bluuebunny/crossref_metadata_2025 huggingface dataset:
15N NMR studies of nitrogen metabolism in intact mycelia of Neurospora crassa.
Authors: Legerton T L , Kanamori K , Weiss R L , Roberts J D
Published: May 2006

Suspensions of intact mycelia of Neurospora crassa grown in medium containing 15NH4Cl have been found to give well-resolved 15N nuclear magnetic resonance spectra for metabolites that play crucial roles in intermediary nitrogen metabolism. These include the amide nitrogen of glutamine, the alpha-amino nitrogens of glutamate and other amino acids, the guanidino nitrogens of arginine, the ureido nitrogen of citrulline, the side-chain nitrogens of ornithine or lysine, or both, and uridine diphosphates. The turnover time of glutamine in vivo was estimated to be less than 1 hr by using nuclear magnetic resonance spectroscopy in conjunction with tracer methodologies. Applications of these techniques to the study of nitrogen metabolism are discussed.
Magnetic modulation of release of macromolecules from polymers.
Authors: Hsieh D S , Langer R , Folkman J
Published: May 2006

Sustained-release systems were made by incorporating bovine serum albumin and magnetic steel beads in an ethylene-vinyl acetate copolymer matrix. When exposed to aqueous medium, the polymer matrix released the albumin slowly and continuously. Application of an oscillating magnetic field increased the release rate by as much as 100%. Intervals of 6-hr periods of magnetic exposure and nonexposure were alternated over a 5-day period, resulting in corresponding increases and decreases in release and establishing a pattern of modulated sustained release.
Enhancement of formation of the esophageal carcinogen benzylmethylnitrosamine from its precursors by
Candida albicans
Authors: Hsia Chu-Chih , Sun Tsung-Tang , Wang Yang-Yuin , Anderson Lucy M. , Armstrong Donald , Good Robert A.
Published: May 2006

Previous studies in Linxian, an area of China with a high incidence of esophageal carcinoma, showed that fungal infections are common in the esophageal epithelium of patients with either premalignant changes or early esophageal carcinoma. Fungi of the genus
Candida
were the most frequent invaders. In these areas nitrate and nitrite are often present in high concentrations in drinking water and staple grains. The present studies have established the ability of
Candida albicans
to augment the nitrosative formation of the esophagus-specific carcinogen, benzylmethylnitrosamine (NBMA;
N
-nitroso-
N
-methylbenzylamine). Stationary
C. albicans
cultures, with pH held at 6.8, were incubated with the precursors of NBMA, benzylmethylamine (BMA;
N
-methylbenzylamine) and NaNO
2
. There was a significant increase in the amount of NBMA formed in these cultures, compared to precursors-only controls. The amount of NBMA synthesized depended on fungal cell number. Exponentially growing cultures were also able to cause NBMA formation. The identity of the NBMA was confirmed by high-performance liquid chromatographic coelution with authentic NBMA in three solvent systems and by mass spectroscopy. Boiled cells and conditioned medium in which cells had been incubated were not effective in enhancing nitrosation. Cultured
Candida
released acidic metabolites that reduced the pH of the medium when only a low concentration of buffer was present. Spontaneous nitrosation of BMA was enhanced under these acidic conditions. Thus,
C. albicans
infecting the esophageal epithelium could cause local formation of NBMA by both cell-mediated catalysis and extracellular decrease in pH.
Synaptic localization of alpha-bungarotoxin binding which blocks nicotinic transmission at frog sympathetic neurons.
Authors: Marshall L M
Published: May 2006

Sympathetic neurons receive direct synaptic input from cholinergic terminal boutons of preganglionic nerve fibers. The distribution of acetylcholine receptors at these synapses is not precisely known. This study shows that alpha-bungarotoxin, which binds specifically to nicotinic receptors on skeletal muscle, also may be useful for localizing postsynaptic nicotinic receptors on principal neurons in the paravertebral sympathetic ganglia of the bullfrog. alpha-Bungarotoxin (1-5 microM) produces a block of nicotinic (fast) excitatory postsynaptic potentials that is fully reversed after 5-8 hr of washing. Dihydro-beta-erythroidine, a nicotinic antagonist, reduces the half-time of recovery from the toxin block to one-third of the control value, presumably by competing for the same receptor sites. Furthermore, the response to applied carbachol is reduced by the toxin, indicating that the block of synaptic transmission is due to a decreased response of the postsynaptic membrane. Peroxidase-labeled alpha-bungarotoxin is localized to small (0.2- to 0.5-micrometers diameter) patches beneath synaptic boutons. Peroxidase reaction product is restricted to regions of the synaptic cleft just opposite the active zones of the presynaptic terminal. In addition, peroxidase-labeled antibodies against Torpedo acetylcholine receptor bind exclusively to these same synaptic regions; evidently these patches are the areas at which nicotinic receptors are concentrated at synaptic contacts on sympathetic neurons.
Method for constructing bijections for classical partition identities
Authors: Garsia A. M. , Milne S. C.
Published: May 2006

We sketch the construction of a bijection between the partitions of
n
with parts congruent to 1 or 4 (mod 5) and the partitions of
n
with parts differing by at least 2. This bijection is obtained by a cut-and-paste procedure that starts with a partition in one class and ends with a partition in the other class. The whole construction is a combination of a bijection discovered quite early by Schur and two bijections of our own. A basic principle concerning pairs of involutions provides the key for connecting all these bijections. It appears that our methods lead to an algorithm for constructing bijections for other identities of Rogers-Ramanujan type such as the Gordon identities.
Distribution of protein I in mammalian brain as determined by a detergent-based radioimmunoassay.
Authors: Goelz S E , Nestler E J , Chehrazi B , Greengard P
Published: May 2006

A radioimmunoassay has been developed for measuring protein I, a basic, neuron-specific protein associated with nerve terminals. The procedure utilizes the detergents NaDodSO4 and Nonidet P-40 to prevent nonspecific adsorption of this highly charged protein to various surfaces. By use of this procedure, it has been possible to show that protein I comprises approximately 0.4% of the total protein in cerebral cortex of several mammalian species. In addition, the amount of protein I was determined in about 40 regions of cat brain. The results suggest that measurement of protein I may provide a quantitative method for estimating the density of nerve terminals in various regions of the mammalian nervous system.
Enzymatic reduction of protein-bound methionine sulfoxide.
Authors: Brot N , Weissbach L , Werth J , Weissbach H
Published: May 2006

An enzyme that catalyzes the reduction of methionine sulfoxide residues in ribosomal protein L12 has been partially purified from Escherichia coli extracts. Methionine sulfoxide present in oxidize [Met]enkephalin is also reduced by the purified enzyme. The enzyme is different from a previously reported E. coli enzyme that catalyzes the reduction of methionine sulfoxide to methionine [Ejiri, S. I., Weissbach, H. & Brot, N. (1980) Anal. Biochem. 102, 393--398]. Extracts of rat tissues, Euglena gracilis, Tetrahymena pyriformis, HeLa cells, and spinach also can catalyze the reduction of methionine sulfoxide residues in protein.
Enhanced phosphorylation of high-mobility-group proteins in nuclease-sensitive mononucleosomes from butyrate-treated HeLa cells.
Authors: Levy-Wilson B
Published: May 2006

The protein composition of nucleosome fractions differing in their sensitivity to micrococcal nuclease and derived from butyrate-treated or untreated HeLa cells has been compared. Most of the high-mobility-group-14 (HMG-14) and HMG-17 content of HeLa cell chromatin is associated with those nucleosomes that are preferentially sensitive to micrococcal nuclease. Furthermore, electrophoresis of these two HMG proteins from the transcriptionally active chromatin fraction MN1 of butyrate-treated cells resolves them into a series of bands. The multiple band pattern of HMG-14 and -17 from butyrate-treated cells results from hyperphosphorylation rather than hyperacetylation. Phosphorylation of these two small nonhistone proteins may play some role in the modulation of the structure of transcriptionally active chromatin.
Effect of vanadate on intracellular distribution and function of 10-nm filaments.
Authors: Wang E , Choppin P W
Published: May 2006

Earlier reports from this laboratory suggested that 10-nm filaments and microtubules act together in the movement and positioning of nuclei and centrioles. Sodium vanadate has been found to alter the distribution of 10-nm filaments and separate them from microtubules in virus-induced syncytia and uninfected cells. Accompanying this change in cytoskeletal elements in an alteration in the distribution of nuclei, centrioles, and other organelles. Nuclei in vanadate-treated syncytia were found in a circle or horseshoe arrangement, and 10-nm filaments were aggregated within the circle, whereas microtubules, were found in a network throughout the cytoplasm. Vanadate also caused a perinuclear aggregation of 10-nm filaments in single uninfected cells, whereas microtubules were throughout the cytoplasm, as in syncytia. Centrioles, mitochondria, rough endoplasmic reticulum, and lysosomes were scattered in the perinuclear area, with mitochondria and rough endoplasmic reticulum frequently closely associated, whereas the peripheral region of vanadate-treated cells contained ribosomes, microfilament bundles, and microtubules, but not 10-nm filaments. Vanadate limited virus-induced fusion of cells to polykaryocytes with 5--20 nuclei, in contrast to the massive syncytia found in untreated cells. These results indicate that vanadate separates 10-nm filaments and microtubules topologically and functionally, and support previous evidence that 10-nm filaments and microtubules act together in the movement and positioning of nuclei and other organelles.
In vitro induction of adult erythropoiesis in early mouse yolk sac.
Authors: Cudennec C A , Thiery J P , Le Douarin N M
Published: May 2006

The capacity of yolk sac hemopoietic cells to produce either primitive or definitive erythrocytes was analyzed in vitro under three different experimental conditions. (i) Before the 28-somite stage (prior to colonization of the liver rudiment by hemopoietic cells), yolk sac explanted alone produced solely primitive erythrocytes and only for a short time. (ii) When allowed to colonize a liver rudiment, hemopoietic cells from the yolk sac gave rise to definitive erythrocytes. (iii) These cells could express the same capacity when stimulated by various intraembryonic organs, even if no direct cell--cell contact was established between stimulating tissue and target hemopoietic cells. These results provide evidence that humoral factors present in embryos past the 28-somite stage act on hemopoietic cells, inducing the onset of definitive erythropoiesis.
Differentiation of regenerating pancreatic cells into hepatocyte-like cells.
Authors: Scarpelli D G , Rao M S
Published: May 2006

Differentiation is the process by which multicellular organisms achieve the specialized functions necessary for adaptation and survival. An in vivo model in the Syrian golden hamster is described in which regenerating pancreatic cells are converted into hepatocyte-like cells, as evidenced by the presence of albumin, peroxisomes, and a variety of morphological markers. These cells are stable after the conversion is triggered by a single dose of the carcinogen N-nitrosobis(2-oxopropyl)amine administered during the S phase in regenerating pancreatic cells. This suggests that, given the proper stimulus, regenerating cells in adult pancreas can be redirected into a totally different pathway of differentiation.
Formation of a covalent complex between the 80,000-dalton adenovirus terminal protein and 5'-dCMP in vitro.
Authors: Lichy J H , Horwitz M S , Hurwitz J
Published: May 2006

An in vitro adenovirus DNA replication system catalyzed the formation of a covalent complex between an 80,000-dalton protein and 5'-dCMP in the presence of [alpha-32P-dCTP, MgCl2, ATP, and adenovirus (Ad) DNA with a protein covalently bound to the 5' end of each strand (Ad DNA-prot). The requirement for Ad DNA-prot in this reaction was similar to that for in vitro DNA replication. When dATP, dTTP, and the 2',3'-dideoxynucleoside triphosphate (ddNTP) ddGTP were included in the reaction mixture, an elongated complex was detected, which consisted of an 80,000-dalton protein bound to a 26-base oligonucleotide. Formation of the elongated product, but not of the protein-dCMP complex, was inhibited by ddATP, ddCTP, or ddTTP. The requirements for formation of the protein-dCMP complex, the nature of the linkage between protein and dCMP, the size of the protein, and the existence of elongated forms indicated that the protein associated with the complex was identical to the 80,000-dalton Ad terminal protein found on replicating DNA molecules as described by Challberg et al. [Challberg, M. D., Desiderio, S. V. & Kelly, T. J., Jr. (1980) Proc. Natl. Acad. Sci. USA 77, 5105-5109].
On the mechanism of selective inhibition of herpesvirus replication by (E)-5-(2-bromovinyl)-2'-deoxyuridine.
Authors: Allaudeen H S , Kozarich J W , Bertino J R , De Clercq E
Published: May 2006

Bromovinyldeoxyuridine (BVdUrd) is a potent antiherpesvirus compound with low cytotoxicity. To gain an insight into its selectivity and mechanism of inhibition, we chemically synthesized the 5'-triphosphate of BVdUrd, BVdUTP, and tested its effect on the activities of DNA polymerases [DNA nucleotidyltransferase (DNA directed), EC 2.7.7.7] of two herpesviruses--i.e., herpes simplex virus type 1 (HSV-1) and Epstein-Barr virus (EBV)--as well as cellular DNA polymerases alpha, beta, and gamma. The effects on the DNA polymerases were determined under assay conditions optimal for the individual polymerases. We found that the BVdUTP was considerably more inhibityory to the utilization of dTTP by the HSV-1 DNA polymerase then by the cellular DNA polymerases. For instance, as little as 1 microM BVdUTP inhibited the utilization of dTTP by HSV-1 DNA polymerase 50%, whereas the same concentration inhibited the DNA polymerase alpha and the DNA polymerase beta activities only 9% and 3%, respectively. The BVdUTP inhibited DNA synthesis by competing with the natural substrate, dTTP. The Km for dTTP and the Ki for the BVdUTP of the HSV-1 DNA polymerase were 0.66 and 0.25 microM, respectively. Kinetic analyses with the DNA polymerases alpha and beta and the EBV DNA polymerase also reflected a similar difference in sensitivity between the HSV-1 enzyme and other enzymes. Increasing the concentration of either the DNA template or the enzyme in the reaction mixture did not bring about a significant change in the extent of inhibition. Preincubation of the inhibitor with the enzyme was not necessary for inhibition. Studies on time course of inhibition revealed that the compound is inhibitory even after the initiation of DNA synthesis. These studies indicate that the ability of BVdUTP to preferentially inhibit the HSV-1 DNA polymerase may contribute towards its selective inhibition of the viral DNA replication in infected cells.
Relationship between glycocalicin and glycoprotein Ib of human platelets.
Authors: Clemetson K J , Naim H Y , Lüscher E F
Published: May 2006

Asialoglycoprotein Ib and asialoglycocalicin have been isolated from the membranes and from the supernatant, respectively, after sonication of neuraminidase-treated platelets, by lectin affinity chromatography on peanut agglutinin. The isolated asialoglycoprotein Ib had an apparent molecular weight of 160,000 when not reduced and 150,000 when reduced, whereas the asialoglycocalicin had an apparent molecular weight of 150,000, both reduced and unreduced, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both preparations contained only trace amounts of impurities. The asialoglycoprotein Ib and asialoglycocalicin in both the unreduced and reduced states were separated by gel electrophoresis, radioiodinated in gel slices, and digested with trypsin, and the digests were analyzed by two-dimensional high-voltage electrophoresis and thin-layer chromatography followed by autoradiography. The tryptic peptide maps showed great similarities between glycocalicin and glycoprotein Ib, with the latter (both unreduced and reduced) containing additional peptides, supporting the idea that glycocalicin is derived from glycoprotein Ib. The unreduced glycoprotein Ib contained additional peptides compared to the reduced due to the disulfide-bond-linked beta component. There were also slight differences between unreduced and reduced glycocalicin, indicating that at least one intramolecular disulfide bond is present.
Alcohol dehydrogenase gene of Drosophila melanogaster: relationship of intervening sequences to functional domains in the protein.
Authors: Benyajati C , Place A R , Powers D A , Sofer W
Published: May 2006

The gene that codes for Drosophila alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase EC 1.1.1.1) was identified in a bacteriophage lambda library of genomic Drosophila DNA by using ADH cDNA cloned DNA as a probe. The DNA sequence of the protein encoding region was shown to be in agreement with the amino acid sequence of the ADH. Two intervening DNA sequences (introns) were identified within the protein encoding region: one was 65 nucleotides and located between the codons for amino acid residues 32 and 33, and one was 70 nucleotides and located between the codons for amino acid residues 167 and 168. Both contained the 5' G-T and 3' A-G dinucleotides characteristic of intron boundaries of eukaryotic genes. On the basis of secondary structure predictions, the first 140 amino acid residues of Drosophila ADH are in an alternating beta-sheet/alpha-helix arrangement which is characteristic of the coenzyme binding domain of dehydrogenases. The smaller of the two introns interrupts the domain predicted to bind the adenine portion of the coenzyme.
Formation of gamma-glutamycyst(e)ine in vivo is catalyzed by gamma-glutamyl transpeptidase.
Authors: Griffith O W , Bridges R J , Meister A
Published: May 2006

These studies indicate that gamma-glutamylcyst(e)ine, found in the urine of a patient with gamma-glutamyl transpeptidase deficiency and also in the urine of experimental animals injected with glutathione or with inhibitors of gamma-glutamyl transpeptidase, is formed by the action of gamma-glutamyltranspeptidase. The evidence demonstrates that transpeptidation between glutathione and cystine occurs in vivo and also that this reaction constitutes a significant physiological function of the enzyme. The appearance of large amounts of gamma-glutamylcyst(e)ine in the urine seems to reflect an inhibitory effect of glutathione on the transport of gamma-glutamylcyst(e)ine into cells. The findings also indicate that conversion of glutathione to gamma-glutamylcysteine by hydrolytic cleavage of the COOH-terminal glycine moiety of glutathione (or analogous cleavage of glutathione disulfide) is not a quantitatively significant pathway. The results reported here show that gamma-glutamyl transpeptidase activity is not completely absent in a patient found to have a deficiency of this enzyme and that the activity of the enzyme is not abolished in experimental animals treated with potent gamma-glutamyl transpeptidase inhibitors.
Identification of hypusine, an unusual amino acid, in a protein from human lymphocytes and of spermidine as its biosynthetic precursor.
Authors: Park M H , Cooper H L , Folk J E
Published: May 2006

When normal human peripheral lymphocytes are treated with mitogen and grown in the presence of [3H]putrescine or [terminal methylenes-3H]spermidine, label is incorporated predominantly into one cellular protein. The radioactive constituent of this protein was identified as the unusual amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. This was accomplished by isolation of the component from proteolytic digests or acid hydrolysates and comparison with authentic hypusine by chromatography, conversion to the 2,4-dinitrophenyl derivative, and oxidative degradation. The observed relationships among intracellular levels of labeled putrescine, polyamines, and protein bound hypusine after growth of cells with the various labeled amines and with or without an inhibitor of polyamine biosynthesis supply evidence that spermidine is the immediate amine precursor of hypusine and that the 4-amino-2-hydroxybutyl portion of hypusine derives from the butylamine moiety of spermidine.
Conformational transitions in Pf3 and their implications for the structure and assembly of filamentous bacterial viruses.
Authors: Thomas G J , Day L A
Published: May 2006

Laser Raman and circular dichroism spectra of filamentous bacteriophage Pf3 show that its coat protein is predominantly alpha-helical, similar to the subunits of bacteriophages Pf1 and fd. Unlike Pf1 and fd, however, the subunits of Pf3 are converted to beta-sheet structures by raising the temperature, the transition temperature depending upon phage and NaCl concentrations. On cooling, the beta structure reverts to an alpha structure the same as or similar to the native structure. On further heating it converts irreversibly to a second alpha-helical form different from the original one. The spectra also show that aromatic amino acid residues of Pf3 undergo dramatic changes in molecular environment during the alpha leads to beta transition. Similar transitions are observed to take place in the filamentous bacteriophage Xf.
An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA
Authors: Deuring Renate , Klotz Günther , Doerfler Walter
Published: May 2006

On purification of human adenovirus type 12 (Ad12) by equilibrium sedimentation in CsCl density gradients, two bands of particles, Ad12-3 and Ad12-3a, are observed. The particles from band Ad12-3a contain a recombinant of human host cell DNA and of Ad12 DNA. The human cell DNA sequences contain repetitive DNA recurring 200 to 500 times in cellular DNA. Ad12 DNA and the recombinant genomes exhibit the same or similar lengths. This finding suggests that a constant amount of DNA is packaged into complete Ad12 particles. On cleavage of KB cellular DNA with
Eco
RI,
Bam
HI,
Hin
fI,
Msp
I,
Mbo
I
Pst
I, or
Bgl
II, the
32
P-labeled cellular DNA from Ad12-3a particles hybridizes on Southern blots to distinct bands of KB DNA. There is also less-specific background hybridization that is not observed in the control. The cellular DNA from Ad12-3a particles is not methylated, whereas the same cellular sequences in KB cell DNA appear to be extensively methylated. On denaturation and renaturation, the recombinant DNA molecules are converted to molecules half as long as Ad12 DNA, as determined by gel electrophoresis and electron microscopy. The recombinant DNA molecules were terminally labeled by exonuclease III treatment and subsequent refilling of the depleted segments with [
32
P]dNTPs by using DNA polymerase I (Klenow fragment). When these molecules were cleaved with
Eco
RI,
Bam
HI,
Msp
I, or
Pst
I, only one terminal DNA fragment was found to be labeled. The results of partial digestion experiments using
Msp
I,
Hin
fI, or
Mbo
I are consistent with a model in which 700-1150 base pairs from the left terminus of Ad12 DNA are linked to host cell DNA containing repetitious sequences, and this structure is symmetrically duplicated as a large inverted repeat of the type ABCDD′C′B′A′. The Ad12 DNA sequences are flanking the entire molecule, which consists mainly of human KB cell DNA. The recombinants appear to be stable on serial passage of the virus preparation for many years, although variations in the sequence of the recombinants occur. These symmetric recombinant (SYREC) molecules suggest a way to use adenovirus DNA as a eukaryotic vector. Their occurrence provides further evidence for the generation of virus-host DNA recombinants and may help elucidate the role this interaction may have in adenovirus replication and oncogenesis.
Expression and synthesis of murine immune response-associated (Ia) antigens by brain cells.
Authors: Ting J P , Shigekawa B L , Linthicum D S , Weiner L P , Frelinger J A
Published: May 2006

This paper provides biochemical and histochemical evidence that a fraction of murine brain cells express and synthesize Ia (Immune response-associated) antigens. Both I-A and I-E subregion products are detected on frozen sections of mouse brains by immunoperoxidase staining. Most of these Ia-bearing cells are located in white matter tracts and appear to be intrafascicular oligodendrocytes. In contrast, cells in the gray matter rarely display detectable Ia antigens on their cell surfaces. Specificity of the staining was confirmed by absorption studies. Biochemical evidence for the active synthesis of Ia antigens by brain cells was obtained by immunoprecipitation of [3H]leucine/tyrosine-labeled, NP-40-extracted cell lysates with monoclonal anti-Ia reagent. Both the alpha and beta subunits of Ia antigens were identified by NaDodSO4 electrophoresis. By contrast, anti-mu serum failed to precipitate any product, thus eliminating contaminant B lymphocytes as a source of Ia antigens.
Analysis of a predominant immunoglobulin population in the cerebrospinal fluid of a multiple sclerosis patient by means of an anti-idiotypic hybridoma antibody.
Authors: Gerhard W , Taylor A , Wroblewska Z , Sandberg-Wollheim M , Koprowski H
Published: May 2006

We have produced hybridoma antibodies directed against immunoglobulins present in the cerebrospinal fluid of a patient with multiple sclerosis (MS). One hybridoma antibody recognized an idiotypic determinant of an immunoglobulin population [an idiotype (Id)] which constituted approximately 1% of the immunoglobulin present in the cerebrospinal fluid. The Id focused in the pH range 8.0-8.4. It was present at roughly 10- to 15-fold higher relative (compared to total immunoglobulin) concentration in cerebrospinal fluid than in serum of the homologous MS patient. The Id could not be detected in three cerebrospinal fluid samples and 28 serum samples of heterologous MS patients or in the serum of 43 optic neuritis patients. The Id persisted in the homologous MS patient at increased concentration over the entire (6 years) observation period. The Id could be shown to react with a Theiler murine encephalomyelitis virus strain WW which was isolated from mice after inoculation with periplaque white matter from brain in a histologically confirmed case of MS.
Lethality of human myeloblasts correlates with the incorporation of arabinofuranosylcytosine into DNA.
Authors: Major P P , Egan E M , Beardsley G P , Minden M D , Kufe D W
Published: May 2006

We recently demonstrated a highly significant relationship between the incorporation of 1-beta-D-arabinofuranosylcytosine (ara-C) into L1210 DNA and the loss of clonogenic survival. These studies have now been extended to the human promyeloblast (HL-60) cell line and myeloblasts from a patient with acute myelogenous leukemia. Our results demonstrate: (i) the specific internucleotide incorporation of ara-C into human myeloblast DNA; (ii) the lability of [3H]ara-C-labeled DNA to alkali which necessitates the use of nondegrading assay conditions; and (iii) a highly significant relationship (P less than 0.0001) between the loss of clonogenicity of these cells and the extent of ara-C incorporation. These findings suggest that the incorporation of ara-C into DNA is one of the initial events leading to cell lethality. This method is applicable to clinical samples of bone marrow and peripheral blood as an in vitro assay for studying the sensitivity of cell populations to this drug.
Dopamine receptor binding in inbred mice: strain differences in mesolimbic and nigrostriatal dopamine binding sites.
Authors: Boehme R E , Ciaranello R D
Published: May 2006

Dopamine receptors were examined by Scatchard analysis in the striatal and olfactory tubercle regions of 11 inbred mouse strains. Simultaneous determinations of the binding characteristics of 3H-labeled 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN), a dopaminergic agonist, and [3H]spiroperidol, a dopaminergic antagonist, were examined. Among the 11 strains, the equilibrium dissociation constant (Kd) for agonist binding did not vary in either the striatum or the olfactory tubercle. Similarly, no strain differences were observed in the Kd for spiroperidol in either region, although the Kd for spiroperidol in the olfactory tubercle was uniformly higher than that in the striatum. Measurement of receptor concentrations revealed strain differences of up to 2-fold for both [3H]ADTN and [3H]spiroperidol binding sites. Within each brain region, the densities of agonist and antagonist binding sites correlated significantly. However, between brain regions there was no correlation in the density of agonist or antagonist binding sites, which suggests that mesolimbic and nigrostriatal dopamine neurons may be under independent genetic control. Analysis of [3H]spiroperidol displacement by clofluperol, aceperone, cinanserin, and mianserin in four inbred mouse strains revealed that 88-90% of the striatal spiroperidol sites are dopaminergic, with the remainder being serotonergic. In contrast, 53-66% of the olfactory tubercle [3H]spiroperidol binding sites are dopaminergic and 34-47% are serotonergic. These data suggest that genetic differences in serotonin receptors and dopamine receptors may exist among inbred mouse strains.
Sequence homology between RNAs encoding rat alpha-fetoprotein and rat serum albumin.
Authors: Jagodzinski L L , Sargent T D , Yang M , Glackin C , Bonner J
Published: May 2006

We have determined the sequences of the recombinant DNA inserts of three bacterial plasmid cDNA clones containing most of the rat alpha a-fetoprotein mRNA. The resultant nucleotide sequence of alpha-fetoprotein was exhaustively compared to the nucleotide sequence of the mRNA encoding rat serum albumin. These two mRNAs have extensive homology (50%) throughout and the same intron locations. The amino acid sequence of rat alpha-fetoprotein has been deduced from the nucleotide sequence, and its comparison to rat serum albumin's amino acid sequence reveals a 34% homology. The regularly spaced positions of the cysteines found in serum albumin are conserved in rat alpha-fetoprotein, indicating that these two proteins may have a similar secondary folding structure. These homologies indicate that alpha-fetoprotein and serum albumin were derived by duplication of a common ancestral gene and constitute a gene family.
In vitro assembly of homopolymer and copolymer filaments from intermediate filament subunits of muscle and fibroblastic cells.
Authors: Steinert P M , Idler W W , Cabral F , Gottesman M M , Goldman R D
Published: May 2006

This paper presents evidence that the intermediate filament (IF) subunits of muscle cells (skeletin or desmin) and fibroblastic cells (decamin or vimentin) separately form homopolymer IF in vitro and, when mixed, prefer to form copolymer IF in vitro. Because they coexist in cells, they may also form copolymers in vivo. The IFs of baby hamster kidney fibroblasts (BHK-21) consist of a major subunit, decamin, and two minor subunits which, on the basis of two-dimensional gel and peptide mapping criteria, are identical to the alpha and beta subunits of smooth muscle desmin. The subunits differ only in their degrees of phosphorylation: alpha desmin contained 2 mol/mol of O-phosphoserine whereas beta desmin contained none. The decamin and desmin subunits assembled into homopolymer IF in vitro in high yield from purified denatured subunits under identical conditions of pH and ionic strength. However, homopolymer decamin IF disassembled into soluble protofilaments in solutions of ionic strength less than 0.05 mol/liter whereas homopolymer desmin IF disassembled at ionic strength less than 0.03 mol/liter. When decamin and desmin were mixed together as denatured subunits or as soluble protofilaments, the IF assembled in vitro had solubility properties intermediate between those of the homopolymer IFs, indicating that the two subunits had formed copolymer IF. The stoichiometry of copolymerization as determined in mixtures in which one subunit was present in excess was suggestive of the formation of three-chain units. The possibility of nonspecific aggregation was eliminated by isolation of stable three-chain alpha-helix-enriched particles from such IF. When tracer amounts of [35S]methionine-labeled decamin were mixed with desmin, labeled IFs were obtained under conditions in which homopolymer decamin IFs were soluble. These in vitro findings may be of physiological significance because native BHK-21 IF also had solubility properties similar to those of the copolymer IF.
Localization of a unique gene by direct hybridization in situ.
Authors: Gerhard D S , Kawasaki E S , Bancroft F C , Szabo P
Published: May 2006

Previous determination of the chromosomal location of unique genes have required that the chromosomes of interest be fractionated, either by species-specific chromosome loss from interspecies hybrids, or by physical fractionation procedures. We have developed a general technique for the localization of a unique gene, which requires no prior chromosome fractionation. The technique involves the use of a labeled hybrid cDNA plasmid for direct hybridization in situ to metaphase cells from the organism under investigation. As a model system for development of this technique, we have employed a human alpha-globin cDNA plasmid (JW101) to localize the corresponding gene cluster. To obtain a sufficiently large autoradiographic signal, we have both labeled this plasmid with 125I to a high specific activity (10(9) dpm/micrograms) and taken advantage of the ability of a double-stranded probe to form networks. To obtain sufficient hybridization specificity, various experimental procedures were used, the most important of which was the choice from among a range of probe concentrations of the highest that does not yield excessive background hybridization. We have shown that, with an autoradiographic exposure time of only 12 days, use of this technique correctly localized the human alpha-globin gene cluster to chromosome 16. This technique should be generally applicable to the localization of any gene for which a corresponding cDNA hybrid plasmid is available.
Identification of the dnaQ gene product and location of the structural gene for RNase H of Escherichia coli by cloning of the genes.
Authors: Horiuchi T , Maki H , Maruyama M , Sekiguchi M
Published: May 2006

By in vitro recombination we have constructed hybrid plasmids capable of complementing a conditional lethal mutator mutation, dnaQ49, in Escherichia coli K12. The dnaQ+ plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase DNA fragment derived from the E. coli chromosome. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the 1.5-kilobase insert codes for two proteins, one whose molecular weight is 25,000 [the 25-kilodalton (kDal) protein] and the other whose molecular weight is 21,000 (the 21-kDal protein). Because insertion of gamma delta sequence into the dnaQ gene of the plasmid resulted in disappearance of the 25-kDal protein, it was concluded that the 25-kDal protein is the dnaQ gene product. The 21-kDal protein was identified as RNase H on the basis of the following evidence. (i) Cells harboring the dnaQ+ plasmids, with or without the gamma delta insertion in the dnaQ gene, had a 5- to 7-fold higher level of RNase H activity than cells harboring pBR322. (ii) After induction of cells that are lysogenized with dnaQ+-transducing lambda phages, RNase H activity increased considerably. A similar high level of RNase H activity was observed with transducing phages whose dnaQ function was inactivated by insertion of a transposon, Tn3, into the gene, (iii) The plasmid-encoded RNase H, labeled with [35S]methionine, was purified in a manner essentially similar to that of the chromosome-encoded enzyme. These results suggest that the dnaQ gene and the structural gene for RNase H, termed gene rnh, are closely linked and located at 5 min on the linkage map.
Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inflammatory response.
Authors: Dahlén S E , Björk J , Hedqvist P , Arfors K E , Hammarström S , Lindgren J A , Samuelsson B
Published: May 2006

Leukotrienes B4, C4, and D4, members of a recently discovered family of substances biosynthesized from arachidonic acid, were found to have potent microvascular actions in the hamster cheek pouch. When applied topically to the vascular network, leukotrienes C4 and D4 caused an intense constriction of arterioles, being similar to angiotensin in potency in this respect. The vasoconstriction induced by leukotrienes C4 and D4 was short-lived, and it was consistently followed by a marked and dose-dependent extravasation of macromolecules from postcapillary venules. Histamine did not constrict arterioles, but it elicited leakage of plasma, although on a molar basis it was no more than 1/1000th as potent as the leukotrienes. When used in the same concentration range as leukotrienes C4 and D4, leukotriene B4 did not evoke vasoconstriction or promote plasma leakage. On the other hand, leukotriene B4 caused a conspicuous and reversible adhesion of leukocytes to the endothelium in postcapillary venules. Our findings that leukotrienes induce microcirculatory alterations in vivo, closely resembling the early events in the acute inflammatory response, imply that leukotrienes, formed in several blood-borne and tissue-bound cells, may mediate important microcirculatory adjustments to noxious stimuli.
Impaired induction and self-catabolite repression of extracellular pectate lyase in
Erwinia chrysanthemi
mutants deficient in oligogalacturonide lyase
Authors: Collmer Alan , Bateman Durward F.
Published: May 2006

The pectate lyase (PL; EC 4.2.2.2) secreted by the plant pathogen
Erwinia chrysanthemi
is induced and catabolite repressed by different concentrations of its own product, digalacturonic acid 4,5-unsaturated at the nonreducing end [u(GalUA)
2
]. Both activities of u(GalUA)
2
depend on its cleavage by oligogalacturonide lyase (OGL; EC 4.2.2.6). This intracellular enzyme converts u(GalUA)
2
to the deoxyketuronic acid 4-deoxy-L-
threo
-5-hexosulose uronic acid, which is then isomerized to 3-deoxy-D-
glycero
-2,5-hexodiulosonic acid. An OGL-deficient mutant unable to grow on u(GalUA)
2
was poorly induced by u(GalUA)
2
or by D-galacturonan but produced wild-type levels of PL when supplied with 3-deoxy-D-
glycero
-2,5-hexodiulosonic acid. PL synthesis in the mutant could also be stimulated by 4,5-unsaturated trigalacturonic acid, from which deoxyketuronic acid is released by another intracellular enzyme. An OGL-deficient mutant that grew slowly on u(GalUA)
2
in comparison with the wild-type parent was hyperinduced by u(GalUA)
2
unless catabolite repression was relieved by cyclic AMP or imposed by logarithmic growth on glycerol. PL synthesis is also stimulated by saturated digalacturonic acid, which is released from D-galacturonan by another extracellular enzyme, exo-poly-α-D-galacturonosidase (EC 3.2.1.82). Because these dimers stimulate PL synthesis at concentrations (wt/vol) 1/1000th of the concentration required by D-galacturonan, and because an OGL-deficient mutant uninducible by dimers was also uninducible by D-galacturonan, we postulate that PL induction by pectic polymers entails extracellular formation of dimers and subsequent intracellular conversion to deoxyketuronic acids, the apparent inducers of PL.
Characterization of a class of cationic proteins that specifically interact with intermediate filaments.
Authors: Steinert P M , Cantieri J S , Teller D C , Lonsdale-Eccles J D , Dale B A
Published: May 2006

We describe a class of cationic structural proteins that associate specifically with intermediate filaments (IF) but not with other types of cytoskeletal proteins. These proteins, for which the term filaggrin is introduced, are isolated from the stratum corneum of mammalian epidermis. They are species-distinct proteins; for example, rat and mouse filaggrin have different molecular weights and amino acid compositions, but are nevertheless chemically and functionally ver similar. They interact in vitro with the IF several different types of cells to form large fibers or macrofibrils in which many IF are highly aligned in parallel arrays. Stoichiometric analyses suggest that two molecules of filaggrin bind to each three-chain building block of the IF, possibly by ionic interactions with the coiled-coil alpha-helical regions of the IF.
Relative orientation of close-packed β-pleated sheets in proteins
Authors: Chothia Cyrus , Janin Joël
Published: May 2006

When β-pleated sheets pack face to face in proteins, the angle between the strand directions of the two β-sheets is observed to be near -30°. We propose a simple model for β-sheet-to-β-sheet packing in concanavalin A, plastocyanin, γ-crystallin, superoxide dismutase, prealbumin, and the immunoglobin fragment V
REI
. This model shows how the observed relative orientation of two packed β-sheets is a consequence of (
i
) the rows of side chains at the interface being approximately aligned and (
ii
) the β-sheet having a right-handed twist. The special amino acid composition of residues at the β-sheet-to-β-sheet interfaces makes the contact surfaces essentially smooth and hydrophobic.
Complete amino acid sequence of a human pituitary glycopeptide: an important maturation product of pro-opiomelanocortin.
Authors: Seidah N G , Chrétien M
Published: May 2006

A glycopeptide isolated in relatively large amounts from human pituitary glands was completely purified, and its sequence was determined. The primary sequence represents the NH2-terminal 76 amino acid residues of pro-opiomelanocortin (POMC). This important secretory product of POMC was shown to possess an interesting aldosterone-stimulating activity on a human adrenal aldosteronoma. It is O-glycosylated at Thr-45 and N-glycosylated at Asn-65. Only one sequence variation with the human genomic DNA was found. Furthermore, comparison with the other preferred cleavage sites of human POMC reveals that the pair of basic residues Lys-Arg represents the major sites of enzymatic maturation of this precursor molecule. This predicts a highly specific type of enzyme involved in the maturation of POMC in the anterior lobe of the human pituitary.
Proton and hydroxide ion permeability of phospholipid vesicles.
Authors: Nozaki Y , Tanford C
Published: May 2006

The apparent permeability of H+ through phospholipid bilayers was determined by measuring H+ efflux from large unilamellar phospholipid vesicles with internal space buffered at pH 4. The value obtained is about 10(-9) cm/sec at room temperature, five orders of magnitude lower than was recently reported for the combined permeability for H+ and OH- [Nichols, J. W. & Deamer, D. W. (1980) Proc. Natl. Acad. Sci. USA, 77, 2038-2042]. The apparent permeability measured in this way is the sum of contributions from the movement of H+ and of uncharged species (HCl or HNO3) in equilibrium with anions in the solution. There is evidence that the uncharged species make the dominant contribution and that the permeability coefficient for H+ per se is no larger than 5 X 10(-12) cm/sec. An attempt to measure OH- permeability by use of vesicles buffered at pH 10 did not give a conclusive result because the vesicle walls appeared to be damaged by exposure to this pH. An apparent permeability coefficient of about 10(-7) cm/sec was estimated for undamaged membranes.
Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells.
Authors: Santerre R F , Cook R A , Crisel R M , Sharp J D , Schmidt R J , Williams D C , Wilson C P
Published: May 2006

A clonal hamster beta cell line (HIT) was established by simian virus 40 transformation of Syrian hamster pancreatic islet cells. Cytoplasmic insulin was detected in all cells by indirect fluorescent antibody staining, and membrane-bound secretory granules were observed ultrastructurally. Acidified-ethanol extracts of HIT cell cultures contained hamster insulin as determined by radioimmunoassay, radioreceptor assay, and bioassay. One subclone at passage 39 contained 2.6 micrograms of insulin per mg of cell protein. [3H]Leucine-labeled HIT insulin and proinsulin were identical to islet-derived proteins when compared by NaDodSO4/polyacrylamide gel electrophoresis of immunoprecipitates. HIT cell insulin secretion was stimulated by glucose, glucagon, and 3-isobutyl-1-methylxanthine. Insulin secretion at optimal glucose concentration (7.5 mM) was 2.4 milliunits per 10(6) cells per hr. Somatostatin and dexamethasone markedly inhibited HIT insulin secretion. The HIT cell line represents a unique in vitro system for studying beta cell metabolism and insulin biosynthesis.
Two-dimensional gel analysis of rat liver nuclear proteins after thyroidectomy and thyroid hormone treatment.
Authors: Nikodem V M , Trus B L , Rall J E
Published: May 2006

The composition of nucleoproteins in normal rat liver, in livers from thyroidectomized animals, and in thyroidectomized animals treated with 3,3',5-triiodo-L-thyronine was examined by two-dimensional gel electrophoresis. A computer program was designed to measure the relative concentrations of each protein. Approximately 500 protein subunits could be distinguished. Of these, 102 either disappeared or were markedly decreased after thyroidectomy. Shortly after administration of the thyronine (6 hr), 13 proteins reappeared and, by 24 hr, 67 additional proteins had reappeared. These changes in protein concentrations are both the greatest in number and have the largest reported as an effect of thyroid hormone. Twenty-four hr after triiodothyronine administration, some 22 proteins seen in normal liver could not be found. Two new proteins appeared in thyroidectomized rat liver nuclei. We conclude that a major and rapid effect of thyroid hormone is exerted on hepatic nucleoproteins.
Human B-cell alloantigens DC1, MT1, and LB12 are identical to each other but distinct from the HLA-DR antigen
Authors: Shackelford Deborah A. , Mann Dean L. , van Rood Jon J. , Ferrara G. B. , Strominger Jack L.
Published: May 2006

Some human B-lymphoblastoid cell lines are shown to express at least two types of Ia-like antigens. One antigen is defined by alloantisera to HLA-DR, and the other antigen is defined by alloantisera and a monoclonal antibody to the specificities DC1, MT1 (MB1), and LB12, which are in linkage disequilibrium with HLA-DR. The subunits of the DC1 molecule differ from those of the DR molecule. The light chains of both molecules are structurally polymorphic.
Transplacental passage of insulin complexed to antibody.
Authors: Bauman W A , Yalow R S
Published: May 2006

The passage of plasma proteins across the placental barrier in humans is known to be highly selective. Thus, free maternal insulin has been reported not to cross the normal maternofetal barrier, although insulin-binding antibodies have been detected in newborn infants whose diabetic mothers received insulin therapy. In this report we demonstrate, with the use of a human antiserum that permits distinction between human and animal insulins, that insulin in the cord blood of each of two neonates of insulin-treated diabetic mothers was, in part, animal insulin. The higher the antibody titer of the mother the greater was the total insulin in the cord plasma and the greater was the fraction that was animal insulin. In case 1 cord plasma insulin was 0.7 unit/liter, of which 10% was animal insulin; in case 2 cord plasma insulin was 3.5 units/liter, of which 25% was animal insulin. The demonstration that antigen restricted from transplacental passage can be transferred while complexed to antibody raises the question whether such fetal exposure would induce partial or total immunologic unresponsiveness subsequently if the fetus were rechallenged with the same antigen.