bluuebunny/crossref_metadata_2025 huggingface dataset:
The loss of female sex pheromone after mating in the corn earworm moth Helicoverpa zea: identification of a male pheromonostatic peptide.
Authors: Kingan T G , Bodnar W M , Raina A K , Shabanowitz J , Hunt D F
Published: May 2006

Female moths often become depleted of sex pheromone after mating as the various components of virgin behavior are switched off. In examining a potential male contribution to these events in the corn earworm moth Helicoverpa zea, we have characterized a basic polypeptide from the tissues producing (accessory glands) and storing (duplex) the seminal fluids. The peptide evokes the depletion of sex pheromone when injected into virgin females. This pheromonostatic peptide (PSP) is 57 amino acids long and contains a single disulfide bridge. It is blocked at the N terminus with pyroglutamate and at the C terminus by amidation. As little as 23 ng of peptide evokes the near-complete depletion of pheromone in decapitated (neck-ligated) females that had been injected with pheromone biosynthesis-activating neuropeptide. Activity is approximately 15-fold less in intact virgins, showing that the head limits the expression of activity in these injected females. Females mated to surgically impaired males, capable of producing a spermatophore but not transferring spermatozoa or seminal fluids, are depleted of pheromone by injected peptide. Females whose abdominal nerve cords have been severed are not depleted of pheromone after mating. Thus, neural signals either descending or ascending via the nerve cord are required for the depletion of pheromone after mating. PSP, from the seminal fluids, may participate in this process by direct or indirect action on the glandular tissue; if so, it represents an unusual mechanism in insects for the regulation by seminal fluids of postmating reproductive behavior.
Neonatal lethality associated with respiratory distress in mice lacking cytochrome P450 1A2.
Authors: Pineau T , Fernandez-Salguero P , Lee S S , McPhail T , Ward J M , Gonzalez F J
Published: May 2006

Cytochrome P450 1A2 (CYP1A2) is a constitutively expressed hepatic enzyme that is highly conserved among mammals. This protein is primarily involved in oxidative metabolism of xenobiotics and is capable of metabolically activating numerous procarcinogens including aflatoxin B1, arylamines, heterocyclic amine food mutagens, and polycylic aromatic hydrocarbons. Expression of CYP1A2 is induced after exposure to certain aromatic hydrocarbons (i.e., 2,3,7,8-tetrachlorodibenzo-p-dioxin). Direct evidence for a role of CYP1A2 in any physiological or developmental pathway has not been documented. We now demonstrate that mice homozygous for a targeted mutation in the Cyp1a-2 gene are nonviable. Lethality occurs shortly after birth with symptoms of severe respiratory distress. Mutant neonates display impaired respiratory function associated with histological signs of lung immaturity, lack of air in alveoli at birth, and changes in expression of surfactant apoprotein in alveolar type II cells. The penetrance of the phenotype is not complete (19 mutants survived to adulthood out of 599 mice). Surviving animals, although lacking expression of CYP1A2, appear to be normal and are able to reproduce. These findings establish that CYP1A2 is critical for neonatal survival by influencing the physiology of respiration in neonates, thus offering etiological insights for neonatal respiratory distress syndrome.
Types of Listeria monocytogenes predicted by the positions of EcoRI cleavage sites relative to ribosomal RNA sequences.
Authors: Hubner R J , Cole E M , Bruce J L , McDowell C I , Webster J A
Published: May 2006

By using taxonomic characters derived from EcoRI restriction endonuclease digestion of genomic DNA and hybridization with a labeled rRNA operon from Escherichia coli, a polymorphic structure of Listeria monocytogenes, characterized by fragments with different frequencies of occurrence, was observed. This structure was expanded by creating predicted patterns through a recursive process of observation, expectation, prediction, and assessment of completeness. This process was applied, in turn, to normalized strain patterns, fragment bands, and positions of EcoRI recognition sites relative to rRNA regions. Analysis of 1346 strains provided observed patterns, fragment sizes, and their frequencies of occurrence in the patterns. Fragment size statistics led to the creation of unobserved combinations of bands, predicted pattern types. The observed fragment bands revealed positions of EcoRI sites relative to rRNA sequences. Each EcoRI site had a frequency of occurrence, and unobserved fragment sizes were postulated on the basis of knowing the restriction site locations. The result of the recursion process applied to the components of the strain data was an extended classification with observed and predicted members.
Emergence of a new epidemic/epizootic Venezuelan equine encephalitis virus in South America.
Authors: Rico-Hesse R , Weaver S C , de Siger J , Medina G , Salas R A
Published: May 2006

One of the most important questions in arbovirology concerns the origin of epidemic Venezuelan equine encephalitis (VEE) viruses; these viruses caused periodic, extensive epidemics/epizootics in the Americas from 1938-1973 (reaching the United States in 1971) but had recently been presumed extinct. We have documented the 1992 emergence of a new epidemic/epizootic VEE virus in Venezuela. Phylogenetic analysis of strains isolated during two outbreaks indicated that the new epidemic/epizootic virus(es) evolved recently from an enzootic VEE virus in northern South America. These results suggest continued emergence of epizootic VEE viruses; surveillance of enzootic viruses and routine vaccination of equines should therefore be resumed.
Structural variety of arginine-rich RNA-binding peptides.
Authors: Tan R , Frankel A D
Published: May 2006

Arginine-rich domains are used by a variety of RNA-binding proteins to recognize specific RNA hairpins. It has been shown previously that a 17-aa arginine-rich peptide from the human immunodeficiency virus Rev protein binds specifically to its RNA site when the peptide is in an alpha-helical conformation. Here we show that related peptides from splicing factors, viral coat proteins, and bacteriophage antiterminators (the N proteins) also have propensities to form alpha-helices and that the N peptides require helical conformations to bind to their cognate RNAs. In contrast, introducing proline mutations into the arginine-rich domain of the human immunodeficiency virus Tat protein abolishes its potential to form an alpha-helix but does not affect RNA-binding affinity in vitro or in vivo. Based on results from several peptide-RNA model systems, we suggest that helical peptides may be used to recognize RNA structures having particularly wide major grooves, such as those found near loops or large bulges, and that nonhelical or extended peptides may be used to recognize less accessible grooves.
Age-related learning deficits in transgenic mice expressing the 751-amino acid isoform of human beta-amyloid precursor protein.
Authors: Moran P M , Higgins L S , Cordell B , Moser P C
Published: May 2006

The beta-amyloid precursor protein (beta-APP), from which the beta-A4 peptide is derived, is considered to be central to the pathogenesis of Alzheimer disease (AD). Transgenic mice expressing the 751-amino acid isoform of human beta-APP (beta-APP751) have been shown to develop early AD-like histopathology with diffuse deposits of beta-A4 and aberrant tau protein expression in the brain, particularly in the hippocampus, cortex, and amygdala. We now report that beta-APP751 transgenic mice exhibit age-dependent deficits in spatial learning in a water-maze task and in spontaneous alternation in a Y maze. These deficits were mild or absent in 6-month-old transgenic mice but were severe in 12-month-old transgenic mice compared to age-matched wild-type control mice. No other behavioral abnormalities were observed. These mice therefore model the progressive learning and memory impairment that is a cardinal feature of AD. These results provide evidence for a relationship between abnormal expression of beta-APP and cognitive impairments.
Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation.
Authors: Colas J F , Launay J M , Kellermann O , Rosay P , Maroteaux L
Published: May 2006

Serotonin, first described as a neurotransmitter in invertebrates, has been investigated mostly for its functions in the mature central nervous system of higher vertebrates. Serotonin receptor diversity has been described in the mammalian brain and in insects. We report the isolation of a cDNA coding for a Drosophila melanogaster serotonin receptor that displays a sequence, a gene organization, and pharmacological properties typical of the mammalian 5-HT2 serotonin receptor subtype. Its mRNA can be detected in the adult fly; moreover, a high level of expression occurs at 3 hr of Drosophila embryogenesis. This early embryonic expression is surprisingly organized in a seven-stripe pattern that appears at the cellular blastoderm stage. In addition, this pattern is in phase with that of the even-parasegment-expressed pair-rule gene fushi-tarazu and is similarly modified by mutations affecting segmentation genes. Simultaneously with this pair-rule expression, the complete machinery of serotonin synthesis is present and leads to a peak of ligand concomitant with a peak of 5-HT2-specific receptor sites in blastoderm embryos.
Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation.
Authors: Missero C , Calautti E , Eckner R , Chin J , Tsai L H , Livingston D M , Dotto G P
Published: May 2006

The mechanism of cell cycle withdrawal during terminal differentiation is poorly understood. We report here that the cyclin-dependent kinase (CDK) inhibitor p21Cip1/WAF1 is induced at early times of both keratinocyte and myoblast differentiation. p21Cip1/WAF1 induction is accompanied by a drastic inhibition of total Cdk2, as well as p21Cip1/WAF1-associated CDK kinase activities. p21Cip1/WAF1 has been implicated in p53-mediated G1 arrest and apoptosis. In keratinocyte differentiation, Cip1/WAF1 induction is observed even in cells derived from p53-null mice. Similarly, keratinocyte differentiation is associated with induction of Cip1/WAF1 promoter activity in both wild-type and p53-negative keratinocytes. Induction of the Cip1/WAF1 promoter upon differentiation is abolished by expression of an adenovirus E1A oncoprotein (d1922/947), which is unable to bind p105-Rb, p107, or cyclin A but which still binds the nuclear phosphoprotein p300. Overexpression of p300 can suppress the E1A effect, independent of its direct binding to E1A. Thus, terminal differentiation-induced growth arrest in both keratinocyte and myoblast systems is associated with induction of Cip1/WAF1 expression. During keratinocyte differentiation, Cip1/WAF1 induction does not require p53 but depends on the transcriptional modulator p300.
Priming of tumor-specific T cells in the draining lymph nodes after immunization with interleukin 2-secreting tumor cells: three consecutive stages may be required for successful tumor vaccination.
Authors: Maass G , Schmidt W , Berger M , Schilcher F , Koszik F , Schneeberger A , Stingl G , Birnstiel M L , Schweighoffer T
Published: May 2006

Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells, (ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction.
Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism.
Authors: Datto M B , Li Y , Panus J F , Howe D J , Xiong Y , Wang X F
Published: May 2006

The transforming growth factor beta s (TGF-beta s) are a group of multifunctional growth factors which inhibit cell cycle progression in many cell types. The TGF-beta-induced cell cycle arrest has been partially attributed to the regulatory effects of TGF-beta on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of small proteins, p21 (WAF1, Cip1), p27Kip1, p16, and p15INK4B, that physically associate with cyclins, cyclin-dependent kinases, or cyclin-Cdk complexes. p21 has been previously shown to be transcriptionally induced by DNA damage through p53 as a mediator. We demonstrate that TGF-beta also causes a rapid transcriptional induction of p21, suggesting that p21 can respond to both intracellular and extracellular signals for cell cycle arrest. In contrast to DNA damage, however, induction of p21 by TGF-beta is not dependent on wild-type p53. The cell line studied in these experiments, HaCaT, contains two mutant alleles of p53, which are unable to activate transcription from the p21 promoter when overexpressed. In addition, TGF-beta and p53 act through distinct elements in the p21 promoter. Taken together, these findings suggest that TGF-beta can induce p21 through a p53-independent pathway. Previous findings have implicated p27Kip1 and p15INK2B as effectors mediating the TGF-beta growth inhibitory effect. These results demonstrate that a single extracellular antiproliferative signal, TGF-beta, can act through multiple signaling pathways to elicit a growth arrest response.
The N-terminal region (A/B) of rat thyroid hormone receptors alpha 1, beta 1, but not beta 2 contains a strong thyroid hormone-dependent transactivation function.
Authors: Tomura H , Lazar J , Phyillaier M , Nikodem V M
Published: May 2006

In this study we have investigated the role of the N-terminal region of thyroid hormone receptors (TRs) in thyroid hormone (TH)-dependent transactivation of a thymidine kinase promoter containing TH response elements composed either of a direct repeat or an inverted palindrome. Comparison of rat TR beta 1 with TR beta 2 provides an excellent model since they share identical sequences except for their N termini. Our results show that TR beta 2 is an inefficient TH-dependent transcriptional activator. The degree of transactivation corresponds to that observed for the mutant TR delta N beta 1/2, which contains only those sequences common to TR beta 1 and TR beta 2. Thus, TH-dependent activation appears to be associated with two separate domains. The more important region, however, is embedded in the N-terminal domain. Furthermore, the transactivating property of TR alpha 1 was also localized to the N-terminal domain between amino acids 19 and 30. Using a coimmunoprecipitation assay, we show that the differential interaction of the N terminus of TR beta 1 and TR beta 2 with transcription factor IIB correlates with the TR beta 1 activation function. Hence, our results underscore the importance of the N-terminal region of TRs in TH-dependent transactivation and suggest that a transactivating signal is transmitted to the general transcriptional machinery via a direct interaction of the receptor N-terminal region with transcription factor IIB.
Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.
Authors: Okazaki Y , Hirose K , Hirotsune S , Okuizumi H , Sasaki N , Ohsumi T , Yoshiki A , Kusakabe M , Muramatsu M , Kawai J
Published: May 2006

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.
Deregulated expression of PAX5 in medulloblastoma.
Authors: Kozmik Z , Sure U , Rüedi D , Busslinger M , Aguzzi A
Published: May 2006

Medulloblatoma is a pediatric brain tumor originating in the human cerebellum. A collection of 23 medulloblastomas was analyzed for expression of the developmental control genes of the PAX and EN gene families by RNase protection and in situ hybridization. Of all nine PAX genes investigated, only PAX5 and PAX6 were consistently expressed in most medulloblastomas (70 and 78% of all cases, respectively), as were the genes EN1 (57%) and EN2 (78%). EN1, EN2, and PAX6 genes were also expressed in normal cerebellar tissue, and their expression in medulloblastoma is consistent with the hypothesis that this tumor originates in the external granular layer of the developing cerebellum. PAX5 transcripts were, however, not detected in the neonatal cerebellum, indicating that this gene is deregulated in medulloblastoma. In the desmoplastic variant of medulloblastoma, PAX5 expression was restricted to the reticulin-producing proliferating tumor areas containing undifferentiated cells; PAX5 was not expressed in the reticulin-free nonproliferating islands undergoing neuronal differentiation. These data suggest that deregulated expression of PAX5 correlates positively with cell proliferation and inversely with neuronal differentiation in desmoplastic medulloblastoma.
The carboxyl-terminal domain of the p53 protein regulates sequence-specific DNA binding through its nonspecific nucleic acid-binding activity.
Authors: Bayle J H , Elenbaas B , Levine A J
Published: May 2006

The murine p53 protein contains two nucleic acid-binding sites, a sequence-specific DNA-binding region localized between amino acid residues 102-290 and a nucleic acid-binding site without sequence specificity that has been localized to residues 364-390. Alternative splicing of mRNA generates two forms of this p53 protein. The normal, or majority, splice form (NSp53) retains its carboxyl-terminal sequence-nonspecific nucleic acid-binding site, which can negatively regulate the sequence-specific DNA-binding site. The alternative splice form of p53 (ASp53) replaces amino acid residues 364-390 with 17 different amino acids. This protein fails to bind nucleic acids nonspecifically and is constitutive for sequence-specific DNA binding. Thus, the binding of nucleic acids at the carboxyl terminus regulates sequence-specific DNA binding by p53. The implications of these findings for the activation of p53 transcriptional activity following DNA damage are discussed.
Insertional mutagenesis and marker rescue in a protozoan parasite: cloning of the uracil phosphoribosyltransferase locus from Toxoplasma gondii.
Authors: Donald R G , Roos D S
Published: May 2006

Nonhomologous integration vectors have been used to demonstrate the feasibility of insertional mutagenesis in haploid tachyzoites of the protozoan parasite Toxoplasma gondii. Mutant clones resistant to 5-fluorouracil were identified at a frequency of approximately 10(-6) (approximately 2 x 10(-5) of the stable transformants). Four independent mutants were isolated, all of which were shown to lack uracil phosphoribosyl-transferase (UPRT) activity and harbor transgenes integrated at closely linked loci, suggesting inactivation of the UPRT-encoding gene. Genomic DNA flanking the insertion point (along with the integrated vector) was readily recovered by bacterial transformation with restriction-digested, self-ligated total genomic DNA. Screening of genomic libraries with the recovered fragment identified sequences exhibiting high homology to known UPRT-encoding genes from other species, and cDNA clones were isolated that contain a single open reading frame predicted to encode the 244-amino acid enzyme. Homologous recombination vectors were exploited to create genetic knock-outs at the UPRT locus, which are deficient in enzyme activity but can be complemented by transient transformation with wild-type sequences--formally confirming identification of the functional UPRT gene. Mapping of transgene insertion points indicates that multiple independent mutants arose from integration at distinct sites within the UPRT gene, suggesting that nonhomologous integration is sufficiently random to permit tagging of the entire parasite genome in a single transformation.
Evolutionary origin of Plasmodium and other Apicomplexa based on rRNA genes.
Authors: Escalante A A , Ayala F J
Published: May 2006

We have explored the evolutionary history of the Apicomplexa and two related protistan phyla, Dinozoa and Ciliophora, by comparing the nucleotide sequences of small subunit ribosomal RNA genes. We conclude that the Plasmodium lineage, to which the malarial parasites belong, diverged from other apicomplexan lineages (piroplasmids and coccidians) several hundred million years ago, perhaps even before the Cambrian. The Plasmodium radiation, which gave rise to several species parasitic to humans, occurred approximately 129 million years ago; Plasmodium parasitism of humans has independently arisen several times. The origin of apicomplexans (Plasmodium), dinoflagellates, and ciliates may be > 1 billion years old, perhaps older than the three multicellular kingdoms of animals, plants, and fungi. Digenetic parasitism independently evolved several times in the Apicomplexa.
T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.
Authors: Gold R , Giegerich G , Hartung H P , Toyka K V
Published: May 2006

Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE.
Meso1, a basic-helix-loop-helix protein involved in mammalian presomitic mesoderm development.
Authors: Blanar M A , Crossley P H , Peters K G , Steingrímsson E , Copeland N G , Jenkins N A , Martin G R , Rutter W J
Published: May 2006

To identify genes involved in the regulation of early mammalian development, we have developed a dominant-negative mutant basic-helix-loop-helix (bHLH) protein probe for interaction cloning and have isolated a member of the bHLH family of transcription factors, Meso1. Meso1-E2A heterodimers are capable of binding to oligonucleotide probes that contain a bHLH DNA recognition motif. In mouse embryos, Meso1 is expressed prior to MyoD1 family members. Meso1 expression is first detected at the neural plate stage of development in the paraxial mesoderm of the head and in presomitic mesodermal cells prior to their condensation into somites. Our findings suggest that Meso1 may be a key regulatory gene involved in the early events of vertebrate mesoderm differentiation.
Identification of a nonneuronal isoform of synaptotagmin.
Authors: Hudson A W , Birnbaum M J
Published: May 2006

Synaptotagmins, which have been found exclusively in neuroendocrine or exocrine tissues, have been implicated in the regulation of secretory vesicle fusion with the plasma membrane. The present paper describes a synaptotagmin isoform (synaptotagmin-5) which exhibits 49% amino acid identity to synaptotagmin-1 and -2. Synaptotagmin-5 mRNA is expressed in rat kidney, adipose tissue, lung, and heart, as well as at higher levels in brain and PC12 cells. Antiserum specific for the synaptotagmin-5 isoform recognizes a protein of about 50 kDa which is about 6-fold more abundant in brain synaptic vesicles than in whole brain membranes.
Correlation of individual papilloma latency time with DNA adducts, 8-hydroxy-2'-deoxyguanosine, and the rate of DNA synthesis in the epidermis of mice treated with 7,12-dimethylbenz[alpha]anthracene.
Authors: Fischer W H , Lutz W K
Published: May 2006

The question was addressed whether the risk of cancer of an individual in a heterogeneous population can be predicted on the basis of measurable biochemical and biological variables postulated to be associated with the process of chemical carcinogenesis. Using the skin tumor model with outbred male NMRI mice, the latency time for the appearance of a papilloma was used as an indicator of the individual cancer risk. Starting at 8 weeks of age, a group of 29 mice was treated twice weekly with 20 nmol of 7,12-dimethylbenz[alpha]anthracene (DMBA) applied to back skin. The individual papilloma latency time ranged from 13.5 to 25 weeks of treatment. Two weeks after the appearance of the first papilloma in each mouse, an osmotic minipump delivering 5-bromo-2'-deoxyuridine was s.c. implanted and the mouse was killed 24 hr later. Levels of DMBA-DNA adducts, of 8-hydroxy-2'-deoxyguanosine, and various measures of the kinetics of cell division were determined in the epidermis of the treated skin area. The levels of 8-hydroxy-2'-deoxyguanosine and the fraction of cells in DNA replication (labeling index for the incorporation of 5-bromo-2'-deoxyuridine) were significantly higher in those mice that showed short latency times. On the other hand, the levels of DMBA-DNA adducts were lowest in animals with short latency times. The latter finding was rather unexpected but can be explained as a consequence of the inverse correlation seen for the labeling index: with each round of cell division, the adduct concentration is reduced to 50% because the new DNA strand is free of DMBA adducts until the next treatment. Under the conditions of this bioassay, therefore, oxygen radical-related genotoxicity and the rate of cell division, rather than levels of carcinogen-DNA adducts, were found to be of predictive value as indicators of an individual cancer risk.
Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.
Authors: Cetin Y , Kulaksiz H , Redecker P , Bargsten G , Adermann K , Grube D , Forssmann W G
Published: May 2006

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.
On the mechanism of skin wound "contraction": a granulation tissue "knockout" with a normal phenotype.
Authors: Gross J , Farinelli W , Sadow P , Anderson R , Bruns R
Published: May 2006

This report explores the mechanism of spontaneous closure of full-thickness skin wounds. The domestic pig, often used as a human analogue for skin wound repair studies, closes these wounds with kinetics similar to those in the guinea pig (mobile skin), even though the porcine dermis on the back is thick and nearly immobile. In the domestic pig, as in the guinea pig, daily full-thickness excisions of the central granulation tissue up to but not including the wound edges in both back and flank wounds do not alter the rate or completeness of wound closure or the final pattern of the scar. A purse-string mechanism of closure was precluded by showing that surgical interruption of wound edge continuity does not alter closure kinetics or wound shape. We conclude that "tightness" of skin is not a key factor nor is the central granulation tissue required for normal wound closure. These data imply that in vitro models such as contraction of isolated granulation tissue or of the cell-populated collagen lattice may not be relevant for understanding the cell biology of in vivo wound closure. Implications for the mechanism for wound closure are discussed.
The Ste locus, a component of the parasitic cry-Ste system of Drosophila melanogaster, encodes a protein that forms crystals in primary spermatocytes and mimics properties of the beta subunit of casein kinase 2.
Authors: Bozzetti M P , Massari S , Finelli P , Meggio F , Pinna L A , Boldyreff B , Issinger O G , Palumbo G , Ciriaco C , Bonaccorsi S
Published: May 2006

Males of Drosophila melanogaster lacking the Y chromosome-linked crystal locus show multiple meiotic alterations including chromosome disorganization and prominent crystal formation in primary spermatocytes. These alterations are due to the derepression of the X chromosome-linked Stellate sequences. To understand how the derepression of the Stellate elements gives rise to these abnormalities, we have expressed the protein encoded by the Stellate sequences in bacteria and produced an antibody against the fusion protein. Immunostaining of crystal- testes has clearly shown that the Stellate protein is a major component of the crystals. Moreover, in vitro experiments have shown that this protein can interact with the catalytic alpha subunit of casein kinase 2 enzyme, altering its activity.
Regulated assembly of tight junctions by protein kinase C.
Authors: Stuart R O , Nigam S K
Published: May 2006

We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.
Interleukin 4 suppresses c-kit ligand-induced expression of cytosolic phospholipase A2 and prostaglandin endoperoxide synthase 2 and their roles in separate pathways of eicosanoid synthesis in mouse bone marrow-derived mast cells.
Authors: Murakami M , Penrose J F , Urade Y , Austen K F , Arm J P
Published: May 2006

Mouse bone marrow-derived mast cells (BMMCs) developed with interleukin 3 (IL-3) can be stimulated by c-kit ligand (KL) and accessory cytokines over a period of hours for direct delayed prostaglandin (PG) generation or over a period of days to prime for augmented IgE-dependent PG and leukotriene (LT) production, as previously reported. We now report that IL-4 is counterregulatory for each of these distinct KL-dependent responses. BMMCs cultured for 4 days with KL + IL-3 or with KL + IL-10 produced 5- to 7-fold more PGD2 and approximately 2-fold more LTC4 in response to IgE-dependent activation than BMMCs maintained in IL-3 alone. IL-4 inhibited the priming for increased IgE-dependent PGD2 and LTC4 production to the level obtained by activation of BMMCs maintained in IL-3 alone with an IC50 of approximately 0.2 ng/ml. IL-4 inhibited the KL-induced increase in expression of cytosolic phospholipase A2 (cPLA2) but had no effect on the incremental expression of PG endoperoxide synthase 1 (PGHS-1) and hematopoietic PGD2 synthase or on the continued baseline expression of 5-lipoxygenase, 5-lipoxygenase activating protein, and LTC4 synthase. BMMCs stimulated by KL + IL-10 for 10 h exhibited a delayed phase of PGD2 generation, which was dependent on de novo induction of PGHS-2. IL-4 inhibited the induction of PGHS-2 expression and the accompanying cytokine-initiated delayed PGD2 generation with an IC50 of approximately 6 ng/ml. IL-4 had no effect on the expression of PGHS-2 and the production of PGD2 elicited by addition of IL-1 beta to the combination of KL + IL-10. IL-4 had no effect on the immediate phase of eicosanoid synthesis elicited by KL alone or by IgE and antigen in BMMCs maintained in IL-3. Thus, the counterregulatory action of IL-4 on eicosanoid generation is highly selective for the induced incremental expression of cPLA2 and the de novo expression of PGHS-2, thereby attenuating time-dependent cytokine-regulated responses to stimulation via Fc epsilon receptor I and stimulation via c-kit, respectively.
Homeostatic regulation of intestinal epithelia by intraepithelial gamma delta T cells.
Authors: Komano H , Fujiura Y , Kawaguchi M , Matsumoto S , Hashimoto Y , Obana S , Mombaerts P , Tonegawa S , Yamamoto H , Itohara S
Published: May 2006

Although T cells bearing gamma delta T-cell receptors have long been known to be present in the epithelial lining of many organs, their specificity and function remain elusive. In the present study, we examined the intestinal epithelia of T-cell-receptor mutant mice, which were deficient in either gamma delta T cells or alpha beta T cells, and of normal littermates. The absence of gamma delta T cells was associated with a reduction in epithelial cell turnover and a downregulation of the expression of major histocompatibility complex class II molecules. No such effects were observed in alpha beta T-cell-deficient mice. These findings indicate that intraepithelial gamma delta T cells regulate the generation and differentiation of intestinal epithelial cells.
Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia.
Authors: Carlsson L M , Jonsson J , Edlund T , Marklund S L
Published: May 2006

Extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is a secreted Cu- and Zn-containing tetrameric glycoprotein, the bulk of which is bound to heparan sulfate proteoglycans in the interstitium of tissues. To test the function of EC-SOD in vivo, mice carrying a targeted disruption of the EC-SOD gene were generated. The EC-SOD null mutant mice develop normally and remain healthy until at least 14 months of age. No compensatory induction of other SOD isoenzymes or other antioxidant enzymes was observed. When stressed by exposure to > 99% oxygen, the EC-SOD null mutant mice display a considerable reduction in survival time compared to wild-type mice and an earlier onset of severe lung edema. These findings suggest that while under normal physiological conditions other antioxidant systems may substitute for the loss of EC-SOD; when the animal is stressed these systems are unable to provide adequate protection.
Recognition by viral and cellular DNA polymerases of nucleosides bearing bases with nonstandard hydrogen bonding patterns.
Authors: Horlacher J , Hottiger M , Podust V N , Hübscher U , Benner S A
Published: May 2006

The ability of DNA polymerases (pols) to catalyze the template-directed synthesis of duplex oligonucleotides containing a nonstandard Watson-Crick base pair between a nucleotide bearing a 5-(2,4-diaminopyrimidine) heterocycle (d kappa) and a nucleotide bearing either deoxyxanthosine (dX) or N1-methyloxoformycin B (pi) has been investigated. The kappa-X and kappa-pi base pairs are jointed by a hydrogen bonding pattern different from and exclusive of those joining the AT and GC base pairs. Reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) incorporates dXTP into an oligonucleotide opposite d kappa in a template with good fidelity. With lower efficiency and fidelity, HIV-1 reverse transcriptase also incorporates d kappa TP opposite dX in the template. With d pi in the template, no incorporation of d kappa TP was observed with HIV reverse transcriptase. The Klenow fragment of DNA pol I from Escherichia coli does not incorporate d kappa TP opposite dX in a template but does incorporate dXTP opposite d kappa. Bovine DNA pols alpha, beta, and epsilon accept neither dXTP opposite d kappa nor d kappa TP opposite d pi. DNA pols alpha and epsilon (but not beta) incorporate d kappa TP opposite dX in a template but discontinue elongation after incorporating a single additional base. These results are discussed in light of the crystal structure for pol beta and general considerations of how polymerases must interact with an incoming base pair to faithfully copy genetic information.
A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.
Authors: Tabor S , Richardson C C
Published: May 2006

Bacteriophage T7 DNA polymerase efficiently incorporates a chain-terminating dideoxynucleotide into DNA, in contrast to the DNA polymerases from Escherichia coli and Thermus aquaticus. The molecular basis for this difference has been determined by constructing active site hybrids of these polymerases. A single hydroxyl group on the polypeptide chain is critical for selectivity. Replacing tyrosine-526 of T7 DNA polymerase with phenylalanine increases discrimination against the four dideoxynucleotides by > 2000-fold, while replacing the phenylalanine at the homologous position in E. coli DNA polymerase I (position 762) or T. aquaticus DNA polymerase (position 667) with tyrosine decreases discrimination against the four dideoxynucleotides 250- to 8000-fold. These mutations allow the engineering of new DNA polymerases with enhanced properties for use in DNA sequence analysis.
Cloning and sequencing of CATR1.3, a human gene associated with tumorigenic conversion.
Authors: Li D , Noyes I , Shuler C , Milo G E
Published: May 2006

The human squamous cell carcinoma cell line SCC83-01-82 (SCC) contains mutations in both the H-ras and p53 genes, but it exhibits a nontumorigenic phenotype in nude mice. This cell line can be converted into a cell line with a tumorigenic phenotype, SCC83-01-82CA (CA), by treatment with the mutagen methyl methanesulfonate (MMS). This indicates that additional genetic events leading to expression of a cooperating tumor susceptibility gene(s) may be required for tumorigenicity. To identify the cooperating gene(s), an expression cDNA library was made from tumorigenic Ca cells. The library DNA was transfected into nontumorigenic SCC cells and the transfected SCC cells were then injected into nude mice for the selection of a tumorigenic phenotype. Tumors developed in 3 of the 18 mice after injection. Several new cell lines were established from these transfected cell-induced tumors and designated as CATR cells. Tumor histology and karyotype analysis of these cells indicated that they were of human epithelial cell origin. All the CATR cells have the library vector sequence integrated in their genome. Cell line CATR1 expressed a single message from the integrated library representing a 1.3-kb cDNA insert that was absent from untransfected SCC cells or MMS-converted CA cells. This 1.3-kb cDNA insert was cloned by PCR amplification of reverse-transcribed CATR1 total RNA and was designated CATR1.3. The nucleotide sequence of CATR1.3 encodes a peptide of 79 amino acids, has a long 3' untranslated region, and represents an unknown gene product that was associated with the tumorigenic conversion due to the transfected expression library.
Different cellular backgrounds confer a marked advantage to either mutant or wild-type mitochondrial genomes.
Authors: Dunbar D R , Moonie P A , Jacobs H T , Holt I J
Published: May 2006

After the introduction of mitochondria with a mixture of mutant and wild-type mitochondrial DNA (mtDNA) into a human rho degree cell line (143B.206), Yoneda et al. [Yoneda, M., Chomyn, A., Martinuzzi, A., Hurko, O. & Attardi, G. (1992) Proc. Natl. Acad. Sci. USA 89, 11164-11168] observed a shift in the proportion of the two mitochondrial genotypes in a number of cybrid clones. In every case where a shift was observed, there was an increase in the proportion of mutant mtDNA. By using the same cell line (143B.206 rho degree), we also generated cybrids that were either stable in their mitochondrial genotype or showed an increase in the proportion of mutant mtDNA. However, temporal analysis of the same mutant mtDNA type in another rho degree cell line revealed a quite distinct outcome. Those clones that showed a change shifted toward higher levels of wild-type rather than mutant mtDNA. These results indicate that the nuclear genetic background of the recipient (rho degree) cell can influence the segregation of mutant and wild-type mitochondrial genomes in cell cybrids.
Naturally processed peptides from two disease-resistance-associated HLA-DR13 alleles show related sequence motifs and the effects of the dimorphism at position 86 of the HLA-DR beta chain.
Authors: Davenport M P , Quinn C L , Chicz R M , Green B N , Willis A C , Lane W S , Bell J I , Hill A V
Published: May 2006

HLA-DR13 has been associated with resistance to two major infectious diseases of humans. To investigate the peptide binding specificity of two HLA-DR13 molecules and the effects of the Gly/Val dimorphism at position 86 of the HLA-DR beta chain on natural peptide ligands, these peptides were acid-eluted from immunoaffinity-purified HLA-DRB1*1301 and -DRB1*1302, molecules that differ only at this position. The eluted peptides were subjected to pool sequencing or individual peptide sequencing by tandem MS or Edman microsequencing. Sequences were obtained for 23 peptides from nine source proteins. Three pool sequences for each allele and the sequences of individual peptides were used to define binding motifs for each allele. Binding specificities varied only at the primary hydrophobic anchor residue, the differences being a preference for the aromatic amino acids Tyr and Phe in DRB1*1302 and a preference for Val in DRB1*1301. Synthetic analogues of the eluted peptides showed allele specificity in their binding to purified HLA-DR, and Ala-substituted peptides were used to identify the primary anchor residues for binding. The failure of some peptides eluted from DRB1*1302 (those that use aromatic amino acids as primary anchors) to bind to DRB1*1301 confirmed the different preferences for peptide anchor residues conferred by the Gly-->Val change at position 86. These data suggest a molecular basis for the differential associations of HLA-DRB1*1301 and DRB1*1302 with resistance to severe malaria and clearance of hepatitis B virus infection.
Capturing genes encoding membrane and secreted proteins important for mouse development.
Authors: Skarnes W C , Moss J E , Hurtley S M , Beddington R S
Published: May 2006

A strategy based on the gene trap was developed to prescreen mouse embryonic stem cells for insertional mutations in genes encoding secreted and membrane-spanning proteins. The "secretory trap" relies on capturing the N-terminal signal sequence of an endogenous gene to generate an active beta-galactosidase fusion protein. Insertions were found in a cadherin gene, an unc6-related laminin (netrin) gene, the sek receptor tyrosine kinase gene, and genes encoding two receptor-linked protein-tyrosine phosphatases, LAR and PTP kappa. Analysis of homozygous mice carrying insertions in LAR and PTP kappa showed that both genes were effectively disrupted, but neither was essential for normal embryonic development.