bluuebunny/crossref_metadata_2025 huggingface dataset:
Dale's hypothesis revisited: different neuropeptides derived from a common prohormone are targeted to different processes.
Authors: Sossin W S , Sweet-Cordero A , Scheller R H
Published: May 2006

In the bag cells of Aplysia californica, the prohormone of egg-laying hormone is processed by means of endoproteolytic cleavage into two sets of peptides. The amino-terminal region of the prohormone gives rise to the bag cell peptides (alpha, beta, and gamma). These serve an autocrine function; they are autoexcitatory on the bag cells and also act locally to alter the firing patterns of neurons in the abdominal ganglion. The carboxyl-terminal portion of the prohormone gives rise to the egg-laying hormone. This peptide acts as a hormone on nearby neurons and by means of the circulation on peripheral tissues to bring about egg-laying. We have previously reported that the first cleavage of the prohormone, which occurs in the trans-Golgi network, results in two intermediates that are sorted into distinct vesicle classes prior to further processing. Here we show that these distinct vesicles are localized to separate processes, thus spatially segregating autocrine and hormonal release sites. The findings of segregation indicate that neurons need not always release the same set of chemical messengers from all of their endings.
A 100-kilodalton protein is associated with the murine interleukin 2 receptor: biochemical evidence that p100 is distinct from the alpha and beta chains.
Authors: Sharon M , Gnarra J R , Leonard W J
Published: May 2006

Two proteins that specifically bind the T-cell growth factor interleukin 2 (IL-2) have been identified previously on the surface of T cells; these proteins have been designated IL-2R alpha and IL-2R beta for the alpha and beta chains of the IL-2 receptor (IL-2R). The association of these independent binding proteins with each other on the surface of activated T cells correlates with the generation of high-affinity binding sites. These high-affinity sites transduce the major mitogenic signal of IL-2, yet the mechanisms of association of the alpha and beta chains with each other as well as signal transduction in response to IL-2 are unknown. Cotransfection experiments of cDNAs encoding the alpha and beta chains in T cells and fibroblasts have suggested functional requirements for other T cell-specific factor(s). We now provide biochemical evidence for a distinct 100-kDa protein that interacts with the alpha or beta chains, or both, on the surface of the IL-2-dependent cell line CTLL-2 as well as activated murine splenocytes. This same 100-kDa protein is capable of being chemically cross-linked to 125I-labeled IL-2.
Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide.
Authors: Yim M B , Chock P B , Stadtman E R
Published: May 2006

Cu,Zn superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) is known to be inhibited slowly by H2O2. Using EPR and the spin traps 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN), we have shown that Cu,Zn-SOD catalyzes the formation of "free" .OH radicals from H2O2 in pH 7.6 bicarbonate buffer. Supporting evidence includes the following: (i) H2O2 and active Cu,Zn-SOD are required to yield significant signals from spin-trap-OH adducts. (ii) With O2-., Cu,Zn-SOD causes the appearance of intense resonance signals due to DMPO-OH adducts. These signals were inhibited strongly by catalase. (iii) With H2O2, Cu,Zn-SOD, and DMPO, radical scavengers formate and azide, but not ethanol, decrease DMPO-OH signals while causing new intense signals due to their corresponding DMPO-radical adducts. Failure of ethanol to quench DMPO-OH signals is discussed in light of the positively charged active channel of the enzyme. (iv) With PBN as a spin trap, ethanol quenches .OH radical signals and yields PBN-trapped hydroxyethyl radical signals. (v) Mn-SOD does not catalyze "free" .OH radical formation and it also exerts no effect on the signals of DMPO-OH adducts when added together with the Cu,Zn-SOD. The capacity of Cu,Zn-SOD to generate "free" .OH radicals from H2O2 may in part explain the biological damage associated with elevated intracellular SOD activity.
Multiple liver-specific factors bind to the hepatitis B virus core/pregenomic promoter: trans-activation and repression by CCAAT/enhancer binding protein.
Authors: López-Cabrera M , Letovsky J , Hu K Q , Siddiqui A
Published: May 2006

The human hepatitis B virus (HBV) is a hepatotropic virus that replicates through an RNA intermediate referred to as the pregenome. The promoter that directs the synthesis of the pregenome and several other transcripts with heterogeneous 5' ends is of particular interest because of its role in regulating key functions during the viral life cycle. We have examined the liver-specific characteristics of this promoter by DNA-protein interactions and by demonstrating the in vivo function of the promoter using the luciferase reporter gene expression system. The DNA-protein interactions in this region appear to be almost entirely liver-specific. Among these, a liver-specific nuclear factor, CCAAT/enhancer binding protein, binds to at least five sites on this promoter. Transient cotransfection experiments using CCAAT/enhancer binding protein expression vectors and the core promoter in the context of either the native hepatitis B virus genome or the luciferase reporter gene demonstrate that CCAAT/enhancer binding protein at low concentration modestly activates expression from the core promoter but represses at high concentration.
Beta-glucoside permease represses the bgl operon of Escherichia coli by phosphorylation of the antiterminator protein and also interacts with glucose-specific enzyme III, the key element in catabolite control.
Authors: Schnetz K , Rak B
Published: May 2006

The beta-glucoside (bgl) operon of Escherichia coli is subject to both positive control by transcriptional termination/antitermination and negative control by the beta-glucoside-specific transport protein, an integral membrane protein known as enzyme IIBgl. Previous results led us to speculate that enzyme IIBgl exerts its negative control by phosphorylating and thereby inactivating the antiterminator protein, BglG. Specifically, our model postulated that the transport protein enzyme IIBgl exhibits protein-phosphotransferase activity in the absence of beta-glucosides. We now present biochemical evidence that the phosphorylation of protein BglG does indeed occur in vivo and that it is accompanied by the loss of antitermination activity. BglG persists in the phosphorylated state in the absence of beta-glucosides but is rapidly dephosphorylated when beta-glucosides become available for transport. Our data also suggested specific interactions between the beta-glucoside transport protein and the glucose-specific enzyme III (enzyme IIIGlc), a component of glucose transport and a key element in regulation of catabolite repression. These observations indicate that enzyme IIIGlc may, in conjunction with enzyme IIBgl, modulate the transport of beta-glucosides and the phosphorylation of the antiterminator protein. In the absence of both sugars, when the catabolite-controlled promoter of the operon is derepressed, enzyme IIIGlc may mediate tight repression of antitermination.
Tumor necrosis factor and immune interferon synergistically increase transcription of HLA class I heavy- and light-chain genes in vascular endothelium.
Authors: Johnson D R , Pober J S
Published: May 2006

Tumor necrosis factor and immune interferon synergistically increase cell-surface expression of class I major histocompatibility complex molecules in cultured human endothelial cells. We report that tumor necrosis factor and interferon gamma each independently increase mRNA levels and together cause a greater-than-additive (i.e., synergistic) increase in steady-state mRNA levels and transcriptional rates of the class I heavy- and light-chain genes. HLA heavy-chain mRNA is equally stable in cytokine-treated and -untreated endothelial cells. Interferon gamma does not increase tumor necrosis factor receptor number or affinity on human endothelial cells. We conclude that the synergistic increase in class I major histocompatibility complex cell-surface expression results principally from the synergistic increase in transcriptional rates. We propose that this increase is caused by the cooperative binding of independently activated transcription factors to the promoter/enhancer sequences of class I genes.
Complete amino acid sequence of bovine glia maturation factor beta.
Authors: Lim R , Zaheer A , Lane W S
Published: May 2006

The protein glia maturation factor beta, isolated from bovine brain, has been sequenced by automated Edman degradation and tandem mass spectrometry of overlapped peptide fragments generated by cyanogen bromide cleavage and enzymatic digestion with trypsin, chymotrypsin, and endoproteinases Asp-N and Lys-C. The protein has 141 amino acid residues and possesses no potential N-glycosylation sites. It contains three cysteines (at positions 7, 86, and 95), three methionines (at positions 33, 101, and 102), and one tryptophan (at position 132). The blocked amino terminus as determined by tandem mass spectrometry is an N-acetylated serine. The carboxyl terminus is a histidine. To our knowledge, the sequence shows no significant homology with other sequenced proteins. The molecular weight calculated from the sequence information is 16,582.
Hypercolumns in primate visual cortex can develop in the absence of cues from photoreceptors.
Authors: Kuljis R O , Rakic P
Published: May 2006

The visual cortex in primates consists of an array of anatomically and chemically identifiable cellular modules (hypercolumns) with distinct physiological properties. For example, layers II/III in the macaque monkey contain a regular array of cytochrome oxidase-rich blobs. Furthermore, the surrounding cytochrome oxidase-poor interblob regions have a higher density of neuropeptide Y-positive aspiny stellate cells. Neurons in the blobs are thought to mediate predominantly low spatial frequencies and color vision, while those in the interblobs appear to be engaged in pattern vision and high spatial frequency analysis. In this study we examined the role of the retina in the development of hypercolumns. A bilateral retinal ablation was performed in embryos at midgestation, before any photoreceptors had established contacts with other retinal neurons and before layers II/III of the cortex--or their synaptic connection--had been generated. We found that the cortex in operated animals had cytochrome oxidase blobs and that their size and spacing were normal. In addition, neuropeptide Y-containing neurons were preferentially distributed in the interblob region as in control animals. Our findings indicate that some basic aspects of the cyto- and chemoarchitectonic organization of the cerebral cortex, which presumably evolved for the analysis of form and color, can emerge in the absence of cues from the retinal photoreceptors that mediate these attributes of vision.
The two functional domains of gamma delta resolvase act on the same recombination site: implications for the mechanism of strand exchange.
Authors: Dröge P , Hatfull G F , Grindley N D , Cozzarelli N R
Published: May 2006

During site-specific recombination by the gamma delta resolvase, four DNA strands are broken, exchanged, and religated. This exchange is carried out within a DNA-protein complex, the synaptosome, in which the recombination sites, res, are aligned. The domain of resolvase that binds to a res site is distinct from the domain that breaks and rejoins the DNA. We tested whether the catalytic domain acts on the res site to which its binding domain is bound (in cis) or on the opposing res site in the synaptic complex (in trans). We constructed a hybrid synaptosome in which one res site is bound to wild-type resolvase and the other is bound to a mutant resolvase that binds normally but is unable to break DNA. From the pattern of strand breakage in the reaction intermediate containing resolvase covalently attached to DNA, we conclude that resolvase attacks predominantly, if not exclusively, in cis. Because cis breakage and reunion per se cannot lead to recombination, our results support a model in which DNA exchange is guided by an exchange of resolvase subunits between the breakage and reunion events.
Autocrine transformation by chimeric signal peptide-basic fibroblast growth factor: reversal by suramin.
Authors: Yayon A , Klagsbrun M
Published: May 2006

NIH 3T3 cells transfected with basic fibroblast growth factor (bFGF) fused to an immunoglobulin signal peptide sequence are transformed in vitro and tumorigenic in vivo. The transformed phenotype of chimeric signal peptide-bFGF (spbFGF) cells is characterized by an enhanced proliferation rate compared to parental NIH 3T3 cells, density- and anchorage-independent growth, a transformed morphology, and lack of cell adhesion. The rate of spbFGF cell proliferation is not diminished by anti-bFGF neutralizing antibodies. 125I-labeled bFGF receptor cross-linking and binding studies suggest that surface FGF receptors in spbFGF cells are unavailable and down-regulated. The FGF receptors are also down-regulated in K-fgf-transformed cells but not in parental 3T3, native bFGF-transfected, and ras-transformed NIH 3T3 cells. The addition of suramin to spbFGF and K-fgf cells rapidly promotes the up-regulation of FGF receptors. Suramin also induces lowering of the proliferation rate to that of parental cells, anchorage-dependent growth, assembly of cytoskeletal filaments, cellular adhesion, and spreading. These results suggest that spbFGF cells undergo autocrine transformation, possibly by an internal autocrine loop, in which there is constitutive activation of the FGF receptor. Suramin inhibits autocrine transformation, leading to a normal untransformed phenotype.
Macrophage-colony-stimulating factor (CSF-1) induces proliferation, chemotaxis, and reversible monocytic differentiation in myeloid progenitor cells transfected with the human c-fms/CSF-1 receptor cDNA.
Authors: Pierce J H , Di Marco E , Cox G W , Lombardi D , Ruggiero M , Varesio L , Wang L M , Choudhury G G , Sakaguchi A Y , Di Fiore P P
Published: May 2006

The c-fms protooncogene encodes the receptor for macrophage-colony-stimulating factor (CSF-1). Expression vectors containing either normal or oncogenic point-mutated human c-fms genes were transfected into interleukin 3 (IL-3)-dependent 32D cells in order to determine the effects of CSF-1 signaling in this murine clonal myeloid progenitor cell line. CSF-1 was shown to trigger proliferation in association with monocytic differentiation of the 32D-c-fms cells. Monocytic differentiation was reversible upon removal of CSF-1, implying that CSF-1 was required for maintenance of the monocyte phenotype but was not sufficient to induce an irrevocable commitment to differentiation. Human CSF-1 was also shown to be a potent chemoattractant for 32D-c-fms cells, suggesting that CSF-1 may serve to recruit monocytes from the circulation to tissue sites of inflammation or injury. Although c-fms did not release 32D cells from factor dependence, point-mutated c-fms[S301,F969] (Leu-301----Ser, Tyr-969----Phe) was able to abrogate their IL-3 requirement and induce tumorigenicity. IL-3-independent 32D-c-fms[S301,F969] cells also displayed a mature monocyte phenotype, implying that differentiation did not interfere with progression of these cells to the malignant state. All of these findings demonstrate that a single growth factor receptor can specifically couple with multiple intracellular signaling pathways and play a critical role in modulating cell proliferation, differentiation, and migration.
MyoD binds cooperatively to two sites in a target enhancer sequence: occupancy of two sites is required for activation.
Authors: Weintraub H , Davis R , Lockshon D , Lassar A
Published: May 2006

MyoD is a master regulatory gene for myogenesis. Its product, the MyoD protein, appears to act by binding to muscle-specific enhancer sequences. We show that MyoD binds cooperatively to two sites in the muscle-specific creatine kinase enhancer; this is dramatically reflected in dissociation-rate measurements. A deletion of the acidic N terminus (residues 3-56) results in a protein that binds normally to single sites but fails to bind cooperatively to two adjacent sites, suggesting a role of the N terminus in cooperative interactions. In transfection assays, a reporter gene flanked by a single MyoD binding site fails to be activated by cotransfected MyoD expression vectors. In contrast, a reporter with two or more MyoD binding sites is activated by wild-type MyoD but not by N-terminally deleted MyoD. A reporter gene with a single binding site, although not activated by MyoD, can nonetheless compete for expression with a reporter gene containing three sites. Thus, in vivo, a single site can bind MyoD, but occupancy of two or more sites is required for subsequent transcriptional activation.
Human homologue of murine tumor rejection antigen gp96: 5'-regulatory and coding regions and relationship to stress-induced proteins.
Authors: Maki R G , Old L J , Srivastava P K
Published: May 2006

Cell-surface glycoproteins of 96 kDa (gp96) have been implicated in immunogenicity of methylcholanthrene-induced mouse sarcomas in syngeneic hosts. In view of the potential immunogenicity of gp96-related molecules in human tumors, we have defined the 5'-regulatory and complete coding regions of a human gp96 transcript. The 5'-regulatory region contains an imperfect heat shock element apart from other regulatory sequences. The amino acid sequence of human gp96 is 96% homologous to its murine counterpart and genes for the two molecules show significant homology between untranslated regions. Comparison of gp96 sequences to other sequences in DNA and protein data bases indicates significant homology with the stress proteins 94-kDa glucose-regulated protein (grp94) and 108-kDa heat shock protein (hsp108) and 99-kDa endoplasmic reticular protein (ERp99). These molecules are either identical or represent a family of closely related molecules. With regard to their role in tumor immunity, it needs to be determined whether gp96 molecules are tumor antigens per se or whether they serve as carriers of other immunogenic moieties.
Penicillin-resistant viridans streptococci have obtained altered penicillin-binding protein genes from penicillin-resistant strains of Streptococcus pneumoniae.
Authors: Dowson C G , Hutchison A , Woodford N , Johnson A P , George R C , Spratt B G
Published: May 2006

Penicillin-resistant strains of Streptococcus pneumoniae possess altered forms of penicillin-binding proteins (PBPs) with decreased affinity for penicillin. The PBP2B genes of these strains have a mosaic structure, consisting of regions that are very similar to those in penicillin-sensitive strains, alternating with regions that are highly diverged. Penicillin-resistant strains of viridans groups streptococci (e.g., S. sanguis and S. oralis) that produce altered PBPs have also been reported. The PBP2B genes of two penicillin-resistant clinical isolates of S. sanguis were identical in sequence to the mosaic class B PBP2B genes found in penicillin-resistant serotype 23 strains of S. pneumoniae. Emergence of penicillin resistance appears to have occurred by the horizontal transfer of an altered PBP2B gene from penicillin-resistant S. pneumoniae into S. sanguis. The PBP2B genes of three penicillin-resistant S. oralis strains were similar to the mosaic class B PBP2B gene of penicillin-resistant strains of S. pneumoniae but possessed an additional block of diverged sequence. Penicillin resistance in S. oralis has also probably arisen by horizontal transfer of this variant form of the class B mosaic PBP2B gene from a penicillin-resistant strain of S. pneumoniae.
FrzE of Myxococcus xanthus is homologous to both CheA and CheY of Salmonella typhimurium.
Authors: McCleary W R , Zusman D R
Published: May 2006

Myxococcus xanthus exhibits multicellular development. The "frizzy" (frz) mutants are unable to complete the developmental pathway. Instead of forming fruiting bodies, these mutants form tangled filaments of cells. We have previously shown that four of the frz gene products are homologous to enteric chemotaxis proteins and have proposed that the frz genes constitute a signal-transduction pathway that controls the frequency at which cells reverse their gliding direction. We show here that frzE encodes a protein with a calculated molecular mass of 83 kDa. FrzE is homologous to both CheA and CheY of Salmonella typhimurium, which are members of a family of "two-component response regulators." It is thought that the modulator components autophosphorylate and transfer a phosphate group to their cognate effector components. FrzE contains an unusual (alanine plus proline)-rich region that might constitute a flexible hinge facilitating phosphate transfer between functional domains. We suggest that FrzE is a second messenger that relays information between the signaling protein FrzCD and the gliding motor.
Linear electric field mass analysis: a technique for three-dimensional high mass resolution space plasma composition measurements.
Authors: McComas D J , Nordholt J E , Bame S J , Barraclough B L , Gosling J T
Published: May 2006

A revolutionary type of three-dimensional space plasma composition analyzer has been developed that combines very high-resolution mass composition measurements on a fraction of the incident ions simultaneously with lower mass resolution but high sensitivity measurements of the remaining population in a single compact and robust sensor design. Whereas the lower mass resolution measurements are achieved using conventional energy/charge (E/q) and linear time-of-flight analysis, the high mass resolution measurements are made by timing reflected E/q analyzed ions in a linear electric field (LEF). In a LEF the restoring (reflecting) force that an ion experiences in the direction parallel to the field is proportional to the depth it travels into the LEF region, and its equation of motion in that direction is that of a simple harmonic oscillator. Consequently, an ion's travel time is independent of its initial angle and energy and is simply proportional to the square root of the ion's mass/charge (m/q). The measured m/q resolution, (m/q)/Delta(m/q), for a small LEF-based prototype that we have developed and tested is approximately 20. In addition, our laboratory measurements with the prototype instrument show that characteristic time-of-flight spectra allow the resolution of atomic and molecular species with nearly identical m/q values. The measured response of the prototype is in excellent agreement with computer simulations of the device. Advanced design work using this computer simulation indicates that three-dimensional plasma composition analyzers with m/q resolutions of at least 50 are readily achievable.
Broad spectrum of in vivo forward mutations, hypermutations, and mutational hotspots in a retroviral shuttle vector after a single replication cycle: deletions and deletions with insertions.
Authors: Pathak V K , Temin H M
Published: May 2006

In the preceding paper we described an experiment that determined the in vivo forward mutation rate in a single replication cycle for spleen necrosis virus. In addition to substitutions, frameshifts, and hypermutations, the mutated proviruses contained two classes of deletions. One class of deletions contained short direct repeats at the deletion junctions. Another class of deletions had short stretches of sequences inserted at the deletion junctions. In this report, we describe the deletion mutations, and we present models for their generation. Detailed analysis of two deletions with insertions indicates that these mutations occurred as a result of template switching during plus-strand DNA synthesis. The analysis also indicates that fragments of viral RNA generated by the viral RNase H endonuclease are used as templates and contribute to the sequences inserted at the deletion junctions.
Carbonic anhydrase isozymes IV and II in urinary membranes from carbonic anhydrase II-deficient patients.
Authors: Sato S , Zhu X L , Sly W S
Published: May 2006

Carbonic anhydrase II (CA II) deficiency has been shown to be the primary defect in the recessively inherited syndrome of osteopetrosis with renal tubular acidosis. Until now, the absence of CA II in kidney of CA II-deficient patients has not been shown directly, and the status of the membrane-associated CA in kidney of CA II-deficient patients has been unclear. To address these questions, we analyzed urinary membranes and soluble fractions from normal and CA II-deficient subjects. The CA activity in membrane fractions of normal urine was found to comprise two components--(i) a vesicle-enclosed, sodium dodecyl sulfate (SDS)-sensitive fraction, which was shown immunochemically to be the 29-kDa CA II, and (ii) an SDS-resistant fraction, which was due to native and cleaved forms of the 35-kDa, membrane-anchored isozyme CA IV. Urinary membranes from CA II-deficient patients showed little or no SDS-sensitive activity and no immunoreactivity for CA II, providing direct evidence that their mutation, which produces CA II deficiency in erythrocytes, also affects CA II in kidney. CA IV activity and immunoreactivity were present in normal amounts in urinary membranes from CA II-deficient patients. We conclude from the enzymatic and immunological evidence presented that both CA II and CA IV are present in urinary membranes from normal subjects, that renal CA IV is present but renal CA II is absent in urinary membranes from patients with the CA II-deficiency syndrome, and that the methods presented should be useful in studying renal CA II and renal CA IV in other disorders of impaired bicarbonate reabsorption.
Cloning and expression of a widely expressed receptor tyrosine phosphatase.
Authors: Sap J , D'Eustachio P , Givol D , Schlessinger J
Published: May 2006

We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid extracellular domain (including signal peptide) of R-PTP-alpha is marked by a high serine/threonine content (32%) as well as eight potential N-glycosylation sites but displays no similarity to known proteins. Genetic mapping assigns the gene for R-PTP-alpha to mouse chromosome 2, closely linked to the Il-1a and Bmp-2a loci. The corresponding mRNA (3.0 kilobases) is expressed in most murine tissues and most abundantly expressed in brain and kidney. Antibodies against a synthetic peptide of R-PTP-alpha identified a 130-kDa protein in cells transfected with the R-PTP-alpha cDNA.
DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase.
Authors: Hollingsworth R E , Sclafani R A
Published: May 2006

The yeast Cdc7 protein is indispensable to initiation of nuclear DNA replication, based on the phenotype of the conditional, temperature-sensitive (ts) cdc7 mutants at the restrictive temperature. This protein has likewise been implicated in commitment to meiotic DNA recombination and induced mutagenesis, which may result from error-prone DNA repair. Our previous work revealed sequence similarity between the Cdc7 protein and known protein kinases. To determine whether it possesses kinase activity, we have immunoprecipitated the protein from Cdc7-overproducing yeast cells by using polyclonal antibodies raised against a nondenatured beta-galactosidase-Cdc7 fusion protein. In this report, we demonstrate that Cdc7 immune complexes are capable of phosphorylating mammalian histone H1 on serine and/or threonine residues. Immune complexes derived from cells harboring the cdc7-2 ts mutant gene on a high copy number plasmid possess a thermolabile kinase activity. Thus, we postulate that Cdc7 may regulate the various DNA metabolic pathways by phosphorylating one or more target substrates. Because Cdc7 kinase acts downstream of Cdc28/cdc2 kinase function at "start," the transition from G1 to S phase in the cell cycle may be the result of a cascade of protein phosphorylation.
Maximizing the natriuretic effect of endogenous atriopeptin in a rat model of heart failure.
Authors: Wilkins M R , Settle S L , Stockmann P T , Needleman P
Published: May 2006

The effect of pharmacological manipulation of atriopeptin (AP) activity on sodium excretion and blood pressure was examined in the rat aortovenocaval (A-V) fistula model of cardiac failure. Introduction of an A-V shunt led to a marked and sustained elevation of plasma AP immunoreactivity and urinary cGMP levels. Further elevation of plasma AP levels by infusion of exogenous peptide induced modest increases in urinary sodium and cGMP excretion and a decrease in blood pressure but these responses were significantly attenuated compared to sham-operated animals. In contrast, low-dose infusion of M + B 22948 (a cGMP phosphodiesterase inhibitor) or thiorphan [a neutral endopeptidase (membrane metallo-endopeptidase, EC 3.4.24.11) inhibitor] induced a natriuresis in A-V fistula rats, which exceeded that seen in control animals given these compounds and matched the peak natriuresis produced in sham-operated animals by high doses of AP. In the doses used, these compounds had little effect on blood pressure. The greater renal efficacy of M + B 22948 in A-V fistula rats is consistent with postreceptor facilitation of AP activity. The effect of thiorphan on sodium excretion was accompanied by a pronounced increase in urinary cGMP and AP immunoreactivity excretion (and was attenuated by anti-AP monoclonal antibody) but could not be explained solely in terms of an increase in circulating AP levels. It is proposed that thiorphan allows filtered AP to reach renal tubule sites that are normally inaccessible to the peptide and are thus protected from down-regulation by high circulating AP levels. The implication of these observations for patients in cardiac failure is the potential for using pharmacological agents to maximize the response to endogenous AP without compromising cardiac function.
Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein.
Authors: Cangelosi G A , Ankenbauer R G , Nester E W
Published: May 2006

Phenolic plant metabolites such as acetosyringone induce transcription of the virulence (vir) genes of Agrobacterium tumefaciens through the transmembrane VirA protein. We report here that certain sugars induce the vir genes synergistically with phenolic inducers by way of a distinct regulatory pathway that includes VirA and a chromosomally encoded virulence protein, ChvE. Sequence comparison showed that ChvE is a periplasmic galactose-binding protein corresponding to the GBP1 protein isolated from Agrobacterium radiobacter. Like homologous sugar-binding proteins in Escherichia coli, ChvE was required for chemotaxis toward galactose and several other sugars. These sugars strongly induced vir gene expression in wild-type cells when acetosyringone was absent or present in low concentrations. Mutations in chvE abolished vir gene induction by sugars and resulted in a limited host range for infection but did not affect vir gene induction by acetosyringone. A mutant lacking the periplasmic domain of VirA exhibited the same regulatory phenotype and limited host range as chvE mutants. These data show that the vir genes are regulated by two separate classes of plant-derived inducers by way of distinct regulatory pathways that can be separated by mutation. Induction by sugars is essential for infection of some but not all plant hosts.
Changed distribution of sodium channels along demyelinated axons.
Authors: England J D , Gamboni F , Levinson S R , Finger T E
Published: May 2006

Voltage-gated sodium channels are largely localized to the nodes of Ranvier in myelinated axons, providing a physiological basis for saltatory conduction. What happens to these channels in demyelinated axons is not known with certainty. Experimentally demyelinated axons were examined by using a well-characterized, polyclonal antibody directed against sodium channels. Immunocytochemical and radioimmunoassay data were consistent with the distribution of an increased number of sodium channels along segments of previously internodal axon. These findings affirm the plasticity of sodium channels in demyelinated axolemma and may be relevant to understanding how axons recover conduction after demyelination.
Sensitization of bleached rod photoreceptors by 11-cis-locked analogues of retinal.
Authors: Corson D W , Cornwall M C , MacNichol E F , Jin J , Johnson R , Derguini F , Crouch R K , Nakanishi K
Published: May 2006

Photoactivation of rhodopsin initiates both excitation and adaptation in vertebrate rod photoreceptors. Bleaching of rhodopsin to free opsin and all-trans-retinal in isolated rods produces a stable desensitization (bleaching adaptation) that is much larger than expected from pigment depletion alone. In our experiments, a 93% bleach produced a 500-fold increase in the light intensity required for saturation of the light response. This component of adaptation was 32-fold larger than the 16-fold increase expected from pigment depletion alone. 11-cis-Retinal, when delivered to isolated rods from liposomes, combines with free opsin to form a bleachable photopigment that fully restores sensitivity. 11-cis-Locked analogues of retinal combine with opsin to form unbleachable pigments in isolated bleached rods from the tiger salamander. They restore sensitivity to a substantial (16- to 25-fold) but incomplete extent. The analogues apparently relieve a stable component of adaptation when they interact with opsin. Because these analogues do not detectably excite rods, the structural requirements of both retinal and opsin for the relief of adaptation are different from those of excitation. The biochemical basis of light adaptation resulting from pigment bleaching and the minimum structural requirements of retinal for its relief remain to be determined.
A single amino acid substitution results in a retinoblastoma protein defective in phosphorylation and oncoprotein binding.
Authors: Kaye F J , Kratzke R A , Gerster J L , Horowitz J M
Published: May 2006

We have previously identified a small-cell lung cancer cell line (NCI-H209) that expresses an aberrant, underphosphorylated form of the retinoblastoma protein RB1. Molecular analysis of RB1 mRNA from this cell line revealed a single point mutation within exon 21 that resulted in a nonconservative amino acid substitution (cysteine to phenylalanine) at codon 706. Stable expression of this mutant RB1 cDNA in a human cell line lacking endogenous RB1 demonstrated that this amino acid change was sufficient to inhibit phosphorylation. In addition, this cysteine-to-phenylalanine substitution also resulted in loss of RB1 binding to the simian virus 40 large tumor and adenovirus E1A transforming proteins. These results confirm the importance of exon 21 coding sequences and suggest that the cysteine residue at codon 706 may play a role in achieving a specific protein conformation essential for protein-protein interactions.
Homeodomain of yeast repressor alpha 2 contains a nuclear localization signal.
Authors: Hall M N , Craik C , Hiraoka Y
Published: May 2006

The yeast repressor alpha 2 is shown, by analysis of deletion-bearing alpha 2-beta-galactosidase hybrid proteins, to have two structurally distinct nuclear localization signals. The cellular location of hybrid proteins was determined by indirect immunofluorescence and optical sectioning of whole fixed yeast cells. The two nuclear localization signals are far apart in the alpha 2 primary structure and do not have any sequence homology. One signal is, as reported previously, within the aminoterminal 13 amino acids of alpha 2. Deletion of only this aminoterminal signal has no evident effect on nuclear localization. The second signal is in a central portion of alpha 2, within the alpha 2 homeodomain. Since this signal is within the amino terminus of the alpha 2 homeodomain, the homeodomain mediates nuclear localization in addition to, and independently of, DNA binding. Deletion of only this second signal results in inefficient localization and accumulation of mutant protein at discrete sites on the nuclear envelope assumed to be nuclear pores. We propose that the two signals in alpha 2 are functionally distinct and act at different steps in a localization pathway.
Cloning of three human tyrosine phosphatases reveals a multigene family of receptor-linked protein-tyrosine-phosphatases expressed in brain.
Authors: Kaplan R , Morse B , Huebner K , Croce C , Howk R , Ravera M , Ricca G , Jaye M , Schlessinger J
Published: May 2006

A human brainstem cDNA library in bacteriophage lambda gt11 was screened under conditions of reduced hybridization stringency with a leukocyte common antigen (LCA) probe that spanned both conserved cytoplasmic domains. cDNA encoding a receptor-linked protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), RPTPase alpha, has been cloned and sequenced. Human RPTPase alpha consists of 802 amino acids. The extracellular domain of 150 residues includes a hydrophobic signal peptide and eight potential N-glycosylation sites. This is followed by a transmembrane region and two tandemly repeated conserved domains characteristic of all RPTPases identified thus far. The gene for RPTPase alpha has been localized to human chromosome region 20pter-20q12 by analysis of its segregation pattern in rodent-human somatic cell hybrids. Northern blot analysis revealed the presence of two major transcripts of 4.3 and 6.3 kilobases. In addition to RPTPase alpha, two other RPTPases (beta and gamma), identified in the same screen, have been partially cloned and sequenced. Analysis of sequence comparisons among LCA, the LCA-related protein LAR, and RPTPases alpha, beta, and gamma reveals the existence of a multigene family encoding different RPTPases, each containing a distinct extracellular domain, a single hydrophobic transmembrane region, and two tandemly repeated conserved cytoplasmic domains.
Prolonged inhibition of luteinizing hormone and testosterone levels in male rats with the luteinizing hormone-releasing hormone antagonist SB-75.
Authors: Bokser L , Bajusz S , Groot K , Schally A V
Published: May 2006

Inhibitory effects of the potent antagonist of luteinizing hormone-releasing hormone N-Ac-[3-(2-naphthyl)-D-alanine1,4-chloro-D-phenylalanine2,3- (3-pyridyl)-D- alanine3,D-citrulline6,D-alanine10]luteinizing hormone-releasing hormone (SB-75) free of edematogenic effects were investigated in male rats. In a study to determine the effect on luteinizing hormone levels in castrated male rats, SB-75 was injected s.c. in doses of 0.625, 1.25, 2.5, 5.0, and 10 micrograms. Blood samples were taken at different intervals for 48 hr. All doses of SB-75 significantly decreased luteinizing hormone levels for greater than 6 hr (P less than 0.01); this inhibition lasted for greater than 24 hr (P less than 0.01) with a dose of 5.0 micrograms and greater than 48 hr with 10 micrograms (P less than 0.05). Serum testosterone levels were also measured in intact male rats injected with SB-75 in doses of 25, 50, and 100 micrograms. All doses produced a dramatic fall in testosterone to castration levels 6 hr after injection (P less than 0.01); this inhibition of serum testosterone was maintained for greater than 72 hr, but only the 100-micrograms dose could keep testosterone in the castration range for greater than 24 hr (P less than 0.01). In another study using a specific RIA, we obtained the pharmacokinetic release pattern of SB-75 from two sustained delivery formulations of SB-75 pamoate microgranules and examined their effect on serum testosterone. After a single i.m. injection of 20 mg of one batch of microgranules, a large peak corresponding to SB-75 at 45.8 ng/ml was observed, corresponding to the "burst" effect. Levels of the analog decreased to 19.6 ng/ml on day 2, gradually reached a concentration of 4.7 ng/ml on day 7, and kept declining thereafter. Testosterone levels were reduced on day 1 (P less than 0.01) and were maintained at low values for greater than 7 days (P less than 0.05). In rats injected with 10 mg of SB-75 pamoate microgranules of the second batch, SB-75 serum levels rose to 33 ng/ml 3 hr after administration and then fell gradually to approximately 3.4 ng/ml on day 16, but a second small peak was seen on day 28. Subsequently, the analog levels decreased slowly to 2.9 ng/ml on day 42. At this time, testosterone serum levels were still significantly lower than in controls. These overall results demonstrate the efficacy of SB-75 in the suppression of the pituitary-gonadal axis. This modern luteinizing hormone-releasing hormone antagonist can possibly be used for treating sex hormone-sensitive cancers and other disorders.
The X chromosome of monotremes shares a highly conserved region with the eutherian and marsupial X chromosomes despite the absence of X chromosome inactivation.
Authors: Watson J M , Spencer J A , Riggs A D , Graves J A
Published: May 2006

Eight genes, located on the long arm of the human X chromosome and present on the marsupial X chromosome, were mapped by in situ hybridization to the chromosomes of the platypus Ornithorhynchus anatinus, one of the three species of monotreme mammals. All were located on the X chromosome. We conclude that the long arm of the human X chromosome represents a highly conserved region that formed part of the X chromosome in a mammalian ancestor at least 150 million years ago. Since three of these genes are located on the long arm of the platypus X chromosome, which is G-band homologous to the Y chromosome and apparently exempt from X chromosome inactivation, the conservation of this region has evidently not depended on isolation by X-Y chromosome differentiation and X chromosome inactivation.
Mechanism of kinase activation in the receptor for colony-stimulating factor 1.
Authors: Lee A W , Nienhuis A W
Published: May 2006

Receptor tyrosine kinases remain dormant until activated by ligand binding to the extracellular domain. Two mechanisms have been proposed for kinase activation: (i) ligand binding to the external domain of a receptor monomer may induce a conformational change that is transmitted across the cell membrane (intramolecular model) or (ii) the ligand may facilitate oligomerization, thereby allowing interactions between the juxtaposed kinase domains (intermolecular model). The receptor for colony-stimulating factor 1 was used to test these models. Large insertions at the junction between the external and transmembrane domains of the receptor, introduced by site-directed mutagenesis of the cDNA, were positioned to isolate the external domain and prevent transmembrane conformational propagation while allowing for receptor oligomerization. Such mutant receptors were expressed on the cell surface, bound ligand with high affinity, exhibited ligand-stimulated autophosphorylation, and signaled mitogenesis and cellular proliferation in the presence of ligand. A second experimental strategy directly tested the intermolecular model of ligand activation. A hybrid receptor composed of the external domain of human glycophorin A and the transmembrane and cytoplasmic domains of the colony-stimulating factor 1 receptor exhibited anti-glycophorin antibody-induced kinase activity that supported mitogenesis. Our data strongly support a mechanism of receptor activation based on ligand-induced receptor oligomerization.
Localization of phosphatidylinositol signaling components in rat taste cells: role in bitter taste transduction.
Authors: Hwang P M , Verma A , Bredt D S , Snyder S H
Published: May 2006

To assess the role of phosphatidylinositol turnover in taste transduction we have visualized, in rat tongue, ATP-dependent endoplasmic reticular accumulation of 45Ca2+, inositol 1,4,5-trisphosphate receptor binding sites, and phosphatidylinositol turnover monitored by autoradiography of [3H]cytidine diphosphate diacylglycerol formed from [3H]cytidine. Accumulated 45Ca2+, inositol 1,4,5-trisphosphate receptors, and phosphatidylinositol turnover are selectively localized to apical areas of the taste buds of circumvallate papillae, which are associated with bitter taste. Further evidence for a role of phosphatidylinositol turnover in bitter taste is our observation of a rapid, selective increase in mass levels of inositol 1,4,5-trisphosphate elicited by low concentrations of denatonium, a potently bitter tastant.
Chronic antidepressant administration decreases the expression of tyrosine hydroxylase in the rat locus coeruleus.
Authors: Nestler E J , McMahon A , Sabban E L , Tallman J F , Duman R S
Published: May 2006

Regulation of tyrosine hydroxylase expression by antidepressant treatments was investigated in the locus coeruleus (LC), the major noradrenergic nucleus in brain. Rats were treated chronically with various antidepressants, and tyrosine hydroxylase levels were measured in the LC by immunoblot analysis. Representatives of all major classes of antidepressant medication-including imipramine, nortriptyline, tranylcypromine, fluvoxamine, fluoxetine, bupropion, iprindole, and electroconvulsive seizures-were found to decrease levels of tyrosine hydroxylase immunoreactivity by 40-70% in the LC. Decreased levels of enzyme immunoreactivity were shown to be associated with equivalent decreases in enzyme mRNA levels. Antidepressant regulation of LC tyrosine hydroxylase appeared specific to these compounds, inasmuch as chronic treatment of rats with representatives of other classes of psychotropic drugs, including haloperidol, diazepam, clonidine, cocaine, and morphine, failed to decrease levels of this protein. The results demonstrate that chronic antidepressants dramatically downregulate the expression of tyrosine hydroxylase in the LC and raise the possibility that such regulation of the enzyme represents an adaptive response of LC neurons to antidepressants that mediates some of their therapeutic actions in depression and/or other psychiatric disturbances.
DNA polymerase II is encoded by the DNA damage-inducible dinA gene of Escherichia coli.
Authors: Bonner C A , Hays S , McEntee K , Goodman M F
Published: May 2006

The structural gene for DNA polymerase II was cloned by using a synthetic inosine-containing oligonucleotide probe corresponding to 11 amino acids, which were determined by sequencing the amino terminus of the purified protein. The labeled oligonucleotide hybridized specifically to the lambda clone 7H9 from the Kohara collection as well as to plasmid pGW511 containing the SOS-regulated dinA gene. Approximately 1400 base pairs of dinA sequence were determined. The predicted amino-terminal sequence of dinA demonstrated that this gene encoded DNA polymerase II. Sequence analysis of the upstream region localized a LexA binding site overlapping the -35 region of the dinA promoter, and this promoter element was found to be only two nucleotides downstream from the 3' end of the araD gene. These results demonstrate that the gene order is thr-dinA (pol II)-ara-leu on the Escherichia coli chromosome and that the DNA polymerase II structural gene is transcribed in the same direction as the araBAD operon. Based on the analysis of the predicted protein, we have identified a sequence motif Asp-Xaa-Xaa-Ser-Leu-Tyr-Pro-Ser in DNA polymerase II that is highly conserved among a diverse group of DNA polymerases, which include those from humans, yeast, Herpes and vaccinia viruses, and phages T4 and PRD1. The demonstration that DNA polymerase II is a component of the SOS response in E. coli suggests that it plays an important role in DNA repair and/or mutagenesis.