bluuebunny/crossref_metadata_2025 huggingface dataset:
Levinthal's paradox.
Authors: Zwanzig R , Szabo A , Bagchi B
Published: May 2006

Levinthal's paradox is that finding the native folded state of a protein by a random search among all possible configurations can take an enormously long time. Yet proteins can fold in seconds or less. Mathematical analysis of a simple model shows that a small and physically reasonable energy bias against locally unfavorable configurations, of the order of a few kT, can reduce Levinthal's time to a biologically significant size.
Specific inflammatory cytokines regulate the expression of human monocyte 15-lipoxygenase.
Authors: Conrad D J , Kuhn H , Mulkins M , Highland E , Sigal E
Published: May 2006

Arachidonate 15-lipoxygenase (arachidonate:oxygen 15-oxidoreductase, EC 1.13.11.33) is a lipid-peroxidating enzyme that is implicated in oxidizing low density lipoprotein to its atherogenic form. Monocyte/macrophage 15-lipoxygenase is present in human atherosclerotic lesions. To pursue a basis for induction of the enzyme, which is not present in blood monocytes, the ability of relevant cytokines to regulate its expression was investigated. Interleukin 4 (IL-4), among 16 factors tested, specifically induced 15-lipoxygenase mRNA and protein in cultured human monocytes. Interferon gamma and hydrocortisone inhibited this induction. High-performance liquid chromatography analysis of lipid extracts from IL-4-treated monocytes detected 15-lipoxygenase products esterified to the cellular membrane lipids, indicating enzymatic action on endogenous substrates. Stimulation of IL-4-treated monocytes with calcium ionophore or opsonized zymosan A enhanced the formation of 15-lipoxygenase products. These data identify IL-4 and interferon gamma as physiological regulators of lipoxygenase expression and suggest an important link between 15-lipoxygenase function and the immune/inflammatory response in atherosclerosis as well as other diseases.
Generation of plasmacytomas with the chromosomal translocation t(12;15) in interleukin 6 transgenic mice.
Authors: Suematsu S , Matsusaka T , Matsuda T , Ohno S , Miyazaki J , Yamamura K , Hirano T , Kishimoto T
Published: May 2006

The mechanisms through which pristane or mineral oil can induce plasmacytomas in BALB/c or NZB mice are not fully understood, but involvement of interleukin 6 (IL-6), a growth factor for plasmacytomas and myelomas, has been strongly suggested. To clarify the role of IL-6 in plasmacytomagenesis, a human IL-6 cDNA was introduced into mouse germ lines under the transcriptional control of the murine major histocompatibility complex class I (H-2Ld) promoter. IL-6 transgenic mice of C57BL/6 origin developed a massive plasmacytosis but not plasmacytomas. However, introduction of BALB/c genetic background into IL-6 transgenic mice could generate monoclonal transplantable plasmacytomas with the chromosomal translocation t(12;15). These results provide firm evidence of the critical role of IL-6 in the plasmacytoma development.
Mutagenic specificities of four stereoisomeric benzo[c]phenanthrene dihydrodiol epoxides.
Authors: Bigger C A , St John J , Yagi H , Jerina D M , Dipple A
Published: May 2006

The pS189 shuttle vector carrying a supF target gene was used to compare the mutagenic specificities of the four configurational isomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide. One of these isomers is the most tumorigenic dihydrodiol epoxide tested to date and another is essentially inactive as a tumorigen. Overall mutagenicities were not correlated with tumorigenicities, but each configurational isomer induced a unique spectrum of mutational hot spots in the supF target gene, which monitors primarily point mutations. It is suggested that the demonstrated isomer-specific selectivity for mutation targets within the supF gene may be indicative of a similar selectivity for one gene versus another and that such selectivity may be one determinant of relative tumorigenicity.
Selection-induced mutations occur in yeast.
Authors: Hall B G
Published: May 2006

Selection-induced mutations are nonrandom mutations that occur as specific and direct responses to environmental challenges and primarily in nondividing cells under conditions of intense prolonged selection. Selection-induced mutations have been shown to occur at six loci in Escherichia coli, but their existence has not previously been demonstrated in any eukaryotic organism. Here it is shown that selection-induced mutations occur at the HIS4 locus in the eukaryotic microorganism Saccharomyces cerevisiae.
2',5'-Bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino-1'',2''- oxathiole-2'',2'-dioxide)pyrimidine (TSAO) nucleoside analogues: highlyselective inhibitors of human immunodeficiency virus type 1 that are targeted at the viral reverse transcriptase.
Authors: Balzarini J , Pérez-Pérez M J , San-Félix A , Schols D , Perno C F , Vandamme A M , Camarasa M J , De Clercq E
Published: May 2006

A series of pyrimidine nucleoside analogues containing [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino- 1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-ribofuranose as the pentose were found to inhibit human immunodeficiency virus type 1 [HIV-1(IIIB)] replication at a concentration of 0.06-0.8 microM but were not cytotoxic at a 1000- to 10,000-fold higher concentration. These nucleoside derivatives were also effective against various other HIV-1 strains, including those resistant to 3'-azido-3'-deoxythymidine, but not against HIV-2, simian immunodeficiency virus, Moloney murine sarcoma virus, or other RNA or DNA viruses. They proved to be highly specific inhibitors of the RNA-dependent DNA polymerase function of the HIV-1 reverse transcriptase, showing no marked inhibition of the HIV-1 reverse transcriptase-associated DNA-dependent DNA polymerase activity, HIV-2 reverse transcriptase, DNA polymerase alpha, herpes simplex virus 1 DNA polymerase, or Thermus aquaticus DNA polymerase.
Approximating Markov chains.
Authors: Pincus S M
Published: May 2006

A common framework of finite state approximating Markov chains is developed for discrete time deterministic and stochastic processes. Two types of approximating chains are introduced: (i) those based on stationary conditional probabilities (time averaging) and (ii) transient, based on the percentage of the Lebesgue measure of the image of cells intersecting any given cell. For general dynamical systems, stationary measures for both approximating chains converge weakly to stationary measures for the true process as partition width converges to 0. From governing equations, transient chains and resultant approximations of all n-time unit probabilities can be computed analytically, despite typically singular true-process stationary measures (no density function). Transition probabilities between cells account explicitly for correlation between successive time increments. For dynamical systems defined by uniformly convergent maps on a compact set (e.g., logistic, Henon maps), there also is weak continuity with a control parameter. Thus all moments are continuous with parameter change, across bifurcations and chaotic regimes. Approximate entropy is seen as the information-theoretic rate of entropy for approximating Markov chains and is suggested as a parameter for turbulence; a discontinuity in the Kolmogorov-Sinai entropy implies that in the physical world, some measure of coarse graining in a mixing parameter is required.
Organization and evolution of the human epidermal keratinocyte transglutaminase I gene.
Authors: Polakowska R R , Eickbush T , Falciano V , Razvi F , Goldsmith L A
Published: May 2006

Transglutaminases (TGases; protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) are calcium-dependent crosslinking enzymes that modify proteins posttranslationally. Several distinct types of TGases have been identified, which appear to be encoded by a family of closely related genes. We isolated the gene encoding human keratinocyte-specific type I TGase (TGase I) and characterized its chromosomal organization. The TGase I gene consists of 15 exons separated by 14 introns and exhibits a restriction fragment length polymorphism. Exons appear to encode functional and/or structural domains: exon I and part of exon XV encode untranslated regions, whereas exons VII and XI contain the active site and a presumptive calcium-binding domain, respectively. Interestingly, exon VI of TGase I contains a consensus Arg-Gly-Asp tripeptide sequence whose presence suggests an intriguing extracellular function for the enzyme. We present a likely phylogenetic tree for seven known members of the TGase family based on amino acid sequence similarity. Arguments presented suggest that the active enzyme evolved first and the structural human erythrocyte membrane protein 4.2 (band 4.2) has undergone a rapid change in amino acid sequence. It follows that band 4.2 evolved from the type II TGases, whereas factor XIII subunit a evolved from the type I group.
Identification of highest-affinity ligands by affinity selection from equimolar peptide mixtures generated by robotic synthesis
Authors: Zuckermann Ronald N. , Kerr Janice M. , Siani Michael A. , Banville Steven C. , Santi Daniel V.
Published: May 2006

A fully automated peptide synthesizer has been constructed that is capable of the simultaneous synthesis of up to 36 individual peptides and the synthesis of equimolar peptide mixtures. The instrument consists of an array of reaction vessels, a series of solenoid valves to control liquid flow, and a Zymark robot to deliver solvents and reagents; all components are computer controlled and coordinated. Equimolar peptide mixtures are obtained by algorithms that automate the mixing and distribution of peptide-resin particles. This technology was used to synthesize a library of 361 peptides, generated by randomizing two critical binding residues of a 10-mer epitope known to bind an anti-human immuno-deficiency virus gp120 monoclonal antibody. Each critical residue was substituted with 19 amino acids consisting of all the natural amino acids except cysteine. The library was synthesized as 19 pools, each containing 19 peptides. Each pool was screened in a solution-phase competition ELISA assay. The 12 most inhibitory peptides in the library were isolated by a rapid affinity-selection method and were identified by mass spectrometry and amino acid analysis. The binding properties of these 12 selected peptides were verified by synthesis and assay of the individual peptides. The two critical residues investigated were found to contribute independently to antibody binding.
Molecular cloning of epididymal and seminal vesicular transcripts encoding a semenogelin-related protein.
Authors: Lilja H , Lundwall A
Published: May 2006

Freshly ejaculated human semen has the appearance of a loose gel in which the predominant structural protein components are the seminal vesicle-secreted semenogelins (Sg). The primary structure of the 439-residue SgI has previously been obtained by cDNA cloning. This cDNA cross-hybridizes to a larger transcript coding for a second secretory protein, SgII. Here we report the almost complete structure of a precursor of SgII established by lambda gt11 clones isolated from epididymal and seminal vesicular cDNA libraries. The deduced amino acid sequence of the 559-residue mature protein has a molecular weight of 62,931 but an increase in weight may be provided by asparagine-linked oligosaccharide attachment at residue 249. SgII, which has 78% overall identity with SgI, contains eight 60-residue regions that display conspicuous internal sequence similarity, whereas SgI only contains six of these regions. The SgII structure is translated from an open reading frame in a polyadenylylated 2.4-kilobase transcript. The message is abundant in the seminal vesicles but rare in the epididymis.
Gamma delta T-cell receptor repertoire in acute multiple sclerosis lesions.
Authors: Wucherpfennig K W , Newcombe J , Li H , Keddy C , Cuzner M L , Hafler D A
Published: May 2006

Gamma delta T cells are a distinct lymphocyte population that can exhibit reactivity with heat shock proteins over-expressed at inflammatory sites. As gamma delta T cells may be involved in the central nervous system (CNS) inflammatory process in multiple sclerosis (MS), we examined T-cell populations in MS plaque tissue by quantitative immunohistochemistry and sequence analysis of T-cell antigen receptor (TCR) delta chains. Gamma delta T cells that express the variable (V) gene segments V delta 1, V delta 2, and V gamma 2 (V gamma 9) were found to accumulate in acute, demyelinating MS plaques and appeared to have undergone clonal expansion, most likely because of recognition of a specific CNS ligand. Further, 60-kDa and 90-kDa heat shock proteins (hsp60 and hsp90), which may be target antigens for autoreactive gamma delta T cells, were found to be expressed in normal CNS tissue and overexpressed in acute MS plaques. In acute plaques, hsp60 was found in foamy macrophages, while hsp90 was detected in reactive astrocytes. These results provide evidence for a role of gamma delta T cells in active stages of MS.
A vaccine carrier derived from Neisseria meningitidis with mitogenic activity for lymphocytes.
Authors: Liu M A , Friedman A , Oliff A I , Tai J , Martinez D , Deck R R , Shieh J T , Jenkins T D , Donnelly J J , Hawe L A
Published: May 2006

Protein carriers vary in their ability to increase the immunogenicity of poorly immunogenic or T-lymphocyte-independent antigens. We examined one such carrier, the outer membrane protein complex derived from Neisseria meningitidis serogroup B strain B11, in an attempt to determine why this outer membrane protein complex was more immunogenic in young infants and in relevant animal models than two other carriers used in conjugates made with Haemophilus influenzae type b polysaccharide, a T-cell-independent antigen. A single protein of the outer membrane protein complex, the class 2 porin protein, was purified and shown to function as a T-helper lymphocyte carrier protein. Unexpectedly, it was also found to have mitogenic activity for lymphocytes that was not due to lipopolysaccharide. This mitogenic activity appears to date to be unique to this carrier protein of the carrier proteins tested and may contribute to the ability of the H. influenzae type b conjugate vaccine made with the outer membrane protein complex to generate IgG anti-polysaccharide antibody responses in mice and infant monkeys and protective immune responses in infants less than 6 months of age.
Metal binding and folding properties of a minimalist Cys2His2 zinc finger peptide.
Authors: Michael S F , Kilfoil V J , Schmidt M H , Amann B T , Berg J M
Published: May 2006

A minimalist Cys2His2 zinc finger peptide, Lys-Tyr-Ala-Cys-Ala-Ala-Cys-Ala-Ala-Ala-Phe-Ala-Ala-Lys-Ala-Ala-Leu-Ala- Ala-His-Ala-Ala-Ala-His-Ala-Lys, has been synthesized. Metal binding studies using Co2+ as a probe indicated that this peptide forms a 1:1 peptide/metal complex with a dissociation constant comparable to that observed for other zinc finger peptides. At high peptide concentrations, a 2:1 peptide/metal complex also forms, with four cysteinates coordinated to Co2+. Additional studies with sequence variants in which the canonical hydrophobic residues were changed to alanine, or in which one of the residues between the cysteines and the histidines was deleted, revealed an even more pronounced formation of the 2:1 complex over the 1:1 complex. In addition, the absorption spectra of the 1:1 peptide/Co2+ complexes of the variant peptides are significantly different from those seen for complexes of the parent peptide or those of more typical zinc finger peptides. NMR studies revealed that the parent peptide folds in the presence of Zn2+ to a structure very similar to that observed for other zinc finger peptides of this class. Taken together, these results suggest that the metal-binding and canonical hydrophobic residues are necessary and sufficient to determine the structure of this class of zinc finger peptides.
The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase.
Authors: Chang H W , Watson J C , Jacobs B L
Published: May 2006

A vaccinia virus-encoded double-stranded RNA-binding protein, p25, has been previously implicated in inhibition of the interferon-induced, double-stranded RNA-activated protein kinase. In this study, we have identified the vaccinia viral gene (WR strain) that encodes p25. Amino acid sequence analysis of a chymotryptic fragment of p25 revealed a close match to the vaccinia virus (Copenhagen strain) E3L gene. The WR strain E3L gene was cloned and expressed either in COS-1 cells or in rabbit reticulocyte lysates in vitro. A M(r) 25,000 polypeptide that could bind to poly(rI).poly(rC)-agarose and that reacted with p25-specific antiserum was produced in each case. In addition, COS cells expressing E3L gene products inhibited activation of the double-stranded RNA-activated protein kinase in extracts from interferon-treated cells. Removal of E3L-encoded products by adsorption with anti-p25 antiserum resulted in loss of kinase inhibitory activity. These results demonstrate that the vaccinia virus E3L gene encodes p25 and that the products of the E3L gene have kinase inhibitory activity. Comparison of the deduced amino acid sequence of the E3L gene products with the protein sequence data base revealed a region closely related to the human interferon-induced, double-stranded RNA-activated protein kinase.
Determination by systematic deletion of the amino acids essential for catalysis by ricin A chain.
Authors: Morris K N , Wool I G
Published: May 2006

The A chain of ricin is a RNA N-glycosidase that inactivates ribosomes by depurination of a single adenosine in 28S rRNA. Of the 267 amino acids in the protein, 222 (83%) could be deleted from one or another of 74 mutants without loss of the capacity of the protein to recognize a single nucleotide from among the 7000 in ribosomes or to catalyze hydrolysis.
Induction of c-jun protooncogene expression and transcription factor AP-1 activity by the polyoma virus middle-sized tumor antigen.
Authors: Schönthal A , Srinivas S , Eckhart W
Published: May 2006

Polyoma virus middle-sized tumor (PymT) antigen is required for neoplastic cell transformation by polyoma virus. We studied changes in gene expression accompanying expression of PymT in murine fibroblasts. These experiments showed that PymT differentially affects several growth-related genes. c-jun protooncogene expression was highly increased, whereas the expression of two growth arrest-specific genes (gas) was reduced, in cells transformed by PymT. Cotransfection experiments showed that the increase in c-jun expression resulted from elevated activity of the transcription factor AP-1 and was mediated through the phorbol 12-tetradecanoate 13-acetate response element in the c-jun promoter. The degree of c-Jun/AP-1 activation by different PymT mutants correlated with their transforming capability, suggesting that regulation of c-Jun/AP-1 activity may play a role in cell transformation by polyoma virus.
Extrachromosomal genetic complementation of surface metalloproteinase (gp63)-deficient Leishmania increases their binding to macrophages.
Authors: Liu X , Chang K P
Published: May 2006

A major surface glycoprotein of 63 kDa (gp63) has been previously identified biochemically and genetically as a zinc proteinase conserved in pathogenic Leishmania spp. The functional significance of this proteinase was analyzed by genetic approaches. A 15-kilobase DNA with a tunicamycin-resistance gene from Leishmania amazonensis was ligated in two different orientations into pBluescript containing a gp63 gene from Leishmania major. These plasmid constructs were used to transfect a variant of L. amazonensis deficient in gp63 expression. Both constructs were found to confer tunicamycin resistance with equal efficiency and remained structurally unchanged in the transfectants. RNA and immunoblot analyses showed over-expression of gp63 in the transfectants with one of the two plasmids constructed. The over-produced products were enzymatically active and expressed on the cell surface. Significantly, the transfectants with over-expressed gp63 increased by 2-fold over controls in their binding to macrophages. Evidence presented thus indicates that the gp63 gene constructed in the plasmid as described and introduced exogenously expresses in the gp63-deficient variants and that the expressed products are functionally active.
Phytochrome requires the 6-kDa N-terminal domain for full biological activity.
Authors: Cherry J R , Hondred D , Walker J M , Vierstra R D
Published: May 2006

Phytochrome is a red/far-red-absorbing photoreceptor that controls many aspects of plant photomorphogenesis. Because proteolytic removal of approximately 6 kDa from the N terminus of 124-kDa oat phytochrome substantially alters many physicochemical properties of the chromoprotein, it has been proposed that the N terminus is required for biological activity. Here we test this hypothesis by comparing tobacco plants expressing full-length oat phytochrome (FL) with plants expressing a 118-kDa oat phytochrome lacking amino acids 7-69 (NA phytochrome). NA phytochrome, like its FL counterpart, exists as a homodimer in solution, is capable of covalently binding chromophore to form a red/far-red-photoreversible product, and is rapidly degraded in vivo after photoconversion to the far-red-absorbing form. However, like proteolytically degraded phytochrome missing the N terminus, the absorption maxima of the red- and far-red-light-absorbing forms of NA phytochrome are blue shifted relative to the maxima of the FL chromoprotein, and the rate of dark reversion of the far-red- to red-light-absorbing form is substantially increased. Tobacco plants producing high levels of NA phytochrome do not exhibit the light-exaggerated phenotype characteristic of FL phytochrome overexpression. By comparison of phytochrome-dose-phenotype-response curves generated by using a series of transgenic lines expressing various levels of FL or NA phytochrome, we demonstrate that NA phytochrome has less than 1/5th the biological activity of FL phytochrome expressed in tobacco. Furthermore, the shape of the dose-response curve for plants expressing FL phytochrome indicates that there is a sharp transition between phenotypically normal and abnormal plants over a relatively narrow range of phytochrome content, demonstrating that precise control of phytochrome levels is critical to photomorphogenesis.
Molecular cloning of the alpha-1 subunit of an omega-conotoxin-sensitive calcium channel.
Authors: Dubel S J , Starr T V , Hell J , Ahlijanian M K , Enyeart J J , Catterall W A , Snutch T P
Published: May 2006

Of the four major types of Ca channel described in vertebrate cells (designated T, L, N, and P), N-type Ca channels are unique in that they are found specifically in neurons, have been correlated with control of neurotransmitter release, and are blocked by omega-conotoxin, a neuropeptide toxin isolated from the marine snail Conus geographus. A set of overlapping cDNA clones were isolated and found to encode a Ca channel alpha-1 subunit, designated rbB-I. Polyclonal antiserum generated against a peptide from the rbB-I sequence selectively immunoprecipitates high-affinity 125I-labeled omega-conotoxin-binding sites from labeled rat forebrain membranes. PCR analysis shows that, like N-type Ca channels, expression of rbB-I is limited to the nervous system and neuronally derived cell lines. This brain Ca channel may mediate the omega-conotoxin-sensitive Ca influx required for neurotransmitter release at many synapses.
Crossmodal changes in the somatosensory vibrissa/barrel system of visually deprived animals.
Authors: Rauschecker J P , Tian B , Korte M , Egert U
Published: May 2006

Cats deprived of vision from birth adapt remarkably well to their situation and show little behavioral impairment. They seem to compensate for their lack of vision by relying more on their auditory and tactile senses. We report here that the facial vibrissae, which are most important for tactile orientation in many animals, show supernormal growth in both cats and mice that have been deprived of vision from birth. Furthermore, the whisker representation in the somatosensory cortical barrel field shows a concomitant enlargement in binocularly enucleated mice: individual barrels are expanded in size by up to one-third. The increased use of the vibrissae in visually deprived animals may stimulate both their own growth and, via activation of the respective neural pathways, the expansion of their central representation.
Chimpanzees immunized with recombinant soluble CD4 develop anti-self CD4 antibody responses with anti-human immunodeficiency virus activity.
Authors: Watanabe M , Boyson J E , Lord C I , Letvin N L
Published: May 2006

In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.
Pregnenolone biosynthesis in C6-2B glioma cell mitochondria: regulation by a mitochondrial diazepam binding inhibitor receptor.
Authors: Papadopoulos V , Guarneri P , Kreuger K E , Guidotti A , Costa E
Published: May 2006

The C6-2B glioma cell line, rich in mitochondrial receptors that bind with high affinity to benzodiazepines, imidazopyridines, and isoquinolinecarboxamides (previously called peripheral-type benzodiazepine receptors), was investigated as a model to study the significance of the polypeptide diazepam binding inhibitor (DBI) and the putative DBI processing products on mitochondrial receptor-regulated steroidogenesis. DBI and its naturally occurring fragments have been found to be present in high concentrations in C6-2B glioma cells, to compete against specific isoquinolinecarboxamide or 4'-chlorodiazepam binding to mitochondrial recognition sites with high affinity, and to stimulate mitochondrial pregnenolone formation. These data suggest that this cell type may express both the receptor and the putative agonist ligand to regulate steroidogenesis. Therefore, we propose to term this mitochondrial receptor MDR (mitochondrial DBI receptor) to indicate its responsiveness to DBI in steroid biosynthesis. In the present work, we show that mitochondria of C6-2B cells convert (22R)-22-hydroxycholesterol to pregnenolone by a mechanism blocked by aminoglutethimide. Immunoblotting confirmed the presence of relatively high levels of cytochrome P-450 cholesterol side-chain-cleavage enzyme in C6-2B cell mitochondria. Furthermore, isoquinolinecarboxamide binding sites associated with the 18-kDa mitochondrial polypeptide subunit of the MDR are abundant in C6-2B glioma cell mitochondria (Bmax approximately 30 pmol/mg protein) and are coupled to the regulation of steroid biosynthesis. Occupancy of MDRs with nanomolar concentrations of the naturally occurring polypeptide, DBI, as well as its naturally occurring processing product tetratriacontaneuropeptide [DBI-(17-50)] increases pregnenolone formation. Clonazepam and octadecaneuropeptide [DBI-(33-50)], which exhibit a higher affinity for gamma-aminobutyric acid type A receptors but a low affinity for MDR, were ineffective in stimulating pregnenolone synthesis. These findings provide evidence that C6-2B cells exhibit a significant steroidogenic activity which resembles that found in peripheral endocrine organs and they suggest that MDRs and DBI are involved in the regulation of glial cell steroidogenesis.
Solvent dielectric effects on protein dynamics.
Authors: Affleck R , Haynes C A , Clark D S
Published: May 2006

Electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations were used to investigate the dynamics of alpha-chymotrypsin in solvents ranging in dielectric constant from 72 to 1.9. EPR measurements showed that motions in the vicinity of two spin-labeled amino acids (Met-192 and Ser-195) decreased dramatically with decreasing solvent dielectric constant, a trend consistent with changes in the electrostatic force between charged residues of the protein. EPR results and MD simulations revealed a very similar functional dependence between rates of motion in the protein and the dielectric constant of the bulk solvent; however, predicted motions of protein atoms were markedly faster than measured motions of the spin labels. MD calculations for dielectric constants of 5 and 72 showed the greatest differences near the outer surface of the protein. In general, at the lower dielectric constant many atoms of the protein move more slowly, and many of the slowest residues are near the exterior. These results suggest that altered dynamics may contribute to the unusual properties--e.g., modified stereoselectivities--of enzymes in nearly dry organic solvents.
Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells.
Authors: Chen M , Pan Z Q , Hurwitz J
Published: May 2006

The elongation of primed DNA templates by DNA polymerase delta and DNA polymerase epsilon requires the action of two accessory proteins, proliferating cell nuclear antigen and activator 1 (A1, also called replication factor C). A1 is an enzyme that contains five different subunits (145, 40, 38, 37, and 36.5 kDa). In this paper, we describe the isolation of the gene encoding the 37-kDa subunit from HeLa cells. This gene was cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence shows a high degree of homology to the 40-kDa subunit of A1; they both contain the identical ATP-binding motif, but in contrast to the bacterial expressed 40-kDa protein, the 37-kDa expressed protein did not bind ATP. Both the 37- and 40-kDa proteins share substantial homology with the phage T4 gene 44 protein and to a lesser extent with the tau and gamma subunits of the E. coli DNA polymerase III holoenzyme. Polyclonal antibodies against the bacterially expressed 37- and 40-kDa proteins do not crossreact and are specific in their interaction. Antibodies against the 37-kDa protein maximally inhibited (by 50%) the A1-dependent synthesis of DNA by DNA polymerase delta; antibodies against the 40-kDa protein quantitatively inhibited the same reaction. When A1-dependent synthesis of DNA was partially inhibited by antibodies against the 40-kDa subunit, the addition of antibodies against the 37-kDa subunit inhibited DNA synthesis to a greater extent than the anti-37-kDa antibody alone. These results suggest that both the 37- and 40-kDa subunits of A1 are required for the biological role of A1 and that they may function differently in this process.
Spontaneous incorporation of the glycosyl-phosphatidylinositol-linked protein Thy-1 into cell membranes.
Authors: Zhang F , Schmidt W G , Hou Y , Williams A F , Jacobson K
Published: May 2006

Thy-1 is a membrane protein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol anchor. Purified rat brain Thy-1 could be reincorporated into the plasma membrane of murine Thy-1- cells directly from aqueous suspension and without the use of detergents. A peripheral staining pattern similar to that observed for endogenous Thy-1 was achieved. Treatment with phosphatidylinositol-specific phospholipase C removed nearly all antibody staining due to either endogenous or inserted Thy-1. Fluorescence recovery after photobleaching (FRAP) was used to compare the lateral mobility of endogenous and inserted Thy-1. Both forms exhibited large lateral diffusion coefficients, but with a substantial immobile fraction (approximately 50%) indicating that the immobile fraction was not due either to chemical differences between inserted and native Thy-1 or to some surface Thy-1 molecules having a protein anchor. However, the inserted Thy-1 failed to activate mouse T lymphocytes upon crosslinking as assayed by [3H]thymidine uptake. Since Thy-1 could be directly labeled with rhodamine, the effect of the size of the labeling ligand on the mobility obtained by the FRAP technique could be explored. Rhodamine-conjugated MRC-OX7 monoclonal antibody or its fragments [R-F(ab)2 or R-Fab] were compared with rhodamine as labels for Thy-1. The measured diffusion coefficients were 1.6 x 10(-9), 2.0 x 10(-9), and 3.2 x 10(-9) cm2/sec for Thy-1 labeled with R-F(ab)2, R-Fab, and rhodamine, respectively; mobile fractions were all in the 40-50% range. Thus, the size of the ligand affects the lateral mobility of this labeled membrane protein to a measurable extent.
A cobalt-porphyrin enzyme converts a fatty aldehyde to a hydrocarbon and CO.
Authors: Dennis M , Kolattukudy P E
Published: May 2006

The final step in hydrocarbon biosynthesis involves loss of CO from a fatty aldehyde. This decarbonylation is catalyzed by microsomes from Botyrococcus braunii. Among the several detergents tested for solubilizing the decarbonylase, octyl beta-glucoside (0.1%) was found to be the most effective and released 65% of the enzyme activity in soluble form. FPLC of the solubilized enzyme preparation with Superose 6 followed by ion-exchange FPLC with Mono Q resulted in 200-fold increase in specific activity with 7% recovery. The purified enzyme released nearly 1 mol of CO for each mol of hydrocarbon. SDS/PAGE of the enzyme preparation showed two protein bands of equal intensity at 66 and 55 kDa. The absorption spectrum of the enzyme with bands at 410 nm, 425 nm, 580 nm, and 620 nm suggests the presence of a porphyrin. Electron microprobe analysis revealed that the enzyme contained Co. Purification of the decarbonylase from B. braunii grown in 57CoCl2 showed that 57Co coeluted with the decarbonylase. These results suggest that the enzyme contains Co that might be part of a Co-porphyrin, although a corrin structure cannot be ruled out. Co-protoporphyrin IX itself caused decarbonylation of octadecanal at 60 degrees C, whereas the metal ion or protoporphyrin alone, or several other metal porphyrins, did not cause decarbonylation. These results strongly suggest that biosynthesis of hydrocarbons is effected by a microsomal Co-porphyrin-containing enzyme that catalyzes decarbonylation of aldehydes and, thus, reveal a biological function for Co in plants.
Selection of intraepithelial lymphocytes with CD8 alpha/alpha co-receptors by self-antigen in the murine gut.
Authors: Rocha B , von Boehmer H , Guy-Grand D
Published: May 2006

We have studied T-cell receptor (TCR) and alpha/alpha CD8 expression in thymus-independent intraepithelial lymphocytes (TI IELs) from the gut of mice bearing transgenic (TG) TCR alpha beta specific for the male antigen, presented by H-2Db class I major histocompatibility complex (MHC) molecules. In contrast to TCR+ alpha beta cells differentiating in the thymus (from CD4+CD8+ precursors to CD4+CD8- or CD4-CD8+ progeny), TI IELs are not deleted by self-antigens, nor are they positively selected in the absence of the specific peptide. On the contrary, recognition of the antigen in the context of self-MHC is required for selection and granular differentiation of CD8+ TI IELs. Our results also show that, in contrast to the thymus, expression of the beta TG does not block expression of endogenous TCR gamma delta genes in TI IELs. The size of this gut IEL subpopulation and its difference in mechanisms of repertoire selection demonstrate the existence of a major extrathymic pathway of T-cell differentiation, the role of which remains to be elucidated.
Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions.
Authors: Parsons C A , Tsaneva I , Lloyd R G , West S C
Published: May 2006

The RuvA, RuvB, and RuvC proteins of Escherichia coli are required for the recombinational repair of ultraviolet light- or chemical-induced DNA damage. In vitro, RuvC protein interacts with Holliday junctions in DNA and promotes their resolution by endonucleolytic cleavage. In this paper, we investigate the interaction of RuvA and RuvB proteins with model Holliday junctions. Using band-shift assays, we show that RuvA binds synthetic Holliday structures to form specific protein-DNA complexes. Moreover, in the presence of ATP, the RuvA and RuvB proteins act in concert to promote dissociation of the synthetic Holliday structures. The dissociation reaction requires both RuvA and RuvB and a nucleotide cofactor (ATP or dATP) and is rapid (40% of DNA molecules dissociate within 1 min). The reaction does not occur when ATP is replaced by either ADP or the nonhydrolyzable analog of ATP, adenosine 5'-[gamma-thio]triphosphate. We suggest that the RuvA and RuvB proteins play a specific role in the branch migration of Holliday junctions during postreplication repair of DNA damage in E. coli.
Zinc regulates the function of two superantigens.
Authors: Fraser J D , Urban R G , Strominger J L , Robinson H
Published: May 2006

Staphylococcal enterotoxins bind with high affinity to class II major histocompatibility complex proteins and subsequently stimulate large numbers of T cells via the V beta portion of the T-cell receptor. Binding of enterotoxin A and enterotoxin E to HLA-DR was completely abolished by low levels of EDTA, whereas binding of toxic shock toxin was unaffected. Addition of Zn2+ to as little as 2 microM excess over EDTA completely reconstituted binding, but Ca2+, Mg2+, Cu2+, Fe2+, and Mn2+ had no effect. The dissociation constant (Kd) of 65Zn2+ binding to a single site on purified enterotoxin A was 2 microM, and addition of purified HLA-DR1 did not alter the Kd, indicating that the binding site was exclusive to enterotoxin A. In the presence of saturating levels of zinc the Kd for enterotoxin A binding to purified HLA-DR1 was 25 nM. Thus, zinc binding is an essential first step in the formation of the major histocompatibility complex binding domain of at least two bacterial superantigens. Given the measured Kd of zinc binding to enterotoxin A, serum levels of free zinc (0.2-1.0 microM) may well regulate the toxic sequelae by these two superantigens.
Immunoelectron microscopic localization of the ubiquitin-activating enzyme E1 in HepG2 cells.
Authors: Schwartz A L , Trausch J S , Ciechanover A , Slot J W , Geuze H
Published: May 2006

As the first enzyme in the ubiquitin system the ubiquitin-activating enzyme E1 plays a pivotal role in all pathways of protein ubiquitination. In an effort to learn more about the cell biology of this pathway, we have purified the 110-kDa enzyme to homogeneity and generated a panel of distinct monoclonal antibodies to it. Using quantitative electron microscopic immunolocalization with these anti-E1 monoclonal antibodies, we find that E1 is abundant both within the cytoplasm and nucleus. Within the cytoplasm, E1 was found throughout the cytoplasmic volume as well as enriched along the cytoplasmic face of the rough endoplasmic reticulum and associated with the dense material along the desmosomal junctions. E1 was also found associated with the cytoplasmic surface of endosomal/lysosomal vacuoles. Interestingly, E1 was also found within the mitochondria. The lumen of rough endoplasmic reticulum, Golgi complex, endosomes, and lysosomes were negative. The specific localization of E1 to distinct subcellular organelles suggests that E1 may play multiple physiological roles within the cell.
Control of excision frequency of maize transposable element Ds in Petunia protoplasts.
Authors: Becker D , Lütticke R , Li M , Starlinger P
Published: May 2006

The complete coding region of maize transposable element Ac and truncated but active derivatives of it were placed under the control of promoters of different strength and tested for the ability to excise transposable element Ds from a beta-glucuronidase reporter gene in a cotransfection assay in Petunia protoplasts. The highest excision values (5% of the protoplasts able to express the beta-glucuronidase gene in a control experiment) were observed with a truncated version of the Ac coding region under the control of the 2' promoter. The weak Ac promoter is sufficient to give rise to excision values not much lower than those found with much stronger promoters such as the 2' and nos promoters. A decrease in excision frequency was observed when translation of the Ac coding region was hindered by out-of-frame ATG codons in addition to the use of weak promoters. Increasing the level of Ac transposase thus does not seem to be sufficient to raise the level of Ds excision observed in this system and possibly also in maize. Therefore another factor limits the excision of Ds elements. Previously, it was reported that in tobacco cells the deletion of Ds sequence between base pairs 186 and 245 led to a decrease of the Ds excision frequency by the full-length but not by the truncated Ac product. In the Petunia assay system, however, deletion of these sequences decreased the excision rate with both the full length and the truncated Ac coding region. A cDNA construct was found similarly active as the corresponding genomic DNA.
Heterodimerization of thyroid hormone (TH) receptor with H-2RIIBP (RXR beta) enhances DNA binding and TH-dependent transcriptional activation.
Authors: Hallenbeck P L , Marks M S , Lippoldt R E , Ozato K , Nikodem V M
Published: May 2006

Steroid/TH receptors mediate transcriptional induction of promoters containing hormone response elements (HREs) through an unclear mechanism that involves receptor binding to both hormone and a HRE. Here we demonstrate that both HRE binding and the transcriptional inducing activities of one member of this family, TH receptor, were markedly enhanced by heterodimerization with H-2RIIBP, a non-TH-binding member of the steroid hormone receptor superfamily. H-2RIIBP, the mouse homologue of human retinoic acid-related receptor, was shown to form stable heterodimers with the TH receptor either in solution or when bound to a TH response element. The results presented indicate that it might be necessary for the TH receptor or other members of this superfamily to have specific partners for heterodimer formation to elicit maximal hormone-specific gene regulation from particular HREs.
cAMP-mediated differential regulation of lignin peroxidase and manganese-dependent peroxidase production in the white-rot basidiomycete Phanerochaete chrysosporium.
Authors: Boominathan K , Reddy C A
Published: May 2006

Lignin peroxidases (LIPs) and manganese-dependent peroxidases (MNPs) are major components of the lignin-degrading enzyme system of Phanerochaete chrysosporium and typically appear during secondary metabolism. The involvement of cAMP in the regulation of production of LIPs and MNPs was investigated in this study. Production of LIPs and MNPs was preceded by a sharp rise in intracellular cAMP concentration. Addition of atropine, theophylline, or histamine to cultures resulted in a drop in intracellular cAMP concentration and a concomitant inhibition of production of LIPs only or of both LIPs and MNPs, depending on the concentration of the inhibitor added. These results were independently confirmed by fast protein liquid chromatographic profiles of the LIPs and MNPs in the extracellular fluid of the inhibitor-treated and untreated control cultures. LIP production was generally more sensitive to the inhibitors than MNP production. Northern blot analyses showed that the inhibitors affect the production of LIPs and MNPs at the level of transcription. Furthermore, LIP and MNP gene expression appears to be differentially regulated depending on the intracellular concentration of cAMP. These results show that cAMP plays a key role in the regulation of production of LIPs and MNPs in P. chrysosporium.
Minimal sequence requirements for ribozyme activity.
Authors: McCall M J , Hendry P , Jennings P A
Published: May 2006

The hammerhead ribozyme, as engineered by J. Haseloff and W. L. Gerlach [(1988) Nature (London) 334, 585-591], is an RNA molecule containing two regions of conserved nucleotides, a double helix, called helix II, which connects the two conserved regions, and flanking arms of variable sequence, which hybridize the ribozyme to its specific target. Here we show that this ribozyme may be reduced in size and still retain cleavage activity by replacing helix II with just a few nucleotides that cannot form Watson-Crick base pairs between themselves. Furthermore, the nucleotides replacing helix II and the nucleotides in the flanking arms may be substituted with DNA, and this small, DNA-containing ribozyme is fully as active as the original, full-size ribozyme. Cleavage activity of the minimized ribozyme depends on the number and sequence of the few nucleotides that replace helix II; optimal activity, thus far, is achieved by four or five deoxyribopyrimidines. The minimized ribozyme, or "minizyme," is active as a monomer, as shown by its nearly constant activity over a concentration range varying 25,000-fold, by the mobility of the minizyme-substrate complex in nondenaturing polyacrylamide gels as compared with other nucleic acid molecules of known size, and by other observations. These minizymes provide an excellent model system for studying the structure and mechanism of catalytic RNA; they might also be useful in a variety of biological applications.
Whole genome amplification from a single cell: implications for genetic analysis.
Authors: Zhang L , Cui X , Schmitt K , Hubert R , Navidi W , Arnheim N
Published: May 2006

We have developed an in vitro method for amplifying a large fraction of the DNA sequences present in a single haploid cell by repeated primer extensions using a mixture of 15-base random oligonucleotides. We studied 12 genetic loci and estimate that the probability of amplifying any sequence in the genome to a minimum of 30 copies is not less than 0.78 (95% confidence). Whole genome amplification beginning with a single cell, or other samples with very small amounts of DNA, has significant implications for multipoint mapping by sperm or oocyte typing and possibly for genetic disease diagnosis, forensics, and the analysis of ancient DNA samples.
Recognition of the Epstein-Barr virus-encoded nuclear antigens EBNA-4 and EBNA-6 by HLA-A11-restricted cytotoxic T lymphocytes: implications for down-regulation of HLA-A11 in Burkitt lymphoma.
Authors: Gavioli R , De Campos-Lima P O , Kurilla M G , Kieff E , Klein G , Masucci M G
Published: May 2006

Evasion from cytotoxic T-lymphocyte (CTL) surveillance may be an important step in the pathogenesis of Epstein-Barr virus (EBV)-carrying Burkitt lymphoma (BL) as suggested by the consistent down-regulation of all transformation-associated viral antigens, except EBV nuclear antigen 1 (EBNA-1), and of certain HLA class I alleles in BL biopsies and cell lines that maintain the tumor cell phenotype in vitro. The most common HLA class I defect recorded in BL lines is a selective down-regulation of HLA-A11. To gain some insight into the role of HLA-A11 down-regulation in pathogenesis of BL, we have investigated the target specificity of HLA-A11-restricted CTLs derived by stimulation of lymphocytes from three EBV-seropositive individuals with autologous EBV-transformed lymphoblastoid cell lines. Recombinant vaccinia viruses carrying the coding sequences for EBNA-1, -2A, -2B, -5, -3, -4, and -6 (also known as EBNA-1, -2A, -2B, -LP, -3a, -3b, and -3c, respectively) and EBV latent membrane protein 1 were used to induce high levels of expression of the relevant EBV antigen in fibroblasts derived from HLA class I-matched individuals. EBNA-4-expressing fibroblasts were the predominant target of HLA-A11-restricted CTLs in all three donors. A less pronounced and less regular EBNA-6-specific cytotoxic component was found in two of the donors.