bluuebunny/crossref_metadata_2025 huggingface dataset:
A herpesvirus trans-activating protein interacts with transcription factor OTF-1 and other cellular proteins.
Authors: Gerster T , Roeder R G
Published: May 2006

Immediate early genes of herpes simplex viruses contain one or more copies of the conserved TAAT-GARAT (where R is purine) DNA motif. A virus-encoded regulatory protein (Vmw65) is believed to stimulate transcription via this element, although the protein does not bind directly to DNA. Overlapping the TAATGARAT element in many cases is an octamer sequence (ATGCAAAT) that is involved both in transcription by RNA polymerases II and III and in adenovirus DNA replication. So far at least two proteins (OTF-1 and OTF-2) have been identified that bind to the octamer. We show that both affinity-purified OTF-1 and OTF-2 bind to the TAATGARAT sequence and that Vmw65 induces the formation of an additional complex that involves OTF-1 and that is further retarded in a band-shift gel assay. Complementation experiments involving addition of purified OTF-1 to nuclear extracts that have been depleted of endogenous OTF-1 show that at least one other cellular factor(s) is required for complex formation. This cellular factor may be involved in recognition of the GARAT sequence.
Malignant transformation of mouse primary keratinocytes by Harvey sarcoma virus and its modulation by surrounding normal cells.
Authors: Dotto G P , Weinberg R A , Ariza A
Published: May 2006

The activated ras oncogene that is present in Harvey sarcoma virus is able to induce malignant transformation of pure cultures of mouse primary keratinocytes. Malignant transformation of these cells is demonstrated by their ability to form carcinomas when grafted back onto syngeneic animals. However, expression of the malignant phenotype by the ras-transformed keratinocytes is drastically inhibited by the presence of normal dermal fibroblasts. This inhibitory effect depends on the ratio of fibroblasts to keratinocytes. It can be observed with mitomycin C-treated growth-arrested dermal fibroblasts and not with other cells, such as normal keratinocytes or established fibroblasts. Thus, a cellular environment approximating normal tissue can suppress tumor formation triggered by a single oncogene.
The 289-amino acid E1A protein of adenovirus binds zinc in a region that is important for trans-activation.
Authors: Culp J S , Webster L C , Friedman D J , Smith C L , Huang W J , Wu F Y , Rosenberg M , Ricciardi R P
Published: May 2006

The E1A gene of adenovirus type 5 encodes two major proteins of 289 and 243 amino acid residues, which are identical except that the larger protein has an internal stretch of 46 amino acids required for efficient trans-activation of early viral promoters. This domain contains a consensus zinc finger motif (Cys-Xaa2-Cys-Xaa13-Cys-Xaa2-Cys) in which the cysteine residues serve as postulated ligands. Atomic absorption spectrophotometry applied to bacterially expressed E1A proteins revealed that the 289-amino acid protein binds one zinc ion, whereas the 243-amino acid protein binds no zinc. Replacing individual cysteine residues of the finger with other amino acids destroyed the trans-activating ability of the 289-amino acid protein, even when structurally or functionally conserved amino acids were substituted. These results strongly suggest that the zinc finger of the 46-amino acid domain is intimately linked to the ability of the large E1A protein to stimulate transcription of E1A-inducible promoters. Furthermore, zinc binding to one of the mutant finger proteins suggests either that only a precise finger structure formed by the tetrahedral coordination of zinc to the four consensus ligands is required for trans-activation or, possibly, that one of several neighboring histidine residues in various combinations with three of the consensus cysteine residues normally coordinates zinc. How the zinc finger in E1A might interact with DNA or protein to bring about trans-activation is discussed.
Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes.
Authors: Nair M P , Pottathil R , Heimer E P , Schwartz S A
Published: May 2006

Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by [3H]thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3+ lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3- lymphocytes. Stimulation of CD3+ and CD3- lymphocyte subpopulations induced by env-gag peptides was specifically inhibited by goat anti-env-gag polyclonal antibodies, demonstrating the specificity of the reaction. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection.
Theoretical comparison of the self diffusion and mutual diffusion of interacting membrane proteins.
Authors: Scalettar B A , Abney J R , Owicki J C
Published: May 2006

Self diffusion and mutual diffusion in two-dimensional membrane systems are analyzed. It is shown that interprotein interactions can produce markedly different density-dependent changes in the diffusion coefficients describing these two processes; the qualitative differences are illustrated by using a theoretical formalism valid for dilute solutions. Results are obtained for three analytical potentials: hard-core repulsions, soft repulsions, and soft repulsions with weak attractions. Self diffusion is inhibited by all three interactions. In contrast, mutual diffusion is inhibited by attractions but is enhanced by repulsions. It is shown that such interaction-dependent differences in self diffusion and mutual diffusion could underlie, among other things, the disparity in protein diffusion coefficients extracted from fluorescence recovery after photobleaching and postelectrophoresis relaxation data.
Photosynthetic characteristics of an amphibious plant,
Eleocharis vivipara
: Expression of C
4
and C
3
modes in contrasting environments
Authors: Ueno Osamu , Samejima Muneaki , Muto Shoshi , Miyachi Shigetoh
Published: May 2006

Eleocharis vivipara
Link, a freshwater amphibious leafless plant belonging to the Cyperaceae can grow in both terrestrial and submersed aquatic conditions. Two forms of
E. vivipara
obtained from these contrasting environments were examined for the characteristics associated with C
4
and C
3
photosynthesis. In the terrestrial form (δ
13
C values = -13.5 to -15.4‰, where ‰ is parts per thousand), the culms, which are photosynthetic organs, possess a Kranz-type anatomy typical of C
4
plants, and well-developed bundle-sheath cells contain numerous large chloroplasts. In the submersed form (δ
13
C value = -25.9‰), the culms possess anatomical features characteristic of submersed aquatic plants, and the reduced bundle-sheath cells contain only a few small chloroplasts.
14
C pulse-
12
C chase experiments showed that the terrestrial form and the submersed form fix carbon by way of the C
4
pathway, with aspartate (40%) and malate (35%) as the main primary products, and by way of the C
3
pathway, with 3-phosphoglyceric acid (53%) and sugar phosphates (14%) as the main primary products, respectively. The terrestrial form showed photosynthetic enzyme activities typical of the NAD-malic enzyme-C
4
subtype, whereas the submersed form showed decreased activities of key C
4
enzymes and an increased ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) activity. These data suggest that this species can differentiate into the C
4
mode under terrestrial conditions and into the C
3
mode under submersed conditions.
Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PK1).
Authors: Haggerty J G , Agarwal N , Reilly R F , Adelberg E A , Slayman C W
Published: May 2006

Proximal tubule cells of the kidney contain, on their apical surface, an amiloride-sensitive Na/H antiporter that functions in Na reabsorption and proton secretion. We have investigated the localization of the antiporter in a cloned cell line of porcine renal origin, LLC-PK1/Cl4, which is often considered to be a useful model of the proximal tubule. Transport measurements were performed with differentiated monolayers grown on Nuclepore filters, permitting independent access to the apical and basolateral cell surfaces. In control experiments with LLC-PK1/Cl4 monolayers, three marker transport systems showed the expected polarity: 87% of ouabain-sensitive Rb uptake was at the basolateral surface, and 99% of Na-dependent alpha-methylglucoside transport and 93% of Na-dependent D-aspartate (L-glutamate) transport were at the apical surface. By contrast, the monolayers displayed significant Na/H antiporter activity (assayed as ethylisopropylamiloride-sensitive 22Na uptake) at both cell surfaces, with an apical uptake rate amounting to 44% and a basolateral rate amounting to 56% of the total. Significantly, the apical and basolateral antiporters could readily be distinguished from one another on the basis of ethylispropylamiloride sensitivity. The apical system had an IC50 of 13 microM, close to that reported for kidney brush border vesicle preparations, whereas the basolateral system had an IC50 of 44 nM, similar to values seen in undifferentiated LLC-PK1 cells and other cultured cell lines. The PKE20 mutant, previously selected from LLC-PK1/Cl4 on the basis of resistance to ethylisopropylamiloride, was found to overexpress the more resistant antiporter both during rapid growth and on its apical cell surface at confluence; normal amounts of the more sensitive antiporter were seen on the basolateral surface of confluent PKE20 cells. Taken together, these results suggest that there are two distinct forms of the Na/H antiporter, which are under separate genetic control.
B-cell-specific enhancer activity of conserved upstream elements of the class II major histocompatibility complex DQB gene.
Authors: Sakurai M , Strominger J L
Published: May 2006

A 95-base-pair immediate upstream sequence of the human class II major histocompatibility complex DQB gene containing the conserved X and Y elements showed enhancer activity in a transient expression assay. An "enhancer test plasmid" harboring the bacterial chloramphenicol acetyltransferase gene under the control of a truncated simian virus 40 enhancerless early promoter was employed. The DQB sequence inserted into this plasmid was active as an enhancer in Raji cells (human Burkitt lymphoma cells) but not active in Jurkat cells (human T-cell leukemia cells) or in HeLa cells (human cervical carcinoma cells). This cell-type specificity suggests that this enhancer activity may be involved in the tissue specificity of the DQB gene that is normally expressed only in mature B cells, macrophages, and thymic epithelial cells. Deletion analysis showed that both X and Y box sequences are essential for the full activity of the enhancer sequence and that these two sequences may function in a cooperative manner as cis-acting elements. Further deletions were used to define the 5' border of the X element. These results suggest that previously characterized protein factors that bind to X and Y include transcription factors involved in the cell-type specificity of this enhancer activity.
Acoustic stimulation causes tonotopic alterations in the length of isolated outer hair cells from guinea pig hearing organ.
Authors: Canlon B , Brundin L , Flock A
Published: May 2006

Isolated outer hair cells from the mammalian cochlea exhibit a motile response to electrical or chemical stimulation. Here we show that isolated outer hair cells can also respond to acoustic stimulation, in the form of a tone burst of 200 Hz, by either shortening or lengthening depending on their cochlear location. Cells from the apical region of the cochlea (long cells) responded by increasing their length, whereas those from more basal regions (short cells) responded by decreasing their length. Cells from intermediate positions showed an equal probability for either elongating or shortening. Both the elongating and shortening response was inhibited by 3 microM poly(L-lysine). It is suggested that this tonotopic and bidirectional acoustic response may be one of the active components underlying the specific phase and frequency displacement of the basilar membrane.
Carotenoid biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene
Authors: Dogbo Odette , Laferriére André , D'Harlingue Alain , Camara Bilal
Published: May 2006

Phytoene is the first C
40
intermediate in the biogenesis of carotenoids. It is formed by two enzyme activities, catalyzing (
i
) the coupling of two molecules of geranylgeranyl diphosphate to yield prephytoene diphosphate and (
ii
) the conversion of prephytoene diphosphate into phytoene. We show now, with
Capsicum
chromoplast stroma, that the overall activity resides in a single protein, which has been purified to homogeneity by affinity chromatography. The monomeric structure and the molecular size (
M
r
47,500) were demonstrated by NaDodSO
4
/PAGE and glycerol gradient centrifugation. Further characterization was achieved by using specific antibodies which allowed immunofractionation and immunoprecipitation of the enzymatic activity from chromoplast stroma. The two reactions followed conventional Michaelis-Menten kinetics, with
K
m
values of 0.30 μM and 0.27 μM, respectively, for geranylgeranyl diphosphate and prephytoene diphosphate. The activity of the enzyme depends strictly upon the presence of Mn
2+
. This selectivity may be one of the factors regulating the competition with potentially rival enzymes converting geranylgeranyl diphosphate into other plastid terpenoids. The two enzymatic reactions were inhibited by inorganic pyrophosphate and by the arginine-specific reagent hydroxyphenylglyoxal. In no instance were the two reactions kinetically uncoupled. These properties strongly suggest that the same enzyme catalyzes the two consecutive reactions, and we propose to name it phytoene synthase.
The B- to Z-DNA equilibrium in vivo is perturbed by biological processes.
Authors: Zacharias W , Jaworski A , Larson J E , Wells R D
Published: May 2006

Right-handed B and left-handed Z conformations coexist in equilibrium in portions of plasmids in Escherichia coli. The equilibria are influenced by the length of the sequences that undergo the structural transitions and are perturbed by biological processes. The composite results of three types of determinations indicate a supercoil density of -0.025 in vivo. The coexistence of alternative DNA conformations in living cells implies the potential of these structures or their transitions for important functions in genetic regulatory processes.
Human papillomavirus type 16 alters human epithelial cell differentiation in vitro.
Authors: McCance D J , Kopan R , Fuchs E , Laimins L A
Published: May 2006

Human papillomavirus (HPV) types 16, 18, 31, and 33 have been implicated as etiologic agents of cervical and penile cancer. Using a cell culture system for keratinocytes which allows stratification and production of differentiation-specific keratins, we have examined the effects of one of these viruses, HPV-16, on the differentiation capabilities of human epithelial cells. A plasmid containing the HPV-16 genome and a neomycin-selectable marker was transfected into primary human epidermal cells and SCC-13 cells, an immortalized squamous cell carcinoma cell line. Cloned neomycin-resistant cell lines were isolated and examined by cell culture on raised collagen rafts. Cell lines containing HPV-16 DNA retained the ability to stratify and express differentiation-specific keratins in the raft system but otherwise failed to differentiate normally. The histological abnormalities induced by HPV-16 closely resembled those seen in genital intraepithelial neoplasia in vivo. Hence, our results support the role of HPV-16 as an etiologic agent in the development of genital neoplasias and suggest a specific system for the study of HPV-16-induced epithelial cancers.
Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil.
Authors: Safa A R
Published: May 2006

Verapamil, a phenylalkylamine calcium channel blocker, has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells. In this study two photoactive radioactive analogs of verapamil, N-(p-azido[3,5-3H]benzoyl)aminomethyl verapamil and N-(p-azido[3-125I]salicyl)aminomethyl verapamil, were synthesized and used to identify the possible biochemical target(s) for verapamil in multidrug-resistant DC-3F/VCRd-5L Chinese hamster lung cells selected for resistance to vincristine. The results show that a specifically labeled 150- to 180-kDa membrane protein in resistant cells was immunoprecipitated with a monoclonal antibody specific for P-glycoprotein. Phenylalkylamine binding specificity was established by competitive blocking of specific photolabeling with the nonradioactive photoactive analogs as well as with verapamil. Photoaffinity labeling was also inhibited by 50 microM concentrations of the calcium channel blockers nimodipine, nifedipine, nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine. Bay K8644, a calcium channel agonist, also inhibited P-glycoprotein photolabeling. Moreover, P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 microM compared to that by verapamil at 8 microM. Photolabeling was also partially inhibited by two of the drugs to which these cells are cross-resistant, doxorubicin and actinomycin D, at 100 microM, but not by colchicine. These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.
Identification of pp120, an endogenous substrate for the hepatocyte insulin receptor tyrosine kinase, as an integral membrane glycoprotein of the bile canalicular domain.
Authors: Margolis R N , Taylor S I , Seminara D , Hubbard A L
Published: May 2006

An endogenous membrane-bound substrate of the insulin receptor beta-subunit tyrosine kinase in liver, pp120, has been identified as HA4, a 110-kDa membrane glycoprotein localized primarily to the bile canalicular domain of the hepatocyte. HA4 has been implicated in bile salt transport and cell adhesion. Monoclonal antibodies to HA4 were used to identify it as a substrate of the insulin receptor kinase. Anti-pp120 and anti-HA4 were found to cross-react, and phosphopeptide maps for each of the corresponding antigens were identical. The identification of pp120 as HA4 serves to link insulin action through the receptor tyrosine kinase activity to bile metabolism and raises questions pertaining to the intracellular site(s) of action of the insulin receptor.
Coexpression and assembly of myosin heavy chain and myosin light chain in Escherichia coli.
Authors: McNally E M , Goodwin E B , Spudich J A , Leinwand L A
Published: May 2006

A fragment of the Dictyostelium discoideum myosin heavy chain gene representing heavy meromyosin was coexpressed in Escherichia coli with the entire essential myosin light chain from the scallop. The expressed myosin heavy chain and essential myosin light chain copurify through ammonium sulfate fractionation, anion exchange, and gel filtration chromatography. The purified complex consists of about 1 mol of light chain per mol of heavy chain. This stoichiometry, which is that of native myosin, suggests that no special eukaryotic machinery is required for coassembly of these two proteins. By coexpressing different myosin heavy chain and myosin light chain combinations, it should be possible to study various isoforms of these two proteins, which are both products of multigene families in mammals. E. coli is thus an ideal system in which to study expression and multimeric assembly of individual components of the eukaryotic contractile apparatus.
Construction of epithelioid sheets by transfection of mouse sarcoma cells with cDNAs for chicken cell adhesion molecules.
Authors: Mege R M , Matsuzaki F , Gallin W J , Goldberg J I , Cunningham B A , Edelman G M
Published: May 2006

Pleiomorphic mouse sarcoma S180 cells were transfected with cDNAs for the liver cell adhesion molecule (L-CAM), the neural cell adhesion molecule (N-CAM), or both CAMs. Transfected cells expressed the appropriate CAMs at their surface and those expressing L-CAM (S180L cells) changed from adjoining spindle or round shapes to a closely linked "epithelioid" sheet when grown to confluence. Cells transfected with cDNA for N-CAM (S180N cells) also expressed this CAM on the cell surfaces and bound brain vesicles containing N-CAM but showed no phenotypic change to an epithelioid state. In S180L cells and doubly transfected (S180L/N) cells, L-CAM was concentrated at regions of cell contact and was codistributed with cortical actin. In S180N cells, N-CAM was uniformly distributed on the cell surface. When S180L cells were cocultured with S180L/N cells, N-CAM was not concentrated at boundaries between the S180L and S180L/N cells but was concentrated at boundaries between pairs of S180L/N cells. Fab' fragments of anti-L-CAM dissociated the epithelioid sheets of S180L or S180L/N cells into cells with shapes resembling those of untransfected cells. Cells in epithelioid sheets were polygonal in shape but, unlike cells in true epithelia, had no basement membrane or polar structure; they also lacked tight junctions and desmosomes. Ultrastructural examination showed that, in contrast to the untransfected phenotype, cells in epithelioid sheets had large increases in adherens junctions and gap junctions. Dye coupling experiments indicated that the gap junctions were functional. The frequency of expression of both kinds of junctions was sharply decreased by treatment with anti-L-CAM Fab' fragments. These experiments provide support for the precedence hypothesis, which proposes that the linkage of cells by means of CAMs is a necessary event for the extensive expression of junctional structures.
Rheoreceptors in the carotid sinus of dog.
Authors: Hajduczok G , Chapleau M W , Abboud F M
Published: May 2006

The arterial baroreceptors are known to be sensitive to changes in pressure but there are no known sensors in the cardiovascular system for changes in flow. We tested the hypothesis that changes in flow at constant pressure alter carotid sinus multi-unit nerve activity. In anesthetized dogs with vascularly isolated carotid sinuses, increases in flow at constant pressure resulted in increases in carotid sinus nerve activity in relation to the increase in flow. The increased activity during flow was not caused by an increase in strain of the sinus wall but was directly related to the increase in shear stress (36.6 +/- 11.7% increase in activity per dyne/cm2; 1 dyne = 0.1 MN). The pressure threshold of single baroreceptor units was determined during a slow pressure ramp with and without flow. Flow caused a significant decrease in pressure threshold from 81.1 +/- 6.1 mmHg (1 mmHg = 1.333 x 10(2) Pa) in the absence of flow to 69.3 +/- 5.7 mmHg with flow. We conclude that there are arterial "rheoreceptors" in the carotid sinus that respond to flow at constant pressure and strain. The results with single baroreceptor units indicate also that baroreceptors may be sensitized by increases in flow. Thus, changes in flow per se in addition to changes in arterial pressure may be important determinants of reflex circulatory adjustments.
Uptake of oleate by isolated rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut.
Authors: Schwieterman W , Sorrentino D , Potter B J , Rand J , Kiang C L , Stump D , Berk P D
Published: May 2006

A portion of the hepatocellular uptake of nonesterified long-chain fatty acids is mediated by a specific 40-kDa plasma membrane fatty acid binding protein, which has also been isolated from the gut. To investigate whether a similar transport process exists in other tissues with high transmembrane fatty acid fluxes, initial rates (Vo) of [3H]oleate uptake into isolated rat adipocytes were studied as a function of the concentration of unbound [3H]oleate in the medium. Vo reached a maximum as the concentration of unbound oleate was increased (Km = 0.30 +/- 0.03 microM; Vmax = 2470 +/- 90 pmol/min per 5 X 10(4) adipocytes) and was significantly inhibited both by phloretin and by prior incubation of the cells with Pronase. A rabbit antibody to the rat liver plasma membrane fatty acid binding protein inhibited adipocyte fatty acid uptake by up to 63% in dose-dependent fashion. Inhibition was noncompetitive; at an immunoglobulin concentration of 250 micrograms/ml Vmax was reduced from 2480 +/- 160 to 1870 +/- 80 pmol/min per 5 X 10(4) adipocytes, with no change in Km. A basic (pI approximately equal to 9.1) 40-kDa adipocyte plasma membrane fatty acid binding protein, isolated from crude adipocyte plasma membrane fractions, reacted strongly in both agar gel diffusion and electrophoretic blots with the antibody raised against the corresponding hepatic plasma membrane protein. These data indicate that the uptake of oleate by rat adipocytes is mediated by a 40-kDa plasma membrane fatty acid binding protein closely related to that in liver and gut.
Functional topography of band 3: specific structural alteration linked to functional aberrations in human erythrocytes.
Authors: Kay M M , Bosman G J , Lawrence C
Published: May 2006

Band 3 is the major anion transport polypeptide of erythrocytes. It appears to be the binding site of several glycolytic enzymes. Structurally, band 3 is the major protein spanning the erythrocyte membrane and connects the plasma membrane to band 2.1, which binds to the cytoskeleton. In the present study, we report an alteration of band 3 molecule that is associated with the following changes: erythrocyte shape change from discoid to "thorny cells" (acanthocytes), restriction of rotational diffusion of band 3 in the membrane, increase in anion transport, and decrease in the number of high-affinity ankyrin-binding sites. Changes in erythrocyte IgG binding, glyceraldehyde-3-phosphate dehydrogenase, fluorescence polarization (indicative of membrane fluidity), and other membrane proteins as determined by polyacrylamide gel electrophoresis were not detected. Cells containing the altered band 3 polypeptide were obtained from individuals with abnormal erythrocyte morphology. Two-dimensional peptide maps revealed differences in the Mr 17,000 anion transport segment of band 3 consistent with additions of tyrosines or tyrosine-containing peptides. The data suggest that (i) this alteration of band 3 does not result in accelerated aging as does cleavage and (ii) structural changes in the anion transport region result in alterations in anion transport.
Site of unequal sister chromatid exchange contains a potential Z-DNA-forming tract.
Authors: Weinreb A , Katzenberg D R , Gilmore G L , Birshtein B K
Published: May 2006

Previous studies from this laboratory have provided evidence that the duplication of the IgG2a heavy chain constant region gene (C gamma 2a) in the murine myeloma cell line MPC-11 occurred via unequal sister chromatid exchange. We now report the determination of the DNA sequences of the germ-line regions in which this exchange has occurred. The two donor sequences have long regions of virtual identity. Furthermore, both chromatids contain stretches of T-C and T-G dinucleotides; the latter has the potential to form Z-DNA. Localization of the actual breakpoints via genomic Southern blot analysis suggests a novel mechanism for the recombination and strongly implicates the simple sequence in the process.
Generation of telomere-length heterogeneity in Saccharomyces cerevisiae.
Authors: Shampay J , Blackburn E H
Published: May 2006

Chromosome ends in the lower eukaryotes terminate in variable numbers of tandem, simple DNA repeats. We tested predictions of a model in which these telomeric repeats provide a substrate for the addition of more repeats by a terminal transferase-like mechanism that, in concert with DNA polymerase and primase, effectively counterbalances the loss of DNA due to degradation or incomplete replication. For individual chromosome ends in yeast, the mean length of any given telomere was shown to vary between different clonal populations of the same strain and to be determined by the initial length of that telomere in the single cell giving rise to the clone. This type of variation was independent of the major yeast recombination pathway. The length heterogeneity at each telomeric end increased with additional rounds of cell division or DNA replication. Lengths of individual telomeres within a single clone varied independently of each other. Thus, this clonal variability is distinct from genetic regulation of chromosome length, which acts on all chromosome ends coordinately. These in vivo phenomena suggest that lengthening and shortening activities act on yeast telomeres during each round of replication.
pH homeostasis in Leishmania donovani amastigotes and promastigotes.
Authors: Glaser T A , Baatz J E , Kreishman G P , Mukkada A J
Published: May 2006

Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment.
Toward a population genetic analysis of Salmonella: genetic diversity and relationships among strains of serotypes S. choleraesuis, S. derby, S. dublin, S. enteritidis, S. heidelberg, S. infantis, S. newport, and S. typhimurium.
Authors: Beltran P , Musser J M , Helmuth R , Farmer J J , Frerichs W M , Wachsmuth I K , Ferris K , McWhorter A C , Wells J G , Cravioto A
Published: May 2006

Variation in the chromosomal genomes of 1527 isolates of eight common serotypes (O and H antigen profiles) of Salmonella was assessed by analysis of electrophoretically demonstrable allelic polymorphism at 23 metabolic enzyme loci. Seventy-one distinctive electrophoretic types, representing multilocus genotypes, were identified. A basically clonal population structure was indicated by the presence of strong linkage disequilibrium among enzyme loci, the association of each serotype with a relatively small number of multilocus enzyme genotypes, and the global distribution of certain genotypes. For each of six of the serotypes, 83-96% of isolates were members of a single clone. The occurrence of each of four serotypes (S. derby, S. enteritidis, S. infantis, and S. newport) in isolates of clones belonging to several evolutionary lineages, some of which are distantly related, suggests that the horizontal transfer and recombination of chromosomal genes mediating expression of cell-surface antigens has been a significant process in the evolution of the salmonellae. Two divergent clone clusters of S. derby differ in the relative frequency with which they cause disease in birds versus mammals, and two major lineages of S. newport differ in the frequency with which their clones are associated with disease in humans versus animals.
Insulin-like growth factor I stimulates erythropoiesis in hypophysectomized rats.
Authors: Kurtz A , Zapf J , Eckardt K U , Clemons G , Froesch E R , Bauer C
Published: May 2006

Stimulation of erythropoiesis during growth is necessary to ensure proportionality between erythrocyte mass and body mass. However, the way by which erythrocyte formation is adapted to body growth is still unknown. Growth arrest in hypophysectomized rats is accompanied by decreased erythropoiesis. We have, therefore, examined whether insulin-like growth factor I (IGF-I), the mediator of growth hormone effects on body growth, is able to restore erythropoiesis in these animals. Subcutaneous infusions of 120 micrograms of recombinant human IGF-I per day in hypophysectomized rats led to increases in body weight, 59Fe incorporation into erythrocytes, and the number of reticulocytes that were similar to increases caused by infusions of 28 milliunits of human growth hormone per day. Body weight gain and 59Fe incorporation were linearly correlated. Like growth hormone, IGF-I also caused a significant rise in serum erythropoietin concentrations. However, the stimulatory effect on erythropoiesis occurred before serum erythropoietin levels had risen. These results demonstrate that IGF-I mediates the stimulatory effect of growth hormone on erythropoiesis in vivo and thus further support the somatomedin concept. They also show that IGF-I can stimulate erythropoiesis in an endocrine manner, and they suggest two possible routes of action: a direct one and an indirect one by means of enhanced erythropoietin production.
Simple and effective purification of a Na+-dependent amino acid transport system from Ehrlich ascites cell plasma membrane.
Authors: McCormick J I , Johnstone R M
Published: May 2006

A reconstitution assay was used to measure transport activity during purification of a Na+-dependent amino acid transporter from Ehrlich cell plasma membrane. Cholate/urea-solubilized membranes were fractionated on a Sepharose 6B column and transport activity was recovered in the column void volume. Centrifugation of the void volume fraction at 105,000 X g and reextraction of the pellet with 1% octyl glucoside led to recovery of an extract whose specific transport activity was nearly 30-fold higher than that of the original solubilized extract with a recovery of 38% of the original activity. The properties of amino acid uptake in the purified reconstituted transporter were identical to those in native plasma membrane vesicles. The major component present in the purified fraction had a molecular mass of 120-130 kDa. Strong evidence that this 120- to 130-kDa peptide contains a component of the amino acid transporter was obtained by immunoprecipitation of transport activity from solubilized membranes with an antibody against the 120- to 130-kDa peptide. This study tentatively identifies a component of the Na+-dependent amino acid transporter as a peptide with an apparent molecular mass of 120-130 kDa.
Deletion in the first cysteine-rich repeat of low density lipoprotein receptor impairs its transport but not lipoprotein binding in fibroblasts from a subject with familial hypercholesterolemia.
Authors: Leitersdorf E , Hobbs H H , Fourie A M , Jacobs M , van der Westhuyzen D R , Coetzee G A
Published: May 2006

The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each approximately 40 amino acids long. Previous studies by van Driel et al. [van Driel, I. R., Goldstein, J. L., Sudhof, T. C. & Brown, M. S. (1987) J. Biol. Chem. 262, 17443-17449] showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated high-affinity cell-surface binding of 125I-labeled LDL but not 125I-labeled IgG-C7. Previous haplotype analysis, using 10 restriction fragment length polymorphic sites, suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new PstI restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate. Thus, deletion of two amino acids within the first cysteine-rich repeat retards receptor transport from the endoplasmic reticulum to the cell surface, in contrast to deletion of the entire first repeat, which has no effect on receptor maturation.
neu protooncogene fused to an immunoglobulin heavy chain gene requires immunoglobulin light chain for cell surface expression and oncogenic transformation.
Authors: Flanagan J G , Leder P
Published: May 2006

The protein encoded by the neu protooncogene (human gene symbol NGL for neuro/glioblastoma-derived) is a member of the surface receptor/tyrosine kinase family. Though its structure suggests that it can transduce a transmembrane signal, neither its extracellular ligand nor its critical intracellular substrates are known. To explore the functional properties of the protein encoded by neu, we created a fusion gene that joins the cytoplasmic domain of neu to the extracellular portion of an immunoglobulin heavy chain. The localization of the fusion polypeptide can then be controlled by coexpression with immunoglobulin light chain. In the absence of light chain, the heavy chain-neu polypeptide is expressed intracellularly and has no transforming activity. By contrast, in the presence of light chain the fusion polypeptide is expressed at the cell surface and produces tumorigenic foci. Thus, transformation apparently requires expression at the cell surface, where the neu intracellular domain can interact with components that are localized to the plasma membrane. The fusion protein is active in cellular transformation when the transmembrane domain is derived either from neu or from immunoglobulin, indicating that the neu transmembrane domain is not specifically required for transformation, although neu activation in tumors is known to result from a point mutation in this region. The extracellular immunoglobulin heavy and light chain domains of the fusion protein form a functional binding site that allows antigen to modulate its activity, reversing the transforming effect.
Aspartic acid at position 57 of the HLA-DQ beta chain protects against type I diabetes: a family study.
Authors: Morel P A , Dorman J S , Todd J A , McDevitt H O , Trucco M
Published: May 2006

One hundred seventy-two members from 27 randomly selected multiple case Caucasian families of patients with insulin-dependent diabetes mellitus (IDDM) were studied at the DNA level to ascertain the reliability of codon 57 of the HLA-DQ beta-chain gene as a disease protection/susceptibility marker. The analysis was carried out by polymerase chain reaction amplification of DNA encoding the first domain of the DQ beta chain and by dot blot analysis of the amplified material with allele-specific oligonucleotide probes. One hundred twenty-three randomly selected healthy Caucasian donors were also tested. The results demonstrated that haplotypes carrying an aspartic acid in position 57 (Asp-57) of their DQ beta chain were significantly increased in frequency among nondiabetic haplotypes (23/38), while non-Asp-57 haplotypes were significantly increased in frequency among diabetic haplotypes (65/69). Ninety-six percent of the diabetic probands in our study were homozygous non-Asp/non-Asp as compared to 19.5% of healthy unrelated controls. This conferred a relative risk of 107 (chi 2 = 54.97; P = 0.00003) for non-Asp-57 homozygous individuals. Even though the inheritance and genetic features of IDDM are complex and are not necessarily fully explained by DQ beta chain polymorphism, this approach is much more sensitive than HLA serolog in assessing risk for IDDM.
Inoculation with BK virus may break immunological tolerance to histone and DNA antigens.
Authors: Flaegstad T , Fredriksen K , Dahl B , Traavik T , Rekvig O P
Published: May 2006

BK virus particles contain histones of host cell origin that combine with viral DNA to form minichromosomes. Data from earlier immunoblotting experiments demonstrated that rabbits inoculated i.v. with purified infectious BK virus produced antibodies not only to the viral structural protein VP1 but also to migrating polypeptides of a molecular mass ranging from 14-16 kDa. These proteins were believed to represent certain histone classes. To examine this hypothesis, sera from five rabbits inoculated with BK virus were analyzed by ELISA for antibodies against polynucleosomes, the individual histone classes, and double-stranded DNA, as well as against antigens carried by the structural viral proteins. Antibodies against polynucleosomes and also against histones H1 and H3 were found in sera from two of five inoculated rabbits. The same sera contained antibodies reacting with double-stranded DNA, whereas no antibodies against H2A, H2B, and H4 were detected. BK virus inoculation may thus lead to a break of tolerance, resulting in autoantibody production against highly conserved antigens that, in this context, may be regarded as "self-antigens", irrespective of the species in which they originate.
Cytoplasmic pH determines K+ conductance in fused renal epithelial cells.
Authors: Oberleithner H , Kersting U , Hunter M
Published: May 2006

The mineralocorticoid hormone aldosterone maintains acid-base balance and K+ homeostasis by regulating H+ and K+ secretory mechanisms in kidney epithelial cells. We have shown recently in the amphibian distal nephron that aldosterone activates a Na+/H+ exchange system in the luminal cell membrane, thus leading to transepithelial H+ secretion and cytoplasmic alkalinization. Since H+ secretory fluxes were paralleled by K+ secretion, it was postulated that the hormone-induced increase of intracellular pH activates the luminally located K+ channels. In "giant" cells fused from individual cells of the distal nephron, we measured simultaneously cytoplasmic pH and cell membrane K+ conductance during acidification of the cell cytoplasm. The experiments show that cell membrane K+ conductance is half-maximal at an intracellular pH of 7.42 and that a positive cooperative interaction exists between K+-channel proteins and H+ (Hill coefficient = 6.5). Moreover, the cellular K+ conductance is most sensitive to cytoplasmic pH in the range modified by aldosterone. This supports the hypothesis that intracellular H+ activity, regulated by the Na+/H+ exchanger, serves as the signal to couple aldosterone-induced K+ secretory flux to H+ secretion in renal tubules.
Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell-surface receptors in the perfused rat liver by the multiple-indicator dilution method.
Authors: Sato H , Sugiyama Y , Sawada Y , Iga T , Sakamoto S , Fuwa T , Hanano M
Published: May 2006

Hepatic elimination of epidermal growth factor (EGF) via receptor-mediated endocytosis was studied by a multiple-indicator dilution method in the isolated perfused rat liver, in which cell polarity and spatial organization are maintained. In this method EGF was given with inulin, an extracellular reference, as a bolus into the portal vein, and dilution curves of both compounds in the hepatic vein effluent were analyzed. Analysis of the dilution curve for EGF, compared with that for somatostatin, which showed no specific binding to isolated liver plasma membranes, resulted as follows: (i) both extraction ratio and distribution volume of 125I-labeled EGF decreased as the injected amount of unlabeled EGF increased; (ii) the ratio plot [ln (inulin/EGF) versus time] of the dilution curve for EGF exhibited an upward straight line initially for a short period of time (approximately equal to 10 sec), whereas the ratio plot [ln (inulin/somatostatin) versus time] of somatostatin gradually decreased. The multiple-indicator dilution method was used for other peptides also. Insulin and glucagon, known to have hepatocyte receptors, behaved similarly to EGF in shape of their ratio plots. Thus, analysis of dilution curves can reveal whether or not the cell surface has receptors for certain peptides. In addition, the dilution curves for EGF at various doses (tracer approximately equal to 30 micrograms) were analyzed simultaneously based on a kinetic model incorporating the perfusion rate, the association rate constant of EGF to surface receptors (kappa on), the dissociation rate constant of EGF from the EGF-receptor complex (kappa off), and the sequestration rate constant of the complex. The kinetic parameters [the dissociation constant (Kd = kappa off/kappa on) and the number of surface receptors] calculated by this analysis were comparable with reported values obtained by in vitro direct binding measurements at equilibrium using liver homogenates. We conclude that the multiple-indicator dilution method is a good tool for analyzing the dynamics of peptide hormones--cell-surface receptor interaction under a condition in which spatial architecture of the liver is maintained.
Receptor interactions through phosphorylation and methylation pathways in bacterial chemotaxis.
Authors: Sanders D A , Koshland D E
Published: May 2006

The effects of messages initiated by one receptor on the covalent modification of a second receptor were studied by use of a technique for rapidly separating the receptors. Methylation of the bacterial-chemotactic serine receptor increases as a result of aspartate binding to the aspartate receptor. The aspartate-induced methylation on the serine receptor is absent in a strain that lacks cheA and cheW genes and is not the result of physical interaction, such as the formation of heterodimers between the aspartate and serine receptors, or of alterations in the affinity of the serine receptor for the methyltransferase and the methylesterase. Serine-induced methylation of the serine receptor did not require cheA and cheW. A model is presented in which the receptor methylation level depends on the combination of (i) a ligand-induced conformational change on the receptor substrate of the methylation enzymes and (ii) an indirect cytoplasmic signal that operates through the methylesterase.