bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.
Authors: Amrein K E , Takacs B , Stieger M , Molnos J , Flint N A , Burn P
Published: May 2006

An Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL was engineered and has been successfully used to produce large quantities of the recombinant human protein-tyrosine kinase p50csk. The co-overproduction of the two chaperones with p50csk results in increased solubility of the kinase and allows purification of milligram amounts of active enzyme. Analysis of the purified protein by SDS/polyacrylamide gel electrophoresis reveals a single band with an apparent molecular mass of 50 kDa, indicating that recombinant human p50csk has been purified to near homogeneity. The purified enzyme displays tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties, including in vitro substrate specificity and enzymatic characteristics of the enzyme, have been assessed and compared with those of members of the Src family of protein-tyrosine kinases. Results indicate that p50csk and p56lck have different substrate specificities and that p50csk and p60c-src have similar kinetic parameters. The successful production and purification of an enzymatically active form of p50csk will enable further characterization of this important kinase and allow clarification of its physiological role. In addition, the results suggest that the approach described may be generally applicable to improve the solubility of recombinant proteins which otherwise are produced in an insoluble form in E. coli.
Identification of presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris.
Authors: Kami J , Velásquez V B , Debouck D G , Gepts P
Published: May 2006

Common bean (Phaseolus vulgaris) consists of two major geographic gene pools, one distributed in Mexico, Central America, and Colombia and the other in the southern Andes (southern Peru, Bolivia, and Argentina). Amplification and sequencing of members of the multigene family coding for phaseolin, the major seed storage protein of the common bean, provide evidence for accumulation of tandem direct repeats in both introns and exons during evolution of the multigene family in this species. The presumed ancestral phaseolin sequences, without tandem repeats, were found in recently discovered but nearly extinct wild common bean populations of Ecuador and northern Peru that are intermediate between the two major gene pools of the species based on geographical and molecular arguments. Our results illustrate the usefulness of tandem direct repeats in establishing the polarity of DNA sequence divergence and therefore in proposing phylogenies.
Immunotoxin-mediated conditional disruption of specific neurons in transgenic mice.
Authors: Kobayashi K , Morita S , Sawada H , Mizuguchi T , Yamada K , Nagatsu I , Fujita K , Kreitman R J , Pastan I , Nagatsu T
Published: May 2006

We have developed a transgenic approach, termed immunotoxin-mediated cell targeting (IMCT), to ablate conditionally selective neurons in the brain with the cytotoxic activity of immunotoxins. Transgenic mice were created that express the human interleukin 2 receptor alpha subunit (IL-2R alpha) under the control of the dopamine beta-hydroxylase (DBH) gene promoter. The animals were treated intracerebroventricularly with a recombinant immunotoxin, anti-Tac(Fv)-PE40, which selectively kills animal cells bearing human IL-2R alpha. The immunotoxin caused a characteristic behavioral abnormality only in the transgenic mice. This was accompanied by a dramatic loss of DBH-containing neurons and a significant decrease in DBH activity and norepinephrine levels in various regions of the brain. IMCT should provide a general technique to create animal models of human neurodegenerative disorders by targeting neurons or other cell types.
Development and characterization of essential fatty acid deficiency in human endothelial cells in culture.
Authors: Lerner R , Lindström P , Berg A , Johansson E , Rosendahl K , Palmblad J
Published: May 2006

We induced an essential fatty acid deficiency (EFAD) in human umbilical vein endothelial cells by culture in medium with 20% (vol/vol) delipidated fetal calf serum. EFAD, reflected by decreased cellular linoleic acid (18:2 omega 6) and arachidonic acid (20:4 omega 6) and emergence of the oleic acid derivative 5,8,11-eicosatrienoic acid (20:3 omega 9; Mead's acid), was evident after 1 week of culture and became pronounced after 2 weeks. Beyond that time point, control cells (cultured in 20% normal fetal calf serum) grew deficient of 18:2 omega 6, and EFAD cells died. 18:2 omega 6 addition to EFAD cells resulted in dose-dependent increases of 18:2 omega 6 and 20:4 omega 6. 20:4 omega 6 or 5,8,11,14,17-eicosapentaenoic acid (20:5 omega 3) additions resulted in normalization of these acids, and conversion of 20:5 omega 3 to 4,7,10,13,16,19-docosahexaenoic acid (22:6 omega 3) was noted. Agonist-induced increases in concentrations of prostacycline (prostaglandin I2; PGI2) and cytosolic Ca2+, [Ca2+]i, were reduced in EFAD cells and not restored by 18:2 omega 6 or 20:4 omega 6 additions. Change of the medium in EFAD cultures 1 day before the experiments decreased 20:3 omega 9 and normalized the PGI2 production and [Ca2+]i changes, whereas addition of 20:3 omega 9 to control cells impaired the [Ca2+]i response, indicating a suppressive effect of 20:3 omega 9. Thus, EFAD in endothelial cells is associated with abnormalities of eicosanoid and second-messenger production partly attributable to 20:3 omega 9 accumulation. Moreover, the gradual emergence of 18:2 omega 6 deficiency in regularly grown control cells underlines the need for careful analysis of fatty acids in long-term cell cultures.
Induction of heme oxygenase 1 in the retina by intense visible light: suppression by the antioxidant dimethylthiourea.
Authors: Kutty R K , Kutty G , Wiggert B , Chader G J , Darrow R M , Organisciak D T
Published: May 2006

The effect of intense visible light (light damage) on the expression of heme oxygenase 1 (HO-1), a protein induced by oxidative stress, was investigated in the rat retina. A sensitive reverse transcription-PCR assay demonstrated the expression of mRNA for HO-1 as well as HO-2, the noninducible HO form, in the normal retina. As analyzed by Northern blotting, however, HO-1 mRNA was barely detectable under normal circumstances. After exposure to intense visible light, retinas had markedly higher HO-1 mRNA levels than unexposed controls, with increases up to 52- and 98-fold at 12 and 24 hr of exposure, respectively. Intense light exposure also resulted in an increase in HO-1 protein. In contrast, no appreciable change in HO-2 mRNA or protein was observed. The increase in HO-1 message was more pronounced in rats previously reared in the dark than in those reared in a weak cyclic-light environment. A marked decrease from the high level of HO-1 mRNA induced by light insult was observed when the animals were allowed to recover in the dark for 24 hr after light exposure. Most important, treatment of animals with 1,3-dimethylthiourea, a synthetic antioxidant, prior to light exposure effectively blocked the increase in HO-1 mRNA. Thus, HO-1 is a sensitive marker for assessing light-induced insult in the retina. Since increased expression of HO-1 is thought to be a cellular defense against oxidative damage, its expression may play an important role in protecting the retina against light damage.
A linear Ricardo model with varying parameters.
Authors: Gomory R E , Baumol W J
Published: May 2006

We show that linear Ricardo models with varying parameters have many of the properties usually associated with models having economies of scale.
Transport of proteins dissolved in organic solvents across biomimetic membranes.
Authors: Bromberg L E , Klibanov A M
Published: May 2006

Using lipid-impregnated porous cellulose membranes as biomimetic barriers, we tested the hypothesis that to afford effective transmembrane transfer of proteins and nucleic acids, the vehicle solvent should be able to dissolve both the biopolymers and the lipids. While the majority of solvents dissolve one or the other, ethanol and methanol were found to dissolve both, especially if the protein had been lyophilized from an aqueous solution of a pH remote from the protein's isoelectric point. A number of proteins, as well as RNA and DNA, dissolved in these alcohols readily crossed the lipidized membranes, whereas the same biopolymers placed in nondissolving solvents (e.g., hexane and ethyl acetate) or in those unable to dissolve lipids (e.g., water and dimethyl sulfoxide) exhibited little transmembrane transport. The solubility of biopolymers in ethanol and methanol was further enhanced by complexation with detergents and poly(ethylene glycol); significant protein and nucleic acid transport through the lipidized membranes was observed from these solvents but not from water.
Yeast glycosylation mutants are sensitive to aminoglycosides.
Authors: Dean N
Published: May 2006

Aminoglycosides are a therapeutically important class of antibiotics that inhibit bacterial protein synthesis and a number of viral and eukaryotic functions by blocking RNA-protein interactions. Vanadate-resistant Saccharomyces cerevisiae mutants with defects in Golgi-specific glycosylation processes exhibit growth sensitivity to hygromycin B, an aminoglycoside [Ballou, L., Hitzeman, R. A., Lewis, M. S. & Ballou, C. E. (1991) Proc. Natl. Acad. Sci. USA 88, 3209-3212]. Here, evidence is presented that glycosylation is, in and of itself, a key factor mediating aminoglycoside sensitivity in yeast. Examination of mutants with a wide range of glycosylation abnormalities reveals that all are sensitive to aminoglycosides. This effect is specific to aminoglycosides and not merely a consequence of increased permeability of the yeast mutants to drugs. Furthermore, inhibition of glycosylation in wild-type cells leads to a marked increase in their sensitivity to aminoglycosides. These results establish that a defect in glycosylation is sufficient to render yeast cells susceptible to these clinically important drugs. Further, they suggest that a molecule which prevents the uptake or mediates removal of aminoglycosides requires glycosylation for its activity. Perhaps more importantly, this finding on drug sensitivity provides the most powerful screen to date to identify mutants and thereby to isolate genes involved in all aspects of N-linked glycosylation.
The dif resolvase locus of the Escherichia coli chromosome can be replaced by a 33-bp sequence, but function depends on location.
Authors: Tecklenburg M , Naumer A , Nagappan O , Kuempel P
Published: May 2006

The dif locus (deletion-induced filamentation) of Escherichia coli is a resolvase site, located in the terminus region of the chromosome, that reduces chromosome multimers to monomers. In strains in which this site has been deleted, a fraction of the cells is filamentous, has abnormal nucleoid structure, and exhibits elevated levels of the SOS repair system. We have demonstrated that a 33-bp sequence, which is sufficient for RecA-independent recombination and which shows similarity to the cer site of pColE1, suppresses the Dif phenotype when inserted in the terminus region. Flanking sequences were not required, since suppression occurred in strains in which dif as well as 12 kb or 173 kb of DNA had been deleted. However, location was important, and insertions at a site 118 kb away from the normal site did not suppress the Dif phenotype. These sites were otherwise still functional, and they exhibited wild-type levels of RecA-independent recombination with dif-containing plasmids and recombined with other chromosomal dif sites to cause deletions and inversions. It is proposed that the functions expressed by a dif site depend on chromosome location and structure, and analysis of these functions provides a way to examine the structure of the terminus region.
Selective boron delivery to murine tumors by lipophilic species incorporated in the membranes of unilamellar liposomes.
Authors: Feakes D A , Shelly K , Hawthorne M F
Published: May 2006

The nido-carborane species K[nido-7-CH3(CH2)15-7,8-C2B9H11] has been synthesized for use as an addend for the bilayer membrane of liposomes. Small unilamellar vesicles, composed of distearoylphosphatidylcholine/cholesterol, 1:1, and incorporating K[nido-7-CH3(CH2)15-7,8-C2B9H11] in the bilayer, have been investigated in vivo. The time-course biodistribution of boron delivered by these liposomes was determined by inductively coupled plasma-atomic emission spectroscopy analyses after the injection of liposomal suspensions in BALB/c mice bearing EMT6 mammary adenocarcinomas. At the low injected doses normally used (approximately 5-10 mg of boron per kg of body weight), peak tumor boron concentrations of approximately 35 micrograms of boron per g of tissue and tumor/blood boron ratios of approximately 8 were achieved. These values are sufficiently high for the successful application of boron neutron capture therapy. The bilayer-embedded boron compound may provide the sole boron source or, alternatively, a concentrated aqueous solution of a hydrophilic boron compound may also be encapsulated within the liposomes to provide a dose enhancement. Thus, the incorporation of both K[nido-7-CH3(CH2)15-7,8-C2B9H11] and the hydrophilic species, Na3[1-(2'-B10H9)-2-NH3B10H8], within the same liposomes demonstrated significantly enhanced biodistribution characteristics, exemplified by maximum tumor boron concentrations of approximately 50 micrograms of boron per g of tissue and tumor/blood boron ratios of approximately 6.
Identification of a Ca2+/calmodulin-dependent protein kinase II regulatory phosphorylation site in non-N-methyl-D-aspartate glutamate receptors.
Authors: Yakel J L , Vissavajjhala P , Derkach V A , Brickey D A , Soderling T R
Published: May 2006

Glutamate receptor ion channels are colocalized in postsynaptic densities with Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II), which can phosphorylate and strongly enhance non-N-methyl-D-aspartate (NMDA) glutamate receptor current. In this study, CaM-kinase II enhanced kainate currents of expressed glutamate receptor 6 in 293 cells and of wild-type glutamate receptor 1, but not the Ser-627 to Ala mutant, in Xenopus oocytes. A synthetic peptide corresponding to residues 620-638 in GluR1 was phosphorylated in vitro by CaM-kinase II but not by cAMP-dependent protein kinase or protein kinase C. The 32P-labeled peptide map of this synthetic peptide appears to be the same as the two-dimensional peptide map of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptors phosphorylated in cultured hippocampal neurons by CaM-kinase II described elsewhere. This CaM-kinase II regulatory phosphorylation site is conserved in all AMPA/kainate-type glutamate receptors, and its phosphorylation may be important in enhancing postsynaptic responsiveness as occurs during synaptic plasticity.
Functional dissection of circadian clock- and phytochrome-regulated transcription of the Arabidopsis CAB2 gene.
Authors: Anderson S L , Kay S A
Published: May 2006

Both the circadian clock and phytochrome regulate expression of the Arabidopsis genes encoding the light-harvesting chlorophyll a/b-binding proteins (CAB genes). Phytochrome activates CAB transcription, and it has been proposed that the circadian clock negatively regulates CAB transcription. The tobacco nuclear proteins CUF-1 (CAB upstream factor 1) and CGF-1 (CAB GATA factor 1) bind the Arabidopsis CAB2 promoter, and the CGF-1 binding site is contained within a minimal clock- and phytochrome-regulated region of the promoter. We have used in vivo cab2::luciferase gene bioluminescence markers containing site-directed mutations in the CUF-1 and CGF-1 binding sites to define the role of these proteins in CAB2 regulation and to further delineate the terminal genomic targets of the phytochrome and circadian clock signal transduction pathways. Results from these studies confirm that CUF-1 is not required to generate the circadian clock- or phytochrome-responsive CAB2 expression pattern but rather functions as a positive factor to increase CAB2 expression levels. CGF-1 interaction with the CAB2 promoter mediates the acute increase in CAB2 expression in response to phytochrome activation and contributes to the light-induced high-amplitude circadian oscillation in CAB2 expression.
Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia.
Authors: Watanabe M , Osada J , Aratani Y , Kluckman K , Reddick R , Malinow M R , Maeda N
Published: May 2006

Studies by various investigators have indicated that elevated levels of plasma homocyst(e)ine are strongly associated with the occurrence of occlusive vascular diseases. With the eventual aim of determining whether or not elevated plasma homocyst(e)ine concentrations are directly causative of cardiovascular diseases, we have generated mice that are moderately and severely homocyst(e)inemic. Homologous recombination in mouse embryonic stem cells was used to inactivate the cystathionine beta-synthase [L-serine hydrolyase (adding homocysteine), EC 4.2.1.22] gene. Homozygous mutants completely lacking cystathionine beta-synthase were born at the expected frequency from matings of heterozygotes, but they suffered from severe growth retardation and a majority of them died within 5 weeks after birth. Histological examination showed that the hepatocytes of homozygotes were enlarged, multinucleated, and filled with microvesicular lipid droplets. Plasma homocyst(e)ine levels of the homozygotes were approximately 40 times normal. These mice, therefore, represent a model for severe homocyst(e)inemia resulting from the complete lack of cystathionine beta-synthase. Heterozygous mutants have approximately 50% reduction in cystathionine beta-synthase mRNA and enzyme activity in the liver and have twice normal plasma homocyst(e)ine levels. Thus, the heterozygous mutants are promising for studying the in vivo role of elevated levels of homocyst(e)ine in the etiology of cardiovascular diseases.
The fungal H(+)-ATPase from Neurospora crassa reconstituted with fusicoccin receptors senses fusicoccin signal.
Authors: Marra M , Ballio A , Battirossi P , Fogliano V , Fullone M R , Slayman C L , Aducci P
Published: May 2006

Fusicoccin affects several physiological processes regulated by the plasma membrane H(+)-ATPase in higher plants while other organisms having P-type H(+)-ATPases (e.g., fungi) are fusicoccin-insensitive. We have previously shown that fusicoccin binding to its receptor is necessary for H(+)-ATPase stimulation and have achieved the functional reconstitution into liposomes of fusicoccin receptors and the H(+)-ATPase from maize. In this paper we show that fusicoccin sensitivity can be conferred on the H(+)-ATPase from Neurospora crassa, a fungus insensitive to fusicoccin. In fact, H+ pumping by purified H(+)-ATPase from Neurospora crassa reconstituted into liposomes containing crude or partially purified fusicoccin receptors from maize was markedly enhanced by fusicoccin. The stimulation of H+ pumping by fusicoccin is dependent upon pH, fusicoccin, and protein concentration, as was reported for the system reconstituted with both proteins from maize.
Two short autoepitopes on the nuclear dot antigen are similar to epitopes encoded by the Epstein-Barr virus.
Authors: Xie K , Snyder M
Published: May 2006

To understand the relationship between antibodies present in patients with anti-nuclear dot (ND) autoimmune disease and the proteins they recognize, epitopes that react with the autoantibodies were mapped. A panel of fusion proteins containing different portions of the ND protein were overproduced in Escherichia coli. Immunoblot analysis with anti-ND antibodies revealed that most (10 of 12) sera recognize two major autoepitopes that are each a maximum of 8 amino acids long. The other two sera recognize one of the two epitopes. In addition to the short linear autoepitopes, a conformational epitope appears to be present on the ND antigen. Each of the two linear epitope sequences shares sequence similarities with those of several viral proteins found in the databases. Furthermore, two fusion proteins containing short Epstein-Barr virus (EBV) protein sequences that are similar to the ND epitopes were recognized by the human autoimmune sera, indicating that the autoepitopes are present in EBV protein sequences. Our results are consistent with the hypothesis that ND autoimmune disease might be associated with EBV infections.
Functional identification of the promoter for the gene encoding the alpha subunit of calcium/calmodulin-dependent protein kinase II.
Authors: Olson N J , Massé T , Suzuki T , Chen J , Alam D , Kelly P T
Published: May 2006

To examine the expression of the alpha subunit of calcium/calmodulin-dependent protein kinase II, various 5' flanking genomic sequences were inserted into a chloramphenicol acetyltransferase (CAT) reporter plasmid and CAT enzyme activities were analyzed in transfected NB2a neuroblastoma cells and mRNA transcription was analyzed by nuclease protection assays. A core promoter was identified which contained an essential TATA element located 162 nt 5' to the transcription start site. Sequences 3' to the transcription start site, as well as 5' to the TATA element, increased levels of CAT activity in transfected cells. The alpha-subunit gene promoter displayed higher CAT activities, relative to a simian virus 40 promoter, in transfected neuronal cell lines than in nonneuronal cell lines. Results also suggested that sequence surrounding the natural alpha-gene transcription initiation site may be important for targeting transcription initiation 162 nt downstream of its TATA element.
Scalp electrical potentials reflect regional cerebral blood flow responses during processing of written words.
Authors: Snyder A Z , Abdullaev Y G , Posner M I , Raichle M E
Published: May 2006

Functional brain imaging studies with positron emission tomography (PET) have identified blood flow changes in widely separated areas of the brain during the performance of word-related tasks. In the present study, we have utilized event-related electrical potentials (ERPs) to investigate the temporal relationships among cortical areas previously identified by PET to be differentially activated when performing a task involving generating the uses of visually presented nouns versus reading aloud. ERPs showed strong task-related differences over left and middle inferior frontal and left parietotemporal regions. Frontal and left parietotemporal channels revealed these differences around 200 and 700 msec, respectively, after word presentation. These results provide the time course for parts of the anatomical circuit involved in generating the meaning of a word. Our results also demonstrate how combining the spatial localization of PET with the temporal resolution of ERPs greatly enhances the capacity to understand the mechanisms involved in human cognition.
Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor.
Authors: Dahlman-Wright K , Baumann H , McEwan I J , Almlöf T , Wright A P , Gustafsson J A , Härd T
Published: May 2006

A 58-amino acid polypeptide containing the functional core region, the tau 1 core, of the major transactivation domain of the human glucocorticoid receptor has been expressed in Escherichia coli and purified to homogeneity. The polypeptide retains 60-70% of the activity of the intact domain when assayed in vivo or in vitro. This report describes a structural characterization of the tau 1 core peptide fragment. Circular dichroism spectroscopy shows that the tau 1 core and a larger fragment encompassing the intact tau 1 domain are largely unstructured in water solution under a variety of pH conditions. The tau 1 core, however, acquires a significant alpha-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structure formation in regions that have propensity for alpha-helical conformation. Two- and three-dimensional NMR spectroscopy of 15N-labeled tau 1 core, in the presence of trifluoroethanol, has allowed sequential assignment of 1H and 15N resonances and identification of three protein segments with alpha-helical character. Potentially helix-breaking proline substitutions, in proposed alpha-helical regions, lead to reduced activity, suggesting that alpha-helices are important for transactivation in vivo.
Mutant farnesyltransferase beta subunit of Saccharomyces cerevisiae that can substitute for geranylgeranyltransferase type I beta subunit.
Authors: Mitsuzawa H , Esson K , Tamanoi F
Published: May 2006

The protein farnesyltransferase (PFT) beta-subunit gene of Saccharomyces cerevisiae, DPR1, was randomly mutagenized by PCR to construct a mutant DPR1 gene library on a high-copy plasmid. The library was screened for suppression of the temperature sensitivity conferred by a mutation in the protein geranylgeranyltransferase type I (PGGT-I) beta-subunit gene, CAL1. A mutant DPR1 gene was identified whose product contained a single amino acid change of Ser-159 to Asn. This mutant gene also suppressed a cal1 disruption even on a low-copy plasmid, suggesting that the product (designated S159N) can substitute for PGGT-I beta subunit in vivo. Its ability to act as a PFT is not drastically reduced, since the mutant gene still complemented a dpr1 disruption. Results of in vitro assays demonstrate that the mutant enzyme has increased activity to farnesylate, a substrate for PGGT-I. On the other hand, the ability to farnesylate its own substrate is reduced. The increased ability to utilize the PGGT-I substrate is due to its increased affinity for the protein substrate. In addition, the mutant enzyme shows a severalfold increase in the sensitivity to a peptidomimetic inhibitor that acts as a competitor of the protein substrate. These results point to the importance of the beta subunit of PFT for the binding of a protein substrate and demonstrate that Ser-159 of DPR1 product is critical for its substrate specificity.
Effects of increased neural activity on brain growth.
Authors: Zheng D , Purves D
Published: May 2006

We have measured the effects of regionally increased metabolic activity--and by inference electrical activity--on cortical growth in the developing rat brain. Cortical growth is significantly and specifically greater in regions of chronically increased activity. This effect of activity on cortical growth may help explain the permanent storage of early experience in the developing nervous system.
Developmentally excised DNA sequences in Euplotes crassus capable of forming G quartets.
Authors: Klobutcher L A
Published: May 2006

Tens of thousands of DNA segments are eliminated by DNA breakage and rejoining events during the formation of a new macronucleus in the hypotrichous ciliated protozoan Euplotes crassus. This study presents evidence for a class of eliminated sequences referred to as telomeric-repeat-like internal eliminated sequences (TelIESs). TelIESs are shorter (< 50 bp) than most previously characterized IESs and their DNA sequences resemble the telomeric repeat sequences of the organism. The TelIESs are excised during the developmental period of chromosome fragmentation/telomere addition, which is later than previously characterized IESs. Additional studies demonstrate that oligonucleotides representing the TelIESs are, like telomeric repeats, capable of forming G-quartet structures in vitro.
Molecular basis of intracistronic complementation in the Passover locus of Drosophila.
Authors: Krishnan S N , Frei E , Schalet A P , Wyman R J
Published: May 2006

The only demonstrated mechanism for intracistronic genetic complementation requires physical interaction of protein subunits to create a functional molecule. We demonstrate another and perhaps quite general mechanism utilizing proteins with unique and shared domains. The Drosophila neural mutant Passover (Pas) disrupts specific synaptic connections. Alleles of a lethal complementation group exhibit a complex pattern of complementation with Pas alleles. Whereas all heterozygotes between these lethal alleles and Pas are viable, only some alleles complement the neural defect of Pas. Lethal and neural functions are separately encoded by two proteins that have distinct N-terminal domains and a common C-terminal portion. Neural-specific and lethal-specific mutations map to unique exons, while neural-lethal mutations map to shared exons. Combinations of lethal and neural alleles result in production of both proteins and demonstrate intracistronic complementation.
A method for probing the topography and interactions of proteins: footprinting of myoglobin.
Authors: Zhong M , Lin L , Kallenbach N R
Published: May 2006

We describe a procedure for mapping residues on the surface of a protein molecule to its sequence, using a scheme that is analogous to nucleic acid footprinting. The protein is end labeled radioactively and subjected to limited proteolysis, and the products are analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. The method is tested with the heme protein myoglobin and applied to mapping the (unknown) surface of the molecule lacking the heme group: apomyoglobin. Sites of protein-protein interaction can be identified, as illustrated by footprinting the association between myoglobin and an anti-myoglobin monoclonal antibody.
Cloning and analysis of murine cDNA that encodes a fibrogenic lymphokine, fibrosin.
Authors: Prakash S , Robbins P W , Wyler D J
Published: May 2006

Tissue fibrosis that complicates chronic inflammation can be a cause of serious morbidity. The molecular links between inflammation and fibrosis appear to be a variety of proteins produced by activated chronic inflammatory cells. Collectively, these fibrogenic cytokines promote the growth of fibroblasts and the production of extracellular matrix that are the characteristic features of fibrotic tissue. In an attempt to clone cDNA for a fibrogenic lymphokine that we had isolated, we transfected COS-7 cells with a cDNA library derived from concanavalin A-stimulated lymphocyte line CDC25. Conditioned medium from the transfected COS-7 cells but not from sham-transfected cells stimulates fibroblast proliferation in vitro. We used heterologous expression in COS-7 cells of pools of CDC25 cDNA and screening for biological activity in conditioned medium to enrich for the cDNA clone(s) that encodes this activity. With this strategy of sib selection we isolated clone 2B3. The culture supernatants of 2B3-transfected COS-7 cells exert maximum growth-stimulating effects on fibroblasts at a dilution of 1:20,000. The isolated cDNA has one open reading frame (216 nucleotides) that has no significant homology with nucleotide sequences that encode other proteins. A synthetic peptide constructed from the deduced amino acid sequence is biologically active in picomolar concentrations, even though it may represent only a portion of the native fibrosin. This lymphokine, which we designate fibrosin, may play a role in regulating fibrogenesis in certain chronic inflammatory diseases.
Systematic screening of an arrayed cDNA library by PCR.
Authors: Munroe D J , Loebbert R , Bric E , Whitton T , Prawitt D , Vu D , Buckler A , Winterpacht A , Zabel B , Housman D E
Published: May 2006

We have developed a PCR-based method for rapid and effective screening of arrayed cDNA libraries. This strategy directly addresses the limitations of conventional hybridization-based schemes and provides a more rapid, cost-effective, and sensitive method compatible with large-scale and routine cDNA clone recovery. To prepare arrayed libraries, 1-2 x 10(6) cDNA clones were propagated as individual plaques on solid medium in 24-well culture dishes at approximately 250 plaque-forming units per well. Phage suspensions were prepared from each well and transferred to a 96-well format. To screen the library, pools were generated that correspond to each individual 96-well plate and to each row and column within "blocks" of six plates each. Library screening for specific cDNA clones was conducted in a systematic and hierarchical fashion beginning with the plate pools. Next, the row/column pools corresponding to each positive plate pool were screened. Finally, isolated clones from within each positive well were identified by hybridization. We have applied this approach to the screening of an arrayed human brain cDNA library resulting in the recovery of cDNAs corresponding to > 25 genes and expressed sequence tags.
Susceptibility to hepatocellular carcinoma is associated with genetic variation in the enzymatic detoxification of aflatoxin B1.
Authors: McGlynn K A , Rosvold E A , Lustbader E D , Hu Y , Clapper M L , Zhou T , Wild C P , Xia X L , Baffoe-Bonnie A , Ofori-Adjei D
Published: May 2006

Aflatoxin B1 (AFB1) has been postulated to be a hepatocarcinogen in humans, possibly by causing p53 mutations at codon 249. AFB1 is metabolized via the phase I and II detoxification pathways; hence, genetic variation at those loci may predict susceptibility to the effects of AFB1. To test this hypothesis, genetic variation in two AFB1 detoxification genes, epoxide hydrolase (EPHX) and glutathione S-transferase M1 (GSTM1), was contrasted with the presence of serum AFB1-albumin adducts, the presence of hepatocellular carcinoma (HCC), and with p53 codon 249 mutations. Mutant alleles at both loci were significantly overrepresented in individuals with serum AFB1-albumin adducts in a cross-sectional study. Mutant alleles of EPHX were significantly overrepresented in persons with HCC, also in a case-control study. The relationship of EPHX to HCC varied by hepatitis B surface antigen status and indicated that a synergistic effect may exist. p53 codon 249 mutations were observed only among HCC patients with one or both high-risk genotypes. These results indicate that individuals with mutant genotypes at EPHX and GSTM1 may be at greater risk of developing AFB1 adducts, p53 mutations, and HCC when exposed to AFB1. Hepatitis B carriers with the high-risk genotypes may be an even greater risk than carriers with low-risk genotypes. These findings support the existence of genetic susceptibility in humans to the environmental carcinogen AFB1 and indicate that there is a synergistic increase in risk of HCC with the combination of hepatitis B virus infection and susceptible genotype.
The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription.
Authors: Churcher M J , Lowe A D , Gait M J , Karn J
Published: May 2006

The human immunodeficiency virus type 1 (HIV-1) trans-activator protein, Tat, specifically stimulates transcription from the viral long terminal repeat. Tat binds to an RNA stem-loop structure encoded by the trans-activation response region (TAR). To test whether TAR is functional when displaced downstream of the start of transcription, we assayed a series of templates carrying duplicated TAR elements in cell-free transcription systems. When the normally positioned TAR element (TAR-1) is inactivated by mutations in either the Tat binding site or the apical loop sequence, which acts as the binding site for a cellular factor, transactivation can be rescued by a wild-type TAR element placed downstream (TAR-2). The TAR-2 element is functional even when placed > 200 nt downstream of TAR-1. TAR complementation experiments have also shown that a functional TAR element requires both an intact Tat binding site and an intact apical loop sequence. For example, if TAR-1 carries a mutation in the loop element it cannot be rescued by a TAR-2 element carrying a mutation in the Tat binding site. Substitution mutations in TAR-1 show that the 5' half of TAR also encodes an essential DNA element which is required for efficient transcription initiation. These results strongly suggest that Tat and cellular cofactors which bind TAR RNA associate with the transcription complex during its transit through TAR.
Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus.
Authors: Okada N , Liszewski M K , Atkinson J P , Caparon M
Published: May 2006

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes.
Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways.
Authors: Scholz G , Felder M P , Hanafusa H
Published: May 2006

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.
Demonstration that polyol accumulation is responsible for diabetic cataract by the use of transgenic mice expressing the aldose reductase gene in the lens.
Authors: Lee A Y , Chung S K , Chung S S
Published: May 2006

Aldose reductase (AR) has been implicated in the etiology of diabetic cataract, as well as in other complications. However, the role of AR in these complications remains controversial because the strongest supporting evidence is drawn from the use of AR inhibitors for which specificity in vivo cannot be ascertained. To settle this issue we developed transgenic mice that overexpress AR in their lens epithelial cells and found that they become susceptible to the development of diabetic and galactose cataracts. When the sorbitol dehydrogenase-deficient mutation is also present in these transgenic mice, greater accumulation of sorbitol and further acceleration of diabetic cataract develop. These genetic studies demonstrated convincingly that accumulation of polyols from the reduction of hexose by AR leads to the formation of sugar cataracts.
A well-differentiated B-cell line is permissive for somatic mutation of a transfected immunoglobulin heavy-chain gene.
Authors: Zhu M , Rabinowitz J L , Green N S , Kobrin B J , Scharff M D
Published: May 2006

pSV2neo plasmids containing an IgM heavy-chain gene with nonsense mutations in either the variable (V) or the constant (C) region were transfected into four differentiated mouse plasma cell lines: S107 and the NSO fusion partner (myeloma cell lines) and 2C3 and 36.65 (hybridoma cell lines). The frequencies of reversion of the nonsense mutations in multiple independent transfectants were determined with the spot ELISA and rates of reversion were calculated by fluctuation analysis. Mutations in both V and C regions were confirmed by sequence analyses. In the S107 cell line, spontaneous point mutations occurred in the V region at a rate of approximately 5 x 10(-5)/bp per cell generation, > 400-fold higher than the rate of V-region mutation in the NSO cell line and considerably higher than the rates in 2C3 and 36.65 hybridoma cell lines. These studies suggest that S107 is a relatively permissive cell line in which V-region mutations can occur constitutively, even though it represents a late stage of B-cell differentiation. Further, the results show that the construct used contains sufficient information in its flanking and coding sequences to allow a relatively high rate of V-region mutation, at least in the S107 cell line.
Cutoff in potency implicates alcohol inhibition of N-methyl-D-aspartate receptors in alcohol intoxication.
Authors: Peoples R W , Weight F F
Published: May 2006

As the number of carbon atoms in an aliphatic n-alcohol is increased from one to five, intoxicating potency, lipid solubility, and membrane lipid disordering potency all increase in a similar exponential manner. However, the potency of aliphatic n-alcohols for producing intoxication reaches a maximum at six to eight carbon atoms and then decreases. The molecular basis of this "cutoff" effect is not understood, as it is not correlated with either the lipid solubility or the membrane disordering potency of the alcohols, which continue to increase exponentially. Since it has been suggested that inhibition of N-methyl-D-aspartate (NMDA) receptors by alcohols may play a role in alcohol intoxication, we investigated whether a series of aliphatic n-alcohols would exhibit a cutoff in potency for inhibition of NMDA receptors. We found that although potency for inhibition of NMDA receptors increased exponentially for alcohols with one to five carbon atoms, potency for inhibition of NMDA receptors reached a maximum at six to eight carbon atoms and then abruptly disappeared. This cutoff for alcohol inhibition of NMDA receptors is consistent with an interaction of the alcohols with a hydrophobic pocket on the receptor protein. In addition, the similarity of the cutoffs for alcohol inhibition of NMDA receptors and alcohol intoxication suggests that the cutoff for NMDA receptor inhibition may contribute to the cutoff for alcohol intoxication, which is consistent with an important role of NMDA receptors in alcohol intoxication.
Defective G protein activation of the cAMP pathway in rat kidney during genetic hypertension.
Authors: Chatziantoniou C , Ruan X , Arendshorst W J
Published: May 2006

The development of hypertension in the spontaneously hypertensive rat (SHR) is associated with renal dysfunction and vasoconstriction. The kidneys of young SHRs exhibit exaggerated reactivity to angiotensin II (Ang-II) and attenuated responses to vasodilators that normally activate the cAMP signal to buffer hormone-induced vasoconstriction. The present study investigates the mechanism(s) responsible for this abnormality in activation of the cAMP second-messenger pathway in hypertensive animals. Renal vascular reactivity was assessed in 7-week-old anesthetized SHRs and normotensive Wistar-Kyoto rats. The animals were pretreated with indomethacin to block prostanoid production throughout an experiment. Ang-II was injected into the renal artery either alone or mixed with the vasodilator fenoldopam, a dopamine-receptor agonist. These two opposing vasoactive agents were administered before and during intrarenal infusion of NaF or cholera toxin, two activators of G proteins that stimulate cAMP production. The results show that Ang-II reduced renal blood flow by 45% in both strains. In Wistar-Kyoto rats, fenoldopam reduced the Ang-II-induced decrease in renal blood flow from -45% to -30%. This protective effect of fenoldopam was increased further during infusion of NaF or cholera toxin (-18% or -19% decrease in renal blood flow). In SHRs, fenoldopam failed to attenuate Ang II-mediated vasoconstriction (-45% vs. -44%). In contrast, fenoldopam effectively blunted the Ang-II-induced vasoconstriction when it was given concurrently with NaF or cholera toxin (-27 or -31% decrease in renal blood flow). These findings provide evidence for defective interaction between receptor coupling and activation of guanine nucleotide stimulatory factor proteins in the renal microcirculation of 7-week-old SHRs. Such a deficiency could play an important role in renal dysfunction associated with the development of genetic hypertension.
A multimerizing transcription factor of sea urchin embryos capable of looping DNA.
Authors: Zeller R W , Griffith J D , Moore J G , Kirchhamer C V , Britten R J , Davidson E H
Published: May 2006

SpGCF1 is a recently cloned sea urchin transcription factor that recognizes target sites in several different sea urchin genes. We find that in gel-shift experiments this factor is able to multimerize. A quantitative simulation of the gel-shift results suggests that SpGCF1 molecules that are bound to DNA target sites may also bind to one another, thus associating several DNA probe molecules. SpGCF1 might therefore be able to loop DNA molecules bearing its target sites at distant locations. We demonstrate this prediction by electron microscopy, and using the well-characterized cis-regulatory domain of the CyIIIa cytoskeletal actin gene, we show that the loop conformations predicted from the known SpGCF1 target site locations are actually formed in vitro. We speculate that the multimerization of this factor in vivo may function to bring distant regions of extended regulatory domains into immediate proximity so that they can interact with one another.
Isolation of an amino-terminal deleted recombinant ADP-ribosylation factor 1 in an activated nucleotide-free state.
Authors: Hong J X , Zhang X , Moss J , Vaughan M
Published: May 2006

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin ADP-ribosyltransferase in vitro and participate in intracellular vesicular membrane trafficking. ARFs are activated when bound GDP is replaced by GTP and inactivated by hydrolysis of bound GTP to yield ARF-GDP. Usually, ARFs are isolated in an inactive GDP-bound state and require addition of GTP along with detergent or phospholipid for activity. Purified mutant recombinant ARF1 lacking the first 13 amino acids (r delta 13ARF1-P) stimulated cholera toxin activity essentially equally with or without added GTP (and phospholipid or detergent), at least in part due to the presence of bound nucleotides, which later were identified as GTP and GDP. Nucleotide-free r delta 13ARF1 (r delta 13ARF1-F), prepared by dialysis against 7 M urea, was active without added GTP in the absence of SDS but inactive without added GTP in its presence. Renaturation of r delta 13ARF1-F in the presence of GTP, ITP, or GDP yielded, respectively, r delta 13ARF1-GTP and r delta 13ARF1-ITP, which were active, and r delta 13ARF1-GDP, which was inactive. Effects of phospholipids and detergents on nucleotide exchangeability evaluated as effects on activity of rARF1 and r delta 13ARF1-F differed. With r delta 13ARF1-F, 100 microM ITP and 100 microM GTP were essentially equally effective in the presence of cardiolipin or SDS. The finding that r delta 13ARF1 differs from rARF1 in the effects of phospholipids and detergents on nucleotide binding is consistent with the conclusion that the ARF amino terminus plays an important role in nucleotide binding and its specificity as well as the molecular conformation and associated activity.