bluuebunny/crossref_metadata_2025 huggingface dataset:
Osmotic regulation of gene action.
Authors: Douzou P
Published: May 2006

Most reactions involved in gene translation
systems are ionic-dependent and may be explained in electrostatic terms.
However, a number of observations of equilibria and rate processes making up the
overall reactions clearly indicate that there is still an enormous gap between
the rough picture of the mechanism of ionic regulation and the detailed behavior
of reactions at the molecular level that hold the key to specific mechanisms.
The present paper deals with possible osmotic contributions arising from the gel
state of gene systems that are complementary to, and interdependent of,
electrostatic contributions. This treatment, although still oversimplified,
explains many previous observations by relating them to a general osmotic
mechanism and suggests experimental approaches to studying the mechanisms of
gene regulation in organelle-free and intact systems.
Members of the low density lipoprotein receptor familymediate cell entry of a minor-group common cold virus.
Authors: Hofer F , Gruenberger M , Kowalski H , Machat H , Huettinger M , Kuechler E , Blass D
Published: May 2006

A protein binding to a minor-group human
rhinovirus (HRV2) was purified from HeLa cell culture supernatant. The amino
acid sequences of tryptic peptides showed identity with the human low density
lipoprotein (LDL) receptor (LDLR). LDL and HRV2 mutually competed for binding
sites on human fibroblasts. Cells down-regulated for LDLR expression yielded
much less HRV2 upon infection than cells with up-regulated LDLR. Virus also
bound to the large subunit of the alpha 2-macroglobulin receptor/LDLR-related
protein (alpha 2MR/LRP). LDLR-deficient fibroblasts yielded considerably less
virus in the presence of receptor-associated protein (RAP), providing evidence
that alpha 2MR/LRP also acts as a minor group HRV receptor.
Ceramide structure predicts tumor gangliosideimmunosuppressive activity.
Authors: Ladisch S , Li R , Olson E
Published: May 2006

Molecular determinants of biological activity of
gangliosides are generally believed to be carbohydrate in nature. However, our
studies of immunomodulation by highly purified naturally occurring tumor
gangliosides provide another perspective: while the immunosuppressive activity
of gangliosides requires the intact molecule (both carbohydrate and ceramide
moieties), ceramide structure strikingly influences ganglioside
immunosuppressive activity. Molecular species of human neuroblastoma GD2
ganglioside in which the ceramide contains a shorter fatty acyl chain (C16:0,
C18:0) were 6- to 10-fold more active than those with a longer fatty acyl chain
(C22:0/C24:1, C24:0). These findings were confirmed in studies of ceramide
species of human leukemia sialosylparagloboside and murine lymphoma GalNAcGM1b.
Gangliosides that contain shorter-chain fatty acids (and are most
immunosuppressive) are known to be preferentially shed by tumor cells.
Therefore, the results suggest that the tumor cell is optimized to protect
itself from host immune destruction by selective shedding of highly active
ceramide species of gangliosides.
Induction of anti-tumor cytotoxic T lymphocytes in normal humans using primary cultures and synthetic peptide epitopes.
Authors: Celis E , Tsai V , Crimi C , DeMars R , Wentworth P A , Chesnut R W , Grey H M , Sette A , Serra H M
Published: May 2006

Cytotoxic T lymphocytes (CTLs) recognize peptide antigens associated with cell surface major histocompatibility complex (MHC) molecules. The identification of tumor cell-derived peptides capable of eliciting anti-tumor CTL responses would enable the design of antigen-specific immunotherapies. Our strategy to identify such potentially therapeutic peptides relies on selecting high-affinity MHC binders from known tumor-associated antigens. These peptides are subsequently tested for their ability to induce CTLs capable of killing tumor cells. With this strategy, we have identified a nine-residue epitope, derived from the product of the tumor-associated gene MAGE-3, which has the capacity to induce in vitro CTLs that kill melanoma and other tumor cell lines. These results show the primary in vitro induction of tumor-specific human CTLs and illustrate the feasibility of ex vivo antigen-specific approaches to the immunological therapy of cancer.
Dominant negative inhibition of tumorigenesis in vivo by human insulin-like growth factor I receptor mutant.
Authors: Prager D , Li H L , Asa S , Melmed S
Published: May 2006

Although insulin-like growth factor I (IGF-I) is a mitogenic growth factor, its role in tumorigenesis is unclear. We therefore transfected wild-type and truncated beta-subunit mutant (952STOP) human IGF-I receptor cDNAs into Rat-1 fibroblasts. Rat-1 transfectants expressed 2.5- to 7-fold increased IGF-I receptor mass, while the Kd for IGF-I binding was unchanged. The Rat-1 cells transfected with wild-type receptor cDNA responded to in vitro IGF-I treatment by increased proliferation and DNA synthesis. Cells overexpressing wild-type receptors were also transformed as evidenced by ligand-dependent colony proliferation in soft agar. After injection into athymic nude mice, all wild-type transfectants formed solid sarcomas within 3 weeks, and ex vivo tumor cell assays confirmed continued overexpression of human IGF-I receptors. In contrast, both DNA synthesis and proliferation of 952STOP-transfected cells were attenuated below that of untransfected cells. 952STOP cells were nonresponsive to IGF-I in vitro and were unable to sustain anchorage-independent growth. No tumors were induced for up to 8 weeks after injection of 952STOP transfectants into athymic mice, despite the presence of demonstrable endogenous IGF-I receptors on the 952STOP-transfected cells. Therefore, 952STOP behaves as a dominant negative inhibitor of endogenous IGF-I receptor function, probably by assembling nonfunctional hybrid rat/mutant human receptor tetramers.
Antagonism of superantigen-stimulated helper T-cell clones and hybridomas by altered peptide ligand.
Authors: Evavold B D , Sloan-Lancaster J , Allen P M
Published: May 2006

T-cell activation by an immunogenic peptide can be antagonized by nonstimulatory analogs of that peptide. We investigated this T-cell receptor antagonism by using staphylococcal enterotoxin superantigen to stimulate hemoglobin-specific helper T (Th) cells because its activation pathway may differ from that of conventional antigen. Interestingly, superantigen activation of these Th cells was antagonized by hemoglobin peptide analogs even though agonist (superantigen) and antagonist (analog peptide) bind at different sites on the major histocompatibility complex-encoded molecule and the T-cell receptor. The antagonism appeared to be a fundamental block in T-cell activation, as phosphoinositol generation, cytokine production, and proliferation were reduced in Th1 clones, and, similarly, proliferative and cytokine responses were inhibited in Th2 cells. Even T-cell hybridoma activation (cytokine production and apoptosis) was inhibited by peptide antagonists. Furthermore, analog peptides that functioned as partial agonists for these Th cells also antagonized superantigen-induced proliferation and thus were a subset of the peptide antagonists. In summary, our results demonstrate that analogs of immunogenic peptide are potent antagonists for Th cell responses induced by superantigen as well as immunogenic peptide.
Selective nature of phorbol 12-myristate 13-acetate-induced neoplastic transformation in NIH 3T3 cells.
Authors: Rubin A L , Rubin H
Published: May 2006

The ability of phorbol 12-myristate 13-acetate (PMA) at 0.02 microgram/ml to induce neoplastic transformation in NIH 3T3 cells is highly dependent on the culture conditions. Optimal transformation, indicated by the saturation density and extent of focus formation in transferred cultures raised under standard conditions, was observed when the original cells were grown in 2% calf serum (CS) and exposed continually to PMA for at least 4 weeks before transfer into the assay. Transformation of stationary cultures in 10% CS occurred later and to a lesser degree than in 2% CS. The same cells subjected to thrice-weekly transfer in 2% or 10% CS at low cell density so that they were in a constant state of exponential growth exhibited no evidence of transformation in response to PMA. This strong condition-dependence of PMA-enhanced transformation is indicative of a selection process similar to that described for spontaneous transformation. In both cases, transformation is promoted by inhibiting multiplication and prevented by maximizing multiplication. Therefore, it has the earmarks of an epigenetic rather than a mutational process and requires phenotypic rather than genotypic variation to supply the states for selection. The concept of "progressive state selection," originally proposed to account for spontaneous transformation, can also account for PMA-enhanced transformation.
Quest for life-long protection by vaccination.
Authors: Dowdle W R , Orenstein W A
Published: May 2006

Life-long protection from disease through immunization can be accomplished through individual or community protection. Individual protection is the goal for vaccination against diseases that have inanimate or animal reservoirs or that pose risks for certain populations. Community protection is the goal for vaccination against diseases that are transmitted only from human to human. Community protection afforded by childhood vaccines has been highly successful against measles, rubella, mumps, and polio. However, outbreaks of measles, rubella, and mumps continue to occur, primarily because of inadequate immunization of children under age 2. Simplification of vaccination regimens, provision of incentives to care providers and parents, and increased access to care should improve vaccination rates in the United States. Better protection requires better use of available vaccines. Eradication of disease through vaccination is the ultimate goal of community protection. Elimination of the infectious agent is the most effective means of achieving life-long protection. The World Health Organization's (WHO) smallpox eradication campaign eliminated a serious disease as well as the need for a vaccine with frequent and severe adverse reactions. The discontinuation of smallpox vaccination in the United States has produced a savings of over $3 billion. Polio has been targeted by WHO for eradication by the year 2000. The eradication of polio and the elimination of the need for polio vaccination in the United States should result in a savings of $110 million per year in vaccine costs alone. Strong United States support is crucial for WHO to reach its goal. Any of the vaccine-preventable childhood virus diseases could be eradicated with sufficient national and international will. Measles and hepatitis B should be high priorities. The ultimate goal of vaccination is life-long protection of all individuals. Any disease of sufficient public health importance to warrant routine vaccination is of sufficient importance to warrant eradication wherever judged to be possible.
Three-dimensional structure of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase: a member of the aldo-keto reductase superfamily.
Authors: Hoog S S , Pawlowski J E , Alzari P M , Penning T M , Lewis M
Published: May 2006

The 3.0-A-resolution x-ray structure of rat liver 3 alpha-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3 alpha-HSD, EC 1.1.1.50) was determined by molecular replacement using human placental aldose reductase as the search model. The protein folds into an alpha/beta or triose-phosphate isomerase barrel and lacks a canonical Rossmann fold for binding pyridine nucleotide. The structure contains a concentration of hydrophobic amino acids that lie in a cavity near the top of the barrel and that are presumed to be involved in binding hydrophobic substrates (steroids, prostaglandins, and polycyclic aromatic hydrocarbons) and inhibitors (nonsteroidal antiinflammatory drugs). At the distal end of this cavity lie three residues in close proximity that have been implicated in catalysis by site-directed mutagenesis--Tyr-55, Asp-50, and Lys-84. Tyr-55 is postulated to act as the general acid. 3 alpha-HSD shares significant sequence identity with other HSDs that belong to the aldo-keto reductase superfamily and these may show similar architecture. Other members of this family include prostaglandin F synthase and rho-crystallin. By contrast, 3 alpha-HSD shares no sequence identity with HSDs that are members of the short-chain alcohol dehydrogenase family but does contain the Tyr-Xaa-Xaa-Xaa-Lys consensus sequence implicated in catalysis in this family. In the 3 alpha-HSD structure these residues are on the periphery of the barrel and are unlikely to participate in catalysis.
Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.
Authors: Challita P M , Kohn D B
Published: May 2006

We describe studies of gene transfer and expression of the human glucocerebrosidase cDNA by a Moloney murine leukemia virus (MoMuLV)-based retroviral vector in a murine gene transfer/bone marrow transplant (BMT) model. Pluripotent hematopoietic stem cells (HSCs) were assayed as the colony-forming units, spleen (CFU-S) generated after serial transplantation. Transcriptional expression from the MoMuLV long-terminal repeat (LTR) was detected at a high level in the primary (1 degree) CFU-S and tissues of reconstituted BMT recipients. However, we observed transcriptional inactivity of the proviral MoMuLV-LTR in > 90% of the secondary (2 degrees) CFU-S and in 100% of the tertiary (3 degrees) CFU-S examined. We have compared the methylation status of the provirus in the 1 degree CFU-S, which show strong vector expression, to that of the transcriptionally inactive provirus in the 2 degrees and 3 degrees CFU-S by Southern blot analysis using the methylation-sensitive restriction enzyme Sma I. The studies demonstrated a 3- to 4-fold increase in methylation of the Sma I site in the proviral LTR of 2 degrees and 3 degrees CFU-S compared to the transcriptionally active 1 degree CFU-S. These observations may have important implications for future clinical applications of retroviral-mediated gene transfer into HSCs, where persistent gene expression would be needed for an enduring therapeutic effect.
Cytolytic and antibacterial activity of synthetic peptides derived from amoebapore, the pore-forming peptide of Entamoeba histolytica.
Authors: Leippe M , Andrä J , Müller-Eberhard H J
Published: May 2006

The pore-forming peptide amoebapore is considered part of the cytolytic armament of pathogenic Entamoeba histolytica. Amoebapore is composed of 77 amino acid residues arranged in four alpha-helical domains. For structure-function analysis, synthetic peptides were constructed corresponding to these four domains: H1 (residues 1-22), H2 (25-39), H3 (40-64), and H4 (67-77). The peptides H1 and H3, representing two highly amphipathic alpha-helical regions of amoebapore, possessed pore-forming activity. Peptide H3 displayed cytolytic and antibacterial functions similar to those of natural amoebapore. The most potent antibacterial activity and the broadest activity spectrum were expressed by H1-Mel, a hybrid molecule composed of the N-terminal alpha-helix of amoebapore and the C-terminal hexapeptide of melittin from the venom of Apis mellifera.
Thorn-like prickles and heterophylly in Cyanea: adaptations to extinct avian browsers on Hawaii?
Authors: Givnish T J , Sytsma K J , Smith J F , Hahn W J
Published: May 2006

The evolution of thorn-like structures in plants on oceanic islands that lack mammalian and reptilian herbivores is puzzling, as is their tendency toward juvenile-adult leaf dimorphism. We propose that these traits arose in Cyanea (Campanulaceae) on Hawaii as mechanical and visual defenses against herbivory by flightless geese and goose-like ducks that were extirpated by Polynesians within the last 1600 years. A chloroplast DNA phylogeny indicates that thorn-like prickles evolved at least four times and leaf dimorphism at least three times during the last 3.7 million years. The incidence of both traits increases from Oahu eastward toward younger islands, paralleling the distribution of avian species apparently adapted for browsing. The effectiveness of visual defenses against avian browsers (once dominant on many oceanic islands, based on the vagility of their ancestors) may provide a general explanation for insular heterophylly: the other islands on which this previously unexplained phenomenon is marked (New Zealand, New Caledonia, Madagascar, Mascarene Islands) are exactly those on which one or more large flightless avian browsers evolved.
Cell adhesion alters gene transcription in chicken embryo brain cells and mouse embryonal carcinoma cells.
Authors: Mauro V P , Wood I C , Krushel L , Crossin K L , Edelman G M
Published: May 2006

To determine whether changes in gene expression occur in embryonic cells as a consequence of changes in cellular aggregation, chicken embryo brain (CEB) cells isolated from 8-day embryos were allowed to aggregate or prevented from aggregating by treatment with anti-neural cell adhesion molecule (N-CAM) Fab' fragments. A subtractive hybridization cloning strategy was employed to identify genes that might show different levels of expression in the two populations of cells. In addition, the transcription rates of a number of genes specifying CAMs and transcription factors were directly estimated by using nuclear run-off transcription assays. The transcription rates of several genes, including those encoding N-CAM, Ng-CAM, alpha-N-catenin, HoxA4 (Hox1.4), a fatty acid-binding protein, and a subunit of the mitochondrially encoded cytochrome-c oxidase enzyme decreased upon CEB cell aggregation. The transcription rates of several previously unidentified genes either increased or decreased upon aggregation, while the transcription of other genes remained unchanged. The transcription rate of the N-CAM gene was 3.3-fold higher in dissociated than in aggregated CEB cells. This rate of transcription also increased when the brain tissue was dissociated into single cells and the increased rate was maintained by keeping the cells dissociated in the presence of Fab' fragments of antibodies to N-CAM. Decreased transcription rates of the N-CAM gene were also observed upon aggregation of P19 cells, a mouse embryonal carcinoma cell line. Primary chicken embryo liver cells, which aggregate primarily by calcium-dependent adhesion mechanisms, did not show changes in the N-CAM gene or in the other genes whose transcription rates changed in CEB cells and P19 cells. These observations suggest that the types of genes regulated by cell aggregation include those for CAMs themselves as well as for transcription factors that may control the expression of CAMs and other molecules significant for morphogenesis.
Crystal structure of human angiogenin reveals the structural basis for its functional divergence from ribonuclease.
Authors: Acharya K R , Shapiro R , Allen S C , Riordan J F , Vallee B L
Published: May 2006

Angiogenin, a potent inducer of neovascularization, is the only angiogenic molecule known to exhibit ribonucleolytic activity. Its overall structure, as determined at 2.4 A, is similar to that of pancreatic ribonuclease A, but it differs markedly in several distinct areas, particularly the ribonucleolytic active center and the putative receptor binding site, both of which are critically involved in biological function. Most strikingly, the site that is spatially analogous to that for pyrimidine binding in ribonuclease A differs significantly in conformation and is "obstructed" by glutamine-117. Movement of this and adjacent residues may be required for substrate binding to angiogenin and, hence, constitute a key part of its mechanism of action.
Dynamics of signal transduction after aggregation of cell-surface receptors: studies on the type I receptor for IgE.
Authors: Kent U M , Mao S Y , Wofsy C , Goldstein B , Ross S , Metzger H
Published: May 2006

Many ligands stimulate cellular responses by aggregating the cell-surface receptors to which they are bound. We investigated several mechanistic questions related to aggregation of receptors by using the high-affinity receptor for IgE (Fc epsilon RI) on mast cells as a model system. We briefly exposed cells to covalently cross-linked oligomers of IgE and then added excess monomeric IgE to prevent further aggregation. Early events were examined by monitoring the phosphorylation of protein tyrosines; later events were examined by monitoring secretion. We found that aggregated receptors continue to signal both late and early events in the absence of formation of new aggregates. Additional experiments suggested that the clustered receptors undergo a dynamic process of phosphorylation and dephosphorylation. Our findings suggest that for these and related receptors that function by aggregation, the persistence of signal transduction is directly related to the intrinsic affinity of the ligand for the individual receptor.
Cytoplasmic phospholipase A2 translocates to membrane fraction in human neutrophils activated by stimuli that phosphorylate mitogen-activated protein kinase.
Authors: Durstin M , Durstin S , Molski T F , Becker E L , Sha'afi R I
Published: May 2006

The addition of the chemotactic peptide formylmethionylleucylphenylalanine (fMet-Leu-Phe) to human neutrophils pretreated with the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) results in a 10-fold enhanced activity of phospholipase A2, measured as the release of arachidonic acid. It is found that GM-CSF increases the tyrosine phosphorylation, enhances the activity of a mitogen-activated protein kinase, and greatly potentiates the fMet-Leu-Phe-induced tyrosine phosphorylation and enhanced activity of this kinase. Stimuli that increase the tyrosine phosphorylation, enhance the activity of the mitogen-activated protein kinase, and cause a rise in the intracellular concentration of free calcium increase the amount of phospholipase A2 associated with the plasma membrane. This increase corresponds to a decrease in the amount found in the cytosol. Whereas GM-CSF alone produces only a small increase in the amount of phospholipase A2 associated with the membrane, it potentiates greatly the fMet-Leu-Phe-induced increase. The total amount (whole cell) of phospholipase A2, as measured by immunoblotting using anti-phospholipase A2 antibody, does not change upon stimulation of human neutrophils with GM-CSF, fMet-Leu-Phe, or both. In addition, the band that corresponds to phospholipase A2 is shifted upward in membrane isolated from neutrophils stimulated with fMet-Leu-Phe, suggesting that the enzyme has been altered, possibly phosphorylated, though not on tyrosine residues. A working hypothesis is presented. Briefly, stimulation of human neutrophils with GM-CSF, in the absence of an additional stimulus, increases the tyrosine phosphorylation and activation of a mitogen-activated protein kinase, which in turn phosphorylates and activates cytoplasmic phospholipase A2. In the presence of an increased intracellular concentration of free calcium the phospholipase A2 is translocated to the plasma membrane where its substrate is located. GM-CSF also potentiates greatly the fMet-Leu-Phe-induced tyrosine phosphorylation and activation of a mitogen-activated protein kinase and, since fMet-Leu-Phe causes an intracellular calcium rise, the amount of the phospholipase A2 that is associated with the membrane fraction.
Functional domains of the receptor-associated protein (RAP).
Authors: Orlando R A , Farquhar M G
Published: May 2006

The receptor-associated protein (RAP) specifically associates with gp330 and the low density lipoprotein (LDL) receptor-related protein (LRP), the two newest members of the LDL receptor gene family. Results obtained by ligand blotting, affinity chromatography, and density-gradient sedimentation demonstrate that RAP binds to both receptors with high affinity and that the binding is Ca2+ dependent. RAP also binds heparin and is identical to a mouse heparin binding protein (HBP-44) identified in a teratocarcinoma cell line (F9). While biochemical studies have shown that RAP is present on the cell surface and is an effective inhibitor of ligand binding to gp330 and LRP, immunocytochemical findings indicate that RAP is most abundant in the endoplasmic reticulum lumen and may function in receptor folding and/or trafficking. To facilitate the characterization of RAP's function(s) we have mapped its gp330 and heparin binding sites by performing direct binding studies on fusion proteins representing overlapping domains of RAP. gp330 was found to bind to two separate sites on RAP--i.e., between amino acids 85-148 and 178-248. Binding studies with radiolabeled heparin indicate that the heparin binding site is between amino acids 261 and 323, which is consistent with our previously proposed site (residues 287-306) based on the amphipathic nature of the C terminus of RAP. These data demonstrate that the gp330 and heparin binding sites and the Heymann nephritis pathogenic epitope (amino acids 1-86) demonstrated earlier are represented by distinct domains of the RAP polypeptide.
Formation of in vivo complexes between the TAL1 and E2A polypeptides of leukemic T cells.
Authors: Hsu H L , Wadman I , Baer R
Published: May 2006

Tumor-specific activation of the TAL1 gene occurs in approximately 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL). The TAL1 gene products possess a basic helix-loop-helix (bHLH) domain that interacts in vitro with the bHLH proteins (E12 and E47) encoded by the E2A locus. We have now applied two independent methods, the two-hybrid procedure and co-immunoprecipitation analysis, to demonstrate that TAL1 and E2A polypeptides also associate in vivo. These studies show that the bHLH domain of TAL1 selectively interacts with the bHLH domains of E12 and E47, but not with the Id1 helix-loop-helix protein. TAL1 does not self-associate to form homodimeric complexes, implying that the in vivo functions of TAL1 depend on heterologous interaction with other bHLH proteins such as E12 and E47. Co-immunoprecipitation analysis revealed the presence of endogenous TAL1/E2A complexes in Jurkat cells, a leukemic line derived from a T-ALL patient. Thus, the malignant properties of TAL1 may be due to obligate interaction with the E2A polypeptides.
In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly.
Authors: Park I S , Carr M B , Hausinger R P
Published: May 2006

The formation of active urease in Klebsiella aerogenes requires the presence of three structural genes for the apoprotein (ureA, ureB, and ureC), as well as four accessory genes (ureD, ureE, ureF, and ureG) that are involved in functional assembly of the metallocenter in this nickel-containing enzyme. Slow and partial activation of urease apoprotein was observed after addition of nickel ion to extracts of Escherichia coli cells bearing a plasmid containing the K. aerogenes urease gene cluster or derivatives of this plasmid with deletions in ureE, ureF, or ureG. In contrast, extracts of cells containing a ureD deletion derivative failed to generate active urease, thus highlighting a key role for UreD in the metallocenter assembly process. Site-directed mutagenesis methods were used to overexpress ureD in the presence of the other urease genes, and the UreD protein was found to copurify with urease. A molecule of native urease apoprotein is capable of binding 0, 1, 2, or 3 molecules of UreD, consistent with a trimeric structure of urease catalytic units. The UreD-urease apoprotein complexes are competent for activation by nickel, with the level of activity obtained being directly related to the number of UreD molecules bound per urease molecule. Activation of the UreD-urease complexes is rapid and accompanied by UreD dissociation. We propose that UreD is a chaperone protein which stabilizes a urease apoprotein conformation that is competent for nickel incorporation.
Neurotrophin 4/5 is a trophic factor for mammalian facial motor neurons.
Authors: Koliatsos V E , Cayouette M H , Berkemeier L R , Clatterbuck R E , Price D L , Rosenthal A
Published: May 2006

The survival of developing motor neurons depends on factors secreted from skeletal muscles and from cells within the central nervous system. Although several members of the nerve growth factor protein family [neurotrophins (NTs)] are able to maintain developing rat motor neurons in vitro, only the brain-derived neurotrophic factor has been shown to have significant effects on the survival of motor neurons in vivo. In the present study, we demonstrate that NT-4/5 also prevents injury-induced death of facial motor neurons in neonatal rats. Furthermore, facial motor neurons express a functional receptor for NT-4/5, whereas mRNA-encoding NT-4/5 can be detected in their environment throughout embryonic and postnatal life. Thus, both NT-4/5 and brain-derived neurotrophic factor may be physiological survival factors for facial motor neurons and may serve as therapeutic agents for motor neuron disease.
Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase.
Authors: Kasten T P , Collin-Osdoby P , Patel N , Osdoby P , Krukowski M , Misko T P , Settle S L , Currie M G , Nickols G A
Published: May 2006

We have examined the effects of modulating nitric oxide (NO) levels on osteoclast-mediated bone resorption in vitro and the effects of nitric oxide synthase (NOS) inhibitors on bone mineral density in vivo. Diaphorase-based histochemical staining for NOS activity of bone sections or highly enriched osteoclast cultures suggested that osteoclasts exhibit substantial NOS activity that may account for basal NO production. Chicken osteoclasts were cultured for 36 hr on bovine bone slices in the presence or absence of the NO-generating agent sodium nitroprusside or the NOS inhibitors N-nitro-L-arginine methyl ester and aminoguanidine. Nitroprusside markedly decreased the number of bone pits and the average pit area in comparison with control cultures. On the other hand, NOS inhibition by N-nitro-L-arginine methyl ester or aminoguanidine dramatically increased the number of bone pits and the average resorption area per pit. In a model of osteoporosis, aminoguanidine potentiated the loss of bone mineral density in ovariectomized rats. Aminoguanidine also caused a loss of bone mineral density in the sham-operated rats. Inhibition of NOS activity in vitro and in vivo resulted in an apparent potentiation of osteoclast activity. These findings suggest that endogenous NO production in osteoclast cultures may regulate resorption activity. The modulation of NOS and NO levels by cells within the bone microenvironment may be a sensitive mechanism for local control of osteoclast bone resorption.
An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.
Authors: Pei G , Tiberi M , Caron M G , Lefkowitz R J
Published: May 2006

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.
Roles of pilin and PilC in adhesion of Neisseria meningitidis to human epithelial and endothelial cells.
Authors: Nassif X , Beretti J L , Lowy J , Stenberg P , O'Gaora P , Pfeifer J , Normark S , So M
Published: May 2006

Pili and pilin antigenic variation play important roles in adhesion of Neisseria meningitidis (MC) to human epithelial and endothelial cells. We recently identified one pilin variant that confers high adhesiveness of MC to human epithelial cells in culture. However, other factor(s) also play a role in MC adhesiveness, since some nonadhesive variants of MC strain 8013 are piliated and produce the same pilin variant as adhesive derivatives. PilC1 and PilC2, high molecular weight outer membrane proteins in Neisseria gonorrhoeae, are proposed to play roles in pilus assembly. Strain 8013 also contains pilC1 and pilC2; their products function in a similar if not identical manner in pilus biogenesis. PilC1 has an additional function in that it also modulates adhesiveness of strain 8013.
A cellular factor is required for transcription of vaccinia viral intermediate-stage genes.
Authors: Rosales R , Sutter G , Moss B
Published: May 2006

The cytoplasmic location of vaccinia virus replication and evidence that the multisubunit DNA-dependent RNA polymerase, early and late stage transcription factors, capping and methylating enzymes, and poly(A) polymerase are virus encoded raised the possibility that all of the proteins needed for viral mRNA synthesis are of viral origin. Previous studies showed that four components from infected cells, the viral RNA polymerase and capping enzyme and two factors called vaccinia virus intermediate transcription factors (VITFs) 1 and 2, can reconstitute transcription of vaccinia virus intermediate-stage genes in vitro. Here, we demonstrate that VITF-2 can be isolated from the nuclei of uninfected HeLa cells as well as from the cytoplasm of infected cells. The proteins with VITF-2 activity from uninfected and infected cells cochromatographed and cosedimented, suggesting that they are identical. VITF-2 activity was found in extracts of other uninfected human and monkey cells but not in nonpermissive Trichoplusia ni insect cells or in conditionally permissive rabbit kidney 13 cells. VITF-2 activity was present, however, in a permissive line of rabbit kidney 13 cells that had been stably transfected with the vaccinia virus K1L host range gene. We suggest that the VITF-2 level acts as a gauge of the permissive state of the cell and thereby regulates the length of the early prereplicative phase of the infection.
The sequence, and its evolutionary implications, of a Thermococcus celer protein associated with transcription.
Authors: Kaine B P , Mehr I J , Woese C R
Published: May 2006

Through random search, a gene from Thermococcus celer has been identified and sequenced that appears to encode a transcription-associated protein (110 amino acid residues). The sequence has clear homology to approximately the last half of an open reading frame reported previously for Sulfolobus acidocaldarius [Langer, D. & Zillig, W. (1993) Nucleic Acids Res. 21, 2251]. The protein translations of these two archaeal genes in turn are homologs of a small subunit found in eukaryotic RNA polymerase I (A12.2) and the counterpart of this from RNA polymerase II (B12.6). Homology is also seen with the eukaryotic transcription factor TFIIS, but it involves only the terminal 45 amino acids of the archaeal proteins. Evolutionary implications of these homologies are discussed.
Left hemisphere dominance for processing vocalizations in adult, but not infant, rhesus monkeys: field experiments.
Authors: Hauser M D , Andersson K
Published: May 2006

In humans, the left hemisphere of the brain is dominant for processing language. To assess the evolutionary origins of this neuropsychological mechanism, playback experiments were conducted on a large population of free-ranging rhesus monkeys (Macaca mulatta). Playbacks provided an equal opportunity to orient the right or left ear toward the speaker. Results revealed that 61 of 80 adult rhesus favored the right ear (left hemisphere) when vocalizations from their own repertoire were heard but favored the left ear when listening to heterospecific vocalizations. In contrast, infants less than a year old showed no perceptual asymmetry for conspecific or heterospecific calls. Thus, like humans, adult rhesus monkeys also evidence left hemisphere dominance for processing species-specific vocalizations. The emergence of such asymmetry, however, may depend on both differential maturation of the two hemispheres and experience with the species-typical vocal repertoire.
Functional synergism between putative gamma-aminobutyrate-containing neurons and pyramidal neurons in prefrontal cortex.
Authors: Wilson F A , O'Scalaidhe S P , Goldman-Rakic P S
Published: May 2006

The responses of putative gamma-aminobutyratergic interneurons (fast-spiking) and pyramidal (regular-spiking) cell pairs were compared in monkeys performing visual and memory-guided oculomotor tasks. Both fast- and regular-spiking neurons had similar receptive fields, indicating that gamma-aminobutyratergic interneurons carry a specific informational signal, as opposed to providing nonspecific modulation. However, the responses of the pairs were inverted and the timing of excitatory and inhibitory responses appeared to be phased, a property consistent with gamma-aminobutyrate-mediated shaping of receptive fields. These observations (i) provide evidence that interneurons and pyramidal cells can be differentiated in vivo and (ii) begin to elucidate the role of gamma-aminobutyratergic mechanisms in cognition.
Different topoisomerase II antitumor drugs direct similar specific long-range fragmentation of an amplified c-MYC gene locus in living cells and in high-salt-extracted nuclei.
Authors: Gromova I I , Thomsen B , Razin S V
Published: May 2006

We have analyzed the long-range distribution of topoisomerase II-mediated cleavages induced in an amplified human c-MYC gene locus in the presence of several antitumor agents. The long-range cleavage patterns were found to be nonrandom and similar for all antitumor drugs tested. Cleavages occurred within several kilobase-long areas (approximately 5 kb) highly accessible to topoisomerase II and separated by extended regions (approximately 70-100 kb) of less accessibility, possibly reflecting the mode of DNA organization into loops along the chromosome. Within the cleavage areas, the patterns of cleavage sites showed a certain dependence on the type of drug used for entrapment of topoisomerase II-DNA complexes. Importantly, distribution of cleavage areas in native chromatin and histone-depleted nuclei was very similar, if not identical, suggesting that the primary target of antitumor agents in vivo is topoisomerase II associated with the high-salt-insoluble nuclear matrix. These data show that matrix-attached DNA is preferentially damaged by topoisomerase II-targeting agents, which may be an important cellular event contributing to drug-induced cell death.
Sequential induction of 5-lipoxygenase gene expression and activity in Mono Mac 6 cells by transforming growth factor beta and 1,25-dihydroxyvitamin D3.
Authors: Brungs M , Rådmark O , Samuelsson B , Steinhilber D
Published: May 2006

5-Lipoxygenase (5-LO; EC 1.13.11.34) activity in the human monocytic cell line Mono Mac 6 was upregulated by combined treatment with transforming growth factor beta 1 (TGF-beta) and 1,25-dihydroxyvitamin D3 (VD3). In undifferentiated cells, 5-LO enzyme activity was undetectable. After the addition of TGF-beta plus VD3, the activity of intact cells was 800 ng per 10(6) cells--500 times more than the assay detection limit. Also 5-LO protein and mRNA expression were induced > 128-fold and 64-fold, respectively, as compared to undifferentiated cells. Both TGF-beta and VD3 were required for these prominent responses. Either agent alone gave small amounts of 5-LO protein and mRNA but very low 5-LO activities. After the addition of TGF-beta and VD3, the induction of 5-LO protein was obvious after 1 day, but the increase in activity was delayed and did not appear until the second day. Pretreatment of cells with TGF-beta or VD3 alone for 2 days led to 5-LO protein expression but very low enzyme activity. Addition of the lacking second inducer was required for full induction of 5-LO protein expression and for upregulation of enzyme activity. Partial purification of 5-LO from Mono Mac 6 cells and recombination with soluble cellular proteins from different sources indicated the presence of cytosolic factors that affect the activity of 5-LO.
Expression of the wild-type p53 antioncogene induces guanine nucleotide-dependent stem cell division kinetics.
Authors: Sherley J L , Stadler P B , Johnson D R
Published: May 2006

The predominant type of cell division in adult mammals is renewal growth. Renewing stem cells in somatic tissues undergo continuous asymmetric divisions. One new daughter cell retains the division potential of the original stem cell, while the other differentiates into a functional constituent of the tissue. Disruptions of this process lead to the development of human cancers. We show that through a guanine nucleotide-dependent mechanism, the p53 antioncogene can induce exponentially dividing cells to switch to an asymmetric stem cell growth pattern. This finding suggests that the observed high frequency of p53 mutations in human cancers reflects a critical function in the regulation of somatic renewal growth.
Altered behavior and long-term potentiation in type I adenylyl cyclase mutant mice.
Authors: Wu Z L , Thomas S A , Villacres E C , Xia Z , Simmons M L , Chavkin C , Palmiter R D , Storm D R
Published: May 2006

The murine Ca(2+)-stimulated adenylyl cyclase (type I) (EC 4.6.1.1), which is expressed predominantly in brain, was inactivated by targeted mutagenesis. Ca(2+)-stimulated adenylyl cyclase activity was reduced 40-60% in the hippocampus, neocortex, and cerebellum. Long-term potentiation in the CA1 region of the hippocampus from mutants was perturbed relative to controls. Both the initial slope and maximum extent of changes in synaptic response were reduced. Although mutant mice learned to find a hidden platform in the Morris water task normally, they did not display a preference for the region where the platform had been when it was removed. These results indicate that disruption of the gene for the type I adenylyl cyclase produces changes in behavior and that the cAMP signal transduction pathway may play an important role in synaptic plasticity.
The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm.
Authors: Schlenstedt G , Saavedra C , Loeb J D , Cole C N , Silver P A
Published: May 2006

Ran/TC4, a Ras-like GTP-binding protein, and its nucleotide exchanger, RCC1, have been implicated in control of protein movement into the nucleus and cytoplasmic accumulation of mRNA. Saccharomyces cerevisiae contains two homologues of the mammalian Ran/TC4, encoded by the GSP1 and GSP2 genes. We have constructed yeast strains that overproduce either wild-type Gsp1 or a form of Gsp1 with glycine-21 converted to valine (Gsp1-G21V), which we show stabilizes the GTP-bound form. Cells producing Gsp1-G21V have defects in localization of nuclear proteins; nuclear proteins accumulate in the cytoplasm following galactose induction of Gsp1-G21V. Similarly, cells producing Gsp1-G21V retain poly(A)+ RNA in their nuclei. These findings suggest that hydrolysis of GTP by Ran/TC4 is necessary for proper import of proteins into the nucleus and appearance of poly(A)+ RNA in the cytoplasm.
O2-sensitive K+ currents in carotid body chemoreceptor cells from normoxic and chronically hypoxic rats and their roles in hypoxic chemotransduction.
Authors: Wyatt C N , Wright C , Bee D , Peers C
Published: May 2006

Carotid body-mediated ventilatory increases in response to acute hypoxia are attenuated in animals reared in an hypoxic environment. Normally, O2-sensitive K+ channels in neurosecretory type I carotid body cells are intimately involved in excitation of the intact organ by hypoxia. We have therefore studied K+ channels and their sensitivity to acute hypoxia (PO2 12-20 mmHg) in type I cells isolated from neonatal rats born and reared in normoxic and hypoxic environments. When compared with cells from normoxic rats, K+ current density in cells from hypoxic rats was significantly reduced, whereas Ca2+ current density was unaffected. Charybdotoxin (20 nM) inhibited K+ currents in cells from normoxic rats by approximately 25% but was without significant effect in cells from hypoxic rats. However, hypoxia caused similar, reversible inhibitions of K+ currents in cells from the two groups. Resting membrane potentials (measured at 37 degrees C using the perforated-patch technique) were similar in normoxic and hypoxic rats. However, although acute hypoxia depolarized type I cells of normoxic rats, it was without effect on membrane potential in type I cells from hypoxic animals. Charybdotoxin (20 nM) also depolarized cells from normoxic rats. Our results suggest that type I cells from chronically hypoxic rats, like normoxic rats, possess O2-sensing mechanisms. However, they lack charybdotoxin-sensitive K+ channels that contribute to resting membrane potential in normoxically reared rats, and this appears to prevent them from depolarizing (and hence triggering Ca2+ influx and neurosecretion) during acute hypoxia.
Binding of an invariant-chain peptide, CLIP, to I-A major histocompatibility complex class II molecules.
Authors: Gautam A M , Pearson C , Quinn V , McDevitt H O , Milburn P J
Published: May 2006

Invariant chain (Ii) associates with major histocompatibility complex (MHC) class II molecules and is crucial for antigen presentation by class II molecules. The exact nature of Ii interaction with MHC class II molecules remains undefined. A nested set of Ii peptides, CLIPs (class II-associated Ii peptides), have been eluted from various MHC class II molecules, suggesting that CLIPs correspond, at least in part, to the Ii motif which blocks the conventional peptide binding site in MHC class II molecules. Here we report how CLIPs interact with class II MHC molecules, I-A. We have identified regions critical for binding of CLIPs and I-A class II molecules. In most cases, the binding of CLIPs to a number of I-A molecules is modulated by the steric bulk of methionine residues at positions 93 and 99. In addition, the binding of CLIPs to an I-A molecule, I-Au, is sensitive to substitutions at aspartic acid-59 in the alpha chain and threonine-86 in the beta chain, whereas the binding of an antigen-derived peptide is not. Taken together, these results provide an insight as to how CLIPs bind to MHC class II heterodimers.