bluuebunny/crossref_metadata_2025 huggingface dataset:
Complementation between Sindbis viral RNAs produces infectious particles with a bipartite genome.
Authors: Geigenmüller-Gnirke U , Weiss B , Wright R , Schlesinger S
Published: May 2006

Sindbis virus, the type member of the alpha-viruses, is an enveloped virus containing a nonsegmented positive-strand RNA genome. We show that the nonstructural and the structural genes can function to produce infectious virus particles when they are expressed on two different RNA segments. The nonstructural genes are translated from an RNA in which the structural genes have been replaced by the chloramphenicol acetyltransferase gene [Xiong, C., Levis, R., Shen, P., Schlesinger, S., Rice, C. M. & Huang, H. V. (1989) Science 243, 1188-1191]. The structural genes are encoded in a defective-interfering RNA but are translated from a subgenomic RNA. Both segments contain the cis-acting sequences required for replication and packaging and are copackaged. This type of genome provides a model for an ancestral intermediate between alphaviruses and the multipartite positive-strand RNA viruses of plants. These different viruses show sequence similarities in their replicative proteins and are thought to have evolved from a common ancestor.
Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase.
Authors: Nunn D N , Lory S
Published: May 2006

The related type IV pilins produced by Pseudomonas aeruginosa, Neisseria gonorrhoeae, Bacteroides nodosus, and Moraxella bovis are synthesized as precursors with short, six- or seven-amino acid N-terminal leader peptides. We have previously observed that P. aeruginosa mutations in pilD, a gene required for pilus biogenesis, result in the accumulation of unprocessed prepilin in the membrane and a general defect in the excretion of a number of extracellular enzymes. An endopeptidase activity has been detected in detergent-solubilized inner membrane of P. aeruginosa and shown to correctly cleave the prepilin of P. aeruginosa and N. gonorrhoeae. It is absent from pilD mutants, increased by pilD overexpression, and conferred on Escherichia coli by the introduction of the pilD gene. The pilD gene product, purified by immunoaffinity chromatography with antibody to a PilD-derived synthetic peptide, was identified with the endopeptidase. PilD appears to be a prototype of a class of enzymes that process not only type IV pilin precursors but also components of a protein-excretion apparatus of Gram-negative bacteria.
Identification and characterization of cellular binding proteins (receptors) for recombinant human bone morphogenetic protein 2B, an initiator of bone differentiation cascade.
Authors: Paralkar V M , Hammonds R G , Reddi A H
Published: May 2006

Bone morphogenetic protein 2B (BMP 2B), is a heparin-binding bone differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously. The molecular mechanism of action of this differentiation factor is not known, and as a first step we have examined BMP 2B-responsive cells for the presence of specific cellular binding proteins. Using 125I-labeled BMP 2B, specific high-affinity binding sites for recombinant human BMP 2B on MC3T3 E1 osteoblast-like cells as well as on NIH 3T3 fibroblasts were identified. Platelet-derived growth factor, insulin-like growth factor 1, basic fibroblast growth factor, epidermal growth factor, and transforming growth factor beta did not compete for the binding of radiolabeled BMP 2B. The binding of BMP 2B is a time- and temperature-dependent process. Chemical crosslinking of radiolabeled BMP showed two components (apparent size, 200 and 70 kDa in MC3T3 E1 cells and 200 and 90 kDa in NIH 3T3 cells). A minor component at 60 kDa was also detected in both cell lines. Scatchard analysis of the binding data showed a high-affinity receptor with an apparent dissociation constant of 128 +/- 40 pM in MC3T3 E1 cells. These data demonstrate specific, high-affinity cell-surface binding proteins for BMP 2B.
Global and local metric geometry of ligand binding thermodynamics.
Authors: Di Cera E , Wyman J
Published: May 2006

An abstract metric representation of ligand binding thermodynamics reveals the basic connections between the classical thermodynamic theory of binding and linkage [Wyman, J. (1964) Adv. Protein Chem. 19, 223-286] and its site-specific formulation [Di Cera, E. (1990) Biophys. Chem. 37, 147-164]. These two approaches are limit cases of a general metric formulation of binding and linkage that also includes a number of intermediate "mixed" representations.
Inactivation of maize transposon Mu suppresses a mutant phenotype by activating an outward-reading promoter near the end of Mu1.
Authors: Barkan A , Martienssen R A
Published: May 2006

We described previously a mutation in maize, hcf106, caused by the insertion of a Mu1 transposon. When the Mu transposon system is in an active phase, hcf106 conditions a nonphotosynthetic, pale green phenotype. However, when the Mu system is inactive (a state correlated with hypermethylation of Mu elements), the plant adopts a normal phenotype despite the continued presence of the transposon within the gene. The molecular mechanisms that mediate this suppression of the mutant phenotype have now been investigated. We show here that the Mu element responsible for the hcf106 lesion lies within sequences encoding the 5'-untranslated leader of the Hcf106 mRNA. When the Mu transposon system is active, this insertion interferes with the accumulation of mRNA from the hcf106 allele. However, when Mu is inactive, mRNA similar in size and abundance to that transcribed from the normal allele accumulates. These transcripts initiate at many sites throughout a 70-base-pair region, within and immediately downstream of the Mu1 insertion. Thus, an unusual promoter spanning the downstream junction between Mu1 and Hcf106 substitutes for the normal Hcf106 promoter but only when Mu is inactive. The pattern of mRNA accumulation in different organs and in response to light suggests that the activity of this promoter is conditional not only upon the phase of Mu activity, but also upon signals that regulate the normal Hcf106 promoter.
Poliovirus can enter and infect mammalian cells by way of an intercellular adhesion molecule 1 pathway.
Authors: Selinka H C , Zibert A , Wimmer E
Published: May 2006

Mouse fibroblast cell lines were transfected with truncated forms of the human poliovirus receptor (PVR) cDNA and tested for the expression of functional receptors for poliovirus. Several receptor constructs, all containing the coding region of the first 143 amino acids of PVR, were able to render mouse cells susceptible to poliovirus infection. A deletion of 65 amino acids in the first extracellular domain of PVR prevented virus attachment and infection. These data suggest that domain 1 is necessary and sufficient for the virus-receptor interaction. A PVR/intercellular adhesion molecule 1 hybrid receptor, expressing the PVR variable domain on a truncated receptor molecule for human rhinovirus 14, was shown to be a functional receptor for poliovirus. This observation indicates that, subsequent to attachment to the PVR-binding domain, poliovirus can use the same pathway as the major receptor group rhinoviruses to enter cells.
Peripherin: an islet antigen that is cross-reactive with nonobese diabetic mouse class II gene products.
Authors: Boitard C , Villa M C , Becourt C , Gia H P , Huc C , Sempe P , Portier M M , Bach J F
Published: May 2006

The nonobese diabetic (NOD) mouse, in which major histocompatibility complex genes may be involved in the susceptibility to diabetes, has been developed as a model of autoimmune diabetes. The NOD mouse expresses I-A-encoded class II major histocompatibility complex antigens, which differ from those of other mouse haplotypes by the presence of a serine at position 57 of the A beta chain. Identifying islet autoantigens may help elucidate the role of class II antigens in the activation of autoreactive T cells and, thus, in the development of diabetes. We have detected autoantibodies directed against a 58-kDa islet cell antigen in NOD mice but not in other strains, including lupus-prone mice. Apart from insulin-secreting cells, the 58-kDa antigen was only found to be expressed by neuroblastoma cells and was identified as peripherin, an intermediate filament protein previously characterized in well-defined neuronal populations. This autoantigen cross-reacted with I-Anod class II antigens, suggesting that it may contribute to defective self-tolerance of islet beta cells in the NOD mouse.
Molecular basis of recognition by the glycoprotein hormone-specific N-acetylgalactosamine-transferase.
Authors: Smith P L , Baenziger J U
Published: May 2006

Lutropin (LH) bears asparagine-linked oligosaccharides terminating with the unique sequence SO4-4GalNAc beta 1-4GlcNAc beta 1-2Man alpha, whereas follitropin (FSH) bears oligosaccharides terminating predominantly with the sequence Sia alpha-Gal beta 1-4GlcNAc beta 1-2Man alpha, where Sia is sialic acid. We previously identified a glycoprotein-hormone-specific N-acetylgalactosamine-transferase (GalNAc-transferase) that recognizes a peptide-recognition marker(s) present on the common glycoprotein hormone alpha subunit and beta subunits of human chorionic gonadotropin and LH but not on the beta subunit of FSH. We have now identified an amino acid sequence motif, Pro-Leu-Arg, that is essential for recognition by the GalNAc-transferase. This tripeptide sequence is found 6-9 residues on the amino-terminal side of a glycosylated asparagine on the alpha subunit and beta subunits of LH and human chorionic gonadotropin but is not present on the beta subunit of FSH. The presence of this motif accounts for the differences in LH and FSH oligosaccharide structures. Additional proteins containing this recognition motif have been identified and were determined to bear sulfated oligosaccharides with the same structures as those on the glycoprotein hormones, indicating that these structures are not restricted to the glycoprotein hormones.
Human immunodeficiency virus (HIV) type 1 can superinfect HIV-2-infected cells: pseudotype virions produced with expanded cellular host range.
Authors: Le Guern M , Levy J A
Published: May 2006

In studies on viral interference, cloned T-cell lines chronically infected with human immunodeficiency virus (HIV) type 1 or HIV-2 were inoculated with several strains of these two AIDS retrovirus subtypes. HIV-2UC1-infected cells, which still express the CD4 receptor, could be superinfected with a variety of HIV-1 and HIV-2 strains. This event was accompanied by cytopathic effects in the cells and production of pseudotype virions with an expanded cellular host range. HIV-1- or HIV-2-infected clonal cell lines, which did not express CD4, could not be superinfected by any HIV strains but were coinfected after transfection of molecular clones into the persistently infected cells. These observations indicate that viral interference with HIV occurs at the cell surface and involves a down-modulation of the CD4 molecule. If the CD4 protein is expressed, superinfection can take place, and phenotypically mixed virus particles are produced. Since HIV-1 and HIV-2 dually infected individuals have been detected, these in vitro observations may have relevance to the in vivo state.
Calcium channel blockers inhibit retinal degeneration in the retinal-degeneration-B mutant of Drosophila.
Authors: Sahly I , Bar Nachum S , Suss-Toby E , Rom A , Peretz A , Kleiman J , Byk T , Selinger Z , Minke B
Published: May 2006

Light accelerates degeneration of photoreceptor cells of the retinal degeneration B (rdgB) mutant of Drosophila. During early stages of degeneration, light stimuli evoke spikes from photoreceptors of the mutant fly; no spikes can be recorded from photoreceptors of the wild-type fly. Production of spike potentials from mutant photoreceptors was blocked by diltiazem, verapamil hydrochloride, and cadmium. Little, if any, effect of the (-)-cis isomer or (+)-cis isomer of diltiazem on the light response was seen. Further, the (+)-cis isomer was approximately 50 times more effective than the (-)-cis isomer in blocking the Ca2+ spikes, indicating that diltiazem action on the rdgB eye is mediated by means of blocking voltage-sensitive Ca2+ channels, rather than by blocking the light-sensitive channels. Application of the Ca(2+)-channel blockers (+)-cis-diltiazem and verapamil hydrochloride to the eyes of rdgB flies over a 7-day period largely inhibited light-dependent degeneration of the photoreceptor cells. Pulse labeling with [32P]phosphate showed much greater incorporation into eye proteins of [32P]phosphate in rdgB flies than in wild-type flies. Retarding the light-induced photoreceptor degeneration in the mutant by Ca(2+)-channel blockers, thus, suggests that toxic increase in intracellular Ca2+ by means of voltage-gated Ca2+ channels, possibly secondary to excessive phosphorylation, leads to photoreceptor degeneration in the rdgB mutant.
Expression of human alpha 1-antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes.
Authors: Kay M A , Baley P , Rothenberg S , Leland F , Fleming L , Ponder K P , Liu T , Finegold M , Darlington G , Pokorny W
Published: May 2006

The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. We have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here we demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. At least 1 x 10(9) hepatocytes or 5% of the liver mass can be transplanted by the portal vasculature. In two animals we have transplanted hepatocytes transduced with a retroviral vector containing the human alpha 1-antitrypsin cDNA under transcriptional control of the cytomegalovirus promoter. Both animals had significant human alpha 1-antitrypsin in the serum for 1 month. Although the serum levels of human alpha 1-antitrypsin eventually fell due to inactivation of the cytomegalovirus promoter, PCR analysis demonstrated that a significant fraction of transduced hepatocytes migrated to the liver and continued to survive in vivo. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstitution with the use of promoters of cellular genes that are active in the normal liver.
The cardiac conduction system in the rat expresses the alpha 2 and alpha 3 isoforms of the Na+,K(+)-ATPase.
Authors: Zahler R , Brines M , Kashgarian M , Benz E J , Gilmore-Hebert M
Published: May 2006

The sodium pump is crucial for the function of the heart and of the cardiac conduction system, which initiates the heartbeat. The alpha (catalytic) subunit of this pump has three isoforms; the alpha 1 isoform is ubiquitous, but the alpha 2 and alpha 3 isoforms are localized to excitable tissue. Because rodent alpha 2 and alpha 3 isoforms are relatively sensitive to ouabain, which also slows cardiac conduction, we studied heart-cell-specific expression of pump isoform genes. Multiple conduction-system structures, including sinoatrial node, bundle branches, and Purkinje strands, had prominent, specific hybridization signal for alpha 2 and alpha 3 isoforms compared with adjacent working myocytes. This gene-expression approach may be useful for labeling conduction tissue and also for localizing specific membrane channels and receptors in this system.
The FixL protein of Rhizobium meliloti can be separated into a heme-binding oxygen-sensing domain and a functional C-terminal kinase domain.
Authors: Monson E K , Weinstein M , Ditta G S , Helinski D R
Published: May 2006

Transcription of nitrogen fixation (nif and fix) genes in Rhizobium meliloti is induced by a decrease in oxygen concentration. The products of two genes, fixL and fixJ, are responsible for sensing and transmitting the low-oxygen signal. The proteins encoded by fixL and fixJ (FixL and FixJ, respectively) are homologous to a family of bacterial proteins that transduce environmental signals through a common phosphotransfer mechanism [David, M., Daveran, M., Batut, J., Dedieu, A., Domergue, O., Ghai, J., Hertig, C., Boistard, P. & Khan, D. (1988) Cell 54, 671-683]. FixL, the oxygen sensor, is a membrane protein. It has previously been shown that a soluble derivative of FixL, FixL*, is an oxygen-binding hemoprotein and a kinase that autophosphorylates and also phosphorylates FixJ [Gilles-Gonzalez, M. A., Ditta, G. S. & Helinski, D. R. (1991) Nature (London) 350, 170-172]. In this work, deletion derivatives of fixL* were constructed and overexpressed in Escherichia coli, and the truncated proteins were purified. We show that a fragment of FixL from amino acid residue 127 to residue 260 binds heme, retains the ability to bind oxygen, and has no detectable kinase activity. A C-terminal fragment of FixL, beginning at residue 260, fails to bind heme but is active as a kinase. We also demonstrate that anaerobiosis results in an enhancement of FixL* autophosphorylation and FixJ phosphorylation activities in vitro. Finally, we show that the heme-binding region of FixL is required in vitro for oxygen regulation of its kinase activities.
Humanization of an anti-p185HER2 antibody for human cancer therapy.
Authors: Carter P , Presta L , Gorman C M , Ridgway J B , Henner D , Wong W L , Rowland A M , Kotts C , Carver M E , Shepard H M
Published: May 2006

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.
Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade.
Authors: Dudek S M , Bear M F
Published: May 2006

We tested a theoretical prediction that patterns of excitatory input activity that consistently fail to activate target neurons sufficiently to induce synaptic potentiation will instead cause a specific synaptic depression. To realize this situation experimentally, the Schaffer collateral projection to area CA1 in rat hippocampal slices was stimulated electrically at frequencies ranging from 0.5 to 50 Hz. Nine hundred pulses at 1-3 Hz consistently yielded a depression of the CA1 population excitatory postsynaptic potential that persisted without signs of recovery for greater than 1 hr after cessation of the conditioning stimulation. This long-term depression was specific to the conditioned input, ruling out generalized changes in postsynaptic responsiveness or excitability. Three lines of evidence suggest that this effect is accounted for by a modification of synaptic effectiveness rather than damage to or fatigue of the stimulated inputs. First, the effect was dependent on the stimulation frequency; 900 pulses at 10 Hz caused no lasting change, and at 50 Hz a synaptic potentiation was usually observed. Second, the depressed synapses continued to support long-term potentiation in response to a high-frequency tetanus. Third, the effects of conditioning stimulation could be prevented by application of NMDA receptor antagonists. Thus, our data suggest that synaptic depression can be triggered by prolonged NMDA receptor activation that is below the threshold for inducing synaptic potentiation. We propose that this mechanism is important for the modifications of hippocampal response properties that underlie some forms of learning and memory.
Long-term agonist exposure induces upregulation of beta 3-adrenergic receptor expression via multiple cAMP response elements.
Authors: Thomas R F , Holt B D , Schwinn D A , Liggett S B
Published: May 2006

During continuous stimulation by agonist, beta 1- and beta 2-adrenergic receptors (ARs) undergo processes that lead to decreases in receptor expression. This receptor down-regulation serves to limit the cellular cAMP response during chronic agonist exposure. In the recently described third subtype of the beta AR, denoted beta 3AR, we found four potential cAMP response elements in the 5' flanking region, suggesting that expression of this receptor might be positively regulated by agonists. These elements were cloned into the vector pA10CAT2, which contains a chloramphenicol acetyltransferase reporter gene, and transiently expressed in VERO cells. Three of these elements, TGACTCCA, TGAGGTCT, and CGAGGTCA (located 518, 622, and 1125 bases upstream of the beta 3AR coding block, respectively) were found to increase transcription of the chloramphenicol acetyltransferase gene in response to cAMP analogues and agents that increase intracellular cAMP. 3T3-F442A cells, when differentiated into the adipocyte phenotype by insulin, expressed beta 3AR, and nuclear runoff studies from such cells confirmed cAMP enhancement of beta 3AR mRNA transcription. In these cells, beta 3AR mRNA increased in response to exposure to the beta 3AR agonist isoproterenol and remained elevated during exposures of up to 24-30 hr. During prolonged exposure to agonist, no downregulation of beta 3AR expression in 3T3-F442A cells occurred. Indeed, beta 3AR expression increased during agonist exposure to approximately 165% of basal expression. In marked contrast, beta 1AR expression declined by approximately 70% in response to chronic agonist exposure. These studies reveal a subtype-specific prolonged transcriptional regulation of a beta AR gene by the end product of its signal transduction pathway. Thus, the beta 3AR undergoes a paradoxical increase in receptor expression during chronic agonist exposure.
Peptidyl-prolyl cis-trans isomerase improves the efficiency of protein disulfide isomerase as a catalyst of protein folding.
Authors: Schönbrunner E R , Schmid F X
Published: May 2006

The cis-trans isomerization of prolyl peptide bonds and the formation of disulfide bonds are both slow steps in protein folding. By using ribonuclease T1 as a model system, we show that these two processes can become linked in the oxidative folding of reduced proteins and that the formation of the correct disulfide bonds is facilitated in the presence of peptidyl-prolyl cis-trans isomerase. In particular, the efficiency of protein disulfide isomerase (EC 5.3.4.1) as a catalyst of disulfide bond formation in the course of oxidative folding is markedly improved when peptidyl-prolyl cis-trans isomerase is present simultaneously. Possibly, unfolded or partially folded protein chains with correct prolyl isomers are better substrates for catalysis by protein disulfide isomerase. The interdependence of the two enzymatic activities detected during in vitro folding experiments could be of importance for the de novo folding and disulfide bond formation of nascent proteins in the endoplasmic reticulum.
Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor.
Authors: Ignar-Trowbridge D M , Nelson K G , Bidwell M C , Curtis S W , Washburn T F , McLachlan J A , Korach K S
Published: May 2006

Epidermal growth factor (EGF) reproduces many of the effects of estrogen on the murine female reproductive tract and may partially mediate estrogen-induced growth and differentiation. This study was performed to investigate the mechanism by which EGF elicits estrogen-like actions in the whole animal. EGF was administered to adult ovariectomized mice by slow release pellets implanted under the kidney capsule. The induction of uterine DNA synthesis and phosphatidylinositol lipid turnover by EGF or administration of diethylstilbestrol (5 micrograms/kg), a potent estrogen, was attenuated by the estrogen receptor antagonist ICI 164,384. Furthermore, EGF mimicked the effects of estrogen on enhanced nuclear localization of the estrogen receptor and the formation of a unique form of the estrogen receptor found exclusively in the nucleus. These results suggest that EGF may induce effects similar to those of estrogen in the mouse uterus by an interaction between the EGF signaling pathway and the classical estrogen receptor. The demonstration of cross-talk between polypeptide growth factors and steroid hormone receptors may be of importance to our understanding of the regulation of normal growth and differentiation as well as the mechanisms of transmission of extracellular mitogen signals to the nucleus.
Purified unitary kainate/alpha-amino-3-hydroxy-5-methylisooxazole-propionate (AMPA) and kainate/AMPA/N-methyl-D-aspartate receptors with interchangeable subunits.
Authors: Henley J M , Ambrosini A , Rodriguez-Ithurralde D , Sudan H , Brackley P , Kerry C , Mellor I , Abutidze K , Usherwood P N , Barnard E A
Published: May 2006

We have purified and characterized two vertebrate excitatory amino acid ionotropic receptors from the Xenopus central nervous system. Each is a unitary receptor (i.e., having more than one class of excitatory amino acid agonist specificity within one protein oligomer). The first is a unitary non-N-methyl-D-aspartate (non-NMDA) receptor and the second is a unitary NMDA/non-NMDA receptor. The specific agonist-activated channel activity and pharmacology of each type were recognized by patch-clamping lipid bilayers in which the isolated protein was reconstituted. In the second case, the NMDA and the non-NMDA sites could not be physically separated and exhibited functional interaction. Parallel evidence for this was obtained when poly(A) RNA from Xenopus brain was translated in oocytes: a noncompetitive inhibition of the response to L-kainate is produced by NMDA to a maximum depression of 30% at 1 mM NMDA. Each isolated oligomer contains 42-kDa subunits of the non-NMDA ligand binding type, but the second type has an additional NMDA-receptor-specific 100-kDa subunit. Thus, a subunit-exchange hypothesis can account for the known multiplicity of excitatory amino acid receptor types.
pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions.
Authors: Schaller M D , Borgman C A , Cobb B S , Vines R R , Reynolds A B , Parsons J T
Published: May 2006

Expression of the Rous sarcoma virus-encoded oncoprotein, pp60v-src, subverts the normal regulation of cell growth, which results in oncogenic transformation. This process requires the intrinsic protein-tyrosine kinase activity of pp60v-src and is associated with an increase in tyrosine phosphorylation of a number of cellular proteins, candidate substrates for pp60v-src. We report here the isolation of a cDNA encoding a protein, pp125, that is a major phosphotyrosine-containing protein in untransformed chicken embryo cells and exhibits an increase in phosphotyrosine in pp60v-src-transformed chicken embryo cells. This cDNA encodes a cytoplasmic protein-tyrosine kinase which, based upon its predicted amino acid sequence and structure, is the prototype for an additional family of protein-tyrosine kinases. Immunofluorescence localization experiments show that pp125 is localized to focal adhesions; hence, we suggest the name focal adhesion kinase.
RecBCD enzyme is altered upon cutting DNA at a chi recombination hotspot.
Authors: Taylor A F , Smith G R
Published: May 2006

During its unidirectional unwinding of DNA, RecBCD enzyme cuts one DNA strand near a properly oriented Chi site, a hotspot of homologous genetic recombination in Escherichia coli. We report here that individual DNA molecules containing two properly oriented Chi sites were cut with about 40% efficiency at one or the other Chi site but not detectably at both Chi sites. Furthermore, initial incubation of RecBCD with Chi-containing DNA reduced its ability both to unwind DNA and to cut at Chi sites on subsequently added DNA molecules much more than did initial incubation with Chi-free DNA; the nuclease activity was less severely affected. These results imply that RecBCD loses its Chi-cutting activity upon cutting at a single Chi site and provide a mechanism for ensuring single genetic exchanges near the ends of DNA molecules.
Potent and selective inhibition of human immunodeficiency virus (HIV)-1 and HIV-2 replication by a class of bicyclams interacting with a viral uncoating event.
Authors: De Clercq E , Yamamoto N , Pauwels R , Baba M , Schols D , Nakashima H , Balzarini J , Debyser Z , Murrer B A , Schwartz D
Published: May 2006

A series of bicyclams have been shown to be potent and selective inhibitors of human immunodeficiency virus (HIV). The compounds are inhibitory to the replication of various HIV-1 and HIV-2 strains in various human T-cell systems, including peripheral blood lymphocytes, at 0.14-1.4 microM, without being toxic to the host cells at 2.2 mM. The bicyclam JM2763 is active against 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant HIV-1 strains and acts additively with AZT. Mechanism of action studies revealed that the bicyclams (i.e., JM2763) interact with an early event of the retrovirus replicative cycle, which could be tentatively identified as a viral uncoating event.
Laser desorption studies of high mass biomolecules in Fourier-transform ion cyclotron resonance mass spectrometry.
Authors: Solouki T , Russell D H
Published: May 2006

Matrix-assisted laser desorption ionization is used to obtain Fourier-transform ion cyclotron resonance mass spectra of model peptides (e.g., gramicidin S, angiotensin I, renin substrate, melittin, and bovine insulin). Matrix-assisted laser desorption ionization yields ions having appreciable kinetic energies. Two methods for trapping the high kinetic energy ions are described: (i) the ion signal for [M+H]+ ions is shown to increase with increasing trapping voltages, and (ii) collisional relaxation is used for the detection of [M+H]+ ions of bovine insulin.
Transient immunoglobulin M antibody response to hepatitis C virus capsid antigen in posttransfusion hepatitis C: putative serological marker for acute viral infection.
Authors: Chen P J , Wang J T , Hwang L H , Yang Y H , Hsieh C L , Kao J H , Sheu J C , Lai M Y , Wang T H , Chen D S
Published: May 2006

The development of serological assays for hepatitis C virus (HCV) has made specific diagnosis possible. However, markers useful in indicating acute-phase HCV infection have not been identified. By an immunoblotting method, we characterized the IgM and IgG antibody response against HCV capsid antigen in patients with HCV infection. Among 88% of patients with acute posttransfusion hepatitis C recruited in a prospective study, there was a transient IgM antibody response. The IgM antibody appeared shortly after onset of hepatitis (average 3.7 weeks), persisted for several months (average 18 weeks), and then disappeared. In contrast, the IgG antibody persisted long-term once it appeared. Among patients with chronic hepatitis C with milder disease activities (serum aminotransferase increase above normal levels of less than 4-fold), the IgM antibody was negative in the majority (72%). In those with acute exacerbations (aminotransferase increase of greater than 10-fold), about 55% were negative for the IgM antibody. The reactivity of the IgM antibody in the rest was weaker or became negative upon further dilution of serum. The results suggest that IgM anti-capsid antibody may serve as a marker indicating acute or active HCV infection.
Beta 2-microglobulin-, CD8+ T-cell-deficient mice survive inoculation with high doses of vaccinia virus and exhibit altered IgG responses.
Authors: Spriggs M K , Koller B H , Sato T , Morrissey P J , Fanslow W C , Smithies O , Voice R F , Widmer M B , Maliszewski C R
Published: May 2006

Transgenic mice lacking an intact beta 2-microglobulin (beta 2m) gene fail to express major histocompatibility complex (MHC) class I proteins on the cell surface and, as a result, are virtually devoid of CD4- CD8+ lymphocytes. These animals provide a unique model system for directly assessing the role of CD8+ lymphocytes in the modulation of viral infection in vivo. beta 2m- CD8- mice and their normal littermates were inoculated at the base of the tail with the WR strain of vaccinia virus and monitored for serum antibody and lesion formation. Both groups developed similar lesions in response to a broad virus dose range, and all animals had completely recovered by day 28 after inoculation. Isotype-specific immunoglobulin levels were determined for each animal on day 7 and day 14 after primary inoculation, and again 7 days after a virus challenge. The virus-specific IgG1, IgG2a, and IgG2b levels were significantly different in the beta 2m-/- group (20-, 9-, and 30-fold lower, respectively, on day 7 after challenge) compared with the beta 2m+/- group. Virus-specific serum IgM levels for both groups remained similar throughout the experiment. In a separate experiment, beta 2m-/- mice were immunized with a nonviral antigen, 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin, and both total and antigen-specific isotype-specific immunoglobulin titers were determined. Total IgG1, IgG2a, IgG2b, and IgG3 tended to be lower overall in the beta 2m-/- mice compared with beta 2m+/- littermates. In contrast, total and antigen-specific IgE titers were similar in the two groups. These data indicate that CD8+ lymphocytes are not required to clear high doses of vaccinia virus, and they suggest that beta 2m-/- mice are less efficient at antigen-specific IgG production than their beta 2m+/- littermates.
Crystal structure of rat liver dihydropteridine reductase.
Authors: Varughese K I , Skinner M M , Whiteley J M , Matthews D A , Xuong N H
Published: May 2006

The structure of a binary complex of dihydropteridine reductase [DHPR; NAD(P)H:6,7-dihydropteridine oxidoreductase, EC 1.6.99.7] with its cofactor, NADH, has been solved and refined to a final R factor of 15.4% by using 2.3 A diffraction data. DHPR is an alpha/beta protein with a Rossmann-type dinucleotide fold for NADH binding. Insertion of an extra threonine residue in the human enzyme is associated with severe symptoms of a variant form of phenylketonuria and maps to a tightly linked sequence of secondary-structural elements near the dimer interface. Dimerization is mediated by a four-helix bundle motif (two helices from each protomer) having an unusual right-handed twist. DHPR is structurally and mechanistically distinct from dihydrofolate reductase, appearing to more closely resemble certain nicotinamide dinucleotide-requiring flavin-dependent enzymes, such as glutathione reductase.
Genetic, cytogenetic, and molecular analyses of mutations induced by melphalan demonstrate high frequencies of heritable deletions and other rearrangements from exposure of postspermatogonial stages of the mouse.
Authors: Russell L B , Hunsicker P R , Cacheiro N L , Rinchik E M
Published: May 2006

Specific-locus experiments have previously shown melphalan to be mutagenic in all male germ-cell stages tested and particularly so in early spermatids. All but 2 of 24 specific-locus mutations recovered were tested genetically, cytogenetically, and/or molecularly. At least 12 of 15 tested mutations recovered from postspermatogonial stages but only 1 of 7 mutations recovered from stem-cell or differentiating spermatogonia gave evidence of being deletions or other rearrangements. Melphalan-induced mutations, thus, confirm the pattern of dependence of mutation structure on germ-cell stage that had been shown earlier for other chemicals. Results of the present investigation illustrate the capabilities of combined genetic, cytogenetic, and molecular analyses for characterizing the nature of specific-locus mutations. Fine-structure molecular mapping of long regions surrounding specific loci has been greatly facilitated by the availability of genetic reagents (particularly, deletion complexes) generated in specific-locus experiments over the course of decades. Reciprocally, this mapping permits increasingly detailed characterization of the nature of lesions induced by mutagenic exposures of germ cells, adding great powers for qualitative analysis of mutations to the specific-locus test. Cytogenetic and genetic investigations also provide evidence on lesion type, especially for loci at which mutations cannot yet be analyzed molecularly. Melphalan, like chlorambucil, can generate many mutations, a high proportion of which are deletions and other rearrangements, making this chemical valuable for generating mutations (at any locus) amenable to molecular access.
CD5 is phosphorylated on tyrosine after stimulation of the T-cell antigen receptor complex.
Authors: Davies A A , Ley S C , Crumpton M J
Published: May 2006

When T cells are activated by the T-cell antigen receptor, a number of cellular proteins are phosphorylated on tyrosine. We investigated whether any of these proteins were present on the surface of activated T cells. Using the human leukemic T-cell line Jurkat and normal peripheral blood lymphocytes, we identified a 67-kDa cell surface glycoprotein in anti-phosphotyrosine immunoprecipitates, after treatment of the cells with CD3 antibody. When cell lysates were depleted of CD5 by sequential immunoprecipitation, the 67-kDa phosphotyrosyl polypeptide was no longer precipitated by the phosphotyrosine antibody. Western blot analysis of anti-phosphotyrosine precipitates confirmed that this glycoprotein was CD5. It was possible that CD5 was present in the anti-phosphotyrosine immunoprecipitates due to its physical association with phosphotyrosyl proteins rather than being directly tyrosine-phosphorylated itself. However, Western blot analysis of anti-CD5 immunoprecipitates with phosphotyrosine antibody and phosphoamino acid analysis demonstrated that CD5 was indeed phosphorylated on tyrosine after stimulation of the cells with CD3 antibody and was concomitantly phosphorylated on serine and threonine. Tyrosine phosphorylation of CD5 was maximal 2 min after CD3 stimulation and returned to baseline levels by 60 min. CD5 is expressed on the cell surface of all mature T cells and a small proportion of B lymphocytes and has recently been identified as the ligand for CD72, a receptor present on the surface of all B cells. The present data suggest that tyrosine phosphorylation may be involved in B-cell-T-cell communication.
A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells.
Authors: Noelle R J , Roy M , Shepherd D M , Stamenkovic I , Ledbetter J A , Aruffo A
Published: May 2006

CD40 is a B-cell surface molecule that has been shown to induce B-cell growth upon ligation with monoclonal antibodies. This report shows that triggering via CD40 is essential for the activation of resting B cells by helper T cells (Th). A soluble fusion protein of CD40 and human immunoglobulin, CD40-Ig, inhibited the induction of B-cell cycle entry, proliferation, and differentiation by activated Th1 and Th2. The ligand for CD40 was identified as a 39-kDa membrane protein that was selectively expressed on activated Th. A monoclonal antibody specific for the 39-kDa protein inhibited CD40-Ig binding and also inhibited the activation of B cells by Th. These data indicate that the 39-kDa membrane protein expressed on activated Th is a binding protein for CD40 and functions to transduce the signal for Th-dependent B-cell activation.
Effects of compact volume and chain stiffness on the conformations of native proteins.
Authors: Hao M H , Rackovsky S , Liwo A , Pincus M R , Scheraga H A
Published: May 2006

An investigation of the statistical properties of the native conformations of proteins, observed from crystal structures, is reported. Protein conformations were analyzed in terms of a bond vector correlation function and molecular volume. It was observed that, while the volume of a protein structure varies nearly linearly with the number of residues, the bond vector correlation function exhibits a universal feature for all sizes of proteins. To interpret the nature of the bond vector correlation function of native protein structures quantitatively, Monte Carlo simulations of realistic polypeptide chains of specific but arbitrary amino acid sequence were carried out. The molecule was constrained in an ellipsoidal volume determined by its chain length, and conformations with unacceptable nonbonded contacts between different amino acid residues were excluded. The interactions within a terminally blocked single residue, which correlate two nearest-neighbor peptide groups in a chain, were taken into account by an energetically biased sampling of its phi-psi space. The simulated chain correlation functions were found to be in good agreement with those of the crystal structures of beta-sheet-type and mixed-type (alpha+beta) proteins of similar length. On the basis of these calculations, it is concluded that the observed conformations of these native proteins may arise from two basic factors: the compactness of structures under hydrophobic interactions and the intrinsic stiffness of polypeptide chains due to the interactions within each terminally blocked residue.
Drug binding to higher ordered DNA structures: netropsin complexation with a nucleic acid triple helix.
Authors: Park Y W , Breslauer K J
Published: May 2006

We have used a combination of spectroscopic and calorimetric techniques to characterize how netropsin, a ligand that binds in the minor groove of DNA, influences the properties of a DNA triple helix. Specifically, our data allow us to reach the following conclusions: (i) netropsin binds to the triplex without displacing the major-groove-bound third strand; (ii) netropsin binding to the triplex exhibits a lower saturation binding density (7.0 base triplets per netropsin bound) than netropsin binding to the corresponding duplex (5.5 base pairs per netropsin bound); (iii) the netropsin-free and the netropsin-bound triplexes each melt in two well-resolved transitions, initial conversion of the triplex to the duplex state followed by duplex melting to the component single-stranded states; (iv) netropsin remains bound to DNA as the triplex melts to the duplex state; (v) netropsin binding thermally destabilizes the triplex in equilibrium with duplex equilibrium dramatically, while thermally stabilizing the duplex to single-strand equilibrium; (vi) netropsin binding to the triplex is enthalpically 4 times more favorable (more exothermic) than netropsin binding to the corresponding duplex; (vii) netropsin binding to the triplex decreases the cooperativity of the triplex----duplex melting event. These results demonstrate that occupancy of the minor groove of a triplex by a ligand such as netropsin can exert a profound impact on the properties of the host triplex, particularly with regard to the equilibrium in which the third strand is expelled from the major groove. Thus, our results reveal considerable major groove/minor groove crosstalk. Such knowledge may prove of practical importance by providing an approach for modulating the affinity and specificity of major-groove-binding third strands in triplex-forming protocols designed to target specific duplex domains. Fundamentally, our results provide insights into the crosstalk that can result when ligands bind to the two major receptor sites of duplex DNA--namely, the major and minor grooves.
Neocarzinostatin acts as a sensitive probe of DNA microheterogeneity: switching of chemistry from C-1' to C-4' by a G.T mismatch 5' to the site of DNA damage.
Authors: Kappen L S , Goldberg I H
Published: May 2006

The diradical form of thiol-activated neocarzinostatin chromophore resides in the minor groove of DNA, where it has access to hydrogen atoms at the C-5', C-1', and C-4' positions of deoxyribose on each strand. In a dodecamer oligodeoxyribonucleotide containing the sequence AGC.GCT, a bistranded lesion staggered two nucleotides in the 3' direction, is generated that consists primarily of an abasic site (2'-deoxyribonolactone) at the C due to 1' chemistry and a direct strand break at the T due to 5' chemistry. Sequencing-gel analysis reveals that 72% of the damage at the C results from 1' chemistry with minor lesions consisting of a strand break due to 5' chemistry (15%) and 4' chemistry (less than 2%) and an abasic site (4'-hydroxylation product) (12%) due to 4' chemistry. Replacement of the G.C base pair 5' to the C by a G.T wobble mismatch results in a remarkable switching of the chemistry of damage at the C from C-1' to C-4'. The 1' chemistry is almost eliminated and replaced by 4' chemistry, so that the latter accounts for 64% of the damage, mainly in the form of the 4'-hydroxylation product (abasic site) and a smaller amount of the DNA fragment with a phosphoglycolate at the 3' end (strand break). Substitution of the radiation sensitizer misonidazole for dioxygen markedly enhances partitioning of the 4' chemistry in favor of the glycolate-containing product. On the complementary strand the G.T mismatch results in an increase in 4' chemistry at the T residue, but 5' chemistry remains the main mechanism. When a G.A mismatch is inserted 5' to the C, there is a marked decrease in all damage at this site without detectable switching of chemistry. These results show that the diradical form of thiol-activated neocarzinostatin chromophore acts as sensitive probe of DNA microheterogeneity.
Cloning and expression of a cAMP-activated Na+/H+ exchanger: evidence that the cytoplasmic domain mediates hormonal regulation.
Authors: Borgese F , Sardet C , Cappadoro M , Pouyssegur J , Motais R
Published: May 2006

The ubiquitous plasma membrane Na+/H+ exchanger (termed NHE1) is activated by diverse hormonal signals, with the notable exception of hormones acting through cAMP as second messenger. Therefore, the Na+/H+ exchanger found in the nucleated trout red cell is of particular interest since it is activated by catecholamines, forskolin, and cAMP analogues. We report here that a cloned cDNA encoding the red cell exchanger restores functional Na+/H+ activity when transfected into Na+/H+ antiporter-deficient fibroblasts (i.e., it regulates intracellular pH in a Na-dependent and amiloride-sensitive manner). This red cell exchanger represents an additional form of Na+/H+ exchanger (termed beta NHE), which is characterized by a specific cytoplasmic domain involved in activation by the cAMP-dependent signaling pathway. After transfection in the same cellular context, beta NHE, but not NHE1, is activated by cAMP or by hormones that increase cAMP levels. Comparison of the amino acid sequences of exchangers shows that beta NHE, but not NHE1, contains two clustered consensus motifs for phosphorylation by a cAMP-dependent protein kinase (protein kinase A; PKA). A deletion mutant devoid of the C-terminal region of the cytoplasmic loop containing the two PKA sites restores Na+/H+ activity but is no longer activated by cAMP analogues or catecholamines. In red blood cells, the Na+/H+ exchanger is also activated by another pathway involving protein kinase C (PKC). Expression of beta NHE in fibroblasts shows that these two independent signaling pathways impinge on two distinct domains of the exchanger. The cytoplasmic segment containing PKA consensus sites, which is crucial for cAMP activation, is unnecessary for stimulation by PKC activators.