bluuebunny/crossref_metadata_2025 huggingface dataset:
Orientation discrimination in the cat: a distributed function.
Authors: Orban G A , Vandenbussche E , Sprague J M , De Weerd P
Published: May 2006

Cats were trained to make fine orientation discriminations with stimuli similar to those used in physiological experiments--narrow, light bars 12 degrees long--before and after various combinations of lesions of areas 17 and 18. Discrimination thresholds were measured at different contrast levels and different bar widths, both pre- and postoperatively, for up to 1.5 years after the lesion. For high contrast stimuli, lesions restricted to area 17 or area 18 had little effect, but those lesions involving area 17 and a substantial part of area 18 raised thresholds. In the latter case there was a relationship between the amount of area 18 spared and the bar width at which discrimination was impaired. At low contrast deficits were seen only for narrow widths. These results lead to the following conclusions. (i) Orientation discrimination is a function distributed within and across areas 17 and 18. (ii) How this function is distributed in this cortex depends on stimulus width. (iii) The X system does not carry the signal necessary for orientation discrimination. (iv) Cells most narrowly tuned for orientation, which reside in the part of area 17 subserving central vision, do not determine the orientation discrimination threshold.
Oxidized low density lipoprotein induces differentiation and adhesion of human monocytes and the monocytic cell line U937.
Authors: Frostegård J , Nilsson J , Haegerstrand A , Hamsten A , Wigzell H , Gidlund M
Published: May 2006

Hypercholesterolemia is a major risk factor for development of atherosclerosis. In experimental animals fed a high-cholesterol diet, monocytes adhere to the arterial endothelium and penetrate into the intima where they differentiate into macrophages and ingest lipids thus giving rise to fatty streaks, the earliest type of atherosclerotic plaque. Macrophages express few receptors for normal low density lipoprotein (LDL) but can take up oxidized LDL by way of a scavenger receptor. The present study was designed to investigate the possible role of oxidized LDL in recruitment of resident intimal macrophages. We found that oxidized LDL induced enhanced expression of major histocompatibility complex class II molecules on human monocytes and U937 cells, a well-established system for studies of monocytic differentiation. Oxidized LDL also induced enhanced expression of the surface antigen LeuM3 but caused decreased expression of CD4 antigen, a pattern compatible with expression of a more-differentiated macrophage-like phenotype. Oxidized LDL also initiated aggregation of monocytes and U937 cells and stimulated adhesion of U937 cells to cultured endothelial cells. The results indicate that oxidized LDL may contribute to development of atherosclerosis by inducing adhesion of monocytes to the arterial intima and by stimulating intimal monocytes to differentiate into resident macrophages.
Differences between pair and bulk hydrophobic interactions.
Authors: Wood R H , Thompson P T
Published: May 2006

It is now well known that the pair interaction between two hydrocarbon molecules in water has distinctly different properties from the bulk hydrophobic interaction familiar to the biochemist, which is modeled by the transfer of a hydrocarbon from aqueous solutions to pure liquid hydrocarbon. We consider experimental data for pair interactions, which have been fitted by a simple empirical potential function, and point out some of their properties. (i) Surface free energy and cosphere overlap models, of the type considered until now, cannot reproduce correctly both the pair and bulk hydrophobic interactions. (ii) Pair interactions though still attractive are strikingly weaker in aqueous solution than in the gas phase, in contrast to the usual view of hydrophobic interactions. (iii) For pair interactions in water, the solvent-separated configuration is less important than the contact configuration if the hydrocarbon has more than two carbon atoms.
Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination.
Authors: Hu W S , Temin H M
Published: May 2006

Retroviruses contain two complete viral genomic RNAs in each virion. A system to study in a single round of replication the products of virions with two different genomic RNAs was established. A spleen necrosis virus-based splicing vector containing both the neomycin-resistance gene (neo) and the hygromycin B phosphotransferase gene (hygro) was used. Two frameshift mutants were derived from this vector such that the neo and the hygro genes were inactivated in separate vectors. Thus, each vector confers resistance to only one selection. The vectors with frameshift mutations were separately propagated and were pooled to infect DSDh helper cells. Doubly resistant cell clones were isolated, and viruses produced from these clones were used to infect D17 cells. This protocol allowed virions containing two different genomic RNAs (heterozygotes) to complete one round of retroviral replication. The molecular nature of progeny that conferred resistance to single or double selection and their ratio were determined. Our data demonstrate that each infectious heterozygous virion produces only one provirus. The rate of retroviral recombination is approximately 2% per kilobase per replication cycle. Recombinant proviruses are progeny of heterozygous virions.
Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.
Authors: Beckman J S , Beckman T W , Chen J , Marshall P A , Freeman B A
Published: May 2006

Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.
Design of a membrane transport protein for fluorescence spectroscopy.
Authors: Menezes M E , Roepe P D , Kaback H R
Published: May 2006

To modify the lac permease of Escherichia coli for fluorescence spectroscopy, six tryptophan residues at positions 10, 33, 78, 151, 171, and 223 were first replaced individually with phenylalanine by using oligonucleotide-directed site-specific mutagenesis. None of the tryptophan residues is critical for activity, as evidenced by the finding that the mutant permease molecules catalyze lactose/H+ symport almost as well as wild-type permease. Subsequently, a permease molecule was designed in which all of the tryptophan residues were replaced with phenylalanine. Remarkably, the lac permease harboring all six mutations catalyzes active lactose transport about 75% as well as wild-type permease. The fluorescence emission spectrum of purified wild-type permease solubilized in octyl beta-D-glucopyranoside and phospholipid exhibits a broad maximum centered at 350 nm, and the peak is almost completely absent from the spectrum of permease devoid of tryptophan. Furthermore, a new maximum centered at about 306 nm is apparent in the spectrum of the modified permease, suggesting that tyrosine fluorescence in the native protein is quenched by internal energy transfer to tryptophan residues. By using site-directed mutagenesis to replace specified residues in the molecule without tryptophan, it should now be possible to utilize tryptophan fluorescence spectroscopy to study static and dynamic aspects of permease structure and function.
Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats.
Authors: Lee F S , Vallee B L
Published: May 2006

Human placental ribonuclease inhibitor (PRI) is a potent protein inhibitor of pancreatic ribonucleases and the homologous blood vessel-inducing protein angiogenin. Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats. These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis. Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit ribonuclease A. Therefore, the angiogenin/ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these. Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity. Hence, the binding site cannot reside exclusively in repeat 5. Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity. These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module. The approach taken may be applicable to other proteins with repeat structures.
Multiple conductance classes of mouse nicotinic acetylcholine receptors expressed in Xenopus oocytes.
Authors: Kullberg R , Owens J L , Camacho P , Mandel G , Brehm P
Published: May 2006

Acetylcholine receptor (AcChoR) subunit mRNAs transcribed from mouse BC3H-1 cDNAs were injected into Xenopus oocytes and the expressed AcChoR channels were examined by single channel recording. Injection of alpha-, beta-, gamma-, and delta-subunit mRNAs produced two predominant channel classes with conductances of approximately 50 and approximately 12 pS, while infrequent openings of approximately 25-pS channels were also observed. Injection of alpha-, beta-, and gamma-subunit mRNAs produced a single class of approximately 12-pS AcChoR channels, which resembled the smallest conductance channels present in alpha beta gamma omega-injected oocytes. Assembly of delta-less channels may thus explain the lowest conductance AcChoR channels in alpha beta gamma delta-injected oocytes and might also account for similar channels that have been observed in vertebrate skeletal muscle.
Glycine decreases desensitization of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes and is required for NMDA responses.
Authors: Lerma J , Zukin R S , Bennett M V
Published: May 2006

In Xenopus oocytes injected with rat brain mRNA, as in neurons, glycine greatly potentiated responses of the N-methyl-D-aspartate (NMDA) type of excitatory amino acid receptor. Injected oocytes generated a partially desensitizing inward current in response to NMDA with 30 nM added glycine. As the added glycine concentration was increased from 30 nM to 1 microM, the NMDA response was increased and exhibited less desensitization. The relationship between the NMDA peak response and added glycine concentration indicated a single component response with apparent affinity of 0.29 microM and a Hill coefficient of 0.77. The desensitized response was also fit by the Hill relation with a lower affinity but similar coefficient. The time course of desensitization at 500 microM NMDA was exponential with a time constant (350 msec) that was independent of glycine concentration between 0.03 and 0.3 microM. At higher glycine concentration a slower component of decay (tau = 1.4 sec) was observed. This component was enhanced by increasing the extracellular Ca2+. NMDA without added glycine evoked a small transient response. However this response was suppressed completely by prewashing with the glycine antagonist 7-chlorokynurenic acid, suggesting that it may have been due to glycine contamination. The dose-response relation for low concentrations of glycine indicated that the measured level of glycine contamination accounted for these responses. These results indicate that glycine has at least two actions at the NMDA receptor: it enables channel opening by the agonist and decreases desensitization.
Amino acid and cDNA sequences of a vascular endothelial cell mitogen that is homologous to platelet-derived growth factor.
Authors: Conn G , Bayne M L , Soderman D D , Kwok P W , Sullivan K A , Palisi T M , Hope D A , Thomas K A
Published: May 2006

Glioma-derived vascular endothelial cell growth factor (GD-VEGF) is a 46-kDa dimeric glycoprotein mitogen with apparently greater specificity for vascular endothelial cells than the well-characterized fibroblast growth factors. The GD-VEGF cDNA sequence encodes a 190-amino acid residue subunit that is converted, by removal of an amino-terminal hydrophobic secretory leader sequence, to the mature 164-residue subunit characterized by direct amino acid sequencing. The GD-VEGF homodimeric subunit is homologous to the platelet-derived growth factor A and B chains and its oncogene homologue v-sis.
Correlation in the discharges of neighboring rat retinal ganglion cells during prenatal life.
Authors: Maffei L , Galli-Resta L
Published: May 2006

The spontaneous discharges of neighboring retinal ganglion cells were recorded simultaneously in anesthetized prenatal rats between embryonic days 18 and 21. We report here that in the majority of cases the firings of neighboring retinal ganglion cells are strongly correlated during prenatal life. Correlation in the discharges of neighboring cells during development has long been suggested as a way to consolidate synaptic connections with a target cell onto which they converge, a model first proposed by Hebb. Correlation in the activities of neighboring neurons in the retina could be the basis of developmental processes such as refinement of retinotopic maps in the brain and segregation of the inputs from the two eyes.
Molecular epidemiology of human hepatitis A virus defined by an antigen-capture polymerase chain reaction method.
Authors: Jansen R W , Siegl G , Lemon S M
Published: May 2006

We describe an immunoaffinity-linked nucleic acid amplification system (antigen-capture/polymerase chain reaction, or AC/PCR) for detection of viruses in clinical specimens and its application to the study of the molecular epidemiology of a picornavirus, hepatitis A virus (HAV). Immunoaffinity capture of virus, synthesis of viral cDNA, and amplification of cDNA by a polymerase chain reaction (PCR) were carried out sequentially in a single reaction vessel. This approach simplified sample preparation and enhanced the specificity of conventional PCR. AC/PCR detected less than one cell culture infectious unit of virus in 80 microliters of sample. Sequencing of AC/PCR reaction products from 34 virus strains demonstrated remarkable conservation at the nucleotide level among most strains but revealed hitherto unsuspected genetic diversity among human isolates. Epidemiologically related strains were identical or closely related in sequence. Virus strains recovered from epidemics of hepatitis A in the United States and Germany were identical in sequence, providing evidence for a previously unrecognized epidemiologic link between these outbreaks.
Rapid neural regulation of muscle urokinase-like plasminogen activator as defined by nerve crush.
Authors: Hantaï D , Rao J S , Festoff B W
Published: May 2006

Muscle plasminogen activators (PAs), such as urokinase-like PA and, to a lesser extent, tissue PA, increase dramatically after denervation induced by axotomy. The PA/plasmin system has also been implicated in degradation of specific components of the muscle fiber basement membrane after local activation of plasminogen. These results suggest that neural regulation of muscle extracellular matrix metabolism accompanies or precedes regeneration after injury and is mediated by activation of PAs. In the present study, we have used nerve crush to explore the neural regulation of muscle PA activities directly after subtotal axon interruption and during the process of reinnervation. Muscle contraction after nerve stimulation and estimation of choline acetyltransferase activity were used to monitor reinnervation. Within 24 hr of nerve crush, muscle urokinase (but not tissue PA) activity rose in soluble and membrane-bound muscle fractions, as shown by an amidolytic assay and a fibrin zymography. Membrane-bound activity was 5-fold higher than cytosol activity, but there was no shift between cellular compartments during the time course of denervation. Coincident with the return of choline acetyltransferase activity and muscle contractility, muscle urokinase returned almost to baseline levels. These results show tight regulation of muscle urokinase levels by some neural influence.
Amino acids in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum involved in cycloguanil resistance differ from those involved in pyrimethamine resistance.
Authors: Foote S J , Galatis D , Cowman A F
Published: May 2006

Cycloguanil, the active metabolite of the antimalarial drug proguanil, is an inhibitor of dihydrofolate reductase as is another antimalarial, pyrimethamine. Its use has been limited by the rapid development of resistance by parasites around the world. We have determined the cycloguanil- and pyrimethamine-sensitivity status of 10 isolates of Plasmodium falciparum and have sequenced in all these isolates the dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) portion of the DHFR-thymidylate synthase (TS; 5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) gene. Instead of the known serine-to-asparagine change at position 108 that is important in pyrimethamine resistance, a serine-to-threonine change at the same position is found in cycloguanil-resistant isolates along with an alanine-to-valine change at position 16. We conclude that pyrimethamine and cycloguanil resistance most commonly involve alternative mutations at the same site. However, we also have identified a parasite with a unique set of changes that results in resistance to both drugs.
Molecular differences among neurons reveal an organization of human visual cortex.
Authors: Hockfield S , Tootell R B , Zaremba S
Published: May 2006

Monoclonal antibody Cat-301 recognizes a cell-surface proteoglycan on subsets of neurons in several areas of the cat and macaque monkey central nervous system. In striate and extrastriate visual cortex of the macaque, the distribution of Cat-301-positive neurons demonstrates features of cellular organization that correlate with previously described functional subdivisions. Here we show that Cat-301 recognizes an antigen in human cortex that is closely related, if not identical, to the antigen in laboratory animals. Further, we use Cat-301 to demonstrate an organization of molecularly defined neurons in primary and secondary visual cortex (cortical areas V1 and V2) of the human. The organization demonstrated with Cat-301 in human area V1 correlates with the organization of ocular dominance columns demonstrated by cytochrome oxidase histochemistry. The organization demonstrated with Cat-301 in human area V2 correlates with the thick stripes of the cytochrome oxidase pattern. The present observations provide evidence for a visual pathway in human cortex homologous to the magnocellular pathway in macaque, a pathway involved in processing the low-contrast, achromatic, and moving components of visual stimuli.
RTA, a candidate G protein-coupled receptor: cloning, sequencing, and tissue distribution.
Authors: Ross P C , Figler R A , Corjay M H , Barber C M , Adam N , Harcus D R , Lynch K R
Published: May 2006

Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. Sequence analysis of the genomic clone further showed that the RTA gene has an intron interrupting the region encoding the amino terminus of the protein. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. RTA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. We cannot detect angiotensin binding to the RTA protein after introducing the cognate cDNA or mRNA into COS cells or Xenopus oocytes, respectively, nor can we detect an electrophysiologic response in the oocyte after application of angiotensin peptides. We conclude that RTA is not an angiotensin receptor; to date, we have been unable to identify its ligand.
Genomic analysis of diploid plants.
Authors: Kimber G , Yen Y
Published: May 2006

The numerical analysis of meiosis in the triploid hybrid between an induced autotetraploid and a related diploid provides the basis for investigations of the genomic relationships of diploid species based on chromosome pairing. It is also possible to make legitimate comparisons between different triploid hybrids of this type so that the genomic relationships can be converted into measures of phylogenetic distance for taxa where only diploids are known.
Revision of the oligosaccharide structures of yeast carboxypeptidase Y.
Authors: Ballou L , Hernandez L M , Alvarado E , Ballou C E
Published: May 2006

The N-linked oligosaccharides from baker's yeast carboxypeptidase Y were analyzed by 1H NMR and specific mannosidase digestion and found to be identical to those from the Saccharomyces cerevisiae mnn9 mutant bulk mannoprotein. The results support the view that the mnn mutants make oligosaccharides that are a true reflection of the normal biosynthetic pathway and confirm that a recently revised yeast oligosaccharide structure is applicable to wild-type mannoproteins.
Human papillomavirus type 16 nucleoprotein E7 is a tumor rejection antigen.
Authors: Chen L P , Thomas E K , Hu S L , Hellström I , Hellström K E
Published: May 2006

It has been speculated that immunological mechanisms play an important role in the control of carcinomas associated with human papillomavirus (HPV), such as cervical cancers. We have now demonstrated that immunization of C3H/HeN mice by syngeneic nontumorigenic fibroblast-like cells that contain the transfected HPV-16 E7 gene conferred protection against transplanted cells from a HPV-16 E7-positive syngeneic tumor. This protection was HPV-16 E7-specific and was mediated by CD8+ lymphocytes, which presumably were cytotoxic T lymphocytes. These results indicate that tumor cells containing HPV-16 E7, either as a result of transfection, as in our studies, or naturally, as occurs in many human carcinomas, can induce a tumor-specific rejection response and serve as targets for such a response. The system described here provides an animal model to further study immune responses to HPV-associated malignancies and to test the efficacy of anti-HPV vaccines toward the therapy and prevention of such tumors.
Increased gene expression of Alzheimer disease beta-amyloid precursor protein in senescent cultured fibroblasts.
Authors: Adler M J , Coronel C , Shelton E , Seegmiller J E , Dewji N N
Published: May 2006

The pathological hallmark of Alzheimer disease is the accumulation of neurofibrillary tangles and neuritic plaques in the brains of patients. Plaque cores contain a 4- to 5-kDa amyloid beta-protein fragment which is also found in the cerebral blood vessels of affected individuals. Since amyloid deposition in the brain increases with age even in normal people, we sought to establish whether the disease state bears a direct relationship with normal aging processes. As a model for biological aging, the process of cellular senescence in vitro was used. mRNA levels of beta-amyloid precursor protein associated with Alzheimer disease were compared in human fibroblasts in culture at early passage and when the same fibroblasts were grown to senescence after more than 52 population doublings. A dramatic increase in mRNA was observed in senescent IMR-90 fibroblasts compared with early-passage cells. Hybridization of mRNA from senescent and early proliferating fibroblasts with oligonucleotide probes specific for the three alternatively spliced transcripts of the gene gave similar results, indicating an increase during senescence of all three forms. A similar, though more modest, increase in message levels was also observed in early-passage fibroblasts made quiescent by serum deprivation; with repletion of serum, however, the expression returned to previous low levels. ELISAs were performed on cell extracts from senescent, early proliferating, and quiescent fibroblasts, and quiescent fibroblasts repleted with serum for over 48 hr, using polyclonal antibodies to a synthetic peptide of the beta-amyloid precursor. The results confirmed that the differences in mRNA expression were partially reflected at the protein level. Regulated expression of beta-amyloid precursor protein may be an important determinant of growth and metabolic responses to serum and growth factors under physiological as well as pathological conditions.
Correlation between amount of virus with altered nucleotide sequence and the monkey test for acceptability of oral poliovirus vaccine.
Authors: Chumakov K M , Powers L B , Noonan K E , Roninson I B , Levenbook I S
Published: May 2006

Production of live attenuated oral poliomyelitis vaccine (OPV) requires rigorous neurovirulence safety testing of each vaccine lot, currently carried out in monkeys. It has been reported that a change from 472-U to 472-C in the type 3 OPV RNA is associated with an increased histologic lesion score produced upon intraspinal inoculation of the mutant virus in monkeys. We have developed a method, based on polymerase chain reaction, for measuring the relative abundance of these mutant sequences directly in vaccine preparations and used this method to evaluate the proportion of 472-C in 40 different lots of type 3 OPV. Six vaccine lots that had failed the intraspinal monkey neurovirulence test contained a higher proportion of 472-C than all other lots that had passed this test. OPV type 3 virus containing 472-C was rapidly selected during serial passages in African green monkey kidney cells that are used for manufacturing of the vaccine. We have also found that the wild-type poliovirus type 3 strain Leon/37, from which the vaccine strain was originally derived, contained a mixture of 472-U and 472-C sequences. No other mutations in OPV type 3 RNA have been detected by similar assays at position 2034, also associated with attenuation, or at several other positions reported to be altered in some vaccine preparations. Our results suggest that molecular diagnostics may provide a supplement or a potential alternative to animal testing of live attenuated vaccines.
Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain.
Authors: Altar C A , Burton L E , Bennett G L , Dugich-Djordjevic M
Published: May 2006

Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity (Kd 52-85 pM) and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.
Cooperative foraging, productivity, and the central limit theorem.
Authors: Wenzel J W , Pickering J
Published: May 2006

The central limit theorem is applied to group foraging to show an automatic and universal benefit to group living. This may explain the paradoxical inverse correlation between group size and per capita brood production in primitively eusocial insects and why only one of the five major lineages of social insects contains species that revert to solitary habit.
The promoter of the latency-associated transcripts of herpes simplex virus type 1 contains a functional cAMP-response element: role of the latency-associated transcripts and cAMP in reactivation of viral latency.
Authors: Leib D A , Nadeau K C , Rundle S A , Schaffer P A
Published: May 2006

A 203-base-pair sequence 5' of the latency-associated transcripts (LATs) of herpes simplex virus type 1 contains a 7-base consensus sequence TGCGTCA that is identical to the cAMP-response element of the proenkephalin gene. This consensus sequence is at -38 relative to the putative 5' end of the LATs with a TATA box at the -24 position. In transient chloramphenicol acetyltransferase assays in rat pheochromocytoma (PC12) cells, this enhancer region stimulated gene expression up to 3-fold in the presence of dibutyryl cAMP, forskolin, nerve growth factor, or phorbol 12-myristate 13-acetate. Mutation of the cAMP-response element to TGCG-CAA resulted in a 4-fold reduction of basal activity and a complete loss of inducible stimulation. In DNA gel retardation assays, purified cAMP-response element-binding protein and a nuclear protein from PC12 cells were shown to bind specifically to this element. Furthermore, it was demonstrated that the reactivation of wild-type herpes simplex virus type 1 from dissociated latently infected murine trigeminal ganglia was significantly accelerated (P less than 0.005) by the addition of cAMP analogs or adenylate cyclase activators. However, these reagents did not accelerate reactivation of a deletion mutant that lacks the putative cAMP-response element-containing promoter region, transcriptional start site, and 1015 base pairs of the LATs. These studies demonstrate that the promoter region of the LATs contains a functional cAMP-response element and that expression of the LATs is likely controlled by second messenger signal transduction and imply a role for cAMP in triggering viral reactivation.
A link between double-strand break-related repair and V(D)J recombination: the scid mutation.
Authors: Hendrickson E A , Qin X Q , Bump E A , Schatz D G , Oettinger M , Weaver D T
Published: May 2006

We show here that mammalian site-specific recombination and DNA-repair pathways share a common factor. The effects of DNA-damaging agents on cell lines derived from mice homozygous for the scid (severe combined immune deficiency) mutation were studied. Surprisingly, all scid cell lines exhibited a profound hypersensitivity to DNA-damaging agents that caused double-strand breaks (x-irradiation and bleomycin) but not to other chemicals that caused single-strand breaks or cross-links. Neutral filter elution assays demonstrated that the x-irradiation hypersensitivity could be correlated with a deficiency in repairing double-strand breaks. These data suggest that the scid gene product is involved in two pathways: DNA repair of random double-strand breaks and the site-specific and lymphoid-restricted variable-(diversity)-joining [V(D)J] DNA rearrangement process. We propose that the scid gene product performs a similar function in both pathways and may be a ubiquitous protein.
Heat shock-regulated transcription in vitro from a reconstituted chromatin template.
Authors: Becker P B , Rabindran S K , Wu C
Published: May 2006

To investigate the mechanisms of transcriptional regulation of Drosophila heat shock genes we studied the activity of a heat shock promoter in vitro after reconstitution into chromatin. Increasing the duration of nucleosome assembly progressively inactivated a plasmid template when it was transcribed with extracts of either unshocked or heat-shocked Drosophila embryos, despite induction of the transcriptional activator heat shock factor. Addition of the general transcription factor IID (TFIID) before nucleosome assembly did not significantly relieve nucleosomal inhibition, but TFIID potentiated the promoter to be responsive to activation by heat shock factor in the heat shock transcription extract. The potentiation by TFIID could be related to the nucleosome-free, hypersensitive state of heat shock promoters previously observed in vivo before heat shock induction and may be necessitated by the need to expedite activation of heat shock genes in response to environmental stress.
Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages.
Authors: Weinstein S L , Gold M R , DeFranco A L
Published: May 2006

Lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, stimulates immune responses by activating macrophages, B lymphocytes, and other cells of the immune system. The mechanisms by which LPS activates these cells are poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signaling event that mediates cellular responses, we examined whether LPS alters tyrosine phosphorylation in macrophages. We found that Escherichia coli K235 LPS increased tyrosine phosphorylation of several proteins in the RAW 264.7 murine macrophage cell line and in resident peritoneal macrophages from C3H/HeSNJ mice. Changes in tyrosine phosphorylation were detectable by 4-5 min, reached a maximum by 15 min, and declined after 30-60 min. Protein tyrosine phosphorylation increased following stimulation with LPS at 100 pg/ml and was maximal with 10 ng/ml. Similar changes in tyrosine phosphorylation were induced by Salmonella minnesota R595 LPS and by the biologically active domain of LPS, lipid A, but not by the inactive lipid A derivative N2-monoacylglucosamine 1-phosphate. Phorbol 12-myristate 13-acetate also stimulated protein tyrosine phosphorylation, but some of the modulated proteins were different than those phosphorylated by LPS. Treatment of RAW 264.7 cells with a tyrosine kinase inhibitor, herbimycin A, inhibited both LPS-stimulated tyrosine phosphorylation and LPS-stimulated release of arachidonic acid metabolites. Thus, increased protein tyrosine phosphorylation is a rapid LPS-activated signaling event that may mediate release of arachidonic acid metabolites in RAW 264.7 cells.
Protein stability: electrostatics and compact denatured states.
Authors: Stigter D , Alonso D O , Dill K A
Published: May 2006

Globular proteins can be denatured by changing pH and ionic strength. Much recent evidence has led to the surprising conclusion that there are two acid-denatured states: one highly unfolded and the other more compact, sometimes called the "molten globule." Here we describe a molecular theory for electrostatic stability of globular proteins based on the properties of the constituent amino acids: oil/water partition coefficients, pK values of the titratable groups, and their temperature dependences. Predicted denaturation temperatures vs. pH are in good agreement with experiments of other workers on myoglobin. The theory also predicts two populations of denatured species, one open and the other more compact, with densities in the range found experimentally for molten globular states. In addition, it predicts a phase diagram (stability vs. pH, ionic strength) in good agreement with experiments of Goto and Fink [Goto, Y. & Fink, A. L. (1989) Biochemistry 28, 945-952; and Goto, Y. & Fink, A. L. (1990) J. Mol. Biol. 214, 803-805]. The well-known salt destabilization of myoglobin has been generally considered evidence for ion pairing, but the present theory, based on smeared charge repulsion, explains the salt destabilization at low pH without ion pairing. In addition, for myoglobin the theory predicts salt stabilization at high pH, as observed for beta-lactamase by Goto and Fink.
Retinoid-mediated transcriptional regulation of keratin genes in human epidermal and squamous cell carcinoma cells.
Authors: Stellmach V , Leask A , Fuchs E
Published: May 2006

Vitamin A and other retinoids profoundly inhibit morphological and biochemical features of epidermal differentiation in vivo and in vitro. To elucidate the molecular mechanisms underlying the differential expression of epidermal keratins and their regulation by retinoids, we examined retinoid-mediated changes in total protein expression, protein synthesis, mRNA expression, and transcription in cultured human keratinocytes and in squamous cell carcinoma (SCC-13) cells of epidermal origin. Our studies revealed that the epidermal keratins, K5, K6, K14, and K16, their mRNAs, and their transcripts were diminished relative to actin as a consequence of retinoic acid (RA) treatment. The effects were most pronounced in SCC-13 and were detected as early as 6 hr post-RA treatment, with enhancement over an additional 24-48 hr. Repression was also observed when 5' upstream sequences of K14 or K5 genes were used to drive expression of a chloramphenicol acetyltransferase reporter gene in SCC-13 keratinocytes. Both cell types were found to express mRNAs for the RA receptors alpha and gamma, which may be involved in the RA-mediated transcriptional changes in these cells. The rapid transcriptional changes in epidermal keratin genes were in striking contrast to the previously reported slow transcriptional changes in simple epithelial keratin genes.
Zone-specific regulation of two messenger RNAs for P450c11 in the adrenals of pregnant and nonpregnant rats.
Authors: Malee M P , Mellon S H
Published: May 2006

Adrenal mitochondria possess two steroidogenic cytochrome P450s. P450c11 converts deoxycorticosterone to corticosterone and aldosterone, and P450scc converts cholesterol to pregnenolone. These P450s receive electrons from NADPH via adrenodoxin reductase and adrenodoxin. A single bovine P450c11 protein has 11-hydroxylase, 18-hydroxylase, and 18-oxidase activities, but this series of enzymatic steps may be mediated by more than one enzyme in rats. Enzymatic assays of purified rat mitochondrial proteins have suggested that one enzyme found in all zones of the adrenal cortex has both 11- and 18-hydroxylase activities, whereas another enzyme, found exclusively in the zona glomerulosa, catalyzes 18-hydroxylation and 18-oxidation of corticosterone. We studied the number and zonal distribution of P450c11 mRNA species in the rat adrenal and how these mRNAs are regulated in the adrenals of normal and pregnant rats. Rats synthesize two similar, but distinct, P450c11 mRNAs. One, P450c11A, is found in both the zona glomerulosa and fasciculata/reticularis, whereas the second, P450c11B, is found only in the zona glomerulosa. The abundance of neither P450c11A mRNA nor P450c11B mRNA is affected by a high-salt diet. However, when rats receive a low-salt diet, P450c11A mRNA decreases and P450c11B mRNA increases. Dexamethasone decreases the amount of P450c11A mRNA without affecting P450c11B mRNA. The combination of a high-salt diet and dexamethasone decreases the amount of both mRNAs further to almost undetectable amounts. Rats given a low-salt diet and dexamethasone have a dramatic increase in the abundance of P450c11B mRNA. Thus both forms of P450c11 mRNA are regulated independently in the rat adrenal cortex. In situ hybridization studies show that only the P450c11 found in the zona glomerulosa is regulated by salt treatment in vivo, whereas glucocorticoid treatment in vivo regulates P450c11 in all zones. In the adrenals of pregnant rats, P450c11B is regulated in a similar fashion to its regulation in the nonpregnant rat adrenal, despite major differences in sodium retention and intravascular volume in pregnant and nonpregnant rats. In the pregnant rat, a low-salt diet increases the abundance of P450c11B to a greater degree than in the nonpregnant rat. By contrast, dexamethasone does not diminish the abundance of P450c11A mRNA in the pregnant rat but reduces it to an almost undetectable amount in the nonpregnant rat. Thus, the regulation of glucocorticoid and mineralocorticoid production in the pregnant and nonpregnant rat occurs by different mechanisms.
Genetic basis of X-Y chromosome dissociation and male sterility in interspecific hybrids.
Authors: Matsuda Y , Hirobe T , Chapman V M
Published: May 2006

A high frequency of X-Y chromosome dissociation (95%) was found at first meiotic metaphase (MI) in spermatocytes of interspecific hybrids between laboratory mice, C57BL/6J (BL/6) and Mus spretus, compared with an X-Y dissociation frequency of only 3-4% in parental mice. The X-Y dissociation in F1 hybrids occurred before diakinesis rather than as a precocious dissociation at MI. The high X-Y dissociation was accompanied by spermatogenic breakdown after MI, resulting in male sterility. All F1 males were sterile and approximately half of the backcross males from fertile F1 females crossed with either BL/6 or M. spretus males were sterile. Male sterility was highly correlated with X-Y dissociation in both backcrosses. All of the mice with high X-Y dissociation were sterile and all of the males with low X-Y dissociation were fertile or subfertile. This correlation suggested that genetic divergence of the X-Y pairing region could contribute to the male sterile phenotype such that the BL/6 X chromosome would not pair with the M. spretus Y chromosome. The segregation of species-type alleles of amelogenin (Amelb and Amels), a distal X chromosome locus adjacent to the X-Y pairing region, was followed in backcross males that were analyzed for X-Y dissociation and sterility (we have used Amel as the designation for the mouse amelogenin locus; the current designation for this locus is Amg). A 95% concordance between Amelb with fertility and Amels with sterility was observed in backcrosses with BL/6, whereas the converse was observed in the backcross to M. spretus. These results imply that X-Y pairing plays an important role in male fertility and suggest that genetic divergence in X-Y pairing region between Mus species can contribute to the reproductive barriers between species and the process of speciation.
Receptor protein-tyrosine phosphatase gamma is a candidate tumor suppressor gene at human chromosome region 3p21.
Authors: LaForgia S , Morse B , Levy J , Barnea G , Cannizzaro L A , Li F , Nowell P C , Boghosian-Sell L , Glick J , Weston A
Published: May 2006

PTPG, the gene for protein-tyrosine phosphatase gamma (PTP gamma), maps to a region of human chromosome 3, 3p21, that is frequently deleted in renal cell carcinoma and lung carcinoma. One of the functions of protein-tyrosine phosphatases is to reverse the effect of protein-tyrosine kinases, many of which are oncogenes, suggesting that some protein-tyrosine phosphatase genes may act as tumor suppressor genes. A hallmark of tumor suppressor genes is that they are deleted in tumors in which their inactivation contributes to the malignant phenotype. In this study, one PTP gamma allele was lost in 3 of 5 renal carcinoma cell lines and 5 of 10 lung carcinoma tumor samples tested. Importantly, one PTP gamma allele was lost in three lung tumors that had not lost flanking loci. PTP gamma mRNA was expressed in kidney cell lines and lung cell lines but not expressed in several hematopoietic cell lines tested. Thus, the PTP gamma gene has characteristics that suggest it as a candidate tumor suppressor gene at 3p21.
Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein.
Authors: Flaherty K M , McKay D B , Kabsch W , Holmes K C
Published: May 2006

Although there is very little sequence identity between the two proteins, the structures of rabbit skeletal muscle actin (375-amino acid residues) and the 44-kDa ATPase fragment of the bovine 70-kDa heat shock cognate protein (HSC70; 386 residues) are very similar. The alpha-carbon positions of 241 pairs of amino acid residues that are structurally equivalent within the two proteins can be superimposed with a root-mean-square difference in distance of 2.3 A; of these, 39 residues are identical, and 56 are conservative substitutions. In addition, the conformations of ADP are very similar in both proteins. A local sequence "fingerprint," which may be diagnostic of the adenine nucleotide beta-phosphate-binding pocket, has been derived. The fingerprint identifies members of the glycerol kinase family as candidates likely to have a similar structure in their nucleotide-binding domains. The structural differences between the two molecules mainly occur in loop regions of actin known to be involved in interactions with other monomers in the actin filament or in the binding of myosin; the corresponding regions in heat shock proteins may have functions that are as yet undetermined. Placing the Ca2+ ATP of actin on the ATPase fragment structure suggests Asp-206 (corresponding to His-161 of actin) as a candidate proton acceptor for the ATPase reaction.