bluuebunny/crossref_metadata_2025 huggingface dataset:
Retention of an idiotypic determinant in a human B-cell lymphoma undergoing immunoglobulin variable-region mutation.
Authors: Kon S , Levy S , Levy R
Published: May 2006

Tumor cells from a patient with B-cell lymphoma were fused with a mouse myeloma cell line. A set of heterohybridomas was thus derived, each of which represented a separate clonal derivative from the tumor cell population. The immunoglobulins secreted by these cell lines reacted variably with a panel of anti-idiotypic antibodies, indicating that the tumor was heterogeneous; however, one antibody, 4D6, reacted strongly with the product of all the heterohybridomas. cDNA for the immunoglobulin heavy chain variable-region genes expressed in these heterohybridomas was cloned and sequenced. Comparison of these sequences indicated that the cells expressing them were clonally related but that they had undergone considerable mutation. Despite mutation, the cells in this tumor population continued to express a functional immunoglobulin molecule and to retain, over a span of 3 years, the idiotypic determinant defined by the 4D6 monoclonal antibody. Thus a selective force existed within the host to retain tumor cells bearing an immunoglobulin molecule with a particular idiotypic structure.
Heterologous transmembrane signaling by a human insulin receptor-v-ros hybrid in Chinese hamster ovary cells.
Authors: Ellis L , Morgan D O , Jong S M , Wang L H , Roth R A , Rutter W J
Published: May 2006

A hybrid receptor molecule composed of the extracellular ligand-binding domain of the human insulin receptor and the transmembrane and cytoplasmic (protein-tyrosine kinase) domains of the chicken sarcoma virus UR2 transforming protein p68gag-ros has been constructed and expressed in Chinese hamster ovary (CHO) cells. The hybrid is processed normally into alpha and hybrid beta subunits, is expressed on the cell surface at high levels, and binds insulin with near-wild-type affinity. Furthermore, insulin stimulates the phosphorylation on tyrosine residues of the hybrid beta subunit in vivo and the phosphorylation of an exogenous substrate [poly(Glu,Tyr)] in vitro. Thus the hybrid is capable of heterologous transmembrane signaling. However, the hybrid mediates neither the insulin-activated uptake of 2-deoxyglucose nor the incorporation of [3H]thymidine into DNA, suggesting that the physiological response(s) mediated by ligand-activated protein-tyrosine kinases may utilize distinct intracellular mechanisms for postreceptor signaling.
Characterization of a panel of somatic cell hybrids for regional mapping of the mouse X chromosome.
Authors: Avner P , Arnaud D , Amar L , Cambrou J , Winking H , Russell L B
Published: May 2006

A panel of five hybrid cell lines containing mouse X chromosomes with various deletions has been obtained by fusing splenocytes from male mice carrying one of a series of reciprocal X-autosome translocations with the azaguanine-resistant Chinese hamster cell line CH3g. These hybrids have been extensively characterized by using the allozymes hypoxanthine/guanine phosphoribosyltransferase (encoded by the Hprt locus) and alpha-galactosidase (Ags) and a series of 11 X-chromosome-specific DNA probes whose localization had been previously established by linkage studies. Such studies have established the genetic breakpoints of the T(X;12)13Rl and T(X;2)14Rl X-autosome translocations on the X chromosome and provided additional information as to the X-chromosome genetic breakpoints of the T(X;16)16H, T(X;4)7Rl, and T(X;7)6Rl translocations. The data establish clearly that both the T(X;4)7Rl and T(X;12)13Rl X-chromosome breakpoints are proximal to Hprt, the breakpoint of the former being more centromeric, lying as it does in the 9-centimorgan interval between the ornithine transcarbamoylase (Otc) and DXPas7 (M2C) loci. Similarly, it is now clear that the T(X;16)16H X-autosome translocation breakpoint lies distal to the DXPas8 (St14-1) locus, narrowing the X-chromosome breakpoint down to a region flanked proximally by this marker and representing, as expected from previous data, the distal quarter of the Hprt-Ta subchromosomal span. These five hybrid cell lines provide, with the previously characterized EBS4 hybrid cell line, a nested series of seven mapping intervals distributed along the length of the mouse X chromosome. Their characterization not only allows further correlation of the genetic and cytological X-chromosome maps but also should permit the rapid identification of DNA probes specific for particular regions of the mouse X chromosome.
The p75 peptide is the receptor for interleukin 2 expressed on large granular lymphocytes and is responsible for the interleukin 2 activation of these cells.
Authors: Tsudo M , Goldman C K , Bongiovanni K F , Chan W C , Winton E F , Yagita M , Grimm E A , Waldmann T A
Published: May 2006

There are at least two interleukin 2 (IL-2) binding peptides: one is the Mr 55,000 peptide (p55) reactive with the anti-Tac monoclonal antibody, and the other is a Mr 75,000 non-Tac IL-2 binding peptide (p75). Independently existing Tac or p75 peptides represent low-affinity IL-2 receptors, whereas high-affinity IL-2 receptors are expressed when both peptides are present and associated in a receptor complex. It has long been known that normal large granular lymphocytes (LGL) or leukemic cells from the patients with abnormal expansions of LGL can be activated by IL-2 not only to more-potent natural killer cells but also to effectors of lymphokine-activated killer (LAK) activity, although they do not express the Tac peptide. In the present study, using cross-linking methodology, we found that normal LGL and leukemic LGL from all individuals tested expressed the p75 IL-2 binding peptide but did not express the Tac peptide. These LGL leukemia cells made proliferative responses to IL-2 but required a much higher concentration than that required for the proliferation of normal phytohemagglutinin-stimulated T lymphoblasts that express high-affinity receptors. Furthermore, the addition of IL-2 to Tac-negative LGL leukemic cells augmented transcription of the Tac gene and induced the Tac peptide. Neither the IL-2-induced proliferation nor the up-regulation of Tac gene expression was inhibited by the addition of anti-Tac. These results strongly suggest that the p75 peptide is responsible for IL-2-induced activation of LGL and that the p75 peptide alone can mediate an IL-2 signal. Thus, the p75 peptide may play an important role in the IL-2-mediated immune response not only by participating with the Tac peptide in the formation of the high-affinity receptor complex on T cells but also by contributing to the initial triggering of LGL activation so that these cells become efficient natural killer and lymphokine-activated killer cells.
T-cell-replacing factor (interleukin 5) induces expression of interleukin 2 receptors on murine splenic B cells.
Authors: Loughnan M S , Takatsu K , Harada N , Nossal G J
Published: May 2006

Small, resting B lymphocytes express few, if any, interleukin 2 (IL-2) receptors, but activated B cells may express such receptors. This paper examines the requirements for receptor expression. Normal murine splenocyte populations were enriched for B cells and cultured at relatively low density. IL-2 receptor expression was studied by measuring the binding of the anti-IL-2 receptor monoclonal antibody PC61. Lymphoblasts arising through stimulation by Escherichia coli lipopolysaccharide failed to express IL-2 receptors. B cells cultured with conditioned medium from concanavalin A-stimulated EL4 thymoma cells, with or without LPS, displayed IL-2 receptors. This bioactivity of EL4 conditioned medium could not be replaced by any concentration of B-cell-stimulatory factor 1 (IL-4), IL-1, IL-2, or IL-3 tested. However, the recently cloned lymphokine T-cell-replacing factor (IL-5) was a potent inducer of IL-2 receptor expression, as was the probably identical material known as eosinophil differentiation factor. The receptors so induced appeared to be functional, as receptor-expressing (but not control) lymphoblasts, responded to IL-2 by proliferation, indicative of high-affinity-receptor expression.
Interaction between the human T-cell lymphotropic virus type IIIB envelope glycoprotein gp120 and the surface antigen CD4: role of carbohydrate in binding and cell fusion.
Authors: Matthews T J , Weinhold K J , Lyerly H K , Langlois A J , Wigzell H , Bolognesi D P
Published: May 2006

Interactions between retroviruses associated with acquired immunodeficiency syndrome and their receptors on lymphocytes represent the initial steps in the process of infection and are also involved in multinucleated giant cell formation, which is one form of virus-mediated cytopathology. The exterior envelope glycoprotein of the retrovirus has been identified as gp120, and we demonstrate here that purified gp120 binds directly to cells expressing the CD4 (T4) surface antigen at a site spatially related to that recognized by the OKT4A monoclonal antibody. The gp120 was also able to temporarily interfere with viral infection and to block the process of multinucleated giant cell formation. However, if the carbohydrate chains were removed from gp120 by enzymatic treatment, CD4 binding and blockade of cell fusion was reduced by about a factor of 50. The significance of these results in relation to preventive and interventive approaches for acquired immunodeficiency syndrome is discussed.
Molecular mechanical simulations on double intercalation of 9-amino acridine into d(CGCGCGC) X d(GCGCGCG): analysis of the physical basis for the neighbor-exclusion principle.
Authors: Rao S N , Kollman P A
Published: May 2006

The neighbor-exclusion principle is one of the most general and interesting rules describing intercalative DNA binding by small molecules. It suggests that such binding can only occur at every other base-pair site, reflecting a very large negative cooperativity in the binding process. We have carried out molecular mechanics and molecular dynamics simulations to study intercalation complexes between 9-amino acridine and the base-paired heptanucleotide d(CGCGCGC) X d(GCGCGCG), in which the neighbor-exclusion principle was both obeyed and violated. Our studies find no stereochemical preference that favors the neighbor-exclusion-obeying structures over the neighbor-exclusion-violating structures. Alternative explanations for the existence of the neighbor-exclusion principle are vibrational entropy effects that we calculate to favor the more flexible neighbor-exclusion models over the more rigid neighbor-exclusion-violating models and polyelectrolyte (counterion release) effects.
Regulation of the activity of platelet-bound C3 convertase of the alternative pathway of complement by platelet factor H.
Authors: Devine D V , Rosse W F
Published: May 2006

The alternative pathway of complement is regulated on the surface of homologous blood cells at the C3 amplification step by the membrane protein decay-accelerating factor, as well as by the plasma protein factor H. We have reported elsewhere that platelets from patients with paroxysmal nocturnal hemoglobinuria regulate the activity of the C3 convertase C3bBb, even though they lack decay-accelerating factor. We now report that normal human platelets contain factor H, which was released from the platelet in response to complement deposition or thrombin stimulation. Factor H was localized to the platelet alpha granules by immunocytochemical techniques. As determined by a solid-phase radioimmunoassay, thrombin-stimulated platelets released approximately equal to 54 ng of factor H per 10(8) platelets. The release of factor H in response to complement or thrombin was inhibited by treating the platelets with metabolic inhibitors. Such inhibition resulted in a 3-fold increase in the activity of C3bBb. Platelets that released factor H bound only half as many molecules of radiolabeled factor B to platelet-bound C3b than platelets that could not release factor H. Treatment of platelets with anti-decay-accelerating factor antibody had no effect on the activity of C3bBb unless the release of factor H was blocked. Therefore, so far as we know, human platelets have a unique mechanism for the regulation of the alternative pathway of complement.
Stable expression of transfected Torpedo acetylcholine receptor alpha subunits in mouse fibroblast L cells.
Authors: Claudio T
Published: May 2006

Torpedo californica electric organ cDNA libraries were constructed in lambda gt10 and lambda gt11. Four acetylcholine receptor (AcChoR) subunit cDNA clones were isolated and shown to contain the entire coding region for each of the subunits. When in vitro synthesized AcChoR mRNA was microinjected into Xenopus laevis oocytes, functional cell surface AcChoRs were expressed. A very simple and fast 22Na-uptake experiment was performed on batches of microinjected oocytes to identify oocytes that were expressing large quantities of functional cell surface AcChoRs for use in single-channel recordings. In addition to the transient expression system, DNA-mediated cotransformation is described, which is a method for stably introducing AcChoR cDNAs into the chromosomes of tissue culture cells. Because the AcChoR is composed of four different subunits, it is necessary to integrate four cDNAs into the chromosomes of the same cell before stable expression of a completely functional receptor complex can be established. We show that 80% of the cells that integrated the selectable marker gene into their chromosomes also integrated all four AcChoR cDNAs. When Torpedo alpha-subunit cDNA inserted into an appropriate expression vector was introduced into cells by transfection, alpha-subunit protein was synthesized that migrated on NaDodSO4/polyacrylamide gels with the same molecular mass as native Torpedo alpha subunits and expressed antigenic determinants similar to those of native Torpedo alpha subunits.
Spontaneous quantal currents in a central neuron match predictions from binomial analysis of evoked responses.
Authors: Korn H , Burnod Y , Faber D S
Published: May 2006

Inhibitory postsynaptic currents occurring spontaneously in the teleost Mauthner cell were analyzed with the single-electrode voltage-clamp technique. They were collected during depolarizing steps and were outward-going; this procedure allowed them to be isolated from possible excitatory currents flowing in the opposite direction. Their amplitude histograms were found to exhibit regularly spaced multiple peaks, each of which had a Gaussian distribution of the same width. These compound inhibitory postsynaptic currents represent responses evoked by background firing of presynaptic neurons, and when tetrodotoxin was applied topically, only the first peak in the frequency histogram, which can be attributed to single exocytotic events, remained. The mean conductance of this quantal unit equalled 46.0 nS, which corresponds to the opening of 1000-2000 Cl- channels activated by glycine--the transmitter at these synapses. Its waveform and those of the larger units were essentially the same. Furthermore, in each set of data provided by a given Mauthner cell, the size of a quantum was quite constant, with its variance and those of further peaks being equivalent to that of background noise. These properties, which characterize the quantal events on the basis of spontaneous synaptic activity, were strikingly similar to those of the basic units derived by the simple binomial to those of the basic units derived by the simple binomial analysis of unitary postsynaptic potentials, thus validating the use of this statistical model to quantify the quantal nature of release at central connections. The quite straightforward method used here to extract single miniature currents from complex signals should be applicable to the other systems of the central nervous system, where the pertinence of this probabilistic model of release has yet to be demonstrated.
Cell specificity of vasopressin binding in renal collecting duct: computer-enhanced imaging of a fluorescent hormone analog.
Authors: Kirk K L , Buku A , Eggena P
Published: May 2006

A noninvasive microscopic method was used to assess the cell specificity of vasopressin binding within the heterogeneous collecting duct. The binding of a fluorescent vasopressin analog (1-desamino-8-rhodamine-L-lysine vasopressin) to cells of the microperfused rabbit cortical collecting tubule was visualized and quantitated with image-intensified video microscopy and digital image processing. Binding to the basolateral membranes of a subpopulation of cells could be detected within 1-2 min of addition of the fluorescent analog (10 nM) to the peritubular bath. Binding could be prevented or reversed by the addition of a 10-fold excess of the native hormone, which indicates that the fluorescent analog binds specifically to vasopressin receptors. The time course of binding paralleled and slightly preceded hyperpolarization of the lumen-negative transepithelial voltage, an electrical response that is also elicited by the native hormone. Double-label experiments in which the intercalated cell population was stained with fluorescein-labeled peanut lectin revealed that binding of the vasopressin analog was localized to the remaining cell type, the principal cell. Our results support the following conclusions. First, the principal cell constitutes the primary target cell for vasopressin in the rabbit cortical collecting tubule, although the intercalated cell may possess a limited number of receptors at a density below the detection limit of this optical approach. Second, computer-enhanced video microscopy is a powerful, noninvasive method for assessing the kinetics and spatial pattern of hormone binding.
The 3' untranslated region of the human interferon-beta mRNA has an inhibitory effect on translation.
Authors: Kruys V , Wathelet M , Poupart P , Contreras R , Fiers W , Content J , Huez G
Published: May 2006

In vitro-transcribed human interferon-beta (IFN-beta) mRNA, which contains all the sequence of the natural molecule, is poorly translated in a reticulocyte lysate or when injected in Xenopus oocytes. This low level of translation is due to an inhibition by the 5' and 3' untranslated regions (UTRs). Indeed, the replacement of these sequences by those of Xenopus beta-globin mRNA dramatically increases the translational efficiency of the mRNA, especially in oocytes. This phenomenon is not due to a difference in mRNA stability since both native and chimeric mRNAs remain undegraded, at least during the translation period considered. Construction of different chimeric molecules having various combinations of 5' and 3' UTRs from IFN-beta or Xenopus beta-globin mRNA or a small sequence of SP6 polylinker as 5' UTR has revealed that the 3' UTR of IFN-beta in itself has a pronounced inhibitory effect on translation in the two translation systems from animal cells. Indeed, the addition of this 3' UTR at the 3' end of the coding region of a chicken lysozyme mRNA also causes a large decrease of its translational capacity in both systems. However, the nature of the 5' noncoding sequence influences the degree of translation inhibition exerted by the 3' UTR. Remarkably, we observed no difference in translation level when the different mRNAs were tested in a wheat germ extract.
Intracellular membrane flow: reconstitution of transition vesicle formation and function in a cell-free system.
Authors: Nowack D D , Morré D M , Paulik M , Keenan T W , Morré D J
Published: May 2006

Transfer of membrane between endoplasmic reticulum and Golgi apparatus in situ is considered to occur via 60-nm transition vesicles derived from part-rough, part-smooth transition elements of the endoplasmic reticulum. A procedure is described for the isolation of a fraction enriched in these transition elements from rat liver. The isolated fraction generates small vesicles morphologically resembling transition vesicles when incubated with nucleoside triphosphate at 37 degrees C. In the cell-free system consisting of a donor fraction enriched in transition elements and an acceptor fraction consisting of intact Golgi apparatus immobilized on nitrocellulose strips, transfer in vitro of radiolabeled membranes was demonstrated. Nucleoside triphosphates were required for transfer, and transfer was facilitated by a cytosol fraction of Mr greater than 10,000. In the presence of both nucleoside triphosphate and cytosol, radiolabeled proteins were transferred in a manner dependent upon both time and temperature. Transfer appeared to be both vectorial and specific in that, with Golgi apparatus (or endoplasmic reticulum) as both donor and acceptor, only negligible time and temperature-dependent transfer was observed. The test system described is expected to facilitate further investigation of the transfer process and to provide a convenient assay to guide transition vesicle isolation and characterization.
Conserved major histocompatibility complex class II boxes--X and Y--are transcriptional control elements and specifically bind nuclear proteins.
Authors: Dorn A , Durand B , Marfing C , Le Meur M , Benoist C , Mathis D
Published: May 2006

A conserved sequence motif exists at the 5' end of all major histocompatibility complex class II genes. This motif consists of the 14-base X and Y boxes separated by a short stretch of variable sequence. In this report, we provide evidence that the X and Y boxes play an important role in controlling transcription of the murine class II gene E kappa alpha. We have developed transgenic mouse lines that carry E alpha genes cleanly deleted for either the X or Y box and have compared the expression of these mutant transgenes with that of a nondeleted control. Both the X and Y segments appear critical for accurate and efficient transcription of E kappa alpha. The most drastic effect is seen with gamma-interferon-treated macrophages, where deletion of the Y box completely abrogates transcription initiated by the normal promoter. In addition, we identify proteins from nuclear extracts that bind specifically to the X or Y box.
Individual variation in serum cholesterol levels.
Authors: Hegsted D M , Nicolosi R J
Published: May 2006

The intraindividual variances in serum/plasma cholesterol levels from a variety of sources have been examined. It is apparent that these are very substantial with mean coefficients of variation usually between 5% and 10%, even when the diet is controlled in metabolic studies. Some subjects show extreme variability from one blood sample to the next. Thus, it is very difficult to assess the degree of risk of individuals according to the guidelines provided by the Consensus Conference on lowering blood cholesterol levels to prevent heart disease, and many individuals will be misclassified unless particular attention is paid to this problem.
17 beta-estradiol acts directly on the clonal osteoblastic cell line UMR106.
Authors: Gray T K , Flynn T C , Gray K M , Nabell L M
Published: May 2006

We studied the effect of 17 beta-estradiol (E) on the proliferation and alkaline phosphatase activity of cultured UMR106 cells, a clonal osteoblastic cell line. Growth rates were reduced and alkaline phosphatase activity was increased in cells incubated for 2 days in medium containing E (10(-8) M). In contrast, E had no effect on the growth rates or alkaline phosphatase of a human fibroblastic cell line, S90E. The effect of E was not observed with low cell density or at confluence. 1,25-Dihydroxyvitamin D3 antagonized the response to E. Preincubation of the cells with dexamethasone, a potent inducer of differentiation, reversed the effect of E or 1,25-dihydroxyvitamin D3. These results indicate that cellular and/or extracellular factors such as cell density, the phase of the cell cycle, the state of differentiation, and the presence or absence of other steroids influenced the response of UMR106 cells to E. Serum was removed from the culture medium to minimize the effect of the steroids, growth factors, and nutrients present in serum. A striking stimulation of alkaline phosphatase by E occurred with serum-free conditions. This stimulation was biphasic over an E concentration from 10(-12) to 10(-8) M, with the peak response at 10(-10) M. The action of E on UMR106 cells was metabolite-specific, since the isomer 17 alpha-estradiol produced no effect on proliferation rates or alkaline phosphatase activity. The cyclic AMP response to parathyroid hormone (residues 1-34) was not altered by E treatment of these cells. In contrast, dexamethasone exposure did increase the cyclic AMP response to parathyroid hormone. These results demonstrate a direct effect of E on an osteoblastic cell line. They also raise the possibility that similar or identical actions of E occur in cultured normal osteoblasts.
Abnormal compact myelin in the myelin-deficient rat: absence of proteolipid protein correlates with a defect in the intraperiod line.
Authors: Duncan I D , Hammang J P , Trapp B D
Published: May 2006

The cervical spinal cords of 23-day-old myelin-deficient (md) rats, an X chromosome-linked myelin mutant, and their normal littermates were studied by light and electron microscopy, immunocytochemistry, and in situ hybridization. Light microscopy showed that there were scattered myelinated fibers in the md rat, particularly in the lateral and ventral columns. Ultrastructural examination of these fibers showed that the myelin often had many lamellae that were tightly compacted, but in which the intraperiod line was abnormally fused at most places, resulting in a minor alteration of the myelin periodicity. Immunocytochemical staining of adjacent sections following a variety of fixation methods showed that the myelinated fibers were positive for myelin basic protein but negative for proteolipid protein (PLP). In situ hybridization using cDNA probes to these proteins showed a severe diminution of the mRNAs for both proteins. These findings provide further support for an abnormality in genetic regulation of PLP as has been described in another X chromosome-linked mutant, the jimpy mouse. Despite the lack of PLP, however, a few myelinated fibers are formed in the md rat, but the myelin formed in general lacks a normal intraperiod line, a site at which this protein is thought to be located.
Shifter circuits: a computational strategy for dynamic aspects of visual processing.
Authors: Anderson C H , Van Essen D C
Published: May 2006

We propose a general strategy for dynamic control of information flow between arrays of neurons at different levels of the visual pathway, starting in the lateral geniculate nucleus and the geniculorecipient layers of cortical area V1. This strategy can be used for resolving computational problems arising in the domains of stereopsis, directed visual attention, and the perception of moving images. In each of these situations, some means of dynamically controlling how retinal outputs map onto higher-level targets is desirable--in order to achieve binocular fusion, to allow shifts of the focus of attention, and to prevent blurring of moving images. The proposed solution involves what we term "shifter circuits," which allow for dynamic shifts in the relative alignment of input and output arrays without loss of local spatial relationships. The shifts are produced in increments along a succession of relay stages that are linked by diverging excitatory inputs. The direction of shift is controlled at each stage by inhibitory neurons that selectively suppress appropriate sets of ascending inputs. The shifter hypothesis is consistent with available anatomical and physiological evidence on the organization of the primate visual pathway, and it offers a sensible explanation for a variety of otherwise puzzling facts, such as the plethora of cells in the geniculorecipient layers of V1.
Structure of the reaction center from Rhodobacter sphaeroides R-26: membrane-protein interactions.
Authors: Yeates T O , Komiya H , Rees D C , Allen J P , Feher G
Published: May 2006

The energetics of membrane-protein interactions are analyzed with the three-dimensional model of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides. The position of the RC in the membrane and the thickness of the membrane were obtained by minimizing the hydrophobic energy with the energy function of Eisenberg and McLachlan. The 2-fold symmetry axis that relates the L and M subunits is, within the accuracy of 5 degrees, parallel to the normal of the membrane. The thickness of the membrane is estimated to be 40-45 A. Residues that are exposed to the membrane are relatively poorly conserved in the sequences of homologous RC proteins. The surface area of the RC is comparable to the surface areas of water-soluble proteins of similar molecular weight. The volumes of interior atoms in the RC are also similar to those of water-soluble proteins, indicating the same compact packing for both types of proteins. The electrostatic potential of the cofactors was calculated. The results show an asymmetry in the potential between the two possible pathways of electron transfer, with the A branch being preferred electrostatically.
Autogenous control of the S10 ribosomal protein operon of Escherichia coli: genetic dissection of transcriptional and posttranscriptional regulation.
Authors: Freedman L P , Zengel J M , Archer R H , Lindahl L
Published: May 2006

The S10 ribosomal protein operon is regulated autogenously by the product of one of the genes of the operon, the gene encoding ribosomal protein L4. We have used site-directed mutagenesis to isolate leader mutations affecting L4 control. The phenotypes of these mutants demonstrate that L4 regulates both transcription and translation of the S10 operon. Several mutations abolish both levels of L4 control; others eliminate either transcriptional or translational control with little or no effect on the other mode of regulation. We conclude that L4-mediated transcriptional and translational control share some sequence requirements, but the two regulatory processes recognize somewhat different features of the S10 leader. Primary as well as secondary structures within the S10 leader appear to be involved.
Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation.
Authors: Nussbaum R L , Lesko J G , Lewis R A , Ledbetter S A , Ledbetter D H
Published: May 2006

Choroideremia, an X-chromosome linked retinal dystrophy of unknown pathogenesis, causes progressive nightblindness and eventual central blindness in affected males by the third to fourth decade of life. Choroideremia has been mapped to Xq13-21 by tight linkage to restriction fragment length polymorphism loci. We have recently identified two families in which choroideremia is inherited with mental retardation and deafness. In family XL-62, an interstitial deletion in Xq21 is visible by cytogenetic analysis and two linked anonymous DNA markers, DXYS1 and DXS72, are deleted. In the second family, XL-45, an interstitial deletion was suspected on phenotypic grounds but could not be confirmed by high-resolution cytogenetic analysis. We used phenol-enhanced reassociation of 48,XXXX DNA in competition with excess XL-45 DNA to generate a library of cloned DNA enriched for sequences that might be deleted in XL-45. Two of the first 83 sequences characterized from the library were found to be deleted in probands from family XL-45 as well as from family XL-62. Isolation of these sequences proves that XL-45 does contain a submicroscopic deletion and provides a starting point for identifying overlapping genomic sequences that span the XL-45 deletion. Each overlapping sequence will be studied to identify exons from the choroideremia locus.
Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage.
Authors: Hamid Q , Wharton J , Terenghi G , Hassall C J , Aimi J , Taylor K M , Nakazato H , Dixon J E , Burnstock G , Polak J M
Published: May 2006

The localization of mRNA encoding preproatrial natriuretic peptide was investigated in tissue sections and cultures of rat heart and in sections of human right atrial appendage using the technique of in situ hybridization with 32P- and 35S-labeled RNA probes. Rat atrial natriuretic peptide (ANP) transcripts were demonstrated in numerous atrial myocytes and, to a lesser extent, in ventricular myocytes in both tissue sections and newborn rat heart cultures. These findings are consistent with those obtained by RNA blot analysis of rat heart total RNA, indicating that a single prepro-ANP transcript of approximately 900 nucleotides was present in the ventricles as well as the atria. Using a 35S-labeled RNA probe for human ANP mRNA, ANP transcripts were also localized to the majority of myocytes in the human right atrial appendage. Only background levels of autoradiographic labeling were obtained when RNA probes identical to the coding sequence of rat or human ANP mRNA were used. A close correlation was found between the distribution of ANP immunoreactivity and prepro-ANP mRNA in these preparations. These results provide unequivocal evidence for the expression of the ANP gene in the rat ventricles, as well as the atria, because myocytes in these tissues have been established as the sites of both ANP localization and precursor biosynthesis. The combined use of cardiac cultures and in situ hybridization may be of value in future studies investigating the regulation of ANP synthesis in cardiac myocytes.
Rat androgen-binding protein: evidence for identical subunits and amino acid sequence homology with human sex hormone-binding globulin.
Authors: Joseph D R , Hall S H , French F S
Published: May 2006

The cDNA for rat androgen-binding protein (ABP) was previously isolated from a bacteriophage lambda gt11 rat testis cDNA library and its identity was confirmed by epitope selection. Hybrid-arrested translation studies have now demonstrated the identity of the isolates. The nucleotide sequence of a near full-length cDNA encodes a 403-amino acid precursor (Mr = 44,539), which agrees in size with the cell-free translation product (Mr = 45,000) of ABP mRNA. Putative sites of N-glycosylation and signal peptide cleavage were identified. Comparison of the predicted amino acid sequence of rat ABP with the amino-terminal amino acid sequence of human sex hormone-binding globulin revealed that 17 of 25 residues are identical. On the basis of the predicted amino acid sequence the molecular weight of the primary translation product, lacking the signal peptide, was 41,183. Hybridization analyses indicated that the two subunits of ABP are coded for by a single gene and a single mRNA species. Our results suggest that ABP consists of two subunits with identical primary sequences and that differences in post-translational processing result in the production of 47,000 and 41,000 molecular weight monomers.
DNA base changes and alkylation following in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.
Authors: Richardson K K , Richardson F C , Crosby R M , Swenberg J A , Skopek T R
Published: May 2006

Dideoxy chain-termination DNA sequencing was used to determine the specific DNA base changes induced after in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. The resultant mutation spectra were compared with the levels of O6-alkylguanine and O4-alkylthymidine in genomic DNA immediately after exposure. All (39/39) of the MNU-induced mutations were G X C----A X T transitions. In contrast, 24/33 point mutations isolated following ENU treatment were G X C----A X T transitions, 7/33 were A X T----G X C transitions, 1/33 was a G X C----C X G transversion, and 1/33 was an A X T----C X G transversion. Three large insertions, probably of spontaneous origin, were also isolated. O4-alkylthymidine/O6-alkylguanine ratios were 0.014 for MNU and 0.28 for ENU. These data suggest that the difference in the mutation spectrum of MNU versus ENU may be attributed, in part, to the different ratio of O6-alkylguanine versus O4-alkylthymidine produced in the DNA. Of the G X C----A X T transitions, 82% of the MNU- and 71% of the ENU-induced mutations occurred at the middle guanine of the sequence 5'-GG(A or T)-3'.
Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family.
Authors: Hanks S K
Published: May 2006

Mixed oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identity with rabbit skeletal muscle phosphorylase kinase gamma-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.
Evolutionary changes in the developmental expression of silkmoth chorion genes and their morphological consequences.
Authors: Hatzopoulos A K , Regier J C
Published: May 2006

Discrete changes in silkmoth choriogenesis have occurred during evolution, as exemplified in the present report in Antheraea polyphemus and Hyalophora cecropia. At the level of morphology, the chorion of A. polyphemus has surface structures, called aeropyle crowns, that are absent from H. cecropia. Aeropyle crowns form during the very late period of choriogenesis and consist of two substructures--lamellae and filler. Filler is present in H. cecropia in greatly reduced amounts. At the level of protein synthesis, overall similarities in the two species are maintained until the very late period of choriogenesis, when synthesis of aeropyle crown components is maximal. In H. cecropia, very late period-specific proteins are reduced in number and abundance. Several of these minor proteins are candidates for E1 and E2, the components of filler. E1 and E2 RNAs are about 35 times more abundant in A. polyphemus, despite very similar gene copy numbers and times of expression in the two species. These results support the hypothesis that evolutionary changes in chorion morphology have resulted from regulatory changes in the expression of chorion genes, either at the level of transcription or mRNA decay. The hypothesis that evolutionary changes in chorion morphology are based on terminal addition onto a preexisting developmental program is discussed.
Induced mRNA stability in Bacillus subtilis.
Authors: Bechhofer D H , Dubnau D
Published: May 2006

We have investigated the induced stability of mRNA encoded by the ermC gene in Bacillus subtilis. Induction of ermC gene expression by erythromycin is known to occur at the translational level. We show that this induction is accompanied by an increase in ermC mRNA half-life from about 2 min to about 40 min. Induced stabilization of ermC mRNA occurs independently of induced translation. The regulatory sequences required for stability are promoter-proximal and can confer induced stability on large mRNAs having diverse 3' ends. Translation of the ermC leader peptide and ribosome-stalling in the leader peptide sequence are necessary for induced stabilization.
Chromosomal location of the human gene for DNA polymerase beta.
Authors: McBride O W , Zmudzka B Z , Wilson S H
Published: May 2006

Inhibition studies indicate that DNA polymerase beta has a synthetic role in DNA repair after exposure of mammalian cells to some types of DNA-damaging agents. The primary structure of the enzyme is highly conserved in vertebrates, and nearly full-length cDNAs for the enzyme were recently cloned from mammalian cDNA libraries. Southern blot analysis of DNA from a panel of human-rodent somatic cell hybrids, using portions of the cDNA as probe, indicates that the gene for human DNA polymerase beta is single copy and located on the short arm or proximal long arm of chromosome 8 (8pter-8q22). A restriction fragment length polymorphism (RFLP) was detected in normal individuals by using a probe from the 5' end of the cDNA, and this RFLP probably is due to an insertion or duplication of DNA in 20-25% of the population. This restriction site can be used as one marker for chromosome 8 genetic linkage studies and for family studies of traits potentially involving this DNA repair gene.
Anterior pituitary gland modulates the release of atrial natriuretic peptides from cardiac atria.
Authors: Zamir N , Haass M , Dave J R , Zukowska-Grojec Z
Published: May 2006

Myocytes in the atria contain a prohormone that gives rise to atrial natriuretic peptides (ANP), which have intrinsic hemodynamic regulatory activity. Little is known about mechanisms regulating ANP release. In rats with indwelling catheters, acute blood volume expansion with 5% (wt/vol) dextrose increases the amount of circulating immunoreactive ANP by a factor of 2-3, as determined by radioimmunoassay. Pithing, which both removes neurogenic influences and interrupts humoral influences of the brain and pituitary gland on the heart, completely blocked stimulus-induced release of ANP. Because our studies using pharmacological blockade of the autonomic nervous system had suggested that neurogenic mechanisms do not play a major role in ANP release, we sought a humoral mechanism involved in ANP secretion. Basal and stimulated release of ANP were significantly blunted in hypophysectomized rats (8 days after operation) but were completely restored when the resected anterior pituitary was reimplanted under the kidney capsule. This suggests that hormones of anterior pituitary origin are required for ANP secretion in response to acute volume loading.
Loss of platelet-derived growth factor-stimulated phospholipase activity in NIH-3T3 cells expressing the EJ-ras oncogene.
Authors: Benjamin C W , Tarpley W G , Gorman R R
Published: May 2006

Data indicating that the 21-kDa protein (p21) Harvey-ras gene product shares sequence homology with guanine nucleotide-binding proteins (G proteins) has stimulated research on the influence(s) of p21 on G-protein-regulated systems in vertebrate cells. Our previous work demonstrated that NIH-3T3 mouse cells expressing high levels of the cellular ras oncogene isolated from the EJ human bladder carcinoma (EJ-ras) exhibited reduced hormone-stimulated adenylate cyclase activity. We now report that in these cells another enzyme system thought to be regulated by G proteins is inhibited, namely phospholipases A2 and C. NIH-3T3 cells incubated in plasma-derived serum release significant levels of prostaglandin E2 (PGE2) as determined by radioimmunoassay when exposed to platelet-derived growth factor (PDGF) at 2 units/ml; the levels of PGE2 released from EJ-ras-transfected cells are only 3% those of controls despite a similar basal (unstimulated) release from control and EJ-ras-transfected cells. The lack of PDGF-stimulated PGE2 release from EJ-ras-transfected cells is not due to a defect in the prostaglandin cyclooxygenase enzyme, since incubation of control cells and EJ-ras-transfected cells in 0.33, 3.3, or 33 microM arachidonate resulted in identical levels of PGE2 release. The lack of PDGF-stimulated PGE2 release from EJ-ras-transfected cells also does not result from the loss of functional PDGF receptors. EJ-ras-transformed cells bind 70% as much 125I-labeled PDGF as control cells and are stimulated to incorporate [3H]thymidine and to proliferate after exposure to PDGF. Moreover, this inhibition is not likely the result of a secondary cellular effect related to the transformed phenotype, since NIH-3T3 cells transformed by v-src released PGE2 at wild-type levels after exposure to PDGF. Determination of total water-soluble inositolphospholipids and changes in the specific activities of phosphatidylcholine in control and EJ-ras-transfected cells demonstrated that PDGF-stimulated phospholipase C and A2 activities are inhibited in the EJ-ras-transfected cells.
Copper amplification of prostaglandin E2 stimulation of the release of luteinizing hormone-releasing hormone is a postreceptor event.
Authors: Barnea A , Cho G
Published: May 2006

We have shown that copper amplifies prostaglandin E2 (PGE2) stimulation of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA) and that this process is calcium-dependent. Since a Ca-cAMP pathway has been implicated in PGE2 action on the LH-RH neuron, in this study we wished to ascertain if copper exerts its effect on the PGE2 receptor or on a postreceptor component involved in PGE2 action. MEA of adult male rats were incubated for 5 min with 200 microM Cu/histidine (CuCl2 mixed with L-histidine at an equimolar ratio) and then incubated for 15 min either with 10 microM PGE2 (Cu/PGE2), 100 microM forskolin (Cu/forskolin), or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (Cu/cAMP). Controls were incubated without Cu/histidine or with Cu/histidine alone. Basal release of LH-RH was 4.6 +/- 0.45 pg/15 min per MEA (mean +/- SEM). Net stimulated release during the 15-min exposure to PGE2, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate was 3.6 +/- 0.52, 3.1 +/- 0.39, and 1.6 +/- 0.42 pg/15 min per MEA, respectively. Net stimulated release after exposure to Cu/PGE2, Cu/forskolin, or Cu/cAMP was 12.7 +/- 2.2, 9.9 +/- 1.46, and 1.4 +/- 1.9 pg/15 min per MEA, respectively, indicating that copper amplifies the action of PGE2 and forskolin but not cAMP action. When MEA were exposed to a mixture of PGE2 and forskolin for 15 min, the effects of these two secretagogues on LH-RH release were not additive, regardless of whether the MEA were pretreated with Cu/histidine. In contrast to PGE2 and forskolin, copper did not amplify K+ stimulation of LH-RH release and, moreover, when Cu/histidine-treated MEA were exposed to a mixture of PGE2 and 30 mM K+, the effects of these two secretagogues were additive. These results are supportive of the proposition that PGE2 stimulation of LH-RH release is mediated by the Ca-cAMP pathway and that copper amplification of PGE2 action is a postreceptor event.
Brief occurrence of a population of presynaptic intramembrane particles coincides with transmission of a nerve impulse.
Authors: Muller D , Garcia-Segura L M , Parducz A , Dunant Y
Published: May 2006

Small pieces of Torpedo electric organ were cryofixed at 1-ms time intervals in a liquid medium at -190 degrees C before, during, and after the passage of a single nerve impulse. In contrast to studies using this or other preparations, these experiments were done without 4-aminopyridine or other drugs that potentiate transmitter release. Freeze-fracture replicas were made from the most superficial layers of the tissue, where the rate of cooling was rapid enough to retain ultrastructure in the absence of chemical fixation. We found that the transmission of an impulse was accompanied by the momentary appearance of a population of large intramembrane particles in both the protoplasmic (P) and the external (E) leaflets of the presynaptic plasma membrane. The change was very brief, appearing soon after the stimulus artifact. It lasted for 2-3 ms. Large pits denoting vesicle openings at the presynaptic membrane were found in a small proportion of nerve terminals; their number did not increase during transmission of the nerve impulse. Reducing the temperature from 16 to 5 degrees C slowed the time course of both the electrophysiological response and the change in intramembrane particles. The number of large particles did not increase when stimulation was applied in a low-Ca medium, a condition where the nerve terminals were still depolarized by the action potential but did not release the neurotransmitter. From these and other observations, we conclude that this transient change of intramembrane particles is closely linked to the mechanism of acetylcholine release at the nerve-electroplaque junction.