bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and evolution of four POU domain genes expressed in mouse brain.
Authors: Hara Y , Rovescalli A C , Kim Y , Nirenberg M
Published: May 2006

Four mouse POU domain genomic DNA clones--Brain-1, Brain-2, Brain-4, and Scip--and Brain-2 cDNA, which are expressed in adult brain, were cloned and the coding and noncoding regions of the genes were sequenced. The amino acid sequences of the four POU domains are highly conserved; sequences in other regions of the proteins also are conserved but to a lesser extent. The absence of introns from the coding regions of the four POU domain genes and the similarity of amino acid sequences of the corresponding proteins suggest that the coding region of the ancestral class III POU domain gene lacked introns and therefore may have originated by reverse transcription of a molecule of POU domain mRNA followed by insertion of the cDNA into germ cell genomic DNA. Additional duplications of the ancestral class III POU domain gene (or mRNA) would create the Brain-1, Brain-2, Brain-4, and Scip genes.
Evolutionarily different alphoid repeat DNA on homologous chromosomes in human and chimpanzee.
Authors: Jørgensen A L , Laursen H B , Jones C , Bak A L
Published: May 2006

Centromeric alphoid DNA in primates represents a class of evolving repeat DNA. In humans, chromosomes 13 and 21 share one subfamily of alphoid DNA while chromosomes 14 and 22 share another subfamily. We show that similar pairwise homogenizations occur in the chimpanzee (Pan troglodytes), where chromosomes 14 and 22, homologous to human chromosomes 13 and 21, share one partially homogenized alphoid DNA subfamily and chromosomes 15 and 23, homologous to human chromosomes 14 and 22, share another extensively homogenized subfamily. Such a pattern of homogenization presumably predates speciation 3-10 million years ago. However, the alphoid DNA on these human and chimpanzee chromosomes is not orthologous but originates from two evolutionarily different repeat families. It follows that dramatic sequence evolution has occurred in a concerted fashion among the chromosomes in one or both species during or after separation.
Ras GTPase-activating protein: a substrate and a potential binding protein of the protein-tyrosine kinase p56lck.
Authors: Amrein K E , Flint N , Panholzer B , Burn P
Published: May 2006

Ras GTPase-activating protein (GAP) is a cytoplasmic factor that regulates the GTPase activity of p21ras. Phosphorylation of GAP on tyrosine has recently been reported by several groups and may be an important step in linking signaling pathways involving p21ras and protein-tyrosine kinases. p56lck, a src-like protein-tyrosine kinase, seems to play a crucial role in T-cell development and T-cell activation. However, the molecular mechanisms of T-cell signaling involving p56lck and the substrates of p56lck have not yet been identified. To test whether GAP is a substrate of p56lck, in vitro kinase reactions were performed with purified, recombinant GAP and p56lck. We found that GAP became specifically phosphorylated on tyrosine within one tryptic peptide. Furthermore, coimmunoprecipitation studies provided evidence that the tyrosine-phosphorylated form of GAP is bound to p56lck. These results suggest that in T cells the function of GAP might be regulated through its phosphorylation on tyrosine and binding to the protein-tyrosine kinase p56lck.
Endogenous oxidative damage of deoxycytidine in DNA.
Authors: Wagner J R , Hu C C , Ames B N
Published: May 2006

Three major oxidation products of 2'-deoxycytidine (dC)--5-hydroxy-2'-deoxycytidine (oh5dC), 5-hydroxy-2'-deoxyuridine (oh5dU), and 5,6-dihydroxy-5,6-dihydro-2'-deoxyuridine (dUg)--were analyzed from enzymatically hydrolyzed DNA with reversed-phase high-performance liquid chromatography coupled to electrochemical detection. oh5dC and oh5dU can be detected with high sensitivity (50 fmol) and selectivity (0-0.2 V) from hydrolyzed DNA. dUg is not electrochemically active but can be measured by dehydrating it into oh5dU. The quantities of oh5dC, dUg, and oh5dU in untreated commercial-grade calf thymus DNA are 10, 10, and 0.75 fmol/micrograms of DNA, respectively. These levels increased substantially when calf thymus DNA was exposed to ionizing radiation, H2O2 alone, H2O2 and combinations of Fe3+ or Cu2+ and ascorbate, near-UV light (365 nm), near-UV light in the presence of menadione, and OsO4, indicating that oh5dC, oh5dU, and dUg are major oxidative DNA damage products. The steady-state levels of these products were determined from freshly extracted rat tissues and ranged from less than 0.5 fmol/micrograms of DNA for oh5dU to about 10 fmol/micrograms of DNA for oh5dC and dUg in liver and kidney and 22 fmol/micrograms of DNA for oh5dC in brain. The levels of oxo8dG were also determined and in general were somewhat lower than the levels of oh5dC. These findings reinforce the link between DNA damage induced by oxidative metabolism and spontaneous mutagenesis leading to cancer and aging.
Immune response to p53 is dependent upon p53/HSP70 complexes in breast cancers.
Authors: Davidoff A M , Iglehart J D , Marks J R
Published: May 2006

Overexpression of the p53 protein, resulting from gene mutations that increase protein stability, has been detected in greater than 25% of primary human breast cancers. In addition, approximately 10% of breast cancer patients have circulating antibodies to the p53 protein. In this study, the anti-p53 humoral response is correlated with the presence and type of mutant p53 protein expressed in the tumor. In a series of 60 breast cancer patients, 0 of 30 tumors with normal, low-level p53 expression induced anti-p53 antibodies, whereas 7 (23%) of 30 tumors with p53 overexpression elicited a specific anti-p53 antibody response. These 7 patients had anti-p53 antibodies that recognized wild-type p53 and a variety of mutant p53 proteins. A comparison of p53 mutations revealed that antibody-negative tumors had mutations exclusively in exons 7 and 8, whereas antibody-positive tumors had mutations primarily in exons 5 and 6. Moreover, all antibody-eliciting tumors contained complexes between p53 and a 70-kDa heat shock protein, whereas none of the antibody-negative tumors contained this complex. This study implicates a 70-kDa heat shock protein in the antigenic presentation of p53.
Integration of human immunodeficiency virus DNA: adduct interference analysis of required DNA sites.
Authors: Bushman F D , Craigie R
Published: May 2006

The integration (IN) protein encoded by human immunodeficiency virus directs the integration of viral DNA into host DNA. We have probed the DNA sites required for the function of IN protein by attaching adducts to model DNA substrates and assaying their effects on integration in vitro. These experiments reveal that modifications in a short region on both DNA strands at the ends of the viral DNA block IN protein function. Modification of the target DNA near the point of DNA strand transfer also blocks IN protein function. Further experiments suggest that distinct subsets of the identified interactions are important for separate steps in the integration process.
Molecular mechanisms of the partial allosteric modulatory effects of bretazenil at gamma-aminobutyric acid type A receptor.
Authors: Puia G , Ducic I , Vicini S , Costa E
Published: May 2006

In central nervous system gamma-aminobutyric acid (GABA) inhibits neuronal activity by acting on GABA type A (GABAA) receptors. These heterooligomeric integral membrane proteins include a GABA-gated Cl- channel and various allosteric modulatory sites where endogenous modulators and anxiolytic drugs act to regulate GABA action. In vivo, various anxiolytic drugs exhibit a wide range of variability in their modulatory efficacy and potency of GABA action. For instance, bretazenil modulatory efficacy is much lower than that of diazepam. Such low efficacy could be due either to a preferential modulation of specific GABAA receptor subtypes or to a low modulatory efficacy at every GABAA receptor subtype. To address these questions we studied drug-induced modifications of GABA-activated Cl- currents in native GABAA receptors of cortical neurons in primary cultures and in recombinant GABAA receptors transiently expressed in transformed human embryonic kidney cells (293) after transfection with cDNAs encoding different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. In cortical neurons the efficacy of bretazenil was lower than that of diazepam, whereas the potency of the two drugs was similar. In cells transfected with gamma 2 subunits and various molecular forms of alpha and beta subunits bretazenil efficacy was always lower than that of diazepam. However, in cells transfected with gamma 1 or gamma 3 subunits and various forms of alpha and beta subunits the efficacy of both diazepam and bretazenil was lower and always of similar magnitude. When bretazenil and diazepam were applied together to GABAA receptors including a gamma 2 subunit, the action of diazepam was curtailed in a manner related to the dose of bretazenil.
Purification and characterization of retrovirally transduced hematopoietic stem cells.
Authors: Spain L M , Mulligan R C
Published: May 2006

We have developed a method for the purification of retrovirally transduced hematopoietic stem cells, based on a modification of the stem cell purification protocols developed by Spangrude et al. [Spangrude, G., Heimfeld, S. & Weissman, I. (1988) Science 241, 58-64] and Spangrude and Scollay [Spangrude, G. & Scollay, R. (1990) Exp. Hematol. 18, 920-926], that depends upon the use of bone marrow cells isolated from 5-fluorouracil-treated mice that have been subsequently cocultivated with recombinant retrovirus-producing cell lines. We found that purified cell populations bearing a Sca 1+ Lin- Thy 1- surface phenotype represent a 50-100% pure population of spleen colony-forming cells (CFU-S day 12). Animals injected with 300 or more purified cells were consistently radioprotected and reconstituted in multiple lineages with donor cells. Sca 1+ Lin- Thy 1- CFU-S day 12 stem cells were shown to be efficiently (100%) transduced by the recombinant retroviruses used in the study. Gene transfer into long-term reconstituting stem cells, as evidenced by Southern blot analysis of mature hematopoietic cell types 3 months after transplantation, was observed only in recipients injected with large numbers (approximately 4000-5000) of the purified cells. The development of methods for purifying retrovirally transduced stem cells should prove extremely useful for various studies in which it is of interest to characterize the activity of a specific gene product (e.g., growth factor, receptor, oncogene) specifically in primitive hematopoietic cell types.
Evidence for peptide transport across microsomal membranes.
Authors: Koppelman B , Zimmerman D L , Walter P , Brodsky F M
Published: May 2006

Antigenic peptides bound to class I molecules of the major histocompatibility complex (MHC) are recognized by T-cell receptors during development of an antiviral immune response. T cells respond to peptides derived from cytoplasmic viral proteins as well as viral membrane proteins, indicating that a pathway exists for the transport of proteins or peptides from the cytosol into the compartment(s) where the MHC class I molecules assemble. To investigate this pathway, we have developed an in vitro assay for the transport of peptides into microsomal vesicles. This assay provides evidence for the transport of chemically synthesized peptides (13-21 amino acids) containing N-linked glycosylation acceptor sequences, which serve as glycosylation substrates. Their transport results in depletion of the pool of available dolichol high-mannose oligosaccharides in the lumen of the microsomal vesicles. We have observed transport of peptides derived from antigenic human immunodeficiency virus gag and influenza B nucleoprotein sequences, but transport of a third randomly selected peptide was not detected, suggesting specificity of the transport process. We were not able to demonstrate ATP dependence of this peptide transport process by using apyrase and an ATPase inhibitor. This result was unexpected in light of the recent identification of MHC-linked genes with homology to ATP-binding cassette transporters, which have been proposed to mediate peptide transport.
Endothelial cells regulate cardiac contractility.
Authors: Ramaciotti C , Sharkey A , McClellan G , Winegrad S
Published: May 2006

Endothelial cells lining the lumen of blood vessels contain the receptors for many substances that alter the contractile tone of smooth muscle in the walls of the blood vessels. In response to their interaction with the signal substances, the endothelial cells release vasoactive factors that modify the contractile state of the vascular smooth muscle. This study was conducted to determine if endothelial cells can also modulate the contraction of cardiac muscle cells and contribute to the physiological regulation of the heart. The venous effluent from the coronary circulation of an isolated perfused working heart was reoxygenated and used to superfuse a trabecula isolated from the right ventricle of another heart. The peak tension and the duration of the contraction of the trabecula were reversibly altered by the effluent fluid. The change in the contraction of the trabecula during its exposure to coronary effluent was inhibited by selectively damaging the endothelial cells in the trabecula before the application of the coronary effluent. The magnitude and direction of the effect of the coronary venous effluent were sensitive to the metabolic and mechanical conditions under which the isolated perfused heart was contracting at the time the effluent was collected. These observations indicate that cardiac tissue can release a substance or substances into the coronary circulation that induce the production of cardioactive factors by endothelial cells.
Coupling of a purified goldfish brain kainate receptor with a pertussis toxin-sensitive G protein.
Authors: Ziegra C J , Willard J M , Oswald R E
Published: May 2006

Goldfish brain has a high density of [3H]kainate-binding sites, a subpopulation of which appears to be coupled to a pertussis toxin-sensitive G protein. We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affinity [3H]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [3H]kainate binding, and kainate promotes [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding and [gamma-32P]GTP hydrolysis. Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) decreases the apparent Stokes radius of the soluble purified receptor preparation, consistent with dissociation of the kainate receptor-G protein complexes. The affinity-purified preparations contain proteins of 45, 41, and 35 kDa. The 45- and 41-kDa proteins crossreact with antibodies against the kainate receptor cloned from frog brain. The 35-kDa protein is recognized by an antiserum (SW) directed against the beta subunit of G proteins. When kainate receptors are purified in the presence of GTP[gamma S], the 35-kDa protein is no longer present. Also, [3H]kainate affinity is decreased and is no longer guanine nucleotide sensitive. Upon reconstitution with purified G proteins, high-affinity guanine nucleotide-sensitive binding and kainate-stimulated GTPase activity can be restored. These observations indicate that a kainate receptor from goldfish brain functionally interacts with a pertussis toxin-sensitive G protein.
Transcriptional regulatory elements stimulate recombination in extrachromosomal substrates carrying immunoglobulin switch-region sequences.
Authors: Leung H , Maizels N
Published: May 2006

We have developed a sensitive genetic assay to analyze DNA sequences and regulatory elements required for immunoglobulin heavy chain isotype switch recombination. Recombination substrates containing mu and gamma 3 chain switch (S)-region sequences, S mu and S gamma 3, are transiently introduced into primary murine B cells cultured with lipopolysaccharide to induce isotype switching. Recombination involving S-region sequences deletes a conditionally lethal marker, the leftward promoter of phage lambda (lambda PL), enabling recovered plasmids to transform Escherichia coli. In substrates carrying S mu-lambda PL-S gamma 3, about 2% of replicated molecules undergo deletion of lambda PL during transfection; insertion of either the immunoglobulin heavy chain promoter and enhancer sequences or cytomegalovirus IE1 promoter region upstream of S mu increases recombination 10-fold or more to 25% of replicated molecules. Guanosine-rich S-region sequences are essential for efficient recombination of these substrates.
Photoaffinity labeling of avermectin binding sites from Caenorhabditis elegans and Drosophila melanogaster.
Authors: Rohrer S P , Meinke P T , Hayes E C , Mrozik H , Schaeffer J M
Published: May 2006

An azido-avermectin analog [4'' alpha-(4-azidosalicylamido-epsilon-caproylamido-beta-alan ylamido)-4''-deoxyavermectin B1a; azido-AVM] was synthesized and used to photoaffinity label avermectin binding sites present in the membranes of Caenorhabditis elegans and Drosophila melanogaster. Azido-AVM was biologically active and behaved like a competitive inhibitor of [3H]ivermectin binding to C. elegans membranes (Ki = 0.2 nM). Radiolabeled azido-AVM bound specifically and with high affinity to C. elegans membranes (Kd = 0.14 nM) and, upon photoactivation, became covalently linked to three C. elegans polypeptides of 53, 47, and 8 kDa. Photoaffinity labeling of a membrane preparation from D. melanogaster heads resulted in labeling of a single major polypeptide of approximately 47 kDa. The proteins that were covalently tagged in these experiments are believed to be associated with avermectin-sensitive chloride channels present in the neuromuscular systems of C. elegans and D. melanogaster. Azido-AVM did not bind to rat brain membranes and therefore was selective for the nematode and insect receptors.
Steroidogenic potential of lyophilized mitochondria from bovine adrenocortical tissue.
Authors: Prasad V V , Mathur C , Welch M , Lieberman S
Published: May 2006

When incubated with [3H]cholesterol, a bovine adrenocortical mitochondrial pellet obtained by centrifugation at 12,000 x g yielded, as expected, only the C21O2 metabolites progesterone and pregnenolone. However, the steroidogenic potential of the 12,000 x g pellet fraction was augmented significantly by lyophilization. After lyophilization, the 12,000 x g pellet converted the sterol into C19 androgens and corticosteroids, in addition to C21O2 pregnane derivatives. Leaching the lyophilized mitochondrial fraction with either hexane or acetone increased substantially the yields of the metabolites. It did not change qualitatively the array of metabolites formed during in vitro incubation, but 5 alpha-reductase activity was unmasked by the washings, particularly with acetone. Thus, the fraction sedimented at 12,000 x g contains the complete complement of steroidogenic enzymes required for the biosynthesis of the aforementioned adrenal hormones. These results cast doubt upon the widely held belief that the various enzymes required for adrenocortical steroidogenesis are distributed between two different subcellular compartments, the mitochondrion and the endoplasmic reticulum.
Ovarian expression of cellular Ki-ras p21 varies with physiological status.
Authors: Palejwala S , Goldsmith L T
Published: May 2006

To elucidate the potential role of the ras protooncogene proteins in a specific tissue, the present study determined the levels of individual c-ras-encoded p21 proteins in the rat ovary during various stages of physiological function. p21 protein was extracted from ovaries taken from immature normal female rats, mature nonpregnant animals in the metestrus stage of the estrus cycle, rats at various stages of pregnancy, and actively lactating animals. Levels of individual p21s were evaluated by immunoblot analysis with specific antibodies to the p21 proteins encoded by the Kirsten, Harvey, and neuroblastoma c-ras protooncogenes, c-Ki-ras, c-Ha-ras, and N-ras. Results showed that c-Ki-ras p21 is at its lowest level in the immature ovary and increases with development of the corpora lutea to its highest levels at day 16 of pregnancy, after which levels decline and then rise again during lactation. This pattern, which mimics that of circulating progesterone levels, suggests that ovarian c-Ki-ras p21 levels are regulated and that c-Ki-ras p21 plays a role in the differentiated function of the rat ovary, likely the luteal compartment. In contrast, levels of c-N-ras p21 did not appear to vary with changes in the physiological function of the ovary but appeared to be constitutive. A preferential role for the c-Ki-ras p21 may be due to the documented unique differences in the structure of the carboxyl terminus of this particular c-ras p21.
Brain weight and life-span in primate species.
Authors: Allman J , McLaughlin T , Hakeem A
Published: May 2006

In haplorhine primates (tarsiers, monkeys, apes, and humans), there is a significant correlation between brain weight and maximum life-span when the effect of body size is removed. There is also a significant correlation in haplorhine primates between brain weight and female age at first reproduction. For strepsirhine primates (lorises and lemurs), there are no significant correlations between brain weight and either life-span or female reproductive age when the effect of body size is removed. This lack of correlation in strepsirhine primates may be related to the fact that these primates are nocturnal and/or natives of the island of Madagascar, both of which conditions may reduce competition for resources and predation pressure. These findings suggest that in haplorhine primates the genetic systems controlling brain growth are linked to the systems governing the life cycle so that species with longer cycles have larger brains. When the effect of body weight is removed, leaf-eating haplorhines have significantly smaller brains and shorter lives than haplorhines with other diets. Harem-living haplorhines also have significantly smaller brains and shorter life-spans than troop-living haplorhines when the effect of body weight is removed. We also sought to test the rate-of-living hypothesis by determining whether primates with basal metabolic rates that are higher than would be expected for their body size have shorter maximum life-spans than would be expected for their body size. Metabolic rate is not correlated with life-span or female age at first reproduction when the effect of body size is removed.
The phylogenetically conserved doublet tertiary interaction in domain III of the large subunit rRNA is crucial for ribosomal protein binding.
Authors: Kooi E A , Rutgers C A , Mulder A , Van't Riet J , Venema J , Raué H A
Published: May 2006

Previous phylogenetic analysis of rRNA sequences for covariant base changes has identified approximately 20 potential tertiary interactions. One of these is present in domain III of the large subunit rRNA and consists of two adjacent Watson-Crick base pairs that, in Saccharomyces cerevisiae 26S rRNA, connect positions 1523 and 1524 to positions 1611 and 1612. This interaction would strongly affect the structure of an evolutionarily highly conserved region that acts as the binding site for the early-assembling ribosomal proteins L25 and EL23 of S. cerevisiae and Escherichia coli, respectively. To assess the functional importance of this tertiary interaction, we determined the ability of synthetically prepared S. cerevisiae ribosomal protein L25 to associate in vitro with synthetic 26S rRNA fragments containing sequence variations at positions 1523 and 1524 and/or positions 1611 and 1612. Mutations that prevent the formation of both base pairs abolished L25 binding completely, whereas the introduction of compensatory mutations fully restored protein binding. Disruption of only the U1524.A1611 pair reduced L25 binding to approximately 30% of the value shown by the wild-type 26S rRNA fragment, whereas disruption of the G1523.C1612 base pair resulted in almost complete loss of protein binding. These results strongly support the existence and functional importance of the proposed doublet tertiary interaction in domain III of the large subunit rRNA.
Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide.
Authors: Thiemermann C , Szabó C , Mitchell J A , Vane J R
Published: May 2006

This study investigates the role of nitric oxide (NO) and the induction of a calcium-independent NO synthase (NOS) in development of vascular hyporeactivity to norepinephrine (NE) and vascular decompensation associated with hemorrhagic shock (HS) in the anesthetized rat. HS for 120 min caused a time-dependent reduction of the pressor responses to NE. This hyporeactivity is mediated by an enhanced release of NO by the constitutive NOS, for it was reversed by NG-nitro-L-arginine methyl ester (NO2Arg), an inhibitor of both constitutive and inducible NOS, but it was not prevented by dexamethasone, an inhibitor of NOS induction. Vascular decompensation following prolonged periods of HS was characterized by a failure of control animals to maintain arterial blood pressures despite reinfusion of blood. This progressive decrease in blood pressure is mediated by enhanced formation of NO by the inducible NOS, for it was prevented by NO2Arg or dexamethasone. A strong increase in calcium-independent (inducible) NOS activity was observed in several organs after 150 and 330 min of HS, being most pronounced in lung, liver, and spleen. HS for 330, but not 150, min also caused hyporeactivity of rat aortic rings to vasoconstrictors, which was associated with induction of calcium-independent NOS activity in this tissue. Aortic hyporeactivity was prevented by dexamethasone pretreatment in vivo and reversed by NO2Arg in vitro. HS was not associated with an increase in plasma endotoxin levels, showing that endotoxin does not account for induction of NOS in this model. Thus, excessive NO formation induces vascular hyporeactivity and decompensation in HS, indicating that NOS inhibitors, particularly of the inducible NOS, may improve the therapeutic outcome of patients suffering from HS.
The mouse intracisternal A particle-promoted placental gene retrotransposition is mouse-strain-specific.
Authors: Chang-Yeh A , Mold D E , Brilliant M H , Huang R C
Published: May 2006

Insertion of a single long terminal repeat (LTR) of an intracisternal A particle (IAP) gene into a placenta-expressed cellular gene (mouse IAP-promoted placental gene, MIPP) has recently been found for the mouse strains Swiss and CF-1. To test whether such a retrotransposition event has also taken place in other strains of mouse, genomic DNA and total RNAs were analyzed from the outbred CF-1 strain and five inbred strains of mouse, AKR/J, C57BL/6J, DBA/2J, C3H/HeJ, and BALB/c. Specific DNA fragments flanking the LTR region were isolated from MIPP cDNA and genomic clones and used as hybridization probes. Three transcripts [two minor, 4.4 kilobases (kb) and 2.2 kb; one major, 1.2 kb] were detected. The 4.4-kb and 2.2-kb species were found in all strains of mouse studied. The 1.2-kb transcript (promoted by IAP LTR) is present only in placentas of C3H/HeJ and BALB/c mice. Both of these strains contain an IAP LTR in the MIPP gene. In contrast, there is no IAP LTR in this cellular gene in strains DBA/2J, C57BL/6J, and AKR/J. Thus, the IAP MIPP retrotransposition is strain-specific. The parents of the outbred CF-1 mice used for the present studies were both heterozygous at the MIPP locus. Restriction fragment length polymorphism studies using inbred strains and recombinant inbred mice have further established the linkage between the MIPP gene (D4Jhu8) and several loci on distal mouse chromosome 4. The symbol Ipp is being used for this gene in all major mouse data bases and in the comparative section of the human genome data base.
Multiple control of Escherichia coli lysyl-tRNA synthetase expression involves a transcriptional repressor and a translational enhancer element.
Authors: Ito K , Kawakami K , Nakamura Y
Published: May 2006

Lysyl-tRNA synthetases [L-lysine:tRNA(Lys) ligase (AMP-forming), EC 6.1.1.6] are synthesized from two distinct genes in Escherichia coli, lysS (constitutively) and lysU (inducibly), but neither the physiological significance nor the mechanism of differential regulation of these two genes is understood. We have constructed a null mutation of lysS that causes cold-sensitive lethality and then used this mutant to acquire and characterize several bypass mutations called als (abandonment of lysS). Cold-resistant survivors were isolated either spontaneously or by transposon-mediated disruption, and all caused derepression of lysU transcription. One class of als mutations is linked to lysU and presumably affects the cis regulatory element. Mutations of the other class map within the lrp gene, which encodes the leucine-responsive regulatory protein (Lrp). A lysU-lacZ gene fusion study revealed that lysU is susceptible to thermal regulation in the absence of lrp and that a small mRNA region immediately downstream of the initiation codon is required for potentially high-level expression. These results suggest that lysU is part of the leucine regulon and is both negatively controlled by Lrp and positively regulated by a potential translational enhancer sequence. This sequence is similar to that of the "downstream box" complementary to nucleotides 1469-1483 of 16S rRNA, which can be universally found in tRNA synthetase genes of E. coli.
The multidrug resistance (mdr1) gene product functions as an ATP channel.
Authors: Abraham E H , Prat A G , Gerweck L , Seneveratne T , Arceci R J , Kramer R , Guidotti G , Cantiello H F
Published: May 2006

The multidrug resistance (mdr1) gene product, P-glycoprotein, is responsible for the ATP-dependent extrusion of a variety of compounds, including chemotherapeutic drugs, from cells. The data presented here show that cells with increased levels of the P-glycoprotein release ATP to the medium in proportion to the concentration of the protein in their plasma membrane. Furthermore, measurements of whole-cell and single-channel currents with patch-clamp electrodes indicate that the P-glycoprotein serves as an ATP-conducting channel in the plasma membrane. These findings suggest an unusual role for the P-glycoprotein.
In vivo response of murine gamma delta T cells to a heat shock protein-derived peptide.
Authors: Fu Y X , Cranfill R , Vollmer M , Van Der Zee R , O'Brien R L , Born W
Published: May 2006

Recent results suggested that a large subset of heat shock protein HSP-60 reactive peripheral lymphoid gamma delta T cells preexists in normal adult mice, all members of which respond to a single segment of this common HSP. However, the experimental evidence supporting this idea involved in vitro peptide responses of gamma delta T-cell hybridomas generated from unprimed spleen cells. Here, we report an attempt to elicit a gamma delta T-cell response in vivo by stimulation of adult C57BL/10 mice with HSP-60 or an HSP-60-derived peptide fragment comprising amino acids 180-196 of mycobacterial HSP-60. Whereas no gamma delta T-cell response was detectable in mice injected with the intact protein, stimulation with the peptide altered the reactive gamma delta T-cell population in vivo. These changes were detected among hybridomas generated with cells restimulated in vitro and included a large increase in hybridizable gamma delta T cells, a nearly maximal increase in the relative frequency of HSP-60-reactive cells, and structural changes in expressed T-cell receptors of HSP-60-reactive cells. Interestingly, we failed to elicit a detectable alpha beta T-cell response to the particular peptide stimulatory for gamma delta T cells, although at least three other HSP-60 epitopes were recognized. Our data show that normal gamma delta T cells can respond in vivo to small peptide antigens. The gamma delta T-cell response to the HSP-60-derived peptide studied here is apparently independent of antigen-specific alpha beta T-cell reactivity.
Soluble CD4 enhances the efficacy of immunotoxins directed against gp41 of the human immunodeficiency virus.
Authors: Pincus S H , McClure J
Published: May 2006

Monoclonal antibodies specific for the gp120 or gp41 portions of the human immunodeficiency virus (HIV) envelope protein gp160 were conjugated to ricin A chain, and their immunotoxic activities against HIV-infected cells were evaluated in the presence or absence of soluble CD4 (sCD4). Immunotoxin activity was measured in vitro as cytotoxicity and inhibition of secretion of infectious HIV. The efficacy of anti-gp41 immunotoxins was enhanced at least 30-fold in the presence of sCD4. This effect was specific for HIV-infected cells, but not for uninfected cells, and was seen at concentrations of sCD4 as low as 0.1 micrograms/ml. Anti-gp120 immunotoxins were marginally inhibited at higher concentrations of sCD4. Flow cytometry analyses showed that sCD4 increased the expression of gp41 on the surface of infected cells and increased internalization of gp120 and gp41. These data suggest that sCD4 alters the cellular trafficking of HIV envelope proteins. These findings also have important implications for the therapeutic use of anti-HIV immunotoxins and may be generalizable to other immunotoxins as well.
Autoxidation of lipids and antioxidation by alpha-tocopherol and ubiquinol in homogeneous solution and in aqueous dispersions of lipids: unrecognized consequences of lipid particle size as exemplified by oxidation of human low density lipoprotein.
Authors: Ingold K U , Bowry V W , Stocker R , Walling C
Published: May 2006

Recent studies on the initial stages in oxidation of low density lipoprotein (LDL) have revealed certain previously unrecognized similarities to emulsion polymerization and some quite unexpected features including the following: (i) ascorbate is an extremely effective antioxidant for LDL containing alpha-tocopherol (alpha-TOH); (ii) in the presence of alpha-TOH and in the absence of both ascorbate and ubiquinol 10 (Q10H2), oxidation of LDL occurs via a free radical chain; (iii) Q10H2 is a much better antioxidant for LDL than alpha-TOH, although the reverse is true in homogeneous systems. We show here that these problems can be solved on the basis of three simple hypothesis, each of which is based on known chemistry: (i) alpha-TOH in LDL can be regenerated from its radical, alpha-TO., by ascorbate; (ii) in the absence of ascorbate and Q10H2, the alpha-TOH in LDL acts as a chain-transfer agent rather than as a radical trap; (iii) Q10H2 is a much more effective chain-breaking antioxidant than alpha-TOH in LDL because the semiquinone radical Q10H. exports its radical character from the LDL into the aqueous phase. Our conclusions imply that the search for better antiatherosclerotic drugs might profitably focus on antioxidants capable of exporting radicals from LDL particles or otherwise increasing the traffic of radicals between particles.
Alteration of Caenorhabditis elegans gene expression by targeted transformation.
Authors: Broverman S , MacMorris M , Blumenthal T
Published: May 2006

We have produced strains carrying a synthetic fusion of parts of two vitellogenin genes, vit-2 and vit-6, integrated into the Caenorhabditis elegans genome. In most of the 63 transformant strains, the plasmid sequences are integrated at random locations in the genome. However, in two strains the transgene integrated by homologous recombination into the endogenous vit-2 gene. In both cases the reciprocal exchange between the chromosome and the injected circular plasmid containing a promoter deletion led to switching of the plasmid-borne promoter and the endogenous promoter, with a reduction in vit-2 expression. Thus in nematodes, transforming DNA can integrate by homologous recombination to result in partial inactivation of the chromosomal locus. The simplicity of the event and its reasonably high frequency suggest that gene targeting by homologous recombination should be considered as a method for directed inactivation of C. elegans genes.
A complementarity-determining region synthetic peptide acts as a miniantibody and neutralizes human immunodeficiency virus type 1 in vitro.
Authors: Levi M , Sällberg M , Rudén U , Herlyn D , Maruyama H , Wigzell H , Marks J , Wahren B
Published: May 2006

A complementarity-determining region (CDR) of the mouse monoclonal antibody (mAb) F58 was constructed with specificity to a neutralization-inducing region of human immunodeficiency virus type 1 (HIV-1). The mAb has its major reactivity to the amino acid sequence I--GPGRA in the V3 viral envelope region. All CDRs including several framework amino acids were synthesized from the sequence deduced by cloning and sequencing mAb F58 heavy- and light-chain variable domains. Peptides derived from the third heavy-chain domain (CDR-H3) alone or in combination with the other CDR sequences competed with F58 mAb for the V3 region. The CDR-H3 peptide was chemically modified by cyclization and then inhibited HIV-1 replication as well as syncytium formation by infected cells. Both the homologous IIIB viral strain to which the F58 mAb was induced and the heterologous SF2 strain were inhibited. This synthetic peptide had unexpectedly potent antiviral activity and may be a potential tool for treatment of HIV-infected persons.
The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways.
Authors: Beck C A , Weigel N L , Moyer M L , Nordeen S K , Edwards D P
Published: May 2006

The protein kinase A stimulator cAMP can potentiate the ability of progestins to induce the transactivation function of the human progesterone receptor (hPR). We questioned in the present study whether cAMP could functionally cooperate with the progestin antagonist RU486. In T47D human breast cancer cells, RU486 behaves as a pure antagonist with respect to induction of the progesterone-responsive mouse mammary tumor virus chloramphenicol acetyltransferase (MMTV-CAT) reporter gene. It fails to stimulate MMTV-CAT expression and completely inhibits induction by the synthetic progestin R5020. However, when RU486 is combined with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), MMTV-CAT is induced to levels approaching that stimulated by R5020 alone. Also, RU486 in the presence of 8-Br-cAMP is only partially effective in antagonizing R5020 action. The agonist activity exhibited under these conditions appears to be due to RU486 acting through hPR as evidenced by the fact that 8-Br-cAMP alone has no effect on MMTV-CAT, whereas induction by the combination of 8-Br-cAMP and RU486 is dose responsive to RU486 in a saturable manner and can be inhibited by the type I antiprogestin (prevents hPR-DNA binding) ZK98299, which does not exhibit positive functional cooperation with cAMP. Acquisition of agonist activity in the presence of 8-Br-cAMP also extends to the type II antiprogestin (permits hPR-DNA binding) ZK112993. Since RU486 is also a type II antagonist, these results suggest that detection of functional synergism between cAMP and antiprogestins may require binding of the hPR-antagonist complex to DNA. We propose that cross-talk between second messenger and steroid receptor signal transduction pathways may be one mechanism for resistance to steroid antagonists that frequently develops in breast cancer.
The retroviral oncogene qin belongs to the transcription factor family that includes the homeotic gene fork head.
Authors: Li J , Vogt P K
Published: May 2006

Avian sarcoma virus 31 contains an oncogene that we have named qin. qin codes for a nuclear protein, Qin, that is a member of the HNF-3/fork head family of transcriptional regulators. Within this family Qin is particularly closely related to rat brain factor 1 (BF-1), a telencephalon-specific gene presumed to play an important role in the development of the mammalian brain.
Transforming growth factor beta enhances integrin expression and type IV collagenase secretion in human monocytes.
Authors: Wahl S M , Allen J B , Weeks B S , Wong H L , Klotman P E
Published: May 2006

Transforming growth factor beta (TGF-beta), secreted within an inflammatory site or injected locally, induces leukocyte margination, chemotaxis, and accumulation. In addition to its potent direct chemotactic activity, TGF-beta may promote this leukocyte response by influencing cell surface integrin expression. At picomolar concentrations, TGF-beta increases steady-state mRNA levels for both the alpha 5 and the beta 1 chain of the fibronectin receptor in human blood monocytes. This increase in gene expression is reflected by selectively enhanced expression of alpha 5 (CDw49e), beta 1 (CDw29), and also alpha 3 (CDw49c) adhesion molecules on the cell surface. Functionally, TGF-beta promotes, in a dose- and time-dependent fashion, monocyte adhesion to type IV collagen, laminin, and fibronectin. Potentially facilitating the movement of monocytes through the extracellular matrix, TGF-beta triggers transcriptional and posttranscriptional regulation of both the 92-kDa and the 72-kDa gelatinase/type IV collagenase. Thus, TGF-beta may play a pivotal role in the early phases of inflammation and repair through its ability to mediate monocyte adhesion, chemotaxis, and enzymatic digestion of extracellular matrix, whereas in chronic lesions, excess TGF-beta may contribute to persistent leukocyte accumulation.
Airway epithelial cells are the site of expression of a mammalian antimicrobial peptide gene.
Authors: Diamond G , Jones D E , Bevins C L
Published: May 2006

We previously reported the isolation and characterization of a broad-spectrum antimicrobial peptide from the bovine tracheal mucosa, which we called tracheal antimicrobial peptide (TAP). We now show the TAP gene is expressed throughout the adult conducting airway, from nasal to bronchiolar tissue, but not in tissues other than airway mucosa, as determined by Northern blot analysis. In situ hybridization of airway sections localizes TAP mRNA to columnar cells of the pseudostratified epithelium. We report the structural organization of the TAP gene and show that TAP is a member of a large family of related sequences with high nucleotide identity in the 5' exon. The data support the hypothesis that antimicrobial peptides contribute to host defense of the respiratory tract.
Nuclear extracts of chicken embryos promote an active demethylation of DNA by excision repair of 5-methyldeoxycytidine.
Authors: Jost J P
Published: May 2006

Here I show that nuclear extracts of chicken embryos can promote the active demethylation of DNA. The evidence shows that in hemimethylated DNA (i.e., methylated on one strand only) demethylation of 5mCpG occurs through nucleotide excision repair. The first step of demethylation is the formation of specific nicks 5' from 5-methyldeoxycytidine. Nicks are also observed in vitro on symmetrically methylated CpGs (i.e., methylated on both strands) but they result in breakage of the oligonucleotide with no repair. No specific nicks are observed on the nonmethylated CpG. Nicks are strictly 5mCpG specific and do not occur on 5mCpC, 5mCpT, 5mCpA, or 6mApT. The effect of nonspecific nuclease(s) has been ruled out. The nicking of mCpG takes place in the presence of 20 mM EDTA irrespective of the nature of the sequence surrounding the 5mCpG. No methylcytosine glycosylase activity could be detected. The repair is aphidicolin and N-ethylmaleimide resistant, suggesting a repair action by DNA polymerase beta. In extracts of chicken embryos, the excision repair of mCpG is highest between the 6th and the 12th day of development, whereas it is barely detectable in nuclear extracts from different organs of adults. The possible implications of 5mCpG endonuclease activity in active demethylation of DNA during differentiation is discussed.
Transfer RNAs with complementary anticodons: could they reflect early evolution of discriminative genetic code adaptors?
Authors: Rodin S , Ohno S , Rodin A
Published: May 2006

In accordance with the hypercycle theory of M. Eigen and P. Schuster [(1979) Hypercycle: A Principle of Natural Self-Organization (Springer, New York)], the ancestors of modern tRNAs appear to have emerged via the shortest possible way, both complementary strands of a short symmetrical double helix serving as pre-tRNAs with complementary anticodons. This conclusion is based upon results of comparative sequence analysis of the 17-base-long anticodon loop and stem of tRNAs totaling 896 and especially of 22 pairs of consensus tRNAs with complementary or quasi-complementary anticodons. With regard to the anticodon loop and stem of pairs of consensus tRNAs, complementary distances were considerably less than direct distances--i.e., antiparallel pairing invariably yielded fewer mismatches than direct pairing. Furthermore, the smallest complementary distance was detected when two antiparallel sequences formed irregular G-U bonds in their anticodon triplets. The above implies that pre-tRNAs in peribiotic times were long hairpin structures having 73 bases or more, the middle base of an anticodon being the center of symmetry. Accordingly, each pair of pre-tRNAs with complementary anticodons should have been almost identical with each other except for their three central bases. The above situation appears to have dictated the early establishment of direct links between anticodons and the type of amino acids with which tRNAs are to be charged. This direct link is still maintained between modern aminoacyl-tRNA synthetases and anticodons. Replication of the double helices concertedly generated new codons for the same pair of amino acids. Thus, occurrence of synonymous as well as certain "palindromic" features of the genetic code table might have been determined by this mechanism.
Preliminary results from the third flight of the Millimeter Anisotropy Experiment (MAX).
Authors: Devlin M , Alsop D , Clapp A , Cottingham D , Fischer M , Gundersen J , Holmes W , Lange A , Lubin P , Meinhold P , Richards P , Smoot G
Published: May 2006

Preliminary results from the June 1991 flight of MAX are presented. Simultaneous observations were made in bands centered at 6, 9, and 12 cm-1 with a bolometric receiver operating at 300 mK. The experimental sensitivities are the highest reported at angular scales of 0.3 degrees to 1.0 degrees. Interstellar dust is observed to have an emissivity [symbol, see text] nu 1.4+/-0.3 and to correlate with the Infrared Astronomical Satellite (IRAS) 100- map. After removal of emission from interstellar dust, 1.3 hr of integration on a 6 degrees scan yields an upper limit of temperature difference Delta T/T < 2.6 x 10(-5) at a Gaussian autocorrelation function centered at 0.5 degrees. The experiment and data analysis are described.
Inflation.
Authors: Guth A H
Published: May 2006

A short review of inflation is given, starting with a description of the underlying mechanism and the scenario of events. Four variations--old inflation, new inflation, chaotic inflation, and extended inflation--are discussed. It is claimed that the inflationary model provides a plausible explanation for (i) the large number of particles in the universe, (ii) the Hubble expansion, (iii) the large-scale uniformity of the universe, (iv) the nearness of the universe to a critical density, (v) the absence of magnetic monopoles, and (vi) the scale-invariant spectrum of microwave background fluctuations observed by COBE (Cosmic Background Explorer). Finally, it is argued that the plausibility of inflation is enhanced by the fact that inflation is eternal.