bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation.
Authors: Whitesell L , Mimnaugh E G , De Costa B , Myers C E , Neckers L M
Published: May 2006

The molecular mechanisms by which oncogenic tyrosine kinases induce cellular transformation are unclear. Herbimycin A, geldanamycin, and certain other benzoquinone ansamycins display an unusual capacity to revert tyrosine kinase-induced oncogenic transformation. As an approach to the study of v-src-mediated transformation, we examined ansamycin action in transformed cells and found that drug-induced reversion could be achieved without direct inhibition of src phosphorylating activity. To identify mechanisms other than kinase inhibition for drug-mediated reversion, we prepared a solid phase-immobilized geldanamycin derivative and affinity precipitated the molecular targets with which the drug interacted. In a range of cell lines, immobilized geldanamycin bound elements of a major class of heat shock protein (HSP90) in a stable and pharmacologically specific manner. Consistent with these binding data, we found that soluble geldanamycin and herbimycin A inhibited specifically the formation of a previously described src-HSP90 heteroprotein complex. A related benzoquinone ansamycin that failed to revert transformed cells did not inhibit the formation of this complex. These results demonstrate that HSP participation in multimolecular complex formation is required for src-mediated transformation and can provide a target for drug modulation.
Phytochelatin production by marine phytoplankton at low free metal ion concentrations: laboratory studies and field data from Massachusetts Bay.
Authors: Ahner B A , Price N M , Morel F M
Published: May 2006

Phytochelatins are small metal-binding polypeptides synthesized by algae in response to high metal concentrations. Using a very sensitive HPLC method, we have quantified phytochelatins from phytoplankton in laboratory cultures at environmentally relevant metal concentrations and in marine field samples. Intracellular concentrations of phytochelatin, in the diatom Thalassiosira weissflogii, exhibit a distinct dose-response relation with free Cd2+ concentration in the medium--not with total Cd(2+)--and are detectable even when the free Cd2+ concentration is less than 1 pM. In Massachusetts Bay, phytochelatin levels (normalized to chlorophyll a) in the particulate fraction are similar to those measured in laboratory cultures exposed to picomolar free Cd2+ concentrations and exhibit a decreasing seaward trend. Incubations of natural samples with added Cd2+ confirmed the induction of the peptides by this metal. Ambient phytochelatin concentrations thus appear to provide a measure of the metal stress resulting from the complex mixture of trace metals and chelators in natural waters.
Structural interaction of natural and synthetic inhibitors with the venom metalloproteinase, atrolysin C (form d).
Authors: Zhang D , Botos I , Gomis-Rüth F X , Doll R , Blood C , Njoroge F G , Fox J W , Bode W , Meyer E F
Published: May 2006

The structure of the metalloproteinase and hemorrhagic toxin atrolysin C form d (EC 3.4.24.42), from the venom of the western diamondback rattlesnake Crotalus atrox, has been determined to atomic resolution by x-ray crystallographic methods. This study illuminates the nature of inhibitor binding with natural (< Glu-Asn-Trp, where < Glu is pyroglutamic acid) and synthetic (SCH 47890) ligands. The primary specificity pocket is exceptionally deep; the nature of inhibitor and productive substrate binding is discussed. Insights gained from the study of these complexes facilitate the design of potential drugs to treat diseases where matrix metalloproteinases have been implicated, e.g., arthritis and tumor metastasis.
Isolation of a human cDNA for heme A:farnesyltransferase by functional complementation of a yeast cox10 mutant.
Authors: Glerum D M , Tzagoloff A
Published: May 2006

We have cloned the human homolog of the Saccharomyces cerevisiae COX10 gene by functional complementation of a yeast cox10 null mutant. The 2.8-kb cDNA encoding the human heme A:farnesyltransferase codes for a 443-aa protein with high homology to the yeast and bacterial farnesylases. The human COX10 homolog, however, does not complement the mutation as efficiently as the yeast COX10 protein, likely due to the heterologous environment. PCR amplification and Southern analysis confirm the existence of a large mRNA for the human protein, with an unusually long 3' untranslated region. This clone can now be used to screen patients with inherited deficiencies in cytochrome oxidase in which the mutations remain unidentified and are likely to reside in a protein influencing the assembly of the enzyme.
Homology-associated nonhomologous recombination in mammalian gene targeting.
Authors: Sakagami K , Tokinaga Y , Yoshikura H , Kobayashi I
Published: May 2006

Nonhomologous (illegitimate) recombination of DNA underlies many changes in the genome. It involves no or little homology between recombining DNAs and has been considered unrelated with homologous recombination, which requires long homology. In mouse cells, however, we found recombination products whose sequences suggest that homologous interaction between DNAs caused nonhomologous recombination with another DNA. The intermediates of homologous recombination were apparently trapped at various stages and shunted to nonhomologous recombination. In one product, the nonhomologous recombination disrupted gene conversion. In another, it took place exactly at the end of long homology shared between two DNAs. This finding explains why gene targeting needs long uninterrupted homology and why mammalian homologous recombination is often nonconservative. We discuss possible consequences and roles of this type of homology-driven gene destruction mechanism.
A vaccine that prevents pregnancy in women.
Authors: Talwar G P , Singh O , Pal R , Chatterjee N , Sahai P , Dhall K , Kaur J , Das S K , Suri S , Buckshee K
Published: May 2006

We report here results of clinical trials on a birth control vaccine, consisting of a heterospecies dimer of the beta subunit of human chorionic gonadotropin (hCG) associated noncovalently with the alpha subunit of ovine luteinizing hormone and conjugated to tetanus and diphtheria toxoids as carriers, that induces antibodies of high avidity (K(a) approximately 10(10) M-1) against hCG. Fertile women exposed to conception over 1224 cycles recorded only one pregnancy at antibody titers of > 50 ng/ml (hCG bioneutralization capacity). The antibody response declines with time; fertility was regained when titers fell to < 35 ng/ml. This study presents evidence of the feasibility of a vaccine for control of human fertility.
Morphine induces c-fos and junB in striatum and nucleus accumbens via D1 and N-methyl-D-aspartate receptors.
Authors: Liu J , Nickolenko J , Sharp F R
Published: May 2006

Morphine induced the c-fos and junB immediate early genes in neurons of the medial and ventral striatum and nucleus accumbens. Induction of c-fos and junB mRNA and Fos protein was blocked by naloxone, the D1 dopamine (DA) receptor antagonists SCH23390 and SCH39166, and the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK801. SCH23390 attenuated morphine induction of AP-1 binding in striatum, suggesting that c-fos and junB contribute to AP-1 binding. SCH23390 and MK801 did not block morphine induction of c-fos and junB in septum. Since the morphine induction of c-fos and junB in striatum and nucleus accumbens (NA) was similar to that observed with cocaine and amphetamine, these data support current concepts that limbic striatum and NA are among the brain regions that mediate drug abuse. Furthermore, since DA and NMDA receptors may mediate opiate reward and opiate induction of c-fos and junB, the DA/NMDA regulation of c-fos and junB and their target genes may produce long-term changes in the striatal and NA circuits that contribute to opiate drug abuse.
Vacuolar H(+)-pumping ATPase variable transport coupling ratio controlled by pH.
Authors: Davies J M , Hunt I , Sanders D
Published: May 2006

The eukaryote endomembrane system contains a class of H(+)-pumping ATPase (H(+)-ATPases) of the vacuolar type (V-ATPases) that are responsible for the acidification of organelles. Their action is critical to numerous physiological processes, but the regulatory mechanisms that may control activity are not yet fully understood. The ratio of H+ transported per ATP hydrolyzed (n) has been determined thermodynamically for the red beet V-ATPase by using patch clamp. The value of n was found to range from 1.75 to 3.28 and was strictly dependent on cytoplasmic and lumenal pH. This suggests a mechanism by which V-ATPases are regulated by and might therefore control cytoplasmic and lumenal pH. Furthermore, the substantial capacity of plant vacuoles for H+ accumulation to pH 3 or lower can only be explained by the finding that n can adopt a value of < 2.
The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase.
Authors: Park P , Copeland W , Yang L , Wang T , Botchan M R , Mohr I J
Published: May 2006

Persistent infection by papillomaviruses involves the maintenance of viral DNA as a nuclear plasmid, the replication of which requires host DNA polymerases. The role of the cellular DNA polymerase alpha-primase holoenzyme was probed by using soluble extracts from rodent cells that replicate bovine papilloma virus 1 and human papilloma virus 6b DNA in the presence of the viral E1 helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of polymerase alpha inhibit DNA synthesis in this system. Addition of purified human polymerase alpha-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids of E1 forms a specific protein complex with the p180 polymerase subunit. Immune complexes can be isolated with antibodies directed against E1 that contain a DNA polymerase activity. Moreover, this polymerase activity can be neutralized by anti-polymerase alpha antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine E1 can interface with the murine and human replication apparatus. Although the large tumor antigens encoded by simian virus 40 and polyoma share limited primary sequence homology with the papillomavirus E1 proteins, the organization of functional motifs at the level of primary protein structure is remarkably similar. In addition to their origin-specific DNA-binding activity, each of these helicases may function to help recruit the cellular polymerase alpha-primase complex to the viral replication origin.
Induction of structural changes in the bovine papillomavirus type 1 origin of replication by the viral E1 and E2 proteins.
Authors: Gillette T G , Lusky M , Borowiec J A
Published: May 2006

Chemical and enzymatic probing techniques were used to examine the interaction of the bovine papillomavirus type 1 E1 and E2 proteins with the viral origin of replication (ori). E1 was found to generate significant distortions to the structure of ori, as assayed by KMnO4 oxidation of DNA. The primary site of ori distortion was located within and adjacent to the AT-element of the core replicator sequence, although a number of minor structural transitions were also detected. The induction of these structural changes required ATP and appeared to require ATP hydrolysis. E2 was found to decrease the amount of E1 required for ori distortion but did not significantly alter the pattern of structural distortion. In contrast, the presence of E2 resulted in a biphasic mechanism for E1 binding to ori, as assayed by nuclease protection. Under these conditions, E1 bound preferentially to the dyad symmetry region containing the conserved Hpa I site. Higher levels of E1 were required for binding to the adjacent ori AT-rich region. Thus, these data suggest that E2 can order the stepwise binding of E1 to ori.
Brain-derived neurotrophic factor increases the electrical activity of pars compacta dopamine neurons in vivo.
Authors: Shen R Y , Altar C A , Chiodo L A
Published: May 2006

Chronic infusions of brain-derived neurotrophic factor (BDNF) immediately above the substantia nigra augment spontaneous locomotion, rotational behavior, and striatal dopamine (DA) turnover, indicating that BDNF increases functions of the nigrostriatal DA system. Because the function of the nigrostriatal DA system is related to the electrical activity of DA neurons, we investigated the effect of BDNF on the electrical activity of DA neurons in the substantia nigra pars compacta in vivo. Chronic supranigral infusions of BDNF (12 micrograms/day), nerve growth factor (11 micrograms/day), or phosphate-buffered saline were started 2 weeks before the electrophysiological recordings. BDNF increased the number of spontaneously active DA neurons by 65-98%, increased the average firing rage by 32%, and increased the number of action potentials contained within bursts. Neither nerve growth factor nor phosphate-buffered saline infusions altered any of these properties relative to unoperated animals. In addition, extremely fast-firing DA neurons (> 10 spikes per sec) were commonly found only in the BDNF-infused animals. These results demonstrate neurotrophin effects on the electrical activity of intact central nervous system neurons in vivo and suggest that the increases in locomotor behavior and striatal dopamine turnover obtained during supranigral BDNF infusions may result from increases in the electrical activity of DA neurons.
Constitutive activation of Mek1 by mutation of serine phosphorylation sites.
Authors: Huang W , Erikson R L
Published: May 2006

A variety of extracellular signals lead to the phosphorylation and activation of mitogen-activated protein kinases (MAP kinases). An activator of MAP kinases, Mek1, phosphorylates MAP kinases at threonine and tyrosine residues and is itself phosphorylated at serine-218 and -222 by the protooncogene product Raf-1. By introducing negatively charged residues that may mimic the effect of phosphorylation at positions 218 and 222, we have activated the capacity of Mek1 to phosphorylate MAP kinase by > 100-fold. The most effective activation by a single substitution resulted from the introduction of aspartate at position 218, whereas the introduction of either aspartate or glutamate at position 222 was ineffective. Expression of the activated Mek1 phosphorylation-site mutants in COS-7 cells led to the activation of MAP kinase in the cells and resulted in an increase in the mass of the transfected COS-7 cell population, suggesting an important role of Mek1 in the transduction of mitogenic signals.
Binding of soluble natural ligands to a soluble human T-cell receptor fragment produced in Escherichia coli.
Authors: Hilyard K L , Reyburn H , Chung S , Bell J I , Strominger J L
Published: May 2006

An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.
Hyperuricemia and urate nephropathy in urate oxidase-deficient mice.
Authors: Wu X , Wakamiya M , Vaishnav S , Geske R , Montgomery C , Jones P , Bradley A , Caskey C T
Published: May 2006

Urate oxidase, or uricase (EC 1.7.3.3), is a purine metabolic enzyme that catalyzes the conversion of uric acid to allantoin in most mammals except humans and certain other primates. The loss of urate oxidase in the human during primate evolution predisposes man to hyperuricemia, a metabolic disturbance that can lead to gouty arthritis and renal stones. To create a mouse model for hyperuricemia and gout, and to address the question of whether urate oxidase is essential in lower mammalian species, we have disrupted the urate oxidase gene in the mouse by homologous recombination in embryonic stem cells. Unlike the human situation, urate oxidase deficiency in mice causes pronounced hyperuricemia and urate nephropathy. More than half of the mutant mice died before 4 weeks of age, indicating that urate oxidase is essential in mice. These mutant mice may also serve as animal models for hyperuricemia and its related nephropathy in humans.
Identification of genes expressed in premalignant breast disease by microscopy-directed cloning.
Authors: Jensen R A , Page D L , Holt J T
Published: May 2006

Histopathologic study of human breast biopsy samples has identified specific lesions which are associated with a high risk of development of invasive breast cancer. Presumably, these lesions (collectively termed premalignant breast disease) represent the earliest recognizable morphologic expression of fundamental molecular events that lead to the development of invasive breast cancer. To study molecular events underlying premalignant breast disease, we have developed a method for isolating RNA from histologically identified lesions from frozen human breast tissue. This method specifically obtains mRNA from breast epithelial cells and has identified three genes which are differentially expressed in premalignant breast epithelial lesions. One gene identified by this method is overexpressed in four of five noncomedo ductal carcinoma in situ lesions and appears to be the human homologue of the gene encoding the M2 subunit of ribonucleotide reductase, an enzyme involved in DNA synthesis.
Neuropathology and apolipoprotein E profile of aged chimpanzees: implications for Alzheimer disease.
Authors: Gearing M , Rebeck G W , Hyman B T , Tigges J , Mirra S S
Published: May 2006

Neuropathological findings in three aged chimpanzees were compared with those in rhesus monkeys and individuals with Alzheimer disease. Senile plaques and blood vessels were immunoreactive for amyloid beta-protein and apolipoprotein E (apoE) in the nonhuman primates, recapitulating findings in human aging and Alzheimer disease. Neurofibrillary tangles, another hallmark of Alzheimer disease, were absent. PCR/restriction-enzyme analysis in chimpanzees revealed an APOE profile similar to the human APOE type 4 allele associated with an increased risk of Alzheimer disease. These findings militate against the hypothesis that the absence of APOE type 3 allele predisposes to neurofibrillary tangle formation and support the value of aged primates for exploring mechanisms of amyloid processing and the role of apoE.
A block in both early T lymphocyte and natural killer cell development in transgenic mice with high-copy numbers of the human CD3E gene.
Authors: Wang B , Biron C , She J , Higgins K , Sunshine M J , Lacy E , Lonberg N , Terhorst C
Published: May 2006

A severe immunodeficiency involving a complete loss of T lymphocytes and natural killer cells was observed in independent lines of transgenic mice containing > 30 copies of the human CD3E gene (pL12). T-cell- natural killer (NK)- mice could also be generated by using a gene fragment pL12 delta 1 (without exons 4A and 5) coding for the CD3-epsilon transmembrane region and its 55-amino acid nonenzymatic cytoplasmic tail. The abnormally small thymus gland in the homozygous transgenic animals, which was approximately 1% the size of a wild-type thymus, contained only a few (2-4%) prethymocytes with a Thy-1+Pgp-1+IL-2R alpha- CD3-4-8- phenotype. In mice with lower copy numbers of the transgene thymocyte development was blocked at the Thy-1+Pgp-1-IL-2R alpha+CD3-4-8- stage, and normal NK activity was detected. Mice generated with high-copy numbers of a transgene pL12 delta 2 (pL12 delta 1 minus exons 6), coding for a truncated protein from which the CD3-epsilon extracellular domain, its transmembrane region, and most of its cytoplasmic region were absent, contained normal numbers of T lymphocytes and NK cells. These transgene effects suggested that recruitment of signal-transduction molecules by the cytoplasmic tail of this protein played an important role in the abrogation of both lineages. Taken together these observations support the notion that T lymphocytes and NK cells stemmed from a common precursor.
Diffusion of inositol 1,4,5-trisphosphate but not Ca2+ is necessary for a class of inositol 1,4,5-trisphosphate-induced Ca2+ waves.
Authors: Jafri M S , Keizer J
Published: May 2006

Combining a realistic model of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ oscillations with the diffusion of IP3 and buffered diffusion of Ca2+, we have found that diffusion of Ca2+ plays only a minor role in a class of agonist-induced Ca2+ wave trains. These waves are primarily kinematic in nature, with variable wavelengths and speeds that depend primarily on the phase differences between oscillators at different spatial points. The period is set by the steady-state value of IP3, while the wave speed approximately equals the wavelength/period. Ca2+ diffusion, which is much slower than that of IP3 because of endogenous buffers, is shown to have only a small effect on the wave trains and not to be necessary for the apparent wave propagation. Diffusion of IP3 sets the phase gradient responsible for these wave trains, which consist primarily of localized cycles of Ca2+ uptake and release. Our results imply a possible previously undisclosed role for IP3 in cell signaling.
A recombinant antibody-interleukin 2 fusion protein suppresses growth of hepatic human neuroblastoma metastases in severe combined immunodeficiency mice.
Authors: Sabzevari H , Gillies S D , Mueller B M , Pancook J D , Reisfeld R A
Published: May 2006

A genetically engineered fusion protein consisting of a human/mouse chimeric anti-ganglioside GD2 antibody (ch14.18) and recombinant human interleukin 2 (rhIL-2) was tested for its ability to target rhIL-2 to tumor sites and stimulate immune effector cells sufficiently to achieve effective tumor cell lysis in vivo. The ch14.18-IL-2 fusion protein proved more effective than equivalent doses of rhIL-2 in suppressing dissemination and growth of human neuroblastoma in an experimental hepatic metastases model of scid (severe combined immunodeficiency) mice reconstituted with human lymphokine-activated killer cells. The ch14.18-IL-2 fusion protein was also more proficient than equivalent doses of rhIL-2 in prolonging the life-span of these animals. This recombinant antibody-cytokine fusion protein may prove useful for future treatment of GD2-expressing human tumors in an adjuvant setting.
Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro affinity selection.
Authors: Tao X , Murphy J R
Published: May 2006

The expression of diphtheria toxin in lysogenic toxigenic strains of Corynebacterium diphtheriae is controlled by the heavy metal ion-activated regulatory protein DtxR. In the presence of divalent heavy metal ions, DtxR specifically binds to the diphtheria tox operator and protects a 27-bp interrupted palindromic sequence from DNase I digestion. To determine the consensus DNA sequence for DtxR binding, we have used gel electrophoresis mobility-shift assay and polymerase chain reaction (PCR) amplification for in vitro affinity selection of DNA binding sequences from a universe of 6.9 x 10(10) variants. After 10 rounds of in vitro affinity selection, each round coupled with 30 cycles of PCR amplification, we isolated and characterized a family of DNA sequences that function as DtxR-responsive genetic elements both in vitro and in vivo. Moreover, these DNA sequences were found to bind activated DtxR with an affinity similar to that of the wild-type tox operator. The DNA sequence analysis of 21 unique in vitro affinity-selected binding sites has revealed the minimal essential nucleotide sequence for DtxR binding to be a 9-bp palindrome separated by a single base pair.
Colocalization of vertebrate lamin B and lamin B receptor (LBR) in nuclear envelopes and in LBR-induced membrane stacks of the yeast Saccharomyces cerevisiae.
Authors: Smith S , Blobel G
Published: May 2006

We have expressed human lamin B and the chicken lamin B receptor (LBR), either separately or together, in yeast and have monitored the subcellular location of the expressed proteins by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. At the light microscopic level, the heterologous lamin B localized to the yeast nuclear rim and at electron microscopic resolution was found subjacent to the yeast inner nuclear membrane. These data indicate that vertebrate lamin B was correctly targeted in yeast. Expression of the heterologous LBR, either alone or together with the heterologous lamin B, resulted in the formation of membrane stacks primarily adjacent to the nuclear envelope, but also projecting from the nuclear envelope into the cytoplasm or under the plasma membrane. Double immunoelectron microscopy showed colocalization of the heterologous lamin B and LBR in the yeast nuclear envelope and in the LBR-induced membrane stacks. Cell fractionation showed the presence of the heterologous lamin B and LBR in a subnuclear fraction enriched in nuclear envelopes. The heterologous lamin B was extracted at 8 M urea, but not at 4 M urea, thus behaving as a peripheral membrane protein and indistinguishable from assembled lamins. The heterologous LBR was not extracted by 8 M urea, indicating that it was integrated into the membrane. The observed colocalization and cofractionation are consistent with previously reported in vitro binding data and suggest that heterologous lamin B and LBR interact with each other when coexpressed in yeast.
Classification of hepatitis C viruses based on phylogenetic analysis of the envelope 1 and nonstructural 5B regions and identification of five additional subtypes.
Authors: Stuyver L , van Arnhem W , Wyseur A , Hernandez F , Delaporte E , Maertens G
Published: May 2006

Genotyping of hepatitis C virus-positive sera by means of a line probe assay indicated that < 3% of European samples, but up to 30% of Gabonese sera, could not be classified as either 1a, 1b, 2a, 2b, 3a, 3b, 4c, 5a, or 6a. Such samples were analyzed in the 5' untranslated region and in the nonstructural 5 (NS5) region. Classification based on phylogenetic analysis of the commonly used 222-bp-long NS5B region was possible for most but not all of the selected sera. Therefore, the core/envelope 1 region (579 bp) and a larger NS5B (340 bp) region were also analyzed. Only the phylogenetic analysis of the 340-bp NS5B region of these newly identified and published isolates provided unambiguous classification into types and subtypes. Furthermore, unequivocal evidence for four subtypes in type 2 and eight subtypes in type 4 was provided. A specific recognition sequence in the 5' untranslated region was observed for every newly identified subtype. Based on 1830 pair-wise comparisons in NS5B, isolates belonging to the same subtype showed evolutionary distances of < 0.127 and isolates of the same type exhibited evolutionary distances of < 0.328. These phylogenetic border distances can be conveniently used for classification of hepatitis C virus isolates into types and subtypes.
Bacterial glutamate racemase has high sequence similarity with myoglobins and forms an equimolar inactive complex with hemin.
Authors: Choi S Y , Esaki N , Ashiuchi M , Yoshimura T , Soda K
Published: May 2006

Glutamate racemase (EC 5.1.1.3), an enzyme of microbial origin, shows significant sequence homology with mammalian myoglobins, in particular in the regions corresponding to the E and F helices, which constitute the heme binding pocket of myoglobins. Glutamate racemase binds tightly an equimolar amount of hemin, leading to loss of racemase activity. Although this enzyme shows homology with aspartate racemase, the latter does not bind hemin. The glutamate racemase gene of Pediococcus pentosaceus has a 795-nt open reading frame and encodes 265-amino acid residues, which form a monomeric protein (M(r) 29,000). Neither racemase has cofactors, but they contain essential cysteine residues [Yohda, M., Okada, H. & Kumagai, H. (1991) Biochim. Biophys. Acta 1089, 234-240].
Lupus susceptibility loci in New Zealand mice.
Authors: Kono D H , Burlingame R W , Owens D G , Kuramochi A , Balderas R S , Balomenos D , Theofilopoulos A N
Published: May 2006

Susceptibility to systemic lupus erythematosus has been unequivocally established to be an inherited trait, but the exact genes and how they confer susceptibility remain largely unknown. In this study of (NZB x NZW)F2 intercross mice, we used linkage analysis of markers covering > 90% of the autosomal genome and identified eight susceptibility loci (Lbw1 to -8, chromosomes 17, 4-7, 18, 1, 11, respectively) associated with antichromatin autoantibody production, glomerulonephritis, and/or mortality. Only one locus, the major histocompatibility complex, was linked to all three traits. Two other loci were associated with both glomerulonephritis and mortality, whereas the remaining loci were linked to one of the above traits. Two additional loci (Sbw1 and -2) that contributed to splenomegaly were also identified. The Sbw2 locus mapped to the identical region as Lbw2, a locus on chromosome 4 linked to glomerulonephritis and mortality, suggesting a single locus with pleiotropic effects. The results indicate that the immunopathologic features of lupus are affected by distinct, but additive, genetic contributions. Studies to determine the nature of the genes associated with these loci should help define the genetic mechanisms involved in this systemic autoimmune disease.
Cloning of the cDNA for a hematopoietic cell-specific protein related to CD20 and the beta subunit of the high-affinity IgE receptor: evidence for a family of proteins with four membrane-spanning regions.
Authors: Adra C N , Lelias J M , Kobayashi H , Kaghad M , Morrison P , Rowley J D , Lim B
Published: May 2006

We report the cloning of the cDNA for a human gene whose mRNA is expressed specifically in hematopoietic cells. A long open reading frame in the 1.7-kb mRNA encodes a 214-aa protein of 25 kDa with four hydrophobic regions consistent with a protein that traverses the membrane four times. To reflect the structure and expression of this gene in diverse hematopoietic lineages of lymphoid and myeloid origin, we named the gene HTm4. The protein is about 20% homologous to two other "four-transmembrane" proteins; the B-cell-specific antigen CD20 and the beta subunit of the high-affinity receptor for IgE, Fc epsilon RI beta. The highest homologies among the three proteins are found in the transmembrane domains, but conserved residues are also recognized in the inter-transmembrane domains and in the N and C termini. Using fluorescence in situ hybridization, we localized HTm4 to human chromosome 11q12-13.1, where the CD20 and Fc epsilon RI beta genes are also located. Both the murine homologue for CD20, Ly-44, and the murine Fc epsilon RI beta gene map to the same region in murine chromosome 19. We propose that the HTm4, CD20, and Fc epsilon RI beta genes evolved from the same ancestral gene to form a family of four-transmembrane proteins. It is possible that other related members exist. Similar to CD20 and Fc epsilon RI beta, it is likely that HTm4 has a role in signal transduction and, like Fc epsilon RI beta, might be a subunit associated with receptor complexes.
Successful passive and active immunization of cynomolgus monkeys against hepatitis E.
Authors: Tsarev S A , Tsareva T S , Emerson S U , Govindarajan S , Shapiro M , Gerin J L , Purcell R H
Published: May 2006

Virtually full protection against hepatitis E and partial or complete protection against infection with hepatitis E virus (HEV) were achieved in passively or actively immunized cynomolgus monkeys. Hepatitis, viremia, and shedding of the virus in feces were detected in all nonimmunized animals that were challenged with HEV. HEV titers detected by reverse transcriptase PCR were higher in feces than in serum of nonimmunized animals. Anti-HEV antibody titers at the time of challenge ranged between 1:40 and 1:200 in animals passively immunized with convalescent plasma from a cynomolgus monkey previously infected with HEV and between 1:100 and 1:10,000 in animals actively immunized with a recombinant 55-kDa open reading frame 2 protein. The estimated 50% protective titer of passively acquired anti-HEV antibodies was 1:40. Although only one of four passively immunized animals showed histopathologic evidence of hepatitis, all four were infected after challenge; however, the titers of HEV in serum and feces were lower in the passively immunized animals than in the nonimmunized group. The actively immunized animals developed neither hepatitis nor viremia when challenged with HEV and virus was either not detected or was present in low titer in feces. The protective response was a function of the ELISA anti-HEV antibody titer at the time of challenge and the immunization schedule.
Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma.
Authors: Herman J G , Latif F , Weng Y , Lerman M I , Zbar B , Liu S , Samid D , Duan D S , Gnarra J R , Linehan W M
Published: May 2006

Mutational inactivation and allelic loss of the von Hippel-Lindau (VHL) gene appear to be causal events for the majority of spontaneous clear-cell renal carcinomas. We now show that hypermethylation of a normally unmethylated CpG island in the 5' region provides another potentially important mechanism for inactivation of the VHL gene in a significant portion of these cancers. This hypermethylation was found in 5 of 26 (19%) tumors examined. Four of these had lost one copy of VHL while one retained two heavily methylated alleles. Four of the tumors with VHL hypermethylation had no detectable mutations, whereas one had a missense mutation in addition to hypermethylation of the single retained allele. As would be predicted for the consequence of methylation in this 5' CpG island, none of the 5 tumors expressed the VHL gene. In contrast, normal kidney and all tumors examined with inactivating VHL gene mutations but no CpG island methylation had expression. In a renal cell culture line, treatment with 5-aza-2'-deoxycytidine resulted in reexpression of the VHL gene. These findings suggest that aberrant methylation of CpG islands may participate in the tumor-suppressor gene inactivations which initiate or cause progression of common human cancers.
Tpl-2 acts in concert with Ras and Raf-1 to activate mitogen-activated protein kinase.
Authors: Patriotis C , Makris A , Chernoff J , Tsichlis P N
Published: May 2006

Mitogenic signals initiated at the plasma membrane by extracellular factors acting on receptor tyrosine kinases or G protein-coupled receptors are transmitted to the nucleus through an intricate signaling network. Components of this network participate, upon stimulation, in a complex array of phosphorylation-dependent protein-protein interactions which leads to the formation of transient multimolecular complexes. Complexes containing products of the protooncogenes ras and raf-1 and the protein kinase MEK-1 activate the mitogen-activated protein kinases (MAPKs), which play a central role in the integration of different mitogenic signals by directly phosphorylating cytoplasmic and nuclear targets. In this report we present evidence that the kinase encoded by the tumor progression locus 2 gene (Tpl-2) contributes to the activation of the MAPK cascade. MAPK activation induced by the Tpl-2 protein is blocked by dominant negative mutants of Ras and Raf-1, whereas a kinase-deficient Tpl-2 mutant down-regulates mitogenic signals induced by v-Ha-Ras or v-Raf. These data suggest that Tpl-2 activates the MAPK cascade, perhaps through its participation in the assembly of Ras/Raf-1-containing multimolecular complexes.
Cloning and characterization of hdlg: the human homologue of the Drosophila discs large tumor suppressor binds to protein 4.1.
Authors: Lue R A , Marfatia S M , Branton D , Chishti A H
Published: May 2006

The Drosophila discs large tumor suppressor protein, dlg, has been shown to regulate the growth of imaginal discs during embryogenesis [Woods, D. F. & Bryant, P. J. (1991) Cell 66, 451-464]. We cloned and sequenced the complete cDNA for a human B-lymphocyte 100-kDa protein that shares 60% amino acid identity with dlg. This human homologue of Drosophila discs large (hdlg) contains a C-terminal domain homologous to the known guanylate kinases, a src homology 3 region motif, and three dlg homology repeats. Two nonhomologous domains that can contain in-frame insertions result in at least four alternatively spliced isoforms of hdlg. Several hdlg RNA transcripts are widely distributed in human and murine tissues, and the protein is localized to regions of cell-cell contact. Protein 4.1, the defining member of a family that includes talin and merlin/schwannomin, has the same cellular localization as hdlg, and two sites within hdlg associate in vitro with the 30-kDa N-terminal domain of protein 4.1.
Mechanism of GTP hydrolysis by G-protein alpha subunits.
Authors: Kleuss C , Raw A S , Lee E , Sprang S R , Gilman A G
Published: May 2006

Hydrolysis of GTP by a variety of guanine nucleotide-binding proteins is a crucial step for regulation of these biological switches. Mutations that impair the GTPase activity of certain heterotrimeric signal-transducing G proteins or of p21ras cause tumors in man. A conserved glutamic residue in the alpha subunit of G proteins has been hypothesized to serve as a general base, thereby activating a water molecule for nucleophilic attack on GTP. The results of mutagenesis of this residue (Glu-207) in Gi alpha 1 refute this hypothesis. Based on the structure of the complex of Gi alpha 1 with GDP, Mg2+, and AlF-4, which appears to resemble the transition state for GTP hydrolysis, we believe that Gln-204 of Gi alpha 1, rather than Glu-207, supports catalysis of GTP hydrolysis by stabilization of the transition state.
Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins.
Authors: Adams D E , Tsaneva I R , West S C
Published: May 2006

The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA. In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay. Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP. These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.
Role of CD26/dipeptidyl peptidase IV in human immunodeficiency virus type 1 infection and apoptosis.
Authors: Morimoto C , Lord C I , Zhang C , Duke-Cohan J S , Letvin N L , Schlossman S F
Published: May 2006

To examine the role of CD26/dipeptidyl peptidase IV (DPPIV; EC 3.4.14.5) in infection by human immunodeficiency virus type 1 (HIV-1), we utilized CD26 cDNA-transfected Jurkat T-cell lines. Both CD26- parental Jurkat cells and mutant CD26+ (DPPIV-) transfected Jurkat cells were readily infected with HIV-1, whereas wild-type CD26+ (DPPIV+) transfected Jurkat cells were more resistant to HIV-1 infection. Our results suggest that CD26 is not essential for HIV-1 infectivity as suggested by others but that DPPIV enzyme activity may decrease the efficiency of HIV-1 infection. Of great interest, we found that mutant CD26+ (DPPIV-) transfectants and CD26- parental Jurkat cells strongly expressed CD95 (Fas/Apo-1) and were more sensitive than wild-type CD26+ (DPPIV+) transfectants to the induction of apoptosis by anti-CD95 monoclonal antibody. These results suggest that CD26 may play a role in HIV-1-associated loss of -CD4+ cells through the process of programmed cell death.
The beta 1 subunit mRNA of the rat brain Na+ channel is expressed in glial cells.
Authors: Oh Y , Waxman S G
Published: May 2006

Although the molecular characteristics of glial Na+ channels are not well understood, recent studies have shown the presence of mRNA for rat brain Na+ channel alpha subunits in astrocytes and Schwann cells. In this study, we asked whether the mRNA for the rat brain Na+ channel beta 1 subunit is expressed in glial cells. We performed in situ hybridization using a complementary RNA probe for the coding regions of the rat brain Na+ channel beta 1 subunit mRNA and detected beta 1 subunit mRNA in cultured rat optic nerve astrocytes and sciatic nerve Schwann cells. The beta 1 subunit was amplified by reverse transcription-polymerase chain reaction in rat optic and sciatic nerves, which lack neuronal somata but contain astrocytes and Schwann cells, respectively. Doublet bands of the beta 1 subunit mRNA were amplified from both optic and sciatic nerves. Through the cloning and sequencing of these bands, we confirmed the amplification of a mRNA highly homologous to the previously cloned rat brain Na+ channel beta 1 subunit (beta 1.1) and a novel form of the beta 1 subunit mRNA (beta 1.2), which is closely homologous to beta 1.1 but contains an additional 86-nucleotide insert in 3' noncoding regions. Two beta 1 subunit mRNAs were also amplified from rat brain and skeletal muscle, but not from rat liver or kidney. These results indicate that rat brain Na+ channel beta 1 subunit mRNAs are expressed in glial cells as well as in neurons.
The nodulation-signaling protein NodO from Rhizobium leguminosarum biovar viciae forms ion channels in membranes.
Authors: Sutton J M , Lea E J , Downie J A
Published: May 2006

The secreted nodulation-signaling protein NodO was purified from the supernatant of cultures of Rhizobium leguminosarum biovar viciae. The native protein has a M(r) of approximately 67,000, suggesting that it exists as a dimer since the DNA sequence predicts a M(r) of 30,002. Pure NodO protein had no protease, pectinase, or cellulase activity, and no binding was observed to lipooligosaccharide nodulation factors. Although NodO is relatively hydrophilic, it appeared to insert into liposomes and was protected by liposomes from proteolytic cleavage. When added to planar lipid bilayers, NodO formed cation-selective channels that allowed the movement of monovalent cations (K+ and Na+) across the membrane. NodO is a Ca(2+)-binding protein; in the presence of high concentrations of Ca2+, channel activity was reduced. We hypothesize that NodO plays a role in nodulation signaling by stimulating uptake of nodulation factors or by forming cation-specific channels that function synergistically with the proposed lipooligosaccharide-induced depolarization of the plasma membrane of leguminous plants.
Frequency-dependent associative long-term potentiation at the hippocampal mossy fiber-CA3 synapse.
Authors: Derrick B E , Martinez J L
Published: May 2006

The mossy fiber-CA3 synapse displays an N-methyl-D-aspartate-receptor-independent mu-opioid-receptor-dependent form of long-term potentiation (LTP) that is thought not to display cooperativity or associativity with coactive afferents. However, because mossy fiber LTP requires repetitive synaptic activity for its induction, we reevaluated cooperativity and associativity at this synapse by using trains of mossy fiber stimulation. Moderate-, but not low-, intensity trains induced mossy fiber LTP, indicating cooperativity. Low-intensity mossy fiber trains that were normally ineffective in inducing LTP could induce mossy fiber LTP when delivered in conjunction with trains delivered to commissural-CA3 afferents. Associative mossy fiber LTP also could be induced with single mossy fiber pulses when delivered with commissural trains in the presence of a mu-opioid-receptor agonist. Our findings suggest a frequency-dependent variation of Hebbian associative LTP induction that is regulated by the release of endogenous opioid peptides.
Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: isolation, structure, synthesis, and endocrine activity.
Authors: Mor A , Chartrel N , Vaudry H , Nicolas P
Published: May 2006

Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three members of a family of structurally related peptides, are mainly expressed in the endocrine pancreas, in endocrine cells of the gut, and in the brain, respectively. In the present study, we have isolated a peptide of the pancreatic polypeptide family from the skin of the South American arboreal frog Phyllomedusa bicolor. The primary structure of the peptide was established as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu10-Asp-Ala-Ser-Pro-Glu-Glu- Met-Asn- Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Val-Thr- Arg-Gln-Arg-Tyr-NH2 . This unusual peptide, named skin peptide tyrosine-tyrosine (SPYY), exhibits 94% similarity with PYY from the frog Rana ridibunda. A synthetic replicate of SPYY inhibits melanotropin release from perifused frog neurointermediate lobes in very much the same way as NPY. These results demonstrate the occurrence of a PYY-like peptide in frog skin. Our data also suggest the existence of a pituitary-skin regulatory loop in amphibians.
Historical development of ornithophily in the western North American flora.
Authors: Grant V
Published: May 2006

The 129 ornithophilous plant species in western North America have floristic affinities with one or the other of four geofloras: the Arcto-Tertiary flora (101 species), Madro-Tertiary flora (19 species), Madrean-Tethyan flora (8 species), and Neotropical flora (1 species). The last three floras have been in continuous contact with hummingbirds since some time early in the Tertiary, and ornithophily is old in this subset of western ornithophilous plants. The Arcto-Tertiary flora had no contact with hummingbirds in Eurasia or in its early history in North America. Ornithophily is a new condition in Arcto-Tertiary plant groups, dating from the first significant contact of these plants with hummingbirds in the Eocene. Buildup of the hummingbird pollination system in the Arcto-Tertiary flora is expected to be gradual and stepwise for several reasons. Ornithophilous plant groups with Arcto-Tertiary affinities in the modern western flora form a graded series with respect to taxonomic rank, taxonomic size, and ecological diversity. The series consists of one large genus (Castilleja), three small genera (Zauschneria, etc.), species groups in several genera (Penstemon, Aquilegia, etc.), single ornithophilous species in otherwise nonornithophilous genera (seven genera--e.g., Pedicularis, Monardella), and ornithophilous races in otherwise nonornithophilous species (known in two species). It is suggested that the gradations in size of the groups approximately reflect stages in their development, with the largest ornithophilous genus being oldest, with single ornithophilous species being relatively recent, and with ornithophilous races being most recent. The observed distribution of numbers of ornithophilous species among genera is in agreement with the expectation of a gradual and stepwise development of ornithophily.