bluuebunny/crossref_metadata_2025 huggingface dataset:
A molecular blueprint for the pore-forming structure of voltage-gated calcium channels.
Authors: Grove A , Tomich J M , Montal M
Published: May 2006

A protein that imitates the sequence of a highly conserved segment predicted to line the pore of dihydropyridine-sensitive L-type calcium channels was designed and synthesized. Single-channel conductance properties were studied in planar lipid bilayers. The synthetic protein emulates the ionic conductance, ionic selectivity, and pharmacological properties of the authentic calcium channel, including the stereospecific action of agonist and antagonist enantiomers of the dihydropyridine BayK 8644. The identified sequence is identical in L-type calcium channels from skeletal muscle and isoforms of cardiac muscle, brain, and aorta. It is plausible that this structural motif represents the molecular blueprint for the pore-forming structure of voltage-gated calcium channels.
Interleukin 6 enhances a cellular activity that functionally substitutes for E1A protein in transactivation.
Authors: Spergel J M , Chen-Kiang S
Published: May 2006

An interleukin 6 (IL-6)-regulated cellular activity in HepG2 cells is found to functionally substitute for the transcriptional transactivator product of the adenovirus transforming gene E1A in transactivating E1A-dependent and E1A-responsive viral early genes. Mutant viruses deficient in E1A expression replicate in HepG2 cells. Induction with IL-6 leads to significant enhancement of synthesis of viral early E1B and E2ae mRNAs by greater than 30-fold and increases viral replication to the wild-type levels. The E1A-substituting activity activates E1A-responsive promoters in transient transfection, and this transcriptional activity is regulated by IL-6 induction. Formation of distinct protein-promoter complexes by binding of proteins in nuclear extracts prepared from HepG2 cells to the E1A-dependent E2ae promoter further supports the possibility that this activity may be a nuclear component in the IL-6 signal transduction pathway.
Loss of heterozygosity and mitotic linkage maps in the mouse.
Authors: Henson V , Palmer L , Banks S , Nadeau J H , Carlson G A
Published: May 2006

Loss of heterozygosity is a significant oncogenetic mechanism and can involve a variety of mechanisms including chromosome loss, deletion, and homologous interchromosomal mitotic recombination. Analysis of H-2 antigen-loss variants from heterozygous murine cell lines provides an experimental system to estimate the relative contributions of different mechanisms for allele loss and to compare the chromosomal patterns of mitotic and meiotic recombination. Cytotoxic anti-H-2D antibodies and complement were used to isolate 161 independent target antigen-negative clones from H-2d/H-2b heterozygous cell lines; of these, 131 (84.5%) lost the allele encoding the target antigen. Allele-loss variants were typed and scored as either heterozygous or homozygous for six H-2D-proximal chromosome 17 markers and for one distal marker by restriction enzyme-site variations and Southern analysis. A single mitotic crossover could account for 50 clones (37%), with heterozygosity for at least one proximal marker and loss of heterozygosity for all markers distal to the putative recombination site. Eighty-two allele-loss variants (60%) were homozygous for all markers; the origin of these clones could be either chromosome loss or mitotic recombination between the centromere and the most proximal marker. Only 4 clones (3%) arose through more complex events such as multiple crossovers or deletion. A mitotic linkage map for mouse chromosome 17 was constructed, and the gene order deduced from somatic recombination was identical to that obtained by conventional transmission genetics. These results demonstrate that mitotic recombination is a common event leading to allele loss, in spite of the lack of evidence for frequent somatic pairing of homologous chromosomes. Mitotic mapping provides a defined system for comparison of mitotic and meiotic recombination and may lead to practical advances for elucidating somatic mechanisms of oncogenesis and for gene therapy in targeting mutations to specific sites through homologous recombination.
Expression of the cystic fibrosis transmembrane conductance regulator gene in the respiratory tract of normal individuals and individuals with cystic fibrosis.
Authors: Trapnell B C , Chu C S , Paakko P K , Banks T C , Yoshimura K , Ferrans V J , Chernick M S , Crystal R G
Published: May 2006

The most common mutation of the cystic fibrosis transmembrane conductance regulator gene, CFTR, associated with the clinical disorder cystic fibrosis (CF) is called "delta Phe508," a triple-base deletion resulting in loss of phenylalanine at residue 508 of the predicted 1480-amino acid CFTR protein. In the context that the lung is the major site of morbidity and mortality in CF, we evaluated airway epithelial cells for CFTR mRNA transcripts in normal individuals, normal-delta Phe508 heterozygotes, and delta Phe508 homozygotes to determine if the normal and delta Phe508 CFTR alleles are expressed in the respiratory epithelium, to what extent they are expressed, and whether there are relative differences in the expression of the normal and abnormal alleles at the mRNA level. Respiratory tract epithelial cells recovered by fiberoptic bronchoscopy with a cytology brush demonstrated CFTR mRNA transcripts with sequences appropriately reflecting the normal and delta Phe508 CFTR alleles of the various study groups. CFTR gene expression quantified by limited polymerase chain reaction amplification showed that in normal individuals, CFTR mRNA transcripts are expressed in nasal, tracheal, and bronchial epithelial cells at approximately 1-2 copies per cell, more than 100-fold greater than in pharyngeal epithelium. Importantly, allele-specific hybridization studies demonstrated that the normal and delta Phe508 CFTR alleles are expressed in the respiratory epithelium in similar amounts.
Signal transduction convergence: phorbol esters and insulin inhibit phosphoenolpyruvate carboxykinase gene transcription through the same 10-base-pair sequence.
Authors: O'Brien R M , Bonovich M T , Forest C D , Granner D K
Published: May 2006

Phosphoenolpyruvate carboxykinase (PEPCK) governs the rate-limiting step in gluconeogenesis. Glucocorticoids and cAMP increase PEPCK gene transcription and gluconeogenesis, whereas insulin and phorbol esters have the opposite effect. Insulin and phorbol esters are dominant, since they prevent cAMP and glucocorticoid-stimulated transcription. Basal promoter elements and hormone response elements for cAMP, glucocorticoids, and insulin have been defined in previous studies. By using stable transfectants containing a variety of different PEPCK-chloramphenicol acetyltransferase fusion gene constructs, a phorbol ester response sequence, located between positions -437 and -402 relative to the transcription start site, was identified. This region coincides with the insulin response sequence that has recently been defined in the PEPCK promoter. Using a vector containing various wild-type and mutated sequences of this region ligated to the heterologous thymidine kinase promoter, we delineated the boundaries of both elements to the 10 base pairs between positions -416 through -407. Thus, although it has been previously shown that insulin and phorbol esters repress PEPCK gene transcription through distinct pathways, the final target of insulin and phorbol ester action is the same DNA element.
Cholinesterase-like domains in enzymes and structural proteins: functional and evolutionary relationships and identification of a catalytically essential aspartic acid.
Authors: Krejci E , Duval N , Chatonnet A , Vincens P , Massoulié J
Published: May 2006

Primary sequences of cholinesterases and related proteins have been systematically compared. The cholinesterase-like domain of these proteins, about 500 amino acids, may fulfill a catalytic and a structural function. We identified an aspartic acid residue that is conserved among esterases and lipases (Asp-397 in Torpedo acetylcholinesterase) but that had not been considered to be involved in the catalytic mechanism. Site-directed mutagenesis demonstrated that this residue is necessary for activity. Analysis of evolutionary relationships shows that the noncatalytic members of the family do not constitute a separate subgroup, suggesting that loss of catalytic activity occurred independently on several occasions, probably from bifunctional molecules. Cholinesterases may thus be involved in cell-cell interactions in addition to the hydrolysis of acetylcholine. This would explain their specific expression in well-defined territories during embryogenesis before the formation of cholinergic synapses and their presence in noncholinergic tissues.
Activation of membrane protein-tyrosine phosphatase involving cAMP- and Ca2+/phospholipid-dependent protein kinases.
Authors: Brautigan D L , Pinault F M
Published: May 2006

Essential to signal transduction are mechanisms of "cross-talk" to coordinate different pathways. This study shows that stimulation of serine/threonine protein kinases activates protein-tyrosine phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). More than 95% of intracellular PTPase was in the particulate fraction of various cell lines and was extracted with detergent as a 150-kDa complex that contained a 55-kDa catalytic subunit. The complex was activated by protease digestion, which changed its substrate specificity coincident with reduction in size. The complex was dissociated by treatment of the membrane fraction with 3 M LiBr. Treatment of intact cells with isoproterenol, forskolin, or cAMP analogues to stimulate cAMP-dependent protein kinase (PKA) or with phorbol ester or dioctanoylglycerol to stimulate Ca2+/phospholipid-dependent protein kinase (PKC) produced activation of membrane PTPase complex without a change in its size. Inhibition of protein-serine/threonine phosphatases with okadaic acid or fluoride also resulted in activation of the membrane PTPase. These results support a model for regulation of PTPase by phosphorylation and dephosphorylation of serine/threonine residues in a regulatory component complexed with the 55-kDa PTPase catalytic subunit. This mechanism may be important in regulating sensitivity to extracellular signals transduced via tyrosine phosphorylation and in the synchronization of events during the cell cycle.
Sequestration of basic fibroblast growth factor in the primate retinal interphotoreceptor matrix.
Authors: Hageman G S , Kirchoff-Rempe M A , Lewis G P , Fisher S K , Anderson D H
Published: May 2006

The interphotoreceptor matrix (IPM) occupies the extracellular space between the photoreceptors of the retina and the apical surface of the retinal pigmented epithelium. A large proportion of the IPM is composed of aqueous-insoluble glycoconjugates, including chondroitin sulfate-containing proteoglycans, the distribution of which exhibits both apical-basal and photoreceptor cell type-specific heterogeneities. The precise function of most insoluble IPM constituents is unknown, although the available evidence suggests some may contribute to retinal adhesion or photoreceptor survival. We have now identified basic fibroblast growth factor (bFGF), or an immunologically related protein from the FGF family, within the IPM. The IPM is labeled on sections of primate retinas by a battery of polyclonal antibodies (Abs) directed against various peptide sequences of bFGF and by an Ab to bovine brain bFGF. bFGF Abs also bind to purified preparations of aqueous-insoluble IPM. All bFGF Abs utilized cross-react with equivalent low molecular mass components of 16.5-17.5 kDa on Western blots of insoluble IPM proteins, purified bFGF, and recombinant bFGF. The Abs do not bind any aqueous-soluble IPM components, suggesting that the bFGF is normally bound to an insoluble IPM constituent(s) in situ. The fact that bFGF is sequestered in the IPM and is located in such close proximity to the photoreceptors, the retinal pigmented epithelium, and Mueller's glia raises the strong possibility that it is synthesized by and regulates the activities of one or more of these three cell types in vivo.
Expression of the human apolipoprotein A-I gene in transgenic mice alters high density lipoprotein (HDL) particle size distribution and diminishes selective uptake of HDL cholesteryl esters.
Authors: Chajek-Shaul T , Hayek T , Walsh A , Breslow J L
Published: May 2006

Transgenic mice carrying the human apolipoprotein (apo) A-I gene (HuAITg mice) were used to examine the effects of overexpression of the human gene on high density lipoprotein (HDL) particle size distribution and metabolism. On a chow diet, control mice had HDL cholesterol and apo A-I levels of 49 +/- 2 and 137 +/- 12 mg/dl of plasma, respectively. HuAITg mice had HDL cholesterol, human apo A-I, and mouse apo A-I levels of 88 +/- 2, 255 +/- 19, and 16 +/- 2 mg/dl, respectively. Nondenaturing gradient gel electrophoresis revealed control mouse plasma HDL to be primarily monodisperse with a particle diameter of 10.2 nm, whereas HuAITg mouse plasma HDL was polydisperse with particles of diameter 11.4, 10.2, and 8.7 nm, which correspond in size to human HDL1, HDL2, and HDL3, respectively. In vivo turnover studies of HDL labeled with [3H]cholesteryl linoleyl ether (representing the cholesteryl ester pool) and 125I-apo A-I were performed. In control animals, the fractional catabolic rate (FCR) for HDL cholesteryl ester (0.197 +/- 0.010 pool/hr) was significantly (P less than 0.0005) more than the apo A-I FCR (0.118 +/- 0.006 pool/hr). In the HuAITg mice, the HDL cholesteryl ester FCR (0.124 +/- 0.008 pool/hr) was the same as the apo A-I FCR (0.126 +/- 0.010 pool/hr). There were no significant differences between control and HuAITg animals in the sites of tissue removal of HDL cholesteryl ester, with the liver extracting most of the injected radioactivity. Control and HuAITg animals had comparable liver and intestinal cholesterol synthesis and LDL FCR. In conclusion, HuAITg mice have principally human and not mouse apo A-I in their plasma. This apparently causes a change in HDL particle size distribution in the transgenic mice to one resembling the human pattern. The replacement of mouse by human apo A-I also apparently causes the loss of the selective uptake pathway of HDL cholesteryl esters present in control mice. These data imply that apo A-I primary structure has a profound influence on HDL particle size distribution and metabolism.
Bidirectional control of phospholipase A2 activity by Ca2+/calmodulin-dependent protein kinase II, cAMP-dependent protein kinase, and casein kinase II.
Authors: Piomelli D , Greengard P
Published: May 2006

In preparations of synaptic terminals (synaptosomes) isolated from rat brain, the activity of phospholipase A2 (PLA2), a phospholipid hydrolase that serves a central function in signal transduction, was inhibited in a Ca(2+)-dependent manner by incubation with 60 mM K+ or with the Ca(2+)-selective ionophore ionomycin. Reversal by alkaline phosphatase treatment suggested that this inhibitory effect resulted from phosphorylation of a synaptosomal protein substrate. When lysed synaptosomes were incubated with Ca2+/calmodulin (CaM), purified Ca2+/CAM-dependent protein kinase II (Ca2+/CaM-dependent PK II) and ATP, PLA2 activity in lysates was nearly abolished within 10 min. This effect was accompanied by a marked decrease in the Vmax of the enzyme and little or no change in the Km. Furthermore, Ca2+/CaM with ATP but without exogenous Ca2+/CaM-dependent PK II partially inhibited PLA2 activity, and this effect was prevented by treating the lysates with a selective peptide inhibitor of Ca2+/CaM-dependent PK II. In contrast, incubation of intact synaptosomes with 4 beta-phorbol 12-myristate 13-acetate or of lysed synaptosomes with purified protein kinase C had little or no effect on PLA2 activity. The results strongly suggest that the Ca(2+)-dependent inhibition of PLA2 activity observed in intact nerve endings was produced by activation of the multifunctional Ca2+/CaM-dependent PK II. A membrane-permeable adenylyl cyclase activator, forskolin, enhanced PLA2 activity in intact synaptosomes, and cAMP-dependent protein kinase potentiated PLA2 activity in lysed synaptosomes. Furthermore, another broad-spectrum protein kinase present in synaptic terminals, casein kinase II, also potentiated PLA2 activity in lysed synaptosomes. The effects of both protein kinases were associated with a decrease in Km and no change in Vmax. The results suggest that PLA2 activity in synaptic terminals is subject to bidirectional control by distinct signal transduction pathways. Moreover, mutually antagonistic effects of the Ca2+/CaM-dependent PK II and PLA2 pathways provide a possible molecular mechanism for bidirectional modulation of neurotransmitter release.
The C-terminal half of UvrC protein is sufficient to reconstitute (A)BC excinuclease.
Authors: Lin J J , Sancar A
Published: May 2006

The UvrC protein is one of three subunits of the Escherichia coli repair enzyme (A)BC excinuclease. This subunit is thought to have at least one of the active sites for nucleophilic attack on the phosphodiester bonds of damaged DNA. To localize the active site, mutant UvrC proteins were constructed by linker-scanning and deletion mutagenesis. In vivo studies revealed that the C-terminal 314 amino acids of the 610-amino acid UvrC protein were sufficient to confer UV resistance to cells lacking the uvrC gene. The portion of the uvrC gene encoding the C-terminal half of the protein was fused to the 3' end of the E. coli malE gene (which encodes maltose binding protein), and the fusion protein MBP-C314C was purified and characterized. The fusion protein, in combination with UvrA and UvrB subunits, reconstituted the excinuclease activity that incised the eighth phosphodiester bond 5' and the fourth phosphodiester bond 3' to a psoralen-thymine adduct. These results suggest that the C-terminal 314 amino acids of UvrC constitute a functional domain capable of interacting with the UvrB-damaged DNA complex and of inducing the two phosphodiester bond incisions characteristic of (A)BC excinuclease.
Human cells contain protein specifically binding to a single 1,N6-ethenoadenine in a DNA fragment.
Authors: Rydberg B , Dosanjh M K , Singer B
Published: May 2006

A human DNA binding protein has been characterized from cell-free extracts of liver, placenta, and cultured cells. This protein, apparent molecular mass approximately 35 kDa, to our knowledge, does not resemble other proteins reported to bind to carcinogen-modified DNA. The probe used for characterization was a 25-base oligonucleotide containing a single site-specifically placed 1,N6-ethenoadenine (epsilon A), a product of vinyl chloride metabolism. When annealed to form an epsilon A.T or epsilon A.C pair, a strong affinity to the protein was observed, with a binding constant of approximately 1 x 10(9) M-1. In contrast, very little binding was found with an epsilon A.A pair and none was found with an epsilon A.G pair. This suggests protein recognition of a specific structural alteration. Other defined probes with alkyl adducts did not bind. In addition, the human cell extracts and a rat liver extract were found to nick specifically at the 5' side of the epsilon A adduct, which could indicate a possible associated repair activity.
Monitoring of intracellular calcium in Saccharomyces cerevisiae with an apoaequorin cDNA expression system.
Authors: Nakajima-Shimada J , Iida H , Tsuji F I , Anraku Y
Published: May 2006

A method is described for measuring cytosolic free Ca2+ and its time-dependent changes in the yeast Saccharomyces cerevisiae by using the luminescent protein aequorin as a Ca(2+)-specific indicator. This method with intact yeast cells is labeled "in vivo" to distinguish it from methods with cell extracts, labeled "in vitro." A plasmid in which the apoaequorin cDNA was joined downstream from the glyceraldehyde-3-phosphate dehydrogenase gene promoter was constructed and introduced into yeast cells. The intracellular concentration of apoaequorin expressed by the cDNA was approximately 1 microM, which was high enough to detect the cytosolic Ca2+. Growth of the transformed cells was normal. In the in vitro method, apoaequorin in crude cell extracts was regenerated into aequorin by mixing with coelenterazine, the substrate for the luminescence reaction, whereas in the in vivo method, aequorin was regenerated by incubating intact cells with coelenterazine. Simultaneous addition of 10 mM CaCl2 and 10 microM A23187, a Ca2+ ionophore, to coelenterazine-incorporated cells generated luminescence. Coelenterazine-incorporated cells also responded to native extracellular stimuli. A mating pheromone, alpha-factor, added to cells of mating type a or alpha, generated extracellular Ca(2+)-dependent luminescence specifically in a mating type cells, with maximal intensity occurring 45-50 min after addition of alpha-factor. Glucose added to glucose-starved G0/G1 cells stimulated an increase in extracellular Ca(2+)-dependent luminescence with maximal intensity occurring 2 min after addition. These results show the usefulness of the aequorin system in monitoring [Ca2+]i response to extracellular stimuli in yeast cells.
Change in the expression of a nuclear matrix-associated protein is correlated with cellular transformation.
Authors: Brancolini C , Schneider C
Published: May 2006

We have identified a monoclonal antibody that recognizes a nuclear matrix-associated protein in NIH 3T3 cells. The immunofluorescence pattern consists predominantly of bright nuclear granule clusters distributed throughout the nucleoplasm, with the exclusion of nucleoli. It recognizes a protein of 190 kDa that is down-regulated to various degrees in a panel of single-oncogene-transformed NIH 3T3 cells. Its localization is similar, but not identical, to the spliceosomal speckles. p190 shows a coordinate expression during the growth cycle of nontransformed NIH 3T3 cells; it is synthesized at the highest level under growth arrest conditions. It is expressed in adult mouse brain and is also present in human IMR-90 fibroblasts.
Repressor to activator switch by mutations in the first Zn finger of the glucocorticoid receptor: is direct DNA binding necessary?
Authors: Ray A , LaForge K S , Sehgal P B
Published: May 2006

Transfection of HeLa cells with cDNA vectors expressing the wild-type human glucocorticoid receptor (GR) enabled dexamethasone to strongly repress cytokine- and second messenger-induced expression of cotransfected chimeric reporter genes containing transcription regulatory DNA elements from the human interleukin 6 (IL-6) promoter. Deletion of the DNA-binding domain or of the second Zn finger or a point mutation in the Zn catenation site in the second finger blocked the ability of GR to mediate repression of the IL-6 promoter. Unexpectedly, deletion of the first Zn finger, a point mutation in the Zn-catenation site in the first finger, or one in the steroid-specificity domain at the base of the first finger converted GR into a dexamethasone-responsive activator that enhanced basal and interleukin 1-induced IL-6 promoter function. These first-finger mutants of GR also mediated dexamethasone-responsive enhancement of expression of the herpesvirus thymidine kinase-chloramphenicol acetyltransferase (TK-105-CAT and TK-80-CAT) reporter genes but not of the murine mammary tumor virus long terminal repeat-CAT or the c-fos-CAT (pFC700) reporter genes. Wild-type GR was able to specifically bind to DNA fragments containing glucocorticoid response element sequences in both the murine mammary tumor virus and IL-6 promoters, albeit weakly to the latter, in a sequential DNA-binding immunoprecipitation assay. The first-finger mutants of GR, however, were inactive in this assay. Thus, mutations in the first Zn finger unmask unusual promoter-specific activation properties of GR that may not require direct high-affinity binding of the mutant GR to target DNA.
Molecular cloning and analysis of small optic lobes, a structural brain gene of Drosophila melanogaster.
Authors: Delaney S J , Hayward D C , Barleben F , Fischbach K F , Miklos G L
Published: May 2006

Mutations in the small optic lobes (sol) gene of Drosophila melanogaster cause specific cells to degenerate in the developing optic lobes, resulting in the absence of certain classes of columnar neurons. These neuronal defects lead to specific alterations in behavioral characteristics, particularly during flight and walking maneuvers. We have isolated the wild-type sol locus by microcloning and chromosomal walking and have established its genetic and molecular limits. Two major transcripts of 5.8 and 5.2 kilobases are produced from this locus by alternative splicing and are present throughout the entire life cycle. Sequence analyses of cDNAs corresponding to these two classes of transcripts predict two proteins of 1597 and 395 amino acids. The first shows similarity in its carboxyl-terminal region to the catalytic domain of a vertebrate calcium-activated neutral protease (calpain), whereas its amino-terminal region contains several zinc-finger-like repeats of the form WXCX2CX10-11CX2C. The second predicted protein contains only the first two of the zinc-finger-like repeats and is missing the calpain domain. By constructing transgenic flies carrying a single wild-type copy of the sol gene in a homozygous sol mutant background, we have restored the normal neuroanatomical phenotype to individuals that would have developed mutant brains.
Both alpha-helices along the major histocompatibility complex binding cleft are required for staphylococcal enterotoxin A function.
Authors: Russell J K , Pontzer C H , Johnson H M
Published: May 2006

The superantigen staphylococcal enterotoxin A (SEA) requires interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously used the synthetic peptide approach to establish one side of the hypothetical class II foreign-antigen binding cleft, alpha-helical region 65-85 of the beta chain, as a binding site involved in accessory cell presentation of SEA to T cells. To further characterize the structural basis for MHC-SEA interaction we have examined the role of the alpha-helical regions of the class II alpha and beta chains in SEA function. Using the synthetic peptide approach, we have found that both alpha-helical regions are required for SEA-induced proliferation. Their corresponding peptides directly bound SEA. Although the beta-chain peptides were able to inhibit SEA binding to human and mouse cells, the alpha-chain peptides were not. The data suggest that the alpha-helices along both sides of the hypothetical class II MHC molecule binding cleft are required for SEA-induced function, whereas the beta-chain alpha-helix is sufficient for SEA binding. A model of superantigen presentation is proposed wherein the MHC beta chain, possibly region 70-80, interacts with SEA region 1-45, whereas another region of SEA binds region 51-80 of the alpha chain.
Autoantigen recognition by thyroid-infiltrating T cells in Graves disease.
Authors: Dayan C M , Londei M , Corcoran A E , Grubeck-Loebenstein B , James R F , Rapoport B , Feldmann M
Published: May 2006

Graves disease is a common form of human autoimmune thyroiditis. It shares many pathological features and HLA associations with other, less easily studied, organ-specific autoimmune conditions such as insulin-dependent diabetes mellitus, and hence it is also a useful model for understanding these other diseases. We have previously shown that thyroid-infiltrating T cells in Graves disease that have been recently activated in vivo specifically recognize autologous thyroid epithelial cells. However, the autoantigens involved were not defined. In this study, we have made use of antigen-independent T-cell cloning techniques to show that at least three different thyroid antigens, three different epitopes on a single antigen, and two HLA class II elements are involved in this recognition process in a single individual. This demonstrates that T cells that are present and activated at the site of a human autoimmune disease may show considerable heterogeneity in their recognition of autoantigen on the target tissue. This contrasts with the limited heterogeneity recently reported in some animal models and has potentially important implications for both our understanding of the autoimmune process in humans and the design of immunotherapies to reverse it.
Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1.
Authors: Weller P F , Rand T H , Goelz S E , Chi-Rosso G , Lobb R R
Published: May 2006

Adherence of human eosinophils to cytokine-stimulated endothelial cells, which was only partially due to CD18-dependent pathways, was also mediated by binding to endothelial leukocyte adhesion molecule 1 (ELAM-1) and vascular cell adhesion molecule 1 (VCAM-1). Eosinophils bound specifically to both recombinant soluble ELAM-1 and recombinant soluble VCAM-1. Eosinophil binding to recombinant soluble VCAM-1 and to transfected CHO cells expressing VCAM-1 was inhibited with anti-VCAM-1 (4B9) and anti-very late activation antigen 4 (anti-VLA-4; HP1/2 or HP2/1) monoclonal antibodies. Eosinophils, but not neutrophils, expressed VLA-4 detected by cytofluorography. Eosinophil adherence to tumor necrosis factor alpha-stimulated human umbilical vein endothelial cells was partially blocked by monoclonal antibodies against ELAM-1 (BB11) and VCAM-1 (4B9) and against VLA-4 (HP2/1). Thus, while both eosinophils and neutrophils can bind to activated endothelial cells by adherence to ICAM-1 and ELAM-1, only eosinophils expressed VLA-4 and adhered to VCAM-1 on activated endothelial cells. Eosinophil adherence to VCAM-1 might provide a mechanism contributing to the selective recruitment of eosinophils into tissue sites of inflammation.
Radiation hybrid mapping.
Authors: Lawrence S , Morton N E , Cox D R
Published: May 2006

A theory is developed to predict marker retention and conditional retention or loss in radiation hybrids. Applied to multiple pairwise analysis of a human chromosome 21 data set, this theory fits much better than proposed alternatives and gives a physical map consistent with other evidence and robust with respect to errors to typing. Radiation hybrids have great promise to provide order and physical location at two levels of resolution, spanning the techniques of linkage and restriction fragments and not limited to polymorphic loci.
Expression of two forms of carp gonadotropin alpha subunit in insect cells by recombinant baculovirus.
Authors: Huang C J , Huang F L , Chang G D , Chang Y S , Lo C F , Fraser M J , Lo T B
Published: May 2006

There are two types of cDNA clones (designated alpha 1 and alpha 2) encoding the alpha subunit of carp gonadotropin. These two cDNAs are derived from different genes and encode proteins that differ by seven amino acid residues (three in the signal peptide and four in the mature polypeptide). Expression of these two cDNAs in insect cells by recombinant baculovirus revealed that the alpha 1 subunit, after noncovalent association with the beta subunit, has the same potency as the native alpha subunit purified from the pituitary. In contrast, the alpha 2 subunit can associate with the beta subunit, but only to form an inactive gonadotropin. Competition of the alpha 2 subunit with the alpha 1 subunit for association with the beta subunit decreases the gonadotropin activity of the alpha/beta complex. In addition, both alpha 1 and alpha 2 subunits are secreted into the culture medium by insect cells and have an apparent molecular mass approximately 5 kDa higher than that of the native alpha subunit. These results indicate that the insect cell-derived alpha 1 subunit is biologically active and that those four amino acid changes in the mature of alpha 2 protein affect the biological activity and thus provide valuable clues for the study of the structure-function relationship of the alpha subunit of glycoprotein hormones.
Dominant expression of a distinctive V14+ T-cell antigen receptor alpha chain in mice.
Authors: Koseki H , Asano H , Inaba T , Miyashita N , Moriwaki K , Lindahl K F , Mizutani Y , Imai K , Taniguchi M
Published: May 2006

A distinctive variable region 14-positive (V14+) alpha chain (V alpha 14+) of the T-cell antigen receptor is predominantly expressed in multiple mouse subspecies. The V alpha 14 family has two members, V alpha 14.1 and V alpha 14.2, which differ by only three amino acids at positions 50-52. Based on the EcoRI restriction fragment length polymorphism of the gene encoding V alpha 14, mice can be divided into three groups: type I with an 11.2-kilobase (kb) fragment, type II with a 2.0-kb fragment, and type III with the 2.0-kb and 11.2-kb fragments. Usage of V alpha 14-J alpha 281, where J alpha 281 is an alpha-chain joining segment, with a one-base N region dominates at the level of 0.02-1.5% of alpha chains in all laboratory strains, Mus musculus castaneus, and Mus musculus domesticus but not in Mus musculus molossinus, Mus musculus musculus, and Mus spicilegus samples. The preferential V alpha 14-J alpha 281 expression seems to be due to positive selection because the V-J junctional region is always glycine, despite the ability of the V alpha 14 gene to associate with J alpha other than J alpha 281. As V alpha 14-J alpha 281 expression is independent of known major histocompatibility complex antigens, including H-2, TLA, Qa, and HMT, the selecting ligand must be a monomorphic molecule of the mouse, expressed in a subspecies-specific manner. Additional observations, such as the expression of homogeneous V alpha 14-J alpha 281 in athymic mice, suggest that the positive selection of V alpha 14+ T cells occurs extrathymically.
Translation and the stability of mRNAs encoding the transferrin receptor and c-fos.
Authors: Koeller D M , Horowitz J A , Casey J L , Klausner R D , Harford J B
Published: May 2006

Turnover of the full-length human transferrin receptor (TfR) mRNA is regulated by iron, and this regulation is mediated by the transcript's 3' untranslated region. Alterations in the sequence of the TfR mRNA regulatory region have been identified that render the mRNA unregulated by iron and intrinsically unstable. When cells expressing this unstable mRNA are treated with inhibitors of protein synthesis (cycloheximide or puromycin), the steady-state level of the encoded human TfR mRNA is increased due to a stabilization of the transcript. A similar set of observations has been made using a chimeric mRNA in which the rapid turnover determinant of the TfR mRNA is replaced by the (A+U)-rich region from the 3' untranslated region of c-fos mRNA. To distinguish between a labile protein participant in the degradation of these mRNAs and a requirement for their translation per se, we introduced a ferritin iron-responsive element into the 5' untranslated region of each of these mRNAs. The presence of the 5' iron-responsive element allowed us to use iron availability to alter the translation of the mRNAs in question without global effects on cellular protein synthesis. Although specific translation of these mRNAs could be inhibited by iron chelation to a degree comparable to that seen with cycloheximide (approximately 95% inhibition), no effects on mRNA turnover were observed. These data support a model in which a trans-acting labile protein is necessary for the turnover of these mRNAs rather than there being a requirement for the translation of the mRNAs themselves.
Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat.
Authors: Slot J W , Geuze H J , Gigengack S , James D E , Lienhard G E
Published: May 2006

The insulin-regulated glucose transporter GLUT4 was immunolocalized in rat cardiac muscle under conditions of basal and stimulated glucose uptake, achieved by fasting and a combined exercise/insulin stimulus, respectively. In basal myocytes there was very little (less than 1%) GLUT4 in the different domains of the plasma membrane (sarcolemma, intercalated disk, and transverse tubular system). GLUT4 was localized in small tubulo-vesicular elements that occur predominantly near the sarcolemma and the transverse tubular system and in the trans-Golgi region. Upon stimulation approximately 42% of GLUT4 was found in the plasma membrane. Each domain of the plasma membrane contributed equally to this effect. GLUT4-positive, clathrin-coated pits were also present at each cell surface domain. The remainder of the labeling was in tubulo-vesicular elements at the same sites as in basal cells and in the intercalated disk areas. The localization of GLUT4 in cardiac myocytes is essentially the same as in brown adipocytes, skeletal muscle, and white adipocytes. We conclude that increased glucose transport in muscle and fat is accounted for by translocation of GLUT4 from the intracellular tubulo-vesicular elements to the plasma membrane. The labeling of coated pits indicates that in stimulated myocytes, as in adipocytes, GLUT4 recycles constantly between the endosomal compartment and the plasma membrane and that stimulation of the exocytotic rate constant is likely the major mechanism for GLUT4 translocation.
A cluster of related zinc finger protein genes is deleted in the mouse embryonic lethal mutation tw18.
Authors: Crossley P H , Little P F
Published: May 2006

We report that a number of related zinc finger protein genes are closely linked on mouse chromosome 17. At least four of these genes are transcribed in the 8.5-day postcoitum embryo and are deleted in the t complex early acting embryonic lethal mutation tw18. We have evidence that additional finger protein genes are located in this region. These findings demonstrate that related finger protein genes can be clustered in the murine genome and identify genes that may be considered as candidates for the tw18 mutation.
Antigen mobility in the combining site of an anti-peptide antibody.
Authors: Cheetham J C , Raleigh D P , Griest R E , Redfield C , Dobson C M , Rees A R
Published: May 2006

The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 57-84), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two-dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, Trp-62, Trp-63, and Ile-78) do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity.
Feed-forward control of prostate growth: dihydrotestosterone induces expression of its own biosynthetic enzyme, steroid 5 alpha-reductase.
Authors: George F W , Russell D W , Wilson J D
Published: May 2006

Dihydrotestosterone, the primary mediator of prostate growth, is synthesized in target tissues from the circulating androgen testosterone through the action of steroid 5 alpha-reductase (EC 1.3.99.5). The expression of 5 alpha-reductase and the level of 5 alpha-reductase messenger RNA in rat ventral prostate are regulated by androgens. To determine whether this control is mediated by dihydrotestosterone or testosterone, we investigated the effect of finasteride, a potent inhibitor of steroid 5 alpha-reductase, on the expression of 5 alpha-reductase in the prostate. The administration of finasteride to intact rats for 7 days caused a 55% decrease in prostate weight and an 87% decrease in 5 alpha-reductase enzyme activity. Furthermore, the restoration of prostate growth after castration and the enhancement in 5 alpha-reductase enzyme activity and 5 alpha-reductase messenger RNA level by testosterone administration were blocked by finasteride, whereas the inhibitor had no effect on dihydrotestosterone-mediated increases in 5 alpha-reductase activity or messenger RNA level. These findings indicate that dihydrotestosterone itself controls prostate growth and 5 alpha-reductase activity. They further suggest that prostate growth is controlled by a feed-forward mechanism by which formation of trace amounts of dihydrotestosterone induces 5 alpha-reductase, thereby increasing dihydrotestosterone synthesis and triggering a positive developmental cascade.
A Ricardo model with economies of scale.
Authors: Gomory R E
Published: May 2006

A model of international trade that resembles the classical Ricardo model but differs from it in admitting economies of scale in production is described.
The position of the microtubule-organizing center in directionally migrating fibroblasts depends on the nature of the substratum.
Authors: Schütze K , Maniotis A , Schliwa M
Published: May 2006

Immunofluorescence and confocal microscopy were used to monitor the positioning of microtubule-organizing centers (MTOCs) during directional migration of chicken embryo fibroblasts on planar substrata and within three-dimensional collagen gels. Homologous assay conditions based on the radial emigration of cells from cell aggregates were used in both cases. Whereas approximately 70% of the cells migrating directionally on glass and at least 60% on other planar substrata have their MTOCs anterior to the nucleus, MTOCs are randomly distributed around the nucleus in cells within collagen gels. The anterior location of the MTOC in cells on glass is attained gradually during the first 4 hr of directional migration. Cells on oriented planar substrata, manufactured by photolithographic etching of narrow parallel grooves into the glass surface, also have a random position of the MTOC, although the cells themselves assume a highly polarized cell shape parallel to the grooves. This environment mimics the partial orientation of the collagen fibers produced by the tractive forces of the cells within collagen networks. These findings demonstrate a difference in MTOC positioning between fibroblasts on planar substrata and within a quasinatural environment.
Stimulation of dihydrofolate reductase promoter activity by antimetabolic drugs.
Authors: Eastman H B , Swick A G , Schmitt M C , Azizkhan J C
Published: May 2006

Dihydrofolate reductase (DHFR; EC 1.5.1.3) is required in folate metabolism for the synthesis of purines, thymidine, and glycine. Although there have been several reports of induction of DHFR enzyme by methotrexate (MTX), a drug that competitively inhibits DHFR, there are no studies reported that examine the effect of MTX on DHFR gene transcription. We have examined the effect of MTX and other inhibitors of DNA synthesis on DHFR transcription using a transient expression assay. MTX stimulates transient expression in a concentration-dependent manner from a hamster DHFR promoter construct containing 150 base pairs 5' to the start of transcription. Addition of either tetrahydrofolate or hypoxanthine plus thymidine prevents the promoter induction in response to MTX, suggesting that stimulation by MTX results from inhibition of these metabolites. Furthermore, two other antimetabolic drugs--fluorodeoxyuridine and hydroxyurea--also stimulate the DHFR promoter in a concentration-dependent manner. In contrast, aphidicolin, which blocks cell growth through inhibition of DNA polymerase alpha, has no effect on the DHFR promoter. The potential relevance of these results to cross-resistance to chemotherapeutic agents and to the process of gene amplification is discussed.
Distinct pathways for basolateral targeting of membrane and secretory proteins in polarized epithelial cells.
Authors: Boll W , Partin J S , Katz A I , Caplan M J , Jamieson J D
Published: May 2006

Polarized epithelial cells target distinct sets of membrane and secretory proteins to their apical and basolateral domains. Here we examine whether constitutively secreted and membrane proteins that are bound for the same domain share the same carrier vesicles. To address the issue, differential effects of microtubule depolymerization on basolateral protein targeting in the polarized Madin-Darby canine kidney II cell line were studied. We find that the basolateral insertion of the active, ouabain-binding Na+,K(+)-ATPase and of a set of very late antigen integrins is little affected by microtubule disruption. Under equivalent conditions, the basolateral secretion of the basement membrane protein laminin is strongly suppressed. More specifically, it is demonstrated that microtubules are involved in targeting laminin, but not integrins, from the compartment related to the accumulation of newly synthesized proteins at 20 degrees C (trans-Golgi network) to the basolateral domain. Our study also reveals that laminin associated with basolateral binding sites interacts with those sites only secondarily to secretion. The data provide evidence for a branch in the basolateral targeting pathway, with secreted and membrane proteins loaded into distinct carrier vesicles.
Stimulation of pheromone biosynthesis in the moth Helicoverpa zea: action of a brain hormone on pheromone glands involves Ca2+ and cAMP as second messengers.
Authors: Jurenka R A , Jacquin E , Roelofs W L
Published: May 2006

Isolated abdomen and pheromone gland bioassays were utilized to determine the physiological action of the pheromone-biosynthesis-activating neuropeptide (PBAN) in the corn earworm moth Helicoverpa (= Heliothis) zea. An isolated pheromone gland bioassay showed that synthetic PBAN was active at 0.02 pmol, with maximal activity occurring at 0.5 pmol and 60 min of incubation. Second-messenger studies demonstrated that extracellular Ca2+ is necessary for PBAN activity on isolated pheromone glands. The Ca2+ ionophore A23187 stimulated pheromone biosynthesis alone, whereas the Ca2+ channel blockers La3+ and Mn2+ inhibited PBAN activity. However, the organic Ca2+ channel blockers verapamil and nifedipine did not inhibit PBAN activity. Both forskolin and two cAMP analogues stimulated pheromone biosynthesis in the absence of extracellular Ca2+, indicating that Ca2+ may activate an adenylate cyclase. The biogenic amine octopamine did not elicit pheromone production in isolated gland or abdomen bioassays or when injected into intact female moths. Removal of the ventral nerve chord, including the terminal abdominal ganglia in isolated abdomens, did not affect PBAN stimulation of pheromone production. Similar levels of stimulation were found when isolated abdomens were treated with PBAN in scotophase or photophase.
A second thrombospondin gene in the mouse is similar in organization to thrombospondin 1 but does not respond to serum.
Authors: Bornstein P , Devarayalu S , Li P , Disteche C M , Framson P
Published: May 2006

A second, expressed thrombospondin (TSP) gene, Thbs2, has been identified in the mouse. The exon/intron organization of Thbs2 is highly conserved in comparison with Thbs1 in that exon size and the pattern of interruption of the reading frame by introns are preserved, but there is a marked divergence in coding sequence, primarily in the first 7 exons. On the other hand, the DNA and translated amino acid sequences of exons coding for the type I, II, and III repeats in the two TSPs are far better conserved. Thbs2 is located on chromosome 17, band A3, whereas Thbs1 was found on chromosome 2, band F. In marked contrast to Thbs1, the Thbs2 gene is not induced by serum in NIH 3T3 cells; promoter sequences in the two genes are also very different. It is therefore likely that the two TSPs perform related but distinct functions.