bluuebunny/crossref_metadata_2025 huggingface dataset:
Different configurational states of beta-amyloid and their distributions relative to plaques and tangles in Alzheimer disease.
Authors: Spillantini M G , Goedert M , Jakes R , Klug A
Published: May 2006

Antibodies have been raised against synthetic peptides corresponding to different parts of the beta-amyloid sequence. These antibodies stain different kinds of amyloid distributions in the hippocampal formation in Alzheimer disease, suggesting the existence of different states of aggregation and/or folding of beta-amyloid molecules. An antibody directed against the middle region of beta-amyloid stained mostly amyloid plaques without cores, whereas an antibody directed against the carboxyl-terminal region of beta-amyloid stained only amyloid plaques with cores. An antiserum directed against the amino terminus of beta-amyloid stained numerous tangle-bearing cells and bodies, as well as the neuritic component of plaques and neuropil threads. These antibodies, in conjunction with anti-tau antibodies, were used to demonstrate a close spatial relationship between amyloid deposits and neurofibrillary tangles.
Expression of microtubule-associated protein 2 by reactive astrocytes.
Authors: Geisert E E , Johnson H G , Binder L I
Published: May 2006

After an injury to the central nervous system, a dramatic change in the astrocytes bordering the wound occurs. The most characteristic feature of this process, termed reactive gliosis, is the upregulation of the intermediate filament protein, glial fibrillary acidic protein. In the present study, we show that reactive astrocytes express high levels of microtubule-associated protein 2 (MAP-2), a protein normally found in the somatodendritic compartment of neurons. When sections of injured brain are double-stained with antibodies directed against MAP-2 and glial fibrillary protein, all of the reactive astrocytes are found to contain MAP-2. The high levels of this protein appear to represent a permanent change in reactive astrocytes. In parallel quantitative studies, an elevated level of MAP-2 in the injured brain is confirmed by an immunoblot analysis of injured and normal white matter. This report demonstrates the direct involvement of a microtubule protein in the process of reactive gliosis.
Interaction and mutual stabilization of the two subunits of vaccinia virus mRNA capping enzyme coexpressed in Escherichia coli.
Authors: Guo P X , Moss B
Published: May 2006

The genes D1 and D2, predicted to encode the 95- and 31-kDa subunits of the vaccinia virus mRNA capping enzyme, were coexpressed from the same plasmid in Escherichia coli. Induction with low concentrations of isopropyl beta-D-thiogalactoside was necessary to obtain soluble enzyme. The active heterodimer was purified by column chromatography and was shown to have both RNA guanylyltransferase and mRNA (guanine-N7-)-methyltransferase activities. Formation of the m7G(5')pppG cap structure was verified by enzyme digestion and thin-layer chromatography. Each subunit was also expressed individually in E. coli. Without the large subunit, the small one was very unstable in some bacterial strains and could only be detected by pulse labeling with radioactive amino acids. The individually expressed large subunit contained the guanylyltransferase domain, but the activity from E. coli was less than 2% of that obtained with both subunits. Two other products of the D1 open reading frame were formed: a 55-kDa subfragment with the GMP binding site and a 38-kDa C-terminal fragment that started at amino acid 498. Expression of this heterodimeric enzyme in E. coli may facilitate the analysis of its functional domains and provide a useful reagent for the specific 5' labeling of uncapped or capped RNA and for enhancing RNA translatability in eukaryotic systems.
Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels.
Authors: Cotten M , Längle-Rouault F , Kirlappos H , Wagner E , Mechtler K , Zenke M , Beug H , Birnstiel M L
Published: May 2006

We have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, we have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We demonstrate here that this procedure is useful for a human leukemic cell line. We enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell-permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, we show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks "transferrinfection," as does incubation of the cells at 18 degrees C.
AIDS Kaposi sarcoma-derived cells produce and respond to interleukin 6.
Authors: Miles S A , Rezai A R , Salazar-González J F , Vander Meyden M , Stevens R H , Logan D M , Mitsuyasu R T , Taga T , Hirano T , Kishimoto T
Published: May 2006

Cell lines derived from Kaposi sarcoma lesions of patients with AIDS (AIDS-KS cells) produce several cytokines, including an endothelial cell growth factor, interleukin 1 beta, and basic fibroblast growth factor. Since exposure to human immunodeficiency virus increases interleukin 6 (IL-6) production in monocytes and endothelial cells produce IL-6, we examined IL-6 expression and response in AIDS-KS cell lines and IL-6 expression in AIDS Kaposi sarcoma tissue. The AIDS-KS cell lines (N521J and EKS3) secreted large amounts of immunoreactive and biologically active IL-6. We found both IL-6 and IL-6 receptor (IL-6-R) RNA by slot blot hybridization analysis of AIDS-KS cells. The IL-6-R was functional, as [3H]thymidine incorporation by AIDS-KS cells increased significantly after exposure to human recombinant IL-6 (hrIL-6) at greater than 10 units/ml. When AIDS-KS cells (EKS3) were exposed to IL-6 antisense oligonucleotide, cellular proliferation decreased by nearly two-thirds, with a corresponding decrease in the production of IL-6. The decrease from IL-6 antisense in AIDS-KS cell proliferation was reversed by the addition of hrIL-6. We confirmed that AIDS-KS cells produced IL-6 in vivo by preparing RNA and tissue sections from involved and uninvolved skin from a patient with AIDS Kaposi sarcoma. We detected immunoreactive IL-6 in the involved tumor areas and to a lesser extent in the surrounding normal epidermis. Slot blot hybridization showed a great excess of IL-6 and IL-6-R RNA in involved skin compared to uninvolved skin. These results show that both IL-6 and IL-6-R are produced by AIDS-KS cells and that IL-6 is required for optimal AIDS-KS cell proliferation, and they suggest that IL-6 is an autocrine growth factor for AIDS-KS cells.
Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV.
Authors: Popoff S C , Spira A I , Johnson A W , Demple B
Published: May 2006

DNA damage generated by oxygen radicals includes base-free apurinic/apyrimidinic (AP) sites and strand breaks that bear deoxyribose fragments. The yeast Saccharomyces cerevisiae repairs such DNA lesions by using a single major enzyme. We have cloned the yeast structural gene (APN1) encoding this AP endonuclease/3'-repair diesterase by immunological screening of a yeast genomic DNA expression library in lambda gt11. Gene disruption experiments confirm that the Apn1 protein accounts for greater than or equal to 97% of both AP endonuclease and DNA 3'-repair diesterase activities in yeast cell-free extracts. The DNA and predicted amino acid sequences for the APN1 gene are homologous to those for the nfo gene encoding DNA endonuclease IV of Escherichia coli. This conservation of structure between a eukaryotic enzyme and its prokaryotic counterpart underscores the fundamental nature of their roles in DNA repair.
The ability of Salmonella to enter mammalian cells is affected by bacterial growth state.
Authors: Lee C A , Falkow S
Published: May 2006

We have examined the effect of different growth conditions on the ability of Salmonella to interact with Madin-Darby canine kidney cells. Two growth conditions that affect the expression of Salmonella adherence and invasiveness have been identified. First, bacteria lose their invasiveness in the stationary phase of growth. Second, bacteria growing in oxygen-limited growth conditions are induced for adherence and invasiveness, whereas those growing aerobically are relatively nonadherent and noninvasive. Salmonella from cultures aerated with gas mixtures containing 0% or 1% oxygen were 6- to 70-fold more adherent and invasive than those from cultures aerated with a gas mixture containing 20% oxygen. The Salmonella typhimurium oxrA gene that is required for the anaerobic induction of many proteins is not involved in the regulation of Salmonella invasiveness. We speculate that oxygen limitation might be an environmental cue that triggers the expression of Salmonella invasiveness within the intestinal lumen and other tissues.
Differential interactions of human choriogonadotropin and its antagonistic aglycosylated analog with their receptor.
Authors: Ji I , Ji T H
Published: May 2006

Human choriogonadotropin (hCG) is a heterodimeric hormone consisting of an alpha subunit and a beta subunit. hCG and aglycosylated hCG (aghCG) have similar receptor binding affinities but differ in their ability to activate hormone-responsive adenylate cyclase. aghCG is an effective antagonist. The mechanisms of this antagonism and interactions of antagonistic aghCG with the receptor are not understood. To address this critical question, we have examined the interaction of this hormone analog with the receptor. The hormone receptor on porcine granulosa cells is a glycoprotein of 86 kDa and thas three domains of 24 kDa, 28 kDa, and 34 kDa, which are disulfide-linked. They undergo proteolysis, particularly when bound to the hormones, to produce three polypeptide components. These three receptor components can readily be identified through the use of affinity labeling with the hormones. Affinity labeling with an amino-specific homobifunctional reagent and subsequent cleavage indicate that hCG is cross-linked directly to the 24-kDa receptor component. In contrast, aghCG is cross-linked directly to the 34-kDa component. The peptide map of the cross-linked aghCG-34-kDa receptor component produced by papain treatment is different from the peptide map of the cross-linked complex of hCG-24-kDa component. This difference in receptor binding may be a factor determining the success or failure of signal transduction from the receptor to the effector system, guanine nucleotide-binding regulatory protein, and adenylate cyclase.
Structural and functional basis of the developmental regulation of human coagulation factor IX gene: factor IX Leyden.
Authors: Hirosawa S , Fahner J B , Salier J P , Wu C T , Lovrien E W , Kurachi K
Published: May 2006

Hemophilia B Leyden is characterized by unusual developmental regulation of factor IX synthesis in affected individuals. One family affected with the hemophilia B Leyden phenotype was found to have a specific single-base mutation (G----A) at nucleotide -6 of the factor IX gene. The mutation site was found in a small region of the 5'-untranslated sequence designated the Leyden-specific region (LS region). This region, approximately 40 base pairs in length, contains the unique mutation sites of all the known factor IX Leyden genes (five families) analyzed to date. This fact strongly suggests that the LS region is directly or indirectly involved in the developmental regulation of factor IX biosynthesis. Base changes at nucleotide -20 as well as at nucleotide -6 and deletions of the 3' half of the LS region reduced expression activity of the factor IX gene to approximately 15-31% that of the normal control, as assessed in a cultured cell (HepG2) expression system. The LS region binds at least two proteins. Androgen significantly increased the transcriptional activities of both mutant and normal factor IX genes in a concentration-dependent manner.
Processing of thyrotropin-releasing hormone prohormone (pro-TRH) generates a biologically active peptide, prepro-TRH-(160-169), which regulates TRH-induced thyrotropin secretion.
Authors: Bulant M , Roussel J P , Astier H , Nicolas P , Vaudry H
Published: May 2006

Rat thyrotropin-releasing hormone (TRH) prohormone contains five copies of the TRH progenitor sequence Gln-His-Pro-Gly linked together by connecting sequences whose biological activity is unknown. Both the predicted connecting peptide prepro-TRH-(160-169) (Ps4) and TRH are predominant storage forms of TRH precursor-related peptides in the hypothalamus. To determine whether Ps4 is co-released with TRH, rat median eminence slices were perifused in vitro. Infusion of depolarizing concentrations of KCl induced stimulation of release of Ps4- and TRH-like immunoreactivity. The possible effect of Ps4 on thyrotropin release was investigated in vitro using quartered anterior pituitaries. Infusion of Ps4 alone had no effect on thyrotropin secretion but potentiated TRH-induced thyrotropin release in a dose-dependent manner. In addition, the occurrence of specific binding sites for 125I-labeled Tyr-Ps4 in the distal lobe of the pituitary was demonstrated by binding analysis and autoradiographic localization. These findings indicate that these two peptides that arise from a single multifunctional precursor, the TRH prohormone, act in a coordinate manner on the same target cells to promote hormonal secretion. These data suggest that differential processing of the TRH prohormone may have the potential to modulate the biological activity of TRH.
Hydroxyl hydrogen conformations in trypsin determined by the neutron diffraction solvent difference map method: relative importance of steric and electrostatic factors in defining hydrogen-bonding geometries.
Authors: Kossiakoff A A , Shpungin J , Sintchak M D
Published: May 2006

Neutron diffraction maps have been used to assign the rotor conformations of the hydroxyl hydrogens in trypsin. Knowledge of these conformations is used to assess the relative importance of steric and electrostatic effects in conferring the H-bonding geometries of these groups. A general finding was that most hydroxyl groups are rotationally ordered with their highest populated conformation near the low-energy staggered orientation. For the low-energy conformers (-60 degrees, 60 degrees, 180 degrees) of serine and threonine, the trans (-180 degrees) position is most highly populated followed by +60 degrees. In trypsin, only 1 of 24 serines was found in the -60 degrees conformer. Serine hydroxyls preferentially act as H-bond acceptors and rarely are observed as H-bond donors alone. Threonines were found to be more likely than serines to participate in two H bonds; tryosines were found to prefer to act as donors. In H-bonding situations in which there was incompatibility between the energies defining the barrier to rotation and the local electrostatics, the electrostatic criteria dominated. Overall, the findings support a model of H bonding where there exists strong inherent complementarity between the low-energy hydroxyl orientations and the local electrostatic environment.
Sequence and structural similarities between the leucine-specific binding protein and leucyl-tRNA synthetase of Escherichia coli.
Authors: Williamson R M , Oxender D L
Published: May 2006

A role for the leucyl-tRNA synthetase (EC 6.1.1.4) has been established for regulating the transport of leucine across the inner membrane of Escherichia coli by the leucine, isoleucine, valine (LIV-I) transport system. This transport system is mediated by interactions of periplasmic binding proteins with a complex of membrane-associated proteins, and transcription of the high-affinity branched-chain amino acid transport system genes is repressed by growth of E. coli on high levels of leucine. We now report results from sequence comparisons and structural modeling studies, which indicate that the leucine-specific binding protein, one of the periplasmic components of the LIV-I transport system, contains a 121-residue stretch, representing 36% of the mature protein, which displays both sequence and structural similarities to a region within the putative nucleotide-binding domain of leucyl-tRNA synthetase. Early fusion events between ancestral genes for the leucine-specific binding protein and leucyl-tRNA synthetase could account for the similarity and suggest that processes of aminoacylation and transport for leucine in E. coli may be performed by evolutionarily interrelated proteins.
Enhancement of bone marrow allografts from nude mice into mismatched recipients by T cells void of graft-versus-host activity.
Authors: Lapidot T , Lubin I , Terenzi A , Faktorowich Y , Erlich P , Reisner Y
Published: May 2006

Transplantation of 8 x 10(6) C57BL/6-Nu+/Nu+ (nude) bone marrow cells into C3H/HeJ recipients after conditioning with 8 Gy of total body irradiation has resulted in a markedly higher rate of graft rejection or graft failure compared to that found in recipients of normal C57BL/6 or C57BL/6-Bg+/Bg+ (beige) T-cell-depleted bone marrow. Mixing experiments using different numbers of nude bone marrow cells with or without mature thymocytes (unagglutinated by peanut agglutinin) revealed that engraftment of allogeneic T-cell-depleted bone marrow is T-cell dependent. To ensure engraftment, a large inoculum of nude bone marrow must be supplemented with a trace number of donor T cells, whereas a small bone marrow dose from nude donors requires a much larger number of T cells for engraftment. Marked enhancement of donor type chimerism was also found when F1 thymocytes were added to nude bone marrow cells, indicating that the enhancement of bone marrow engraftment by T cells is not only mediated by alloreactivity against residual host cells but may rather be generated by growth factors, the release of which may require specific interactions between T cells and stem cells or between T cells and bone marrow stroma cells.
Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y.
Authors: Nurse P , DiGate R J , Zavitz K H , Marians K J
Published: May 2006

Escherichia coli replication factor Y (protein n') functions in the assembly of a mobile multiprotein replication-priming complex called the primosome. Although the role of factor Y in primosome assembly during replication in vitro of bacteriophage phi X174 and plasmid pBR322 DNA is clear, its role in E. coli chromosomal replication is not. To address this issue, the gene for factor Y has been cloned molecularly and its DNA sequence has been determined. The cloned fragment of DNA contained an open reading frame capable of encoding a polypeptide of 81.7 kDa. This open reading frame contains amino acid sequences identical to 13 N-terminal amino acids of purified factor Y, as well as to a 10-amino acid internal sequence (from a cyanogen bromide fragment) as determined by gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame using a bacteriophage T7 transient expression system resulted in the accumulation of a polypeptide with an apparent molecular mass of 78 kDa that comigrated with bona fide factor Y during SDS/polyacrylamide gel electrophoresis. Soluble extracts made from cells overexpressing the product of the putative factor Y open reading frame showed a 2000-fold increase in factor Y activity during bacteriophage phi X174 complementary-strand DNA synthesis in vitro when compared to control extracts. The gene encoding factor Y, which maps to 88.5 min on the E. coli chromosome, has been designated primosome A (priA).
Identification of low density lipoprotein as a regulator of Fc receptor-mediated phagocytosis.
Authors: Bigler R D , Khoo M , Lund-Katz S , Scerbo L , Esfahani M
Published: May 2006

Optimal expression of the high-affinity Fc receptor for IgG (FcRI) by the human monocyte cell line U-937 requires the presence of low density lipoprotein (LDL), and neither cholesterol nor high density lipoprotein can provide the component necessary for optimal FcRI expression. Here we show that FcR-mediated phagocytosis also requires LDL. U-937 cells were cultured in medium containing interferon gamma and either fetal calf serum (FCS) or delipidated FCS (DLFCS). The phagocytosis of IgG-coated erythrocytes was measured by a colorimetric assay. U-937 cells cultured in DLFCS medium had less than 16% of the phagocytic activity of cells cultured in normal FCS medium. Phagocytosis of IgG-coated erythrocytes could be inhibited 85% by the addition of murine IgG2a myeloma protein (5 micrograms/ml). U-937 cells cultured in DLFCS medium supplemented with pure cholesterol in ethanol (10 micrograms/ml) had only 30% of the phagocytic activity of cells grown in FCS medium. Addition of very low density lipoprotein (0.2 mg of protein per ml) to DLFCS medium also failed to increase phagocytosis. However, the addition of LDL (0.2 mg of protein per ml) to DLFCS medium restored 90% of the phagocytic activity. Since neither pure cholesterol nor very low density lipoprotein restored normal phagocytic function to U-937 cells despite a normalization of cellular cholesterol content, the restoration of phagocytosis observed with LDL replacement cannot be explained by mere delivery of cholesterol by LDL. Thus, LDL is required for the expression of FcRI and FcR-mediated phagocytosis by U-937 cells and may be an important regulator of phagocytic activity of monocytes and macrophages in vivo.
Symplastic spermatids (sys): a recessive insertional mutation in mice causing a defect in spermatogenesis.
Authors: MacGregor G R , Russell L D , Van Beek M E , Hanten G R , Kovac M J , Kozak C A , Meistrich M L , Overbeek P A
Published: May 2006

A line of transgenic mice that carries an insertional mutation in a gene essential for spermatogenesis is described. Males homozygous for the transgenic insert are sterile, while female homozygotes and both male and female heterozygotes exhibit normal fertility. Developing spermatids in homozygous males form prominent abnormal multinucleated syncytia (symplasts) and do not complete maturation. In addition, abnormal cytoplasmic vacuolation is commonly seen in Sertoli cells. One flank of the transgenic integration site within the genome has been cloned and used to show linkage between homozygosity for the transgene and the mutant phenotype. The flank maps to mouse chromosome 14 approximately 4 centimorgans proximal to the gene encoding esterase-10 (Es-10). As no other gene that is known to be essential for spermatogenesis has been mapped to this region of the genome and as the mutant phenotype is unique, the transgenic insert appears to affect a previously unidentified gene. We have named the mutation "symplastic spermatids" (sys).
Alpha-tubulin influences nucleotide binding to beta-tubulin: an assay using picomoles of unpurified protein.
Authors: Farr G W , Yaffe M B , Sternlicht H
Published: May 2006

Tubulin binds guanine nucleotides tightly within its beta subunit. Whether the alpha subunit influences binding to this site has been unknown. This question was addressed by comparing the nucleotide binding properties of the free beta subunit with those of the heterodimer. The free beta subunit was obtained from an in vitro expression system and its nucleotide binding properties were determined by an assay that requires approximately 100-fold less protein than conventional assays. This assay exploits the observation that the recovery of beta-tubulin from Mono Q anion-exchange columns is dependent on added nucleotide. Our results demonstrate that the newly synthesized beta subunit and the heterodimer bind nucleotides with similar specificity. We found that in the presence of magnesium the alpha subunit enhances GTP binding to the beta subunit approximately 4-fold. However, in the absence of magnesium the alpha subunit appears to specifically weaken GTP binding to the beta subunit. Thus, nucleotide binding to the E site in the heterodimer may not be solely defined by the beta subunit.
Real-time imaging of calcium influx in mammalian cerebellar Purkinje cells in vitro.
Authors: Sugimori M , Llinás R R
Published: May 2006

Real-time visualization of intracellular calcium concentration ([Ca2+]i) changes in mammalian Purkinje cells in vitro, utilizing the dye Fura-2, indicates that calcium action potentials are generated in the dendritic tree and follow a particular activation sequence. During spontaneous oscillations or after direct current injection, dendritic spikes are initiated as slow and graded plateau potentials at the level of the tertiary or spiny branchlets of the dendrite. As the plateau potentials become sufficiently high to reach the firing threshold for full dendritic spike generation, calcium entry is observed at the more proximal branches of the dendritic tree. These action potentials are then conducted orthodromically toward the soma and may invade other branches in the arbor antidromically. Simultaneous recording of the intracellular electrical activity and the Fura-2 fluorescent signal indicates that the intracellular calcium transients are accompanied by a very rapid increase in intracellular calcium concentration. This increase in [Ca2+]i exhibits an almost equally fast return to baseline after the termination of the action potential, indicating the presence of very efficient calcium sequestering and extruding mechanisms in the dendrites. Iontophoretic application of glutamate at the dendritic level provided a further demonstration of the spatial separation of plateau potentials from dendritic spikes and gives further insights into the details of dendritic integration in this neuron.
Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells.
Authors: Pachnis V , Pevny L , Rothstein R , Costantini F
Published: May 2006

To test the feasibility of transferring yeast artificial chromosomes (YACs) into mammalian cells, we modified a YAC that carries approximately 450 kilobases (kb) of human DNA, by inserting a neomycin-resistance gene. Saccharomyces cerevisiae cells carrying this YAC were fused by polyethylene glycol to mouse L cells and G418-resistant colonies were obtained. A high percentage of these clones contained virtually intact YAC sequences as revealed by "Alu fingerprint" analysis and restriction enzyme analysis using pulsed-field gel electrophoresis. Furthermore, the YAC sequences were stably integrated into the mouse chromosomes, as shown by in situ hybridization and by the stability of the G418 resistance. These results establish that large segments of the mammalian genome, cloned in yeast, can be efficiently transferred into cultured mammalian cells.
Determination of fructose metabolic pathways in normal and fructose-intolerant children: a 13C NMR study using [U-13C]fructose.
Authors: Gopher A , Vaisman N , Mandel H , Lapidot A
Published: May 2006

An inborn deficiency in the ability of aldolase B to split fructose 1-phosphate is found in humans with hereditary fructose intolerance (HFI). A stable isotope procedure to elucidate the mechanism of conversion of fructose to glucose in normal children and in HFI children has been developed. A constant infusion of D-[U-13C]fructose was given nasogastrically to control and to HFI children. Hepatic fructose conversion to glucose was estimated by examination of 13C NMR spectra of plasma glucose. The conversion parameters in the control and HFI children were estimated on the basis of doublet/singlet values of the plasma beta-glucose C-1 splitting pattern as a function of the rate of fructose infusion (0.26-0.5 mg/kg per min). Significantly lower values (approximately 3-fold) for fructose conversion to glucose were obtained for the HFI patients as compared to the controls. A quantitative determination of the metabolic pathways of fructose conversion to glucose was derived from 13C NMR measurement of plasma [13C]glucose isotopomer populations. The finding of isotopomer populations of three adjacent 13C atoms at glucose C-4 (13C3-13C4-13C5) suggests that there is a direct pathway from fructose, by-passing fructose-1-phosphate aldolase, to fructose 1,6-bisphosphate. The metabolism of fructose by fructose-1-phosphate aldolase activity accounts for only approximately 50% of the total amount of hepatic fructose conversion to glucose. It is suggested that phosphorylation of fructose 1-phosphate to fructose 1,6-bisphosphate by 1-phosphofructokinase occurs in human liver (and intestine) when fructose is administered nasogastrically; 47% and 27% of the total fructose conversion to glucose in controls and in HFI children, respectively, takes place by way of this pathway. In view of the marked decline by 67% in synthesis of glucose from fructose in HFI subjects found in this study, the extent of [13C]glucose formation from a "trace" amount (approximately 20 mg/kg) of [U-13C]fructose infused into the patient can be used as a safe and noninvasive diagnostic test for inherent faulty fructose metabolism.
Isolation of mutants of human immunodeficiency virus protease based on the toxicity of the enzyme in Escherichia coli.
Authors: Baum E Z , Bebernitz G A , Gluzman Y
Published: May 2006

The protease encoded by the pol gene of human immunodeficiency virus was expressed in Escherichia coli and found to be toxic to strain BL21(DE3). This toxicity provided a convenient selection for isolating mutants of the protease that are nontoxic and enzymatically inactive. This strong correlation between functional protease and toxicity resulted in rapid identification of several protease mutations, including mutations that exhibit temperature sensitivity. A total of 24 missense mutations and 7 nonsense mutations were identified. The described selection procedure may have wider applications for isolating mutants of other eukaryotic proteins that exhibit a toxic phenotype in E. coli.
Interleukin 2 prevents graft-versus-host disease while preserving the graft-versus-leukemia effect of allogeneic T cells.
Authors: Sykes M , Romick M L , Sachs D H
Published: May 2006

We have recently demonstrated that interleukin 2 (IL-2), when administered in high doses for several days beginning on the day of allogeneic bone marrow transplantation (BMT), markedly diminishes graft-versus-host disease (GVHD) mortality in lethally irradiated mice. An optimal anti-GVHD effect was attained by coadministering T-cell-depleted (TCD) syngeneic marrow. We demonstrate here that the full graft-versus-leukemia effect of allogeneic T lymphocytes is obtained even when GVHD is markedly diminished by the coadministration of IL-2 and TCD syngeneic marrow. This methodology represents an approach to the treatment of leukemia in which the beneficial effects of allogeneic T cells can be exploited while their major deleterious effect, GVHD, is avoided. These results may thus have an impact on the clinical use of BMT for the treatment of hematologic malignancies.
Genetic variation in clonal vertebrates detected by simple-sequence DNA fingerprinting.
Authors: Turner B J , Elder J F , Laughlin T F , Davis W P
Published: May 2006

The measurement of clonal heterogeneity is central to understanding the evolutionary and population genetics of the roughly 50 species of vertebrates that lack effective genetic recombination. Simple-sequence DNA fingerprinting with oligonucleotide probes (CAC)5 and (GACA)4 is a sensitive and efficient means of detecting this heterogeneity in natural populations of two clonal fishes, Poecilia formosa, an apomictic unisexual, and Rivulus marmoratus, a selfing hermaphrodite. The fingerprints are clonally stable for at least three generations. The technique clearly differentiates allozymically identical laboratory lines of R. marmoratus that were previously distinguishable only by histocompatibility analysis. The technique also reveals apparent cases of shifts in clonal composition of a natural population of each species. Clonal variation in most natural populations is quite high. For example, a sample of 19 specimens of P. formosa from one station on the Rio Soto la Marina contained 16 clones (average clonal frequency = 0.07). This level of clonal diversity implies that mutation, subsequent to the founding of clonal lineages, is an important source of variation in these populations. It also suggests that chance (sampling error) has a previously unappreciated role in determining the clonal composition of populations even though some of the clones may be divergent in biologically significant features.
Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules.
Authors: Darchen F , Zahraoui A , Hammel F , Monteils M P , Tavitian A , Scherman D
Published: May 2006

The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells. Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine beta-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTP-binding proteins, is involved in their biogenesis or in the regulated secretion process.
Definition of a physiologic aging autoantigen by using synthetic peptides of membrane protein band 3: localization of the active antigenic sites.
Authors: Kay M M , Marchalonis J J , Hughes J , Watanabe K , Schluter S F
Published: May 2006

Senescent cell antigen (SCA), an aging antigen, is a protein that appears on old cells and marks them for removal by the immune system in mammals. It is derived from band 3, a ubiquitous membrane transport protein found in diverse cell types and tissues. We have used synthetic peptides to identify aging antigenic sites on band 3, using a competitive inhibition assay and immunoblotting with IgG directed against the aging antigen on old cells. Results indicate that: (i) the active antigenic sites of the aging antigen reside on membrane protein band 3 residues that are extracellular regions implicated in anion transport (residues 538-554 and 788-827); (ii) a putative ankyrin-binding-region peptide is not involved in SCA activity; and (iii) carbohydrate moieties are not required for the antigenicity or recognition of SCA because synthetic peptides alone abolish binding of senescent cell IgG to erythrocytes. One of the putative transport sites that contributes to the aging antigen is located toward the carboxyl terminus. A model of band 3 is presented. Localization of the active antigenic site on the band 3 molecule facilitates definition of the molecular changes occurring during aging that initiate molecular as well as cellular degeneration.
Rescue of a paralyzed-flagella mutant of Chlamydomonas by transformation.
Authors: Diener D R , Curry A M , Johnson K A , Williams B D , Lefebvre P A , Kindle K L , Rosenbaum J L
Published: May 2006

The biflagellate alga Chlamydomonas has been used extensively in the genetic and biochemical analysis of flagellar assembly and motility. We have restored motility to a paralyzed-flagella mutant of Chlamydomonas by transforming with the corresponding wild-type gene. A nitrate reductase-deficient paralyzed-flagella strain, nit1-305 pf-14, carrying mutations in the genes for nitrate reductase and radial spoke protein 3, was transformed with wild-type copies of both genes. Two-thirds of the cells that survived nitrate selection also regained motility, indicating that they had been transformed with both the nitrate reductase and radial spoke protein 3 genes. Transformants typically contained multiple copies of both genes, genetically linked to each other, but not linked to the original mutant loci. Complementation of paralyzed-flagella mutants by transformation is a powerful tool for investigating flagellar assembly and function.
Lipid composition is important for highly efficient target binding and retention of immunoliposomes.
Authors: Maruyama K , Kennel S J , Huang L
Published: May 2006

By taking advantage of a monoclonal IgG antibody, 34A, which is highly specific to pulmonary endothelial cells, we have prepared liposomes containing various amounts of antibody molecules (immunoliposomes). These immunoliposomes accumulate specifically in the lung when injected i.v. Two lipid compositions were used: phosphatidylcholine/cholesterol/phosphatidylserine (PS), 10:5:1 (mol/mol), a composition that allows liposomes to be readily taken up by the reticuloendothelial system (RES) (liver and spleen), and phosphatidylcholine/cholesterol/ganglioside GM1, 10:5:1 (mol/mol), a composition that allows liposomes to avoid or delay the RES uptake (the so-called stealth liposomes). Although an increase in the number of antibody molecules per liposome was accompanied by an increased level of lung binding of the immunoliposomes, differences due to the lipid composition were more profound. For example, stealth immunoliposomes containing an antibody/lipid ratio = 1:37 (wt/wt) accumulated in lung to a level of 60% of the injected dose, whereas PS-containing immunoliposomes with a higher antibody/lipid ratio (1:8) only accumulated 50% of the injected dose in the lung. Conjugation of antibody to the stealth liposome did not increase the rate of liposome uptake by liver; this rate was approximately 10-fold lower than that of the PS-containing liposomes without antibody. Stealth immunoliposomes with high antibody content also showed long retention in the lung. The t1/2 of lung residence for the stealth immunoliposomes with an antibody/lipid ratio = 1:11 (wt/wt) was approximately 24 hr. The fact that stealth immunoliposomes showed a longer retention time in the lung than the PS-containing immunoliposomes of similar antibody content suggests that macrophages may play a role in the removal of the bound immunoliposomes from the pulmonary endothelium. Alternatively, dissociated stealth immunoliposomes may reenter the circulation and rebind to the lung target, causing an apparent slow overall dissociation rate. These results can be understood on the basis of two competing kinetic processes: lung binding whose rate is directly proportional to the antibody content of the immunoliposomes and uptake by RES whose rate is significantly reduced in the case of the stealth liposomes. Even for a modest level of antibody content, the half-life for target binding of immunoliposomes was significantly shorter than the half-life of liver uptake of the liposomes, resulting in a favorable target binding. Significant immunoliposome binding to the lung is not due to the fact that tail vein-injected liposomes flow through the lung capillary bed before they encounter the liver, because portal vein-injected immunoliposomes showed the same rate and extent of target binding as the tail vein-injected ones.
Oxidation-reduction sensitive interaction of a cellular 50-kDa protein with an RNA hairpin in the 5' noncoding region of the poliovirus genome.
Authors: Najita L , Sarnow P
Published: May 2006

Genetic and biochemical analyses of the 5' noncoding region of poliovirus have indicated the importance of this region in both translation and amplification of the viral RNA. The role of the cellular machinery required for these events is just beginning to be revealed. Using an RNA gel retention assay, we have identified a cellular 50-kDa protein that forms a specific complex with a stable stem-loop structure present in the viral 5' noncoding region. The formation of the RNA-protein complex is dependent on the availability of free sulfhydryl groups in the protein. The possible involvement of this RNA-protein complex in the regulation of viral gene expression is discussed.
Mechanisms of p53 loss in human sarcomas.
Authors: Mulligan L M , Matlashewski G J , Scrable H J , Cavenee W K
Published: May 2006

An important role for the p53 gene in neoplastic transformation in vitro and in vivo has been imputed by functional studies and identification of tumor-acquired gene defects or alterations in its expression. To study the generality and mechanisms of p53 alteration in human cancer, we examined 241 tumors of several types for structural aberrations of the locus. Alterations of the gene or its RNA or protein products consistent with loss of function by either recessive or dominant mechanisms were identified among this set uniquely in rhabdomyosarcomas and osteosarcomas. The alterations of p53 in rhabdomyosarcoma tumors included cases with complete deletion of both p53 alleles, complete deletions of one allele with or without point mutation of the remaining allele, and absence of detectable RNA. Similarly, we detected homozygous deletion and lack of expression of p53 RNA or aberrant expression of p53 protein in osteosarcomas. These observations provide strong support for the inclusion of the p53 locus in the group of loci whose functional inactivation by either dominant or recessive modes plays a significant role in human cancer.
Fourier-transform Raman spectroscopy applied to photobiological systems.
Authors: Sawatzki J , Fishcer R , Scheer H , Siebert F
Published: May 2006

Fluorescence and initiation of photoreactions are problems frequently encountered with resonance Raman spectroscopy of photobiological systems. These problems can be circumvented with Fourier-transform Raman spectroscopy by using the 1064-nm wavelength of a continuous wave neodymium-yttrium/aluminum-garnet laser as the probing beam. This wavelength is far from the absorption band of most pigments. Yet, the spectra of the investigated systems--bacteriorhodopsin, rhodopsin, and phycocyanin--show that these systems are still dominated by the chromophore, or that preresonant Raman scattering is still prevalent. Only for rhodopsin were contributions of the protein and the membrane discernible. The spectra of phycocyanin differ considerably from those obtained by excitation into the UV-absorption band. The results show the usefulness of this method and its wide applicability. In addition, analysis of the relative preresonant scattering cross sections may provide a detailed insight into the scattering mechanism.
Structural and functional comparisons of the Drosophila virilis and Drosophila melanogaster rough genes.
Authors: Heberlein U , Rubin G M
Published: May 2006

We have isolated the homeobox gene rough (ro) from Drosophila virilis. Comparison of the predicted amino acid sequences of the D. melanogaster and D. virilis rough proteins reveals that domains of high conservation, including the homeodomain, are interspersed with highly diverged regions. Stretches of significant sequence conservation are also observed in the 5' promoter region and in the introns. The D. virilis rough gene rescues the rough mutant phenotype and is properly regulated when introduced into the D. melanogaster genome. Thus the rough protein as well as the cis-regulatory elements that ensure proper temporal and spatial regulation are functionally conserved between these Drosophila species.
Priming of CD4+ T cells specific for conserved regions of human immunodeficiency virus glycoprotein gp120 in humans immunized with a recombinant envelope protein.
Authors: Abrignani S , Montagna D , Jeannet M , Wintsch J , Haigwood N L , Shuster J R , Steimer K S , Cruchaud A , Staehelin T
Published: May 2006

A nonglycosylated denatured form of human immunodeficiency virus (HIV) 1 glycoprotein gp120 (Env 2-3), which does not bind to CD4, was used with muramyl tripeptide as adjuvant to immunize HIV-seronegative healthy volunteers. In all the volunteers, three 50-micrograms injections of Env 2-3 induced priming of CD4+ T cells specific for conserved regions of the native glycosylated gp120. Moreover, we found that several major histocompatibility complex class II (DR) alleles can function as restriction molecules for presentation of conserved epitopes of gp120 to T cells, implying that a T-cell response to these epitopes can be obtained in a large fraction of the population. The possibility to prime CD4+ T cells specific for conserved epitopes of a HIV protein is particularly important in view of the lack of such cells in HIV-infected individuals and of a possible role that CD4+ T cells may play in the development of protective immunity against AIDS.
Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53.
Authors: Mercer W E , Shields M T , Amin M , Sauve G J , Appella E , Romano J W , Ullrich S J
Published: May 2006

To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation we constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastoma multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. We show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits G0/G1 progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the G0/G1 phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.