bluuebunny/crossref_metadata_2025 huggingface dataset:
"E3/19K" protein of adenovirus type 2 inhibits lysis of cytolytic T lymphocytes by blocking cell-surface expression of histocompatibility class I antigens.
Authors: Burgert H G , Maryanski J L , Kvist S
Published: May 2006

The E3 19,000-dalton protein termed "E3/19K" of adenovirus type 2 binds to human class I histocompatibility antigens (HLA antigens). Human 293.12 cultured cells that express a cloned gene for the E3/19K protein show reduced levels of HLA antigens on the cell surface compared to parental 293 cells. We have transfected these cell lines with plasmid DNA containing the murine histocompatibility H-2Kd allele to demonstrate that this antigen binds also to the E3/19K protein. The resulting association prevents the H-2Kd antigen from being terminally glycosylated and inhibits its cell-surface expression. Two murine cytolytic T-lymphocyte clones specific for HLA antigens and restricted by the H-2Kd antigen lyse the human 293Kd cells. In the presence of the E3/19K protein, a dramatically reduced cell surface density of both HLA and H-2Kd antigens was shown. This decreased amount of cell-surface HLA/H-2Kd antigens correlated with a reduction in susceptibility to lysis of the target cells. In particular, the cell-surface level of the H-2Kd antigen, which is the restricting element, was crucial for efficient lysis. Thus, the E3/19K protein of adenovirus type 2 indirectly reduces the cellular immune recognition in the in vitro system. This might be the mechanism involved in latent and persistent infections caused by adenoviruses in vivo.
Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma model.
Authors: Katre N V , Knauf M J , Laird W J
Published: May 2006

Recombinant human interleukin 2 purified from Escherichia coli has limited solubility at neutral pH and a short circulatory half-life. This recombinant interleukin 2 was chemically modified by an active ester of polyethylene glycol. The modified interleukin 2 was purified by hydrophobic interaction chromatography and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and isoelectric focusing. This conjugate was compared to unmodified recombinant interleukin 2 in vitro and in vivo. Covalent attachment of the hydrophilic polymer polyethylene glycol enhanced the solubility of interleukin 2, decreased its plasma clearance, and increased its antitumor potency in the Meth A murine sarcoma model.
Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells.
Authors: Zimmerberg J , Curran M , Cohen F S , Brodwick M
Published: May 2006

Mast cells show dramatic morphological changes when undergoing exocytosis. We have investigated whether the first of those morphological changes, swelling of the secretory granule, precedes--and therefore possibly initiates--secretion or whether it occurs after fusion of the granule and plasma membranes. We used cell membrane capacitance to detect the moment when granule and plasma membrane become continuous. We measured large capacitance increases, often preceded by transients in capacitance. The rise-times of the capacitance increases were half-maximal at 2-59 msec. We observed cells with high-resolution video microscopy while these measurements were done. The capacitance increase always preceded the granular swelling that leads to exocytosis. To rule out the possibility that fusion was induced by a mechanical stress imparted by the internal pressure of a taut granule, we performed control experiments using cells in which vesicles were shrunken with hyperosmotic solutions. With these flaccid granules, again, the capacitance rise always preceded the swelling of the granules. We conclude that swelling cannot be the driving force for membrane fusion in this system.
Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes.
Authors: Hershko A , Rose I A
Published: May 2006

The generation and characterization of ubiquitin (Ub)-aldehyde, a potent inhibitor of Ub-C-terminal hydrolase, has previously been reported. We now examine the action of this compound on the Ub-mediated proteolytic pathway using the system derived from rabbit reticulocytes. Addition of Ub-aldehyde was found to strongly inhibit breakdown of added 125I-labeled lysozyme, but inhibition was overcome by increasing concentrations of Ub. The following evidence shows the effect of Ub-aldehyde on protein breakdown to be indirectly caused by its interference with the recycling of Ub, leading to exhaustion of the supply of free Ub: Ub-aldehyde markedly increased the accumulation of Ub-protein conjugates coincident with a much decreased rate of conjugate breakdown. release of Ub from isolated Ub-protein conjugates in the absence of ATP (and therefore not coupled to protein degradation) is markedly inhibited by Ub-aldehyde. On the other hand, the ATP-dependent degradation of the protein moiety of Ub conjugates, which is an integral part of the proteolytic process, is not inhibited by this agent. Direct measurement of levels of free Ub showed a rapid disappearance caused by the inhibitor. The Ub is found to be distributed in derivatives of a wide range of molecular weight classes. It thus seems that Ub-aldehyde, previously demonstrated to inhibit the hydrolysis of Ub conjugates of small molecules, also inhibits the activity of a series of enzymes that regenerate free Ub from adducts with proteins and intermediates in protein breakdown.
Interrelationship between growth factor-induced pH changes and intracellular Ca2+.
Authors: Ives H E , Daniel T O
Published: May 2006

Many mitogens cause rapid changes in intracellular pH and Ca2+. We studied the patterns of pH and Ca2+ changes after exposure of murine fibroblasts to platelet-derived growth factor (PDGF), bombesin, phorbol 12-myristate 13-acetate (PMA), and the vasoactive peptide bradykinin. Intracellular pH and Ca2+ were measured by using the fluorescent dyes 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2. Three distinct patterns of intracellular pH change were observed. PDGF and bombesin caused a rapid (maximum change, less than 2 min) cytoplasmic acidification of 0.03 pH unit followed by a slower (5-10 min) alkalinization of approximately 0.11 pH unit above the resting pH of 6.88. PMA caused alkalinization without causing the early acidification. Bradykinin caused rapid acidification without the slower net alkalinization. Ionomycin also caused acidification without subsequent alkalinization. All acidification responses were amiloride resistant. Patterns of intracellular Ca2+ response were also determined for each agent. PDGF and bombesin caused a transient increase in cytoplasmic Ca2+ from a resting level of 85 +/- 12 nM to 190 +/- 12 nM within 2 min and return to baseline within 5 min. PMA caused no change in intracellular Ca2+. Bradykinin caused the most rapid (maximum response, less than 20 sec) increase in intracellular Ca2+. For each agonist, the Ca2+ transient could be blocked by buffering intracellular Ca2+ with quin-2. In Ca2+-buffered cells, PDGF, bombesin, bradykinin, and ionomycin failed to induce cellular acidification, but alkalinization responses to PDGF, bombesin, and PMA persisted. We propose that the transient acidification seen with PDGF, bombesin, and other agents is the result of increased intracellular Ca2+. However, growth factor-induced alkalinization via the Na+/H+ exchanger is independent of changes in Ca2+.
On the transfer of integral proteins into membranes.
Authors: Singer S J , Maher P A , Yaffe M P
Published: May 2006

We have earlier proposed a molecular mechanism for the translocation of hydrophilic proteins across membranes that accounts for the experimental facts and meets the restrictions that we stipulate for such a mechanism. In particular, the restrictions are that translocation occurs by successive segments of the polypeptide chain and that the ionic groups of the polypeptide remain in contact with water throughout the translocation process. The evidence indicates that the transfer of integral proteins into membranes very likely uses the same molecular machinery as does the translocation of hydrophilic proteins across membranes. Here we show how the mechanism we have proposed for translocation can also be utilized in the intercalation of known types of integral proteins, accounting for their specific topologies in the membrane.
Intrahypothalamic injection of insulin decreases firing rate of sympathetic nerves.
Authors: Sakaguchi T , Bray G A
Published: May 2006

Injection of picomolar quantities of insulin into the ventromedial hypothalamus of rats significantly reduced the firing rate of sympathetic nerves that supply interscapular brown adipose tissue. The minimal firing rate was reached in 2 min, and the effect was gone within 4 min. The effect of insulin was dose-related and did not occur when comparable volumes of physiological saline were injected into the ventromedial hypothalamus. Destruction of neurons in the ventromedial hypothalamus by injection of kainic acid abolished the inhibitory effects of insulin. These data suggest that insulin may play a role in modulating the sympathetic firing rate to thermogenically important tissues.
Modulation of type beta transforming growth factor activity in bone cultures by osteotropic hormones.
Authors: Pfeilschifter J , Mundy G R
Published: May 2006

Type beta transforming growth factor (TGF-beta) activity was found in conditioned medium harvested from fetal rat and neonatal mouse calvariae by using the anchorage-independent growth of normal rat kidney fibroblasts as an indicator system. Calvariae incubated with parathyroid hormone, 1,25-dihydroxyvitamin D3, and interleukin 1, all factors that stimulate bone resorption showed a concentration-dependent increase in TGF-beta activity in the culture medium. Increases in TGF-beta activity persisted undiminished for 48 hr after removal of these factors. The increases in TGF-beta activity from the resorbing bone cultures were relatively greater than the increases in bone resorption. Calcitonin inhibition of bone resorption correlated with a decrease in TGF-beta activity. Thus, agents that modulate bone resorption also affect TGF-beta activity in the bone culture medium. Changes in local concentrations of TGF-beta activity by osteotropic hormones may be important in the regulation of normal bone remodeling.
Induction of differentiation of human promyelocytic leukemia cell line HL-60 by retinoyl glucuronide, a biologically active metabolite of vitamin A.
Authors: Zile M H , Cullum M E , Simpson R U , Barua A B , Swartz D A
Published: May 2006

We examined the differentiation activity of retinoyl beta-D-glucuronide, a biologically active physiological metabolite of vitamin A, using the human promyelocytic leukemic cell line HL-60, which can be induced to differentiate with retinoic acid. Retinoyl beta-D-glucuronide (1 microM) inhibited HL-60 cell proliferation by 55-75%, inhibited tritiated thymidine incorporation into DNA by 63-80%, and induced 38-50% of the cells to differentiate into mature granulocytes. The potency of growth inhibition and induction of differentiation by retinoyl beta-D-glucuronide was similar to that of all-trans-retinoic acid. The continuous presence of either retinoyl beta-D-glucuronide or all-trans-retinoic acid was not required to obtain maximum growth arrest and differentiation: a 1-hr exposure of HL-60 cells to the retinoids gave the same response (measured after a total incubation time of 48 hr) as a 24-hr or 48-hr continuous treatment. Retinoyl beta-D-glucuronide (0.1-0.2 mM) was 50% less cytotoxic to HL-60 cells than all-trans-retinoic acid at an equimolar concentration. Retinoyl beta-D-glucuronide was not significantly metabolized to other retinoids; retinoic acid was not formed during incubation. We conclude that retinoyl beta-D-glucuronide can arrest HL-60 cell proliferation and induce their differentiation into mature granulocytes; it may act by itself or by being hydrolyzed to retinoic acid, which could be immediately utilized and metabolized. The therapeutic use of this retinoid as an antineoplastic agent is suggested.
Primary structure of human neutrophil elastase.
Authors: Sinha S , Watorek W , Karr S , Giles J , Bode W , Travis J
Published: May 2006

The complete amino acid sequence of human neutrophil elastase has been determined. The protein consists of 218 amino acid residues, contains two asparagine-linked carbohydrate side chains, and is joined together by four disulfide bonds. Comparison of the sequence to other serine proteinases indicates only moderate homology with porcine pancreatic elastase (43.0%) or neutrophil cathepsin G (37.2%). In particular, many of the residues suggested to play important roles in the mechanism by which the pancreatic elastase functions are significantly changed in the neutrophil enzyme, indicating alternative types of binding with the human proteinase.
Pressure-sensitive ion channel in Escherichia coli.
Authors: Martinac B , Buechner M , Delcour A H , Adler J , Kung C
Published: May 2006

We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherichia coli and have discovered pressure-activated ion channels. The channels have the following properties: activation by slight positive or negative pressure; voltage dependence; large conductance; selectivity for anions over cations; dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.
Three clonal types of keratinocyte with different capacities for multiplication.
Authors: Barrandon Y , Green H
Published: May 2006

Colony-forming human epidermal cells are heterogeneous in their capacity for sustained growth. Once a clone has been derived from a single cell, its growth potential can be estimated from the colony types resulting from a single plating, and the clone can be assigned to one of three classes. The holoclone has the greatest reproductive capacity: under standard conditions, fewer than 5% of the colonies formed by the cells of a holoclone abort and terminally differentiate. The paraclone contains exclusively cells with a short replicative lifespan (not more than 15 cell generations), after which they uniformly abort and terminally differentiate. The third type of clone, the meroclone, contains a mixture of cells of different growth potential and is a transitional stage between the holoclone and the paraclone. The incidence of the different clonal types is affected by aging, since cells originating from the epidermis of older donors give rise to a lower proportion of holoclones and a higher proportion of paraclones.
Interferons regulate the in vitro differentiation of multilineage lympho-myeloid stem cells in hairy cell leukemia.
Authors: Michalevicz R , Revel M
Published: May 2006

In vitro 14-day cultures of peripheral blood mononuclear cells from hairy cell leukemia patients consistently showed the presence of hematopoietic stem cells giving rise to multilineage colonies containing a high proportion of lymphoid cells associated with the myeloid and erythroid progenitors. These stem cells are not the hairy cells but appear to be pluripotent lymphomyeloid primitive stem cells persisting in this leukemia. Interferon alpha c or beta 1 did not inhibit the growth of these colonies, as they did the growth of colonies of normal hematopoietic progenitors, but markedly decreased the ratio of lymphoid to myelomonocytic cells, by increasing the formation of monocytes and other nonlymphoid cell types in these multilineage colonies. Interferon gamma did not have the same effects on differentiation.
Construction of multilocus genetic linkage maps in humans.
Authors: Lander E S , Green P
Published: May 2006

Human genetic linkage maps are most accurately constructed by using information from many loci simultaneously. Traditional methods for such multilocus linkage analysis are computationally prohibitive in general, even with supercomputers. The problem has acquired practical importance because of the current international collaboration aimed at constructing a complete human linkage map of DNA markers through the study of three-generation pedigrees. We describe here several alternative algorithms for constructing human linkage maps given a specified gene order. One method allows maximum-likelihood multilocus linkage maps for dozens of DNA markers in such three-generation pedigrees to be constructed in minutes.
Dual control mechanism for heme oxygenase: tin(IV)-protoporphyrin potently inhibits enzyme activity while markedly increasing content of enzyme protein in liver.
Authors: Sardana M K , Kappas A
Published: May 2006

Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in vitro and in vivo, a property that confers upon this synthetic compound the ability to suppress a variety of experimentally induced and naturally occurring forms of jaundice in animals and humans. Utilizing rat liver heme oxygenase purified to homogeneity together with appropriate immunoquantitation techniques, we have demonstrated that Sn-protoporphyrin possesses the additional property of potently inducing the synthesis of heme oxygenase protein in liver cells while, concurrently, completely inhibiting the activity of the newly formed enzyme. Substitution of tin for the central iron atom of heme thus leads to the formation of a synthetic heme analogue that regulates heme oxygenase by a dual mechanism, which involves competitive inhibition of the enzyme for the natural substrate heme and simultaneous enhancement of new enzyme synthesis. Cobaltic(III)-protoporphyrin (Co-protoporphyrin) also inhibits heme oxygenase activity in vitro, but unlike Sn-protoporphyrin it greatly enhances the activity of the enzyme in the whole animal. Co-protoporphyrin also acts as an in vivo inhibitor of heme oxygenase; however, its inducing effect on heme oxygenase synthesis is so pronounced as to prevail in vivo over its inhibitory effect on the enzyme. These studies show that certain synthetic heme analogues possess the ability to simultaneously inhibit as well as induce the enzyme heme oxygenase in liver. The net balance between these two actions, as reflected in the rate of heme oxidation activity in the whole animal, appears to be influenced by the nature of the central metal atom of the synthetic metalloporphyrin.
Molecular structure of mammalian neuropeptide Y: analysis by molecular cloning and computer-aided comparison with crystal structure of avian homologue.
Authors: Allen J , Novotný J , Martin J , Heinrich G
Published: May 2006

Identification and characterization of the cDNA encoding rat neuropeptide Y revealed the nucleotide sequence coding for a 98-amino acid precursor. The deduced amino acid sequence for rat neuropeptide Y is identical to the human peptide and is highly homologous to avian pancreatic polypeptide. The tertiary structure of avian pancreatic polypeptide has been previously derived from crystallographic data by Blundell and coworkers. The homology between neuropeptide Y and avian pancreatic polypeptide preserves all of the residues essential for the maintenance of the tertiary structure. Thus, it has been possible to compute a three-dimensional model of the mammalian neuropeptide, neuropeptide Y, based on the known structure of the avian homologue. This model suggest that neuropeptide preserves a compact tertiary structure characterized by extensive hydrophobic interactions between an N-terminal polyproline-II-like helix and a C-terminal alpha-helix. The model has been used to identify amino acids residing in key positions within this structure and, thereby, to direct future analysis of neuropeptide Y structure-function relationships.
Cloning and heterologous expression of glycosidase genes from Saccharomyces cerevisiae.
Authors: Kuranda M J , Robbins P W
Published: May 2006

Genomic clones were isolated that code for three glycosidases proposed to be involved in the catabolism of cell wall components in Saccharomyces cerevisiae. alpha-Mannosidase (AMS1), exoglucanase (BGL1), and endochitinase (CTS1) genes were isolated with the aid of filter assays based on the hydrolysis of 4-methylumbelliferyl glycosides, which permitted the in situ monitoring of these glycosidase activities in yeast colonies. Uracil prototrophs resulting from transformation with a multicopy YEp24 yeast genomic library were screened, leading to the identification of transformants possessing high levels of glycosidase activity. Restriction maps of plasmids from multiple isolates were used to localize glycosidase-overproduction genes, which were subcloned into a Schizosaccharomyces pombe/S. cerevisiae shuttle vector. Transformation of Sch. pombe with BGL1 and CTS1 subclones resulted in the appearance of these activities in this organism, and an AMS1 plasmid caused a 2-fold increase in endogenous alpha-mannosidase levels. Insertion of the marker gene LEU2 into putative AMS1 sequences disrupted plasmid-encoded alpha-mannosidase overproduction. S. cerevisiae strains that incorporated a restriction fragment containing ams1::LEU2 into their chromosomal DNA by homologous recombination expressed no detectable alpha-mannosidase activity in either the haploid or homozygous recessive diploid states, whereas heterozygous and wild-type cells exhibited levels proportional to AMS1 gene dosage. No readily apparent phenotype was associated with the alpha-mannosidase deficiency; however, labeling experiments utilizing [2-3H]mannose suggest that alpha-mannosidase may function in mannan turnover.
Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase.
Authors: Towler D A , Adams S P , Eubanks S R , Towery D S , Jackson-Machelski E , Glaser L , Gordon J I
Published: May 2006

Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of approximately equal to 500 in the apparent Km in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.
Stimulation of proliferation, differentiation, and function of human cells by primate interleukin 3.
Authors: Lopez A F , To L B , Yang Y C , Gamble J R , Shannon M F , Burns G F , Dyson P G , Juttner C A , Clark S , Vadas M A
Published: May 2006

Cloned gibbon interleukin 3 (gIL-3) was found to stimulate the proliferation and differentiation of human bone marrow cells to produce day-14 granulocyte, macrophage, granulocyte-macrophage, and eosinophil colonies in semisolid agar. In the presence of normal human plasma, gIL-3 stimulated megakaryocytes. In methylcellulose cultures, it stimulated erythroid colonies in the presence, but not in the absence, of erythropoietin. When mature human leukocytes were used, gIL-3 stimulated the function of purified mature eosinophils as measured by the capacity to kill antibody-coated target cells, to produce superoxide anions, and to phagocytize opsonized yeast particles in a manner similar to recombinant human granulocyte-macrophage colony-stimulating factor. In contrast, gIL-3 did not significantly stimulate any of the neutrophil functions tested, whereas human recombinant granulocyte-macrophage colony-stimulating factor was active in these assays. Among cytokines that are active on human hematopoietic cells, gIL-3 thus has a distinct set of functions and may predict the range of actions of the human molecule.
Translational control of photo-induced expression of the Cat2 catalase gene during leaf development in maize.
Authors: Skadsen R W , Scandalios J G
Published: May 2006

In dark-grown leaves of Zea mays, the catalase (H2O2:H2O2 oxidoreductase; EC 1.11.1.6; CAT) isozyme CAT-2 is absent. With continuous white light, CAT-2 protein levels increase (due to de novo synthesis) and plateau after 24 hr. When total poly(A)+ RNA (mRNA), polysomes, or isolated polysomal mRNA from light- and dark-treated leaves was translated in vitro, CAT-2 was detected only among the light-treated leaf products. A Cat2 clone was used to probe blots of total mRNA and polysomal mRNA from light- and dark-treated leaves. Cat2 mRNA was found in approximately equal quantities in both. Cat2 mRNA was equally distributed in identical high molecular weight fractions in polysomes from light- and dark-treated leaves and, therefore, was probably not sequestered in ribonucleoprotein particles in dark-grown leaves. The control of Cat2 expression appears to involve a unique form of translational inhibition in dark-grown leaves, preventing translation of the isolated mRNA and preventing polypeptide elongation. These results may have important implication in studies of translational control in other systems.
The progesterone receptor gene maps to human chromosome band 11q13, the site of the mammary oncogene int-2.
Authors: Law M L , Kao F T , Wei Q , Hartz J A , Greene G L , Zarucki-Schulz T , Conneely O M , Jones C , Puck T T , O'Malley B W
Published: May 2006

Progesterone is involved in the development and progression of breast cancers, and progesterone receptors (PR) are important markers of hormone dependence and disease prognosis. We have used a human PR cDNA probe, genomic DNA blotting of a series of Chinese hamster-human cell hybrids, and in situ hybridization to map the human PR gene to chromosome 11, band q13. This band also contains the human homolog of the mouse mammary tumor virus integration site, int-2, which surrounds a protooncogene thought to be involved in the development of murine mammary cancers. That these two genes share the same chromosomal location raises important questions about their possible linkage and about the relationship between the mammary-specific oncogene and the steroid hormone in the development, growth, and hormone dependence of human breast cancers.
Human epidermal Langerhans cells cointernalize by receptor-mediated endocytosis "nonclassical" major histocompatibility complex class I molecules (T6 antigens) and class II molecules (HLA-DR antigens).
Authors: Hanau D , Fabre M , Schmitt D A , Garaud J C , Pauly G , Tongio M M , Mayer S , Cazenave J P
Published: May 2006

HLA-DR and T6 surface antigens are expressed only by Langerhans cells and indeterminate cells in normal human epidermis. We have previously demonstrated that T6 antigens are internalized in Langerhans cells and indeterminate cells by receptor-mediated endocytosis. This process is induced by the binding of BL6, a monoclonal antibody directed against T6 antigens. In the present study, using a monoclonal antibody directed against HLA-DR antigens, on human epidermal cells in suspension, we show that the surface HLA-DR antigens are also internalized by receptor-mediated endocytosis in Langerhans and indeterminate cells. Moreover, using immunogold double labeling, we demonstrate that T6 and HLA-DR antigens are internalized through common coated regions of the membrane of Langerhans or indeterminate cells. The receptor-mediated endocytosis that is induced involves coated pits and vesicles, receptosomes, lysosomes, and also, in Langerhans cells, the Birbeck granules. Thus, T6 antigens, which are considered to be "unusual" or "nonclassical" major histocompatibility complex class I molecules, and the major histocompatibility complex class II molecules, HLA-DR, are internalized in Langerhans and indeterminate cells through common receptor-mediated endocytosis organelles.
Molecular cloning of a gene belonging to the carcinoembryonic antigen gene family and discussion of a domain model.
Authors: Thompson J A , Pande H , Paxton R J , Shively L , Padma A , Simmer R L , Todd C W , Riggs A D , Shively J E
Published: May 2006

Carcinoembryonic antigen (CEA) is a glycoprotein important as a tumor marker for colonic cancer. Immunological and biochemical studies have shown it to be closely related to a number of other glycoproteins, which together make up a gene family. We have cloned a member of this gene family by using long oligonucleotide probes (42-54 nucleotides) based on our protein sequence data for CEA and NCA (nonspecific cross-reacting antigen) and on human codon usage. The clone obtained (lambda 39.2) hybridizes with six probes and has a 15-kilobase insert. The 5' end of the gene is contained within a 2700-base-pair EcoRI fragment, which hybridizes with five of the six synthetic probes. Sequencing of the 5' end region revealed the location and structure of one exon and two putative intron boundaries. The exon encodes part of the leader sequence and the NH2-terminal 107 amino acids of NCA. Southern blot analysis of human normal and tumor DNA, using as probes two lambda 39.2 fragments that contain coding sequences, suggests the existence of 9-11 genes for the CEA family. One of the restriction fragments described here has been used by Zimmermann et al. [Zimmermann, W., Ortlieb, B., Friedrich, R. & von Kleist, S. (1987) Proc. Natl. Acad. Sci. USA 84, 2960-2964] to isolate partial cDNA clones for CEA. The identity of this clone was verified with our protein sequence data [Paxton, R., Mooser, G., Pande, H., Lee, T.D. & Shively, J.E. (1987) Proc. Natl. Acad. Sci. USA 84, 920-924]. We discuss a domain structure for CEA based on the CEA sequence data and the NCA exon sequence data. It is likely that this gene family evolved from a common ancestor shared with neural cell adhesion molecule and alpha 1 B-glycoprotein and is perhaps a family within the immunoglobulin superfamily.
Oxidatively modified low density lipoproteins: a potential role in recruitment and retention of monocyte/macrophages during atherogenesis.
Authors: Quinn M T , Parthasarathy S , Fong L G , Steinberg D
Published: May 2006

Previous studies in this laboratory established that low density lipoprotein (LDL) incubated with cultured endothelial cells, smooth muscle cells, or macrophages undergoes free radical-catalyzed oxidative modification that generates lipid peroxides and extensive structural changes in the LDL molecule. The oxidatively modified LDL strongly inhibited chemotactic responses of the mouse resident peritoneal macrophage. The present studies show that this oxidized LDL does not inhibit the motility of mouse monocytes and actually exhibits a chemotactic activity for human monocytes; the chemotactic activity of the oxidized LDL resides in the lipid fraction. These findings allow us to propose a pathogenetic sequence by which elevated plasma LDL levels, followed by oxidative modification in the arterial wall, could sufficiently account for the generation of the lipid-laden foam cells and the initiation of the fatty streak, the earliest well-defined lesion in atherogenesis.
Screening an expression library with a ligand probe: isolation and sequence of a cDNA corresponding to a brain calmodulin-binding protein.
Authors: Sikela J M , Hahn W E
Published: May 2006

The use of cloning vectors that express inserted cDNA as fusion protein has led to the isolation of genes encoding a variety of eukaryotic proteins. In these instances antisera or monoclonal antibodies were used as probes to screen expression libraries. Since fusion proteins sometimes display biological activity reflective of the insert-specified portion, we tested the possibility that ligand-binding sites might exist in fusion proteins. Specifically we used 125I-labeled calmodulin as a probe to screen a mouse brain lambda gt11 library. One clone, lambda ICM-1 isolated using this approach, produces fusion protein that binds calmodulin with high affinity (Kd, 3-10 nM) in a Ca2+-dependent manner. Molecular genetic mapping experiments and deduction of the predicted higher-order structure from sequence data indicate the binding site is, or is within, a basic, amphiphilic alpha-helical domain composed of approximately 20 amino acids. lambda ICM-1 hybridizes with brain mRNA of 2.1 and 3.5 kb but not with mRNA from liver or kidney, suggesting possible restriction of the protein to brain. We discuss several observations that suggest lambda ICM-1 corresponds to Ca2+/calmodulin-dependent protein kinase II, an enzyme that phosphorylates several neuronal proteins, some of which apparently play a role in synaptic function. Our results suggest certain types of ligands may be useful probes to isolate genes encoding various receptor proteins, particularly when the protein is very rare or when it is difficult to obtain antibodies suitable for screening libraries.
Expression of human epidermal growth factor precursor cDNA in transfected mouse NIH 3T3 cells.
Authors: Mroczkowski B , Reich M , Whittaker J , Bell G I , Cohen S
Published: May 2006

Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 microM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approximately 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.
Expression of the insulin-like growth factor II gene in the choroid plexus and the leptomeninges of the adult rat central nervous system.
Authors: Stylianopoulou F , Herbert J , Soares M B , Efstratiadis A
Published: May 2006

The rat insulin-like growth factor II gene, encoding a fetal somatomedin, expresses a multitranscript family in embryonic/fetal tissues and in the adult brain and spinal cord. By performing in situ hybridization on tissue sections of adult brain and spinal cord, we have found that these transcripts are not expressed in neural or glial cells but are expressed in the epithelium of the choroid plexus of each cerebral ventricle and in the leptomeninges. We propose that the choroidal epithelial cells synthesize and secrete insulin-like growth factor II into the cerebrospinal fluid.
Sodium-coupled glycine uptake by Ehrlich ascites tumor cells results in an increase in cell volume and plasma membrane channel activities.
Authors: Hudson R L , Schultz S G
Published: May 2006

The addition of 10 mM glycine to a physiological saline bathing Ehrlich ascites tumor cells is followed by a slow increase in cell volume that plateaus between 15 and 30 min at a level approximately equal to 17% greater than the control volume; this increase is not observed when glycine is added to cells suspended in a Na+-free saline. The results of studies using the patch-clamp technique in the cell-attached mode indicate that, 0.5-3 min after the addition of glycine to the bathing solution, there is a marked increase in the activity of single channels, which is almost all instances were previously present and operant in the plasma membrane. Successfully excised patches of membrane that contained a channel stimulated by glycine fell into two categories. Some became inactive within 15 sec in spite of the fact that the G omega seal remained intact. Others persisted for the lifetime of the seal. All of the persistent channels had an 11-fold selectivity for Cl- over K+ and a conductance of 23 pS when bathed by symmetrical 150 mM KCl solutions. Although the ionic specificities of the other channels have not been identified, there is reason to suspect that they might be K+ channels whose activities are dependent on factors lost when the patch is excised. Swelling induced by exposing these cells to a 50% hypotonic perfusate stimulated the activities of Cl- channels whose properties closely resemble those stimulated by the addition of glycine to the perfusate, strongly suggesting that the glycine-induced stimulation of Cl- channel activity is part of a volume-regulatory response to cell swelling. If the increase in channel activity induced by the addition of glycine to the perfusate is indeed a response to cell swelling, then this perfusate is indeed a response to cell swelling, then this volume-regulatory response must be extremely sensitive inasmuch as it appears to be "triggered" by an average increase in cell volume that does not exceed 5%.
Potassium translocation by the Na+/K+ pump is voltage insensitive.
Authors: Bahinski A , Nakao M , Gadsby D C
Published: May 2006

The voltage dependence of steady and transient changes in Na+/K+ pump current, in response to step changes in membrane potential, was investigated in guinea pig ventricular myocytes voltage clamped and internally dialyzed under experimental conditions designed to support four separate modes of Na+/K+ pump activity. Voltage jumps elicited transient pump currents when the pump cycle was running forward or backward, or when pumps were limited to Na+ translocation, but not when they were made to carry out K+/K+ exchange. This result indicates that K+ translocation involves no net charge movement across the membrane field and is therefore voltage insensitive. The transient pump currents seen during Na+/K+ transport demonstrate that both forward and reverse pump cycles are rate limited not by the voltage-dependent step but by a voltage-independent step, probably K+ translocation. These findings severely constrain kinetic models of Na+/K+ pump activity.
Rearrangement and expression of endogenous immunoglobulin genes occur in many murine B cells expressing transgenic membrane IgM.
Authors: Stall A M , Kroese F G , Gadus F T , Sieckmann D G , Herzenberg L A , Herzenberg L A
Published: May 2006

Transgenic mice carrying immunoglobulin genes coding for mu heavy chain and kappa light chain have been used to study the mechanisms involved in allelic and isotypic exclusion. We report here that individual cells from transgenic mice carrying a functionally rearranged mu heavy chain gene (capable of generating both membrane and secreted forms of IgM) can rearrange an endogenous mu heavy chain gene and simultaneously produce both transgenic and endogenous IgM. These "double-producing" cells express both endogenous and transgenic IgM in the cytoplasm (detected by immunohistology) and on the cell surface (detected by multiparameter fluorescence-activated cell sorter analysis). In addition, they secrete mixed IgM molecules containing both transgenic and endogenous mu heavy chains (detected in serum by radioimmune assay). The transgenic mice studied also have relatively large numbers of cells that produce endogenous immunoglobulin in the absence of detectable transgenic immunoglobulin ("endogenous-only cells"). The mechanisms that generate double-producing cells and endogenous-only cells appear to be under genetic control because the frequencies of these B-cell populations are characteristic for a given transgenic line. Thus, our findings indicate that more is involved in triggering allelic exclusion than the simple presence or absence of membrane mu heavy chains (as has been previously postulated).
Neural plasticity without postsynaptic action potentials: less-active inputs become dominant when kitten visual cortical cells are pharmacologically inhibited.
Authors: Reiter H O , Stryker M P
Published: May 2006

Models of synaptic plasticity in the nervous system have conventionally assumed a mechanism in which spike activity of a postsynaptic cell enhances the efficacy of recently active presynaptic inputs. Making use of the prompt and dramatic response of the visual cortex to occlusion of vision in one eye during the critical period, we tested the role of postsynaptic activity in ocular dominance plasticity. To do so, we selectively blocked cortical cell discharges with a continuous intracortical infusion of the inhibitory neurotransmitter agonist muscimol during a period of monocular deprivation. This drug inhibits cortical cell discharges with no apparent effect on the activity of their presynaptic geniculocortical inputs. Recording from single cortical cells after they had recovered from the muscimol-induced blockade, we found a consistent shift in the responsiveness of the visual cortex in favor of the less-active, closed eye, while the normal shift in favor of the more-active, open eye was evident in regions not affected by the treatment. Such an inhibition-coupled expression of plasticity in favor of the less-active, closed eye cannot be explained by a nonspecific disruption of cortical function. We interpret these results to indicate (i) that the postsynaptic cell is crucially involved in plasticity of the visual cortex, (ii) that the direction of cortical plasticity depends on postsynaptic membrane conductance or polarization, and (iii) that plasticity can occur in the absence of postsynaptic spike activity.
Icosahedral quasicrystals as twins of cubic crystals containing large icosahedral clusters of atoms: The 1012-atom primitive cubic structure of Al
6
CuLi
3
, the C-phase Al
37
Cu
3
Li
21
Mg
3
, and GaMg
2
Zn
3
Authors: Pauling Linus
Published: May 2006

Single-grain precession x-ray diffraction photographs of Al
6
CuLi
3
have been successfully indexed on the basis of icosahedral twinning of cubic crystals with a 1012-atom primitive cubic unit with edge 25.70 Å, giving support to the proposal that the so-called icosahedral quasicrystals are twins of crystals containing eight large icosahedral clusters in the β-W arrangement. In this compound two of the clusters consist of 104 atoms and six consist of 136 atoms, with 24 atoms shared. The same structure is assigned to the C-phase, Al
37
Cu
3
Li
21
Mg
3
, and to GaMg
2
Zn
3
. A theory of icosahedral quasicrystals and amorphous metals is described.
Mammalian tyrosinase: biosynthesis, processing, and modulation by melanocyte-stimulating hormone.
Authors: Jiménez M , Kameyama K , Maloy W L , Tomita Y , Hearing V J
Published: May 2006

We have examined the rate of synthesis and degradation of tyrosinase (monophenol, 3,4-dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1), the critical enzyme involved in mammalian pigmentation, using pulse-chase metabolic labeling of murine melanoma cells and immunoprecipitation of protein extracts with antibodies directed specifically against the enzyme. We have found that tyrosinase is synthesized and glycosylated within melanocytes rapidly, since significant quantities of pulse-labeled enzyme could be detected within 30 min. The maximum amount of enzyme was processed within 4 hr, and the t1/2 of tyrosinase in vivo was 10 hr (compared to 120 hr with purified enzyme), suggesting that tyrosinase activity in melanocytes is at least in part regulated by rapid synthesis and active degradation. We also have examined the melanogenic stimulation caused by melanocyte-stimulating hormone, using metabolic labeling, radiometric assays, and immunofluorescence techniques; responding cells increased their melanogenic potential more than 7-fold within 4 days without increasing their levels of tyrosinase synthesis. The results demonstrate that a pool of inactive tyrosinase exists in melanocytes and that rapid increases in enzyme activity elicited by melanocyte-stimulating hormone reflect an alteration in the activity of a preexisting pool of intracellular tyrosinase.
Glucose stimulates proinsulin biosynthesis by a dose-dependent recruitment of pancreatic beta cells.
Authors: Schuit F C , In't Veld P A , Pipeleers D G
Published: May 2006

Glucose is a well-known stimulus of proinsulin biosynthesis. In purified beta cells, the sugar induces a 25-fold increase in the synthesis of insulin immunoreactive material over 60-min incubation. Autoradiographic analysis of the individual cells shows that this effect is achieved via dose-dependent recruitment of pancreatic beta cells to biosynthetic activity. Recruitment of beta cells is also seen in isolated islets exposed to glucose. The sigmoidal dose-response curve for glucose-induced proinsulin biosynthesis thus reflects a heterogeneous responsiveness of pancreatic beta cells rather than a progressively increasing activity of functionally homogeneous cells. Dose-dependent recruitment of functionally diverse cells may be a ubiquitous mechanism in tissue function.
Expression of modified rye ribosomal RNA genes in wheat
Authors: Gustafson J. P. , Dera A. R. , Petrovic S.
Published: May 2006

Several wheats (
Triticum aestivum
L. em Thell.) containing the short arm of chromosome 1R from rye (
Secale cereale
L.) translocated to the long arm of wheat chromosome 1B were examined, by
in situ
hybridization with a biotinylated probe, for the expression of rye rRNA genes located in the nucleolus-organizing region (NOR) present on the short arm of 1R. The wheats analyzed contain a reduced rye NOR band, identified by a C-banding technique, that was assumed to have resulted from a deletion that reduced the number of rRNA genes and spacer units. Genes being actively transcribed could be recognized as dispersed label on DNA within the nucleolus, while those genes that were inactive were seen as condensed label. After
in situ
hybridization with a biotinylated rye NOR spacer probe, no evidence of rye rRNA gene activity was detected in the plants containing a reduced rye NOR band. Biotin-labeling of chromosomes containing a normal-sized rye NOR locus showed a degree of rye activity consistent with the results of a previous study that used a radioactive probe detected by autoradiography. It was determined previously that rye rRNA genes in a wheat background show significantly less activity than in diploid rye because the spacer units between the rRNA genes of rye are smaller than those of wheat, and in cereals the ribosomal locus with the larger spacer unit is preferentially transcribed. The present results indicate that the number of spacer units is also important in determining which NOR locus (i.e., rye or wheat) will be expressed when several ribosomal loci are present in a wheat background.