bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-surface heparan sulfate: an intercalated membrane proteoglycan.
Authors: Kjellén L , Pettersson I , Höök M
Published: May 2006

Two pools of heparan sulfate proteoglycans have been selectively solubilized from rat liver plasma membranes by successive incubations with heparin and detergent. The two populations of proteoglycans have similar polyanionic properties as indicated by identical elution positions on ion-exchange chromatography on DEAE-Sephacel but differ in buoyant density in CsCl when analyzed by density gradient centrifugation in the presence of 4 M guanidine. The detergent-extracted proteoglycan has a lower buoyant density (less than or equal to 1.40 g/ml) and is, as determined by gel chromatography, slightly larger than the heparin-released proteoglycan (buoyant density, greater than or equal to 1.55 g/ml). Furthermore, in contrast to the heparin-released proteoglycan, the detergent-extracted proteoglycan is able to bind detergent micelles, shows affinity for the hydrophobic gel octyl-Sepharose, and can be inserted into liposomes. We conclude that the detergent-extracted heparan sulfate represents a proteoglycan species that has its core protein rooted in the lipid bilayer of the plasma membrane.
In vitro reactions of aflatoxin B1-adducted DNA.
Authors: Groopman J D , Croy R G , Wogan G N
Published: May 2006

The chemical stability of aflatoxin B1 bound to calf thymus DNA was studied over a 48-hour exposure to phosphate buffers at pH 6.8-8.0 (37 degrees C). During this time, aliquots of the aflatoxin B1-modified DNA were acid-hydrolyzed and analyzed for the presence of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyflatoxin B1, 2,3-dihydro-2,3-dihydroxy-aflatoxin B1, and the tentatively identified, 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl-3-hydroxyflatoxin B1 and 2,3-dihydro-2-(8,9-dihydro-8-hydroxy-N7-guanyl)-3-hydroxyaflatoxin B1. Initial experiments determined the stability of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 in DNA at levels of modification of one residue per 60 and 1500 nucleotides. The acid-hydrolysis products obtained from these modified nucleic acids were qualitatively similar, but their proportional concentrations were different. These quantitative differences were dependent upon both pH and the initial level of modification of the DNA. During the first 6 hr of incubation, under all conditions examined, the formation of 2,3-dihydro-2,3-dihydroxyaflatoxin B1 was responsible for the initial decrease of the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 adduct in DNA. After 48 hr of incubation at pH 7.0, the major reaction of the modified DNA was depurination of the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 adduct. However, at pH 8.0, the predominant reaction product formed in 48 hr was the putative 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxy-aflatoxin B1. The putative DNA-bound products resulting from the elimination of the positive charge in the imidazole ring of the aflatoxin-N7-guanine adduct [2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxy-aflatoxin B1 and 2,3-dihydro-2-(8,9-dihydro-8-hydroxy-N7-guanyl)-3-hydroxyaflatoxin B1] were found to be stable in DNA for at least 24 hr at both pH 6.8 and 7.4. Taken together with observed patterns of stability of aflatoxin B1 adducts in vivo, these observations strongly suggest the involvement of enzymatic repair processes in removal of the N7-guanyl adduct and also emphasize the possible biological significance of the stable imidazole ring-opened adduct.
Identification of the uvrC gene product.
Authors: Sancar A , Kacinski B M , Mott D L , Rupp W D
Published: May 2006

We have constructed a multicopy plasmid that carries the uvrC gene of Escherichia coli. By inserting the transposon Tn1000 (previously designated gamma delta) into this plasmid, we obtained many derivatives that fail to complement uvrC34. The proteins synthesized by the original plasmid and the uvrC::Tn1000 derivatives were labeled in maxicells and analyzed on gels, demonstrating that a protein of Mr 70,000 encoded by the original uvrC+ plasmid was absent from the mutated noncomplementing derivatives; this protein is presumed to be the uvrC gene product. We found that this protein of Mr 70,000 binds to DNA and have partially purified the uvrC protein by DNA-cellulose chromatography. Because some of the uvrC::Tn1000 derivatives produce truncated polypeptides, the orientation of expression and the location of the promoter were determined by correlating the sizes of the truncated polypeptides with the sites of insertion of Tn1000.
Rearrangements in the chloroplast genomes of mung bean and pea
Authors: Palmer Jeffrey D. , Thompson William F.
Published: May 2006

We have mapped all the cleavage sites for the restriction endonucleases
Bst
EII,
Kpn
I,
Pst
I,
Pvu
II,
Sac
I,
Sal
I,
Sma
I, and
Xho
I on the circular chloroplast chromosomes from mung bean and pea. The mung bean chloroplast genome measures 150 kilobase pairs (kb) in length; it includes two identical sequences of 23 kb that contain the ribosomal genes and are arranged as an inverted repeat separated by single-copy regions of 21 and 83 kb. The pea chloroplast genome is only 120 kb in size, has only one set of ribosomal genes, and does not possess any detectable repeated sequences. The mung bean inverted repeat structure is common to all other nonleguminous higher plant chloroplast genomes studied, whereas the pea structure has been found only in the closely related legume
Vicia faba
. We conclude from these data that loss of one copy of the inverted repeat sequence has occurred only rarely during the evolution of the Angiosperms, and in the case of the legumes after the divergence of the mung bean line from the pea-
Vicia
line. We present hybridization data indicating that rearrangements that change the linear order of homologous sequences within the chloroplast genome have been quite frequent during the course of legume evolution.
Photoreception in Neurospora crassa: correlation of reduced light sensitivity with flavin deficiency.
Authors: Paietta J , Sargent M L
Published: May 2006

The effect of flavin deficiency on blue light responses in Neurospora crassa was studied through the use of two riboflavin mutants (rib-1 and rib-2). The photoresponses assayed were the suppression of circadian conidiation, the phase shifting of the circadian conidiation rhythm, and the induction of carotenoid synthesis. Flavin deficiency was induced in the rib-1 mutant by restrictive growth temperatures and in the rib-2 mutant by low levels of supplemental riboflavin. At 26 degrees C, a semirestrictive growth temperature, the rib-1 mutant is about 1/80th as sensitive to light for the photosuppression of circadian conidiation. Flavin deficiency in the rib-1 and rib-2 strains was effective in reducing the photosensitivity for phase shifting and carotenogenesis to about 1/16th and 1/4th of normal, respectively. Experiments with permissive temperatures, riboflavin supplementation, and revertants at the rib locus all indicated that the effects on light sensitivity were due solely to the presence of the rib mutations. These results provide evidence that one or more flavin photoreceptors are involved in the blue light responses of Neurospora.
Mitogenic factors present in serum but not in plasma.
Authors: Balk S D , Levine S P , Young L L , LaFleur M M , Raymond N M
Published: May 2006

In culture medium containing heparinized, heat-inactivated, chicken plasma, normal chicken heart mesenchymal cells do not proliferate but their Rous sarcoma virus-infected counterparts proliferate maximally. In medium containing serum derived from chicken whole blood or plasma, on the other hand, normal chicken heart mesenchymal cells proliferate actively, at similar overall rates and to similar extents. The rate and extent of normal cell proliferation are decreased by a factor of approximately 1/2 with whole blood-derived serum that is heparinized and inactivated; proliferation ceases in plasma-derived serum that is heparinized and inactivated. Heparinization and inactivation of serum does not affect the proliferation of Rous sarcoma virus-infected cells, indicating that this combined treatment eliminates a mitogenic (regulatory) rather than a supportive (nutrient) factor(s) for cell replication. We hypothesize that mitogen(s) is released from plasma protein precursors when plasma clots in the presence of formed elements of the blood or when plasma-derived serum is exposed to cultured cells; heparinization and inactivation, within the framework of this hypothesis, would render nonfunctional the plasma protein precursor(s) from which the mitogen(s) is generated. Alternatively, our data are consistent with the release of two mitogens during blood clotting, one from plasma protein precursors and the other from formed elements of the blood. We also have studied the proliferative behavior of Swiss and BALB/c 3T3 cells in whole blood-derived and plasma-derived human serum. Our studies suggest that the platelet-derived growth factor has an artifactual supportive (nutrient) role, rather than an authentic mitogenic role, in cell replication.
Ethnic variation in Hpa 1 endonuclease cleavage patterns of human mitochondrial DNA.
Authors: Denaro M , Blanc H , Johnson M J , Chen K H , Wilmsen E , Cavalli-Sforza L L , Wallace D C
Published: May 2006

The mtDNAs of 235 individuals from five ethnic groups were analyzed for restriction site variation by digestion with restriction endonuclease Hpa I, Southern transfer, and hybridization with 32P-labeled human mtDNA. Six different cleavage patterns (morphs) were found, all of which could be related to each other by single nucleotide substitutions. Differences were found in the frequency of these morphs among the populations. The largest difference observed was in the frequency of the morph most common in Caucasians and Orientals compared to the frequency of that found in Africans. This difference apparently originated by the sequence change G-T-C-A-A-C to G-T-T-A-A-C. This alteration permitted recognition by Hpa I but did not alter the amino acid sequence. Two other observed differences were due to separate substitutions occurring in the ribosomal RNA genes. Comparison with primate data shows that the morph with two fragments, found in 12.5% of Oriental and 4% of Bantu samples, might be the ancestral type common to all hominoids. These two conserved sites were localized in tRNA genes in the anticodon loop. Assuming that the two-fragment morph is ancestral, this finding is consistent with previous data suggesting that Asia is genetically central to the radiations that are thought to have given rise to the human ethnic groups.
Long-term culture of normal mouse B lymphocytes.
Authors: Howard M , Kessler S , Chused T , Paul W E
Published: May 2006

A procedure is described for the preparation of long-term lines of normal mouse B lymphocytes. Surface immunoglobulin-bearing splenic B lymphocytes were purified with the fluorescence-activated cell sorter and then cultured with lipopolysaccharide for 1-4 wk. The cells were then transferred into medium supplemented with a T-hybridoma-derived supernatant containing interleukin 2 (IL2). Continuous feeding with this supernatant led to the establishment of cell lines that also could be propagated to IL 2-free medium containing interleukin 1 but not in culture medium alone. Cell lines have been propagated in this manner for as long as 10 mo. The cells in these lines have the appearance for small, dense lymphocytes, which all bear surface IgM detectable by immunofluorescence, rosetting, and surface radiolabeling and immunoprecipitation. The cells express Ia and lack Thy 1. These cultured B lymphocytes are unresponsive to lipopolysaccharide but can be activated to become more rapidly dividing, immunoglobulin-secreting cells by exposure to culture supernatants containing both T-cell-replacing factor and IL 2.
Plasmacytomas with more than one immunoglobulin kappa mRNA: implications for allelic exclusion.
Authors: Bernard O , Gough N M , Adams J M
Published: May 2006

Although only one allele of an immunoglobulin gene is thought to be expressed as a polypeptide by a given lymphocyte ("allelic exclusion"), three murine plasmacytomas were found to contain more than one kappa light chain mRNA species, as evidenced by the sequences of distinct kappa cDNA clones. Two different kappa cDNA sequences were cloned from BFPC 61 microsomal mRNA, two from MOPC 173, and three from S107. One cDNA sequence from each tumor matches the known secreted polypeptide, while the variant sequences differ in the variable (V) region. Hence fusion of a V kappa gene to a joining (J kappa) gene has occurred independently on separate homologous chromosomes and each allele is transcriptionally competent. The BFPC 61 variant sequence contained a normal V kappa sequence linked out of phase to J kappa 2; hence allelic exclusion in this line is accounted for by an error in DNA rearrangement. One S107 variant cDNA has an untranslatable sequence linked to J kappa--C kappa and may derive from a non-V kappa or pseudo-V kappa gene fused to J kappa. Another S107 variant cDNA, however, has a proper V kappa linked in phase to J kappa (albeit missing the first two germ-line J kappa codons) and the MOPC 173 variant sequence also contains a proper V kappa--J kappa join, although it does not encode a tryptophan residue common to all immunoglobulin chains. The presence of two potentially expressible kappa mRNAs in both S107 and MOPC 173 suggests that allelic exclusion does not hold in all lymphocytes, or that it sometimes reflects events subsequent to mRNA production, such as inability of certain kappa chains to assemble properly with the heavy chain. These observations are compatible with a stochastic model for allelic exclusion in which productive and nonproductive V--J recombination events occur at a certain frequency for each allele.
In vitro assembly of the Bacillus subtilis bacteriophage phi 29.
Authors: Bjornsti M A , Reilly B E , Anderson D L
Published: May 2006

In vitro assembly of the Bacillus subtilis bacteriophage phi 29 that approaches the efficiency of assembly in vivo has been demonstrated. Proheads, DNA, and gene 16 product (gp16) were essential for DNA encapsidation, and the average yield in extracts was 180 phage per prohead donor cell. The in vitro maturation was very similar to in vivo assembly in terms of yield, intermediates, and abortive structures. More that 30% of the proheads in the extract were converted to phage, and about 20% of DNA--protein extracted from phage could be repackaged. In vitro assembly was blocked by the addition of DNase I, EDTA, pyrophosphatase, or the ATP analogues adenosine 5'-[alpha, beta-methylene]triphosphate and adenosine 5'-[beta, gamma-methylene]triphosphate. Less than 1% of the proheads isolated in sucrose gradients can accept DNA--protein in packaging in vitro.
Benzodiazepine receptor increases after repeated seizures: evidence for localization to dentate granule cells.
Authors: Valdes F , Dasheiff R M , Birmingham F , Crutcher K A , McNamara J O
Published: May 2006

Repeated seizures, whether induced by kindling or electroshock, result in increased numbers of benzodiazepine receptors in hippocampal formation membranes. We sought to determine the cellular constituents containing the receptor increases. Binding studies of microdissected samples localized the receptor increases to fascia dentata. [3H]Flunitrazepam autoradiographic studies showed increases of silver grain density over the granule cell and molecular layers of fascia dentata but not in other regions of hippocampal formation. Destruction of granule cells by colchicine or neonatal x-irradiation was associated with marked decrease of benzodiazepine receptor binding. Together, these results provide evidence for localization of the receptor increases to the somata and dendritic tree of the granule cells. We suggest that this cellular localization may provide a clue to the network of altered neural circuitry underlying amygdala kindling.
Chemospecificity of ontogenetic units in the striatum: demonstration by combining [3H]thymidine neuronography and histochemical staining.
Authors: Graybiel A M , Hickey T L
Published: May 2006

Neurons being generated in the striatum of 10 fetal cats were pulse labeled by injection of [3H]thymidine directly into the maternal uterus at times ranging between the 22nd and 30th days (E22-E30) of the 65-day gestational period. Many of the striatal neurons labeled during this interval were found, at adolescence, to form 100- to 600-mum-wide cell clusters in the caudate nucleus. In E24-E30 specimens, we compared the distributions of these cell clusters with the locations of patches of low acetylcholinesterase activity and high enkephalin immunoreactivity (the "striosomes') visualized in serially adjoining sections. We found precise matches between most of the cell clusters and the acetylcholinesterase-poor enkephalin-rich zones, regardless of the embryonic age at which exposure to the [3H]thymidine had occurred. We conclude that the histochemically distinct striosomal patch-works observed in the acetylcholinesterase and enkephalin preparations correspond to ontogenetic units of the striatum.
Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts.
Authors: Li G C , Werb Z
Published: May 2006

Synthesis of a family of proteins called "heat shock" proteins is induced or enhanced in cells in response to various environmental stresses, suggesting that these proteins may perform functions essential to cell survival. Because a brief, nonlethal heat treatment can dramatically induce a transient resistance to a subsequent lethal heat treatment (thermotolerance), we examined the effect of heat treatment (41-46 degrees C) on protein synthesis and cell survival in plateau-phase Chinese hamster fibroblast (HA-1) cells. After heat treatments that either drastically inhibited total protein synthesis (46 degrees C) or did not suppress it (41 degrees C), the synthesis of heat shock proteins was greatly enhanced over that in unheated cells, and cell survival was increased 10(2)- to 10(6)-fold when cells were challenged by a subsequent lethal heat treatment. The synthesis of heat shock proteins correlated well with the development of thermotolerance, and the stability of these proteins correlated well with the persistence of thermotolerance up to 36 hr. Sodium arsenite, hypoxia, and ethanol also induced both the synthesis of heat shock proteins and transient thermotolerance. A qualitative analysis of individual proteins suggests that the synthesis and persistence of polypeptides of Mr 70,000 or 87,000 most closely conformed to the kinetics of thermotolerance.
cAMP and spore differentiation in Dictyostelium discoideum.
Authors: Kay R R
Published: May 2006

The individual amoebae composing of Dictyostelium aggregate can differentiate into either stalk or spore cells according, it is believed, to the extracellular signals they receive. By inducing the differentiation of isolated cells we hope to identify these signals. It is shown here that wild-type cells can differentiate into prespore cells in a solution of cAMP plus salts supplemented by conditioned medium. Cell-to-cell contact is not required. More important, isolated cells of various sporogenous mutant strains form spores in similar conditions without needing conditioned medium at all. For these strains at least, cAMP is the sole inducer necessary for spore formation. Earlier work has shown that stalk cells are induced by a combination of cAMP and a low molecular weight factor, differentiation inducing factor (DIF). DIF now appears to be the only pathway-specific inducer involved in the differentiation of sporogenous amoebae and DIF levels in the aggregate may therefore determine whether an amoeba becomes a stalk or a spore cell. In suitable conditions some sporogenous mutants form migrating slugs having an anterior/posterior pattern of prestalk and prespore cells. This pattern could be generated by the localized activity of DIF.
Use of un-derivatized thyroid hormones for photoaffinity labeling of binding proteins.
Authors: van der Walt B , Nikodem V M , Cahnmann H J
Published: May 2006

Irradiation of the thyroid hormones thyroxine and 3,5,3'-triiodothyronine in the near UV (greater than 300 nm) causes homolytic fission of C--I bonds in both rings. In the presence of hormone-binding proteins, the phenyl radical thus formed, and possibly also the iodine radical, can establish a covalent bond with certain amino acid residues in the binding site. Most if not all of the iodine radicals, however, appear to be reduced to iodide. Incubation of purified carrier proteins for the thyroid hormones in human serum as well as of an extract of rat liver nuclei or of whole nuclei with trace amounts of 125I- or 14C-labeled hormone, followed by irradiation, resulted in covalent binding. This was proven by gel filtration after boiling with guanidine:HCl and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the irradiated solutions or of the excluded-peak material obtained after gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of 125I-labeled irradiated nuclear extracts showed a prominent peak (Mr approximately equal to 45,000), sometimes with a shoulder or small peak at Mr approximately equal to 56,000, and a fast-moving peak (Mr approximately equal to 12,000). Similar patterns were obtained with N-bromoacetylthyroxine or N-bromoacetyltriiodothyronine without irradiation. When a suspension of whole nuclei was irradiated instead of nuclear extracts, the shoulder also became a prominent peak.
Immunocytochemical localization of gamma-glutamyltransferase in rat kidney with protein A-horseradish peroxidase.
Authors: Spater H W , Poruchynsky M S , Quintana N , Inoue M , Novikoff A B
Published: May 2006

Immunocytochemistry with a specific antiserum and protein A-horseradish peroxidase permits visualization of the sites of gamma-glutamyltransferase [(5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] at both the light- and the electron-microscope levels. As seen by light microscopy, the enzyme is localized only in the proximal convolutions of the renal tubules. Electron microscopy reveals dense deposits of 3,3'-diaminobenzidine reaction product embedded in the glycocalyx along the entire luminal surface of the microvilli and in the basolateral membranes.
gamma-Aminobutyric acid antagonists decrease junctional communication between L-horizontal cells of the retina.
Authors: Piccolino M , Neyton J , Witkovsky P , Gerschenfeld H M
Published: May 2006

The antagonists of gamma-aminobutyric acid, bicuculline and picrotoxin, were found to narrow the receptive field profile of the large field horizontal cell (L1HC) in the turtle retina when added to the perfusion medium in micromolar concentrations. The coupling resistance between neighboring L1HCs was increased by bicuculline or picrotoxin. Under control conditions, the dye Lucifer yellow injected into one L1HC diffused into a large number of neighboring L1HCs; bicuculline or picrotoxin greatly restricted dye passage between these same cells. We conclude that antagonists of gamma-aminobutyric acid decrease the conductance of gap junctions between L1HCs.
A major calmodulin-binding protein common to various vertebrate tissues.
Authors: Palfrey H C , Schiebler W , Greengard P
Published: May 2006

A major calmodulin-binding protein (CaM-BP) of Mr 240,000 was demonstrated in various rat tissues by using a 125I-labeled CaM gel overlay technique. This protein (designated p240) was detected in the particulate fraction and to a lesser extent in the cytosol of all tissues studied. Binding of CaM to p240 was completely dependent on Ca2+. A second, exclusively soluble, CaM-BP (Mr115,000) common to several tissues and a number of other CaM-BPs with a more restricted tissue distribution were also observed by using this technique. CaM binding to p240 occurred in high amounts in plasma membranes from avian erythrocytes but was absent from mammalian erythrocyte membranes. Antibodies prepared against turkey erythrocyte p240 (anti-Tp240) crossreacted with p240 in other tissues. Identity between the proteins recognized by anti-Tp240 and CaM was confirmed by demonstrating that 125I-labeled CaM could bind to p240 specifically immunoprecipitated from either rat brain or turkey erythrocytes by anti-Tp240. The p240 may be related to a previously described actin-binding protein and may represent a major site of action of CaM on the cytoskeleton.
Temperature-compensated oscillations in respiration and cellular protein content in synchronous cultures of Acanthamoeba castellanii.
Authors: Lloyd D , Edwards S W , Fry J C
Published: May 2006

Synchronous cultures of the soil amoeba Acanthamoeba castellanii, established by a selection procedure, show significant oscillations of respiration and total cell protein. There was little difference between the period of these oscillations, which averaged 76 min, although the five incubation temperatures used varied between 20 degrees C and 30 degrees C and the cell division time increased from 7.8 to 16 hr. The phase of these oscillations also corresponded approximately at all incubation temperatures. Similar observations made over the whole division cycle at three temperatures indicated that similar oscillations occurred, with a constant period of 65 min, although these data were too variable to show this unequivocally. Control (asynchronous) cultures show that the oscillations are not a consequence of metabolic perturbation produced by the centrifugal selection procedure. It is suggested that these temperature-compensated epigenetic oscillations serve a dual role in cell cycle and circadian timekeeping and that cell cycle time is quantized.
Base sequence of a cloned snake W-chromosome DNA fragment and identification of a male-specific putative mRNA in the mouse.
Authors: Epplen J T , McCarrey J R , Sutou S , Ohno S
Published: May 2006

A 2.5-kilobase fragment of a sex-specific satellite DNA from the Colubrid snake species Elaphe radiata has been cloned, and its sequence has been determined. It contains 26 and 12 copies, respectively, of two base quadruplets, G-A-T-A and G-A-C-A, as its sole highly repetitious elements. Southern hybridization experiments with genomic DNA of the chicken, the mouse, and man indicated male sex-specific conservation of at least parts of this cloned DNA. In situ hybridization experiments with metaphase chromosomes of the mouse showed that elements that can cross-hybridize with parts of the cloned snake DNA are concentrated in the pericentric region of the Y chromosome. In blot hybridization experiments with liver poly(A)+ polysomal RNAs of male and female mice, a probe consisting of the first 1,224 bases of the cloned snake DNA singled out a male-specific RNA of 1,250-1,400 bases. Inasmuch as the proximal end of this probe contained an open reading frame (44 consecutive amino acid-specifying codons), the male-specific putative mRNA so detected may specify H-Y antigen. By contrast, a probe consisting of bases 1,480-1,906, containing the simple repeats of the quadruplets, singled out a shorter (approximately 1,000-base) RNA from males and females alike. Although this RNA is poly(A)+, we have yet to establish its attachment to ribosomes.
Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.
Authors: Kraus J P , Rosenberg L E
Published: May 2006

We have purified three low-abundance hepatic mRNAs to near homogeneity by polysome immunoadsorption. The mRNAs coding for the precursor of ornithine transcarbamoylase [carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3], the precursor of the beta-subunit of propionyl-CoA carboxylase [propionyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3], and cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], representing approximately 0.20, 0.02, and 0.015% of total hepatic mRNA, respectively, were purified 450- to 6,300-fold. We used the following steps: interaction of rat liver polysomes with an IgG fraction of monospecific antisera raised against each polypeptide; immobilization of polysome-antibody complexes on a protein A-Sepharose column; removal of the bulk of polysomes by extensive washing; dissociation of ribosomal subunits and elution of specific mRNA with EDTA; and isolation of the eluted mRNA by chromatography on an oligo(dT)-cellulose column. It seems likely that this procedure will permit isolation of other low-abundance mRNAs and subsequent cloning of their respective cDNAs.
Hyperpolarization of neuroblastoma-glioma hybrid NG108-15 by vanadium ions.
Authors: Lichtstein D , Mullikin-Kilpatrick D , Blume A J
Published: May 2006

Vanadate hyperpolarizes mouse neuroblastoma-glioma hybrid NG108-15 by 20-30 mV. These changes in membrane potential (delta psi) are observed by monitoring the equilibrium distribution (intracellular/extracellular) of the lipophilic cation [3H]tetraphenylphosphonium (TTP+) and by directly measuring delta psi with intracellular microelectrodes. In physiological media (i.e., 135 mM NaCl/5 mM KCl), the half-maximal effective concentrations of sodium orthovanadate (Na3VO4) and sodium metavanadate (NaVO3) are 35 and 160 microM, respectively. The maximal effects for both these ions are quantitatively indistinguishable. The hyperpolarizing responses to vanadate occur without any observable lag, have t1/2 less than or equal to 30 sec, and are always accompanied by simultaneous decreases in membrane resistance. Neither ouabain nor media containing high K (i.e., 120 mM) devoid of Na and K (isotonicity maintained by choline) prevent the change in delta psi induced by vanadate. Therefore, vanadate produces a unique hyperpolarization which does not depend upon Na, K, or the Na/K pump. Furthermore, the accompanying decreases in membrane resistance indicate that vanadate must increase the permeability of the membrane to some ion. Our data are consistent with it being an anion, such as chloride or vanadate itself. Finally, vanadate hyperpolarizes many different types of cultured cells, only some of which are of neuronal origin. This indicates that a hyperpolarization of delta psi must be considered in any assessment of the physiological actions of the vanadates.
Ca2+ -activated K+ conductance in internally perfused snail neurons is enhanced by protein phosphorylation.
Authors: de Peyer J E , Cachelin A B , Levitan I B , Reuter H
Published: May 2006

Depolarizing voltage steps induce inward and outward currents in voltage-clamped, internally perfused neurons from the snail Helix roseneri. Addition of the catalytic subunit of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) to the internal perfusing medium results in an increase in the net outward current, with no apparent effect on the inward current. Catalytic subunit inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) is without effect, indicating that the increase in net outward current results from protein phosphorylation rather than an unspecific effect of protein perfusion. Decreasing the external Ca2+ concentration from 10 to 1 mM eliminates the effect of catalytic subunit, suggesting that Ca2+ plays an important role in this response. This suggestion is supported by the fact that the stimulation by catalytic subunit can be mimicked by increasing the Ca2+ concentration in the internal perfusion medium and can be prevented by intracellular perfusion with 10 mM EGTA. The results are consistent with the hypothesis that cyclic AMP-dependent protein phosphorylation regulates the Ca2+-activated K+ conductance in these cells.
Sagittal cerebellar microbands of taurine neurons: Immunocytochemical demonstration by using antibodies against the taurine-synthesizing enzyme cysteine sulfinic acid decarboxylase
Authors: Chan-Palay Victoria , Palay Sanford L. , Wu Jang-Yen
Published: May 2006

Taurine neurons in the cerebellum of rabbit, rat, and mouse were localized at the light microscope level by using polyclonal antibodies against cysteine sulfinic acid decarboxylase (CSADCase; EC 4.1.1.29), the enzyme responsible for the conversion of cysteine sulfinic acid to hypotaurine and of cysteic acid to taurine. The indirect peroxidase-antiperoxidase method was used on Vibratome sections and on serial sections of paraffin-embedded tissue. Intensification of CSADCase immunoreactivity was achieved by pretreatment of the animal with L-cysteine or L-cysteic acid intravenously 1-2 hr prior to perfusion. A combination of L-cysteic acid and demecolcine, which retards axoplasmic flow, was most effective in maximizing CSADCase immunoreactivity. Although these treatments intensified immunoreactivity in neurons, no more cells were reactive than in untreated controls. L-Glutamic acid did not increase CSADCase immunoreactivity but did increase immunoreactivity with antibodies against L-glutamic acid decarboxylase (GAD; EC 4.1.1.15), the synthetic enzyme for γ-aminobutyric acid. Specificity was established by negative results obtained with various control incubations including the use of CSADCase antiserum preabsorbed with the antigen. Taurine neurons of the cerebellar cortex are arranged in sagittal microbands, defined by intensely CSADCase-reactive Purkinje neurons and their axons and dendrites, together with stellate, basket, and Golgi cells and their processes. In the vermis there is a narrow midline band, flanked laterally by three wider bands on either side, each separated from the next by an unreactive zone. Although the zonal borders are sharp, the interzonal areas contain some CSADCase-immunoreactive axons but no cell bodies. The seven vermal bands are best observed in the anterior lobe. Others exist in the lateral hemispheres. The paraflocculus and flocculus contain numerous intensely immunoreactive neurons, and banding is difficult to discern. Lobule X of the vermis is also heavily endowed with taurine neurons. Numerous large and medium-sized deep cerebellar and vestibular nuclei are also immunoreactive. These observations indicate that cerebellar neurons are chemically heterogeneous but that neurons of similar chemical signature in the cerebellar cortex are organized into sagittal microbands. This corroborates our earlier evidence that Purkinje cells containing motilin and those containing both motilin and γ-aminobutyric acid are also arranged in vermal sagittal microbands. The midline vermal band contains Purkinje neurons with multiple neuroactive substances—taurine, γ-aminobutyric acid, and motilin. It remains to be determined how this chemical zonation in the cerebellar cortex relates to the banded afferent innervation from spinal, vestibular, reticular, and olivary sources.
Conformation of
cyclo
(-L-Pro-Gly-)
3
and its Ca
2+
and Mg
2+
complexes
Authors: Kartha Gopinath , Varughese Kottayil I. , Aimoto Saburo
Published: May 2006

The synthetic hexapeptide
cyclo
(-L-Pro-Gly-)
3
is an ionophore that shows interesting conformational changes upon binding metal ions. X-ray crystallographic studies of this peptide show that when it is crystallized from an ethanol/ethyl acetate mixture the ring takes up an asymmetric conformation containing one
cis
peptide bond. In crystals of a Ca
2+
complex, the cation is sandwiched between two peptide molecules that differ markedly in conformation. However, both exhibit threefold symmetric forms, with all six peptide bonds in the molecule occurring in the usual
trans
conformation. The Ca
2+
is octahedrally surrounded by six glycyl carbonyl oxygens from the two peptides at an average distance of 2.26 Å and can easily be released by the disruption of the peptide sandwich. In the magnesium complex, the peptide forms a 1:1 complex with the ion. The Mg
2+
is octahedrally coordinated to three glycyl carbonyls and three water oxygens. The average coordination distance between magnesium and the peptide oxygens is 2.03 Å and that between magnesium and water oxygen is 2.11 Å. The two peptide molecules in the asymmetric unit have similar conformations and have approximate threefold symmetry.
endo-beta-N-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins.
Authors: Elder J H , Alexander S
Published: May 2006

We have detected an endoglycosidase activity produced by Flavobacterium meningosepticum. This enzyme, named endo F, cleaves glycans of both the high-mannose and the complex type linked through asparagine to the protein backbone. The data indicate that cleavage occurs via hydrolysis of the glycosidic bond of the N,N'-diacetylchitobiose core structure adjacent to asparagine, similar to that due to endo H and endo D. Extreme variability was noted in the availability of this cleavage site among N-linked glycoproteins. Glycoproteins of retrovirus, lymphocytic choriomeningitis virus, Pichinde virus, and HLA-A and -B antigens were readily cleaved in the presence of nonionic detergent. Others, such as ovalbumin, fetuin, bromelain, ovomucoid, alpha 1-acid glycoprotein, immunoglobulin G, and influenza virus hemagglutinin became susceptible only after reduction and alkylation or when cleavage was performed in the presence of 1% 2-mercaptoethanol. Endo F should prove useful in the study of glycans and protein backbones as discrete entities and for defining the nature of the glycan-protein interface.
Concerning 1e- transfer in reduction by dihydronicotinamide: reaction of oxidized flavin and flavin radical with N-benzyl-1,5-dihydronicotinamide.
Authors: Powell M F , Wong W H , Bruice T C
Published: May 2006

The rates of reaction of 5-ethyl-3-methyllumiflavinium perchlorate and 5-ethyl-3-methyllumiflavinyl radical with a NADH analogue, N-benzyl-1,4-dihydronicotinamide, were measured anaerobically in tert-butanol and 5% acetonitrile/95% tert-butanol solutions at 30 degrees C. The biphasic kinetics observed for the reaction of flavin radical with dihydronicotinamide were interpreted in terms of both a 1e- and a 2e- mechanism; the former was found to be inadequate based on experimental requirements of the mechanism. The dihydronicotinamide reacts preferentially with oxidized flavin rather than flavin radical, even when the concentration of oxidized flavin is at a concentration 5 orders of magnitude less than that of radical. These studies show that the "hydride" reduction of oxidized flavin by BNAH is more facile than is the 1e- reduction of flavin radical by BNAH.
A unique cell-surface protein phenotype distinguishes human small-cell from non-small-cell lung cancer.
Authors: Baylin S B , Gazdar A F , Minna J D , Bernal S D , Shaper J H
Published: May 2006

We have used radioiodination (125I) and two-dimensional polyacrylamide gel electrophoresis to determine that small- (oat) cell lung carcinoma (SCC)--a tumor with neuroendocrine features--possesses a surface protein pattern distinct from the other types of lung cancer cells (squamous, adeno-, and large-cell undifferentiated carcinoma). Twelve distinguishing proteins, 40 to 70 kilodaltons (kDal), characterized four separate lines of SCC; three of these, designated E (60 kDal; pI = 7.3), S (30 kDal; pI = 6.0), and U (57 kDal; pI = 5.6), may be unique SCC gene products and were identified only in [35S]methionine labeling of SCC and not in non-SCC or human fibroblasts. Two lines of adeno-, one of squamous, and one of undifferentiated large-cell lung carcinoma exhibited similar surface protein patterns to one another. Nine distinguishing proteins (40 to 100 kDal) and at least five large proteins (greater than 100 kDal) were unique to these lines. The surface protein phenotypes for SCC and non-SCC were distinct from those for human lymphoblastoid cells and fibroblasts. However, the neuroendocrine features of SCC were further substantiated because 6 of the 12 distinguishing SCC surface proteins, including E and U, were identified on human neuroblastoma cells. The proteins identified should (i) help define differentiation steps for normal and neoplastic bronchial epithelial cells, (ii) prove useful in better classifying lung cancers, and (iii) be instrumental in tracing formation of neuroendocrine cells.
Pigment particle translocation in detergent-permeabilized melanophores of Fundulus heteroclitus.
Authors: Clark T G , Rosenbaum J L
Published: May 2006

Melanophore preparations of Fundulus heteroclitus that have been treated with the detergent Brij 58 can aggregate their pigment in response to epinephrine. On the basis of several criteria, it appears that cell lysis occurs under the detergent conditions used. Electron microscopic examination of detergent-treated cells shows progressive disruption of the melanophore plasma membrane during the time in which pigment aggregation occurs. Brij-treated cells are accessible to ferritin, a large electron-dense probe that is effectively excluded from non-detergent-treated controls. In cells incubated with detergent, fixed, and treated first with rat monoclonal antibodies against tubulin and then with fluorescein-isothiocyanate-labeled goat anti-rat IgG, a characteristic radical pattern of microtubule staining can be visualized by fluorescence microscopy. Control preparations treated similarly, but without detergent, do not stain. Vanadate, an inhibitor of ciliary and flagellar dynein ATPase, blocks melanosome aggregation in response to epinephrine in detergent-treated preparations but has no effect on intact melanophores. erythro-9-[3-(2-hydroxynonyl)]Adenine, another inhibitor of dynein ATPase, also inhibits pigment aggregation in Fundulus melanophores. The possibility that a dynein-like molecule plays a role in pigment aggregation is discussed.
Treatment of perennial allergic rhinitis by local hyperthermia.
Authors: Yerushalmi A , Karman S , Lwoff A
Published: May 2006

Ninety-five patients with documented perennial allergic rhinitis have been treated with local hyperthermia of the nasal passages in a randomized double-blind trial. The treatment consisted of one series of three 30-min insufflations of humidified air at 43 degrees C at a 2-hr interval. In the active treatment group, 75% (P less than 0.00003) and 68% (P less than 0.00001) of the patients were free of symptoms 1 week and 1 month, respectively, after treatment, compared with a 28% and 17% response in the placebo group. We conclude that local hyperthermia is effective in the treatment of perennial allergic rhinitis.
Human genome contains four genes homologous to transforming genes of Harvey and Kirsten murine sarcoma viruses.
Authors: Chang E H , Gonda M A , Ellis R W , Scolnick E M , Lowy D R
Published: May 2006

Harvey and Kirsten murine sarcoma viruses each encode a structurally and functionally related 21-kilodalton protein (p21), which is the transforming protein of each virus. Using probes from the 0.9-kilobase (kb) p21-coding region of each virus (called v-Ha-ras and v-Ki-ras, respectively), we have molecularly cloned from normal human genomic DNA the sequences that hybridize to these probes. Four evolutionarily divergent restriction endonuclease fragments were isolated. Two hybridized preferentially to v-Ha-ras and were designated human c-Ha-ras1 and c-Ha-ras2; two hybridized preferentially to v-Ki-ras and were called c-Ki-ras1 and c-Ki-ras2. Human c-Ha-ras1 contained 0.9 kb of sequence homologous with v-Ha-ras interspersed with three intervening sequences; this gene was closely related to a previously cloned rat c-Ha-ras gene that also contained intervening sequences. Human c-Ha-ras2 was more divergent from v-Ha-ras and also hybridized poorly to human c-Ha-ras1. One c-Ki-ras gene contained 0.9 kb homologous to v-Ki-ras and had one intervening sequence, whereas the other contained only 0.3 kb homologous to v-Ki-ras. The results indicated that human DNA contains several copies of the c-ras family and that c-Ha-ras1 (with intervening sequences) was more highly conserved evolutionarily than was c-Ha-ras2.
Amino acid sequence of mouse submaxillary gland renin.
Authors: Misono K S , Chang J J , Inagami T
Published: May 2006

The complete amino acid sequences of the heavy chain and light chain of mouse submaxillary gland renin have been determined. The heavy chain consists of 288 amino acid residues having a Mr of 31,036 calculated from the sequence. The light chain contains 48 amino acid residues with a Mr of 5,458. The sequence of the heavy chain was determined by automated Edman degradations of the cyanogen bromide peptides and tryptic peptides generated after citraconylation, as well as other peptides generated therefrom. The sequence of the light chain was derived from sequence analyses of the peptides generated by cyanogen bromide cleavage or by digestion with Staphylococcus aureus protease. The sequences in the active site regions in renin containing two catalytically essential aspartyl residues 32 and 215 were found identical with those in pepsin, chymosin, and penicillopepsin. Comparison of the amino acid sequence of renin with that of porcine pepsin indicated a 42% sequence identity of the heavy chain with the amino-terminal and middle regions and a 46% identity of the light chain with the carboxyl-terminal region of the porcine pepsin sequence. Residues identical in renin and pepsin are distributed throughout the length of the molecules, suggesting a similarity in their overall structures.
Nucleotide sequences involved in bacteriophage T4 gene 32 translational self-regulation.
Authors: Krisch H M , Allet B
Published: May 2006

We have determined the nucleotide sequence of a cloned segment of the bacteriophage T4D chromosome, which contains the regulatory sequences and the structural gene (gene 32) for the single-stranded DNA binding protein (gp32). The amino acid sequence predicted by translation of the structural gene agrees well with that published for gp32 [Williams, K. R., Lo-Presti, M. B., Setoguchi, M. & Konigsberg, W. H. (1980) Proc. Natl. Acad. Sci. USA 77, 4614-4617]. To localize the nucleotide sequence involved in translational self-regulation of gene 32, we have constructed a series of plasmids in which gene 32 is fused to an amino-terminal deletion mutant of the beta-galactosidase gene of Escherichia coli. Expression of a beta-galactosidase fusion protein that contains only the first seven amino acids of gp32 is still repressed by gp32. The ribosomal binding site of gene 32 is flanked by a repetitive A+T-rich sequence. Preferential and cooperative binding of gp32 to this region of its mRNA could inhibit translation initiation and, thus, would account for the autoregulation.
Presynaptic dendrites and perikarya in deafferented cerebellar cortex.
Authors: Hámori J , Somogyi J
Published: May 2006

Two to 30 days after complete isolation of the cerebellar cortex of adult rats, small Golgi II type neurons in the granular layer develop presynaptic sites on their dendrites and perikarya. The newly developed presynaptic dendrites established dendrodendritic synaptic contacts with dendritic digits of granule cells. In addition, some granule cells were observed to develop presynaptic sites on their somata and dendritic digits, resulting in the formation of (sometimes reciprocal) dendrodendritic synapses between granule cells or between granule cells and Golgi neurons. The development of these unusual synaptic formations indicates a persisting synaptic plasticity of these two cerebellar cell types. It is suggested that this unorthodox synapse formation develops as part of a reactive compensatory process to synaptic desaturation brought about by the deafferentation of the cerebellum.
Ribosomal protein S4 is an internal protein: Localization by immunoelectron microscopy on protein-deficient subribosomal particles.
Authors: Winkelmann D A , Kahan L , Lake J A
Published: May 2006

The location of protein S4 in the small ribosomal subunit has been identified by immunoelectron microscopy. Although intact small subunits are not reactive with antibodies directed against protein S4, subribosomal particles reconstituted without proteins S5 and S12 are reactive. By using these "incomplete" subparticles, we have mapped the position of S4. It is located at a single site on the exterior (cytoplasmic) side of the subunit, at the partition that separates the one-third, or head, from two-thirds, or base, of the subunit. In this location, protein S4 is "beneath" proteins S5 and S12. All three proteins are members of a complex on, or near, the external surface of the small ribosomal subunit that plays an important role in regulation of translational fidelity.