bluuebunny/crossref_metadata_2025 huggingface dataset:
Two genetically identical antigen-presenting cell clones display heterogeneity in antigen processing.
Authors: Michalek M T , Benacerraf B , Rock K L
Published: May 2006

Evidence from various antigen systems suggests that antigen processing can be one factor that determines the repertoire of immunogenic peptides. Thus, processing events may account for some of the disparity between the available and expressed helper T-cell repertoires. In this report, we demonstrate that the immunodominant T-cell determinant in ovalbumin [p323-339; ovalbumin-(323-339) heptadecapeptide] is processed differently by two genetically identical antigen-presenting cell lines, M12 and A20. The ovalbumin-specific T-cell-T-cell hybridomas, DO-11.10 and 3DO-54.8, were used to detect processed antigen. These T-T hybridomas have different fine specificities for the p323-339 determinant. A20 cells presented native ovalbumin well to both T-T hybridomas, whereas M12 cells presented native ovalbumin well to 3DO-54.8 but very inefficiently to DO-11.10. M12 and A20 cells effectively stimulated both T-T hybridomas with the same concentrations of the immunogenic synthetic peptide p323-339. Therefore, M12 cells and DO-11.10 can interact with each other, and both T-T hybridomas have similar sensitivities for the same immunogenic peptide. We conclude that genetically identical antigen-presenting cells can display heterogeneity in the fine processing of an immunodominant T-cell determinant, and synthetic model peptides that represent the minimal stimulatory sequence of a T-cell determinant are not necessarily identical to the structure of in vivo processed antigen. Heterogeneity in antigen processing by individual antigen-presenting cells would serve to increase the repertoire of immunogenic peptides that are presented to T cells.
Reversible sodium pump defect and swelling in the diabetic rat erythrocyte: effects on filterability and implications for microangiopathy.
Authors: Kowluru R , Bitensky M W , Kowluru A , Dembo M , Keaton P A , Buican T
Published: May 2006

We have found a defect in the ouabain-sensitive Na+, K+-ATPase (Na+ pump, EC 3.6.1.37) of erythrocytes from streptozocin diabetic rats. This defect was accompanied by an increase in cell volume and osmotic fragility and a decrease in the cytosolic K+/Na+ ratio. There was also a doubling in the time needed for diabetic erythrocytes to pass through 4.7-micron channels in a polycarbonate filter. Our data are consistent with a primary defect in the erythrocyte Na+ pump and secondary changes in cell volume, osmotic fragility, K+/Na+ ratio, and cell filterability. All were reversed or prevented in vivo by insulin or the aldose reductase inhibitor Sorbinil. Protein kinase C agonists (phorbol ester and diacylglycerol) and agonist precursor (myoinositol) reversed the Na+ pump lesion, suggesting that protein kinase C-dependent phosphorylation of the 100-kDa subunit regulates Na+ pump activity and that insulin can influence erythrocyte protein kinase C activity. Ouabain inhibition of the erythrocyte Na+ pump also produced increases in cell size and reductions in rates of filtration. Theoretical treatment of the volume changes also predicts reduction in filterability as a consequence of cell swelling. We suggest that enlarged erythrocytes could play a role in the evolution of the microvascular changes of diabetes mellitus.
Spectroscopic studies of wild-type and mutant "zinc finger" peptides: determinants of domain folding and structure.
Authors: Párraga G , Horvath S , Hood L , Young E T , Klevit R E
Published: May 2006

The "zinc finger" model [Miller, J., McLachlan, A. D. & Klug, A. (1985) EMBO J. 4, 1609-1614; Brown, R. S., Sander, C. & Argos, P. (1985) FEBS Lett. 186, 271-274] makes both specific structural and specific functional predictions about zinc finger consensus sequences that can be tested with a combination of genetic, molecular biological, and biophysical techniques. The yeast transcription factor ADR1 contains two adjacent zinc finger domains; genetic and deletion analyses showed that amino acid substitutions and deletions in the zinc finger domains resulted in the loss of protein activity. To test the structural and folding predictions of the zinc finger model, peptides encompassing each of the ADR1 fingers were synthesized (ADR1a and ADR1b) as well as a mutant finger peptide (del138) deleted for a single amino acid residue. The folding and metal-binding characteristics of these were assessed by 1H nuclear magnetic resonance (NMR) and visible spectroscopy. While a single unique conformational species was detected for the two wild-type peptides upon tetrahedral binding of zinc, the deletion peptide did not bind zinc with tetrahedral geometry, nor did it fold into a zinc finger domain. The metal-binding and folding results found with the mutant peptide were similar to those obtained when thiol alkylation or imidazole protonation of the wild-type peptides was performed. These data indicate that ligand spacing and both thiol and imidazole participation in zinc binding are specific and necessary requirements for zinc finger folding, which provides direct support for the initial predictions of the model.
Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes.
Authors: Kagan B L , Selsted M E , Ganz T , Lehrer R I
Published: May 2006

Defensins are cationic, cysteine-rich peptides (Mr = 3500-4000) found in the cytoplasmic granules of neutrophils and macrophages. These peptides possess broad antimicrobial activity in vitro against bacteria, fungi, tumor cells, and enveloped viruses, and they are believed to contribute to the "oxygen-independent" antimicrobial defenses of neutrophils and macrophages. Pathophysiologic studies in vitro have pointed to the plasma membrane as a possible target for the cytotoxic action of defensins. We report here that defensins form voltage-dependent, weakly anion-selective channels in planar lipid bilayer membranes, and we suggest that this channel-forming ability contributes to their antimicrobial properties observed in vitro.
Femtosecond dynamics of energy transfer in B800-850 light-harvesting complexes of Rhodobacter sphaeroides.
Authors: Trautman J K , Shreve A P , Violette C A , Frank H A , Owens T G , Albrecht A C
Published: May 2006

We report femtosecond transient absorption studies of energy transfer dynamics in the B800-850 light-harvesting complex (LHC) of Rhodobacter sphaeroides 2.4.1. For complexes solubilized in lauryldimethylamine-N-oxide (LDAO), the carotenoid to bacteriochlorophyll (Bchl) B800 and carotenoid to Bchl B850 energy transfer times are 0.34 and 0.20 ps, respectively. The B800 to B850 energy transfer time is 2.5 ps. For complexes treated with lithium dodecyl sulfate (LDS), a carotenoid to B850 energy transfer time of less than or equal to 0.2 ps is seen, and a portion of the total carotenoid population is decoupled from Bchl. In both LDAO-solubilized and LDS-treated complexes an intensity-dependent picosecond decay component of the excited B850 population is ascribed to excitation annihilation within minimal units of the LHC.
Albumin Redhill (-1 Arg, 320 Ala----Thr): a glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site.
Authors: Brennan S O , Myles T , Peach R J , Donaldson D , George P M
Published: May 2006

Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind 63Ni2+ and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni2+ was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation (since proalbumin contains adjacent Arg residues at -1 and -2) or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala----Thr. This introduces an Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg----Cys. This introduces a prepro sequence, Met-Lys-Trp-Val-Thr-Phe-Ile-Ser-Leu-Leu-Phe-Leu-Phe-Ser-Ser-Ala-Tyr- Ser-Arg-Gly-Val-Phe-Cys-Arg (cf.-Tyr-Ser-Arg-Gly-Val-Phe-Arg-Arg- in normal human pre-proalbumin). We propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase.
Inhibition by Ca2+ of inositol trisphosphate-mediated Ca2+ liberation: a possible mechanism for oscillatory release of Ca2+.
Authors: Parker I , Ivorra I
Published: May 2006

Light-flash photolysis of caged inositol 1,4,5-trisphosphate (InsP3) was used to generate reproducible transients of free InsP3 in Xenopus oocytes, and the resulting liberation of Ca2+ from intracellular stores was monitored by recording Ca2+-activated membrane currents and by use of the fluorescent Ca2+ indicator fluo-3. InsP3-mediated Ca2+ release was inhibited by elevating the intracellular free Ca2+ level, either by microinjecting Ca2+ into the cell or by applying conditioning light flashes to liberate Ca2+. This inhibition followed a slow time course, being maximal after about 2 s and subsequently declining over several seconds. Negative feedback of Ca2+ ions on InsP3-mediated Ca2+ liberation may explain the oscillatory release of Ca2+ seen during activation of inositol phospholipid signaling in the oocyte, and the time course of the inhibition is consistent with the period of the oscillations.
Enantiomers of HA-966 (3-amino-1-hydroxypyrrolid-2-one) exhibit distinct central nervous system effects: (+)-HA-966 is a selective glycine/N-methyl-D-aspartate receptor antagonist, but (-)-HA-966 is a potent gamma-butyrolactone-like sedative.
Authors: Singh L , Donald A E , Foster A C , Hutson P H , Iversen L L , Iversen S D , Kemp J A , Leeson P D , Marshall G R , Oles R J
Published: May 2006

The antagonist effect of (+/-)-3-amino-1-hydroxypyrrolid-2-one (HA-966) at the N-methyl-D-aspartate (NMDA) receptor occurs through a selective interaction with the glycine modulatory site within the receptor complex. When the enantiomers of (+/-)-HA-966 were resolved, the (R)-(+)-enantiomer was found to be a selective glycine/NMDA receptor antagonist, a property that accounts for its anticonvulsant activity in vivo. In contrast, the (S)-(-)-enantiomer was only weakly active as an NMDA-receptor antagonist, but nevertheless it possessed a marked sedative and muscle relaxant action in vivo. In radioligand binding experiments, (+)-HA-966 inhibited strychnine-insensitive [3H]glycine binding to rat cerebral cortex synaptic membranes with an IC50 of 12.5 microM, whereas (-)-HA-966 had an IC50 value of 339 microM. In electrophysiological experiments, (+)-HA-966 selectively antagonized NMDA receptor responses in rat cortical slices, whereas the (-)-enantiomer was much weaker. On cultured cortical neurones (+)-HA-966 inhibited glycine-potentiated NMDA responses with an IC50 = 13 microM compared with (-)-HA-966, which has an IC50 = 708 microM. In agreement with findings with racemic HA-966, even high concentrations of (+)-HA-966 did not completely inhibit NMDA responses, suggesting that (+)-HA-966 is a low-efficacy partial agonist. (+)-HA-966 produced parallel shifts to the right of the glycine concentration curve for potentiation of NMDA responses, resulting in an estimated pKb = 5.6. In mice, (+)-HA-966 antagonized sound and N-methyl-DL-aspartic acid (NMDLA)-induced seizures with ED50 values of 52.6 mg/kg of body weight (i.p.) and 900 mg/kg (i.v.), respectively. The coadministration of D-serine dose-dependently (10-100 micrograms into the cerebral ventricles per mouse) antagonized the anticonvulsant effect of a submaximal dose of (+)-HA-966 (100 micrograms administered directly into the cerebral ventricles) against NMDLA-induced seizures. The sedative/ataxic effect of racemic HA-966 was mainly attributable to the (-)-enantiomer, which was greater than 25-fold more potent than the (+)-enantiomer. It is suggested that, as in the case of the sedative gamma-butyrolactone, disruption of striatal dopaminergic mechanisms may be responsible for this action.
DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3.
Authors: Demay M B , Gerardi J M , DeLuca H F , Kronenberg H M
Published: May 2006

The 5' flanking region of the rat osteocalcin gene has been shown to confer responsiveness to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after transfection of fusion genes into ROS 17/2.8 cells. Deletion analysis has demonstrated that there are at least two domains in this 5' flanking region that contribute to 1,25(OH)2D3 responsiveness; however, only the downstream region is able to confer 1,25(OH)2D3 responsiveness to either the native osteocalcin promoter or to a heterologous viral promoter (herpes simplex virus thymidine kinase). The proximal region responsible for 1,25(OH)2D3 induction of the rat osteocalcin gene lies 458 base pairs upstream from the transcription start site of this gene. A 25-base-pair oligonucleotide corresponding to the sequences in this region is able to confer 1,25(OH)2D3 responsiveness to the thymidine kinase promoter in an orientation-independent fashion. This sequence contains three copies of a short sequence that are homologous to "half-sites" of steroid response elements. Gel-retardation assays using porcine intestinal nuclear extract as a rich source of 1,25(OH)2D3 receptor demonstrated retardation in the migration of probes containing the sequence noted above. A monoclonal antibody directed against the 1,25(OH)2D3 receptor caused further retardation in the migration of these protein-DNA complexes. Therefore, the sequences represented in this oligonucleotide encompass the sequences necessary for binding of the 1,25(OH)2D3 receptor to DNA as well as those sequences necessary for 1,25(OH)2D3 to induce osteocalcin gene transcription.
Manganese-dependent disproportionation of hydrogen peroxide in bicarbonate buffer.
Authors: Stadtman E R , Berlett B S , Chock P B
Published: May 2006

At physiological concentrations of HCO3- and CO2, Mn(II) catalyzes disproportionation of H2O2. This catalase-like activity is directly proportional to the concentrations of Mn(II) and H2O2, and it increases exponentially with increases in pH. The effect of increasing pH is almost completely attributable to the concomitant increase in HCO3- concentration. The rate is proportional to the third power of the HCO3- concentration, suggesting that 3 equivalents of HCO3- combine with 1 equivalent of Mn(II) to form the catalytic complex. It is presumed that the redox potential of the Mn(II) in equilibrium with Mn(III) couple in such a complex permits H2O2 to carry out facile reactions with Mn(II) comparable to those that occur with Fe(III) and Cu(II) chelate complexes, in which OH. and O2-. are established intermediates. The Mn-catalyzed disproportionation of H2O2 does not occur at physiological pH in the absence of HCO3-. Hepes, inorganic phosphate, and inorganic pyrophosphate inhibit the reaction catalyzed by the Mn/HCO3- system. These results are similar to those of Sychev et al. [Sychev, A.Y., Pfannmeller, U. & Isak, V.G. (1983) Russ. J. Phys. Chem. 57, 1690-1693]. The catalase-like activity of Mn(II)-bicarbonate complexes reported here, together with the superoxide dismutase activity of Mn complexes demonstrated by Archibald and Fridovich [Archibald, F.S. & Fridovich, I. (1982) Arch. Biochem. Biophys. 214, 452-463], strengthen the proposition that Mn may play an important role in the protection of cells against oxygen radical-mediated damage.
Regulation of surface expression of the granulocyte/macrophage colony-stimulating factor receptor in normal human myeloid cells.
Authors: Cannistra S A , Groshek P , Garlick R , Miller J , Griffin J D
Published: May 2006

Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) exerts stimulatory effects on hematopoietic cells through binding to specific, high-affinity receptors (Kd = 30-100 pM). By using radiolabeled GM-CSF with high specific activity, we have investigated the factors and mechanisms that regulate GM-CSF receptor expression in normal human neutrophils, monocytes, and partially purified bone marrow myeloid progenitor cells. The neutrophil GM-CSF receptor was found to rapidly internalize in the presence of ligand through a mechanism that required endocytosis. Out of a large panel of naturally occurring humoral factors tested, only GM-CSF itself, tumor necrosis factor, and formyl-Met-Leu-Phe were found to down-regulate neutrophil GM-CSF receptor expression after a 2-hr exposure at biologically active concentrations (95% +/- 1%, 34% +/- 5%, 48% +/- 8% receptor down-regulation, respectively). GM-CSF also down-regulated its own receptor on monocytes and myeloid progenitor cells. Since formyl-Met-Leu-Phe is known to stimulate neutrophil protein kinase C activity, we also tested the ability of protein kinase C agonists to modulate GM-CSF receptor expression. Phorbol 12-myristate 13-acetate, bryostatin-1, and 1,2-dioctanoylglycerol were found to induce rapid down-regulation of the GM-CSF receptor in neutrophils, monocytes, and partially purified myeloid progenitor cells, suggesting that this effect may be at least partially mediated by protein kinase C. These data suggest that certain activators of neutrophil function may negatively regulate their biological effects by inducing down-regulation of the GM-CSF receptor.
Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco.
Authors: Payne G , Ahl P , Moyer M , Harper A , Beck J , Meins F , Ryals J
Published: May 2006

Complementary DNA clones encoding two isoforms of the acidic endochitinase (chitinase, EC 3.2.1.14) from tobacco were isolated. Comparison of amino acid sequences deduced from the cDNA clones and the sequence of peptides derived from purified proteins show that these clones encode the pathogenesis-related proteins PR-P and PR-Q. The cDNA inserts were not homologous to either the bacterial form of chitinase or the form from cucumber but shared significant homology to the basic form of chitinase from tobacco and bean. The acidic isoforms of tobacco chitinase did not contain the amino-terminal, cysteine-rich "hevein" domain found in the basic isoforms, indicating that this domain, which binds chitin, is not essential for chitinolytic activity. The accumulation of mRNA for the pathogenesis-related proteins PR-1, PR-R, PR-P, and PR-Q in Xanthi.nc tobacco leaves following infection with tobacco mosaic virus was measured by primer extension. The results indicate that the induction of these proteins during the local necrotic lesion response to the virus is coordinated at the mRNA level.
Activation of ara operons by a truncated AraC protein does not require inducer.
Authors: Menon K P , Lee N L
Published: May 2006

The araC gene of Escherichia coli encodes a protein that binds the inducer L-arabinose to activate the transcription of three ara operons. In a study to determine the functional domains within the AraC protein, we have generated a set of overlapping deletions from the proximal end of the araC gene. We found that the removal of up to nearly 60% of the coding sequence of this protein still allows transcriptional activation of the ara operons in vivo, up to 27% that of the wild type. These truncated proteins, however, no longer require arabinose for induction. The ligand-induced conformational change apparently either releases or unmasks an existing functional domain within AraC, rather than generating a new conformation that is required for activation of the promoter of araBAD. Since the truncated protein of the mutant C154 (which lacks 153 amino acid residues from the N terminus) retains DNA binding specificity, the DNA-recognition domain is localized in the C-terminal half of the AraC protein. Truncated proteins were unable to repress araBAD or araC in vivo, even though they were able to bind all ara operators. We propose that the N-terminal half of AraC is essential for the formation of the DNA loops that are responsible for repression of araBAD and for autoregulation of araC.
Corn metabolites affect growth and virulence of Agrobacterium tumefaciens.
Authors: Sahi S V , Chilton M D , Chilton W S
Published: May 2006

Homogenates of corn seedlings inhibit both growth of Agrobacterium tumefaciens and induction of its Ti plasmid virulence (vir) genes by acetosyringone (AS). The heat-labile inhibitor has been identified as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), present in 2-week-old seedlings (B73) at a concentration of 1.5 mM or greater. A concentration of 0.3 mM DIMBOA is sufficient to block growth of A. tumefaciens completely for 220 hr. DIMBOA at 0.1 mM concentration completely inhibited vir gene induction by 100 microM AS and reduced growth rate by 50%. Thus, DIMBOA can be expected to have a significant effect on attempts to transform corn by using A. tumefaciens as a vector.
Tandem duplication within a type II collagen gene (COL2A1) exon in an individual with spondyloepiphyseal dysplasia.
Authors: Tiller G E , Rimoin D L , Murray L W , Cohn D H
Published: May 2006

We have characterized a mutation in the type II collagen gene (COL2A1) that produces a form of spondyloepiphyseal dysplasia. The mutation is an internal tandem duplication of 45 base pairs within exon 48 and results in the addition of 15 amino acids to the triple-helical domain of the alpha 1 chains of type II collagen derived from the abnormal allele. Although the repeating (Gly-Xaa-Yaa)n motif that characterizes the triple-helical domain is preserved, type II collagen derived from cartilage of the affected individual contains a population with excessive posttranslational modification, consistent with a disruption in triple-helix structure. The mutation is not carried by either parent, indicating that the phenotype in the affected individual is due to a new dominant mutation. DNA sequence homology in the area of the duplication suggests that the mutation may have arisen by unequal crossover between related sequences, a proposed mechanism in the evolution and diversification of the collagen gene family.
Specific reduction of calcium-binding protein (28-kilodalton calbindin-D) gene expression in aging and neurodegenerative diseases.
Authors: Iacopino A M , Christakos S
Published: May 2006

The present studies establish that there are specific, significant decreases in the neuronal calcium-binding protein (28-kDa calbindin-D) gene expression in aging and in neurodegenerative diseases. The specificity of the changes observed in calbindin mRNA levels was tested by reprobing blots with calmodulin, cyclophilin, and B-actin cDNAs. Gross brain regions of the aging rat exhibited specific, significant decreases (60-80%) in calbindin mRNA and protein levels in the cerebellum, corpus striatum, and brain-stem region but not in the cerebral cortex or hippocampus. Discrete areas of the aging human brain exhibited significant decreases (50-88%) in calbindin protein and mRNA in the cerebellum, corpus striatum, and nucleus basalis but not in the neocortex, hippocampus, amygdala, locus ceruleus, or nucleus raphe dorsalis. Comparison of diseased human brain tissue with age- and sex-matched controls yielded significant decreases (60-88%) in calbindin protein and mRNA in the substantia nigra (Parkinson disease), in the corpus striatum (Huntington disease), in the nucleus basalis (Alzheimer disease), and in the hippocampus and nucleus raphe dorsalis (Parkinson, Huntington, and Alzheimer diseases) but not in the cerebellum, neocortex, amygdala, or locus ceruleus. Since calbindin gene expression decreased specifically in brain areas known to be particularly affected in aging and in each of the neurodegenerative diseases, these findings suggest that decreased calbindin gene expression may lead to a failure of calcium buffering or intraneuronal calcium homeostasis, which contributes to calcium-mediated cytotoxic events during aging and in the pathogenesis of neurodegenerative diseases.
Meander tail reveals a discrete developmental unit in the mouse cerebellum.
Authors: Ross M E , Fletcher C , Mason C A , Hatten M E , Heintz N
Published: May 2006

Analyses of phenotypic mutations with altered patterns of cellular organization in invertebrate systems have lead to the identification of genes important to histogenesis. Efforts to identify genes defining segments or compartments in mammalian systems and demonstrate a role for such genes in the establishment of the cellular architectonics of the brain have been hampered by the absence of phenotypic mutations that reveal compartments. Evidence is presented that in the cerebellum of the mutant mouse, meander tail, there is an abrupt transition from the normal cytoarchitecture seen in the posterior lobes to a severely disorganized cellular pattern. Thus, in the anterior lobes the Purkinje cells are positioned randomly throughout the cortex, and other cellular layers, including the internal granule cell layer, do not form. In addition, radial Bergmann glial processes are virtually absent. One explanation for the discrete boundary of the affected area in the anterior portion of this mutant cerebellar cortex is that the meander tail gene influences compartmental cellular organization in mammalian brain.
Rapid preimplantation detection of mutant (shiverer) and normal alleles of the mouse myelin basic protein gene allowing selective implantation and birth of live young.
Authors: Gomez C M , Muggleton-Harris A L , Whittingham D G , Hood L E , Readhead C
Published: May 2006

As a model for the detection of human genetic disease in preimplantation embryos, we describe a method in which trophectoderm biopsy samples from viable mouse blastocysts are simultaneously analyzed for the presence of a normal or mutant allele of the myelin basic protein gene by the polymerase chain reaction. The biopsied embryos are kept in culture during analysis of biopsied material and later reintroduced to a foster mother. Prenatal diagnosis can be completed in less than 7 hr. The identity of either amplification product was proved conclusively by direct sequence analysis of amplified products. Ninety-six percent of recovered blastocysts survived biopsy, as judged by re-formation of a blastocyst cavity in culture. Fifty-nine percent of the biopsied embryos established pregnancy by day 6.5, compared to 88% of unmanipulated controls. This approach can be applied to preimplantation diagnosis of human genetic diseases by using extraembryonic cells from blastocysts obtained after in vitro fertilization or uterine lavage. It will make possible the elimination of a mutant allele from a family in a single generation.
Bovine and mouse serum beta inhibitors of influenza A viruses are mannose-binding lectins.
Authors: Anders E M , Hartley C A , Jackson D C
Published: May 2006

Normal bovine and mouse sera contain a component, termed beta inhibitor, that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. To investigate the nature of the interaction of beta inhibitors with influenza A viruses we isolated a mutant of the virus Mem71H-BelN (H3N1) that could grow in the presence of bovine serum. The mutant virus was resistant to hemagglutination inhibition by mouse serum as well as by bovine serum and had undergone changes in the receptor-binding and the antigenic properties of its hemagglutinin (HA) molecule. Sequence analysis of the HA genes of parent and mutant viruses revealed a single nucleotide change in the mutant, resulting in the substitution Thr----Asn at residue 167 of the HA1 chain of HA. This change leads to loss of the potential glycosylation site Asn-165-Val-166-Thr-167 at the tip of the HA spike, which in viruses of the H3 subtype is known to bear a high-mannose (type II) carbohydrate side chain N-linked to Asn-165. The association of beta inhibitor resistance with loss of this carbohydrate side chain suggested that beta inhibitors may be lectins. In support of this hypothesis, treatment of the beta inhibitor-sensitive parent virus Mem71H-BelN with periodate converted it to the resistant state. Furthermore, the inhibitory activity of both bovine and mouse sera for the parental virus was abrogated by D-mannose. We conclude that the beta inhibitors in bovine and mouse sera are mannose-binding lectins that inhibit hemagglutination and neutralize virus infectivity by binding to carbohydrate at the tip of the HA spike, blocking access of cell-surface receptors to the receptor-binding site on HA.
Cell proliferation in human coronary arteries.
Authors: Gordon D , Reidy M A , Benditt E P , Schwartz S M
Published: May 2006

Despite the lack of direct evidence for cell multiplication, proliferation of smooth muscle cells in human atherosclerotic lesions has been assumed to play a central role in ontogeny of the plaque. We used antibodies to cell cycle-related proteins on tissue sections of human arteries and coronary atherosclerotic plaques. Specific cell types were identified by immunochemical reagents for smooth muscle, monocyte-macrophages, and other blood cells. Low rates of smooth muscle cell proliferation were observed. Macrophages were also observed with rates of proliferation comparable to that of the smooth muscle. Additional replicating cells could not be defined as belonging to specific cell types with the reagents used in this study. These findings imply that smooth muscle replication in advanced plaques is indolent and raise the possibility of a role for proliferating leukocytes.
Features of the formate dehydrogenase mRNA necessary for decoding of the UGA codon as selenocysteine.
Authors: Zinoni F , Heider J , Böck A
Published: May 2006

The fdhF gene encoding the 80-kDa selenopolypeptide subunit of formate dehydrogenase H from Escherichia coli contains an in-frame TGA codon at amino acid position 140, which encodes selenocysteine. We have analyzed how this UGA "sense codon" is discriminated from a UGA codon signaling polypeptide chain termination. Deletions were introduced from the 3' side into the fdhF gene and the truncated 5' segments were fused in-frame to the lacZ reporter gene. Efficient read-through of the UGA codon, as measured by beta-galactosidase activity and incorporation of selenium, was dependent on the presence of at least 40 bases of fdhF mRNA downstream of the UGA codon. There was excellent correlation between the results of the deletion studies and the existence of a putative stem-loop structure lying immediately downstream of the UGA in that deletions extending into the helix drastically reduced UGA translation. Similar secondary structures can be formed in the mRNAs coding for other selenoproteins. Selenocysteine insertion cartridges were synthesized that contained this hairpin structure and variable portions of the fdhF gene upstream of the UGA codon and inserted into the lacZ gene. Expression studies showed that upstream sequences were not required for selenocysteine insertion but that they may be involved in modulating the efficiency of read-through. Translation of the UGA codon was found to occur with high fidelity since it was refractory to ribosomal mutations affecting proofreading and to suppression by the sup-9 gene product.
The expected equilibrium of the CpG dinucleotide in vertebrate genomes under a mutation model.
Authors: Sved J , Bird A
Published: May 2006

The CpG dinucleotide is present at approximately 20% of its expected frequency in vertebrate genomes, a deficiency thought due to a high mutation rate from the methylated form of CpG to TpG and CpA. We examine the hypothesis that the 20% frequency represents an equilibrium between rate of creation of new CpGs and accelerated rate of CpG loss from methylation. Using this model, we calculate the expected reduction in the equilibrium frequency of the CpG dinucleotide and find that the observed CpG deficiency can be explained by mutation from methylated CpG to TpG/CpA at approximately 12 times the normal transition rate, the exact rate depending on the ratio of transitions to transversions. The observed rate of CpG dinucleotide loss in a human alpha-globin nonprocessed pseudogene, psi alpha 1, and the apparent replenishment of the CpG pool in this sequence by new mutations, agree with the above parameters. These calculations indicate that it would take 25 million years or less, a small fraction of the time for vertebrate evolution, for CpG frequency to be reduced from undepleted levels to the current depleted levels.
Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors.
Authors: Paria B C , Dey S K
Published: May 2006

We have established a model that shows cooperative interaction among preimplantation embryos and the role of growth factors on their development and growth. Two-cell mouse embryos cultured singly in 25-microliters microdrops had inferior development to blastocysts and lower cell numbers per blastocyst compared with those cultured in groups of 5 or 10. The inferior development of singly cultured embryos was markedly improved by addition of epidermal growth factor (EGF) or transforming growth factor alpha or beta 1 (TGF-alpha or TGF-beta 1) to the culture medium. The stage of embryonic development, primarily affected by these treatments, was between eight-cell/morula and blastocyst. Furthermore, blastocysts developed from eight-cell embryos cultured in groups or singly in the presence of EGF showed a higher incidence of zona hatching compared with those cultured singly in the absence of EGF. Detection of EGF receptors on the embryonic cell surface at eight-cell/morula and blastocyst stages suggests beneficial effects of EGF or TGF-alpha on preimplantation embryo development and blastocyst functions. Insulin-like growth factor I (IGF-I) had no influence on embryo development. To further document the cooperative interactions among embryos, the volume of the culture medium was doubled to 50 microliters. This increase in culture volume was even more detrimental to the development of singly cultured embryos. However, this detrimental effect was significantly reversed by EGF and reversed even more markedly by a combination of EGF and TGF-beta 1 but not by TGF-beta 1 alone. Although TGF-beta 1 plus IGF-I caused a modest improvement of embryo development, the response was not as great as shown by EGF alone. Furthermore, IGF-I had no additive effect on EGF-induced embryonic development. The study presents clear evidence that specific growth factors of embryonic and/or reproductive tract origin participate in preimplantation embryo development and blastocyst functions in an autocrine/paracrine manner.
Interleukin 1 receptor blockade attenuates the host inflammatory response.
Authors: Gershenwald J E , Fong Y M , Fahey T J , Calvano S E , Chizzonite R , Kilian P L , Lowry S F , Moldawer L L
Published: May 2006

Cytokines, including interleukin 1 (IL-1), tumor necrosis factor alpha, and interleukin 6, are often produced in response to tissue injury and contribute to several host responses such as weight loss, anorexia, and acute-phase protein synthesis. However, the role of IL-1 in specific tissue responses is unclear. To test our hypothesis that specific in vivo blockade of IL-1's action might inhibit the catabolic host changes associated with inflammation, mice were passively immunized with a monoclonal antibody directed against the murine IL-1 receptor prior to initiation of a turpentine-induced sterile abscess. This antibody prevents IL-1-mediated proliferation of murine thymocytes in vitro by inhibiting IL-1 alpha and IL-1 beta by way of competition for a common receptor. Weight loss following turpentine challenge was prevented by daily injections of anti-IL-1 receptor monoclonal IgG. Body composition analysis confirmed that lean tissue and fat were preserved by passive immunization. Furthermore, pretreatment with an anti-IL-1 receptor monoclonal antibody significantly attenuated the plasma amyloid P and interleukin 6 responses but did not affect the decline in plasma albumin or the increase in circulating corticosterone. Passive immunization of similar mice with polyclonal antisera against another cytokine, tumor necrosis factor alpha, failed to prevent either the weight loss or hepatic acute-phase protein changes observed in this inflammatory model. These findings suggest that IL-1 orchestrates weight loss and body compositional changes during inflammation and contributes to the induction of interleukin 6 and acute-phase protein synthesis.
CD4-T-cell antigen receptor complexes on human leukemia T cells.
Authors: Chuck R S , Cantor C R , Tse D B
Published: May 2006

CD4 and T-cell antigen receptor (TCR) comodulate from the surface of human and murine T cells following exposure to monoclonal anti-CD4 or anti-TCR. This comodulation may occur because expression of CD4 and TCR is regulated by similar transmembrane signals or because CD4 and TCR are physically associated. To study multimolecular assemblies on the plasma membrane, we developed a flow cytometric method for detecting singlet-singlet energy transfer between fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated monoclonal antibodies as sensitized TRITC emission on intact, single cells. Using this procedure, we detected CD4-TCR complexes on the surface of the transformed human leukemia T cells, HPB-ALL, in the absence of stimulation. More than one CD4 were found in association with one TCR. CD4-TCR complexes were not in rapid equilibrium with free CD4 and free TCR, and they were not induced by the dye-labeled anti-CD4 or anti-TCR.
Interleukin 1 pretreatment decreases ischemia/reperfusion injury.
Authors: Brown J M , White C W , Terada L S , Grosso M A , Shanley P F , Mulvin D W , Banerjee A , Whitman G J , Harken A H , Repine J E
Published: May 2006

Hearts isolated from rats treated 36 hr before with interleukin 1 (IL-1) had increased glucose-6-phosphate dehydrogenase (G6PD) activity and decreased hydrogen peroxide levels and injury after global ischemia (I, 20 min)/reperfusion (R, 40 min) compared with hearts from untreated rats. Hearts isolated from rats treated 6 hr earlier with IL-1 also had increased polymorphonuclear leukocytes (PMN), H2O2 levels, and oxidized glutathione (GSSG) contents compared with hearts from untreated rats. Depletion of circulating blood PMN by prior treatment with vinblastine prevented both early (from treatment 6 hr before study) IL-1-induced increases in myocardial PMN accumulation, H2O2 levels, and GSSG contents and late (from treatment 36 hr before study) increases in myocardial G6PD activity and protection against I/R. Our results indicate that IL-1 pretreatment causes an early (6 hr after IL-1 treatment) myocardial PMN accumulation and most likely an H2O2-dependent oxidative stress, which contributes to late (36 hr after IL-1 treatment) increases in myocardial G6PD activity and decreases in I/R injury.
Expression of a mutated bovine growth hormone gene suppresses growth of transgenic mice.
Authors: Chen W Y , Wight D C , Wagner T E , Kopchick J J
Published: May 2006

To determine the importance of the third alpha-helix in bovine growth hormone (bGH) relative to growth-related biological activities, the following experimental approach was used: (i) mutagenesis of helix III of bGH to generate an idealized amphiphilic helix; (ii) in vitro expression analyses of the mutated bGH gene in cultured mouse L cells; (iii) mouse liver membrane binding studies of wild-type and mutated bGH; and (iv) expression of the mutated gene in the transgenic mouse. An altered bGH gene (pBGH10 delta 6-M8) was generated that encodes the following changes: glutamate-117 to leucine, glycine-119 to arginine, and alanine-122 to aspartate. The plasmid pBGH10 delta 6-M8 was shown to be expressed in, and its protein product secreted by, mouse L cells. The altered hormone possessed the same binding affinity to mouse liver membrane preparations as wild-type bGH. Transgenic mice containing the mutated bGH gene, however, showed a significant growth-suppressed phenotype. The degree of suppression was directly related to serum levels of the altered bGH molecule.
Involvement of tyrosine residues located in the carboxyl tail of the human beta 2-adrenergic receptor in agonist-induced down-regulation of the receptor.
Authors: Valiquette M , Bonin H , Hnatowich M , Caron M G , Lefkowitz R J , Bouvier M
Published: May 2006

Chronic exposure of various cell types to adrenergic agonists leads to a decrease in cell surface beta 2-adrenergic receptor (beta 2AR) number. Sequestration of the receptor away from the cell surface as well as a down-regulation of the total number of cellular receptors are believed to contribute to this agonist-mediated regulation of receptor number. However, the molecular mechanisms underlying these phenomena are not well characterized. Recently, tyrosine residues located in the cytoplasmic tails of several membrane receptors, such as the low density lipoprotein and mannose-6-phosphate receptors, have been suggested as playing an important role in the agonist-induced internalization of these receptors. Accordingly, we assessed the potential role of two tyrosine residues in the carboxyl tail of the human beta 2AR in agonist-induced sequestration and down-regulation of the receptor. Tyr-350 and Tyr-354 of the human beta 2AR were replaced with alanine residues by site-directed mutagenesis and both wild-type and mutant beta 2AR were stably expressed in transformed Chinese hamster fibroblasts. The mutation dramatically decreased the ability of the beta 2AR to undergo isoproterenol-induced down-regulation. However, the substitution of Tyr-350 and Tyr-354 did not affect agonist-induced sequestration of the receptor. These results suggest that tyrosine residues in the cytoplasmic tail of human beta 2AR are crucial determinants involved in its down-regulation.
Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase 1B.
Authors: Brown-Shimer S , Johnson K A , Lawrence J B , Johnson C , Bruskin A , Green N R , Hill D E
Published: May 2006

The inactivation of growth suppressor genes appears to play a major role in the malignant process. To assess whether protein phosphotyrosyl phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) function as growth suppressors, we have isolated a cDNA clone encoding human protein phosphotyrosyl phosphatase 1B for structural and functional characterization. The translation product deduced from the 1305-nucleotide open reading frame predicts a protein containing 435 amino acids and having a molecular mass of 49,966 Da. The amino-terminal 321 amino acids deduced from the cDNA sequence are identical to the empirically determined sequence of protein phosphotyrosyl phosphatase 1B. [Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C. D., Harrylock, M., Cool, D. E., Krebs, E. G., Fischer, E. H. & Walsh, K. A. (1989) Proc. Natl. Acad. Sci. USA 86, 5252-5256]. A genomic clone has been isolated and used in an in situ hybridization to banded metaphase chromosomes to determine that the gene encoding protein phosphotyrosyl phosphatase 1B maps as a single-copy gene to the long arm of chromosome 20 in the region q13.1-q13.2.
Transactivation of interleukin 2 and its receptor induces immune activation in human T-cell lymphotropic virus type I-associated myelopathy: pathogenic implications and a rationale for immunotherapy.
Authors: Tendler C L , Greenberg S J , Blattner W A , Manns A , Murphy E , Fleisher T , Hanchard B , Morgan O , Burton J D , Nelson D L
Published: May 2006

A state of T-cell activation, reflected by a marked degree of spontaneous proliferation in vitro, exists among patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) but not in those with retroviral-induced adult T-cell leukemia (ATL). We wished to define the mechanism by which the immune activation of circulating cells from HAM/TSP is driven, thus gaining insight into the pathogenesis of this HTLV-I-associated disease. By using a modification of the polymerase chain reaction, we compared the levels of interleukin 2 (IL-2) and IL-2 receptor alpha chain (IL-2R alpha) mRNA expression to the transcription of the HTLV-I transactivator gene, pX, in peripheral blood mononuclear cells of HAM/TSP and ATL patients as well as seropositive carriers. Up-regulation of IL-2 and IL-2R alpha transcripts was detected in HAM/TSP and seropositive carriers that paralleled the coordinate mRNA expression of the pX transactivator. In addition, IL-2 and soluble IL-2R alpha serum levels in HAM/TSP and seropositive carriers were elevated. Despite markedly elevated levels of soluble IL-2R alpha in ATL, transcripts for IL-2 and pX were not demonstrable in the circulating cells. Finally, the marked degree of in vitro spontaneous proliferation present in HAM/TSP was profoundly inhibited by specific anti-IL-2R or anti-IL-2 blocking antibodies. Collectively, these results suggest that immune activation in HAM/TSP, in contrast to ATL, is virally driven by the transactivation and coordinate expression of IL-2 and IL-2R alpha. This deregulated autocrine process may contribute to the evolution of inflammatory nervous system damage in HAM/TSP.
Functional borders, genetic fine structure, and distance requirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter.
Authors: Block A , Dangl J L , Hahlbrock K , Schulze-Lefert P
Published: May 2006

The genetic fine structure of cis-acting sequences previously shown to be necessary for light-regulated expression in the promoter of the parsley (Petroselinum crispum) chalcone synthase gene was analyzed. Site-directed mutations and changes in spacing between cis elements were measured in transient expression assays in parsley protoplasts. Clustered point mutations allowed assignment of functional borders. Single-base substitutions within a highly conserved cis element (box II/G box) defined a critical core of seven bases, 5'-ACGTGGC-3'. It is functionally equivalent to a second sequence-related element (box III), which could replace box II in an orientation-dependent manner. The activity of box II required the presence of another juxtaposed element (box I) at a defined distance. No distance requirement was observed between the two large separable promoter regions known to independently confer light-regulated expression. These data support our hypothesis that a cis-acting sequence that is present in a limited number of diversely regulated plant genes gains its functional capacity and specificity by combinatorial diversity involving flanking partner elements.