bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional antagonism between the retinoic acid receptor and the viral transactivator BZLF1 is mediated by protein-protein interactions.
Authors: Pfitzner E , Becker P , Rolke A , Schüle R
Published: May 2006

The Epstein-Barr virus-encoded protein BZLF1 is a member of the basic leucine zipper (bZip) family of transcription factors. Like several other members of the bZip family, transcriptional activity of BZLF1 is modulated by retinoic acid receptors (RARs). We present evidence that the RAR alpha and BZLF1 can reciprocally repress each other's transcriptional activation by a newly discovered mechanism. Analysis of RAR alpha mutants in transfection studies reveals that the DNA binding domain is sufficient for inhibition of BZLF1 activity. Analysis of BZLF1 mutants indicates that both the coiled-coil dimerization domain and a region containing the transcriptional activation domain of BZLF1 are required for transrepression. Coimmunoprecipitation experiments demonstrate physical interactions between RAR alpha and BZLF1 in vivo. Furthermore, glutathione S-transferase-pulldown assays reveal that these protein-protein interactions are mediated by the coiled-coil dimerization domain of BZLF1 and the DNA binding domain of RAR alpha. While RAR alpha is unable to recognize BZLF1 binding sites, the RAR alpha can be tethered to the DNA by forming a heteromeric complex with BZLF1 bound to DNA. Tethering RARs via protein-protein interactions onto promoter DNA suggest a mechanism through which RARs might gain additional levels of transcriptional regulation.
Differential recognition of the type I interferon receptor by interferons tau and alpha is responsible for their disparate cytotoxicities.
Authors: Subramaniam P S , Khan S A , Pontzer C H , Johnson H M
Published: May 2006

Interferon tau (IFN tau), originally identified as a pregnancy recognition hormone, is a type I interferon that is related to the various IFN alpha species (IFN alpha s). Ovine IFN tau has antiviral activity similar to that of human IFN alpha A on the Madin-Darby bovine kidney (MDBK) cell line and is equally effective in inhibiting cell proliferation. In this study, IFN tau was found to differ from IFN alpha A in that is was > 30-fold less toxic to MDBK cells at high concentrations. Excess IFN tau did not block the cytotoxicity of IFN alpha A on MDBK cells, suggesting that these two type I IFNs recognize the type I IFN receptor differently on these cells. In direct binding studies, 125I-IFN tau had a Kd of 3.90 x 10(-10) M for receptor on MDBK cells, whereas that of 125I-IFN alpha A was 4.45 x 10(-11) M. Consistent with the higher binding affinity, IFN alpha A was severalfold more effective than IFN tau in competitive binding against 125I-IFN tau to receptor on MDBK cells. Paradoxically, the two IFNs had similar specific antiviral activities on MDBK cells. However, maximal IFN antiviral activity required only fractional occupancy of receptors, whereas toxicity was associated with maximal receptor occupancy. Hence, IFN alpha A, with the higher binding affinity, was more toxic than IFN tau. The IFNs were similar in inducing the specific phosphorylation of the type I receptor-associated tyrosine kinase Tyk2, and the transcription factors Stat1 alpha and Stat2, suggesting that phosphorylation of these signal transduction proteins is not involved in the cellular toxicity associated with type I IFNs. Experiments using synthetic peptides suggest that differences in the interaction at the N terminal of IFN tau and IFN alpha with the type I receptor complex contribute significantly to differences in high-affinity equilibrium binding of these molecules. It is postulated that such a differential recognition of the receptor is responsible for the similar antiviral but different cytotoxic effects of these IFNs. Moreover, these data imply that receptors are "spare'' with respect to certain biological properties, and we speculate that IFNs may induce a concentration-dependent selective association of receptor subunits.
A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone.
Authors: Kaiser U B , Sabbagh E , Katzenellenbogen R A , Conn P M , Chin W W
Published: May 2006

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.
Liver-specific expression of the human factor VII gene.
Authors: Greenberg D , Miao C H , Ho W T , Chung D W , Davie E W
Published: May 2006

Promoter and silencer elements of the immediate 5' flanking region of the gene coding for human factor VII were identified and characterized. The major transcription start site, designated as +1, was determined by RACE (rapid amplification of cDNA ends) analysis of human liver cDNA and was found to be located 50 bp upstream from the translation start site. Two minor transcription start sites were found at bp +32 bp and +37. Progressive deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase reporter gene and transient expression in HepG2 and HeLa cells was measured. Two promoter elements that were essential for hepatocyte-specific transcription were identified. The first site, FVIIP1, located at bp -19 to +1, functioned independently of orientation or position and contributed about one-third of the promoter activity of the factor VII gene. Electrophoretic mobility-shift, competition, and anti-hepatocyte nuclear factor 4 (HNF4) antibody supershift experiments demonstrated that this site contained an HNF-4 binding element homologous to the promoters in the genes coding for factor IX and factor X. The second site, FVIIP2, located at bp -50 to -26, also functioned independent of orientation or position and contributed about two thirds of the promoter activity in the gene for factor VII. Functional assays with mutant sequences demonstrated that a 10-bp G + C-rich core sequence which shares 90% sequence identity with the prothrombin gene enhancer was essential for the function of the second site. Mobility-shift and competition assays suggested that this site also binds hepatic-specific factors as well as the transcription factor Sp1. Two silencer elements located upstream of the promoter region spanning bp -130 to -103 (FVIIS1 site) and bp -202 to -130 (FVIIS2) were also identified by reporter gene assays.
Differentiation of canine bone marrow cells with hemopoietic characteristics from an adherent stromal cell precursor.
Authors: Huss R , Hong D S , McSweeney P A , Hoy C A , Deeg H J
Published: May 2006

Stromal cell lines were established from canine long-term marrow cultures, cloned by limiting dilution, and maintained in stromal cell-conditioned medium. These cells grew adherent, maintained stable growth rate and morphology under standard conditions (in 20-30% conditioned medium; confluency, 70-90%), and supported hemopoiesis in long-term marrow cultures. In the presence of exogenous recombinant canine stem cell factor (rcSCF), round cells developed from the adherent layer, detached, and remained in culture as viable floating cells. Round floating cells also appeared when cultures were grown to > 90% confluency without rcSCF. Round cells were smaller than adherent cells, expressed CD34, showed basophilic plasma, and stained positive for c-kit, MHC-class II markers, and myeloid markers. In standard assays for colony formation, the detached cells produced granulocyte-macrophage colony-forming units (CFU-GM), fibroblast colony-forming units (CFU-F), and less well-defined colony-forming units. In addition, on allogeneic feeder cells in long-term cultures, these cells generated hemopoietic colonies. Strikingly, the differentiation was reversible: when nonadherent cells were resuspended at lower density in serum-containing medium, they reattached and grew to confluence when, once again, round cells detached. Detached cells from this secondary cycle produced mainly CFU-F and few CFU-GM when placed in clonal assays. These results suggest that some fibroblast-like stromal cells have the potential to differentiate into cells with hemopoietic characteristics. These observations provide evidence for the existence of a quiescent precursor of hemopoietic progenitors in the bone marrow stroma of the adult dog.
Praxic and nonverbal cognitive deficits in a large family with a genetically transmitted speech and language disorder.
Authors: Vargha-Khadem F , Watkins K , Alcock K , Fletcher P , Passingham R
Published: May 2006

A pronounced speech and language disorder affecting half of the 30 members of the four-generational KE family has been attributed by some researchers to a specific defect in the generation of morphosyntactic rules. The reported selectivity of the impairment has led to the view that the affected members suffer from a grammar-specific disorder. Our investigations of the same KE family indicate that the inherited disorder has a broad phenotype which transcends impaired generation of syntactical rules and includes a striking articulatory impairment as well as defects in intellectual, linguistic, and orofacial praxic functions generally. Although the evidence from this family thus provides no support for the existence of "grammar genes," their linguistic difficulties do constitute a prominent part of their phenotype. Investigations of the neural and genetic correlates of their disorder could therefore uncover important clues to some of the bases of the primary human faculties of speech and language.
Cooperative action of cellular proteins YB-1 and Pur alpha with the tumor antigen of the human JC polyomavirus determines their interaction with the viral lytic control element.
Authors: Chen N N , Chang C F , Gallia G L , Kerr D A , Johnson E M , Krachmarov C P , Barr S M , Frisque R J , Bollag B , Khalili K
Published: May 2006

Human JC polyomavirus (JCV) is the etiologic agent of the neurodegenerative disease progressive mulifocal leukoencephalopathy. By using JCV as a model, we investigated the role of the viral early protein tumor antigen (TAg) in the binding of two cellular proteins, Pura alpha and YB-1, to JCV regulatory sequences. Results from band-shift assays with purified YB-1, Pur alpha, and TAg indicated that efficient binding of Pur alpha, a strong activator of early gene transcription, to a single-stranded target sequence corresponding to the viral lytic control element, is diminished in the presence of the late gene activator YB-1, which recognizes the opposite strand of the Pur alpha binding site. Of particular interest was the ability of Pur alpha and TAg to enhance binding of YB-1 to DNA molecules without being associated with this complex. Binding studies using a mutant peptide encompassing the N terminus of YB-1 indicated that the C terminus of YB-1 is important for its DNA binding activity. The ability of Pur alpha and TAg to increase binding of YB-1 to DNA is independent of the YB-1 C terminus. Similarly, results from band-shift assays using Pur alpha variants indicated that two distinct regions of this protein contribute either to its ability to bind DNA or to its ability to enhance YB-1 DNA binding activity. Based on the interaction of Pur alpha, YB-1, and TAg, and their binding to DNA, a model is proposed for the role of these proteins in transcription of viral early and late genes during the lytic cycle.
Sexual dimorphism in the architecture of the lung's gas-exchange region.
Authors: Massaro G D , Mortola J P , Massaro D
Published: May 2006

The lung's only vital function is to provide sufficient gas-exchange surface area (Sa) to meet the organism's needs for oxygen uptake (VO2) and carbon dioxide elimination. A direct linear relation between Sa and VO2 and an inverse linear relation between the size of the lung's gas-exchange units and the species mass-specific VO2 are strongly conserved across species. Within species, Sa increases in response to prolonged (weeks) elevation of VO2. We now report sex-dependent deviations from these relationships that seem to anticipate the need for increased gas-exchange capacity engendered in females by the metabolic demands of pregnancy and lactation. We found that although VO2 almost doubled in rats during pregnancy and lactation, Sa was the same in age-matched virgin, pregnant, and lactating females. However, at the onset of sexual maturity, virgin female rats and mice had higher mass-specific Sa than males of the same species although mass-specific VO2 was identical, within species, in both sexes. In addition, even though mass-specific VO2 was identical in males and females, alveoli were 30% and 50% smaller in female rats and mice, respectively, than males of the same species. We suggest the greater mass-specific Sa and smaller alveoli in females in spite of identical mass-specific VO2 as males were selected for evolutionarily; they help females meet the metabolic demands of reproduction without adding to the energy demands of these periods a requirement to form additional lung.
Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene.
Authors: von der Leyen H E , Gibbons G H , Morishita R , Lewis N P , Zhang L , Nakajima M , Kaneda Y , Cooke J P , Dzau V J
Published: May 2006

It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as beta-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessels. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.
cDNA cloning of prophenoloxidase from the freshwater crayfish Pacifastacus leniusculus and its activation.
Authors: Aspán A , Huang T S , Cerenius L , Söderhäll K
Published: May 2006

Prophenoloxidase (proPO), an enzyme that is the terminal component of the so-called proPO activating system, a defense and recognition system in crustaceans and insects, has been purified and cloned from a crayfish blood cell cDNA library. The deduced amino acid sequence codes for a polypeptide with a mass of 80,732 Da, which is close to 76 kDa, the apparent mass of the purified enzyme. proPO contains two copper atoms, and two putative copper-binding sites were found in the deduced amino acid sequence. Sequence comparisons show that these putative copper-binding sites are similar to the corresponding sites in arthropod hemocyanins and also, although the sequence similarities are less extensive, similar to tyrosinases from vertebrates and microorganisms. The purified enzyme is a typical tyrosinase because it hydroxylates monophenols and oxidizes o-diphenols but does not oxidize p-diphenols. If a homogeneous preparation of crayfish proPO were incubated with a homogeneous sample of the proPO activating enzyme, a serine proteinase, the cleavage of proPO by this trypsin-like enzyme was found to occur between Arg-176 and Thr-177.
An early increase in somatostatin mRNA expression in the frontal cortex of rhesus monkeys infected with simian immunodeficiency virus.
Authors: Da Cunha A , Rausch D M , Eiden L E
Published: May 2006

Motor and cognitive impairment is common in human immunodeficiency virus disease in humans and simian immunodeficiency virus (SIV) disease in rhesus monkeys. We have examined peptide neurotransmitter expression in the frontal cortex of SIV-infected rhesus monkeys to identify alterations in cortical neurons that might explain this impairment. A 2-fold higher number of preprosomatostatin (SRIF) mRNA-positive interneurons was observed in layer IV of frontal cortex in two separate cohorts of SIV-infected animals compared to uninfected controls. Increased SRIF mRNA expression in layer IV was independent of clinical signs of immunodeficiency disease and was associated with both motor and cognitive impairment. Altered SRIF mRNA expression in deeper cortical layers was associated specifically with motor impairment. Increased SRIF mRNA expression occurred without detectable changes in cortical cell density. These data suggest two mechanisms for cortical dysfunction associated with lentivirus infection. Increased SRIF mRNA expression in layer IV may be due to altered patterns of activity in cortical afferents that project to layer IV, while increased SRIF mRNA expression in deeper cortical layers could reflect susceptibility to locally generated mediators in response to primate lentivirus infection of the brain. Altered function of somatostatinergic interneurons may contribute to primate lentivirus-induced encephalopathy.
A mutation in the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results in impaired lactation.
Authors: Fowler K J , Walker F , Alexander W , Hibbs M L , Nice E C , Bohmer R M , Mann G B , Thumwood C , Maglitto R , Danks J A
Published: May 2006

The mutant mouse waved-2 (wa-2) is strikingly similar to transforming growth factor alpha-deficient mice generated by gene targeting in embryonic stem cells. We confirm that wa-2 is a point mutation (T-->G resulting in a valine-->glycine substitution at residue 743) in the gene encoding the epidermal growth factor (EGF) receptor. wa-2 fibroblastic cells lack high-affinity binding sites for EGF, and the rate of internalization of EGF is retarded. Although the tyrosine kinase activity of wa-2 EGF receptors is significantly impaired, NIH 3T3 cells lacking endogenous EGF receptors but overexpressing recombinant wa-2 EGF receptor cDNA are mitogenically responsive to EGF. While young and adult wa-2 mice are healthy and fertile, 35% of wa-2 mice born of homozygous wa-2 mothers die of malnutrition because of impaired maternal lactation.
Long-lived testosterone esters in the rat.
Authors: Borg W , Shackleton C H , Pahuja S L , Hochberg R B
Published: May 2006

Over the past decade it has become increasingly clear that steroid hormones are enzymatically esterified with fatty acids. These steroidal esters are the natural analogs of synthetic esters that are used therapeutically. One such family of pharmacological steroids is the synthetic alkyl esters of testosterone, androgens with great hormonal potency. We have investigated whether testosterone esters exist naturally by using the rat as a model. Most tissues of male rats, including blood, have very little if any ester (quantified by immunoassay as a nonpolar saponifiable metabolite), but fat and testes have sizable quantities, approximately 3 ng of testosterone equivalents per g of tissue. Testosterone in fat averages 9 ng/g. The fat from female rats and long-term (> 2 weeks) castrated males has no detectable testosterone ester. The presence of testosterone esters was confirmed by GC/MS, which clearly showed the presence of testosterone in the hydrolyzed ester fraction of fat from intact males but not long-term castrates. Upon castration, testosterone levels in the fat completely disappear within 6 hr. To the contrary, it is not until 48 hr after castration that a measurable fall in the testosterone ester fraction was observed; even after 10 days a small amount of ester is still present in the fat. These experiments demonstrate the existence of a previously unknown androgen with a potentially important physiological impact; testosterone esters, natural analogs of potent therapeutic agents, occur in the fat where they can serve as a reservoir of preformed androgen to stimulate neighboring target tissues.
Caveolae from luminal plasmalemma of rat lung endothelium: microdomains enriched in caveolin, Ca(2+)-ATPase, and inositol trisphosphate receptor.
Authors: Schnitzer J E , Oh P , Jacobson B S , Dvorak A M
Published: May 2006

A distinctive feature of many endothelia is an abundant population of noncoated plasmalemmal vesicles, or caveolae. Caveolae have been implicated in many important cellular processes, including transcytosis, endocytosis, potocytosis, and even signal transduction. Because caveolae have not been purified from endothelial cell surfaces, little is known directly about their structure and function in the endothelium. To delineate the transport role of these caveolae, we purified them from isolated luminal endothelial plasma membranes of rat lung. The rat lung luminal endothelial cell surfaces were isolated after coating them, in situ, with positively charged colloidal silica. The caveolae were then separated from these coated membranes and purified to yield a homogeneous population of morphologically distinct vesicles enriched in the structural protein caveolin. As with caveolae found on the endothelial cell surface in vivo, these highly purified caveolae contained the plasmalemmal Ca(2+)-ATPase and inositol 1,4,5-trisphosphate surface receptors. By contrast, other plasma membrane proteins were excluded from the caveolae, including angiotensin-converting enzyme, beta-actin, and band 4.1. The purified caveolae appeared to represent specific microdomains of the cell surface with their own unique molecular topography.
Identification of a gene, spoIIR, that links the activation of sigma E to the transcriptional activity of sigma F during sporulation in Bacillus subtilis.
Authors: Karow M L , Glaser P , Piggot P J
Published: May 2006

Sporulation of Bacillus subtilis requires the coordinated expression of two separate developmental programs in the mother cell and forespore compartments by sigma E and sigma F, respectively. This coordination is maintained through the action of cross-regulatory factors that control the activities of the various sporulation-specific sigma factors. We present here the isolation and characterization of one such cross-regulatory factor, the spoIIR gene. Using a genetic screen, we have isolated four mutant alleles of spoIIR. These mutants were isolated as expressing sigma F-directed genes but not sigma E-directed genes. The block in sigma E-directed gene expression in spoIIR mutants was caused by an inability to process pro-sigma E to its active form. Cloning and characterization of the spoIIR gene determined that its transcription is directed by sigma F. Thus, SpoIIR is required for linking the activation of sigma E to the activation of sigma F and coordinating the initiation of the two developmental programs required to form a spore.
A phylogeny of cockroaches and related insects based on DNA sequence of mitochondrial ribosomal RNA genes.
Authors: Kambhampati S
Published: May 2006

Cockroaches are among the most ancient winged insects, the earliest fossils dating back to about 400 million years. Several conflicting phylogenies for cockroach families, subfamilies, and genera have been proposed in the past. In addition, the relationship of Cryptocercidae to other cockroach families and the relationship between the cockroach, Cryptocercus punctulatus, and the termite, Mastotermes darwiniensis, have generated debate. In this paper, a phylogeny for cockroaches, mantids, and termites based on DNA sequence of the mitochondrial ribosomal RNA genes is presented. The results indicated that cockroaches are a monophyletic group, whose sister group is Mantoidea. The inferred relationship among cockroach families was in agreement with the presently accepted phylogeny. However, there was only partial congruence at the subfamily and the generic levels. The phylogeny inferred here does not support a close relationship between C. punctulatus and M. darwiniensis. The apparent synapomorphies of these two species are likely a manifestation of convergent evolution because there are similarities in biology and habitat.
Primary picosecond molecular events in the photoreaction of the BR5.12 artificial bacteriorhodopsin pigment.
Authors: Delaney J K , Brack T L , Atkinson G H , Ottolenghi M , Steinberg G , Sheves M
Published: May 2006

The picosecond dynamics of the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a retinal in which a five-membered ring spans the C-12 to C-14 positions of the polyene chain (BR5.12) is examined by using time-resolved absorption and fluorescence and resonance Raman spectroscopy. The ring within the retinal chromophore of BR5.12 blocks the C-13 = C-14 isomerization proposed to be a primary step in the energy storage/transduction mechanism in the BR photocycle. Relative to the native BR pigment (BR-570), the absorption spectrum of BR5.12 is red-shifted by 8 nm. The fluorescence spectrum of BR5.12 closely resembles that of BR-570 although the relative fluorescence yield is higher (approximately 10-fold). Picosecond transient absorption (4-ps pulses, 568-662 nm) measurements reveal an intermediate absorbing to the red side of BR5.12. Kinetic fits show that the red-absorbing intermediate appears within < 3 ps and decays with a time constant of 17 +/- 1 ps to form only BR5.12. No emission in the 650- to 900-nm region can be attributed to the red-absorbing species. Since rotation around C-12 - C-13 and isomerization around C-13 = C-14 are prevented in BR5.12, these results demonstrate that motion in these regions of the retinal is (i) necessary to form the K-like intermediate observed in the native BR-570 photocycle and (ii) not necessary to form a red-absorbing intermediate that has spectral and kinetic properties analogous to those of J-625 in the native BR photocycle. Discussions of the excited and ground electronic state assignments for the intermediate observed in the BR5.12 photoreaction are presented.
Unexpectedly high occurrence of catalytic antibodies in MRL/lpr and SJL mice immunized with a transition-state analog: is there a linkage to autoimmunity?
Authors: Tawfik D S , Chap R , Green B S , Sela M , Eshhar Z
Published: May 2006

Upon testing the ability of several strains of mice to elicit esterolytic antibodies after immunization with a p-nitrobenzyl phosphonate hapten, we have found that the occurrence of catalytic antibodies in SJL and MRL/lpr autoimmune mice is dramatically higher than in normal mouse strains (e.g., the wild-type MRL/++ or BALB/c). Fewer than 10 catalytic clones are usually obtained from a single fusion of lymphocytes taken from normal mice, whereas several hundred catalytic clones are obtained in SJL or MRL/lpr mice. Differences in the numbers of hapten-binding clones do not account for the high occurrences of catalytic clones in these strains. This phenomenon prevailed in the early responses; in both SJL and MRL/lpr mice a significant decline in the appearance of catalytic clones was observed after multiple immunizations. Esterolytic antibodies were not found in MRL/lpr mice immunized with haptens that do not mimic the transition state for the hydrolysis of the ester substrate (e.g., with a substrate analog). The catalytic antibodies manifest high specificity to the antigen and variability in their binding and catalytic properties. The use of autoimmunity-prone mice may greatly expand the repertoire of catalytic clones elicited against a transition-state analog hapten. More intriguing is the possible linkage between autoimmunity and the appearance of catalytic antibodies. These results suggest that there is normally a selection against the expression of certain variable genes encoding antibodies with catalytic activity.
Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes.
Authors: Haaf T , Golub E I , Reddy G , Radding C M , Ward D C
Published: May 2006

Rad51 protein of Saccharomyces cerevisiae is a structural homolog of the Escherichia coli recombination enzyme RecA. In yeast, the Rad51 protein is required for mitotic and meiotic recombination and for repair of double-strand breaks in DNA. We have used antibodies raised against the homologous human protein, HsRad51, expressed in E. coli, to visualize the spatial distribution of the protein in mammalian somatic and meiotic cells. In cultured human cells, the HsRad51 protein is concentrated in multiple discrete foci in the nucleoplasm; it is largely absent from cytoplasm and nucleoli. After treatment of cells with methyl methanesulfonate, ultraviolet irradiation, or 137Cs irradiation, the percentage of cells with HsRad51 protein immunofluorescence increases; the same cells show unscheduled DNA synthesis. Induction of Rad51 foci is blocked by inhibitors of transcription. In mouse pachytene spermatocytes, the mouse homolog of Rad51 protein is highly enriched in synaptonemal complexes that are formed between the paired homologous chromosomes during meiotic prophase. We conclude that the mammalian proteins homologous to yeast Rad51 are involved in repair of DNA damage and recombinational repair during meiosis.
Selective labeling of intracellular parasite proteins by using ricin.
Authors: Gurnett A M , Dulski P M , Darkin-Rattray S J , Carrington M J , Schmatz D M
Published: May 2006

Studies focused on the synthesis by intracellular parasites of developmentally regulated proteins have been limited due to the lack of a simple method for selectively labeling proteins produced by the parasite. A method has now been developed in which ricin is employed to selectively inhibit host-cell protein synthesis. Ricin is a heterodimer composed of two subunits, a lectin and a glycosidase, and it binds to terminal galactose residues on the cell surface via the lectin. Following endocytosis of the intact molecule, a disulfide bond linking the two subunits is cleaved, and only the glycosidase subunit enters the cytoplasm, where it inhibits cytoplasmic protein synthesis by catalyzing the cleavage of the 28S rRNA. Due to the loss of the receptor-binding lectin subunit, ricin cannot permeate host-cell mitochondria or intracellular parasites, and, therefore, protein synthesis within these compartments continues uninterrupted. This system has been used to selectively label parasite proteins from Eimeria tenella and Toxoplasma gondii by using the avian cell line DU-24. In these cells, mitochondrial protein synthesis was inhibited by using chloramphenicol. The use of the avian rho0 cell line DUS-3 provided an additional advantage, because these cells lack mitochondrial DNA. Therefore, those proteins radiolabeled with [35S]methionine/cysteine in ricin-treated, parasite-infected rho0 cells are exclusively those of the intracellular parasite. This technique should be applicable for studying protein synthesis by other intracellular parasites.
Visual motion perception.
Authors: Albright T D , Stoner G R
Published: May 2006

The primate visual motion system performs numerous functions essential for survival in a dynamic visual world. Prominent among these functions is the ability to recover and represent the trajectories of objects in a form that facilitates behavioral responses to those movements. The first step toward this goal, which consists of detecting the displacement of retinal image features, has been studied for many years in both psychophysical and neurobiological experiments. Evidence indicates that achievement of this step is computationally straightforward and occurs at the earliest cortical stage. The second step involves the selective integration of retinal motion signals according to the object of origin. Realization of this step is computationally demanding, as the solution is formally underconstrained. It must rely--by definition--upon utilization of retinal cues that are indicative of the spatial relationships within and between objects in the visual scene. Psychophysical experiments have documented this dependence and suggested mechanisms by which it may be achieved. Neurophysiological experiments have provided evidence for a neural substrate that may underlie this selective motion signal integration. Together they paint a coherent portrait of the means by which retinal image motion gives rise to our perceptual experience of moving objects.
Human autoantibody recognition of DNA.
Authors: Barbas S M , Ditzel H J , Salonen E M , Yang W P , Silverman G J , Burton D R
Published: May 2006

Combinatorial IgG Fab phage display libraries prepared from a systemic lupus erythematosus (SLE) donor and a healthy donor were affinity selected against human placental DNA. Human monoclonal antibody Fab fragments specific for DNA were isolated from both libraries, although Fabs of the highest affinity were isolated only from the lupus library. Generally, apparent affinities of the Fabs for human placental DNA, purified double-stranded DNA, and denatured DNA were approximately equivalent. Surface plasmon resonance indicated Fab binding constants for a double-stranded oligodeoxynucleotide of 0.2-1.3 x 10(8) M-1. The higher-affinity Fabs, as ranked by binding to human placental DNA or to the oligonucleotide probe, tested positive in the Crithidia luciliae assay commonly used in the diagnosis of SLE, and interestingly the genes encoding the heavy-chain variable regions of these antibodies displayed evidence of only minimal somatic hypermutation. The heavy chains of the SLE Fabs were characterized by a predominance of basic residues toward the N terminus of complementarity-determining region 3 (CDR3). The crucial role of heavy-chain CDR3 (HCDR3) in high-affinity DNA recognition was suggested by the creation of DNA binding in an unrelated antibody by HCDR3 transplantation from SLE antibodies. We propose that high-affinity DNA-binding antibodies can arise in SLE without extensive somatic hypermutation in the variable-region genes because of the expression of inappropriate HCDR3s.
The rapid assembly of synaptic sites in photoreceptor terminals of the fly's optic lobe recovering from cold shock.
Authors: Brandstätter J H , Meinertzhagen I A
Published: May 2006

When a housefly, Musca domestica, is subject to cold exposure (0 degrees C for 24 hr), a number of obvious changes are seen in the first optic neuropil, or lamina, beneath the compound eye. In particular, the number of afferent photoreceptor synapses declines by about 30%. This loss is dramatically restored after warm recovery at 23 degrees C for 24 hr. Synapses disappear at an average rate of 2-3/hr during cold exposure and reappear at a maximal rate of more than 20/hr during the first 2 hr of warm recovery. Thereafter their number temporarily overshoots control values, to increase at 6 hr of warm recovery to 60% above their cold-exposed minimum. The number subsequently returns more or less to normal. These changes demonstrate the lability of synaptic sites under these conditions, with individual sites forming and disappearing rapidly. The changes also interrupt the close correlation between synaptic number and the surface area of the receptor terminal, a correlation that normally conserves synaptic spacing density. The density is preserved during cold exposure but increases during warm recovery at a time when the addition of newly formed synapses exceeds the slower increase in receptor terminal size.
Two forms of acetylcholine receptor gamma subunit in mouse muscle.
Authors: Mileo A M , Monaco L , Palma E , Grassi F , Miledi R , Eusebi F
Published: May 2006

Nicotinic acetylcholine receptors (nAcChoRs) of skeletal muscle are heterosubunit ligand-gated channels that mediate signal transmission from motor nerves to muscle. While cloning murine nAcChoR subunits, to gain an insight into the receptor diversity across species, we detected two forms of gamma subunits in the myogenic C2C12 cell line. Both forms are functional when expressed in Xenopus oocytes. One gamma subunit [long gamma (gamma 1)] was almost identical to that previously cloned in the murine BC3H-1 tumor cell line. The second form of gamma subunit [short gamma (gamma s)] lacked 156 bp (52 amino acids) in the extracellular N terminus, adjoining the hydrophobic segment M1, which corresponds to the fifth exon of the gamma-subunit gene. The two forms of gamma subunit coexist during myogenesis in vitro and in 17-day embryonic and denervated adult muscle fibers in vivo. However, the gamma s variant was the only form of gamma subunit in newborn muscle. In dissociated muscle fibers of newborn mice, AcCho-evoked channel openings were more prolonged when compared with C2C12 myotubes or denervated adult muscle fibers. The gamma s subunit may, thus, contribute to the structural and functional diversity of nAcChoRs in muscle cells.
Mutated K65R recombinant reverse transcriptase of human immunodeficiency virus type 1 shows diminished chain termination in the presence of 2',3'-dideoxycytidine 5'-triphosphate and other drugs.
Authors: Gu Z , Arts E J , Parniak M A , Wainberg M A
Published: May 2006

A lysine-to-arginine substitution at amino acid 65 (K65R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is associated with resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC). To further characterize the molecular basis of such resistance, we expressed the pp6/p51 heterodimer of wild-type RT, K65R mutated RT, and a doubly mutated (K65R/M184V) RT in Escherichia coli and assessed the characteristics of nucleotide incorporation and chain termination in cell-free reverse transcription reactions in the presence and absence of various nucleoside triphosphate analogs. These reactions employed a HIV RNA template (HIV-PBS) that contained the primer binding sequence (PBS) and the U5 and R regions of HIV-1 genomic RNA and an oligodeoxynucleotide (dPR) complementary to the HIV-1 PBS as primer. The K65R and K65R/M184V RTs showed significantly decreased chain-termination effects during polymerization with the 5'-triphosphates of ddC, 3TC, 2',3'-dideoxyadenosine, and AZT (3'-azido-3'-deoxythymidine) in comparison with wild-type RT. Detailed analysis with ddCTP and wild-type RT revealed that chain termination occurred at all guanines in the RNA template. However, the frequency of dideoxynucleoside triphosphate (ddNTP)-induced chain termination was decreased at certain guanines but not others in reactions catalyzed by K65R RT. Both the K65R mutant RT and wild-type RT had similar processive activity. These results indicate that decreased chain termination of K65R RT in the presence of ddNTPs is consistent with data obtained in viral replication assays.
Decreased expression of the embryonic form of the neural cell adhesion molecule in schizophrenic brains.
Authors: Barbeau D , Liang J J , Robitalille Y , Quirion R , Srivastava L K
Published: May 2006

The regulated expression of neural cell adhesion molecule (NCAM) isoforms in the brain is critical for many neurodevelopmental processes including neurulation, axonal outgrowth, and the establishment of neuronal connectivity. We have investigated the expression of the major adult isoforms of NCAM (NCAM-180, NCAM-140, and NCAM-120) and its embryonic highly polysialylated isoform (PSA-NCAM) in the hippocampal region of postmortem brains from 10 schizophrenic and 11 control individuals. Immunohistochemical analysis with a monoclonal antibody recognizing the PSA-NCAM revealed immunoreactivity primarily in the dentate gyrus and in a subset of cells in the hilus region. We have observed a 20-95% reduction in the number of hilar PSA-NCAM-immunoreactive cells in the great majority of schizophrenic brains. The change in PSA-NCAM immunoreactivity is not obvious in other hippocampal subfields. Western blots of tissues from the hippocampal region (as well as from the frontal cortex) probed with a polyclonal antibody recognizing all NCAM isoforms did not reveal significant changes in the overall expression of NCAM, suggesting that the decrease in PSA-NCAM-immunoreactive cells may be related to post-translational processing of the molecule. The expression of this embryonic form of NCAM has been proposed to be related to synaptic rearrangement and plasticity. Therefore, the decrease in PSA-NCAM immunoreactivity in schizophrenic hippocampi may suggest an altered plasticity of this structure in a large proportion of schizophrenic brains. These findings may bear significance to the "neurodevelopmental hypothesis" of schizophrenia.
Crystal structure of bovine angiogenin at 1.5-A resolution.
Authors: Acharya K R , Shapiro R , Riordan J F , Vallee B L
Published: May 2006

The capacity of angiogenin (Ang) to induce blood vessel growth is critically dependent on its ribonucleolytic activity. Crystallography and mutagenesis of human Ang have previously shown that its pyrimidine binding site is obstructed by Gln-117, implying that a conformational change is a key part of the mechanism of Ang action. The 1.5-A-resolution crystal structure of bovine Ang, in which glutamic acid is substituted for Gln-117, now confirms that a blocked active site is characteristic of these proteins. Indeed, the inactive conformation of bovine Ang is stabilized by a more extensive set of interactions than is that of human Ang. The three-dimensional structure of the putative receptor binding site is also well conserved in the two proteins. The Arg-Gly-Asp segment of this site in bovine Ang, which is replaced by Arg-Glu-Asn in human Ang, does not have a conformation typical of an integrin recognition site.
Primary structure of a potent endogenous dopa-containing inhibitor of phenol oxidase from Musca domestica.
Authors: Daquinag A C , Nakamura S , Takao T , Shimonishi Y , Tsukamoto T
Published: May 2006

The complete amino acid sequence of a low molecular weight peptide from the hemolymph of the housefly Musca domestica L., which had been determined to competitively inhibit phenol oxidase (PO; monophenol, dihydroxy-phenylalanine:oxygen oxidoreductase; EC 1.14.18.1) in the nM range, was unambiguously established by employing both automatic Edman degradation and mass spectrometry. The physiologically active peptide, which was designated phenol oxidase inhibitor (POI), has an observed molecular weight of 4213.1 +/- 0.2 by electrospray ionization mass spectrometry. The relatively short and structurally dense peptide contained 38 amino acid residues rich in cysteine and lysine. Comparison of the observed and calculated molecular mass indicates that apparently all six cysteine residues form disulfide bridges. Interestingly, sequence analyses of both the intact and protease-digested S-pyridylethylated POI showed that one of the two tyrosine residues (Tyr-32) is hydroxylated to a 3,4-dihydroxyphenylalanine (dopa) residue. This agreed with the increase of 16 mass units observed in mass spectrometric measurements. This was further verified by submission of free L-dopa to the sequencer, which gave a retention time consistent with the atypical peak observed at the Edman cycle of the peptide containing dopa. This study demonstrates the existence of a biologically active, dopa-containing peptide among the insects. Since the POI activity was most prominent in aged pupae, especially pharate adults, the POI may play an important role in smoothing the way of adult emergence through hindering excessive melanization, as well as hardening, of cuticular proteins under the epicuticle.
Alzheimer and beta-amyloid-treated fibroblasts demonstrate a decrease in a memory-associated GTP-binding protein, Cp20.
Authors: Kim C S , Han Y F , Etcheberrigaray R , Nelson T J , Olds J L , Yoshioka T , Alkon D L
Published: May 2006

The two proteins most consistently identified in the brains of patients with Alzheimer disease (AD) have been beta-amyloid and tau, whose roles in the physiology or pathophysiology of brain cells are not fully understood. To identify other protein(s) involved in AD that have been implicated in physiological contexts, we undertook to analyze a specific memory-associated protein, Cp20, in fibroblasts from AD and control donors. Cp20, a GTP-binding protein that is a member of the ADP-ribosylation factor family, was significantly decreased in fibroblasts from AD patients. Normal control fibroblasts exposed to 10 nM beta-amyloid, the same concentration that induced AD-like K+ changes in control fibroblasts, showed a similar decrease in Cp20. Since it has been previously demonstrated that Cp20 is a potent regulator of K+ channels, these findings suggest that changes in this memory-associated protein may explain previously observed differences in AD K+ channels and suggest a pathophysiologic involvement linked to soluble beta-amyloid metabolism that could contribute to the characteristic memory loss of AD.
The retinoblastoma-susceptibility gene product binds directly to the human TATA-binding protein-associated factor TAFII250.
Authors: Shao Z , Ruppert S , Robbins P D
Published: May 2006

RB, the protein product of the retinoblastoma tumor-suppressor gene, regulates the activity of specific transcription factors. This regulation appears to be mediated either directly through interactions with specific transcription factors or through an alternative mechanism. Here we report that stimulation of Sp1-mediated transcription by RB is partially abrogated at the nonpermissive temperature in ts13 cells. These cells contain a temperature-sensitive mutation in the TATA-binding protein-associated factor TAFII250, first identified as the cell cycle regulatory protein CCG1. The stimulation of Sp1-mediated transcription by RB in ts13 cells at the nonpermissive temperature could be restored by the introduction of wild-type human TAFII250. Furthermore, we demonstrate that RB binds directly to hTAFII250 in vitro and in vivo. These results suggest that RB can confer transcriptional regulation and possibly cell cycle control and tumor suppression through an interaction with TFIID, in particular with TAFII250.