bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties.
Authors: Mannervik B , Alin P , Guthenberg C , Jensson H , Tahir M K , Warholm M , Jörnvall H
Published: May 2006

The major isoenzymes of cytosolic glutathione transferase (EC 2.5.1.18) from rat, mouse, and man are shown to share structural and catalytic properties that can be used for species-independent classification. Rat, mouse, and human isoenzymes were grouped with respect to amino-terminal amino acid sequences, after correlation of seven structures analyzed in the present investigation with structures determined earlier. The isoenzymes were also characterized by substrate specificities and sensitivities to inhibitors, and the data were subjected to pattern recognition analysis. In addition, the various isoenzymes were tested for cross-reactivity by immunoprecipitation with antibodies raised against rat and human transferases. The different types of data were clearly correlated and afforded an unambiguous division of the isoenzymes into three classes named alpha, mu, and pi. Each of the three mammalian species studied contains at least one isoenzyme of each class. It is suggested that the similarities of the isoenzymes in a class reflect evolutionary relationships and that the classification applies generally.
Molecular cloning and regulated expression of the human c-myc gene in Escherichia coli and Saccharomyces cerevisiae: comparison of the protein products.
Authors: Miyamoto C , Chizzonite R , Crowl R , Rupprecht K , Kramer R , Schaber M , Kumar G , Poonian M , Ju G
Published: May 2006

mRNA from human HL-60 cells was used to prepare a cDNA library, from which two full-length clones that encompass the complete c-myc coding region were isolated. One clone, pM1-11, contains all three exons of human c-myc. The second clone, pM4-10, represents a relatively rare transcript that initiated in the first intron and includes the coding exons 2 and 3. The cDNA insert in pM1-11 was used to express the human c-myc protein in both prokaryotic and eukaryotic cells. Insertion of the coding sequences in exons 2 and 3 into the appropriate expression vectors yielded detectable c-myc protein in Escherichia coli lacking the Lon protease and in Saccharomyces cerevisiae upon induction. The protein produced in E. coli has an apparent size of 60 kDa and appears to be unmodified, as it is identical in size to the protein synthesized in an in vitro system. In contrast, yeast cells synthesize two myc proteins, of 60 kDa and 62 kDa. The difference in apparent molecular mass between the two proteins appears to be due, in part, to phosphorylation. Subcellular fractionation of yeast cells showed that the c-myc phosphoprotein is located predominantly in the nuclear fraction.
The ets sequence from the transforming gene of avian erythroblastosis virus, E26, has unique domains on human chromosomes 11 and 21: both loci are transcriptionally active.
Authors: Watson D K , McWilliams-Smith M J , Nunn M F , Duesberg P H , O'Brien S J , Papas T S
Published: May 2006

Human DNA segments homologous to the ets region from the transforming gene of avian erythroblastosis virus, E26, were molecularly cloned and shown to be closely related to the viral equivalent by hybridization and partial sequence analysis. The transforming gene of E26 has a tripartite origin with the structure delta gag [1.2 kilobases (kb) from the viral gag gene]-myb(0.9 kb from the chicken myb gene)-ets (1.6 kb from the chicken ets gene). Human ets DNA is located on two distinct human chromosomes. The human ets-1 locus on chromosome 11 encodes a single mRNA of 6.8 kb; the second locus, ets-2 on chromosome 21, encodes three mRNAs of 4.7, 3.2, and 2.7 kb. The ets-related sequences of human DNA on chromosomes 11 and 21 are discontiguous, except for a small overlap region encoding 14 amino acids, where 12 are conserved between these two loci. By contrast, the chicken homolog has contiguous ets-1 and ets-2 sequences and is primarily expressed in normal chicken cells as a single 7.5-kb mRNA. We conclude that the ets sequence shared by the virus, the chicken, and humans is likely to contain at least two dissociable functional domains, ets-1 and ets-2. Thus, the tripartite transforming gene of E26 includes four distinct domains that may be functionally relevant for the transforming function of the virus (delta gag, myb, ets-1, and ets-2).
Functional mapping of the human cerebellum with positron emission tomography.
Authors: Fox P T , Raichle M E , Thach W T
Published: May 2006

Alterations of local neuronal activity induced within the human cerebellum by tactile stimulation and voluntary movement were mapped with positron emission tomographic measurements of brain blood flow. Finger movements produced bilateral, parasagittal blood-flow increases in anterior, superior hemispheric cortex of the cerebellum. Responses to tactile finger stimulation were coextensive with responses to voluntary finger movements but were less intense. Saccadic eye movements produced midline blood-flow increases in the posterior vermis of the cerebellum. Positron emission tomography now permits investigation of functional-anatomical relations within the human cerebellum.
Cryobaroenzymic studies as a tool for investigating activated complexes: creatine kinase.ADP.Mg.nitrate.creatine as a model.
Authors: Balny C , Travers F , Barman T , Douzou P
Published: May 2006

By combining cryoenzymology with baroenzymology (a technique we term "cryobaroenzymology") one can obtain "stop-action" pictures of the intermediates in an enzyme reaction pathway and then observe their structural and energetic features ("motion features"). We illustrate the potential of this approach by considering the formation of a transient state analogue complex of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2): enzyme.ADP.nitrate.creatine, where nitrate mimics the transferable gamma-phosphate of ATP. Formation of the analogue complex is accompanied by a conformational change that manifests itself by tryptophan perturbation and thus allows kinetic studies by the stopped-flow method. We studied the formation of the analogue complex under cryoenzymic conditions as a function of pressure and solvent composition. This allowed a detailed description of the structural and energetic features of the activation process of an elementary step in an enzyme pathway.
Genetic manipulation of membrane phospholipid composition in Escherichia coli: pgsA mutants defective in phosphatidylglycerol synthesis.
Authors: Miyazaki C , Kuroda M , Ohta A , Shibuya I
Published: May 2006

Unique mutants of Escherichia coli K-12, defective in phosphatidylglycerol synthesis, have been isolated from a temperature-sensitive strain incubated at its nonpermissive temperature. The parent strain had excess phosphatidylglycerol by harboring both the pss-1 allele [coding for a temperature-sensitive phosphatidylserine synthase (EC 2.7.8.8)] and the cls- allele (responsible for a defective cardiolipin synthase). The newly acquired mutations caused better growth at higher temperatures. One of the mutations (pgsA3) has been identified in the structural gene for phosphatidylglycerophosphate synthase [glycerophosphate phosphatidyltransferase (EC 2.7.8.5)]. Phospholipid compositions of these mutants were remarkable; phosphatidylethanolamine was the sole major lipid. In media with low osmotic pressures, these cells grew more slowly than the wild-type cells. They grew normally without recovering from the phospholipid abnormality in media appropriately supplemented with sucrose and MgCl2. Formation of cardiolipin and phosphoglycerol derivatives of membrane-derived oligosaccharides was reduced in a pgsA3 mutant. E. coli strains having the pgsA3, pss-1, and cls- mutations, either individually or in combination, constitute an empirical system in which the molar ratio of three major membrane phospholipids can be variously altered.
Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells.
Authors: Tsujimoto M , Yip Y K , Vilcek J
Published: May 2006

Highly purified, Escherichia coli-derived recombinant human tumor necrosis factor (TNF) was labeled with 125I and employed to determine receptor binding, internalization, and intracellular degradation in murine L929 cells (highly sensitive to the cytotoxic action of TNF) and in diploid human FS-4 cells (resistant to TNF cytotoxicity). 125I-labeled TNF bound specifically to high-affinity receptors on both L929 and FS-4 cells. Scatchard analysis of the binding data indicated the presence of 2200 binding sites per L929 cell and 7500 binding sites per FS-4 cell. The calculated dissociation constants are 6.1 X 10(-10) M and 3.2 X 10(-10) M for L929 and FS-4 cells, respectively. In both L929 and FS-4 cells, incubation at 37 degrees C resulted in a rapid internalization of the bulk of the cell-bound TNF, followed by the appearance of trichloroacetic acid-soluble 125I radioactivity in the tissue culture medium, due to degradation of TNF. Degradation but not cellular uptake of TNF was inhibited in the presence of chloroquine (an inhibitor of lysosomal proteases) in both L929 and FS-4 cells, suggesting that degradation occurs intracellularly, probably within lysosomes. These results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF.
Three restriction points in the cell cycle of activated murine B lymphocytes.
Authors: Melchers F , Lernhardt W
Published: May 2006

The cell cycle of activated B lymphocytes was found to be controlled by three restriction points. The first occurs immediately after mitosis and was found to be controlled by the binding of Sepharose-bound, immunoglobulin-specific antibodies to surface membrane-bound immunoglobulin. Exposure to this stimulus as short as 15 min or as long as 36 hr allowed B cells to move into the G1 phase up to the next restriction point. The second restriction point was observed to be approximately equal to 4 hr after mitosis, in the G1 phase of the cycle and 3-4 hr before the B cells entered S phase, and was found to be controlled by alpha-type B-cell growth factors produced by the P388D1 macrophage line. A third restriction point occurs in the G2 phase, 2-4 hr before mitosis, and is apparently controlled by beta-type B-cell growth factors that are likely to be produced by helper T lymphocytes.
Prevention of type I diabetes in nonobese diabetic mice by allogenic bone marrow transplantation.
Authors: Ikehara S , Ohtsuki H , Good R A , Asamoto H , Nakamura T , Sekita K , Muso E , Tochino Y , Ida T , Kuzuya H
Published: May 2006

An animal model [the nonobese diabetic (NOD) mouse] for type I diabetes features a striking infiltration of T cells into the pancreatic islets. This infiltration selectively destroys beta cells. Most of the T cells are Lyt-1+, but some are Lyt-2+,3+. Transfer experiments using parabiosis revealed that insulitis can be transferred within 2 weeks after parabiosis to immunoincompetent thymectomized mice. When NOD mice (6 mo old) were irradiated and reconstituted with bone marrow cells from young BALB/c nu/nu mice (less than 2 mo old), the NOD mice exhibited neither insulitis nor overt diabetes. Deposits of immunoglobulin in mesangial areas of the glomeruli disappeared within 3 mo after bone marrow transplantation in such irradiated allogeneic bone marrow reconstituted mice. Assays for immunological functions, including mitogen response and mixed lymphocyte reaction, revealed that both T- and B-cell functions were increased in NOD mice with overt diabetes. NOD mice reconstituted with BALB/c nu/nu bone marrow cells displayed normal T- and B-cell functions. The newly developed T cells in the allogeneic bone marrow recipients are tolerant to cells with both donor- and host-type major histocompatibility complex determinants. These results suggest that bone marrow transplantation may ultimately be developed as a component of a strategy to be employed for treatment of type I diabetes in humans.
Dendritic sprouting and compensatory synaptogenesis in an identified interneuron follow auditory deprivation in a cricket.
Authors: Hoy R R , Nolen T G , Casaday G C
Published: May 2006

We examined the effect of chronic afferent deprivation on an identified interneuron (Int-1) in the auditory system of the Australian field cricket Teleogryllus oceanicus. In normal intact crickets, the auditory afferents from each ear terminate ipsilaterally onto a single Int-1. Each bilaterally paired Int-1 is excited by ultrasound stimulation of its ipsilateral ear but not by the contralateral ear. Unilateral removal of an ear early in postembryonic development deprives the developing Int-1 of ipsilateral auditory innervation. Consequently, the ipsilateral dendrites of the deprived interneuron sprout, grow aberrantly across the ganglionic midline, and terminate specifically in the intact auditory neuropile of the contralateral (unlesioned) side, where they form functional synapses with the contralateral afferents. This unusual compensatory dendritic sprouting restores auditory function to the neuron. Thus, it is demonstrated that the dendritic shape of an identified Int, as well as its synaptic connectivity, is altered as a consequence of chronic sensory deprivation.
Effects of intravenous and intraventricular injection of antisera directed against corticotropin-releasing factor on the secretion of anterior pituitary hormones.
Authors: Ono N , Samson W K , McDonald J K , Lumpkin M D , Bedran de Castro J C , McCann S M
Published: May 2006

To determine the physiological significance of corticotropin-releasing factor (CRF) in the control of pituitary hormone secretion, highly specific antibodies directed against the peptide were injected either intravenously or intraventricularly (third ventricle) and the effect on plasma levels of pituitary hormones was determined before and after application of ether stress for 1 min. The intravenous injection of CRF antiserum (0.5 ml) did not significantly alter basal corticotropin (ACTH) levels in freely moving ovariectomized rats but largely blocked the increase in plasma ACTH resulting from ether stress. These antibodies had no effect on the ether-induced decline in plasma growth hormone (GH), and they failed to modify plasma luteinizing hormone levels. In a second experiment, CRF antiserum (3 microliter) or normal rabbit serum was injected into the third ventricle. A blood sample was drawn 24 hr later and immediately thereafter another injection of CRF antiserum or normal rabbit serum was made. There was no modification in the level of any of the hormones 24 hr after the first injections, and they were similar in CRF antiserum and normal rabbit serum-injected animals. After imposition of ether stress, the response of plasma ACTH was nearly completely blocked by the intraventricular CRF antiserum, but the degree of blockade was slightly less than that obtained by intravenous injection. The decline in plasma GH after ether stress was blocked by the intraventricular CRF antiserum. There was no effect of the intraventricular injection of the antiserum on the levels of the other pituitary hormones. The results with intravenous injection of the antisera indicate that CRF plays an extremely important but probably not completely indispensable role in the release of ACTH after ether stress. The results of the intraventricular injection of the antiserum suggest strongly that endogenous CRF may also modify its own release in response to stress, augmenting it by a positive ultrashort loop feedback, and that the antisera against the peptide blocked this action; however, an action at the pituitary of these intraventricularly injected antibodies cannot be completely ruled out. The blockade of the stress-induced suppression of GH release by the CRF antibodies suggests that CRF released intrahypothalamically during ether stress brings about an alteration in the hypothalamic control of GH secretion such that the stress-induced inhibition of GH release is blocked.
A muscle-type tropomyosin in human fibroblasts: evidence for expression by an alternative RNA splicing mechanism.
Authors: MacLeod A R , Houlker C , Reinach F C , Smillie L B , Talbot K , Modi G , Walsh F S
Published: May 2006

We have isolated a cDNA clone from a human fibroblast cDNA library that contains the entire protein-coding region of a 1.1-kilobase mRNA. This mRNA encodes a 284-amino acid tropomyosin, the primary structure of which most closely resembles smooth muscle tropomyosin. Thus, the expression of both 284-amino acid muscle-type and 247-amino acid non-muscle-type tropomyosins appears to be a normal feature of human non-muscle cells. We also present evidence to suggest that this cytoskeletal tropomyosin and a human skeletal muscle beta-tropomyosin are derived from a common structural gene by an alternative RNA splicing mechanism.
Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.
Authors: de Wet J R , Wood K V , Helinski D R , DeLuca M
Published: May 2006

A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.
Repair-defect mutations inhibit rDNA magnification in Drosophila and discriminate between meiotic and premeiotic magnification.
Authors: Hawley R S , Marcus C H , Cameron M L , Schwartz R L , Zitron A E
Published: May 2006

We have examined rDNA magnification in Drosophila melanogaster males carrying one of 11 recombination- or repair-defective mutations representing seven loci. We show that mutations defined by a defect in postreplication repair (mus-101, mei-41, and mus-108) are also defective in rDNA magnification, whereas mutations that do not affect postreplication repair have little or no effect on magnification. mei-41 inhibits only premeiotic magnification events, while mus-108 blocks both premeiotic and meiotic events. This suggests that meiotic and premeiotic events share some but not all functions. A molecular analysis of rDNA magnification reveals that in mus-108 males, changes in the rDNA restriction pattern can occur within one or a few generations under magnifying conditions. We interpret these data in terms of the role of DNA repair systems in rDNA magnification and in terms of stable maintenance of tandemly repeated genes.
Intracellular class II HLA antigens are accessible to transferrin-neuraminidase conjugates internalized by receptor-mediated endocytosis.
Authors: Cresswell P
Published: May 2006

Newly synthesized class II HLA antigens being transported to the surface of human B-lymphoblastoid cell lines (B-LCL) interact with transferrin-neuraminidase conjugates internalized by means of receptor-mediated endocytosis. Class II antigens, isolated from [35S]methionine-labeled B-LCL after incubation with the conjugates at 37 degrees C, showed extensive desialylation of associated invariant chain and detectable loss of beta-subunit sialic acid on analysis by two-dimensional gel electrophoresis. An equal amount of unconjugated neuraminidase had no effect, and desialylation of class II antigen components was blocked when access of transferrin-neuraminidase conjugates to the B-LCL transferrin receptors was competitively inhibited by the addition of excess iron-saturated transferrin. The conjugates were shown to cycle through the cells in the same way as unconjugated transferrin, being first internalized and then rapidly secreted in an undegraded form. The data suggest that the exocytic pathway taken by class II antigens intersects the route followed by recycling transferrin receptors and that the interaction occurs prior to the dissociation of the invariant chain from the class II antigen complex. Similar intracellular interactions between class II molecules and foreign proteins internalized by antigen-presenting cells may be important in class II antigen-restricted recognition by helper T lymphocytes.
Keratin gene expression in simian virus 40-transformed human keratinocytes.
Authors: Morris A , Steinberg M L , Defendi V
Published: May 2006

Previous reports from this laboratory indicate that cultured simian virus 40 (SV40)-transformed human keratinocytes express keratin proteins characteristic of simple epithelia that are not found in their untransformed counterparts. In this study we show by in vitro translation and RNA transfer blot analysis that the altered keratin synthesis reflects changes in the abundance of specific keratin mRNAs. SV40-transformed keratinocytes have a reduced abundance of transcripts for 58-, 56-, 52-, 50-, 48-, and 46-kDa keratin species, compared with uninfected cultured keratinocytes, but express significant levels of transcripts for 52-, 45-, and 40-kDa keratins, typical of simple epithelia. The SV40-transformed cells also express mRNA for a 48-kDa keratin that is unique to SV40-transformed keratinocytes. Analysis of the keratin genome with keratin-specific cDNAs as probes indicates that the changes in keratin transcription are not correlated with gross rearrangements of the keratin genome. These results suggest that analysis of viral transformation of cultured keratinocytes affords a novel approach to study mechanisms regulating keratin gene expression.
Genes selectively expressed in proliferating Dictyostelium amoebae.
Authors: Kopachik W , Bergen L G , Barclay S L
Published: May 2006

Few eukaryotic genes are expressed only during cell growth and division. We found that the slime mold Dictyostelium discoideum is unusual in that it expresses many genes only during proliferation. Thirty-two percent (304/950) of the sequences in a cDNA library made from vegetative mRNA were homologous to RNAs that are present at high levels during growth but at low or undetectable levels during differentiation when no cell growth occurs. In vitro translation assays confirmed that one-third of the vegetative cell mRNAs decreased in steady-state levels during differentiation. These vegetative cell-specific transcripts identified a diverse coordinately regulated class of genes: (i) 9 of the 10 cDNAs tested hybridized to unique small transcripts ranging from 400 to 620 bases long; (ii) the sequences showed various degrees of homology to related species; (iii) transcript levels synchronously fell by a factor of greater than 20 during development and synchronously increased during germination. This class of genes may play important roles in normal cell proliferation.
HLA-B locus polymorphism: studies with a specific hybridization probe.
Authors: Coppin H L , Denny D W , Weissman S M , McDevitt H O
Published: May 2006

The large number of class I histocompatibility genes (HLA) and their extensive homology has made it difficult to assign bands on genomic Southern blots to known genes. Therefore, we have tried to obtain nucleic acid probes for class I genes that are locus specific or have restricted locus specificity. Computer sequence-homology analysis was used to compare the nucleic acid sequences of two genomic clones, one coding for the HLA-B7 antigen (JY150) and one containing a class I pseudogene (pHLA12.4). A sequence in the 3' untranslated region with very low homology was identified. This sequence from the HLA-B7 gene was subcloned into M13 phage. This fragment, JY150/C5, hybridized with two genomic bands in DNA from human HLA homozygotes--presumably the HLA-B locus gene and a closely related gene. The probe was used to assess restriction fragment polymorphism at the HLA-B locus in homozygous consanguineous cell lines. This analysis permitted the association of certain polymorphic restriction enzyme fragments with some alleles of this locus. However, many HLA-B alleles have identical restriction fragments produced by a number of restriction endonucleases.
Administration of recombinant interferon gamma to cancer patients enhances monocyte secretion of hydrogen peroxide.
Authors: Nathan C F , Horowitz C R , de la Harpe J , Vadhan-Raj S , Sherwin S A , Oettgen H F , Krown S E
Published: May 2006

Recombinant interferon gamma (rIFN-gamma) activates macrophage antimicrobial and antitumor functions and related metabolic processes, including secretion of reactive oxygen intermediates in mice and in cultured mouse and human macrophages. To look for similar actions in man, we monitored the H2O2 secretory capacity of monocytes from cancer patients receiving intravenous rIFN-gamma at 0.1, 0.5, or 1.0 mg/m2 of body area over 6 hr daily or over 1 hr on alternate days. Monocytes taken just before the first infusion served as controls and were comparable to normal donor monocytes in secretion of H2O2. Monocytes from 11 of the 13 subjects (85%) studied through 20 treatment cycles responded to rIFN-gamma with elevation in H2O2 secretion in greater than or equal to 67% of the tests conducted greater than 1 hr after the start of treatment. Five of the five subjects tested had monocytes with diminished H2O2 secretory capacity when tested immediately after a 1-hr infusion of rIRN-gamma, at which time the amount of adherent mononuclear cell protein recovered from the blood averaged only 24% of the control. At all other times tested (from 6 hr to 5 days after infusion) combined results for all subjects showed enhancement of H2O2 releasing capacity. Statistically significant mean increases ranged from 1.4- to 2.8-fold above the control and included the sets in which monocytes collected 24 hr following a single infusion were assayed the same day or the next. By the criterion of enhanced H2O2 secretory capacity, the ability of rIFN-gamma to activate mononuclear phagocytes is manifest upon its administration to patients with advanced malignancy.
Neuritic plaques and cerebrovascular amyloid in Alzheimer disease are antigenically related.
Authors: Wong C W , Quaranta V , Glenner G G
Published: May 2006

A synthetic peptide (Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr), homologous to the amino terminus of a protein purified from cerebrovascular amyloid (beta protein), induced antibodies in BALB/c mice that were used immunohistochemically to stain not only amyloid-laden cerebral vessels but neuritic plaques as well. These findings suggest that the amyloid in neuritic plaques shares antigenic determinants with beta protein of cerebral vessels. Since the amino acid compositions of plaque amyloid and cerebrovascular amyloid are similar, it is likely that plaque amyloid also consists of beta protein. This possibility suggests a model for the pathogenesis of Alzheimer disease involving beta protein.
The neostriatal mosaic: compartmental distribution of calcium-binding protein and parvalbumin in the basal ganglia of the rat and monkey.
Authors: Gerfen C R , Baimbridge K G , Miller J J
Published: May 2006

Calcium-binding protein (CaBP) and parvalbumin are two proteins that are expressed in brain and bind calcium in the micromolar range. The immunohistochemical distribution of these two proteins was examined in the basal ganglia of rats and rhesus monkeys. In the striatum, CaBP immunoreactivity is localized to a subset of striatonigral projection neurons; CaBP-positive neurons are distributed in areas containing somatostatin-immunoreactive fibers and not in the complementary areas containing dense mu opiate-receptor binding. These biochemical labels mark, respectively, the matrix and patch compartments of the striatum. Previous studies have shown that striatal matrix neurons project to the substantia nigra pars reticulata, whereas striatal patch neurons project to the substantia nigra pars compacta. Consistent with the restricted localization of CaBP in the matrix projection neurons is the confinement of CaBP-immunoreactive afferent fibers to the pars reticulata. CaBP is also localized to a portion of dopaminergic and a few nondopaminergic neurons in the substantia nigra pars compacta and in most dopaminergic neurons in the ventral tegmental area. Parvalbumin immunoreactivity is localized to a subset of substantia nigra pars reticulata neurons and their axons. In the lateral striatum, some medium-sized aspiny interneurons are also parvalbumin immunoreactive. The distinct distributions of CaBP and parvalbumin in the basal ganglia are discussed in terms of their possible roles as intracellular calcium buffer systems related to the physiologic response properties of the neurons in which they are contained.
Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments.
Authors: Zimmerman A L , Yamanaka G , Eckstein F , Baylor D A , Stryer L
Published: May 2006

cGMP opens cation-selective channels when applied to the cytoplasmic side of excised patches of membrane from retinal rod outer segments (ROS). If the light-sensitive channel in intact rods is gated only by cGMP, it should be possible to find a hydrolysis-resistant analog of cGMP that blocks the normal response to light by holding the channel open independent of the degree of illumination. We have studied the interaction of 8-bromo-cGMP (8-Br-cGMP) and the SP and RP phosphorothioate derivatives of cGMP [(Sp)-cGMP[S] and (RP)-cGMP[S]) with the cGMP phosphodiesterase (PDEase) of ROS, the cGMP-sensitive channel of excised ROS patches, and the light-sensitive channel of intact rods. All three analogs were hydrolyzed by PDEase much more slowly than was cGMP. The maximal rates of hydrolysis of 8-Br-cGMP, (SP)-cGMP[S], and (RP)-cGMP[S] were 7.3, 3.7, and less than 0.2 s-1, respectively, compared with 4000 s-1 for cGMP. These analogs are effective competitive inhibitors of the PDEase, with Ki values of 48, 25, and 90 microM, respectively. The nucleotide-activated conductances of excised patches were half-maximal at concentrations of 1.6, 210, and 1200 microM, respectively, compared with 17 microM for cGMP. Thus, 8-Br-cGMP is a highly potent channel agonist. The effects of these analogs on the dark current and photoresponses of intact rod cells were also measured. A suction electrode monitored membrane current across the ROS, while a patch electrode sealed on the inner segment was used to introduce a cGMP analog and to control membrane potential. All three analogs increased the dark current and markedly slowed the response to light flashes. 8-Br-cGMP increased the dark current of the outer segment as much as 48-fold. After the concentration of this analog had risen sufficiently, little of the current could be shut off by light, as expected of a direct effect on the light-sensitive channel of the plasma membrane. These results are consistent with the notions that (i) the light-sensitive channel of rods is controlled solely by the instantaneous concentration of cGMP and (ii) the cGMP-sensitive channel of excised patches is identical to the light-sensitive channel of intact rods.
Differences between two fractions of glycosaminoglycans of the corneal stroma in their structural relation to collagen.
Authors: Dische Z , Cremer-Bartels G , Kaye G I
Published: May 2006

Glycosaminoglycans (GAG) of bovine cornea were sequentially extracted by 0.15 M NaCl and by 1 M CaCl2, pH 8. The amounts of hexosamine (HexN) and hexuronic acid (HexUA), specific hexoses (Hex), and protein were determined in the extracts. The ultrastructure of the corneal stroma after NaCl and NaCl/CaCl2 extraction was also studied. Approximately 70% of the Hex-HexN extractable by 0.15 M NaCl is removed in the first NaCl extract, with the amount decreasing rapidly to the fifth NaCl extract. Only 20-30% of the extractable HexUA-HexN is removed in the first NaCl extract; subsequent extraction removes successively less HexUA-HexN. There is a sharp increase, however, in both Hex-HexN and HexUA-HexN removed in the first 1 M CaCl2 extract, ranging from 25 to 40% of the total extractable with NaCl. The stroma retained lamellar organization and normal spacing, diameter, and length of collagen fibers after NaCl extraction. Visibility of the 64-nm periodicity was enhanced because of loss of density in the ground substance. CaCl2 extraction led to loss of lamellar organization and apparent disruption of the collagen fibers. Only short thin fibrils remained, embedded in a material having the density and very fine fibrillar organization of basal lamina. Disruption of the collagen fibers is probably due to removal of a specific GAG component necessary to maintain the collagen as an extended morphologic structure but may also be due to a specific degradative effect of CaCl2 on collagen no longer covered by a protective layer of GAG and glycoproteins.
Diacylglycerols mimic phorbol diester induction of leukemic cell differentiation.
Authors: Ebeling J G , Vandenbark G R , Kuhn L J , Ganong B R , Bell R M , Niedel J E
Published: May 2006

Activation of cellular protein kinase C appears to be involved in the mechanism by which phorbol diesters induce differentiation of human myeloid leukemia cells (HL-60). Protein kinase C is thought to be physiologically activated by diacylglycerol derived from receptor-mediated phosphatidylinositol hydrolysis. sn-1,2-diacylglycerols with short saturated acyl side chains (C4-C10) were synthesized and found to be potent activators of protein kinase C partially purified from HL-60 cells. These diacylglycerols were also competitive inhibitors of [3H]phorbol dibutyrate binding to the soluble phorbol diester receptor. The most potent diacylglycerol, sn-1,2-dioctanoylglycerol, displaced greater than 90% of [3H]phorbol dibutyrate from the phorbol diester receptor of intact HL-60 cells. Because of probable cellular metabolism of sn-1,2-dioctanoylglycerol, hourly doses were required to maintain persistent occupancy of the phorbol diester binding site. Treatment of HL-60 cells with either phorbol 12-myristate 13-acetate or sn-1,2-dioctanoylglycerol produced identical phosphoprotein changes. Finally, sn-1,2-dioctanoylglycerol induced differentiation of the HL-60 cells into cells with morphologic characteristics of macrophages. Substitution of the hydroxyl group at position 3 with a hydrogen, chloro, or sulfhydryl moiety inactivated sn-1,2-dioctanoylglycerol. These data strengthen the hypothesis that protein kinase C activation plays a role in macrophage differentiation.
Association of the N-formyl-Met-Leu-Phe receptor in human neutrophils with a GTP-binding protein sensitive to pertussis toxin.
Authors: Lad P M , Olson C V , Smiley P A
Published: May 2006

Pertussis toxin inhibits the N-formyl-Met-Leu-Phe (fMet-Leu-Phe) mediated human neutrophil functions of enzyme release, superoxide generation, aggregation, and chemotaxis. As pertussis toxin modifies the GTP binding receptor-regulatory protein "Ni," the association of the fMet-Leu-Phe receptor with such a protein was further examined in purified neutrophil plasma membranes. Both fMet-Leu-Phe-mediated guanine nucleotide exchange and nucleotide-mediated regulation of the fMet-Leu-Phe receptor are inhibited by pertussis toxin. In addition, membrane pretreatment with pertussis toxin abolishes the fMet-Leu-Phe-mediated inhibition of adenylate cyclase. Actions of pertussis toxin are due to the ADP-ribosylation of a single subunit at 41 kDa in the neutrophil plasma membrane, which comigrates on NaDodSO4 gels with the Ni GTP-binding protein in the platelet plasma membrane. Our results suggest that (i) the fMet-Leu-Phe receptor is associated with a Ni GTP regulatory protein, and (ii) a fMet-Leu-Phe-Ni complex is important in the control of several neutrophil functions, probably involving multiple transduction systems, including adenylate cyclase.
Proapolipoprotein A-I conversion kinetics in vivo in human and in rat.
Authors: Ghiselli G , Gotto A M , Tanenbaum S , Sherrill B C
Published: May 2006

We have determined the turnover rates for proapolipoprotein A-I (proapoA-I) and mature apolipoprotein A-I (apoA-I) in vivo in human and in rat. For the human study, high density lipoprotein (HDL)-associated 125I-labeled proapoA-I (A-I isoform 1) and 131I-labeled mature apoA-I (A-I isoforms 3, 4, and 5) were injected simultaneously into two normal volunteers. Blood samples were obtained serially and HDL proapoA-I and mature apoA-I were separated by isoelectrofocusing for the determination of the associated radioactivity. Residence times of proapoA-I and mature apoA-I were 0.13 and 3.9 days and production rates were 9.0 and 9.3 mg/kg per day, respectively. Analysis of the specific activity curves suggested a complete proapoA-I to mature apoA-I precursor-product relationship. Proprotein conversion was virtually completed in 24 hr. For the rat study, HDL- and lymph chylomicron- (flotation constant Sf greater than 400 S) associated 125I-labeled proapoA-I and 131I-mature apoA-I were injected into fasted rats. Residence times of proapoA-I and mature apoA-I injected in association with HDL were 0.13 and 0.28 day, respectively, and for injection associated with chylomicrons the residence times were 0.08 and 0.26 day. When associated with chylomicrons, proapoA-I was converted more efficiently to the mature form. As in the human study, the results demonstrated a complete proapoA-I to mature apoA-I precursor-product relationship. Our data are consistent with the concepts that (i) conversion to mature apoA-I is the major proapoA-I catabolic fate in plasma; (ii) mature apoA-I is conceivably derived solely from proapoA-I in plasma and proapoA-I is the major apoA-I form secreted in vivo; and (iii) a putative proapoA-I-chylomicron complex is the preferred substrate for the apoA-I propeptidase.
Activation of an N-ras gene in acute myeloblastic leukemia through somatic mutation in the first exon.
Authors: Gambke C , Hall A , Moroni C
Published: May 2006

A transforming N-ras gene has been cloned from acute myeloblastic leukemia bone marrow cells, in parallel with the N-ras gene derived from fibroblasts of the same patient. N-ras derived from fibroblasts lacked focus-forming activity in NIH/3T3 cells, indicating that gene activation in the leukemia cells must have occurred by a somatic event. Construction of chimeric molecules between the transforming and the normal N-ras genes and subsequent biological and sequence analysis of these constructs revealed that the transforming gene was altered by a point mutation changing amino acid 12 of the N-ras protein from glycine to aspartic acid.
Nucleotide sequence of F
0
-ATPase proteolipid (subunit 9) gene of maize mitochondria
Authors: Dewey R. E. , Schuster A. M. , Levings C. S. , Timothy D. H.
Published: May 2006

The F
0
-ATPase proteolipid, also referred to as subunit 9 or the dicyclohexylcarbodiimide-binding protein, is encoded by a mitochondrial gene in maize that we have designated
atp
9. The clone containing
atp
9 was selected for investigation from a mitochondrial DNA library because of its abundant transcript in total maize mitochondrial RNA preparations. Sequence analysis of the clone revealed an open reading frame that was readily identified by its nucleotide homology with the ATPase subunit 9 gene of yeast. As deduced from the nucleotide sequence, the maize ATPase subunit 9 protein contains 74 amino acids with a molecular weight of 7368. Substantial amino acid sequence homology is conserved among maize, yeast, bovine, and
Neurospora
mitochondrial ATPase subunit 9 proteins, regardless of whether the gene is nuclearly encoded (bovine and
Neurospora
) or mitochondrially encoded (yeast and maize). RNA transfer blot analysis indicated that the gene sequence is actively transcribed, producing an initial transcript that is large and extensively processed.
Presence of two distinct regions in the coiled-coil structure of the streptococcal Pep M5 protein: relationship to mammalian coiled-coil proteins and implications to its biological properties.
Authors: Manjula B N , Trus B L , Fischetti V A
Published: May 2006

The complete amino acid sequence of Pep M5, a biologically active 197-residue fragment comprising nearly half of the group A streptococcal M5 protein, has structural features characteristic of an alpha-helical coiled-coil protein. Fourier analyses of the nonpolar residues show strong periodicities based on repeats of 7 residues (7/2 and 7/3). Except for the nonhelical NH2-terminal 12-residue segment, the 7-residue periodicity in the distribution of nonpolar residues extends through the remainder of the Pep M5 molecule, with some discontinuities and irregularities. The molecule contains two distinct regions that differ in the pattern of distribution of the nonpolar and charged residues. The 7-residue pattern "a, b, c, d, e, f, g" in region 13-121 is atypical in that position "a" is predominantly occupied by asparagine, rather than nonpolar residues. On the other hand, the periodicity in region 122-196 is more typical of that found in other coiled-coil proteins, such as the myosin rod region, keratin, desmin, and vimentin, rather than tropomyosin. Although the periodicity in nonpolar residues is not highly regular, the predominance of basic and acidic residues in the inner "e" and "g" positions, respectively, suggests that ionic interactions between chains may contribute significantly to the stability of the coiled-coil. The distribution of charged residues in the outer positions within the two regions of the molecule is also distinct. The NH2-terminal region carries a significantly higher net negative charge than the COOH-terminal region, suggesting that the former region may play an important role in some of the biological functions of the Pep M5 molecule.
Circulating hydroxy fatty acids in familial Mediterranean fever.
Authors: Aisen P S , Haines K A , Given W , Abramson S B , Pras M , Serhan C , Hamberg M , Samuelsson B , Weissmann G
Published: May 2006

Episodes of fever, serositis, and arthritis in familial Mediterranean fever (FMF) suggested circulating mediators of acute inflammation (e.g., neutrophil activation). The mean serum neutrophil-aggregating activity of 51 FMF patients was 2.5 +/- 0.2 cm2/min, compared to 1.0 +/- 0.1 cm2/min in 20 normal controls (P less than 0.0002). Lipid extracts of FMF sera retained neutrophil-aggregating activity and had UV absorbance peaks at 269 and 279 nm, indicating the presence of lipids with a conjugated triene structure. Chromatography of extracts yielded peaks that were coeluted with reference dihydroxyicosatetraenoic acids, had UV absorbance peaks at 259, 269, and 279 nm, and possessed neutrophil-aggregating activity. The presence of leukotriene B4 was excluded by chromatography following methyl-esterification. Monohydroxy compounds identified in FMF extracts by gas chromatography/mass spectrometry included 5-hydroxyicosatetraenoic acid, and 9- and 13-hydroxyoctadecadienoic acids. Hydroxy acids were present in 19 of 31 FMF sera and absent in extracts of sera from 8 patients with active systemic lupus erythematosus, 7 with fever from infection, and 12 normal controls. The finding of circulating mono- and dihydroxy fatty acids in FMF suggests that defects in the formation or elimination of these compounds might play a role in the pathogenesis of FMF.
Establishment of a line of human fetal glial cells that supports JC virus multiplication.
Authors: Major E O , Miller A E , Mourrain P , Traub R G , de Widt E , Sever J
Published: May 2006

Primary cultures of human fetal brain cells were transfected with plasmid DNA pMK16, containing an origin-defective mutant of simian virus 40 (SV40). Several weeks after DNA treatment, proliferation of glial cells was evident in the culture, allowing passage of the cells at low split ratios. Initially, only 10% of the cells demonstrated nuclear fluorescence staining using a hamster tumor antibody to the SV40 T protein. By the sixth passage, however, 100% of the cells reacted positively to the same antibody. During these early passages, the cells designated SVG began growing very rapidly and acquired a homogeneous morphology. Cell division required only low serum concentrations, was not contact-inhibited, and remained anchorage dependent. These characteristics of the SVG cells have been stable through 25 passages or approximately equal to 80 cell generations. The SV40 T protein is continuously produced in the cells and can direct the replication of DNA inserts in the pSV2 vector, determined by in situ hybridization using biotin-labeled DNA probes, which contains the SV40 replication origin. More importantly, SVG cells support the multiplication of the human papovavirus JCV at levels comparable to primary cultures of human fetal glial cells, producing infectious virus as early as 1 week after viral adsorption. Their brain-cell derivation has been established as astroglial, based on their reactivity with a monoclonal antibody to glial fibrillary acid protein and lack of activity with an anti-galactocerebroside antibody, which identifies oligodendroglial cells. The SVG cells represent a unique line of continuous rapidly growing human fetal astroglial cells that synthesizes a replication-proficient SV40 T protein. Their susceptibility to JC virus (JCV) infection obviates a host restriction barrier that limited JCV studies to primary cultures of human fetal brain and thus should allow for more detailed molecular studies of human brain cells and JCV that infects them.
Precise localization of genes on large animal virus genomes: use of lambda gt11 and monoclonal antibodies to map the gene for a cytomegalovirus protein family.
Authors: Mocarski E S , Pereira L , Michael N
Published: May 2006

We describe an efficient procedure, which uses monoclonal antibodies directed against specific viral proteins, for the precise mapping of genes on large DNA virus genomes. We have used the technique to locate the gene encoding a family of antigenically related DNA-binding proteins on the 240-kilobase-pair human cytomegalovirus (CMV) genome. A random library of CMV DNA fragments was generated using the prokaryotic vector lambda gt11, which expresses open reading frames as beta-galactosidase fusion proteins in infected Escherichia coli. The library was screened with a mixture of monoclonal antibodies directed against the gene products of interest. The coding sequence for infected cell protein 36 (ICP36) was localized to a 2800-base-pair EcoRI fragment (map coordinates 0.228-0.240) on the CMV(Towne) and CMV(AD169) genomes by using DNA from immunoreactive lambda gt11 as probe. A 5000-nucleotide transcript from this region was detected during the early and late phases of the CMV growth cycle. This transcript directed the synthesis of the predominant member of the ICP36 family when hybrid-selected and translated in vitro. Immunoprecipitation of the in vitro translation product with the same monoclonal antibodies used in the initial mapping confirmed the location of the ICP36 gene. These studies establish the utility of the lambda gt11 expression system for rapid and precise mapping of CMV genes (or other large animal virus genes) that encode proteins for which serological reagents exist.
Axonal transport of a heat shock protein in the rabbit visual system.
Authors: Clark B D , Brown I R
Published: May 2006

Intraocular injection of [35S]methionine was used to demonstrate the pronounced induction of a 74-kDa heat shock protein in the rabbit retina after a 3 degrees C increase in body temperature was generated by intravenous administration of D-lysergic acid diethylamide. Two-dimensional polyacrylamide gel electrophoresis and fluorography revealed that the induced heat shock protein underwent axonal transport from retinal ganglion cells into the optic nerve and subsequently down the contralateral optic tract to synaptic termini in the visual projection area. Since the heat shock protein took more than 8 days to move down the optic nerve to the superior colliculus, it is transported by slow rather than by fast axonal transport.