bluuebunny/crossref_metadata_2025 huggingface dataset:
Location of the cyclohexene ring of the chromophore of bacteriorhodopsin by neutron diffraction with selectively deuterated retinal
Authors: Seiff F. , Westerhausen J. , Wallat I. , Heyn M. P.
Published: May 2006

We report on the location of the cyclohexene ring of the retinylidene chromophore of bacteriorhodopsin projected onto the plane of the membrane. For this purpose, partially deuterated retinal was synthesized containing 11 deuterons at the following positions of the cyclohexene ring: one at C-2, two at C-4, three at C-16, three at C-17, and two at C-18. The partially deuterated retinal was incorporated biosynthetically during growth of the bacteria by using the mutant JW5, which is deficient in the synthesis of retinal. Undeuterated samples were prepared in the same way. Characterization by x-ray diffraction and absorption spectroscopy showed that these samples are identical to native purple membranes as judged by these criteria. A Fourier difference map was calculated from the differences in in-plane diffraction intensities between the deuterated and undeuterated dark-adapted membrane samples. Model calculations showed that the observed difference density had the amplitude expected for a label containing 11 deuterons. At 8.7 Å resolution, the map shows one major peak with the center of mass of the deuterated ring in the interior of the molecule between helices 3, 4, 5, and 6. Based on this result and on our previous work on the location of the middle of the polyene chain, we conclude that the COOH-terminal helix G, to which retinal is attached at lysine-216, is either helix 2 or helix 6.
Inherited polymorphism of the human T-cell antigen receptor detected by a monoclonal antibody.
Authors: Posnett D N , Wang C Y , Friedman S M
Published: May 2006

Three different murine monoclonal antibodies to the human clonotypic T-cell antigen receptor immunoprecipitate the alpha-beta chain heterodimer; induce comodulation of the clonotypic molecule with the T3 molecular complex; stain small populations of normal polyclonal T cells, suggesting that they react with variable or joining region determinants of the clonotypic receptor; and induce proliferation of resting T cells. While two of these antibodies detect the clonotypic receptor in all individuals studied, the third antibody (OT145), described herein, does not detect the T-cell antigen receptor on T cells of all individuals. By indirect immunofluorescence, three groups can be distinguished within a population of individuals (n = 138) by OT145. Individuals lacking T cells reactive with OT145 have a homozygous OT145-phenotype. T cells from such individuals fail to proliferate in the presence of OT145 in contrast to T cells from OT145+ individuals. Individuals with a relatively large percentage of OT145+ T cells, 4.5 +/- 1.54% (mean +/- 2 SEM) are homozygous OT145+, while those with an intermediate percentage, 2.04 +/- 0.9%, have a heterozygous phenotype. Family studies suggest autosomal codominant inheritance of the OT145 phenotype. The distribution of the three OT145-defined phenotypes varies considerably in populations of different ethnic background. Taken together these data suggest that the polymorphism detected by OT145 may represent a variable or joining region allotypic system of the human T-cell antigen receptor. In addition, our results indicate that allelic exclusion governs the expression of the clonotypic receptor by human T cells.
Inhibition of the development of radiation-induced leukemia in mice by reduction of food intake.
Authors: Gross L , Dreyfuss Y
Published: May 2006

We have reported previously that the incidence of tumors induced in Sprague-Dawley rats by total-body gamma-ray irradiation can be considerably reduced by restriction of food intake [Gross, L. & Dreyfuss, Y. (1984) Proc. Natl. Acad. Sci. USA 81, 7596-7598]. In experiments reported here we investigated the influence of reduced food intake on the development of radiation-induced leukemia in C3H(f) mice. The incidence of spontaneous leukemia in mice of this strain does not exceed 0.5%, but it can be considerably increased by total-body x-irradiation. In our study, two groups of C3H(f) mice were submitted to fractionated total-body gamma-irradiation (150 rads, five times at weekly intervals; 1 rad = 0.01 gray). The first group received a full ad lib diet (4.5-5.4 g of Purina Rodent Lab Chow pellets per day, each). In this group 31 out of 58 females (53.4%) and 24 out of 50 males (48%) developed leukemia at an average age of 8 months. In the second group, consisting of sisters and brothers of the first group, and submitted to the same gamma-irradiation but receiving a restricted diet (2 g of Purina Lab Chow pellets each, followed by 3 g on alternate days), only 2 out of 55 females (3.6%), and 1 out of 36 males (2.8%), developed leukemia at an average age of 9 and 12 months, respectively. Leukemia in both groups was predominantly of the lymphatic or lymphoblastic form, the leukemic cells infiltrating most organs, particularly the thymus, mesenteric and peripheral lymph nodes, spleen, liver, kidneys, and bone marrow; in most instances the peripheral blood was also leukemic.
Inhibition of N-methyl-N-nitrosourea-induced mutagenicity and DNA methylation by ellagic acid.
Authors: Dixit R , Gold B
Published: May 2006

Ellagic acid, a naturally occurring plant phenol, inhibits the activity of the direct-acting mutagen N-methyl-N-nitrosourea (MeNU) in Salmonella typhimurium TA100. Ellagic acid at 0.10, 0.25, 0.50, and 1.00 mM inhibited the mutagenicity of MeNU (0.40 mM) by 3%, 13%, 45%, and 60%, respectively. Ellagic acid (3 mM) also inhibited the mutagenic activity of N,N-dimethylnitrosamine (25-200 mM) in the presence of pyrazole-induced rat liver fraction S-9. The effect of ellagic acid on DNA methylation was studied by incubating 0, 0.72, 1.32, 2.64, and 6.60 mM ellagic acid with DNA (0.9 mM nucleotide) and [3H]MeNU (0.66 mM). HPLC analysis of DNA hydrolysates showed that ellagic acid caused a dose-dependent 36-84% decrease in O6-methylguanine but only a 20% decrease in the 7-methylguanine adduct. Under conditions where methylation at the O6 position of guanine in double-stranded DNA was inhibited 65% by ellagic acid, no significant inhibition of either O6- or 7-methylguanine formation was detected in single-stranded DNA. Affinity-binding studies revealed that [3H]ellagic acid binds equally to double-stranded or single-stranded DNA but that poly(dA X dT) binds 1.5 times as much ellagic acid as does poly(dG X dC). The binding of ellagic acid to DNA is dependent on the concentration of both ellagic acid and DNA. The specific inhibition of O6-methylguanine formation only in double-stranded DNA and the relatively low inhibition of 7-methylguanine formation rule out the possibility that ellagic acid prevents DNA alkylation by scavenging the electrophilic intermediate generated in the hydrolysis of MeNU. The results suggest that ellagic acid inhibition of MeNU-induced mutagenicity is due to specific inhibition of methylation at the O6 position of guanine through an ellagic acid-duplex DNA affinity-binding mechanism.
Linking functional domains of the human insulin receptor with the bacterial aspartate receptor.
Authors: Ellis L , Morgan D O , Koshland D E , Clauser E , Moe G R , Bollag G , Roth R A , Rutter W J
Published: May 2006

A hybrid receptor has been constructed that is composed of the extracellular domain of the human insulin receptor fused to the transmembrane and cytoplasmic domains of the bacterial aspartate chemoreceptor. This hybrid protein can be expressed in rodent (CHO) cells and displays several functional features comparable to wild-type insulin receptor. It is localized to the cell surface, binds insulin with high affinity, forms oligomers, and is recognized by conformation-specific monoclonal antibodies. Although most of the expressed protein accumulates as a 180-kDa proreceptor, some processed 135-kDa receptor can be detected on the cell surface by covalent cross-linking. Expression of the hybrid receptor inhibits the insulin-activated uptake of 2-deoxyglucose by CHO cells. Thus, this hybrid is partially functional and can be processed; however, it is incapable of native transmembrane signaling. The results indicate that the intact domains of different types of receptors can retain some of the native features in a hybrid molecule but specific requirements will need to be satisfied for transmembrane signaling.
Radiofrequency hyperthermia with successive monitoring of its effects on tumors using NMR spectroscopy.
Authors: Naruse S , Higuchi T , Horikawa Y , Tanaka C , Nakamura K , Hirakawa K
Published: May 2006

Radiofrequency (rf) hyperthermia was generated on rat glioma inoculated s.c. in CD Fisher rats by applying the rf pulse using the surface coil in the NMR spectrometer, and the effect was monitored successively in the same spectrometer by measuring 31P NMR spectra and 1H NMR images. In the 31P NMR spectrum at the preirradiation stage, nucleoside triphosphate peaks and a phosphomonoester peak were high and a Pi peak was low. After a rf pulse at a power of 5 W was applied continuously for 60 min, the nucleoside triphosphate peaks decreased and the Pi peak increased immediately, resulting finally in a dominant Pi peak pattern within 30 min in all 10 cases examined. These spectral changes occurred much earlier than the histological changes and lasted for at least 7 days. By the 1H NMR imaging, the necrotic region was detected as a high-intensity lesion in spin echo and inversion recovery images 2 days after the irradiation. There were no changes either in the spectrum or in 1H NMR images in any of 8 cases after irradiation with a rf pulse of less than 3 W. Thus, we could generate rf hyperthermia with the NMR spectrometer and the effects were monitored sensitively with the same spectrometer. It can be concluded that the NMR device can be used not only for diagnosis but also as a therapeutic tool.
DNase I-hypersensitive sites surround the mouse acetylcholine receptor delta-subunit gene.
Authors: Crowder C M , Merlie J P
Published: May 2006

The mouse skeletal muscle acetylcholine receptor is regulated, at least in part, by transcriptional mechanisms. As a first step toward defining the cis- and trans-acting factors involved in acetylcholine receptor transcriptional regulation, we examined mouse muscle and fibroblast cells for the presence of DNase I-hypersensitive sites in the chromatin surrounding the delta-subunit gene. We detect six DNase I-hypersensitive sites in muscle DNA blots hybridized with a mouse delta-subunit cDNA. Two hypersensitive sites lie distantly 5' to the delta gene, one lies near the 5' end of the delta gene, one lies near its 3' end, and two hypersensitive sites lie distantly 3'. The pair of distantly 3' hypersensitive sites should by analogy to chicken and human genomes lie near the gamma-subunit gene. Only the distantly 5' pair of hypersensitive sites is present in fibroblasts; the remainder is muscle specific. Two hypersensitive sites, one at the 3' end of the delta gene and one distantly 3', appear only after terminal muscle differentiation and, therefore, are developmentally regulated, similarly to receptor subunit mRNAs. All four muscle-specific hypersensitive sites are excellent candidates for regulatory loci for the delta- and gamma-subunit genes of mouse acetylcholine receptor.
A Drosophila mutation that eliminates a calcium-dependent potassium current.
Authors: Elkins T , Ganetzky B , Wu C F
Published: May 2006

A mutation of Drosophila, slowpoke (slo), specifically abolishes a Ca2+-dependent K+ current, IC, from dorsal longitudinal flight muscles of adult flies. Other K+ currents remain normal, providing evidence that IC is mediated by a molecularly distinguishable set of channels. The pharmacological properties of IC are similar to those of Ca2+-dependent currents in some vertebrate cells. The muscle action potential was significantly lengthened in slo flies, indicating that IC plays the major role in its repolarization.
Functional analysis of regulatory elements in a plant embryo-specific gene.
Authors: Chen Z L , Schuler M A , Beachy R N
Published: May 2006

Previously we demonstrated the expression of a plant embryo-specific gene encoding the alpha' subunit of beta-conglycinin, a seed storage protein of soybean (Glycine max), in transgenic petunia plants. To examine the regulatory elements that control the expression of this embryo-specific gene (Gmg17.1), a series of deletion mutants was made that contain the alpha'-subunit gene flanked in the 5' direction from +14 nucleotides to -8.5 kilobases (kb) relative to the site of transcription initiation. Each of these deletion mutants was introduced into the genome of petunia cells with the help of Ti-plasmid-derived vectors. Petunia plants were regenerated from transformed cells and expression of the introduced soybean gene was examined. When the alpha'-subunit gene was flanked by 159 nucleotides upstream (Gmg17.1 delta-159), the gene was expressed at a low level in immature embryos. When the gene was flanked by 257 nucleotides upstream of the site of transcription initiation (Gmg17.1 delta-257), a high level of expression was obtained. An additional 8 kb of DNA sequence (which includes the sequence GTGGATAG at -560, which is identical to the core enhancer sequence of simian virus 40 and some animal genes) did not significantly increase the level of expression. The increase in expression level between the delta-159 and delta-257 mutants was at least 20-fold. Analysis of the nucleotides between delta-159 and delta-257 reveals four repeats of a 6-base-pair (G + C)-rich sequence (see formula in text). The deletion Gmg17.1 delta-159 contains a single AACCCA sequence. We suggest that the (G + C)-rich repeats play a critical role in determining the level of expression of the transgenic plants.
cAMP induction of prespore and prestalk gene expression in Dictyostelium is mediated by the cell-surface cAMP receptor.
Authors: Gomer R H , Armstrong D , Leichtling B H , Firtel R A
Published: May 2006

Extracellular adenosine 3',5'-cyclic monophosphate (cAMP) is required for cell-type-specific gene expression in developing Dictyostelium discoideum. We have developed a microassay for the expression of these genes, using antibodies directed against their protein products. To characterize the transduction mechanism, we have used in this assay cAMP analogues that preferentially activate either the cell-surface cAMP receptor or the internal cAMP-dependent protein kinase. N6-(aminohexyl) cAMP activates the Dictyostelium cAMP-dependent protein kinase but does not bind to the cell-surface cAMP receptor and does not cause cell-type-specific gene expression. 2'-Deoxy-cAMP does not activate the cAMP-dependent protein kinase but binds to the receptor and causes cell-type-specific gene expression. Cyclic AMP-induced accumulation of prestalk mRNA in shaking cultures still occurs in the presence of caffeine, which blocks the receptor-coupled activation of adenyl cyclase. This suggests that the extracellular cAMP induction of cell-type-specific gene expression in developing Dictyostelium cells is mediated by the cell-surface cAMP receptor and that activating adenyl cyclase by this receptor is not essential. Using the N6-(aminohexyl) cAMP to competitively inhibit phosphodiesterase, we show that 30 nM cAMP is sufficient to induce prestalk or prespore gene expression.
Characterization of the boundaries between adjacent rapidly and slowly evolving genomic regions in Drosophila.
Authors: Martin C H , Meyerowitz E M
Published: May 2006

The site of a dramatic change in the rate of DNA sequence evolution exists near the 68C glue gene clusters of several Drosophila species. We have previously determined the approximate location of this transition site by comparison of restriction maps of the regions flanking the 68C-like glue gene cluster of five members of the melanogaster species subgroup. In the present work we report the sequence of the transition region in three of these Drosophila species: D. melanogaster, D. yakuba, and D. erecta. Using a best-fit alignment of these sequences, we find that the site of transition from slowly to rapidly evolving sequences occurs abruptly within a region less than 50 nucleotides in length. Although frequency of nucleotide substitutions changes as much as 10-fold across this boundary, frequency of small insertion/deletion events stays nearly constant.
Genes for the tumor necrosis factors alpha and beta are linked to the human major histocompatibility complex.
Authors: Spies T , Morton C C , Nedospasov S A , Fiers W , Pious D , Strominger J L
Published: May 2006

The human major histocompatibility complex (MHC) includes the closely linked genes for the tumor necrosis factors alpha and beta. Their location is within the chromosomal segment between HLA-DR and HLA-A or centromeric of HLA-DP. This assignment is based on Southern blot analysis of a number of different MHC deletion mutants and is corroborated by chromosome in situ hybridization.
From basic network principles to neural architecture: emergence of orientation columns.
Authors: Linsker R
Published: May 2006

Orientation-selective cells--cells that are selectively responsive to bars and edges at particular orientations--are a salient feature of the architecture of mammalian visual cortex. In the previous paper of this series, I showed that such cells emerge spontaneously during the development of a simple multilayered network having local but initially random feedforward connections that mature, one layer at a time, according to a simple development rule (of Hebb type). In this paper, I show that, in the presence of lateral connections between developing orientation cells, these cells self-organize into banded patterns of cells of similar orientation. These patterns are similar to the "orientation columns" found in mammalian visual cortex. No orientation preference is specified to the system at any stage, none of the basic developmental rules is specific to visual processing, and the results emerge even in the absence of visual input to the system (as has been observed in macaque monkey).
Identification and characterization of a vasopressin isoreceptor in porcine seminal vesicles.
Authors: Maggi M , Kassis S , Malozowski S , Guardabasso V , Rodbard D
Published: May 2006

Neurohypophysial hormones stimulate the motility of tunica albuginea, epididymis, and vas deferens acting through oxytocin (OT) and V1 vasopressin receptors. To test the hypothesis that these hormones are involved also in the regulation of seminal vesicle physiology, we studied binding of [3H]OT and [3H] arginine vasopressin ([3H]AVP) to porcine seminal vesicle membranes. Neurohypophysial hormones bind to two different classes of sites. The first class shows low capacity (35 fmol per mg of protein) and a very high affinity (Kd less than 1 nM) for both the labeled ligands. The second class is characterized by a high capacity (2000 fmol per mg of protein) and a high affinity for AVP (Kd approximately equal to 2.5 nM), whereas OT has 160 times lower affinity. Lysine vasopressin and the V1 antagonist [1-deaminopenicillamine, 2-(O-methyl)tyrosine]Arg8-vasopressin compete with high affinity with [3H]AVP binding, whereas the V2 agonist [1-deamino,4-valine]D-Arg8-vasopressin (dVDAVP) is 110 times less potent than AVP. The OT agonist [Thr4,Gly7]OT and the OT antagonist [1(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)tyrosine, 8-ornithine]vasotocin failed to affect [3H]AVP binding. These findings seem to suggest that AVP interacts with the V1 vasopressin isoreceptor in porcine seminal vesicle membranes. However, AVP stimulates adenylate cyclase activity in a dose-dependent fashion with an EC50 of 14 nM, whereas OT or dVDAVP has no effect at 100 nM. Moreover, a well-characterized V1 vasopressin antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine]Arg8-vasopressin [d(CH2)5Tyr(Me)AVP], competes with [3H]AVP binding with an IC50 of 0.17 microM. These pharmacological properties are distinct from the previously described V1 and V2 vasopressin receptors and indicate the presence of a new class of AVP receptors. Although this vasopressin isoreceptor shares some pharmacological characteristics with the V1 (pressor) isoreceptor, it has low affinity for the V1 antagonist d(CH2)5-Tyr(Me)AVP and is linked to the adenylate cyclase system. The extremely high density of AVP receptors in porcine seminal vesicles (2 pmol per mg of protein) is comparable to the density of V2 vasopressin receptors in porcine renal medulla, suggesting a physiological role for vasopressin in the seminal vesicle.
Characterization of a thrombomodulin cDNA reveals structural similarity to the low density lipoprotein receptor.
Authors: Jackman R W , Beeler D L , VanDeWater L , Rosenberg R D
Published: May 2006

We have isolated a partial-length cDNA for bovine thrombomodulin from a lambda gt11 bovine adrenal capillary endothelial cell expression library. This was accomplished by immunoscreening with rabbit anti-thrombomodulin IgG heteroantibody and then rescreening with the initial positive recombinant insert. The cDNA obtained was authenticated by showing that it coded for the primary structure of two separate regions of bovine thrombomodulin. The nucleotide sequence of the largest cDNA allowed us to establish the structure of about 80% of the mature thrombomodulin transcript, which encodes the C-terminal half of the polypeptide. This membrane component is structurally similar to coated-pit receptors and is organized into domains that resemble those of the low density lipoprotein receptor.
Lipid requirements for reconstitution of the proton-translocating complex of clathrin-coated vesicles.
Authors: Xie X S , Tsai S J , Stone D K
Published: May 2006

We have recently reported the reconstitution of the clathrin-coated vesicle proton-translocating complex with liposomes prepared from ethanol-extracted crude bovine brain lipids. Reconstitution of proton pumping by the isolated proton ATPase has now been achieved with liposomes prepared from pure lipids. Optimal proton pumping, as assessed by ATP-generated acridine orange quenching and 32Pi-ATP exchange, is achieved with liposomes prepared from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol at a weight ratio of 40:26.5:7.5:26, and a lipid-to-protein weight ratio of 200:1. Under such conditions, the extent of decrease of ATP-generated acridine orange quenching is 150-fold greater than that of native clathrin-coated vesicles. Cholesterol is required to stabilize the proteoliposome. Phosphatidylserine, which is most effective in activating ATP-hydrolytic activity of the solubilized enzyme, is not obligatory for reconstitution of proton pumping.
Stoichiometric model of the photosynthetic unit of
Ectothiorhodospira halochloris
Authors: Engelhardt Harald , Engel Andreas , Baumeister Wolfgang
Published: May 2006

A stoichiometric model of the photosynthetic unit of
Ectothiorhodospira halochloris
has been obtained by means of scanning transmission electron microscope mass determination and mass mapping in conjunction with polyacrylamide gel electrophoresis. One reaction center, consisting of four single polypeptides, including one cytochrome, is surrounded by six identical light-harvesting complexes, each containing three polypeptides with 2:2:2 stoichiometry. This stoichiometric model was incorporated into the three-dimensional structure of the photosynthetic unit as derived from surface relief reconstructions of the two surfaces of shadowed membranes. The reaction center protrudes substantially from both membrane surfaces and has the cytochrome attached to the periplasmic face in a noncentrosymmetric fashion. The reaction center may assume various orientations within the photosynthetic complexes.
Escherichia coli K-12 restricts DNA containing 5-methylcytosine.
Authors: Raleigh E A , Wilson G
Published: May 2006

We have observed that plasmids containing certain cloned modification methylase genes of type II restriction-modification systems cannot be transformed into many laboratory strains of Escherichia coli K-12. The investigation of this phenomenon, reported here, has revealed (i) DNA containing 5-methylcytosine is biologically restricted by these strains, while DNA containing 6-methyladenine is not; (ii) restriction is due to two genetically distinct systems that differ in their sequence specificities, which we have named mcrA and mcrB (for modified cytosine restriction). Since 5-methylcytosine containing DNA is widespread in nature, the Mcr systems probably have a broad biological role. Mcr restriction may seriously interfere with molecular cloning of 5-methylcytosine-containing foreign DNAs. The Mcr phenotypes of some commonly used strains of E. coli K-12 are reported.
Theory of an immune system retrovirus.
Authors: Cooper L N
Published: May 2006

Human immunodeficiency virus (HIV; formerly known as human T-cell lymphotropic virus type III/lymphadenopathy-associated virus, HTLV-III/LAV), the retrovirus that infects T4-positive (helper) T cells of the immune system, has been implicated as the agent responsible for the acquired immune deficiency syndrome. In this paper, I contrast the growth of a "normal" virus with what I call an immune system retrovirus: a retrovirus that attacks the T4-positive T cells of the immune system. I show that remarkable interactions with other infections as well as strong virus concentration dependence are general properties of immune system retroviruses. Some of the consequences of these ideas are compared with observations.
Expression of insulin-like growth factor II in human placentas from normal and diabetic pregnancies.
Authors: Shen S J , Wang C Y , Nelson K K , Jansen M , Ilan J
Published: May 2006

The growth and development of the placenta is critical to fetal growth and development; however, little is known regarding the mechanisms controlling placental growth and development. Human placental membranes are known to possess receptors for insulin-like growth factors I and II (IGF-I and IGF-II) from early gestation, and increasing evidence supports a major role for IGF-I and/or IGF-II in fetal growth and development. Therefore, the IGFs may also play a significant role in regulating placental growth and development. We report here that an adult human liver IGF-II cDNA hybridizes to poly(A)+ RNAs of human placentas from different gestational ages. There are four placental poly(A)+ RNA species that hybridize to IGF-II cDNA, the major one of which is about 6000 bases. The sizes of the hybridized transcripts are the same for placentas of different gestational ages. Furthermore, the IGF-II sequences expressed in the human placenta were quantitated by dot blot hybridization. The second trimester placenta expresses more IGF-II mRNA sequences than placenta of first trimester and term. Interestingly, the term placentas from diabetic pregnancies also express more of these sequences than those from normal pregnancies. These results suggest that there are developmental changes in the expression of the IGF-II gene in the placenta and that IGF-II may promote placental growth by way of an autocrine and/or paracrine mechanism. Moreover, fetuses developing in diabetic pregnancies receive a large influx of glucose, which in turn may stimulate the expression of IGF-II sequences in placenta, resulting in higher utilization of glucose and overgrowth of placenta. This may explain the macrosomia and high incidence of malformations and stillbirths known to result from pregnancies in diabetics.
Specific human cytotoxic T cells recognize B-cell lines persistently infected with respiratory syncytial virus.
Authors: Bangham C R , McMichael A J
Published: May 2006

The T-lymphocyte response to respiratory syncytial (RS) virus has been invoked to explain the bronchiolitis and pneumonia caused by RS virus in human infants. However, T cells also appear to play a role in protection against RS virus infection. Although RS virus-specific human lymphocytes have been demonstrated, neither the phenotype nor the function of the lymphocytes was characterized. We describe here the induction of anti-RS virus cytotoxic T lymphocytes, in both bulk culture and restimulated cell lines, from human peripheral blood. Infection of Epstein-Barr virus-transformed human B-cell lines with RS virus in vitro readily caused a persistent infection; these cells continued to synthesize RS viral proteins and secrete infectious RS virus 4 months after infection. The persistently infected cells were used both to restimulate cytotoxic-T-cell precursors and as targets for RS virus-specific cytotoxic T cells.
Sequence homology between acquired immunodeficiency syndrome virus envelope protein and interleukin 2.
Authors: Reiher W E , Blalock J E , Brunck T K
Published: May 2006

A region of homology has been found between the envelope (env) protein of the acquired immunodeficiency syndrome (AIDS) virus and a portion of interleukin 2 (IL-2) that purportedly binds to the IL-2 receptor. This homology, between two proteins associated with opposing biological functions, suggests possible mechanisms for the immunosuppressive activity of the AIDS virus. Two mechanisms are proposed in which the AIDS virus env protein interferes with IL-2 activity either directly or indirectly. A region of similarity to the purported IL-2 receptor binding site on IL-2 and AIDS virus env is present in the env proteins of other retroviruses associated with immunosuppression. A synthetic peptide vaccine for AIDS is suggested based on the IL-2 receptor binding sequence in AIDS virus env.
Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection.
Authors: Williams L R , Varon S , Peterson G M , Wictorin K , Fischer W , Bjorklund A , Gage F H
Published: May 2006

Neurons in the rat medial septum (MS) and vertical limb of the diagonal band of Broca (VDB) undergo a rapid and severe cell death after transection of their dorsal projection to the hippocampus by aspiration of the ipsilateral fimbria fornix and supracallosal striae. By 2 weeks posttransection, the extent of neuronal loss was 50% of the total neurons and 70% of the cholinergic neurons in the MS and 30% of the total neurons and 40% of the cholinergic neurons in the VDB. We hypothesized that (i) the death was due to the loss of a hippocampus-derived neuronotrophic factor, and (ii) exogenous nerve growth factor (NGF) might provide trophic support to the MS/VDB cholinergic neurons, in light of recent reports that the septal diagonal band cholinergic neurons are responsive to NGF and that NGF is present and produced in the hippocampus. In the present study, we attempted to prevent the transection-induced neuronal death by continuous infusion of exogenous 7S NGF (1 microgram/wk) through an intraventricular cannula device. We report here that NGF treatment significantly reduces both the total neuronal and cholinergic neuronal death found 2 weeks after fimbria fornix transection; there was a sparing of 50% of the neurons in the MS and essentially 100% of those in the VDB that otherwise would have died. We conclude that NGF also has a protective effect on noncholinergic neurons since calculations indicate that 80% of the NGF-affected neurons are noncholinergic.
Identification of an activated c-Ki-ras oncogene in rat liver tumors induced by aflatoxin B1.
Authors: McMahon G , Hanson L , Lee J J , Wogan G N
Published: May 2006

Weanling male Fischer rats were administered 40 intraperitoneal injections of aflatoxin B1 (25 micrograms per animal per day) over a 2-month period. This chronic dosing regimen resulted in the sequential formation of hyperplastic foci, preneoplastic nodules, and hepatocellular carcinomas in all of the animals treated. The presence of transforming DNA sequences was detected by formation of anchorage-independent foci after transfection of tumor-derived DNA in NIH 3T3 mouse fibroblasts. Transfection of genomic DNA isolated from individual tumors from eight animals resulted in specific transforming activities ranging from 0.05 to 0.2 foci per micrograms of DNA. Primary transfectant DNAs were analyzed by Southern blot hybridization with DNA probes homologous to c-Ha-ras, c-Ki-ras, and N-ras oncogenes. A highly amplified c-Ki-ras oncogene of rat origin was detected in transformants derived from tumors in two of the eight animals tested. There was no evidence to suggest the presence of c-Ha-ras or N-ras sequences in any of the transformants. Analysis of primary liver tumor DNA showed no Ki-ras DNA amplification when compared to control liver DNA samples. Increased levels of c-Ki-ras p21 proteins were detected in 3T3 transformants containing activated rat c-Ki-ras genes. The presence of c-Ki-ras sequences of rat origin capable of inducing transformed foci can be taken as evidence that the c-Ki-ras gene has been activated in the primary liver tumors.
Insulin and growth factor effects on c-fos expression in normal and protein kinase C-deficient 3T3-L1 fibroblasts and adipocytes.
Authors: Stumpo D J , Blackshear P J
Published: May 2006

We investigated the expression of the protooncogene c-fos in 3T3-L1 fibroblasts and adipocytes in response to a variety of growth-promoting agents in normal cells and in cells preincubated with phorbol esters to deplete them of protein kinase C. There was a rapid accumulation of c-fos mRNA in fibroblasts and adipocytes treated with phorbol 12-myristate 13-acetate, platelet-derived growth factor, fibroblast growth factor, fetal calf serum, bombesin, and insulin, especially in the adipocytes. Phorbol 12-myristate 13-acetate pretreatment abolished the increase in c-fos mRNA due to additional phorbol 12-myristate 13-acetate treatment and decreased but did not eliminate the ability of platelet-derived growth factor, fibroblast growth factor, fetal calf serum, bombesin, and insulin to stimulate c-fos mRNA. These data suggested that c-fos mRNA could be induced in serum-deprived 3T3-L1 fibroblasts and adipocytes by at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C. In the very insulin-sensitive 3T3-L1 adipocytes, insulin rapidly and transiently increased c-fos expression (c-fos mRNA appeared by 15 min and disappeared after 60 min) via interaction with its own cellular receptor, rather than by interacting with receptors for one of the insulin-like growth factors. Cycloheximide treatment in combination with insulin or phorbol 12-myristate 13-acetate resulted in superinduction of c-fos mRNA. We conclude that insulin can rapidly stimulate c-fos mRNA accumulation in 3T3-L1 adipocytes and that part of the growth factor-stimulated increase in this mRNA that occurs in protein kinase C-deficient cells may be due to activation of a pathway similar or identical to that activated by insulin.
Negative regulation of the major histocompatibility class I gene in undifferentiated embryonal carcinoma cells.
Authors: Miyazaki J , Appella E , Ozato K
Published: May 2006

Murine embryonal carcinoma F9 cells, which do not express appreciable levels of major histocompatibility complex (MHC) class I mRNA, start to express the mRNA and proteins upon differentiation induced by retinoic acid (RA). To investigate the molecular mechanism of this regulation, we examined in F9 cells transient expression of the chloramphenicol acetyltransferase (CAT) gene directed by the 5' flanking region of a MHC class I gene, H-2Ld. The native 1.4-kilobase H-2Ld 5' upstream region gave very low CAT activity in undifferentiated F9 cells. Deletion between positions -210 and -135 relative to the cap site resulted in a 4- to 5-fold increase in CAT activity as compared with constructs containing the region. However, all of these constructs, regardless of the deletion, expressed comparable CAT activity in differentiated F9 cells. These data suggest the presence of a negative cis-acting element that is under developmental control. Further analysis revealed that the sequence conferring the negative regulation resides between positions -195 and -161. This region, highly conserved among the MHC class I genes, is found to be capable of increasing CAT activity in NIH 3T3 cells that express the class I genes constitutively. Further, this regulatory sequence, when connected to the simian virus 40 promoter, produced repressive and enhancing effects in F9 and NIH 3T3 cells, respectively. Based on these results, we suggest that the expression of MHC class I genes during development involves switching from negative to positive regulation dictated by the class I regulatory element located between positions -195 and -161.
2-(N-acetoxy-N-acetylamino)fluorene mutagenesis in mammalian cells: sequence-specific hot spot.
Authors: Gentil A , Margot A , Sarasin A
Published: May 2006

Mutations induced by 2-(N-acetoxy-N-acetylamino)fluorene were studied using temperature-sensitive simian virus 40 (SV40) mutants as probe in monkey kidney cells. In vitro treatment of the SV40 virions with 2-(N-acetoxy-N-acetylamino)fluorene increased mutagenesis and decreased survival in the viral progeny. A lethal hit of approximately 85 acetylaminofluorene adducts per SV40 genome was calculated. UV irradiation of cells prior to infection did not modify the results. Molecular analysis of independent SV40 revertants showed that 2-(N-acetoxy-N-acetylamino)fluorene induces base substitutions that are located not opposite putative acetylaminofluorene adducts but next to them. Moreover, a hot spot of mutation restoring a true wild-type genotype was observed in 10 of the 16 revertants analyzed. This hot spot, not targeted opposite a major DNA lesion, was not observed using UV light as damaging agent in the same genetic assay. Two models involving the stabilization, by acetylaminofluorene adducts, of the secondary structure of a specific quasipalindromic SV40 sequence are proposed to explain this sequence-specific hot spot.
Allelic variation in human mitochondrial genes based on patterns of restriction site polymorphism.
Authors: Whittam T S , Clark A G , Stoneking M , Cann R L , Wilson A C
Published: May 2006

Restriction maps of 145 human mtDNAs representing samples from five geographic regions were used to construct multilocus genotypes for 28 genetic loci of the mitochondrial genome. Alleles were defined as distinct combinations of the presence or absence of polymorphic restriction sites within each locus. The 28 loci included 13 genes encoding proteins, 10 genes specifying tRNAs, 2 genes specifying rRNAs, and 3 noncoding regions consisting of the D loop, the light strand origin of replication, and the 5' noncoding sequence. In 35 comparisons of allele frequency distributions to expected distributions predicted by neutral mutation theory (assuming an infinite alleles model), the results revealed that most genetic diversity values (71%) fell within the range predicted by the neutral model; however, excesses in the frequencies of common alleles and in the number of singleton alleles within populations were observed at specific loci. Departures from the neutral mutation model are most readily explained by the effects of the recent expansion of the human population and the action of purifying selection. Coefficients of population differentiation suggest that gene flow of mtDNA types between certain geographic regions may be limited.
Systemic resistance to methotrexate in transgenic mice carrying a mutant dihydrofolate reductase gene.
Authors: Isola L M , Gordon J W
Published: May 2006

A full-length cDNA coding for a mutant dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3), cloned from a mouse fibroblast cell line grown in high concentrations of methotrexate (MTX), was microinjected into mouse embryos to produce transgenic mice. The DHFR cDNA product is 270-fold more resistant to MTX than the wild-type enzyme. Seventeen transgenic mouse lines, identified by Southern blotting of tail or spleen DNA, carried between 1 and 400 copies of the foreign gene per cell. Eight lines have thus far been tested for resistance to MTX. Control mice were treated until death; MTX was withdrawn from transgenic mice when a cumulative MTX dose uniformly fatal for controls was reached. The major site of MTX toxicity was the gastrointestinal tract, with death of controls resulting from fluid and weight loss. Transgenic animals were relatively resistant to these symptoms and tolerated significantly more MTX than control animals. These results show that genes conferring resistance to chemotherapeutic agents can, after transfer into intact organisms, produce systemic drug resistance.
Demonstration of a non-Tac peptide that binds interleukin 2: a potential participant in a multichain interleukin 2 receptor complex.
Authors: Tsudo M , Kozak R W , Goldman C K , Waldmann T A
Published: May 2006

The interleukin 2 (IL-2) receptor system plays a key role in the T-cell immune response. Although IL-2 binding was reported to be restricted to the Tac peptide, we have identified an IL-2 binding peptide that does not react with anti-human IL-2 receptor monoclonal antibodies, including anti-Tac on MLA 144, a gibbon ape T-cell line. The MLA 144 cell line expressed 6800 IL-2 binding sites per cell with a low (Kd = 14 nM) affinity for human recombinant IL-2. Using cross-linking methodology, we demonstrated that the IL-2 binding peptide on MLA 144 is larger (Mr 75,000) than the Tac peptide, which has a Mr of 55,000. An IL-2 binding peptide of similar size (Mr 75,000) was also identified in addition to the Tac peptide (Mr 54,000-57,000) on Hut 102, a human T-cell lymphotrophic virus I-induced T-cell leukemia line, and phytohemagglutinin-activated normal human and gibbon ape lymphoblasts. Anti-Tac antibody did not block the binding of 125I-labeled IL-2 to MLA 144 cells. However, this antibody abolished the binding of 125I-labeled IL-2 not only to the Tac peptide on Hut 102 cells and normal lymphoblasts but also to the Mr 75,000 IL-2 binding peptide, suggesting that this latter peptide is associated with the Tac peptide to form the high-affinity IL-2 receptor complex.
Peripheral neural serotonin receptors: identification and characterization with specific antagonists and agonists.
Authors: Mawe G M , Branchek T A , Gershon M D
Published: May 2006

Serotonin (5-hydroxytryptamine, 5-HT) has been shown to be a neurotransmitter in the enteric nervous system (ENS). Although 5-HT is a mediator of slow excitatory postsynaptic potentials evoked by stimulation of interganglionic connectives, the precise role it plays in the physiology of the gut is unclear. Research has been hampered by an inadequate knowledge of the types of 5-HT receptor in the ENS and thus the lack of well-characterized antagonists. We now report the identification of two classes of enteric neural 5-HT receptor, the effects of activating these receptors on myenteric type II/AH neurons, and their characterization with specific agonists and antagonists. One class, which we propose to call 5-HT1P, is characterized by a high affinity for [3H]5-HT in radioligand binding assays. This class of receptor mediates a slow depolarization of myenteric type II/AH neurons associated with an increase in input resistance. Agonists at this receptor include, in addition to 5-HT (in order of potency), 5- and 6-hydroxyindalpine and 2-methyl-5-HT. 5-HT1P-mediated responses are specifically antagonized by 5-hydroxytryptophyl-5-hydroxytryptophan amide. The other class of 5-HT receptor, which we propose to call 5-HT2P, appears not to have a high affinity for [3H]5-HT. This receptor mediates a brief depolarization of myenteric II/AH neurons associated with a fall in input resistance. 2-Methyl-5-HT, at low concentrations, is a specific agonist at this receptor and ICS 205-930 is a specific antagonist. Binding of [3H]5-HT to enteric membranes is inhibited by 5-HT1P receptor agonists and antagonists but not by the 5-HT2P receptor antagonist ICS 205-930 or by MDL 72222, another compound reported to be an antagonist of 5-HT at peripheral receptors.
Interaction of vanadate with phenol and tyrosine: implications for the effects of vanadate on systems regulated by tyrosine phosphorylation.
Authors: Tracey A S , Gresser M J
Published: May 2006

The interaction of vanadate with phenol and N-acetyltyrosine ethyl ester in aqueous solution has been studied by using 51V nuclear magnetic resonance spectroscopy. On the basis of these studies, it has been concluded that vanadate rapidly esterifies the hydroxyl group of the aromatic ring to yield a phenyl vanadate. For phenol, the equilibrium constant for this reaction in terms of the convention that the activity of liquid water is 1.0 is K1 = [phenyl vanadate]/[phenol][vanadate] = 0.97 +/- 0.02. This value is well over 4 orders of magnitude larger than estimates from the literature for the corresponding equilibrium constant for the esterification of phenol by phosphate. The equilibrium constant for esterification of the phenol moiety of N-acetyltyrosine ethyl ester is similar to that for esterification of phenol. The relevance of these observations to processes that are regulated by reversible phosphorylation/dephosphorylation of tyrosine residues is discussed, in particular the insulin-like effect of vanadate.
Mapping of the human APOB gene to chromosome 2p and demonstration of a two-allele restriction fragment length polymorphism.
Authors: Huang L S , Miller D A , Bruns G A , Breslow J L
Published: May 2006

ApoB is a large glycoprotein with an apparent molecular mass of 550 kDa on NaDodSO4/PAGE. It is a major constituent of most lipoproteins and plays an important role in their metabolism. Recently, apoB cDNA clones have been isolated from an expression library made with mRNA from a human hepatoma cell line. These clones, which were all 1.5-1.6 kilobases (kb) long and corresponded to the 3' end of apoB mRNA, were used to demonstrate that hepatic apoB mRNA is approximately 22 kb long. In the current report, a probe derived from one of these cDNA clones, pB8, was used for in situ hybridization experiments to map the human gene for apoB, APOB, to the distal half of the short arm of chromosome 2. This probe was also used to analyze somatic cell hybrids and, in agreement with the in situ hybridization studies, concordancy was demonstrated with chromosome 2. In addition, two hybrids with chromosome 2 translocations that contain only the short arm reacted with the pB8 probe. A third hybrid with a complex rearrangement of chromosome 2, which deleted an interstitial region and the tip of the short arm of chromosome 2, did not react. These data indicate that APOB maps to either 2p21-p23 or 2p24-pter. In further studies, DNA from normal individuals, digested with the restriction endonuclease EcoRI and subjected to Southern blot analysis with the pB8 probe, revealed a two-allele restriction fragment length polymorphism (RFLP). The major allele was 11 kb, and the minor allele was 13 kb. The minor allele was present with a frequency of 20-25%. The inheritance of the two alleles was studied in an informative family, and they segregated in a typical autosomal Mendelian fashion. The mapping studies provide the means for understanding the relationship of the APOB locus to others in the human genome, whereas the demonstration of an APOB RFLP increases our ability to assess the role of this locus in determining plasma lipoprotein levels.