bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial-frequency organization in primate striate cortex.
Authors: Silverman M S , Grosof D H , De Valois R L , Elfar S D
Published: May 2006

We measured the spatial-frequency tuning of cells at regular intervals along tangential probes through the monkey striate cortex and correlated the recording sites with the cortical cytochrome oxidase (CytOx) patterns to address three questions with regard to the cortical spatial-frequency organization. (i) Is there a periodic anatomical arrangement of cells tuned to different spatial-frequency ranges? We found there is, because the spatial-frequency tuning of cells along tangential probes changed systematically, varying from a low frequency to a middle range to high frequencies and back again repeatedly over distances of about 0.6-0.7 mm. (ii) Are there just two populations of cells, low-frequency and high-frequency units, at a given eccentricity (perhaps corresponding to the magno- and parvocellular geniculate pathways) or is there a continuum of spatial-frequency peaks? We found a continuum of peak tuning. Most cells are tuned to intermediate spatial frequencies and form a unimodal rather than a bimodal distribution of cell peaks. Furthermore, the cells with different peak frequencies were found to be continuously and smoothly distributed across a module. (iii) What is the relation between the physiological spatial-frequency organization and the regions of high CytOx concentration ("blobs")? We found a systematic correlation between the topographical variation in spatial-frequency tuning and the modular CytOx pattern, which also varied continuously in density. Low-frequency cells are at the center of the blobs, and cells tuned to increasingly higher spatial frequencies are at increasing radial distances.
Cloning of the cDNA encoding the myosin heavy chain of a vertebrate cellular myosin.
Authors: Shohet R V , Conti M A , Kawamoto S , Preston Y A , Brill D A , Adelstein R S
Published: May 2006

The complete amino acid sequence of a vertebrate cellular myosin heavy chain (MHC; 1,959 amino acids, 226 kDa) has been deduced by using cDNA clones from a chicken intestinal epithelial cell library. RNA blot analysis of kidney, spleen, brain, liver, and intestinal epithelial cells as well as smooth muscle cells from the aorta and gizzard indicates the presence of a 7.3-kilobase (kb) message that is larger than the message for chicken smooth and striated muscle MHC. The chicken intestinal epithelial cell MHC shows overall similarity in primary structure to other MHCs in the areas of the reactive thiol residues and in areas contributing to the ATP binding site and actin binding site. The globular head domain is followed by an alpha-helical coiled-coil region, and as in smooth muscle MHC there is a short uncoiled sequence at the carboxyl terminus of the molecule. Comparison of amino acid sequences in the rod regions between human and chicken cellular MHCs shows a remarkable 92% identity.
Structural changes in bacteriorhodopsin during proton translocation revealed by neutron diffraction.
Authors: Dencher N A , Dresselhaus D , Zaccai G , Büldt G
Published: May 2006

A neutron diffraction study of spectroscopic states for the light-energized proton pump bacteriorhodopsin (BR) is presented. The photocycle states BR-568 and M were generated at temperatures above 4 degrees C and were measured after trapping at--180 degrees C. In the BR-568 to M-state transition, which is known to be a key step in transmembrane proton pumping, reversible structural changes of the protein were detected. These structural alterations occur in the neighborhood of the cyclohexene ring and at the Schiff's base end of the chromophore retinal. They are interpreted as a 1-2 degree tilt of three or four of the transmembrane alpha-helices or as positional changes of four or five amino acids. The structural changes observed are inherent in the transport mechanism of bacteriorhodopsin.
Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins.
Authors: McEachern M J , Bott M A , Tooker P A , Helinski D R
Published: May 2006

The gamma origin binding sites of the replication initiator pi protein, composed of seven 22-base-pair (bp) direct repeats and previously shown to be essential for replication of plasmid R6K, can also act as an inhibitor of R6K replication in Escherichia coli cells if provided in trans. Inhibition is dependent upon the ability of these repeats to bind the R6K-encoded pi protein but is not overcome by increasing the intracellular pi level. The insertion of a second repeat cluster in close proximity to the gamma origin also can markedly inhibit replication. The severity of this effect is dependent upon the position, orientation, and number of repeats present in the extra cluster. As few as six extra repeats can result in a completely nonfunctional gamma origin. However, this inactive gamma origin plasmid containing the six extra repeats is functional when placed in a strain that underproduces the wild-type pi protein or when placed in the presence of any of several copy-up mutant pi proteins. On the basis of these observations, we propose that the nucleoprotein structures formed by the binding of pi protein to the seven 22-bp direct repeats at the gamma origin are capable of coupling with each other in vivo and that replication initiation is prevented at such coupled origins. In support of this model of replication control, we demonstrate by electron microscopy analysis that the pi protein has the ability to associate two DNA molecules containing gamma origin sequences and also show that pi enhances the DNA ligase-catalyzed multimerization of a DNA fragment containing the gamma origin.
Nucleotide sequence analysis of feline immunodeficiency virus: genome organization and relationship to other lentiviruses.
Authors: Olmsted R A , Hirsch V M , Purcell R H , Johnson P R
Published: May 2006

We determined the complete nucleotide sequence of an infectious proviral molecular clone (FIV-14) of the feline immunodeficiency virus (FIV). FIV-14 has a genome organization similar in complexity to other lentiviruses. In addition to three large open reading frames representing the gag, pol, and env genes, at least four small open reading frames are present in the pol-env intergenic, env, and env-3' long terminal repeat regions. Nucleotide and deduced amino acid sequence alignments of the FIV coding sequences with analogous sequences of other lentiviruses revealed significant identities only in the gag and pol genes. Phylogenetic tree analyses of gag and pol gene-encoded protein sequences demonstrate that FIV is more closely related to the ungulate lentiviruses, equine infectious anemia virus and visna virus, than to the primate lentiviruses, human and simian immunodeficiency viruses.
In vivo synthesis of adenylylated bis(5'-nucleosidyl) tetraphosphates (Ap4N) by Escherichia coli aminoacyl-tRNA synthetases.
Authors: Brevet A , Chen J , Lévêque F , Plateau P , Blanquet S
Published: May 2006

The role of aminoacyl-tRNA synthetases in the in vivo synthesis of adenylylated bis(5'-nucleosidyl) tetraphosphates (Ap4N) was studied by measuring the concentration of these nucleotides in Escherichia coli cells overproducing lysyl-, methionyl- phenylalanyl-, or valyl-tRNA synthetase. Overproduction of each aminoacyl-tRNA synthetase (20- to 80-fold) was accompanied by a significant increase in intracellular Ap4N concentration (3- to 14-fold). As expected, non-adenylylated bis(5'-nucleosidyl) tetraphosphate concentration was not changed by synthetase overproduction. It was also verified that overproduction of an inactive methionyl-tRNA synthetase mutant did not modify Ap4N concentration. Ap4N accumulation during heat shock occurred in all strains studied. The increase factor (approximately 50-fold after 1 hr at 48 degrees C) was not changed by overproduction of any of the aminoacyl-tRNA synthetases studied, including that of the heat-inducible form of lysyl-tRNA synthetase from the lysU gene. Together, these results establish that aminoacyl-tRNA synthetases are involved in Ap4N biosynthesis during exponential growth as well as during heat shock.
Transcription terminates near the poly(A) site in the CYC1 gene of the yeast Saccharomyces cerevisiae.
Authors: Russo P , Sherman F
Published: May 2006

A 38-base-pair region required for normal CYC1 mRNA 3' end formation in Saccharomyces cerevisiae was shown to be necessary for the termination of transcription in vivo by examining the stability of CEN3 plasmids. CEN3 plasmids were stably maintained during vegetative growth, unless a GAL1 transcript impinged on the CEN3 region. Transcription from the GAL1 promoter was terminated, and plasmid stability was restored by the insertion of a fragment containing the 38-base-pair region of CYC1. In contrast, a similar fragment lacking the 38-base-pair region had no such stabilizing effect. Furthermore, CYC1 mRNA transcription terminated in a region less than 100 nucleotides downstream from the normal poly(A) site, thus establishing that CYC1 mRNA 3' end formation does not involve overly extended precursors as are observed in higher eukaryotes.
Copper and the ACE1 regulatory protein reversibly induce yeast metallothionein gene transcription in a mouse extract.
Authors: Culotta V C , Hsu T , Hu S , Fürst P , Hamer D
Published: May 2006

We describe a cell-free system in which the transcription of the yeast metallothionein gene is inducible by the addition of metal ions plus a specific regulatory protein. Efficient transcription requires the complete yeast ACE1 metalloregulatory protein, including both its DNA-binding and transactivation domains; a mouse nuclear extract providing RNA polymerase and general transcription factors; a template containing the ACE1 binding site; and Cu(I). Because the binding of ACE1 to DNA is dependent on Cu, it is possible to inhibit transcription by the use of Cu-complexing agents such as CN-. We have used this specific inhibition to show that the ACE1 regulatory protein is required for the maintenance as well as the formation of a functional preinitiation complex. The ability to reversibly induce yeast metallothionein gene transcription in vitro provides a powerful system for determining the molecular mechanism of a simple eukaryotic regulatory circuit.
Direct observations of ligand dynamics in hemoglobin by subpicosecond infrared spectroscopy.
Authors: Anfinrud P A , Han C , Hochstrasser R M
Published: May 2006

The photodissociation of CO from HbCO at ambient temperature is studied by means of a femtosecond IR technique. The bleaching of the FeCO absorption and the appearance of a new IR absorption near that of free CO are both observed at 300 fs after optical excitation. The bleach does not recover on the time scale of a few picoseconds but does recover by approximately 4% within 1 ns, which suggests that a barrier to recombination is formed within a few picoseconds. The CO spectrum does not change significantly between 300 fs and 1 ns, suggesting that the CO quickly finds some locations in the heme pocket that are not more than a few angstroms from the iron. The de-ligated CO appears in its ground vibrational level. There is evidence that 85 +/- 10% of this CO remains in the heme pocket at 1 ns; it probably resides there for 50 ns. The flow of excess vibrational energy from the heme to the solvent was directly observed in the IR experiments. The heme cools within 1-2 ps while thermal disruption of the surrounding solvent structure requires approximately 30 ps.
Concordant evolution of coding and noncoding regions of DNA made possible by the universal rule of TA/CG deficiency-TG/CT excess.
Authors: Yomo T , Ohno S
Published: May 2006

The universal rule of TA/CG deficiency-TG/CT excess previously proposed as the construction principle of coding sequences applies to noncoding regions of the gene as well. Analysis of a 1989-base-long gene sequence for mouse immunoglobulin gamma 2a heavy-chain constant region as well as the 19,002-base-long gene sequence for human serum albumin revealed deficiency and overabundance of very similar sets of base trimers and tetramers in the coding and noncoding regions of the same gene, in spite of the fact that noncoding regions were considerably richer in A + T. Inasmuch as this universal rule does not discriminate one strand of DNA double helix from another, two complementary DNA strands of the entire gene maintained nearly perfect symmetry. That is to say, the degrees of excesses, deficiencies of the 64-base trimers remained nearly identical between two complementary strands, and this symmetry was only slightly disturbed in the coding region. It would thus appear that the universal rule as an intrinsic force has been exerting far greater influence than natural selection in the evolution of genes.
A nuclear protein associated with human cancer cells binds preferentially to a human repetitive DNA sequence.
Authors: Law M L , Gao J Z , Puck T T
Published: May 2006

A protein (Rp66) of 66 kDa was shown by DNA-binding protein blot assay to bind to a human repetitive DNA sequence (low-repeat sequence; LRS) in each of 10 transformed human cell lines examined. This protein-DNA interaction was not observed in 11 normal human cell cultures or in the Chinese hamster cell line CHO-K1. Gel retardation assay confirmed the specificity of the protein-DNA binding between Rp66 and LRS. In a histiocytic lymphoma human cell line, U937, that can be induced to differentiate in the presence of phorbol ester, this binding disappeared after cell differentiation. These together with other results cited suggest a regulatory role for these repetitive sequences in the human genome, with particular application to cancer.
The same 50-kDa cellular protein binds to the negative regulatory elements of the interleukin 2 receptor alpha-chain gene and the human immunodeficiency virus type 1 long terminal repeat.
Authors: Smith M R , Greene W C
Published: May 2006

We have investigated the biochemical basis for negative regulation of interleukin 2 receptor alpha-chain (IL-2R alpha) gene expression. Transient transfection studies employing internally deleted forms of the IL-2R alpha promoter localized a negative regulatory element (NRE) between nucleotides -400 and -368 relative to the major distal transcription start (cap) site. This 31-base-pair (bp) element is involved in the attenuation of both basal and inducible IL-2R alpha promoter activity. Comparison of this IL-2R alpha NRE with other known regulatory motifs revealed an 11-bp core element (TTCATCCCAGG) that was strikingly similar to a protein-binding domain within the long terminal repeat of the type 1 human immunodeficiency virus (HIV-1). This viral domain has been previously implicated in the negative control of HIV-1 gene expression. In vitro protein-DNA binding studies demonstrated that the same constitutively expressed approximately 50-kDa protein (SP-50) specifically bound to both the IL-2R alpha and HIV-1 NRE core elements. Mutation of the 11-bp IL-2R alpha NRE core element, which disrupted protein binding, significantly augmented basal as well as Tax protein- or phorbol ester-induced IL-2R alpha promoter activity in vivo, suggesting that SP-50 functions as a transcriptional silencer.
Localization of the mRNA for the dopamine D2 receptor in the rat brain by in situ hybridization histochemistry.
Authors: Mengod G , Martinez-Mir M I , Vilaró M T , Palacios J M
Published: May 2006

32P-labeled oligonucleotides derived from the coding region of rat dopamine D2 receptor cDNA were used as probes to localize cells in the rat brain that contain the mRNA coding for this receptor by using in situ hybridization histochemistry. The highest level of hybridization was found in the intermediate lobe of the pituitary gland. High mRNA content was observed in the anterior lobe of the pituitary gland, the nuclei caudate-putamen and accumbens, and the olfactory tubercle. Lower levels were seen in the substantia nigra pars compacta and the ventral tegmental area, as well as in the lateral mammillary body. In these areas the distribution was comparable to that of the dopamine D2 receptor binding sites as visualized by autoradiography using [3H]SDZ 205-502 as a ligand. However, in some areas such as the olfactory bulb, neocortex, hippocampus, superior colliculus, and cerebellum, D2 receptors have been visualized but no significant hybridization signal could be detected. The mRNA coding for these receptors in these areas could be contained in cells outside those brain regions, be different from the one recognized by our probes, or be present at levels below the detection limits of our procedure. The possibility of visualizing and quantifying the mRNA coding for dopamine D2 receptor at the microscopic level will yield more information about the in vivo regulation of the synthesis of these receptors and their alteration following selective lesions or drug treatments.
The human protooncogene product p33pim is expressed during fetal hematopoiesis and in diverse leukemias.
Authors: Amson R , Sigaux F , Przedborski S , Flandrin G , Givol D , Telerman A
Published: May 2006

We measured the human pim-1 protooncogene (PIM) expression during fetal development and in hematopoietic malignancies. Our data indicate that during human fetal hematopoiesis the 33-kDa pim product, p33pim, is highly expressed in the liver and spleen. In contrast, at the adult stage it is only slightly expressed in circulating granulocytes. Out of 70 hematopoietic malignancies analyzed, 51 patients and 19 cell lines, p33pim was overexpressed in approximately 30% of the samples, particularly in myeloid and lymphoid acute leukemias. This overexpression was unrelated to any stage of cellular differentiation and was not due to gene rearrangement or amplification. These results imply a physiological role of the pim-1 protooncogene during hematopoietic development and a deregulation in various leukemias.
A long interspersed repetitive element--the I factor of Drosophila teissieri--is able to transpose in different Drosophila species.
Authors: Abad P , Vaury C , Pélisson A , Chaboissier M C , Busseau I , Bucheton A
Published: May 2006

Long interspersed repetitive elements (LINEs) are transposable elements present in many species. In mammals they are difficult to study because most of them are defective and their transposition frequency is low. The I factor of Drosophila melanogaster is a LINE element that is particularly interesting because its transposition occurs at high frequency during I-R hybrid dysgenesis. This phenomenon occurs when males from the class of inducer strains are crossed with females from the class of reactive strains. Inducer strains contain several complete 5.4-kilobase I factors at various sites on the chromosomal arms. Reactive strains are devoid of complete I factors. Many results indicate that active I factors have invaded the D. melanogaster genome recently. To study the evolutionary history of I elements, we have cloned and sequenced a potentially active I factor from Drosophila teissieri. It is flanked by a target-site duplication and terminates at the 3' end by tandem repeats of the sequence TAA. When introduced into the germ line of a reactive strain of D. melanogaster by P element-mediated transformation, it is able to transpose and induces hybrid dysgenesis. This strengthens the hypothesis of a recent reinvasion of the D. melanogaster genome by active I factors giving rise to the inducer strains. They could have originated by horizontal transfer from another species. Such events also could occur for other LINE elements and might explain the spread of new variants in mammalian genomes. Moreover, the results give a further insight into I factor functional organization.
Definition of a bacterial virulence factor: sialylation of the group B streptococcal capsule.
Authors: Wessels M R , Rubens C E , Benedí V J , Kasper D L
Published: May 2006

Sialylation of bacterial capsules has been proposed as an important virulence factor for several species of encapsulated pathogens, including group B Streptococcus. We have constructed a transposon mutant strain of type III group B Streptococcus that expresses a capsular polysaccharide differing from the wild type only in that the mutant strain's capsule lacks sialic acid. The mutant polysaccharide is antigenically identical to the capsular polysaccharide of type 14 Streptococcus pneumoniae, as predicted by the structures of the type III group B Streptococcus and S. pneumoniae polysaccharides. Loss of capsular sialic acid was associated with loss of virulence in the mutant strain in a neonatal rat model of lethal group B Streptococcus infection. These studies demonstrate directly that capsular sialic acid is a critical virulence determinant for type III group B Streptococcus and support the general hypothesis that surface sialylation aids pathogenic microorganisms in evading host defenses.
Thymine dimer formation as a probe of the path of DNA in and between nucleosomes in intact chromatin.
Authors: Pehrson J R
Published: May 2006

Photo-induced thymine dimer formation was used to probe nucleosome structure in nuclei. The distribution of thymine dimers in the nucleosome and recent studies of the structure of thymine dimer-containing DNA suggest that the rate of thymine dimer formation is affected by the direction and degree of DNA bending. This premise was used to construct a model of the path of DNA in the nucleosome, which has the following features. (i) There are four regions of sharp bending, two which have been seen previously by x-ray crystallography of the core particle. (ii) The DNA in H1-containing nucleosomes deviates from its superhelical path near the midpoint; this is not seen with H1-stripped chromatin. (iii) The internucleosomal (linker) DNA appears to be relatively straight.
Detection of specific DNA sequences by fluorescence amplification: a color complementation assay.
Authors: Chehab F F , Kan Y W
Published: May 2006

We have developed a color complementation assay that allows rapid screening of specific genomic DNA sequences. It is based on the simultaneous amplification of two or more DNA segments with fluorescent oligonucleotide primers such that the generation of a color, or combination of colors, can be visualized and used for diagnosis. Color complementation assay obviates the need for gel electrophoresis and has been applied to the detection of a large and small gene deletion, a chromosomal translocation, an infectious agent, and a single-base substitution. DNA amplification with fluorescent oligonucleotide primers has also been used to multiplex and discriminate five different amplified DNA loci simultaneously. Each primer set is conjugated to a different dye, and the fluorescence of each dye respective to its amplified DNA locus is scored on a fluorometer. This method is valuable for DNA diagnostics of genetic, acquired, and infectious diseases, as well as in DNA forensics. It also lends itself to complete automation.
A tissue-specific change in repetitive DNA in rats.
Authors: Yokota H , Iwasaki T , Takahashi M , Oishi M
Published: May 2006

From a genomic library constructed from an EcoRI digest of Wistar rat brain DNA, we isolated a clone (BL-1) that gave a 0.6-kilobase restriction fragment only in brain (or lens) DNA upon Southern hybridization. The tissue-specific fragment was present in Wistar and other strains (Sprague-Dawley and Donryu) of rats regardless of their sex and age. Sequencing of the clone indicated that it is closely related to a part of the LINE 3 sequence, one of the highly repetitive sequences present throughout mammalian genomes. Polymerase chain reaction using primer sequences in the BL-1 clone indicated that it is derived from an amplified (rear-ranged) sequence, although other explanations are possible. These results suggest that there are tissue-specific changes in DNA primary structure during mammalian developmental processes.
Expression cloning of the murine interferon gamma receptor cDNA.
Authors: Munro S , Maniatis T
Published: May 2006

A cDNA encoding a receptor for murine interferon gamma (IFN-gamma) was isolated from an expression library made from murine thymocytes. The clone was identified by transfecting the library into monkey COS cells and probing the transfected monolayer with radiolabeled murine IFN-gamma. Cells expressing the receptor were identified by autoradiography and plasmids encoding the receptor were directly rescued from those cells producing a positive signal. A partial cDNA so obtained was used to isolate a full-length cDNA from mouse L929 cells by conventional means. When this cDNA was expressed in COS cells it produced a specific binding site for murine IFN-gamma with an affinity constant similar to that of the receptor found on L929 cells. The predicted amino acid sequence of the murine IFN-gamma receptor shows homology to that previously reported for the human IFN-gamma receptor. However, although the two proteins are clearly related, they show less than 60% identity in both the putative extracellular domain and the intracellular domain.
Predictions of thermodynamic efficiency in a pumped biochemical reaction.
Authors: Hervagault J F , Lazar J G , Ross J
Published: May 2006

We propose and analyze a possible experimental system for the investigation of the thermodynamic efficiency of generating biochemical gradients. We investigate the efficiency of a model pump that uses 6-phosphofructokinase (EC 2.7.1.11, an enzyme that exhibits highly nonlinear kinetics), chromatophores from Rhodobacter sphaeroides, and light to generate a biochemical gradient. We analyze the experimental system and an equivalent alternative configuration and show that the establishment and maintenance of a concentration gradient across a membrane is thermodynamically equivalent to the establishment and maintenance of a stationary state in a single-phase, isothermal, open, homogeneous reaction system. With a constant input of light, the system can exist in a stable node (a stable steady state), a stable focus (upon perturbation from its steady state, the system returns to its steady state with an oscillatory component), and a stable limit cycle (at steady state the system exhibits stable oscillations). We investigate the efficiency of the system with both steady and oscillatory light input and observe efficiency changes that depend upon the autonomous state of the system and the frequency and amplitude of the periodic light input. When the system is in a stable focus, an efficiency maximum is seen when the system is perturbed at its resonant frequency. When the system is in a stable limit cycle, efficiency increases are seen near the 1:3, 1:2, and 2:1 entrainment regions and an efficiency decrease is seen near the 1:1 entrainment region. We further calculate various contributions to the efficiency: the phase shift of the force and flux, the magnitude of the response to the perturbation, and changes in the average values of the force and flux during a perturbation. We show that all three changes contribute to the overall changes in efficiency, but increases and decreases in the average force make the largest contributions.
DNA flexibility variation may dominate DNase I cleavage.
Authors: Hogan M E , Roberson M W , Austin R H
Published: May 2006

In a previous experimental study, we proposed that the bending and torsional stiffness of DNA display a systematic sequence dependence. Subsequently, we developed an elastic strain model to quantify the sequence dependence of the bending and torsional rigidity in terms of nearest neighbor interactions and used that model to analyze the sequence dependence of the 434 repressor binding to its operator. The analysis presented here shows that, in the absence of significant local variation of DNA secondary structure, DNase I cleavage is strongly correlated with local variation in the bending flexibility as calculated from our elastic strain model and that the agreement is also quantitatively significant. It is proposed that analysis using elastic strain models will provide a preliminary set of biochemical and chemical tools to explore the relation between DNA flexibility and the binding of other proteins.
Gamma-aminobutyrate type B receptor modulation of L-type calcium channel current at bipolar cell terminals in the retina of the tiger salamander.
Authors: Maguire G , Maple B , Lukasiewicz P , Werblin F
Published: May 2006

Bipolar-cell axon terminals receive direct synaptic input from amacrine-cell processes, suggesting a possible pathway for modulation of transmitter release. In retinal slices, bath-applied baclofen, a gamma-aminobutyrate type B (GABAB) receptor agonist, reduced a patch-clamp-recorded L-type calcium channel current in a population of bipolar cells with axon terminals that ramify along the midline of the inner plexiform layer. Lucifer yellow staining revealed that this current was found only in bipolar cells that retain axon terminals and their associated telodendria, suggesting that the current is generated at the terminal and also possibly modulated there. T-type calcium currents were found in all bipolar cells, including those without axon terminals, but were not modulated by baclofen. The baclofen-induced reduction of calcium current was enhanced by guanosine 5'-[gamma-thio]triphosphate and eliminated by guanosine 5'-[beta-thio]diphosphate added to the cytoplasm by the patch recording electrode, suggesting that the GABAB receptors act through a guanine nucleotide-binding regulatory protein (G protein). Baclofen also reduced an excitatory synaptic input to a population of amacrine cells with processes that ramify along the midline of the inner plexiform layer--cells probably postsynaptic to the bipolar terminals. This suggests that GABAB receptors modulate not only the calcium current but also transmitter release by a pathway involving G proteins and L-type calcium channels.
Substitution at position 116 of Schizosaccharomyces pombe calmodulin decreases its stability under nitrogen starvation and results in a sporulation-deficient phenotype.
Authors: Takeda T , Imai Y , Yamamoto M
Published: May 2006

We constructed Schizosaccharomyces pombe strains that carry phenylalanine, instead of arginine, as residue 116 of calmodulin by site-directed mutagenesis of the cam1 gene. Whereas haploid strains carrying the mutant allele, designated cam1-F116, exhibit no defects in growth and mating, diploid strains homozygous for cam1-F116 are deficient in sporulation. The four nuclei generated by the two serial meiotic divisions are not encapsulated in these diploids. The mutation is recessive. Semiquantitative analysis using polyclonal antibodies showed that vegetatively growing cam1-F116 cells have a smaller amount of calmodulin than wild-type cells. The quantitative difference becomes more remarkable if the cells are starved for nitrogen, which is a condition for induction of sporulation. In addition to this in vivo observation, we showed in vitro that the mutant protein is susceptible to a proteolytic activity induced by nitrogen starvation that hardly affects the wild-type calmodulin. Thus, the sporulation deficiency of the cam1-F116 mutant may be ascribed to shortage of calmodulin due to proteolysis of the mutant molecules under nitrogen starvation. Two other mutations at position 116 resulted in similar but leakier Spo- phenotypes.
Nucleolar targeting signal of human T-cell leukemia virus type I rex-encoded protein is essential for cytoplasmic accumulation of unspliced viral mRNA.
Authors: Nosaka T , Siomi H , Adachi Y , Ishibashi M , Kubota S , Maki M , Hatanaka M
Published: May 2006

The posttranscriptional regulator (rex) of human T-cell leukemia virus type I is known to be located predominantly in the cell nucleolus and to induce the accumulation of gag and env viral mRNAs. The N-terminal 19 amino acids of rex-encoded protein (Rex) has been shown to be sufficient to direct hybrid proteins to the cell nucleolus. We have studied the function of the nucleolar targeting signal (NOS) of rex by using full-length proviral DNA and mutant rex expression plasmids. Partial deletions of the NOS sequence abolished the accumulation of unspliced cytoplasmic mRNA, although the gene products of rex mutants were found in the nucleoplasm. These results indicate that NOS sequence, or nucleolar localization of Rex, is essential for Rex function.
Mitochondrial benzodiazepine receptors regulate steroid biosynthesis.
Authors: Mukhin A G , Papadopoulos V , Costa E , Krueger K E
Published: May 2006

Recent observations on the steroid synthetic capability within the brain open the possibility that benzodiazepines may influence steroid synthesis in nervous tissue through interactions with peripheral-type benzodiazepine recognition sites, which are highly expressed in steroidogenic cells and associated with the outer mitochondrial membrane. To examine this possibility nine molecules that exhibit a greater than 10,000-fold difference in their affinities for peripheral-type benzodiazepine binding sites were tested for their effects on a well-established steroidogenic model system, the Y-1 mouse adrenal tumor cell line. 4'-Chlorodiazepam, PK 11195, and PK 14067 stimulated steroid production by 2-fold in Y-1 cells, whereas diazepam, flunitrazepam, zolpidem, and PK 14068 displayed a lower (1.2- to 1.5-fold) maximal stimulation. In contrast, clonazepam and flumazenil did not stimulate steroid synthesis. The potencies of these compounds to inhibit 3H-labeled PK 11195 binding to peripheral-type benzodiazepine recognition sites correlated (r = 0.985) with their potencies to stimulate steroid production. Similar findings were observed in bovine and rat adrenocortical cell preparations. These results suggest that ligands of the peripheral-type benzodiazepine recognition site acting on this mitochondrial receptor can enhance steroid production. This action may contribute specificity to the pharmacological profile of drugs preferentially acting on the benzodiazepine recognition site associated with the outer membrane of certain mitochondrial populations.
A major insertion accounts for a significant proportion of mutations underlying human lipoprotein lipase deficiency.
Authors: Langlois S , Deeb S , Brunzell J D , Kastelein J J , Hayden M R
Published: May 2006

Lipoprotein lipase (LPL; triacylglyceroprotein acylhydrolase, EC 3.1.1.34) is an important enzyme involved in triacylglycerol metabolism. Primary LPL deficiency is a genetic disorder that is usually manifested by a severe elevation in triacylglycerol levels. We have used a recently isolated LPL cDNA clone to study 15 probands from 11 families with this inherited disorder. Surprisingly, 7 of the probands from 4 families, of different ancestries, had a similar insertion in their LPL gene. In contrast to other human genetic disorders, where insertions are rare causes of mutation, this insertion accounts for a significant proportion of the alleles causing LPL deficiency. Detailed restriction mapping of the insertion revealed that it was unlikely to be a duplication of neighboring DNA and that it was not similar to the consensus sequence of human L1 repetitive elements. This suggests that there must be other mechanisms of insertional mutagenesis in human genetic disease besides transposition of mobile L1 repetitive elements.
Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format.
Authors: Kwoh D Y , Davis G R , Whitfield K M , Chappelle H L , DiMichele L J , Gingeras T R
Published: May 2006

The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 x 10(6)-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 10(6) uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily be accomplished by means of a bead-based sandwich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.
Characterization of cDNAs encoding human leukosialin and localization of the leukosialin gene to chromosome 16.
Authors: Pallant A , Eskenazi A , Mattei M G , Fournier R E , Carlsson S R , Fukuda M , Frelinger J G
Published: May 2006

We describe the isolation and characterization of cDNA clones encoding human leukosialin, a major sialoglycoprotein of human leukocytes. Leukosialin is very closely related or identical to the sialophorin molecule, which is involved in T-cell proliferation and whose expression is altered in Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disease. Using a rabbit anti-serum to leukosialin, a cDNA clone was isolated from a lambda gt11 cDNA library constructed from human peripheral blood cells. This lambda gt11 clone was used to isolate longer cDNA clones that correspond to the entire coding sequence of leukosialin. DNA sequence analysis reveals three domains in the predicted mature protein. The extracellular domain is enriched for Ser, Thr, and Pro and contains four contiguous 18-amino acid repeats. The transmembrane and intracellular domains of the human leukosialin molecule are highly homologous to the rat W3/13 molecule. RNA gel blot analysis reveals two polyadenylylated species of 2.3 and 8 kilobases. Southern blot analysis suggests that human leukosialin is a single-copy gene. Analysis of monochromosomal cell hybrids indicates that the leukosialin gene is not X chromosome linked and in situ hybridization shows leukosialin is located on chromosome 16. These findings demonstrate that the primary mutation in WAS is not a defect in the structural gene for leukosialin.
Sequence-specific 1H-NMR assignments and identification of two small antiparallel beta-sheets in the solution structure of recombinant human transforming growth factor alpha.
Authors: Montelione G T , Winkler M E , Burton L E , Rinderknecht E , Sporn M B , Wagner G
Published: May 2006

Transforming growth factor alpha (TGF alpha) is a small mitogenic protein with about 35% sequence identity with epidermal growth factor (EGF). TGF alpha-like proteins have been proposed to play a role in oncogenesis and wound healing. This report describes sequence-specific 1H-NMR resonance assignments for recombinant human TGF alpha (hTGF alpha). These assignments provide the basis for interpreting NMR data which demonstrate that the solution structure of hTGF alpha includes an antiparallel beta-sheet involving residues Gly-19 to Leu-24 and Lys-29 to Cys-34 and a second, smaller, antiparallel beta-sheet involving residues Tyr-38 and Val-39 and His-45 and Ala-46. These data, together with constraints imposed by the disulfide bonds, are combined to construct a molecular model of the polypeptide chain fold for residues Cys-8 to Ala-46. The resulting structure is similar to that of mouse and human EGF. Human TGF alpha and mouse EGF, however, differ with respect to their structural dynamics, since amide proton/deuteron exchange is much faster for hTGF alpha than for mouse EGF at pH 3.5.
Vaccine T-cell epitope selection by a peptide competition assay.
Authors: Kilgus J , Romagnoli P , Guttinger M , Stuber D , Adorini L , Sinigaglia F
Published: May 2006

The binding of several peptides derived from the Plasmodium falciparum circumsporozoite protein (CS protein) to the human major histocompatibility complex class II proteins HLA-DR5 and -DRw6 was examined in a competition assay. Fixed antigen-presenting cells (APCs) were incubated with various concentrations of each peptide and suboptimal concentrations of stimulator peptides. The binding of the CS peptides to DR5 or DRw6 proteins was then determined in a proliferation assay using two established DR5 or DRw6-restricted T-cell clones with specificity for the stimulator peptides as responder cells. One of five CS peptides, comprising together about 50% of the CS protein sequence, was found to compete with the binding of the stimulator peptides to DR5 and DRw6. The CS peptide CS-(378-398), binding to DR5 and DRw6, was then shown to be able to induce primary in vitro responses of T cells from donors with DR5 and DRw6 haplotypes. CS-(378-398)-induced T-cell clones responded not only to the homologous peptide but also to the native CS protein in the presence of appropriate APCs. The strategy we have applied is of considerable general interest for the engineering of vaccines against any pathogen, since it greatly facilitates the selection of appropriate T-cell epitopes to be incorporated in the vaccine.
Molecular characterization and expression in Escherichia coli of the gene complex encoding the polysaccharide capsule of Neisseria meningitidis group B.
Authors: Frosch M , Weisgerber C , Meyer T F
Published: May 2006

The gene complex encoding all determinants of the biosynthesis pathway of the capsule of group B meningococci (cps) has been cloned in Escherichia coli. A 24-kilobase large chromosomal fragment is necessary for capsule expression on the E. coli surface. By transposon and deletion mutagenesis, two separate steps in transport of the polysaccharide from the cytoplasm to the periplasm and further to the cell surface became evident. Mutants were also isolated that accumulate soluble poly(sialic acid) in the cytoplasm. The cloned cps complex conferred to E. coli strain GC6 sensitivity for E. coli K1-specific phages; phage sensitivity was enhanced in two distinct classes of cps mutants. Southern blot experiments revealed homology to some or all other Neisseria meningitidis capsular types and other Neisseria species, depending on the fragment of the cps complex used as probe.
Calcium channel block by cadmium in chicken sensory neurons.
Authors: Swandulla D , Armstrong C M
Published: May 2006

Cadmium block of calcium channels was studied in chicken dorsal root ganglion cells by a whole-cell patch clamp that provides high time resolution. Barium ion was the current carrier, and the channel type studied had a high threshold of activation and fast deactivation (type FD). Block of these channels by 20 microM external Cd2+ is voltage dependent. Cd2+ ions can be cleared from blocked channels by stepping the membrane voltage (Vm) to a negative value. Clearing the channels is progressively faster and more complete as Vm is made more negative. Once cleared of Cd2+, the channels conduct transiently on reopening but reequilibrate with Cd2+ and become blocked within a few milliseconds. Cd2+ equilibrates much more slowly with closed channels, but at a holding potential of -80 mV virtually all channels are blocked at equilibrium. Cd2+ does not slow closing of the channels, as would be expected if it were necessary for Cd2+ to leave the channels before closing occurred. Instead, the data show unambiguously that the channel gate can close when the channel is Cd2+ occupied.
Purification and analysis of RNA polymerase II transcription factors by using wheat germ agglutinin affinity chromatography.
Authors: Jackson S P , Tjian R
Published: May 2006

We recently found that many RNA polymerase II transcription factors are modified with N-acetylglucosamine residues. These sugar moieties confer upon transcription factors an ability to bind the lectin wheat germ agglutinin. We have taken advantage of this interaction to devise a purification procedure for the "GC-box" binding transcription factor Sp1. Crude nuclear extracts are first subjected to wheat germ agglutinin affinity chromatography and then subjected to sequence-specific DNA affinity chromatography. The Sp1 protein purified by this procedure is at least 95% pure, and the overall recovery is greater than 80%. In addition to yielding larger quantities of Sp1 than conventional schemes, the new purification procedure is also simpler and more rapid. We show that wheat germ agglutinin affinity chromatography can also be used to purify the glycosylated forms of the CCAAT-binding transcription factor. Thus, wheat germ agglutinin affinity chromatography may aid the purification of other transcription factors that bear N-acetylglucosamine residues. Furthermore, the ability to separate glycosylated forms of transcription factors from their unglycosylated counterparts by wheat germ agglutinin affinity chromatography should facilitate investigations into the role of N-acetylglucosamine residues in the functioning of transcription factor proteins.
One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.
Authors: Chung C T , Niemela S L , Miller R H
Published: May 2006

We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.