D‐responsive gene promoters. Molecular mechanisms that integrate the activities at promoter elements contributing to vitamin D‐related transcriptional control include overlapping transcription factor binding domains within regulatory elements and cooperative activities at independent regulatory sequences that determine the level of vitamin D responsiveness.
Sharing of antigenic epitopes between synaptophysin and granulophysin
Authors: Shalev Abraham , Gerrard Jon M. , Robertson Catherine , Greenberg Arnold H. , Linial Michal
Published: May 2005

The immunological crossreactivity between the two granule‐specific membrane glycoproteins, synaptophysin and granulophysin, was studied using a series of site‐specific monoclonal and polyclonal antibodies. The epitope relatedness of six monoclonal antibodies against granulophysin was examined by competitive ELISA. The antibodies are shown to recognize distinct, but overlapping epitopes within a compact region that is constructed by the three‐dimensional configuration of the molecule. All these antibody clones also recognize rat neuronal synaptophysin. Two monoclonal antibodies against synaptophysin, of which one is the well‐characterized SY38 antibody, directed against the carboxy terminal of the molecule, are also shown to react with granulophysin. Characterized polyclonal antibodies against different peptide antigens of synaptophysin failed to recognize granulophysin. Synaptophysin and granulophysin are distinctly recognized in brain cell (white matter) and the pituitary both qualitatively and quantitatively. Based on these and other observations, it is suggested that the repeat motif in the cytoplasmic tail of synaptophysin represents an immunodominant construct that is the target for the observed crossreactive antibodies and that a similar tertiary construct has been preserved in granulophysin and in other transmembrane proteins.