bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA sequencing and melting curve.
Authors: Azbel M Y
Published: May 2006

The dependence of DNA absorbance (for light at about 260 nm) on temperature is related to a specific DNA sequence structure in the vicinity of DNA thermal denaturation (the so-called DNA melting or coiling). A straightforward analysis of the experimental DNA melting curve allows us to determine the lengths, the A+T content, and the location in DNA of certain domains. In the case of a specific DNA fragmentation, the order of fragments in DNA can be learned from this analysis, nondestructively and quickly, without fractionating the fragments and other methods of fragmentation. If the DNA nucleotide sequence is known except for some sites and uncertain portions, the analysis determines these sites and the accuracy of the sequence at the portions. This information may complement exact methods of DNA sequencing. The proposed analysis is applied to bacteriophage phiX174, whose melting curve is known. The results are compared to and found to be in an excellent agreement with the known phiX174 nucleotide sequence.
Lateral diffusion in binary mixtures of cholesterol and phosphatidylcholines.
Authors: Rubenstein J L , Smith B A , McConnell H M
Published: May 2006

The lateral diffusion of a fluorescent-labeled phospholipid, phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3,-diazole)ethanolamine, has been measured in binary mixtures of cholesterol and dimyristoyl phosphatidylcholine at temperatures both above and below 23.8 degrees C, the chain-melting transition temperature of this phosphatidylcholine. There is a temperature-composition region, approximately temperature less than 23 degrees C and mole fraction of cholesterol (X) less than 0.20, in which the lateral diffusion coefficient of the fluorescent probe is at least an order of magnitude smaller than it is at points outside of this temperature-composition region. At temperatures above approximately 23 degrees C there is a significant increasing cholesterol concentration, for X greater than 0.2.
Partial primary structure of bacteriorhodopsin: sequencing methods for membrane proteins.
Authors: Gerber G E , Anderegg R J , Herlihy W C , Gray C P , Biemann K , Khorana H G
Published: May 2006

The sequence of 102 amino acid residues from the NH2 terminus and that of 39 amino acid residues from the COOH terminus of bacteriorhodopsin have been determined. These results are in agreement with those recently published by Ovchinnikov and coworkers [Ovchinnikov, Y.A., Abdulaey, N.G., Feigina, M.Y., Kiselev, A.V. & Lobanov, N.A. (1977) FEBS Lett. 84, 1-4]. Chymotryptic cleavage of bacteriorhodopsin produced two fragments, C-1 (Mr 19,000) and C-2 (Mr 6900), the latter containing the blocked NH2 terminus (pyroglutamic acid). Further fragmentation with CNBr gave mostly hydrophobic fragments, which were separated by gel permeation and reverse-phase high-pressure liquid chromatography in formic acid/ethanol/water mixtures. The fragments were sequenced by a judicious combination of mass spectrometric peptide sequencing and automated Edman degradation. The C-2 fragments were ordered on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry, while C-1 and C-2 were arranged by analysis of an overlapping CNBr fragment.
beta-Adrenergic receptor agonists increase phospholipid methylation, membrane fluidity, and beta-adrenergic receptor-adenylate cyclase coupling.
Authors: Hirata F , Strittmatter W J , Axelrod J
Published: May 2006

The beta-adrenergic agonist L-isoproterenol stimulated the enzymic synthesis of phosphatidyl-N-monomethylethanolamine and phosphatidylcholine in rat reticulocyte ghosts containing the methyl donor S-adenosyl-L-methionine. The stimulation was stereospecific, dose-dependent, and inhibited by the beta-adrenergic agonist propranolol. The addition of GTP inside the resealed ghosts shifted the dose-response of phospholipid methylation by L-isoproterenol to the left by 2 orders of magnitude. Direct stimulation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with sodium fluoride or cholera toxin did not increase the methylation of phospholipids. At a concentration of S-adenosyl-L-methionine that stimulates synthesis of phosphatidyl-N-monomethylethanolamine, the activity of isoproterenol-sensitive adenylate cyclase was increased 2-fold without changes in the basal activity of adenylate cyclase and the number of beta-adrenergic receptors. The increase of phospholipid methylation by L-isoproterenol decreased membrane viscosity and increased translocation of methylated lipids. These findings indicate that enhancement of phospholipid methylation by L-isoproterenol decreases membrane microviscosity and thus increases lateral movement of the beta-adrenergic receptors and coupling with adenylate cyclase.
Genetic lesions induced by chemicals in spermatozoa and spermatids of mice are repaired in the egg.
Authors: Generoso W M , Cain K T , Krishna M , Huff S W
Published: May 2006

Conclusive proof that the mouse egg is capable of carrying out repair of genetic lesions present in the male genome was obtained through dominant-lethal studies of chemically treated spermatozoa and spermatids and through cytological analysis of first-cleavage metaphases. The maximum difference in repair capability between stocks of females, found for isopropyl methanesulfonate treatment, was large; considerably smaller differences were found for ethyl methanesulfonate, triethylenemelamine, and benzo[a]pyrene treatments; and no difference was found for x-ray treatment.
Human heavy chain disease protein WIS: implications for the organization of immunoglobulin genes.
Authors: Franklin E C , Prelli F , Frangione B
Published: May 2006

Protein WIS is a human gamma3 heavy (H) chain disease immunoglobulin variant whose amino acid sequence is most readily interpreted by postulating that three residues of the amino terminus are followed by a deletion of most of the variable (VH) domain, which ends at the variable-constant (VC) joining region. Then there is a stretch of eight residues, three of which are unusual, while the other five have striking homology to the VC junction sequence. This is followed by a second deletion, which ends at the beginning of the quadruplicated hinge region. These findings are consistent with mutations resulting in deletions of most of the gene coding for the V region and CH1 domain followed by splicing at the VC joining region and at the hinge. These structural features fit well the notion of genetic discontinuity between V and C genes and also suggest similar mechanisms of excision and splicing in the interdomain regions of the C gene of the heavy chain.
Novel protein kinase, AUT-PK 85, isolated from adrenocortical carcinoma: purification and characterization.
Authors: Shanker G , Ahrens H , Sharma R K
Published: May 2006

We describe the purification to apparent homogeneity of a protein kinase (designated AUT-PK 85) from adrenocortical carcinoma 494, as evidenced by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme binds cyclic AMP (cAMP) and autophosphorylates but does not use histone, casein, or polysomes as substrates in the presence or absence of cAMP. Stoichiometry of phosphate incorporation was 0.71 mol/mol of enzyme. The enzyme was found to have a molecular weight of 85,000 based on gel filtration. The protein was composed of polypeptides having the same molecular weight 42,000, and thus it appears to consist of two subunits of equal size. The enzyme bound two cAMP molecules, indicating that each subunit binds one molecule of cAMP. The homogeneous enzyme did not inhibit the protein kinase activity of the free catalytic subunit of normal adrenal cAMP-dependent protein kinase under conditions such that recombination with the free regulatory subunit occurred. cAMP bound specifically to the enzyme with an apparent dissociation constant (cfKd) of 1.2 X 10(-8) M. Scatchard plot data indicated one type of binding sites for cAMP. The enzyme did not bind adenosine. This novel autophosphorylating, cAMP-binding, protein kinase may be a characteristic of certain adrenal neoplasms.
Chicken lysozyme gene contains several intervening sequences.
Authors: Nguyen-Huu M C , Stratmann M , Groner B , Wurtz T , Land H , Giesecke K , Sippel A E , Schütz G
Published: May 2006

The organization of the chicken lysozyme gene and its neighboring sequences was examined by a comparison of the restriction map of the lysozyme structural gene with the map of the lysozyme gene in genomic DNA. Chicken DNA was cleaved with restriction endonucleases and the DNA fragments were separated by agarose gel electrophoresis. After transfer of the fragments onto nitrocellulose filters, those fragments that contain lysozyme mRNA sequences were detected by hybridization of the filters to labeled probes generated from pls-1, a recombinant plasmid carrying the lysozyme structural gene. This analysis revealed the presence of at least three intervening sequences, two of which interrupt the protein coding region and one of which is located in the 3' untranslated region. When oviduct DNA and sperm DNA were compared, no difference was observed in the size and number of restriction fragments that contain either lysozyme or ovalbumin structural gene sequences.
Float trajectories in simple kinematic flows
Authors: Regier Lloyd , Stommel Henry
Published: May 2006

A simple kinematic model has been used to compute Lagrangian trajectories. Although it is certainly too simple to model geophysical flows, it has provided insights into the behavior of Lagrangian tracers. In particular, the existence of trapping regions has been shown to greatly increase the dispersion rate of tracers and to lead to net tracer displacements when the Eulerian mean flow is zero. In general, the spectrum of spatial scales present in the trajectories is wider than the Eulerian spectrum and is biased towards shorter wavelengths; the disparity between the 100-km Eulerian scale and the much shorter length scales experienced by the tracers is demonstrated. The estimation of the Eulerian parameters of the field from Lagranian observations must be done with a great deal of care, particularly if the eddy flow velocities are comparable to or exceed the mean flow. With a limited number of tracers it is extremely difficult to estimate, with any degree of confidence, the properties of either the mean Eulerian flow or the eddy field. Clearly, more effort must be spent to better understand the behavior of tracers in more realistic flows, to devise data analysis techniques, and to relate the Eulerian and Lagrangian spectra.
An interferon-induced phosphodiesterase degrading (2'-5') oligoisoadenylate and the C-C-A terminus of tRNA.
Authors: Schmidt A , Chernajovsky Y , Shulman L , Federman P , Berissi H , Revel M
Published: May 2006

A phosphodiesterase characterized by a generally higher activity on 2'-5' than on 3'-5' phosphodiester bonds was isolated from mouse L cells treated with interferon. A similar enzyme was purified from mouse reticulocytes. The phosphodiesterase 2'-PDi splits the 2'-phosphate bond of pppA2'p5'A2'p5'A, the oligonucleotide activator of ribonuclease F. The level of phosphodiesterase 2'-PDi is increased by interferon treatment of L cells. The phosphodiesterase was also shown to degrade the C-C-A terminus of tRNA and to reduce the amino acid acceptance of tRNA in cell-free extracts, thereby causing a tRNA-reversible inhibition of mRNA translation.
leu operon of Salmonella typhimurium is controlled by an attenuation mechanism.
Authors: Gemmill R M , Wessler S R , Keller E B , Calvo J M
Published: May 2006

The nucleotide sequence of the control region of the leu operon of Salmonella typhimurium was determined. A prominent feature of this region is a signal for termination of transcription. In vitro, transcription does terminate at this site, yielding a leader RNA of about 160 nucleotides as a major product. This leader RNA is potentially translatable into a peptide containing 28 amino acids, 4 of which are adjacent leucine residues. Several regions of base complementarity exist within the leader, positioned such that pairing of one region precludes pairing of another. The position of the four leucine codons relative to two regions of base complementarity suggest a model for the regulation of the leu operon similar to that proposed by Yanofsky and coworkers for the trp operon. In addition, a third region of base complementarity was identified which, when incorporated into the model, explains why premature termination is the usual outcome when transcription is initiated in vitro by purified RNA polymerase.
Reassessment of histone gene expression during cell cycle in human cells by using homologous H4 histone cDNA.
Authors: Detke S , Lichtler A , Phillips I , Stein J , Stein G
Published: May 2006

The representation of H4 histone mRNA sequences in RNAs isolated from G1 and S phase HeLa cells was assessed by use of a homologous H4 histone cDNA. S phase cells were obtained by double thymidine block, and G1 cells were obtained by double thymidine block or mitotic selective detachment. Nuclear and cytoplasmic RNAs from S phase cells hybridized with H4 histone cDNA as did nuclear and cytoplasmic RNAs from G1 cells synchronized by double thymidine block. In contrast, significant levels of hybridization were not observed between H4 histone cDNA and nuclear, polysomal, or postpolysomal cytoplasmic RNAs of G1 cells synchronized by mitotic selective detachment. Double thymidine block yields a G1 cell population containing 20-25% S phase cells whereas the G1 population obtained by mitotic detachment contains less than 0.1% S phase cells. The ability of H4 histone cDNA to hybridize with the RNAs from G1 cells obtained after release from double thymidine block can therefore be explained by the presence of S phase cells in such a G1 population--an artifact of the synchronization procedure. We interpret these results to be consistent with the presence of H4 histone mRNA sequences during the S but not G1 phase of the cell cycle in continuously dividing HeLa S3 cells.
Localization of transcribed regions on extrachromosomal ribosomal RNA genes of Tetrahymena thermophila by R-loop mapping.
Authors: Cech T R , Rio D C
Published: May 2006

R-loop hybridization and electron microscopy were used to map the RNA transcription products of the extrachromosomal rRNA genes of Tetrahymena thermophila. The mature 17S and 26S rRNAs and the nuclear 35S pre-rRNA and pre-26S rRNA were located with a precision of approximately 100 base pairs. A 370-base pair intervening sequence was found in the 26S coding region. It has the same size and relative location as that found in Tetrahymena pigmentosa [Wild, M.A. & Gall, J.G. (1979) Cell 16, 565-573]. One class of R-loop structures formed by nuclear pre-rRNA provided preliminary evidence for a primary transcript that contains the intervening sequence. The results suggested a processing scheme in which splicing of the intervening sequences is followed by a series of strand cleavages to give the mature 17S and 26S rRNAs. Analysis of the data also showed that RNA . DNA and DNA . DNA duplexes, when mounted for electron microscopy by the R-loop procedure, have the same length per base pair within 4%.
Autoimmune and lymphoproliferative disease in (B6-G
IX
+
× 129) F
1
mice: Relation to naturally occurring antibodies against murine leukemia virus-related cell surface antigens
Authors: Obata Yuichi , Tanaka Toshio , Stockert Elisabeth , Good Robert A.
Published: May 2006

G
IX
congeneic mouse strains, C57BL/6-G
IX
+
(B6-G
IX
+
) and 129-G
IX
-
, have been derived from the prototype strains, B6(G
IX
-
) and 129(G
IX
+
). The hybrids, (B6-G
IX
+
× 129)F
1
(G
IX
+
F
1
) and (B6 × 129-G
IX
-
)F
1
(G
IX
-
F
1
), differ only in regard to genetic loci controlling G
IX
antigen expression. G
IX
+
F
1
mice spontaneously produce G
IX
antibody and often show signs of autoimmune disease and lymphoproliferative disease. G
IX
-
F
1
mice and mice of the two parental strains (B6-G
IX
+
and 129) of G
IX
+
F
1
do not produce G
IX
antibody and seldom show signs of these diseases. G
(ERLD)
, and G
(RADA1)
, antibodies, natural thymocytotoxic autoantibody, and antinuclear antibodies were produced by G
IX
+
F
1
mice. However, these four antibodies were also found in the other strains. G
IX
+
F
1
mice develop pronounced diffuse glomerulonephritis similar to that found in systemic lupus erythematosus in man. Incidence studies in which mice were examined according to age rather than state of health showed that the lesions occurred in 38% of G
IX
+
F
1
mice but not in G
IX
-
F
1
, B6-G
IX
+
, or 129 mice. Lymphoproliferative lesions were either reticulum cell sarcoma (RCS) type A or reactive lymphoid hyperplasia (RLH). RCS occurred more often in G
IX
+
F
1
(38%) than in G
IX
-
F
1
(12%) or B6-G
IX
+
(8%). No RCS occurred in mice of the 129 strain. RLH occurred in G
IX
+
F
1
mice (10%) but not in the other strains. From these results, the following conclusions are drawn: (
i
) Severe glomerulonephritis and the increased occurrence of lymphoproliferative lesions in these animals depend on the presence of G
IX
antigen; (
ii
) besides genes controlling G
IX
antigen expression, other genes from both parental strains are required to create the basis in the progeny F
1
mice for the development of these diseases; and (
iii
) the chronic production of G
IX
antibody may be necessary for the development of the severe glomerulonephritis and for the increased occurrence of lymphoproliferative diseases in G
IX
+
F
1
mice.
Uroporphyrinogen I synthase induction in normal human bone marrow cultures: an early and quantitative response of erythroid differentiation.
Authors: Sassa S , Urabe A
Published: May 2006

Uroporphyrinogen I (URO) synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] activity increased when cultures of normal human bone marrow cells were incubated with erythropoietin. The increase of URO synthase activity was a linear function of erythropoietin concentration in the culture medium and was proportional to the extent of heme synthesis as determined by 55Fe incorporation into heme. The onset of the increase in URO synthase activity, which occurred on day 4 of incubation with erythropoietin, preceded by 3 days the appearance of hemoglobin in colonies derived from erythroid progenitor cells. These results indicate that induction of URO synthase in normal human bone marrow cells incubated with erythropoietin is an early event and that URO synthase activity is a useful quantitative index of erythroid differentiation.
Experimental autoimmune model of nerve growth factor deprivation: Effects on developing peripheral sympathetic and sensory neurons
Authors: Gorin Pamela Dolkart , Johnson Eugene M.
Published: May 2006

An experimental autoimmune model of nerve growth factor (NGF) deprivation has been used to assess the role of NGF in the development of various cell types in the nervous system. Adult rats immunized with 2.5S mouse NGF in complete Freund's adjuvant produced antibodies that crossreacted with their own NGF and that were transferred
in utero
to the fetus and in milk to the neonate. Cross-fostering experiments were carried out to separate the effects of exposure to anti-NGF
in utero
from those due to exposure through the milk. Anti-NGF transferred
in utero
and in milk resulted in the destruction of peripheral sympathetic neurons assessed by morphological methods (light microscopy) and biochemical methods (tyrosine hydroxylase activity, choline acetyltransferase activity, and protein content). No effects were observed on the adrenal medulla. Offspring of NGF-immunized females exposed to anti-NGF
in utero
had a decreased protein content in the dorsal root ganglia and were unable to transport
125
I-labeled NGF injected in the forepaw to the dorsal root ganglia. These results suggest that a subpopulation of sensory neurons is dependent on NGF for survival during some period of fetal development. This model offers the potential for determining the degree and time of dependence of various cell types on NGF.
Evidence for a cholinergic projection to neocortex from neurons in basal forebrain.
Authors: Johnston M V , McKinney M , Coyle J T
Published: May 2006

Unilateral stereotaxic injection of 3.5 nmol of kainic acid into the ventral globus pallidus of rats reduced biochemical cholinergic neuronal markers by 45-50% and virtually eliminated histochemical staining for acetylcholinesterase in neocortex ipsilateral to the lesion. At the lesion site, the large, multipolar neurons that stain densely for acetylcholinesterase were absent when compared with the uninjected side. Kainate was as effective as electrocoagulation for reducing cholinergic markers although it did not affect aminergic projections ascending through the lesioned area. The conclusion that the cholinergic projection originated in neuronal perikarya at the lesion site was supported by the failure of kainate or electrolytic lesions in contiguous regions to produce similar effects. These studies provide strong evidence for a cholinergic projection to neocortex from neurons in the forebrain in the nucleus basalis.
Solubilization of the low density lipoprotein receptor
Authors: Schneider Wolfgang J. , Basu Sandip K. , McPhaul Michael J. , Goldstein Joseph L. , Brown Michael S.
Published: May 2006

The low density lipoprotein (LDL) receptor was solubilized from membranes of bovine adrenal cortex and cultured human cells by incubation with the nonionic detergent octyl-β-D-glucoside. Receptor activity released into the 100,000 ×
g
supernatant was assayed by a solid-phase procedure: an aliquot of the soluble extract was removed, the detergent was diluted below its critical micellar concentration, causing the receptor to precipitate as a lipid—protein aggregate; the precipitate was collected by centrifugation and incubated with
125
I-labeled LDL (
125
I-LDL); and the receptor-bound
125
I-LDL was separated from free
125
I-LDL by filtration. The
125
I-LDL binding site that was precipitated from the soluble extract of bovine adrenocortical membranes appeared to be the same as the functional LDL receptor of cultured bovine adrenocortical cells and human fibroblasts. It exhibited high affinity and specificity (affinity for LDL more than 200-fold greater than for acetylated LDL, methylated LDL, or high density lipoprotein), dependence on calcium, and susceptibility to destruction by Pronase. The amount of
125
I-LDL binding activity in solubilized membranes from cultured cells was proportional to the number of receptors on the surface of the intact cells. Thus, the number of solubilized receptors was 1/20th of normal in mutant fibroblasts from a subject with homozygous familial hypercholesterolemia and was 1/4th of normal in human epithelioid carcinoma A-431 cells when they were grown in the presence of 25-hydroxycholesterol plus cholesterol. While in the soluble form in the presence of octyl-β-D-glucoside, the LDL receptor can be carried through several steps of purification.
Purification and partial characterization of human lymphoblast interferon.
Authors: Zoon K C , Smith M E , Bridgen P J , zur Nedden D , Anfinsen C B
Published: May 2006

One component of human lymphoblastoid interferon obtained from Namalwa cultures induced by Newcastle disease virus has been purified to a specific activity of 2.5 x 10(8) interferon units per mg of protein (protein content based on amino acid analysis). A single polypeptide species with an apparent molecular weight of 18,500 comigrating with the antiviral activity was observed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Preliminary amino-terminal sequencing results support the conclusion that the interferon species is essentially homogeneous.
Interactions of tumor cells with vascular endothelial cell monolayers: a model for metastatic invasion.
Authors: Kramer R H , Nicolson G L
Published: May 2006

The interactions of tumorigenic and nontumorigenic human and rodent cells with vascular endothelial cells and their underlying extracellular matrix were studied in culture. The abilities of various cells to attach to endothelial monolayers and cause morphologic changes, such as rupture of endothelial-endothelial cell interactions leading to retraction of endothelial cells and exposure of extracellular matrix, as well as their propensities to invade and underlap retracted endothelial monolayers and continue migration were assessed by time-lapse and phase-contrast microscopy as well as scanning and transmission electron microscopy. In general, highly malignant or highly invasive cells in vivo were capable of attachment, invasion, and migration under endothelial cells in vitro. This system may be useful for elucidating mechanisms of tumor cell arrest and extravasation.
Relationship between movement and aggregation of centrioles in syncytia and formation of microtubule bundles.
Authors: Wang E , Connolly J A , Kalnins V I , Choppin P W
Published: May 2006

Previous reports from this laboratory have provided evidence suggesting that microtubules and 10-nm filaments serve both cytoskeletal and force-generating functions in the intracellular movement and positioning of nuclei in syncytia. It has been found that, during the process of cell fusion and nuclear migration in syncytia induced by the paramyxovirus simian virus 5, centrioles are transported in the cytoplasm and form large aggregates. These aggregates are usually found in regions adjacent to rows of aligned nuclei and large bundles of microtubules and 10-nm filaments. Colchicine prevents the translocation and aggregation of centrioles, but cytochalasin B has little effect on this process. These results suggest that the same cytoskeletal elements that are involved in nuclear migration and positioning--i.e., microtubules and 10-nm filaments--are also involved in the transport of centrioles. The possibility that aggregates of centrioles may serve as centers for the organization of microtubules and 10-nm filaments into the large bundles observed in the syncytia is discussed.
In vitro synthesis of a putative precursor of mitochondrial ornithine transcarbamoylase.
Authors: Conboy J G , Kalousek F , Rosenberg L E
Published: May 2006

Ornithine transcarbamoylase (OTCase; ornithine carbamoyltransferase; carbamoyl phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), a major mitochondrial matrix enzyme in ureotelic animals, is synthesized on cytoplasmic ribosomes and translocated across both mitochondrial membranes to the matrix. In an attempt to identify the primary translation product (or an early intermediate) that is the substrate for this transport process, we translated rat liver polysomal RNA in vitro by using the rabbit reticulocyte lysate system. Immunoprecipitation of the [35S]methionine-labeled translation mixture was performed by using monospecific OTCase antiserum and the immunoadsorbent Staphylococcus aureus. Approximately 0.3% of total trichloroacetic acid-insoluble 35S-labeled material was specifically precipitated. Analysis of the precipitate by fluorography of a dried sodium dodecyl sulfate/polyacrylamide gel showed a single major translation product whose mobility corresponded to a polypeptide of 43,000 daltons, a value approximately 4000 daltons greater than that noted for the "mature" OTCase subunit isolated from rat liver. This translation product was not precipitated by preimmune rabbit serum, and excess unlabeled mature OTCase competed with it for interaction with OTCase antiserum. These results suggested that rat liver OTCase, like a number of other cytoplasmically synthesized organellar proteins, is initially made as a larger precursor that contains an amino acid sequence necessary to confer on OTCase its transport properties. The potential application of these findings to the study of inherited complete OTCase deficiency in humans is discussed.
Increased expression of a eukaryotic gene in Escherichia coli through stabilization of its messenger RNA.
Authors: Hautala J A , Bassett C L , Giles N H , Kushner S R
Published: May 2006

The expression of a cloned eukaryotic gene [catabolic dehydroquinase (3-dehydroquinate hydro-lyase, EC 4.2.1.10) (qa-2+) from Neurospora crassa] is dramatically increased (as much as 100-fold) in Escherichia coli strains deficient in polynucleotide phosphorylase (pnp) (polynucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) and RNase I (rna). The increased expression is controlled primarily by the absence of polynucleotide phosphorylase and appears to be specific for the eukaryotic gene. No increase in the specific activity of either chromosomal or plasmid-borne prokaryotic genes has been observed. In polynucleotide phosphorylase-deficient strains of E. coli the half-life of plasmid (pVK88, ampr qa-2+)-encoded mRNAs increases from 1.0 to 2.8 min. This increase must be due primarily to stabilization of the aq-2 mRNA because no increase in the half-lives of pBR322 vehicle mRNAs was observed in polynucleotide phosphorylase-deficient strains. These results suggest that there are inherent structural differences between prokaryotic and eukaryotic mRNAs.
Antibody-induced modulation of Friend virus cell surface antigens decreases virus production by persistent erythroleukemia cells: influence of the Rfv-3 gene.
Authors: Chesebro B , Wehrly K , Doig D , Nishio J
Published: May 2006

The Rfv-3 gene was found to influence the level of Friend leukemia virus production in spleens of leukemic mice later than 30 days after virus inoculation. Rfv-3r/s mice [(B10.A X A)F1 and (B10.A X A.BY)F1] had decreased spleen virus levels 30-90 days after virus inoculation compared to Rfv-3s/s mice [A.BY, A, BALB.B, and (BALB/c X A)F1)]. In (B10.A X A)F1 X A backcross mice the spleen virus titer segregated with the level of viremia. The Rfv-3 gene appeared to act by controlling anti-Friend virus antibody production. The interaction of antiviral antibody with infected cells led to a decrease in release of infectious virus by late leukemic spleen cells in Rfv-3r/s mice to 1/300th that in Rfv-3s/s mice. This decrease in virus release appeared to be due to interference with the virus budding process due to antibody-mediated modulation of virus-induced cell surface antigens.
Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse.
Authors: Russell W L , Kelly E M , Hunsicker P R , Bangham J W , Maddux S C , Phipps E L
Published: May 2006

Use of the specific-locus test to measure the frequency of transmitted gene mutations induced in mouse spermatogonia has shown ethylnitrosourea to be by far the most potent mutagen yet discovered in the mouse. The dose used, 250 mg/kg, gave a mutation rate 5 times as high as had been obtained with 600 R, the most effective acute dose of x-rays. Compared to procarbazine, heretofore the most mutagenic chemical known in the mouse, ethylnitrosourea proved to be 15 times more mutagenic than the peak effect obtained with the most effective dose of procarbazine. Because of its high mutagenicity, ethylnitrosourea can serve as a model compound in exploring the effect of such factors as dose response, dose fractionation, sex, and cell stage on the mutagenic action of a chemical. Ethylnitrosourea is clearly the mutagen of choice for the production of any kind of desired new gene mutations in the mouse.
DNA-mediated gene transfer of a circular plasmid into murine cells.
Authors: Huttner K M , Scangos G A , Ruddle F H
Published: May 2006

We have used DNA-mediated gene transfer to introduce a recombinant plasmid containing the human beta-globin gene (H beta 1) into cells of a mouse tissue culture line, Ltk-. DNA isolated from independent transfer lines was analyzed by restriction endonuclease digestion, gel electrophoresis, modified Southern blotting, and filter hybridization using H beta 1 as a probe. H beta 1 sequences were present in 80% of the lines at 1-30 copies per cell. Many of the lines gave a hybridization pattern indicative of H beta 1 sequences integrated into high molecular weight DNA. DNA from three cell lines, digested with several restriction enzymes, produced a pattern providing evidence for the presence of circular H beta 1 molecules in the murine recipient cells.
Spontaneous tumors in Sprague-Dawley and Long-Evans rats and in their F1 hybrids: carcinogenic effect of total-body x-irradiation.
Authors: Gross L , Dreyfuss Y
Published: May 2006

Rats frequently develop various tumors, many of them malignant; the majority of tumors in the females develop in the mammary glands. In primary spontaneous tumors and lymphomas virus particles cannot be found on electron microscopic examination; transmission of the tumors by filtered extracts has not been successful. In our colonies of Sprague-Dawley rats the incidence of tumors was 22% in females and 5% in males; in Long-Evans rats the incidence of tumors and 28% in females and 10% in males. In (Sprague-Dawley x Long-Evans) F1 hybrids the incidence of tumors was 67% in females and 32% in males, about twice as high as in the parental strains. Fractionated total-body x-irradiation (150 rads five times at weekly intervals) (1 rad = 0.01 gray) increased the incidence of tumors in Sprague-Dawley rats from 22% to 93% in females and from 5% to 59% in males. In Long-Evans rats, irradiation increased the incidence of tumors from 28% to 63% in females and from 10% to 42% in males. The incidence of malignant tumors was almost twice as high in irradiated Sprague-Dawley and Long-Evans rats as compared with nonirradiated animals of the same strains. Partial shielding during irradiation had no significant effect on the incidence or on the forms of tumors developing in the irradiated animals. In striking contrast to results of experiments carried out on mice, the incidence of leukemia and lymphomas was not increased in the irradiated rats, as compared with control animals.
Persistence of circadian rhythmicity in a mammalian hypothalamic "island" containing the suprachiasmatic nucleus.
Authors: Inouye S T , Kawamura H
Published: May 2006

The experimental work described tested the prosposition that the suprachiasmatic nucleus of the hypothalamus is an autonomous circadian pacemaker. Simultaneous recording from two extracellular electrodes indicated neural (multiple unit) activity at two sites in the brain, one of which is in or near the suprachiasmatic nucleus and the other in one of many other brain locations. Both sites in intact rats displayed clear circadian rhythmicity of spontaneous neural activity. In experimental animals, a Halasz knife was used to create an island of hypothalamic tissue that contained the suprachiasmatic nuclei. In such animals that were also blinded by bilateral ocular enucleation, circadian rhythmicity was lost at all brain locations recorded outside the island, but it persisted within the island that contained the suprachiasmatic nuclei. The rhythmicity of the island is thus not dependent on afferent inputs from elsewhere in the brain.
Localization of neurophysin within organelles associated with protein synthesis and packaging in the hypothalamoneurohypophysial system: an immunocytochemical study.
Authors: Broadwell R D , Oliver C , Brightman M W
Published: May 2006

Electron microscopic immunocytochemical localization of neurophysin in the hypothalamo-neurohypophysial system of mice has been studied by using the pre-embedding staining approach to the unlabeled antibody-enzyme technique of Sternberger. In supraoptic cell bodies, peroxidase-antiperoxidase reaction product was localized within cisternae of the nuclear envelope, rough endoplasmic reticulum, and Golgi saccules but not in GERL (Golgi-associated smooth endoplasmic reticulum from which lysosomes arise). Reaction product was also present in secondary lysosomes. Secretory granules in supraoptic perikarya and posterior pituitary Herring bodies were likewise immunoreactive. These findings with the unlabeled antibody-enzyme technique provide conclusive evidence for the localization of an antigen within cellular organelles associated with protein synthesis and packaging.
On the lowest eigenvalue of a pseudo-differential operator
Authors: Fefferman C. , Phong D. H.
Published: May 2006

Positive lower bounds for pseudo-differential operators with nonnegative symbols are derived; the bounds in particular yield subelliptic estimates for operators arising as sums of squares of vector fields.
Discontinuous translation of silk fibroin in a reticulocyte cell-free system and in intact silk gland cells.
Authors: Lizardi P M , Mahdavi V , Shields D , Candelas G
Published: May 2006

Silk fibroin mRNA was translated in a rabbit reticulocyte cell-free system. Addition of tRNA from silk glands was essential for complete translation of the fibroin polypeptide. (Mr approximately 400,000). Synthesis of full-sized product took at least 85 min. In addition to full-size product, a large number of smaller polypeptides were observed upon analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Evidence is presented that these smaller polypeptides are growing fibroin chains that transiently accumulate as discrete size classes due to discontinuities in the translation process. These discontinuities, or pauses, occur at specific sites in the fibroin mRNA template. The relative duration of the pauses can be experimentally modulated by changing the source of the supplementary tRNA added to the in vitro system. Silk glands were incubated in organ culture under conditions where essentially exclusive labeling of newly synthesized fibroins was attained. Analysis in sodium dodecyl sulfate gels showed that the labeling pattern of nascent silk fibroins is similar to the pattern observed in the reticulocyte cell-free system. This result suggests that discontinuities or pauses in polypeptide chain elongation also occur in vivo under conditions of organ culture.
Both chains of HLA-DR bind to the membrane with a penultimate hydrophobic region and the heavy chain is phosphorylated at its hydrophilic carboxyl terminus.
Authors: Kaufman J F , Strominger J L
Published: May 2006

The HLA-DR antigen, a complex of two glycoproteins of 29,000 and 34,000 daltons, can be isolated from the membranes of human B-lymphoblastoid cell lines. Extensive proteolysis releases only 5-10% of the antigen, whereas detergent solubilizes all of it. Detergent solubilization after papain proteolysis of membranes produces antigen with chains cleaved near the carboxyl termini. Comparison of these three preparations demonstrates that each chain contains a carboxyl-terminal hydrophilic region that is sensitive to proteolytic degradation and a penultimate hydrophobic region, responsible for membrane binding, that is more resistant to papain. This two-step cleavage of each chain is also observed during the proteolysis of detergent-solubilized HLA-DR antigen. Both chains of HLA-DR in the membrane can be labeled with the lipophilic photoactivatable carbene reagent adamantane diazirine. This label is released from both chains during the second cleavage. The heavy chain can be reduced and alkylated under mild conditions, and this label is also lost during the second cleavage. The heavy chain is phosphorylated in vivo, and this label is lost upon the first cleavage. This observation suggests that the carboxyl terminus of the heavy chain is intracellular. Cumulatively, these data suggest that both chains of HLA-DR antigens are comprised of large extracellular NH2-terminal regions, small penultimate intramembranous regions, and small carboxyl-terminal intracellular regions.
Patterned acquisition of the antibody repertoire: diversity of the hemagglutinin-specific B-cell repertoire in neonatal BALB/c mice.
Authors: Cancro M P , Wylie D E , Gerhard W , Klinman N R
Published: May 2006

The B-cell response of 12- to 14-day old BALB/c mice to the hemagglutinin molecule of influenzae virus A/PR/8/34(H0N1) has been examined with monoclonal antibodies obtained by the splenic focus technique. An analysis of the specificity of these antibodies with a panel of heterologous viruses indicates that the antibody repertoire is highly restricted at an intermediate stage in postnatal development of the immune system. In toto, only 10 distinct reactivity patterns have been observed in an analysis of 72 antibodies derived from 28 donors. This contrasts with a substantially more diverse repertoire present in nonimmune and immune adult populations. The neonatal antibody specificities do not appear to be a random sampling of adult specificities, because several clonotypes (as defined by reactivity pattern) frequently found in neonates are rare or absent in adults. Most importantly, the vast majority of adult clonotypes are absent from the neonatal repertoire. These findings indicate that, at a developmental stage when the B-cell repertoire contains at least 10(6) clonotypes, the repertoire of genetically identical individuals is shared. This is consistent with a diversification process that is highly patterned and genetically determined. Furthermore, because 12- to 14-day-old neonates exhibit a diversified but definable hemagglutinin-specific B-cell repertoire, this experimental system should enable precise analyses of genetic and environmental influences on repertoire expression.
Thymic control of proliferation of T cell precursors in bone marrow.
Authors: Cohen J J , Fairchild S S
Published: May 2006

The activity of T lymphocyte precursors (pre-T cells) in the bone marrow of mice was measured by the concanavalin A response synergy assay. Pre-T cell levels were low in marrow of neonatally thymectomized mice and could be restored to control values by treatment in vivo with an extract of mouse thymus. Levels of activity were also low in aging mice and again could be restored by thymic extract treatment. The most profound fall with aging was in the proliferating pre-T cell compartment as detected by tritiated thymidine suicide; and this compartment was restored by thymic extract treatment. Irradiation to the thymus, with the bone marrow shielded, caused a fall in resting pre-T cells in the bone marrow and a concomitant rise in proliferating cells. These results are consistent with a model of control of pre-T cell maturation in which the thymus senses the number of developing lymphocytes within it and responds to a fall in this number by increasing production of hormone. The hormone acts on resting pre-T cells in the marrow, stimulating some of them to proliferate, leave the bone marrow, and repopulate the thymus.
Inhibition of cis-platinum nephrotoxicity by diethyldithiocarbamate rescue in a rat model.
Authors: Borch R F , Pleasants M E
Published: May 2006

The nephrotoxic effects of cis-dichlorodiammineplatinum(II) (NSC-119875) administered to male F344 rats at the median lethal dose (LD50; 7.5 mg/kg) were inhibited by treatment with sodium diethyldithiocarbamate (500 or 750 mg/kg) between 1 and 4 hr after cis-platinum administration. Those animals receiving cis-platinum alone had mean serum blood urea nitrogen levels of 234 mg/dl at the time of maximal toxicity (day 5); kidney sections revealed large areas of degeneration and necrosis. When dithiocarbamate rescue was carried out after cis-platinum treatment, mean blood urea nitrogen levels were in the range 56-95 mg/dl; kidney sections were grossly normal with a barely discernible band of degeneration at the corticomedullary junction. Gastrointestinal toxicity was observed in greater than 95% of the cis-platinum-treated rats but was totally absent in those receiving subsequent rescue treatment. A significant decrease in weight loss was also observed in the dithiocarbamate-rescued rats. Based on the chemistry of platinum-sulfur interactions and the observed time-dependence of the rescue treatment, it is suggested that dithiocarbamate exerts its effects via competitive chelation and removal of platinum coordinated to protein-bound sulfhydryl groups of the kidney tubule cells.
Human cell lines containing Epstein-Barr virus but distinct from the common B cell lymphoblastoid lines.
Authors: Fu S M , Hurley J N
Published: May 2006

A group of very similar cell lines was established from peripheral blood or bone marrow of 12 patients with a variety of disorders. The cells in these cell lines were uniform and round in shape. They grew as single-cell suspensions or as aggregates of small numbers of cells in stationary culture. The most striking characteristic of these lines was the lack of cells with surface immunoglobulin or with demonstrable immunoglobulin synthesis. This lack of immunoglobulin synthesis and their special growth characteristics distinguished them from the lymphoblastoid B cell lines previously described. The cells of these unusual cell lines had strong Fc receptors and C3 receptors and expressed Ia antigens. They did not form rosettes with sheep erythrocytes and did not have detectable levels of terminal deoxynucleotidyltransferase. They did not secrete lysozyme and failed to stain for peroxidase. The presence of the Epstein-Barr virus nuclear antigen in the cells indicated the presence of Epstein-Barr viral genome. The possibility that these cells represent some type of precursor cell in the B cell lineage is discussed, but the exact cellular origin remains to be ascertained.
Immunoglobulin heavy chain gene organization in mice: analysis of a myeloma genomic clone containing variable and alpha constant regions.
Authors: Early P W , Davis M M , Kaback D B , Davidson N , Hood L
Published: May 2006

We have isolated a myeloma genomic DNA clone containing the variable and constant regions of a mouse alpha chain. Restriction enzyme analyses and electron microscopic R loop mapping have demonstrated that the variable region is separated from the constant region by 6.8 kilobases of intervening DNA. In addition, two intervening DNA sequences of 100--200 bases separate the constant region into three approximately equal units. These intervening sequences may separate each of the segments coding for the three constant region domains of the alpha heavy chain. Southern blot analysis of embryo and myeloma DNA suggests that DNA rearrangement of heavy chain variable and constant regions occurs during the differentiation of antibody-producing cells.
Identification of 7,12-dimethylbenz[
a
]anthracene metabolites that lead to mutagenesis in mammalian cells
Authors: Huberman Eliezer , Chou Ming W. , Yang Shen K.
Published: May 2006

The mutagenicity of 7,12-dimethylbenz[
a
]-anthracene (DMBA) and 11 of its enzymatically derived metabolites was tested with Chinese hamster V79 cells for identification of mutagenic metabolites. The metabolites consisted of 7-hydroxymethyl-12-methylbenz[
a
]anthracene, 7-methyl-12-hydroxymethylbenz[
a
]anthracene, 7,12-dihydroxymethylbenz[
a
]anthracene, three
trans
-3,4-diols, two
trans
-5,6-diols, and three
trans
-8,9-diols, all of which derived from DMBA or from the hydroxymethyl derivatives. Mutations were characterized by resistance to ouabain and 6-thioguanine. None of the tested metabolites were mutagenic in V79 cells, which do not metabolize polycyclic aromatic hydrocarbons. Therefore, mutagenesis in the V79 cells was tested in the presence of golden hamster cells capable of metabolizing polycyclic aromatic hydrocarbons (cell-mediated assay). In this assay, DMBA, 7-hydroxymethyl-12-methylbenz[
a
]anthracene, 7-methyl-12-hydroxymethylbenz[
a
]anthracene, and their
trans
3,4-diols were mutagenic for both genetic markers, and the mutagenic response increased as a function of the hydrocarbon dose. All other metabolites were either inactive or showed up to a 4-fold higher mutation frequency than the untreated V79 cells for ouabain and 6-thioguanine resistance. The DMBA-
trans
-3,4-diol was the only metabolite that was more active than DMBA itself; at 0.05 μM it was 6-8 times more active than DMBA itself; at 0.05 μM it was 6-8 times more active than DMBA in inducing both ouabain and 6-thioguanine resistance. This diol was mutagenic at a dose as low as 0.01 μM. Mutagenesis by DMBA and the
trans
-3,4-diols was inhibited by 7,8-benzoflavone, an inhibitor of mixed-function oxidases. Analysis of DMBA metabolism in intact golden hamster cells indicated that DMBA-
trans
-3,4-diol is one of the major metabolites produced. Our results therefore suggest that DMBA-
trans
-3,4-diol may be metabolized to a diol-epoxide, presumably the
trans
-3,4-diol-1,2-epoxide, which may be a major reactive metabolite responsible for DMBA mutagenicity in mammalian cells.