bluuebunny/crossref_metadata_2025 huggingface dataset:
Human glucokinase gene: isolation, characterization, and identification of two missense mutations linked to early-onset non-insulin-dependent (type 2) diabetes mellitus.
Authors: Stoffel M , Froguel P , Takeda J , Zouali H , Vionnet N , Nishi S , Weber I T , Harrison R W , Pilkis S J , Lesage S
Published: June 2006

DNA polymorphisms in the glucokinase gene have recently been shown to be tightly linked to early-onset non-insulin-dependent diabetes mellitus in approximately 80% of French families with this form of diabetes. We previously identified a nonsense mutation in exon 7 in one of these families and showed that it was the likely cause of glucose intolerance in this dominantly inherited disorder. Here we report the isolation and partial sequence of the human glucokinase gene and the identification of two missense mutations in exon 7, Thr-228----Met and Gly-261----Arg, that cosegregate with early-onset non-insulin-dependent diabetes mellitus. To assess the molecular mechanism by which mutations at these two sites may affect glucokinase activity, the crystal structure of the related yeast hexokinase B was used as a simple model for human beta-cell glucokinase. Computer-assisted modeling suggests that mutation of Thr-228 affects affinity for ATP and mutation of Gly-261 may alter glucose binding. The identification of mutations in glucokinase, a protein that plays an important role in hepatic and beta-cell glucose metabolism, indicates that early-onset non-insulin-dependent diabetes mellitus may be primarily a disorder of carbohydrate metabolism.
The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins.
Authors: Rao L , Debbas M , Sabbatini P , Hockenbery D , Korsmeyer S , White E
Published: June 2006

Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis.
Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein.
Authors: Petranka J G , Fleenor D E , Sykes K , Kaufman R E , Rosse W F
Published: June 2006

The gene for CD59 [membrane inhibitor of reactive lysis (MIRL), protectin], a phosphatidylinositol-linked surface glycoprotein that regulates the formation of the polymeric C9 complex of complement and that is deficient on the abnormal hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria, consists of four exons spanning 20 kilobases. The untranslated first exon is preceded by a G+C-rich promoter region that lacks a consensus TATA or CAAT motif. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the amino-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence necessary for glycosyl-phosphatidylinositol anchor attachment. The structure of the CD59 gene is very similar to that encoding Ly-6, a murine glycoprotein with which CD59 has some structural similarity. The striking similarity in gene structure is further evidence that the two proteins belong to a superfamily of proteins that may also include the urokinase plasminogen-activator receptor and a squid glycoprotein of unknown function.
A monomeric von Willebrand factor fragment, Leu-504--Lys-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX [corrected]
Authors: Gralnick H R , Williams S , McKeown L , Kramer W , Krutzsch H , Gorecki M , Pinet A , Garfinkel L I
Published: June 2006

von Willebrand factor interaction with glycoprotein Ib alpha (GPIb alpha) plays a critical role in the initial phase of platelet adhesion at high shear rates, and it may also play a role in platelet thrombus formation in partially occluded arteries. Previous studies have indicated that two peptides, Cys-474--Pro-488 (peptide 153) and Ser-692--Pro-708 (peptide 154), inhibit von Willebrand factor--GPIb alpha interaction. We have expressed a recombinant fragment of von Willebrand factor, Leu-504--Lys-728 [corrected], with a single intrachain disulfide bond linking residues Cys-509--Cys-695 and examined its ability to inhibit von Willebrand factor--GPIb alpha interactions and platelet adhesion at high shear forces. This recombinant fragment, named VCL, inhibits ristocetin-induced, botrocetin-induced, and asialo-von Willebrand factor-induced platelet aggregation and binding to platelets at an IC50 = 0.011-0.260 microM, significantly lower than the IC50 of peptide 153 or 154, IC50 = 86-700 microM. Peptides 153 and 154 did not result in any inhibition of platelet adhesion (IC50 greater than 500 microM). In contrast, VCL inhibited 50% of platelet adhesion at 0.94 microM and at 7.6 microM inhibited greater than 80% of platelet adhesion to human umbilical artery subendothelium at high shear forces. VCL inhibited the contact and spreading of platelets and also caused a marked decrease in thrombus formation. These studies indicate that VCL may be an effective antithrombotic agent in preventing arterial thrombus formation in areas of high shear force.
Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme.
Authors: McIntosh E M , Ager D D , Gadsden M H , Haynes R H
Published: June 2006

Two functional human dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) cDNAs were isolated from a cDNA expression library by genetic complementation in Escherichia coli. These cDNAs differed in size but exhibited a common overlapping DNA sequence. Contained within this sequence was a single long open reading frame sufficient to encode a polypeptide of 141 amino acids with a calculated molecular mass of 16.6 kDa. The amino acid sequence of this protein exhibits 35% identity with the E. coli dUTPase and 53% identity with the Saccharomyces cerevisiae enzyme. The human dUTPase was found to contain five characteristics amino acid sequence motifs that are common to the dUTPases of E. coli, yeast, and herpesviruses and to dUTPase-like sequences encoded by some retrovirus gag and pol genes. A high degree of amino acid sequence identity (greater than 60%) was also observed between the human dUTPase and the putative pseudoproteases of two poxviruses, indicating that these virus proteins are dUTPases. Northern hybridization analysis reveals that dUTPase is encoded by at least two species of poly(A)+ mRNA and possibly a third, smaller species. All of these mRNAs are present in a variety of human tissues but their relative levels vary between tissues. Southern analysis indicates that the dUTPase gene has been conserved to some extent throughout vertebrate evolution; however, the gene may be very large, or its organization somewhat complex in some systems. We suggest that dUTPase may generally perform an essential role in DNA replication and therefore could serve as a target enzyme for the development of chemotherapeutic compounds.
Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells.
Authors: Petersen O W , Rønnov-Jessen L , Howlett A R , Bissell M J
Published: June 2006

Normal human breast epithelial cells show a high degree of phenotypic plasticity in monolayer culture and express many traits that otherwise characterize tumor cells in vivo. Paradoxically, primary human breast carcinoma cells are difficult to establish in culture: most outgrowths arise from the normal tissue surrounding the tumor. These characteristics have posed major obstacles to the establishment of simple reliable criteria for mammary epithelial transformation in culture. In the present study, we show that a reconstituted basement membrane (BM) can be used to culture all normal human breast epithelial cells and a subset of human breast carcinoma cells. The two cell types can be readily distinguished by virtue of the ability of normal cells to reexpress a structurally and functionally differentiated phenotype within BM. Twelve specimens of normal breast tissue and 2 normal breast epithelial cell lines (total 14 samples) embedded in BM as single cells were able to form multicellular spherical colonies with a final size close to that of true acini in situ. Sections of mature spheres revealed a central lumen surrounded by polarized luminal epithelial cells expressing keratins 18 and 19 and sialomucin at the apical membrane. Significantly, two-thirds of normal spheres deposited a visible endogenous type IV collagen-containing BM even though they were in contact with exogenously provided BM. Growth was arrested completely within the same time period. In contrast, none of 6 carcinoma cell lines or 2 cultures of carcinoma from fresh samples (total 8 samples) responded to BM by growth regulation, lumen formation, correct polarity, or deposition of endogenous BM. These findings may provide the basis of a rapid assay for discriminating normal human breast epithelial cells from their malignant counterparts. Furthermore, we propose that the ability to sense BM appropriately and to form three-dimensional organotypic structures may be the function of a class of "suppressor" genes that are lost as cells become malignant.
Protein oxidation associated with aging is reduced by dietary restriction of protein or calories.
Authors: Youngman L D , Park J Y , Ames B N
Published: June 2006

The accumulation of unrepaired oxidative damage products may be a major factor in cellular aging. Both oxidative lesions in DNA and oxidatively damaged proteins have been shown to accumulate during aging. The accumulation of oxidized proteins in Fischer 344 rats was compared for animals consuming protein-restricted and calorically restricted diets--both of which have been shown to extend lifespan. Rats were fed diets restricted in either protein (5% or 10% of the diet as compared with the normal 20% casein), or calories (25% or 40% less than normal), or total diet (40% less than normal). In addition, some of the rats fed a diet providing 5% or 20% protein were irradiated twice weekly (125 rads per exposure; 1 rad = 0.01 Gy). The level of oxidative damage to proteins (protein carbonyls) was determined in rats sacrificed at various times. The oxidative damage to proteins increased with aging and with radiation. Either protein or calorie restriction markedly inhibited the accumulation of oxidatively damaged proteins. Protein restriction reduced the accumulation of oxidatively damaged proteins during the oxidative stress of chronic irradiation.
Molecular cloning of a calmodulin-dependent phosphatase from murine testis: identification of a developmentally expressed nonneural isoenzyme.
Authors: Muramatsu T , Giri P R , Higuchi S , Kincaid R L
Published: June 2006

A unique isoform of the catalytic subunit of calmodulin-dependent protein phosphatase (CaM-PrP) was cloned from a murine testis library. The cDNA sequence of 1964 base pairs contained an open reading frame encoding a protein of 513 amino acids (Mr approximately 58,706), the predicted isoelectric point of which (pI 7.1) was much more basic than those of brain isoforms (pI 5.6-5.8). The deduced amino acid sequence was 77-81% identical to two other murine CaM-PrP genes and displayed a distinct Southern blot hybridization pattern, indicating that it was derived from a separate gene (type 3). High amounts of a 2800-nucleotide mRNA transcript were observed in testis, whereas mRNA species were not detectable in brain; thus, it seems likely that this CaM-PrP represents a nonneural isoenzyme. Measurements of CaM-PrP mRNA during testicular development showed a dramatic increase in expression during weeks 4-6, correlating with the later stages of spermatogenesis. These data suggest that this phosphatase isoform may be involved in germ-cell function and are consistent with the report of a flagellum-associated form of CaM-PrP that may regulate sperm motility [Tash, J. S., Krinks, M., Patel, J., Means, R. L., Klee, C. B. & Means, A. R. (1988) J. Cell Biol. 106, 1625-1633].
Inflammatory leukocytes and cytokines in the peptide-induced disease of experimental allergic encephalomyelitis in SJL and B10.PL mice.
Authors: Merrill J E , Kono D H , Clayton J , Ando D G , Hinton D R , Hofman F M
Published: June 2006

Experimental allergic encephalomyelitis (EAE) was generated in SJL and B10.PL mice by using the synthetic myelin basic protein peptides. Inflammation in brain and spinal cord preceded clinical signs of disease. Infiltrating lymphocytes were predominantly Lyt1+ (CD5+), L3T4+ (CD4+) T cells, until day 18. After that, F4/80+ monocyte/macrophages outnumbered T cells. Ia+ cells were microglia, macrophages, and endothelial cells, but Ia was not detectable on astrocytes in this EAE model. Ia+ endothelial cells appeared later in the disease than Ia+ microglia and macrophages, suggesting that antigen presentation at the blood-brain barrier is not initially responsible for inflammation. Cells staining for interferon gamma, interleukin 2 (IL-2), and IL-2 receptors were more prominent than IL-4, IL-5, lymphotoxin (LT), and tumor necrosis factor alpha (TNF-alpha), which occurred transiently in the second week and were associated with fewer cells. TNF-alpha and LT were never seen in spinal cord, suggesting that these cytokines are not responsible for initiation of clinical disease. Few or no cells stained for IL-6, IL-1, or transforming growth factor beta. Control animals injected with complete Freund's adjuvant in saline or control antigen demonstrated no inflammatory cell infiltration or cytokine production. Thus, our findings suggest a peptide-induced EAE model in which Th1 T-cell-macrophage interactions result in the disease process.
Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein.
Authors: Tiley L S , Malim M H , Tewary H K , Stockley P G , Cullen B R
Published: June 2006

Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev response element (RRE). Nucleotides critical for Rev monomer binding have been mapped by chemical interference to a single site flanking the base of an RNA helix (stem IIB) located within the 234-nucleotide RRE. Binding of additional Rev molecules to an RRE probe did not require any RNA primary sequence information detectable by modification interference beyond that required for binding of a single Rev protein molecule. A synthetic 29-nucleotide RNA molecule designed to incorporate nucleotides identified as critical for Rev binding retained the ability to bind Rev specifically and, therefore, represents a minimal Rev-binding site. We propose that Rev binding to the RRE initiates with the direct interaction of a Rev monomer with a high-affinity binding site located at the base of the IIB stem of the RRE. The subsequent formation of Rev multimers on the RRE appears, in contrast, primarily driven by specific protein-protein interactions.
Switch in glutamate receptor subunit gene expression in CA1 subfield of hippocampus following global ischemia in rats.
Authors: Pellegrini-Giampietro D E , Zukin R S , Bennett M V , Cho S , Pulsinelli W A
Published: June 2006

Severe, transient global ischemia of the brain induces delayed damage to specific neuronal populations. Sustained Ca2+ influx through glutamate receptor channels is thought to play a critical role in postischemic cell death. Although most kainate-type glutamate receptors are Ca(2+)-impermeable, Ca(2+)-permeable kainate receptors have been reported in specific kinds of neurons and glia. Recombinant receptors assembled from GluR1 and/or GluR3 subunits in exogenous expression systems are permeable to Ca2+; heteromeric channels containing GluR2 subunits are Ca(2+)-impermeable. Thus, altered expression of GluR2 in development or following a neurological insult or injury to the brain can act as a switch to modify Ca2+ permeability. To investigate the molecular mechanism underlying delayed postischemic cell death, GluR1, GluR2, and GluR3 gene expression was examined by in situ hybridization in postischemic rats. Following severe, transient forebrain ischemia GluR2 gene expression was preferentially reduced in CA1 hippocampal neurons at a time point that preceded their degeneration. The switch in expression of kainate/AMPA receptor subunits coincided with the previously reported increase in Ca2+ influx into CA1 cells. Timing of the switch indicates that it may play a causal role in postischemic cell death.
Model for the role of macromolecular crowding in regulation of cellular volume.
Authors: Minton A P , Colclasure G C , Parker J C
Published: June 2006

A simple model is proposed to account for large increases in transporter-mediated ion flux across cell membranes that are elicited by small fractional changes of cell volume. The model is based upon the concept that, as a result of large excluded volume effects in cytoplasm (macromolecular crowding), the tendency of soluble macromolecules to associate with membrane proteins is much more sensitive to changes in cell water content than expected on the basis of simple considerations of mass action. The model postulates that an ion transporter may exist in either an active dephosphorylated state or an inactive phosphorylated state and that the steady-state activity of the transporter reflects a balance between the rates of phosphatase-catalyzed activation and kinase-catalyzed inactivation. Cell swelling results in the inhibition of kinase relative to phosphatase activity, thereby increasing the steady-state concentration of the active form of the transporter. Calculated volume-dependent stimulation of ion flux is comparable to that observed experimentally.
Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation and reversal of hypogonadism in mutant mice.
Authors: Silverman A J , Roberts J L , Dong K W , Miller G M , Gibson M J
Published: June 2006

GT1 is an immortalized cell line that synthesizes and secretes the neurohormone gonadotropin-releasing hormone (GnRH). We have placed these cells into the brains of adult mutant hypogonadal (hpg) mice, which lack a functional GnRH gene, to determine whether such cells could differentiate in situ and support gonadal development. Immunocytochemical detection of GnRH revealed that these cells migrated widely in the central nervous system and elaborated axonal processes which on rare occasion projected to the normal target, the median eminence. Using a battery of antibodies, we demonstrated that these cells could cleave the GnRH precursor and that the amidated decapeptide as well as other cleavage products were present. The presence of biologically active material and its appropriate secretion were further documented by gonadal growth in both males and females. The morphological differentiation of the GT1 cells correlated with the density of cells injected. Those remaining within the injection site and/or forming a tumor retained a simple, rounded or fibroblastic appearance. Those cells that migrated into the host away from such tumors assumed the simple fusiform shape of normal GnRH neurons with dendrites extending from one or both poles. When cell density was drastically reduced a much more complex dendritic arbor was elaborated. These data suggest that such cell lines can be useful in reversing genetic defects and in studying such processes as GnRH neuronal migration, axonal targeting, and cytological differentiation.
Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein.
Authors: Kennedy M C , Mende-Mueller L , Blondin G A , Beinert H
Published: June 2006

In recent reports attention has been drawn to the extensive amino acid homology between pig heart, yeast, and Escherichia coli aconitases (EC 4.2.1.3) and the iron-responsive element binding protein (IRE-BP) of mammalian cells [Rouault, T. A., Stout, C. D., Kaptain, S., Harford, J. B. & Klausner, R. D. (1991) Cell 64, 881-883.; Hentze, M. W. & Argos, P. (1991) Nucleic Acids Res. 19, 1739-1740.; Prodromou, C., Artymiuk, P. J. & Guest, J. R. (1992) Eur. J. Biochem. 204, 599-609]. Iron-responsive elements (IREs) are stem-loop structures located in the untranslated regions of mRNAs. IRE-BP is required in the posttranscriptional regulation of ferritin mRNA translation and stabilization of transferrin receptor mRNA. In spite of substantial homology between the amino acid sequences of mammalian mitochondrial aconitase and IRE-BP, the mitochondrial protein does not bind IREs. However, there is a second aconitase, found only in the cytosol of mammalian tissues, that might serve as an IRE-BP. To test this possibility, we have prepared sufficient quantities of the heretofore poorly characterized beef liver cytosolic aconitase. This enzyme is isolated largely in its active [4Fe-4S] form and has a turnover number similar to that of mitochondrial aconitase. The EPR spectra of the two enzymes are markedly different. The amino acid composition, molecular weight, isoelectric point, and the sequences of six random peptides clearly show that these physicochemical and structural characteristics are identical to those of IRE-BP, and that c-aconitase is distinctly different from m-aconitase. In addition, both cytosolic aconitase and IRE-BP can have aconitase activity or function as IRE-BPs, as shown in the following paper and elsewhere [Zheng, L. Kennedy, M. C., Blondin, G. A., Beinert, H. & Zalkin, H. (1992) Arch. Biochem. Biophys., in press]. This leads us to the conclusion that cytosolic aconitase is IRE-BP.
Calcium ions are involved in Escherichia coli chemotaxis.
Authors: Tisa L S , Adler J
Published: June 2006

Escherichia coli regulates intracellular free Ca2+ at about 90 nM [Gangola, P. & Rosen, B. P. (1987) J. Biol. Chem. 262, 12570-12574]. To increase intracellular free Ca2+, nitr-5/Ca2+, a "caged" Ca2+ compound, was electroporated into cells and then its affinity for Ca2+ was reduced by exposure to 370-nm light. Upon release of the Ca2+ ions, the cells tumbled. Studies on mutant strains showed that the receptor proteins (methyl-accepting chemotaxis proteins, MCPs) were not required for the Ca(2+)-induced tumbling but that CheA, CheW, and CheY proteins were required. Similar results were obtained with DM-nitrophen/Ca2+, another caged calcium compound that releases Ca2+ upon illumination at 340 nm. Diazo-2, a caged Ca2+ chelator that takes up Ca2+ upon illumination at 340 nm, was used to decrease intracellular free Ca2+, and this caused smooth swimming.
Direct measurement of brain glucose concentrations in humans by 13C NMR spectroscopy.
Authors: Gruetter R , Novotny E J , Boulware S D , Rothman D L , Mason G F , Shulman G I , Shulman R G , Tamborlane W V
Published: June 2006

Glucose is the main fuel for energy metabolism in the normal human brain. It is generally assumed that glucose transport into the brain is not rate-limiting for metabolism. Since brain glucose concentrations cannot be determined directly by radiotracer techniques, we used 13C NMR spectroscopy after infusing enriched D-[1-13C]glucose to measure brain glucose concentrations at euglycemia and at hyperglycemia (range, 4.5-12.1 mM) in six healthy children (13-16 years old). Brain glucose concentrations averaged 1.0 +/- 0.1 mumol/ml at euglycemia (4.7 +/- 0.3 mM plasma) and 1.8-2.7 mumol/ml at hyperglycemia (7.3-12.1 mM plasma). Michaelis-Menten parameters of transport were calculated to be Kt = 6.2 +/- 1.7 mM and Tmax = 1.2 +/- 0.1 mumol/g.min from the relationship between plasma and brain glucose concentrations. The brain glucose concentrations and transport constants are consistent with transport not being rate-limiting for resting brain metabolism at plasma levels greater than 3 mM.
Structure and dynamics of des-pentapeptide-insulin in solution: the molten-globule hypothesis.
Authors: Hua Q X , Kochoyan M , Weiss M A
Published: June 2006

Structures of insulin in different crystal forms exhibit significant local and nonlocal differences, including correlated displacement of elements of secondary structure. Here we describe the solution structure and dynamics of a monomeric insulin analogue, des-pentapeptide-(B26-B30)-insulin (DPI), as determined by two-dimensional NMR spectroscopy and distance geometry/restrained molecular dynamics (DG/RMD). Although the solution structure of DPI exhibits a general similarity to its crystal structure, individual DG/RMD structures in the NMR ensemble differ by rigid-body displacements of alpha-helices that span the range of different crystal forms. These results suggest that DPI exists as a partially folded state formed by coalescence of distinct alpha-helix-associated microdomains. The physical reality of this model is investigated by comparison of the observed two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy (NOESY) spectrum with that predicted from crystal and DG/RMD structures. The observed NOESY spectrum contains fewer tertiary contacts than predicted by any single simulation, but it matches their shared features; such "ensemble correspondence" is likely to reflect the effect of protein dynamics on observed NOE intensities. We propose (i) that the folded state of DPI is analogous to that of a compact protein-folding intermediate rather than a conventional native state and (ii) that the molten state is the biologically active species. This proposal (the molten-globule hypothesis) leads to testable thermodynamic predictions and has general implications for protein design.
Demonstration of processing and recycling of biologically active V1 vasopressin receptors in vascular smooth muscle.
Authors: Briner V A , Williams B , Tsai P , Schrier R W
Published: June 2006

The present study examines the binding and postbinding cellular processing and recycling of the V1 arginine vasopressin (AVP) receptor in cultured vascular smooth muscle cells (VSMCs). The surface binding of AVP to VSMCs was temperature dependent and reached equilibrium within 60 min at 4 degrees C. Displacement studies with unlabeled AVP or a specific V1 AVP antagonist revealed a single class of V1 receptors (Bmax, 1.99 pmol [corrected] per mg of protein; Kd, 2.15 nM). Incubation of VSMCs with unlabeled 10 nM AVP to promote receptor internalization resulted in a time- and temperature-dependent loss of AVP surface binding. At 37 degrees C, maximum loss of binding sites (65%) occurred within 20 min. Recovery of AVP binding occurred rapidly (t1/2, 15-20 min at room temperature) and was uninfluenced by inhibiting protein synthesis with cycloheximide. Pretreating VSMCs with chloroquine prevented AVP receptor recycling, indicating that the AVP-receptor complex requires endosomal processing. The biological competence of the recycled AVP receptor was shown by AVP-induced Ca2+ uptake. The results of these studies therefore indicate that, after surface binding, the AVP-receptor complex internalizes and dissociates in an endosomal compartment. It is demonstrated that in VSMCs biologically active V1 AVP receptors recycle back to the cell surface, thus attenuating the loss of AVP surface binding sites.
Reverse gyrase: a helicase-like domain and a type I topoisomerase in the same polypeptide.
Authors: Confalonieri F , Elie C , Nadal M , de La Tour C , Forterre P , Duguet M
Published: June 2006

Reverse gyrase is a type I DNA topoisomerase able to positively supercoil DNA and is found in thermophilic archaebacteria and eubacteria. The gene coding for this protein was cloned from Sulfolobus acidocaldarius DSM 639. Analysis of the 1247-amino acid sequence and comparison of it with available sequence data suggest that reverse gyrase is constituted of two distinct domains: (i) a C-terminal domain of approximately 630 amino acids clearly related to eubacterial topoisomerase I (Escherichia coli topA and topB gene products) and to Saccharomyces cerevisiae top3; (ii) an N-terminal domain without any similarity to other known topoisomerases but containing several helicase motifs, including an ATP-binding site. These results are consistent with those from our previous mechanistic studies of reverse gyrase and suggest a model in which positive supercoiling is driven by the concerted action of helicase and topoisomerase in the same polypeptide: this constitutes an example of a composite gene formed by a helicase domain and a topoisomerase domain.
Expression of functional trk protooncogene in human monocytes.
Authors: Ehrhard P B , Ganter U , Stalder A , Bauer J , Otten U
Published: June 2006

There is increasing evidence that neurotrophins, including nerve growth factor (NGF), exert specific effects on cells of the immune system in addition to their neurotrophic actions. This report shows that human monocytes express the trk protooncogene, encoding the signal-transducing receptor unit for NGF. This receptor is functional, since interaction of NGF with monocytes triggered a respiratory burst, the major component of monocyte cytotoxic activity. During in vitro differentiation of human blood monocytes to macrophages trk expression decreased, suggesting a maturation-dependent trk expression decreased, suggesting a maturation-dependent trk regulation. Treatment of monocytes with Staphylococcus aureus Cowan I, a potent activator of monocytes, stimulated trk mRNA synthesis in a time-dependent way, implying a modulatory role for NGF in immune functions. The finding that dibutyryl cAMP elicited a time-dependent trk induction in monocytes as well as in phorbol ester-differentiated promonocytic U937 cells indicates that adenylate cyclase is involved in monocytic trk regulation. These results suggest that NGF, in addition to its neurotrophic function, is an immunoregulatory cytokine acting on monocytes.
Alzheimer disease and the prion disorders amyloid beta-protein and prion protein amyloidoses.
Authors: Price D L , Borchelt D R , Sisodia S S
Published: June 2006

Alzheimer disease and the prion disorders/spongiform encephalopathies share many common features. These chronic, progressive, sometimes familial diseases of the central nervous system are characterized by the presence of different types of amyloid deposits in the brain. This review provides a perspective on these two types of neurodegenerative disorders.
Substitution of the erbB-2 oncoprotein transmembrane domain activates the insulin receptor and modulates the action of insulin and insulin-receptor substrate 1.
Authors: Cheatham B , Shoelson S E , Yamada K , Goncalves E , Kahn C R
Published: June 2006

The mechanism through which insulin binding to the extracellular domain of the insulin receptor activates the intrinsic tyrosine kinase in the intracellular domain of the protein is unknown. For the c-neu/erbB-2 (c-erbB-2) protooncogene, a single point mutation within the transmembrane (TM) domain converting Val-664 to Glu (erbB-2V-->E) results in elevated levels of tyrosine kinase activity and cellular transformation. We report the construction of a chimeric insulin receptor in which the TM domain of the receptor has been substituted with that encoded by erbB-2V-->E. When expressed in Chinese hamster ovary cells this chimeric receptor displays maximal levels of autophosphorylation and kinase activity in the absence of insulin. This activity results in an increase in the level of insulin-receptor substrate 1 phosphorylation but a down-regulation in insulin-receptor substrate 1 protein and desensitization to insulin stimulation of glycogen synthesis. By contrast, basal levels of DNA synthesis are elevated to levels approximately 60% of those observed in serum-stimulated cells. Over-expression of chimeric insulin receptors containing the c-erbB-2 TM domain or a single point mutation in the insulin receptor TM domain of Val-938-->Asp, on the other hand, shows none of these alterations. Thus, the TM domain encoded by erbB-2V-->E contains structural features that can confer ligand-independent activation in a heterologous protein. Constitutive activation of the insulin receptor results in a relative increase in basal levels of DNA synthesis, but an apparent resistance to the metabolic effects of insulin.
Characterization of a gene encoding a Ca(2+)-ATPase-like protein in the plastid envelope.
Authors: Huang L , Berkelman T , Franklin A E , Hoffman N E
Published: June 2006

By screening an Arabidopsis expression library with an antiserum against chloroplast envelope proteins, we have isolated a partial cDNA with an open reading frame that encodes a polypeptide similar to P-type cation-transporting ATPases. The corresponding genomic clone was isolated and the complete coding sequence was deduced after identification and mapping of introns. The gene has been designated PEA1 (plastid envelope ATPase) and the predicted polypeptide PEA1p. PEA1p has 946 amino acids and a molecular mass of 104 kDa. This protein is 40-44% identical to various mammalian plasma membrane Ca(2+)-ATPases but lacks the C-terminal calmodulin binding domain present in the mammalian polypeptides. When aligned with mammalian plasma membrane Ca(2+)-ATPases, PEA1p has a 70- to 80-amino acid N-terminal region that extends beyond the N terminus of these enzymes. This extension has some similarity to the transit peptide of the plastid envelope phosphate translocator and may function to target the protein to the plastid. Antibodies raised against a portion of PEA1p recognize a single 90- to 95-kDa polypeptide in chloroplast inner envelope preparations. Transcript abundance as determined by RNase protection was found to be 7- to 9-fold higher in roots than in leaves. Possible roles for a plastid envelope calcium pump are suggested.
Selective erythroid replacement in murine beta-thalassemia using fetal hematopoietic stem cells.
Authors: Bethel C A , Murugesh D , Harrison M R , Mohandas N , Rubin E M
Published: June 2006

We have explored the application of fetal hematopoietic stem cell (HSC) transplants for cellular replacement in a murine model of beta-thalassemia. Liver-derived HSCs from nonthalassemic syngeneic murine fetal donors were transplanted into nonirradiated neonatal beta-thalassemic recipients. Significant erythrocyte chimerism (9-27%) was demonstrated in the majority of recipients at 1 month and remained stable or increased (up to 55%) during long-term follow-up in almost all cases. Chimeras had improved phenotypes, as evidenced by decreased reticulocyte counts, increased mean erythrocyte deformability, and decreased iron deposits in comparison to controls. To investigate whether the high degree of peripheral blood chimerism was predominantly a feature of erythroid elements or was a general feature of all hematopoietic elements, chimeras were created using donor HSCs "tagged" with a DNA transgene. Whereas donor hemoglobin comprised > 30% of total hemoglobin, nucleated tagged nonerythroid donor cells comprised < 1% of peripheral blood elements. Explanations for the observed selective increase in erythroid chimerism include longer survival of normal donor red cells compared to that of thalassemic red cells and the effective maturation of the donor erythroid elements in the bone marrow in chimeric animals. The latter explanation bears consideration because it is consistent with the process of ineffective erythropoiesis, well documented to occur in thalassemia, in which the majority of thalassemic erythroid cells are destroyed during erythropoiesis prior to release from the bone marrow. Overall, these data demonstrate the potential for significant erythroid chimerism and suggest that fetal HSC transplantation may play a significant role in future treatment.
PAS10 is a tetratricopeptide-repeat protein that is essential for the import of most matrix proteins into peroxisomes of Saccharomyces cerevisiae.
Authors: Van der Leij I , Franse M M , Elgersma Y , Distel B , Tabak H F
Published: June 2006

pas mutants of Saccharomyces cerevisiae are disturbed in peroxisome assembly (pas) and proliferation. Here we report the characterization of the PAS10 gene and its product (PAS10) that is essential for the import of a large subset of proteins into the peroxisomal matrix. PAS10, a protein of 69 kDa, is a member of the tetratricopeptide repeat, or snap helix, protein family, characterized by several direct repeats of a degenerate 34-amino acid motif (Sikorski, R. S., Boguski, M. S., Goebl, M. & Hieter, P. (1990) Cell 60, 307-317). Other members of this family are MAS70 (S. cerevisiae) and MOM72 (Neurospora crassa), which are mitochondrial receptors for protein import. A pas10 null mutant accumulates peroxisomal, leaflet-like membrane structures and exhibits deficient import of a number of peroxisomal matrix enzymes, particularly of proteins with an SKL-like import signal. In contrast, 3-ketoacyl-CoA thiolase associated with these membranes is resistant in vitro to degradation by proteinase K, indicating true protein import. These results suggest that PAS10 is an essential component of a peroxisomal import machinery which mediates the translocation of a specific subset of proteins to the peroxisomal matrix with an SKL-like import signal.
Substoichiometric amounts of the molecular chaperones GroEL and GroES prevent thermal denaturation and aggregation of mammalian mitochondrial malate dehydrogenase in vitro.
Authors: Hartman D J , Surin B P , Dixon N E , Hoogenraad N J , Høj P B
Published: June 2006

The molecular chaperones GroEL and GroES were produced at very high levels in Escherichia coli, purified, and shown to protect pig mitochondrial malate dehydrogenase (MDH) against thermal inactivation in vitro. The apparent rate of MDH inactivation at 37 degrees C was reduced by a factor of at least 5 in a process which required only GroEL, GroES, and ATP. GroEL alone did not protect MDH against thermal inactivation but kept the denatured protein soluble and thereby prevented its aggregation. Reactivation of this soluble and inactive form of MDH could be achieved by addition of GroES even after 120 days of storage at -20 degrees C. Protection could be extended for more than 24 hr at 37 degrees C and was observed at molar ratios of chaperones to MDH as low as 1:4, suggesting that GroEL and GroES perform multiple turnovers in the absence of auxiliary chaperones. The availability of these chaperones in large quantities combined with the apparent promiscuity of GroEL binding shows great potential for stabilization of many proteins for which thermostable variants are not available. We speculate that GroEL and GroES perform similar protective roles in vivo and thereby increase the half-life of proteins which otherwise might aggregate under physiological conditions.
Bovine papilloma virus (BPV)-encoded E2 protein enhances binding of E1 protein to the BPV replication origin.
Authors: Seo Y S , Müller F , Lusky M , Gibbs E , Kim H Y , Phillips B , Hurwitz J
Published: June 2006

The replication of bovine papilloma virus (BPV) DNA in vivo requires two viral-encoded proteins, E1 and E2, while all other proteins are derived from the host. We described previously the isolation of the E1 protein and showed that it contains multiple functions required for BPV DNA replication. The BPV transcription factor E2 was shown by others to stimulate BPV DNA replication in vitro. Here, we present results that account for the role of the E2 protein. The E1 protein bound selectively to the BPV minimal origin of replication. This process required MgCl2 and ATP for maximal efficiency. The E1 protein also catalyzed a BPV origin-dependent DNA unwinding reaction. In this report, we show that at low levels of E1 protein, origin binding could be stimulated up to 40-fold by the E2 protein, provided that the DNA contained an E2 binding site. Consistent with this result, the E2 protein stimulated the origin-specific unwinding reaction catalyzed by E1, but it had no effect on the nonspecific E1-catalyzed helicase activity. In the absence of an E2 binding site, both origin-dependent binding and unwinding reactions with the E1 protein were unaffected by the E2 protein. These results suggest that E2 participates in the initiation of BPV DNA replication by enhancing E1 binding to the BPV origin via DNA-protein and protein-protein interactions.
In vivo expression of inducible nitric oxide synthase in experimentally induced neurologic diseases.
Authors: Koprowski H , Zheng Y M , Heber-Katz E , Fraser N , Rorke L , Fu Z F , Hanlon C , Dietzschold B
Published: June 2006

The purpose of this study was to investigate the induction of inducible nitric oxide synthase (iNOS) mRNA in the brain tissue of rats and mice under the following experimental conditions: in rats infected with borna disease virus and rabies virus, in mice infected with herpes simplex virus, and in rats after the induction of experimental allergic encephalitis. The results showed that iNOS mRNA, normally nondetectable in the brain, was present in animals after viral infection or after induction of experimental allergic encephalitis. The induction of iNOS mRNA coincided with the severity of clinical signs and in some cases with the presence of inflammatory cells in the brain. The results indicate that nitric oxide produced by cells induced by iNOS may be the toxic factor accounting for cell damage and this may open the door to approaches to the study of the pathogenesis of neurological diseases.
The p53 protein is an unusually shaped tetramer that binds directly to DNA.
Authors: Friedman P N , Chen X , Bargonetti J , Prives C
Published: June 2006

We have analyzed the size and structure of native immunopurified human p53 protein. By using a combination of chemical crosslinking, gel filtration chromatography, and zonal velocity gradient centrifugation, we have determined that the predominant form of p53 in such preparations is a tetramer. The behavior of purified p53 in gels and sucrose gradients implies that the protein has an extended shape. Wild-type p53 has been shown to bind specifically to sites in cellular and viral DNA. We show in this study by Southwestern ligand blotting and by analysis of DNA-bound crosslinked p53 that p53 monomers, dimers, and tetramers can bind directly to DNA.
Heterogeneity of the principal sigma factor in Escherichia coli: the rpoS gene product, sigma 38, is a second principal sigma factor of RNA polymerase in stationary-phase Escherichia coli.
Authors: Tanaka K , Takayanagi Y , Fujita N , Ishihama A , Takahashi H
Published: June 2006

The rpoS gene of Escherichia coli encodes a putative RNA polymerase sigma factor that is considered to be the central regulator of gene expression in stationary phase. The gene product (sigma 38) was overproduced using the cloned rpoS gene and purified to homogeneity. Reconstituted RNA polymerase holoenzyme (E sigma 38) was found to recognize in vitro a number of typical sigma 70-type promoters, including the lacUV5 and trp promoters. Some, however, were recognized exclusively or preferentially by E sigma 70, whereas at least one, fic, was favored by E sigma 38. Thus E. coli promoters can be classified into three groups: the first group is recognized by E sigma 70 and E sigma 38, but the second and third groups are recognized substantially by either E sigma 70 or E sigma 38 alone. In contrast to other minor sigma factors, sigma 38 shares a set of amino acid sequences common among the principal sigma factors of eubacteria and is therefore a member of the RpoD-related protein family. The intracellular level of sigma 38 was demonstrated to increase in vivo upon entry into stationary phase. These results together indicate that sigma 38 is a second principal sigma factor in stationary-phase E. coli.
Prolonged and effective blockade of tumor necrosis factor activity through adenovirus-mediated gene transfer.
Authors: Kolls J , Peppel K , Silva M , Beutler B
Published: June 2006

A chimeric protein capable of binding and neutralizing tumor necrosis factor (TNF) and lymphotoxin was expressed in mice transduced with a replication-incompetent adenoviral vector into which a TNF inhibitor gene had been engineered. Within 3 days following the injection of 10(9) infectious particles, the TNF inhibitor concentration exceeded 1 mg/ml of plasma; this level of expression was maintained for at least 4 weeks, and detectable TNF inhibitory activity was measured 6 weeks after injection of the recombinant virus. Introduction of the artificial gene produced a phenotypic effect comparable to homozygous deletion of the 55-kDa TNF receptor, in that animals were rendered highly susceptible to infection by Listeria monocytogenes, whereas control animals receiving a replication-incompetent virus coding for beta-galactosidase were capable of resisting Listeria challenge. Adenovirus-mediated transfer of a gene encoding a TNF inhibitor offers a practical means of imposing effective, long-term blockade of TNF activity in vivo for investigational and therapeutic purposes.
Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody.
Authors: Duan L , Bagasra O , Laughlin M A , Oakes J W , Pomerantz R J
Published: June 2006

Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.
Analysis of gene expression in the preimplantation mouse embryo: use of mRNA differential display.
Authors: Zimmermann J W , Schultz R M
Published: June 2006

The analysis of differential gene expression during preimplantation embryogenesis has been hindered by the paucity of biological material. We report modifications of the recently described mRNA differential display method (Liang, P. & Pardee, A. B. (1992) Science 257, 967-971) to analyze differential gene expression during mouse preimplantation development. The method detects the appropriate changes in the temporal pattern of expression of an amplicon that by DNA sequence analysis is the cytokeratin endo A, a gene whose temporal pattern of expression has been previously determined by S1 nuclease digestion. In addition, this method identifies amplicons that likely represent genes (i) that encode maternal mRNAs, (ii) that are products of early and late zygotic gene activation, (iii) whose expression is greatest during the eight-cell stage (i.e., expressed in a stage-specific manner), and (iv) whose expression is greatest in the blastocyst. In addition to endo A, sequence analysis of these amplicons reveals that an amplicon that displays a temporal pattern of expression consistent with it being a maternal mRNA is the alpha subunit of the mitochondrial F1 ATP synthase.