bluuebunny/crossref_metadata_2025 huggingface dataset:
Norepinephrine and isoproterenol increase the phosphorylation of synapsin I and synapsin II in dentate slices of young but not aged Fisher 344 rats.
Authors: Parfitt K D , Hoffer B J , Browning M D
Published: May 2006

A number of recent reports have suggested that norepinephrine (NE) produces a form of synaptic enhancement that resembles long-term potentiation (LTP). LTP, thought to be an electrophysiological correlate of memory, in part involves an augmentation of transmitter release. Although the effects of NE have not been unequivocally linked to LTP, it is clear that NE can produce increased transmitter release in the dentate gyrus of the hippocampus. The purpose of this study was to determine whether NE was capable of enhancing the phosphorylation of synapsin I and synapsin II, two homologous phosphoproteins thought to be involved in modulation of neurotransmitter release. NE (10 microM) and isoproterenol (250 nM) produced an increase in the phosphorylation of synapsin I and synapsin II in dentate slices from young rats. Phosphorylation site analysis of synapsin I, performed by limited proteolysis, indicated that NE and isoproterenol increased the phosphorylation of synapsin I at sites modified by Ca2+/calmodulin-dependent protein kinase II as well as cAMP-dependent protein kinase. These data demonstrate that NE stimulates the phosphorylation of synapsin I at its Ca2+/calmodulin-dependent protein kinase II site, which is a site that has been shown to regulate the effect of synapsin I on neurotransmitter release. We have also examined the effects of NE and isoproterenol on synapsin phosphorylation in dentate slices prepared from aged animals. Such animals have previously been shown to exhibit deficits in NE sensitivity as well as significant impairment in their ability to exhibit LTP. Neither NE nor isoproterenol stimulated synapsin phosphorylation in slices prepared from aged animals. Interestingly, the basal level of phosphorylation of the synapsin proteins was higher in slices prepared from aged animals. This higher basal level of phosphorylation may underlie the failure of aged animals to exhibit NE-stimulated increases in phosphorylation of the synapsin proteins. We hypothesize that the beta-adrenergic agonist-stimulated phosphorylation of synapsin I and synapsin II in young rats plays a role in the increase in transmitter release produced by NE in the dentate. Thus, the failure of the aged rats to show such phosphorylation may underlie, in part, their failure to exhibit normal responsiveness to NE. Moreover, these deficits in synapsin phosphorylation may also play some role in the deficits in plasticity seen in aged rats.
TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.
Authors: Leskiw B K , Lawlor E J , Fernandez-Abalos J M , Chater K F
Published: May 2006

In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bldA, which specifies a tRNA(Leu)-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bldA. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bldA-independent expression; hence the bldA product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bldA mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation.
Restricted T-cell receptor V beta gene usage by myelin basic protein-specific T-cell clones in multiple sclerosis: predominant genes vary in individuals.
Authors: Ben-Nun A , Liblau R S , Cohen L , Lehmann D , Tournier-Lasserve E , Rosenzweig A , Zhang J W , Raus J C , Bach M A
Published: May 2006

Recent studies in experimental autoimmune encephalomyelitis as a model for multiple sclerosis (MS) have demonstrated limited heterogeneity in T-cell antigen receptors (TCR) specific for myelin basic protein (MBP). To investigate restricted beta-chain variable-region (V beta) gene usage in humans, we analyzed TCR gene rearrangements in two lines and 34 MBP-specific T-cell clones that were isolated from five MS patients and two healthy subjects. The T cells were characterized for their specificity to MBP epitopes and HLA-restricting molecules. We demonstrate here that MBP-specific T-cell clones from these different MS patients and healthy individuals, in contrast to T cells from rodents, display a more diverse V beta gene usage as evidenced by their TCR V beta gene rearrangements. However, the different MBP-specific T-cell clones isolated from each individual MS patient showed a common V beta gene usage, suggesting individual-specific TCR restriction. Out of 16 MBP-specific clones derived from a single MS patient, 12 clones (75%) utilized the V beta 15 gene for their TCR gene rearrangement. MBP-specific clones isolated from four other MS patients also showed a consistent tendency for a predominant, but different, TCR V beta gene rearrangement. These results suggest a TCR heterogeneity among MBP-specific T-cell clones from different individuals but a limited TCR V beta gene usage among MBP-specific T-cell clones of the same individual. The predominant V beta gene used by the MBP-specific T-cell clones studied here was not found to correlate with the epitope specificity of T cells or with their restricting HLA molecule. These findings may support the possibility of intervention with monoclonal antibodies to specific V beta gene products as an approach to immune therapy of MS but also imply the necessity for an individual-specific immunotherapeutic approach.
Proton transfer is rate-limiting for translocation of precursor proteins by the Escherichia coli translocase.
Authors: Driessen A J , Wickner W
Published: May 2006

The protonmotive force stimulates translocation in vivo, in crude in vitro reactions, and in a purified, reconstituted reaction. Translocation activity is a function of the pH at the inner face of the membrane. Both the transmembrane pH gradient and the transmembrane electrical potential stimulate translocation. A late-stage translocation intermediate of the proOmpA preprotein completes its translocation in the absence of ATP when a protonmotive force is imposed. This completion of translocation is retarded by a factor of greater than 3 in deuterium oxide relative to water, demonstrating that translocation involves proton-transfer reactions in rate-limiting steps.
Characterization of a nuclear protein that binds to three elements within the silencer region of a bean chalcone synthase gene promoter.
Authors: Harrison M J , Lawton M A , Lamb C J , Dixon R A
Published: May 2006

The chalcone synthase (EC 2.3.1.74) gene promoter from the bean Phaseolus vulgaris L. contains a silencer element between positions -140 and -326 fro the transcription start site that is functional in electroporated soybean protoplasts. This element contains three binding sites for a bean nuclear factor (SBF-1) with DNA sequence recognition properties that are very similar to those of nuclear factor GT-1. By using a synthetic tetramer of one of the binding sites as probe, we have purified sequence-specific SBF-1 activity approximately 1750-fold from suspension-cell nuclei, by using a combination of ammonium sulfate precipitation, gel filtration, heparin-agarose chromatography, and sequence-specific DNA affinity chromatography. The factor exhibited an apparent molecular weight of 160,000-200,000 on the basis of gel filtration. A subunit molecular weight of approximately 95,000 was determined from SDS/polyacrylamide gel electrophoretic analysis of purified fractions, followed by Southwestern blot analysis (a protein blot probed with oligonucleotide probes), and from UV-cross-linking experiments. The factor lost DNA-binding activity on treatment with alkaline phosphatase. We discuss the properties of SBF-1 in relation to the functionality of GT-1 binding sequences in plant genes.
Interspecific gene flow in sympatric oaks.
Authors: Whittemore A T , Schaal B A
Published: May 2006

Variation of chloroplast DNA and nuclear ribosomal DNA (DNA encoding ribosomal RNA) was studied for five species of white oak native to the eastern United States. Although these species differ in many morphological characters and have different (though overlapping) geographical ranges and ecological tolerances, they are interfertile and often grow in mixed stands, and hybrids are occasionally found in nature. All individuals studied were morphologically typical members of their respective species-i.e., showed no evidence of recent hybrid ancestry. Restriction site markers in the chloroplast DNA reveal several clear cases of localized gene exchange between species, showing that there is appreciable gene flow between sympatric species in this group. One length variant of the nuclear ribosomal DNA, however, is species specific. The sharp morphological and ecological differences between the species, together with the one ribosomal DNA variant, suggest that nuclear genes may be exchanged less freely between species than are chloroplast genotypes.
Receptors of the serotonin 1C subtype expressed from cloned DNA mediate the closing of K+ membrane channels encoded by brain mRNA.
Authors: Panicker M M , Parker I , Miledi R
Published: May 2006

The modulation of K+ channels by serotonin (5-HT) receptors was studied by coinjecting Xenopus oocytes with mRNA transcribed in vitro from a cloned 5-HT 1C subtype (5-HT1C) receptor gene, together with size-fractionated mRNA isolated from rat cerebral cortex that expresses K+ channels. After intracellular loading with EGTA to block Ca2(+)-dependent chloride currents, these oocytes responded to 5-HT with an inward current associated with a decrease in membrane conductance. Membrane current responses were small or absent in oocytes injected with either mRNA alone. We conclude that 5-HT1C receptors are able to cause the closing of a class of K+ channels expressed by cortex mRNA in a Ca2(+)-independent manner. The coupling between the receptors and channels appears to be mediated by the inositol phospholipid second messenger pathway, since activation of this pathway by application of serum evoked a similar closing current.
Cortisol rapidly reduces prolactin release and cAMP and 45Ca2+ accumulation in the cichlid fish pituitary in vitro.
Authors: Borski R J , Helms L M , Richman N H , Grau E G
Published: May 2006

During in vitro incubation, prolactin release is inhibited in a dose-related manner by cortisol. This action is mimicked by the synthetic glucocorticoid agonist dexamethasone but not by other steroids tested. Perifusion studies indicate that the inhibition of [3H]prolactin release by cortisol occurs within 20 min. Cortisol (50 nM) also inhibits cAMP accumulation and reduces 45Ca2+ accumulation in the tilapia rostral pars distalis within 15 min. Cortisol's action on prolactin release is blocked in the presence of either the Ca2+ ionophore A23187 or a combination of dibutyryl cAMP and 3-isobutyl-1-methylxanthine, which increase intracellular Ca2+ and cAMP, respectively. Taken together, these findings suggest that cortisol may play a physiologically relevant role in the rapid modulation of prolactin secretion in vivo. Our studies also suggest that the inhibition of prolactin release by cortisol is a specific glucocorticoid action that may be mediated, in part, through cortisol's ability to inhibit intracellular cAMP and Ca2+ metabolism.
Neuronal NADPH diaphorase is a nitric oxide synthase.
Authors: Hope B T , Michael G J , Knigge K M , Vincent S R
Published: May 2006

NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affinity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPH-dependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.
Retroviral sequences related to human T-lymphotropic virus type II in patients with chronic fatigue immune dysfunction syndrome.
Authors: DeFreitas E , Hilliard B , Cheney P R , Bell D S , Kiggundu E , Sankey D , Wroblewska Z , Palladino M , Woodward J P , Koprowski H
Published: May 2006

Chronic fatigue immune dysfunction syndrome (CFIDS) is a recently recognized illness characterized by debilitating fatigue as well as immunological and neurological abnormalities [Straus, S.E. (1988) J. Inf. Dis. 157, 405-412]. Once thought to be caused by Epstein-Barr virus, it is now thought to have a different but unknown etiology. We evaluated 30 adult and pediatric CFIDS patients from six eastern states for the presence of human T-lymphotropic virus (HTLV) types I and II by Western immunoblotting, polymearse chain reaction, and in situ hybridization of blood samples. The majority of patients were positive for HTLV antibodies by Western blotting and for HTLV-II gag sequences by polymerase chain reaction and in situ hybridization. Twenty nonexposure healthy controls were negative in all assays. These data support an association between an HTLV-II-like virus and CFIDS.
Sequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability.
Authors: McKenna E , Hardy D , Pastore J C , Kaback H R
Published: May 2006

Previous experiments are consistent with the notion that residues 396-401 (... SVFTLS ...) at the carboxyl terminus of the last putative transmembrane helix of the lactose (lac) permease of Escherichia coli are important for protection against proteolytic degradation and suggest that this region of the permease may be necessary for proper folding. Stop codons (TAA) have now been substituted sequentially for amino acid codons 396-401 in the lacY gene, and the termination mutants were expressed from the plasmid pT7-5. With respect to transport, permease truncated at residue 396 or 397 is completely defective, while molecules truncated at residues 398, 399, 400, and 401, respectively, exhibit 15-25%, 30-40%, 40-45%, and 70-100% of wild-type activity. As judged by pulse-chase experiments with [35S]methionine, wild-type permease or permease truncated at residue 401 is stable, while permease molecules truncated at position 400, 399, 398, 397, or 396 are degraded at increasingly rapid rates. The findings indicate that either the last turn of putative helix XII or the region immediately distal to helix XII is important for proper folding and protection against proteolytic degradation.
Helical repeat and chirality effects on DNA gel electrophoretic mobility.
Authors: Drak J , Crothers D M
Published: May 2006

We determined a value of 10.34 +/- 0.04 base pairs (bp) per turn for the helical repeat of bent DNA sequences of the form A6N4-A6N5 by estimating the sequence repeat required to produce a planar curve, as judged from the maximum in the electrophoretic mobility anomaly of multimers containing different sequence repeats (10.00, 10.33, 10.50, 10.67, and 11.00 bp per turn). This result provides the basis for a method to evaluate the helical repeat of any DNA segment by comparative electrophoresis measurements. The sequence of interest is placed between two A-tract bends and the phasing is varied over an entire helical turn. Knowledge of the number of base pairs between the bends in the cis isomer, which has the lowest electrophoretic mobility, allows calculation of the average helical repeat of the inserted sequence. In the course of these experiments we observed an unexpected dependence of electrophoretic mobility on the shape of DNA molecules: in high-percentage polyacrylamide gels, those bent molecules for which we deduced a right-handed superhelical form are less retarded than their homologous left-handed isomers. To explain this finding we propose that superhelical chirality influences the choice of DNA migration pathway, leading to rotation of the DNA molecule relative to the local coordinate frame in the gel. High-percentage gels have sufficiently close contact with the right-handed DNA helical twist to differentiate the frictional consequences of right- and left-handed twisting motions.
Localization of the estrogen-binding site of alpha-fetoprotein in the chimeric human-rat proteins.
Authors: Nishi S , Matsue H , Yoshida H , Yamaoto R , Sakai M
Published: May 2006

Rat alpha-fetoprotein (AFP) has been demonstrated to bind estrogen, whereas human AFP lacks the activity. We constructed four chimeric molecules from cDNAs encoding these AFPs with the use of two restriction sites common to them and expressed them as well as rat and human AFP cDNA in yeast. The recombinant molecules were purified, characterized, and found to have the predicted structures. Analyses of estrogen binding indicated that a rat AFP sequence composed of residues 423-506 that contains 31 rat-specific amino acids is essential for the activity.
Plasmodium falciparum appears to have arisen as a result of lateral transfer between avian and human hosts.
Authors: Waters A P , Higgins D G , McCutchan T F
Published: May 2006

It has been proposed that the acquisition of Plasmodium falciparum by man is a relatively recent event and that the sustained presence of this disease in man is unlikely to have been possible prior to the establishment of agriculture. To establish phylogenetic relationships among the Plasmodium species and to unravel the mystery of the origin of P. falciparum, we have analyzed and compared phylogenetically the small-subunit ribosomal RNA gene sequences of the species of malaria that infect humans as well as a number of those sequences from species that infect animals. Although this comparison confirmed the three established major subgroups, broadly classed as avian, simian, and rodent, we find that the human pathogen P. falciparum is monophyletic with the avian subgroup, indicating that P. falciparum and avian parasites share a relatively recent avian progenitor. The other important human pathogen, P. vivax, is very similar to a representative of the simian group of Plasmodium. The relationship between P. falciparum and the avian parasites, and the overall phylogeny of the genus, provides evidence of an exception to Farenholz's rule, which propounds synchronous speciation between host and parasite.
Identification of two forms of the RNA polymerase I transcription factor UBF.
Authors: O'Mahony D J , Rothblum L I
Published: May 2006

The structure of the rat homologue of the RNA polymerase I transcription factor UBF was investigated. The sequence of the protein was deduced from the sequence of overlapping cDNAs isolated from a cDNA library and from clones of the products generated by the polymerase chain reaction from random-primed, first-strand cDNA. The sequences of these clones indicated that there were two mRNAs for UBF and that the encoded proteins were similar but not identical. One form of rat UBF was essentially identical to human UBF. The second class of UBF mRNA contained an in-frame "deletion" in the coding region that results in the deletion of 37 amino acids from the predicted protein sequence. This deletion reduces the predicted molecular size of the encoded form of UBF by approximately 4400 from 89.4 kDa to 85 kDa and significantly alters the structure of one of the four HMG-1 homology regions (HMG box-2) in that form of UBF. Evidence for the existence of two mRNAs in rat cells was confirmed by a probe protection assay, and we provide evidence that other vertebrate cells contain these same two forms of UBF mRNA. These results are consistent with the observation that UBF purified from four different vertebrates migrates as two bands upon SDS/PAGE. It has been hypothesized that the HMG motifs are the DNA-binding domains of UBF. Altering one of these "boxes," as in the second form of UBF, may alter the functional characteristics of the transcription factor. Thus, the existence of different forms of UBF may have important ramifications for transcription by RNA polymerase I.
Genetic linkage of autosomal recessive canine narcolepsy with a mu immunoglobulin heavy-chain switch-like segment.
Authors: Mignot E , Wang C , Rattazzi C , Gaiser C , Lovett M , Guilleminault C , Dement W C , Grumet F C
Published: May 2006

Identification of genes determining narcolepsy susceptibility is important not only for understanding that disorder but also for possible clues to general sleep-control mechanisms. Studies in humans reveal at least one such gene related to the major histocompatibility complex and in dog an as-yet-unmapped single, autosomal recessive gene canarc-1. Gene markers for canarc-1 were therefore sought by DNA restriction fragment length polymorphisms in our colony of narcoleptic dogs. A human mu-switch immunoglobulin probe and the enzyme Hae III identified a gene cosegregating with canarc-1 in backcrossed animals (logarithm of odds scores: m = 24, Z max = 7.2 at theta = 0%). canarc-1 was also shown not to be tightly linked with the dog major histocompatibility complex (m = 40, Z less than -2 at theta less than 4.8%). These results represent the mapping of a non-major histocompatibility complex narcolepsy gene and strongly suggest involvement of the immune system in the pathophysiology of that disease.
Generation of sensory neurons is stimulated by leukemia inhibitory factor.
Authors: Murphy M , Reid K , Hilton D J , Bartlett P F
Published: May 2006

The processes that regulate the development of peripheral neurons from their precursors in the embryonic neural crest are essentially unknown. In this report, we show that leukemia inhibitory factor stimulates the generation of neurons in cultures of mouse neural crest. These neurons have the morphology of sensory neurons and contain neuropeptides found in mammalian sensory neurons. Consistent with these neurons being of the sensory lineage is the finding that they arise from nondividing precursors within the neural crest. In addition, we show that leukemia inhibitory factor supports the generation and/or maturation of sensory neurons in cultures of cells obtained from embryonic dorsal root ganglia. In cultures of postnatal dorsal root ganglia, which contain mature sensory neurons, leukemia inhibitory factor acts directly as a survival molecule on the majority of neurons.
Functional analysis of human cardiac gap junction channel mutants.
Authors: Fishman G I , Moreno A P , Spray D C , Leinwand L A
Published: May 2006

The connexins form a family of membrane spanning proteins that assemble into gap junction channels. The biophysical properties of these channels are dependent upon the constituent connexin isoform. To begin identifying the molecular basis for gap junction channel behavior in the human heart, a tissue that expresses connexin43, we used site-directed mutagenesis to generate mutant cDNAs of human connexin43 with shortened cytoplasmic tail domains. Premature stop codons were inserted, resulting in proteins corresponding in length to the mammalian isoforms connexin32 and connexin26, which are expressed primarily in liver. All constructs restore intercellular coupling when they are transfected into SKHep1 cells, a human hepatoma line that is communication deficient. Whereas wild-type connexin43 transfectants display two distinct unitary conductance values of about 60 and 100 pS, transfectants expressing the mutant proteins, from which 80 and 138 amino acids have been deleted, exhibit markedly different single-channel properties, with unitary conductance values of about 160 and 50 pS, respectively. Junctional conductance of channels composed of wild-type connexin43 is less voltage-sensitive compared with transfectants expressing wild-type connexin32. However, neither of the connexin43 truncation mutants alters this relative voltage insensitivity. These results suggest that the cytoplasmic tail domain is an important determinant of the unitary conductance event of gap junction channels but not their voltage dependence. Furthermore, since the mutant connexins are missing several consensus phosphorylation sites, modification of these particular sites may not be required for membrane insertion or assembly of human connexin43 into functional channels.
Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity.
Authors: Hai T , Curran T
Published: May 2006

The Fos/Jun and ATF/CREB families of transcription factors function in coupling extracellular signals to alterations in expression of specific target genes. Like many eukaryotic transcription factors, these proteins bind to DNA as dimers. Dimerization is mediated by a structure known as the "leucine-zipper" motif. Although Fos/Jun and ATF/CREB were previously thought to interact preferentially with different DNA regulatory elements (the AP-1/TRE and ATF/CRE sites, respectively), we find that members of these two families form selective cross-family heterodimers. The resulting heterodimers display distinguishable DNA binding specificities from each other and from their parental homodimers. These findings indicate that the Fos/Jun and ATF/CREB families of transcription factors are not as distinct as was previously thought. We suggest that they can be grouped into a superfamily of transcription factors.
UGA is translated as cysteine in pheromone 3 of Euplotes octocarinatus.
Authors: Meyer F , Schmidt H J , Plümper E , Hasilik A , Mersmann G , Meyer H E , Engström A , Heckmann K
Published: May 2006

Pheromone 3 mRNA of the ciliate Euplotes octocarinatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. No UAG codon was found. In connection with results reported for other ciliates, this suggests that the role of the classic termination codons had not yet been established when the ciliates started to diverge from other eukaryotes.
Protein kinase A- and protein kinase C-regulated interaction of plectin with lamin B and vimentin.
Authors: Foisner R , Traub P , Wiche G
Published: May 2006

Solid-phase binding assays with protein species purified from cultured rat glioma C6 cells and Ehrlich ascites revealed that plectin bound specifically to lamin B but not to lamins A and C. Lamin B interaction was significantly decreased upon in vitro phosphorylation of either lamin B or plectin with protein kinase A or C. In contrast, phosphorylation of plectin with kinase A increased its binding to vimentin, suggesting a different regulation of plectin interactions by this kinase. 32P-radiolabeling of rat glioma C6 cells revealed plectin as a major in vivo target of protein kinase A and protein kinase C. Plectin, present in lysates of dibutyryladenosine 3',5'-cyclic monophosphate-treated cells, showed a 2.5 times higher binding affinity to vimentin than plectin from phorbol ester-treated cells. Furthermore, the relative amounts of plectin in 1% Triton X-100/high salt-insoluble cell fractions decreased to one-fourth of control values upon treating cells with phorbol esters, whereas vimentin was unaffected. This finding suggested a protein kinase C-dependent weakening of plectin interaction with intermediate filaments in vivo. Taken together, these results point to a role of plectin in interlinking cytoskeletal and nuclear elements and suggest that specific protein kinases are involved in regulating these interactions.
Direct spread of reovirus from the intestinal lumen to the central nervous system through vagal autonomic nerve fibers.
Authors: Morrison L A , Sidman R L , Fields B N
Published: May 2006

A crucial event in the pathogenesis of systemic enteric virus infections is entry of virus into the nervous system. Whether enteric virus spreads from the intestinal tract to the central nervous system through nerves or through the bloodstream was examined using a serotype 3 reovirus strain. After peroral inoculation of newborn mice with reovirus, serial histologic sections of small intestine, brain and spinal cord were prepared and stained by immunoperoxidase to detect viral antigen. Three days after inoculation, viral antigen was observed in mononuclear cells of ileal Peyer's patches and in neurons of the adjacent myenteric plexus. Infection first appeared in the central nervous system 1-2 days later in neurons of the dorsal motor nucleus of the vagus nerve. Endothelial cells, meninges, choroid plexus, hypothalamus, and area postrema were not infected, indicating neural rather than bloodborne spread from the intestine. Staining of neurons in the dorsal motor nucleus of the vagus nerve depended on the route of virus inoculation and was independent of the amount of virus in the bloodstream. These results demonstrate that an enteric virus entering a host from the intestinal lumen can spread to the central nervous system through nerve fiber innervating the intestine.
Characterization of melanotropin-release-inhibiting factor (melanostatin) from frog brain: homology with human neuropeptide Y.
Authors: Chartrel N , Conlon J M , Danger J M , Fournier A , Tonon M C , Vaudry H
Published: May 2006

A polypeptide was purified from frog brain extracts on the basis of its ability to inhibit alpha-melanotropin release from perifused frog neurointermediate lobes. Based on Edman degradation, amino acid analysis, and peptide mapping, the primary structure of this frog melanotropin-release-inhibiting factor (melanostatin) was determined to be H-Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met- Ala-Lys-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg- Tyr-NH2 . Frog melanostatin belongs to the pancreatic polypeptide/neuropeptide Y/peptide YY family, and the structure of this peptide differs from that of human neuropeptide Y by only one amino acid substitution in position 19. A synthetic replicate of frog melanostatin is coeluted with the native peptide on HPLC and is highly potent in inhibiting alpha-melanotropin secretion in vitro (IC50 = 60 nM).
Evidence that nerve growth factor dependence of sympathetic neurons for survival in vitro may be determined by levels of cytoplasmic free Ca2+.
Authors: Koike T , Tanaka S
Published: May 2006

Developing sympathetic neurons established in the presence of nerve growth factor (NGF) die in vitro after acute withdrawal of NGF. This in vitro model mimics the physiological situation in which neurons die during development or after axotomy when trophic support becomes insufficient. We have previously shown that depolarizing agents including high K+ and cholinergic agonists prevent neuronal death induced by acute deprivation of NGF in vitro. Based on this finding, a Ca2+ set-point hypothesis was proposed for the degree of neuronal dependence on tropic factor in vitro. Here we have examined the validity of this hypothesis by measuring the level of cytoplasmic free Ca2+ ([Ca2+]i) with fura-2 as a probe for monitoring Ca2+. (i) There was a good correlation between cell survival in the absence of NGF and [Ca2+]i levels of young sympathetic neurons (1 week in vitro) chronically exposed to various concentrations of extracellular K+, which shows that 50% survival occurred at approximately 184 nM [Ca2+]i and complete survival, independent of trophic support, occurred at approximately 240 nM [Ca2+]i. (ii) The basal level of [Ca2+]i of sympathetic neurons was relatively low (93.0 +/- 10.5 nM) at days 6-8, then increased with incubation time, and finally reached a plateau level of 241 +/- 7 nM at around week 3, when the neurons became independent of NGF for survival. (iii) Sympathetic neurons maintained in the presence of high or low concentrations of Ca2+ displayed altered trophic dependence. Thus, these findings are consistent with this Ca2+ set-point hypothesis for the degree of NGF dependence of sympathetic neurons for survival in vitro.
Intracellular injection of Ca2+ chelators blocks induction of long-term depression in rat visual cortex.
Authors: Bröcher S , Artola A , Singer W
Published: May 2006

In a variety of brain structures repetitive activation of synaptic connections can lead to long-term potentiation (LTP) or long-term depression (LTD) of synaptic transmission, and these modifications are held responsible for memory formation. Here we examine the role of postsynaptic Ca2+ concentration in the induction of LTD in the neocortex. In layer III cells of the rat visual cortex, LTD can be induced by tetanic stimulation of afferent fibers ascending from the white matter. We show that LTD induction is reliably blocked by intracellular injection of either EGTA or BAPTA [bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate], two different Ca2+ chelators. This confirms that the processes underlying the induction of LTD in neocortex are located postsynaptically and indicates that they depend on intracellular Ca2+ concentration. Thus, both LTP and LTD induction appear to involve calcium-mediated processes in the postsynaptic neuron. We propose that LTD is caused by a surge of calcium either through voltage-gated Ca2+ conductances and/or by transmitter-induced release of calcium from intracellular stores.
Selective killing of CD4+ cells harboring a human immunodeficiency virus-inducible suicide gene prevents viral spread in an infected cell population.
Authors: Caruso M , Klatzmann D
Published: May 2006

We have stably expressed in CD4+ lymphoid cells the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene under the control of the human immunodeficiency virus (HIV) promoter and transactivation response element sequences. Upon HIV infection these regulatory sequences were transactivated, switching on high-level expression of HSV1-TK. This in turn caused the death of HIV-infected cells when they were cultured in the presence of acyclovir, a nucleoside analog that becomes toxic after phosphorylation by HSV1-TK. The elimination of HIV-infected cells resulted in the arrest of HIV spreading in the culture. Complete protection of HSV1-TK-expressing cells was obtained using acyclovir concentrations that are commonly detected in the plasma of patients treated for HSV1 infection. Thus, expression of this DNA construct generates a pool of CD4+ booby-trapped cells that, as a population, are resistant to HIV infection. Our data provide a rationale for the use of suicide genes in the design of gene therapy of HIV infection.
Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor/CD3 complex.
Authors: Reiser H , Freeman G J , Razi-Wolf Z , Gimmi C D , Benacerraf B , Nadler L M
Published: May 2006

We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex.
Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains.
Authors: Hohman V S , Schluter S F , Marchalonis J J
Published: May 2006

A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.
Actin as the generator of tension during muscle contraction.
Authors: Schutt C E , Lindberg U
Published: May 2006

We propose that the key structural feature in the conversion of chemical free energy into mechanical work by actomyosin is a myosin-induced change in the length of the actin filament. As reported earlier, there is evidence that helical actin filaments can untwist into ribbons having an increased intersubunit repeat. Regular patterns of actomyosin interactions arise when ribbons are aligned with myosin thick filaments, because the repeat distance of the myosin lattice (429 A) is an integral multiple of the subunit repeat in the ribbon (35.7 A). This commensurability property of the actomyosin lattice leads to a simple mechanism for controlling the sequence of events in chemical-mechanical transduction. A role for tropomyosin in transmitting the forces developed by actomyosin is proposed. In this paper, we describe how these transduction principles provide the basis for a theory of muscle contraction.
Crystallographic detection of a second ligand binding site in influenza virus hemagglutinin.
Authors: Sauter N K , Glick G D , Crowther R L , Park S J , Eisen M B , Skehel J J , Knowles J R , Wiley D C
Published: May 2006

X-ray crystal structures have been determined for several complexes between influenza virus hemagglutinin and derivatives of its cell-surface receptor, sialic acid (Neu5Ac). Difference electron density maps establish the existence of a second binding site in addition to the primary site characterized previously. Three compounds bind to both sites: Neu5Ac(alpha 2-3)Gal(beta 1-4)Glc [(alpha 2-3)sialyllactose], alpha-2-O-(4'-benzylamidocarboxybutyl)-5-N-acetylneuraminic acid, and alpha-2-O-(4'-methylamidocarboxybutyl)-5-N-acetylneuraminic acid; and four other compounds bind only to the primary site: Neu5Ac(alpha 2-6)Gal(beta 1-4)Glc [(alpha 2-6)sialyllactose], alpha-2-O-methyl-5-N-acetylneuraminic acid, 4-]-acetyl-alpha-2-O-methyl-5-N-acetylneuraminic acid, and 9-amino-9-deoxy-alpha-2-O-methyl-5-N-acetylneuraminic acid. The maps also extend earlier results by showing the location of all three sugar residues of (alpha 2-3)sialyllactose in the primary binding site. The affinity of (alpha 2-3)sialyllactose for the second site was estimated by collecting x-ray diffraction data at various ligand concentrations and was found to be at least four times weaker than its affinity for the primary site. Although it is not yet known whether the second binding site participates in the infection process, it nevertheless offers a potential target for the design of antiviral drugs.
Semisynthetic combinatorial antibody libraries: a chemical solution to the diversity problem.
Authors: Barbas C F , Bain J D , Hoekstra D M , Lerner R A
Published: May 2006

The properties of naiveté and large diversity are considered to be essential starting features for combinatorial antibody libraries that eschew immunization by evolution in vitro. We have prepared large libraries with such properties by using random oligonucleotide synthesis, which has the potential to create approximately 10(20) complementarity-determining regions for antibody heavy chains. When combined with light chains and expressed on phage surfaces, high-affinity antibodies could be selected from 5.0 x 10(7) Escherichia coli transformants. Remarkably, antibodies selected only for binding displayed both general structural features known to be important in nature's own antibodies and specific consensus sequences thought to be critical for interaction with the hapten against which the library was selected. Semisynthetic and ultimately totally synthetic combinatorial libraries when coupled with mutation and selection procedures should replace immunization for generation of reagent, therapeutic, and catalytic antibodies.
Excretion of putrescine by the putrescine-ornithine antiporter encoded by the potE gene of Escherichia coli.
Authors: Kashiwagi K , Miyamoto S , Suzuki F , Kobayashi H , Igarashi K
Published: May 2006

Excretion of putrescine from Escherichia coli was assessed by measuring its uptake into inside-out membrane vesicles. The vesicles were prepared from wild-type E. coli or E. coli transformed with plasmids containing one of the three polyamine transport systems. The results indicate that excretion of putrescine is catalyzed by the putrescine transport protein, encoded by the potE gene located at 16 min on the E. coli chromosome. Loading of ornithine (or lysine) inside the vesicles was essential for the uptake of putrescine, indicating that the protein exchanges putrescine and ornithine (or lysine) by an antiport mechanism. The Km and Vmax values for the putrescine uptake by inside-out membrane vesicles were 73 microM and 0.82 nmol/min per mg of protein, respectively. The antiport protein (potE protein) also catalyzed putrescine-putrescine and ornithine-ornithine exchange. The transport activity was not disturbed by inhibitors of energy production such as KCN and carbonyl cyanide m-chlorophenylhydrazone. When intact E. coli was used instead of the inside-out membrane vesicles, excretion of putrescine was also catalyzed by the antiport protein in the presence of ornithine in the medium.
Finding errors in DNA sequences.
Authors: Posfai J , Roberts R J
Published: May 2006

An algorithm is described that can detect certain errors within coding regions of DNA sequences. The algorithm is based on the idea that an insertion or deletion error within a coding sequence would interrupt the reading frame and cause the correct translation of a DNA sequence to require one or more frameshifts. If the coding sequence shows similarity to a known protein sequence then such errors can be detected by comparing the conceptual translations of DNA sequences in all six reading frames with every sequence in a protein sequence data base. We have incorporated these ideas into a computer program, called DETECT, that can serve as an aid to the experimentalist who is determining new DNA sequences so that obvious errors may be located and corrected. The program has been tested using raw experimental data and against sequences from the European Molecular Biology Laboratory data base, annotated as containing frameshifts. We have also tested it using unidentified open reading frames that flank known, annotated genes in the GenBank data base. Many potential errors are apparent and in some cases functions can be suggested for the "corrected" versions of these reading frames leading to the identification of new genes. As more sequences are determined the power of this method will increase substantially.
Quantitation of reversible binding by particle counting: hapten-antibody interaction as a model system.
Authors: Sykulev Y K , Sherman D A , Cohen R J , Eisen H N
Published: May 2006

With a view toward developing a general method for measuring intrinsic equilibrium constants for the reversible interactions between two ligands, we used an antibody-hapten model system [2,4-dinitrophenyl (DNP) hapten and anti-DNP antibody] to explore an approach based on particle counting of uniform polystyrene spheres to which the hapten is coupled covalently. This approach was made possible by an optical pulse particle size analyzer that accurately counts individual sphere clusters and quantitates with high precision specific aggregation of spheres crosslinked by antibody. The reduction in crosslinking that results from competition for antibody binding sites between a soluble DNP ligand and immobilized DNP groups on the spheres provides the basis for measuring the intrinsic equilibrium constant for the soluble ligand-antibody interaction. The binding constants measured in this way for several DNP ligands and an anti-DNP antibody (2A1) agreed with the values obtained by conventional methods. The range of intrinsic equilibrium constants that can be determined by particle counting is likely to be exceptionally wide and a value as low as 10(3) liters/mol has been measured. And since all soluble antigens, regardless of their mass, acquire the same ability to scatter light as a result of their immobilization on the much larger uniform spheres (0.36 microns), the approach described here should be applicable to virtually any molecularly dispersed antigen and its monoclonal antibody.
Sobolev inequalities, the Poisson semigroup, and analysis on the sphere Sn.
Authors: Beckner W
Published: May 2006

Hypercontractive estimates are obtained for the Poisson semigroup on Lp(Sn). Such estimates are determined by a sharp logarithmic Sobolev inequality that relates the entropy of a function on Sn to its smoothness.