bluuebunny/crossref_metadata_2025 huggingface dataset:
The hemodynamic destruction of intravascular cancer cells in relation to myocardial metastasis.
Authors: Weiss L , Dimitrov D S , Angelova M
Published: May 2006

A variety of observations in humans and experimental animals indicate that large numbers of circulating cancer cells are killed in the microvasculature. It is suggested that this occurs when friction or adhesion between individual cancer cells and capillary walls results in an increase of tension in the cancer cell peripheries above a critical level because of (blood) pressure differentials between their free ends. Hemodynamic and anatomic data relating to the myocardial circulation and deformability measurements on four types of rat cancer cells have been reported previously by others. Novel calculations based on these data suggest that the increased tension at the peripheries of cancer cells passing through the myocardial capillaries will exceed the critical levels for rupture. Analysis of autopsy data for solid tumors reveals a low (less than 3%) incidence of myocardial metastases in the absence of lung metastases and a higher (15%) incidence in their presence. One explanation for these observations is that, in the absence of lung metastases, relatively few of the cancer cells enter the coronary arteries from primary tumors with systemic venous drainage because many are retained or destroyed in transit through the pulmonary vasculature, and most of those delivered to the myocardium then suffer hemodynamic destruction. In the presence of pulmonary metastases, large numbers of viable cancer cells are liberated directly into the pulmonary venules and subsequently are delivered to the myocardium without prior exposure to the arterial side of the microcirculation. The combined effects of increased delivery and the protective effects of arrested cells on those preceding them in files along the capillaries account for the higher incidence of myocardial metastases. It is proposed that hemodynamic destruction of circulating cancer cells may be an important underlying cause of metastatic inefficiency, together with other cytocidal mechanisms.
Long terminal repeat sequences impart hematopoietic transformation properties to the myeloproliferative sarcoma virus.
Authors: Stocking C , Kollek R , Bergholz U , Ostertag W
Published: May 2006

The myeloproliferative sarcoma virus not only transforms fibroblasts but also causes extensive expansion of the hematopoietic stem cell compartment on infection of adult mice. Similar to the Moloney sarcoma virus, it carries the mos oncogene. Moloney sarcoma virus, however, does not induce myeloproliferation and leukemia in adult mice. The difference between the two viruses was explored by using their molecularly cloned genomes and the cellular mos oncogene to construct recombinant genomes. It was shown that the U3 region of the viral long terminal repeat (LTR) has a decisive function in determining the target cell specificity of the myeloproliferative sarcoma virus. Any mos gene, whether of cellular or viral origin, is sufficient in conjunction with the proper LTR to induce myeloproliferation. Our results indicate that the pathogenicity of acutely transforming viruses is determined not only by the oncogene but also by sequences in the viral LTR.
A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin.
Authors: Pytela R , Pierschbacher M D , Ruoslahti E
Published: May 2006

Affinity chromatography was used to identify a cell surface receptor for the adhesive protein vitronectin. Detergent extracts of human osteosarcoma (MG-63) cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronectin cell attachment sequence Arg-Gly-Asp. Two cell surface proteins with apparent molecular mass of 125 and 115 kDa bound to both columns and were specifically eluted with a solution containing the Gly-Arg-Gly-Asp-Ser-Pro peptide. These proteins could be incorporated into phosphatidylcholine liposomes and mediated the specific binding of these liposomes to vitronectin but not to fibronectin. In contrast, liposomes containing a previously identified 140-kDa fibronectin receptor, which interacts with the Arg-Gly-Asp sequence in fibronectin, did not bind to vitronectin. Thus, the fibronectin and vitronectin receptors each recognize the Gly-Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive reactivities toward fibronectin and vitronectin. These receptors appear to belong to a family of proteins that mediate cell substratum adhesion via related but subtly different specificities.
Chromosomal localization of the human Thy-1 gene.
Authors: van Rijs J , Giguère V , Hurst J , van Agthoven T , Geurts van Kessel A , Goyert S , Grosveld F
Published: May 2006

We have isolated the gene coding for human Thy-1. Introduction of this gene into HeLa cells by DNA-mediated transfer results in the expression of Thy-1 antigen on the cell surface. Chromosomal mapping of the Thy-1 gene by hybridization to metaphase chromosomes and Southern blots of DNA from hybrid cells indicate that the Thy-1 gene is located on the long arm of chromosome 11.
Prazosin, an alpha 1-adrenergic receptor antagonist, suppresses experimental autoimmune encephalomyelitis in the Lewis rat.
Authors: Brosnan C F , Goldmuntz E A , Cammer W , Factor S M , Bloom B R , Norton W T
Published: May 2006

Prazosin, an antagonist of alpha 1-adrenergic receptors, has been found to suppress the clinical and histological expression of experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. Suppression was more significant in females than in males and was a dose-dependent phenomenon. Analysis of the effect of other adrenergic receptor antagonists supports the conclusion that the suppressive effect of prazosin is a consequence of blockade of the alpha 1-receptor since treatment with either the alpha 2-antagonist yohimbine or the beta-antagonist propranolol exacerbated the disease, whereas treatment with the long-acting mixed alpha 1/alpha 2-antagonist phenoxybenzamine had some suppressive activity. Treatment with prazosin was also able to suppress clinical and histological signs of EAE in animals sensitized by adoptive transfer with activated spleen or lymph node cells. Whether prazosin acts through altering vascular permeability or the immune response, or both, remains to be determined.
Genetic dissection of Drosophila adenylate cyclase.
Authors: Livingstone M S
Published: May 2006

The Drosophila learning mutant rutabaga is missing calcium/calmodulin activation of adenylate cyclase (EC4.6.1.1). The mutation was mapped at a finer resolution to X chromosome bands 12F5-7. By comparing wild-type and mutant cyclase activities, the relative responsiveness of the calcium-sensitive and calcium-insensitive components to different ligands could be determined; the calcium-sensitive fraction of the total cyclase activity was significantly less responsive to guanyl nucleotides and monoamines. The results suggest that the component of cyclase activity that is stimulated by calcium/calmodulin, possibly a genetically distinct catalytic subunit, is not coupled to the G subunit or the G subunit/monoamine receptor complex.
Preferential fertilization in
Plumbago
: Ultrastructural evidence for gamete-level recognition in an angiosperm
Authors: Russell Scott D.
Published: May 2006

Gametic fusion patterns in the angiosperm
Plumbago zeylanica
were determined by using cytoplasmically dimorphic sperm cells differing in mitochondrion and plastid content and then identifying paternal organelles through their ultrastructural characteristics within the maternal cytoplasm at the time of fertilization. The virtual absence of plastids within the sperm cell that is physically associated with the vegetative nucleus allows paternal plastids to be used to trace the fate of the two male gametes after fusion. Such paternal plastids were present in the egg in >94% of the observed cases, indicating the preferential fusion of the plastid-rich, mitochondrion-poor sperm cell with the egg. In only one instance did the opposite pattern occur. Since the possibility of this result occurring as the consequence of chance in random fusions is <1 in 7000, this represents strong evidence for the presence of a final putative recognition event occurring at the gametic level.
The formation of fenestrations and channels by capillary endothelium in vitro.
Authors: Milici A J , Furie M B , Carley W W
Published: May 2006

Microvascular endothelial cells isolated from fenestrated capillaries have been shown to form tubes in vitro, thereby demonstrating that they retain the ability to express some degree of their in vivo differentiated phenotype. However, some of their physiologically important structural features, such as transendothelial openings (i. e., diaphragmed fenestrations and transendothelial channels) are lost or are greatly reduced in number. In this study, cloned bovine adrenal cortex endothelial cells were cultured on plastic or on a basal lamina produced by Madin-Darby canine kidney (MDCK) cells for up to 17 days postconfluence. All cultures were then routinely fixed and processed for electron microscopic morphometry. For cells grown on plastic for 17 days postconfluence, the linear density of transendothelial openings in endothelial profiles less than 400 nm thick was found to be 0.007 openings per micron. On MDCK matrix, however, the linear density of transendothelial openings in endothelial profiles less than 400 nm thick was found to be 0.157 per micron. Occasionally some cells formed "tube-like" structures that also contained diaphragmed fenestrations and transendothelial channels on both sides of the tubes. These findings suggest that the substrate on which endothelial cells are grown can affect their differentiation.
Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA.
Authors: Schneider J W , Mercer R W , Caplan M , Emanuel J R , Sweadner K J , Benz E J , Levenson R
Published: May 2006

We have isolated a cDNA clone for the rat brain Na,K-ATPase alpha subunit. A lambda gt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, lambda rb5, containing a 1200-base-pair cDNA insert expressed a beta-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the alpha subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of lambda rb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney greater than brain greater than liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to lambda rb5 cDNA were amplified approximately 40-fold. Analysis of the lambda rb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase alpha-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the lambda rb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase alpha subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.
Peptide mixture sequencing by tandem Fourier-transform mass spectrometry.
Authors: Cody R B , Amster I J , McLafferty F W
Published: May 2006

Picomole samples of the linear peptide gramicidin D and cyclic peptide gramicidin S are shown to be impure by the laser-desorption formation of multiple groups of molecular adduct peaks by using Fourier-transform mass spectrometry. Selective excitation of the molecular peaks of the major sample component followed by collisionally activated dissociation provides complete sequence information for the cyclic decapeptide and for 12 of the 15 amino acids of the linear peptide. This instrumentation shows striking advantages in sensitivity, resolution, and mass accuracy in comparison to tandem mass spectrometers used previously.
Nucleotide sequences of the
Pseudomonas savastanoi
indoleacetic acid genes show homology with
Agrobacterium tumefaciens
T-DNA
Authors: Yamada Tetsuji , Palm Curtis J. , Brooks Bob , Kosuge Tsune
Published: May 2006

We report the nucleotide sequences of
iaaM
and
iaaH
, the genetic determinants for, respectively, tryptophan 2-monooxygenase and indoleacetamide hydrolase, the enzymes that catalyze the conversion of L-tryptophan to indoleacetic acid in the tumor-forming bacterium
Pseudomonas syringae
pv.
savastanoi
. The sequence analysis indicates that the
iaaM
locus contains an open reading frame encoding 557 amino acids that would comprise a protein with a molecular weight of 61,783; the
iaaH
locus contains an open reading frame of 455 amino acids that would comprise a protein with a molecular weight of 48,515. Significant amino acid sequence homology was found between the predicted sequence of the tryptophan monooxygenase of
P. savastanoi
and the deduced product of the T-DNA
tms-1
gene of the octopine-type plasmid pTiA6NC from
Agrobacterium tumefaciens
. Strong homology was found in the 25 amino acid sequence in the putative FAD-binding region of tryptophan monooxygenase. Homology was also found in the amino acid sequences representing the central regions of the putative products of
iaaH
and
tms
-2 T-DNA. The results suggest a strong similarity in the pathways for indoleacetic acid synthesis encoded by genes in
P. savastanoi
and in
A. tumefaciens
T-DNA.
Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and delta pH determinations.
Authors: Lomax T L , Mehlhorn R J , Briggs W R
Published: May 2006

Closed and pH-tight membrane vesicles prepared from hypocotyls of 5-day-old dark-grown seedlings of Cucurbita pepo accumulate the plant growth hormone indole-3-acetic acid along an imposed proton gradient (pH low outside, high inside). The use of electron paramagnetic spin probes permitted quantitation both of apparent vesicle volume and magnitude of the pH gradient. Under the experimental conditions used, hormone accumulation was at minimum 20-fold, a value 4 times larger than what one would predict if accumulation reflected only diffusional equilibrium at the measured pH gradient. It is concluded that hormone uptake is an active process, with each protonated molecule of hormone accompanied by an additional proton. Experiments with ionophores confirm that it is the pH gradient itself which drives the uptake.
Peroxisomal organization in normal and cerebrohepatorenal (Zellweger) syndrome fibroblasts.
Authors: Santos M J , Ojeda J M , Garrido J , Leighton F
Published: May 2006

The reported absence of morphologically detectable peroxisomes in liver and kidney tissue cells from patients affected by the autosomic recessive, inherited metabolic disease known as cerebrohepatorenal, or Zellweger, syndrome was studied in fibroblasts, assuming it to be a generalized defect. Normal cultured fibroblasts were shown to contain peroxisomes according to morphological, biochemical, and subcellular fractionation criteria: particle-bound catalase and fatty acyl-CoA oxidase copurify in subcellular fractionation by differential centrifugation or isopycnic equilibrium in continuous density gradients and peroxidase-positive organelles of approximately equal to 0.1 micron in diameter are detected in the cytoplasm. In Zellweger cultured fibroblasts, these peroxisomal enzymes are present; however, they behave as cytosolic enzymes in the different subcellular fractionation procedures employed and peroxisomes are not detected cytochemically. These findings support the hypothesis that the lack of peroxisomes in this genetic disease is the consequence of a defect in the assembly of the peroxisomal constituents. Furthermore, the value of fibroblasts for subcellular analysis of peroxisomal defects is illustrated.
Ultraviolet light-induced crosslinking reveals a unique region of local tertiary structure in potato spindle tuber viroid and HeLa 5S RNA.
Authors: Branch A D , Benenfeld B J , Robertson H D
Published: May 2006

The positions of intramolecular crosslinks induced by irradiation with ultraviolet light were mapped into potato spindle tuber viroid RNA and HeLa 5S rRNA. Crosslinking in each of these molecules occurred at a single major site, which was located by RNA fingerprinting and secondary analysis (and additional primer extension studies in the case of the viroid). Various lines of evidence suggest that these crosslinks identify a previously undescribed element of local tertiary structure common to these two widely divergent RNA molecules: (i) both crosslinks occur in an identical eight-base context, with the sequence 5' GGGAA 3' on one side and the sequence 5' UAC 3' on the other; (ii) both crosslinks connect bases that are not thought to be involved in conventional hydrogen bonding, within regions usually depicted as single-stranded loops flanked by short helical segments; and (iii) both crosslinks connect a purine and a pyrimidine residue, and both may generate the same G-U dimer. Furthermore, it is likely that the crosslinking site is of functional significance because it is located within the most highly conserved region of the viroid sequence and involves bases that are essentially invariant among eukaryotic 5S rRNA molecules.
Visualization of mu1 opiate receptors in rat brain by using a computerized autoradiographic subtraction technique.
Authors: Goodman R R , Pasternak G W
Published: May 2006

We have developed a quantitative computerized subtraction technique to demonstrate in rat brain the regional distribution of mu1 sites, a common very-high-affinity binding site for both morphine and the enkephalins. Low concentrations of [D-Ala2, D-Leu5]enkephalin selectively inhibit the mu1 binding of [3H]dihydromorphine, leaving mu2 sites, while low morphine concentrations eliminate the mu1 binding of [3H][D-Ala2, D-Leu5]enkephalin, leaving delta sites. Thus, quantitative differences between images of sections incubated in the presence and absence of these low concentrations of unlabeled opioid represent mu1 binding sites. The regional distributions of mu1 sites labeled with [3H]dihydromorphine were quite similar to those determined by using [3H][D-Ala2, D-Leu5]enkephalin. High levels of mu1 binding were observed in the periaqueductal gray, medial thalamus, and median raphe, consistent with the previously described role of mu1 sites in analgesia. Other regions with high levels of mu1 binding include the nucleus accumbens, the clusters and subcallosal streak of the striatum, hypothalamus, medial habenula, and the medial septum/diagonal band region. The proportion of total specific binding corresponding to mu1 sites varied among the regions, ranging from 14% to 75% for [3H][D-Ala2, D-Leu5]enkephalin and 20% to 52% for [3H]dihydromorphine.
Transferrin gene expression visualized in oligodendrocytes of the rat brain by using in situ hybridization and immunohistochemistry.
Authors: Bloch B , Popovici T , Levin M J , Tuil D , Kahn A
Published: May 2006

The presence and production of transferrin in the adult rat brain have been investigated using both immunohistochemistry and in situ hybridization in tissue sections. Indirect immunofluorescence with four distinct antisera against rat and human transferrin and one monoclonal antibody against human transferrin demonstrated labeling of the cytoplasm of oligodendrocytes (a category of glial cells) in most parts of the brain, especially in the white matter. In situ hybridization using rat transferrin 32P-labeled cDNA as a probe revealed the presence of transferrin mRNA in glial cells whose appearance, distribution, and organization exactly matched those of the cells decorated with the transferrin antibodies. These results provide evidence that the transferrin gene is expressed in the central nervous system and that transferrin is synthesized by and stored within oligodendrocytes in the adult rat brain. These data suggest that this molecule could have a specific function in nervous system activity.
Quantitative in vivo receptor binding III: Tracer kinetic modeling of muscarinic cholinergic receptor binding.
Authors: Frey K A , Hichwa R D , Ehrenkaufer R L , Agranoff B W
Published: May 2006

A tracer kinetic method is developed for the in vivo estimation of high-affinity radioligand binding to central nervous system receptors. Ligand is considered to exist in three brain pools corresponding to free, nonspecifically bound, and specifically bound tracer. These environments, in addition to that of intravascular tracer, are interrelated by a compartmental model of in vivo ligand distribution. A mathematical description of the model is derived, which allows determination of regional blood-brain barrier permeability, nonspecific binding, the rate of receptor-ligand association, and the rate of dissociation of bound ligand, from the time courses of arterial blood and tissue tracer concentrations. The term "free receptor density" is introduced to describe the receptor population measured by this method. The technique is applied to the in vivo determination of regional muscarinic acetylcholine receptors in the rat, with the use of [3H]scopolamine. Kinetic estimates of free muscarinic receptor density are in general agreement with binding capacities obtained from previous in vivo and in vitro equilibrium binding studies. In the striatum, however, kinetic estimates of free receptor density are less than those in the neocortex--a reversal of the rank ordering of these regions derived from equilibrium determinations. A simplified model is presented that is applicable to tracers that do not readily dissociate from specific binding sites during the experimental period. In this instance, specific tracer binding may be accurately determined by measuring tissue ligand concentration at a single time point after bolus intravenous injection, providing that regional cerebral blood flow is known. This derivation has potential clinical application, because it will permit construction of quantitative pictorial maps of regional free receptor densities in the human brain by means of positron emission tomographic imaging.
Light-harvesting chlorophyll
a
/
b
-protein: Three-dimensional structure of a reconstituted membrane lattice in negative stain
Authors: Li Jade
Published: May 2006

The three-dimensional structure of a negatively stained hexagonal membrane lattice containing the light-harvesting chlorophyll
a
/
b
-protein complex and phospholipids has been determined to 30-Å resolution by image reconstruction from electron micrographs. This lattice has
p
321 symmetry, a lattice constant of 125 Å and a thickness of 75 Å. The monomer is shown to be an elongated molecule about 65 Å long in the dimension perpendicular to the plane of the membrane. It spans the hydrophobic domain of the membrane in an asymmetric fashion, projecting [unk]20 Å from one surface and less from the other. On the basis of this image and available biochemical data, the structure of the complex in the native thylakoid membrane is proposed.
Mammalian reticulocytes lose adhesion to fibronectin during maturation to erythrocytes.
Authors: Patel V P , Ciechanover A , Platt O , Lodish H F
Published: May 2006

We describe three situations in which a large fraction of circulating red blood cells attach tightly and specifically to fibronectin: (i) rabbits made anemic by repeated bleeding, (ii) patients with hemolytic anemia and functional asplenia and splenectomized normal humans, and (iii) splenectomized mice. Upon induction of anemia in rabbits, the proportion of circulating red blood cells capable of specifically attaching to fibronectin-coated plastic increased in parallel with the number of reticulocytes. Fibronectin-adherent red cells were barely detectable when the rabbit had recovered from the anemia. Attachment of reticulocytes to fibronectin was specific; cells did not attach to dishes coated with albumin, laminin, or collagen. None of these proteins promoted the attachment of normal erythrocytes. About 75% of the erythrocytes from splenectomized mice (but not control mice) also attached specifically to fibronectin 40 days after surgery. The effect of splenectomy was incomplete and transient; adherent cells were not detectable 8 weeks after splenectomy. As judged by labeling studies with [35S]methionine, newly emergent reticulocytes preferentially attached to fibronectin. We suggest that about half of the reticulocytes in erythropoietically unstressed mice lose their ability to attach to fibronectin, possibly due to loss of fibronectin-adhesive components, during passage through the spleen. The others lose their ability to interact with fibronectin before release, in the bone marrow, or in some extrasplenic site.
The Ly-15 alloantigenic system: a genetically determined polymorphism of the murine lymphocyte function-associated antigen-1 molecule.
Authors: Hogarth P M , Walker I D , McKenzie I F , Springer T A
Published: May 2006

Serological and biochemical studies using monoclonal antibodies have demonstrated that the Ly-15 cell membrane alloantigens are polymorphic sites on the lymphocyte function-associated antigen-1 (LFA-1) molecule. Ly-15.2 and LFA-1 show identical tissue distributions, being present on all thymocytes, lymphocytes, and neutrophils, and flow cytofluorometric analysis indicated identical cell surface expression of these molecules. Identity of Ly-15.2 and LFA-1 was confirmed by immunochemical analysis. The Ly-15.2 and LFA-1 molecules have an identical heterodimeric structure of Mr 180,000 alpha chain and Mr 94,000 beta chain, which coelectrophorese on two-dimensional NaDodSO4/PAGE. Furthermore, anti-Ly-15.2 and anti-LFA-1 antibodies coprecipitate the same molecule from thymocyte lysates, and peptide mapping studies show that the Ly-15.2 and LFA-1 alpha chains are identical, as are the beta chains.
Structure of the human phosphoglycerate kinase gene and the intron-mediated evolution and dispersal of the nucleotide-binding domain.
Authors: Michelson A M , Blake C C , Evans S T , Orkin S H
Published: May 2006

The human X-linked phosphoglycerate kinase (PGK) gene, which is expressed in all somatic cells, was cloned and its structure was determined. The gene is interrupted by 10 introns and spans 23 kilobases. When projected on the three-dimensional structure of the PGK protein molecule, splice junctions are located between established peptide domains. In particular, an intron separates the two mononucleotide subdomains of the ATP-binding region, and additional introns divide each of these subdomains between their characteristic beta-strands. Similar correlations are found in the bipartite NAD-binding domains of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, in each case the nucleotide-binding domain is separated from the catalytic domain by at least one intron. The homology of the exon organization in structurally similar regions of these three enzymes suggests that a nucleotide-binding domain evolved by gene duplication and was subsequently dispersed to different proteins through a process of intron-mediated recombination.
The murine bm12 gene conversion provides evidence that T cells recognize predominantly Ia conformation.
Authors: Tse H Y , Kanamori S , Walsh W D , Hansen T H
Published: May 2006

The structure of the highly polymorphic Ia dimer is the genetically determined factor that controls the immune response to foreign antigens, albeit the mechanism remains unresolved. However, it is clear that, in diverse immune responses, effector T lymphocytes require recognition of self-Ia and foreign antigenic determinants on the surface of an antigen-presenting cell or an antibody-secreting B cell. Furthermore, a single Ia molecule has been found to possess several independently acting functional domains. In this report T-cell recognition of Ia was limited to a single, defined structure by using the Ia mutant mouse strain B6.C-H-2bm12 (bm12). The Ia determinant being recognized is the site of the mutation that represents a difference in three of five amino acid residues in a hypervariable region of its beta chain. This mutation has been proposed to have resulted from a gene conversion-like event and is known to have functional importance. Recognition of the bm12 mutation site was studied here in in vitro cultures of T cells generated against Iabm12 antigens. The specificity of these alloreactive T cells was tested by using stimulator cells expressing various Ia alloantigens of known structure. Our findings provide direct genetic evidence that T cells recognize predominantly conformational determinants on Ia molecules and not their primary structure. The implications of these findings on our understanding of the genetic control of the immune response and the potential to modulate these responses in an antigen-specific way are discussed.
Selection of unique antigenic variants of Newcastle disease virus with neutralizing monoclonal antibodies and anti-immunoglobulin.
Authors: Iorio R M , Bratt M A
Published: May 2006

Monoclonal antibodies were used to isolate nonneutralizable antigenic variants in the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus. It had been found that a large percentage of virus retains infectivity despite binding neutralizing antibody. This high persistent fraction of nonneutralized virus precluded the isolation of variants by the standard treatment with antibody alone. Rabbit anti-mouse immunoglobulin was used to reduce the percentage of virus that remains infectious despite the presence of bound antibody. This procedure made possible the isolation of variants of two distinct types: classical variants, not neutralized because they do not bind the antibody used to select them; and unique variants that, although still capable of binding the selecting antibody, are only slightly neutralized. The general applicability of this method for the isolation of antigenic variants in nonneutralizing epitopes is also discussed.
Tyrosine sulfation of proteins from the human hepatoma cell line HepG2.
Authors: Liu M C , Yu S , Sy J , Redman C M , Lipmann F
Published: May 2006

[35S]Sulfate labeling of the human hepatoma cell line HepG2 showed it to contain many sulfated proteins of diverse molecular weight range. The isolation of tyrosine O-sulfate indicated the supernatant fraction to contain a 5- to 7-fold higher level than the cellular fraction at the end of a 24-hr incubation. The proteins in the supernatant fraction were immunoprecipitated and examined for sulfation. Of 15 proteins tested, 7 were found to be sulfated as indicated by [35S]sulfate incorporation into proteins separated by NaDodSO4/PAGE and detected by autoradiography. The 35S-labeled bands were excised from the dried gel and subjected to extensive Pronase hydrolysis and the hydrolysates were analyzed for tyrosine [35S]sulfate by a two-dimensional procedure combining high-voltage electrophoresis and thin-layer chromatography [Liu, M. C. & Lipmann, F. (1984) Proc. Natl. Acad. Sci. USA 81, 3695-3698]. Of the sulfated proteins, three--fibrinogen, alpha-fetoprotein, and fibronectin--were found to contain tyrosine O-sulfate. The simultaneous presence of carbohydrate-bound sulfate, however, could not be exactly determined, but the other four [35S]sulfate-containing proteins--alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-macroglobulin, and transferrin--did not reveal any tyrosine O-sulfate and might be sulfated on their carbohydrate moieties.
Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148.
Authors: Carroll S F , McCloskey J A , Crain P F , Oppenheimer N J , Marschner T M , Collier R J
Published: May 2006

Irradiation of mixtures of diphtheria toxin fragment A and [carbonyl-14C]NAD with UV light (253.7 nm) is known to induce efficient transfer of the radiolabel to position 148, corresponding to glutamic acid in the unmodified protein. Here we report the structure of the photoproduct at position 148, as determined by chemical and photochemical methods, fast-atom-bombardment mass spectrometry, and nuclear magnetic resonance. The photoproduct [an alpha-amino-gamma-(6-nicotin-amidyl)butyric acid residue] contains the entire nicotinamide moiety of NAD linked via its number 6 carbon to the decarboxylated gamma-methylene carbon of Glu-148. No portion of the ADP-ribosyl group of NAD is present. These findings are consistent with the idea that Glu-148 lies at or near the catalytic center of diphtheria toxin.
Identification and expression of a nuclear antigen from the genomic region of the Jijoye strain of Epstein-Barr virus that is missing in its nonimmortalizing deletion mutant, P3HR-1.
Authors: Rowe D , Heston L , Metlay J , Miller G
Published: May 2006

An Epstein-Barr virus (EBV) deletion mutant, HR-1, cannot immortalize lymphocytes. HR-1 was derived from a virus strain, Jijoye, that is immortalization competent. Using human antiserum from certain patients with chronic active EBV infection, we have identified in Jijoye cells a protein of apparent mass of 78-80 kDa that is missing in cells with the HR-1 genome. A protein of identical size and antigenicity has been stably expressed in mouse LTK- cells by gene transfer with cloned Jijoye EBV DNA that encompasses the deletion in the HR-1 genome. The expressed product is a nuclear neoantigen. The polypeptide we have identified is likely to be essential in the immortalization process.
Spermidine is bound to a unique protein in early sea urchin embryos.
Authors: Canellakis Z N , Bondy P K , Infante A A
Published: May 2006

Spermidine is rapidly taken up and becomes bound to protein during the very early hours of sea urchin embryogenesis. During the first 6 hr after fertilization of freshly obtained sea urchin eggs (Strongylocentrotus purpuratus), which are incubated in the presence of exogenous [3H]-spermidine, up to 7% of the total cell-associated spermidine appears uniquely as spermidine bound in macromolecular form. This unique protein containing spermidine migrates as a single radioactive band in gel electrophoresis. It has a Mr of approximately equal to 30,000 and is readily distinguishable from the protein initiation factor eIF-4D, which has a Mr of 18,000, the only other identifiable protein known to date to be posttranslationally modified by polyamines.
Natural effector T lymphocytes in normal mice.
Authors: Pereira P , Larsson E L , Forni L , Bandeira A , Coutinho A
Published: May 2006

The "natural" T-cell activity in normal unimmunized mice was studied. By double-parameter fluorescence-activated cell sorter analysis, it was found that 5-10% of all splenic Lyt-2+ and L3T4+ lymphocytes are large, of which more than half are in mitotic cycle. In contrast with small resting cells of the same phenotype, activated (large) T cells isolated from normal mice are functional effector cells: L3T4+ large cells induce normal B lymphocytes into proliferation and antibody secretion, while large Lyt-2+ cells efficiently suppress B-lymphocyte responses. No effector cell cytolytic activity could be detected among naturally activated T cells. The significance of these findings for the internal activity in the normal immune system is discussed.
Identification of a partial cDNA clone for the human receptor for complement fragments C3b/C4b.
Authors: Wong W W , Klickstein L B , Smith J A , Weis J H , Fearon D T
Published: May 2006

Redundant oligonucleotides were synthesized based on amino acid sequences of tryptic peptides from the purified receptor for human complement fragments C3b/C4b (CR1). These probes were used to screen a size-selected human tonsilar cDNA library. A single positive clone was identified that hybridized to three oligonucleotide probes. The cDNA insert was 1.5 kilobases in length and contained sequences homologous to those of the oligonucleotide probes as well as nucleotide sequences corresponding to another independent CR1 tryptic peptide. Blot-hybridization analysis using fragments of the cDNA insert as probes revealed two distinct species of the CR1 message of 9 and 11 kilobases in human tonsil mRNA. The two EcoRI fragments of the CR1 cDNA insert hybridized to each other, suggesting the presence of homologous sequences. When used as probes in Southern blot analysis of human DNA, each fragment identified similar but not identical patterns of multiple restriction fragments, indicating either a series of homologous domains in a single CR1 gene or the presence of multiple CR1 genes. Furthermore, an additional BamHI fragment was found to segregate with the expression of the S allotype of the CR1 protein in a family. Thus, the molecular weight difference in the polymorphic variants of the CR1 protein is based on differences in nucleotide sequences.
Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide.
Authors: Stuehr D J , Marletta M A
Published: May 2006

Escherichia coli lipopolysaccharide (LPS)-induced nitrate biosynthesis was studied in LPS-sensitive C3H/He and LPS-resistant C3H/HeJ mice. Intraperitoneal injection of 15 micrograms of LPS led to a temporary 5- to 6-fold increase in blood nitrate concentration in the C3H/He strain. Levels of nitrate excreted in the urine were also increased. In contrast, no increase was observed in the C3H/HeJ strain with LPS injections up to 175 micrograms. Furthermore, thioglycolate-elicited peritoneal macrophages from C3H/He, but not from C3H/HeJ mice, produced nitrite (60%) and nitrate (40%) when cultured with LPS (10 micrograms/ml). T-lymphocyte addition/depletion experiments showed the presence of T cells enhanced this response. However, LPS did not cause nitrite or nitrate production in cultures of spleen lymphocytes from either strain. LPS-induced nitrate synthesis was also observed with nude mice and CBA/N mice, indicating that neither functional T lymphocytes nor LPS-responsive B lymphocytes were required for the response in vivo. This was consistent with the in vitro results showing macrophages alone were competent. Mycobacterium bovis infection of C3H/He and C3H/HeJ mice resulted in a large increase in nitrate production over the course of the infection for both strains, suggesting T-lymphocyte-mediated activation of macrophages as a potent stimulus for nitrate biosynthesis. The synthesis of nitrite is significant in that it can directly participate in the endogenous formation of nitrosamines and may also be involved in some aspect of the chemistry of cytotoxicity.
Heat-resistant variants of Chinese hamster fibroblasts altered in expression of heat shock protein.
Authors: Laszlo A , Li G C
Published: May 2006

Heat-resistant variants of the Chinese hamster HA-1 line have been isolated after repeated heat treatments. The heat-resistant phenotype has been stable for over 70 passages. One of the members of the 70-kDa heat shock protein family was found to be synthesized at greater levels in the heat-resistant variants under normal growth conditions. Mild heat treatment of the variant lines induced a transient thermotolerance that was accompanied by additional increase in the synthesis of the 70-kDa heat shock proteins. Cell-free translation of total cellular RNA revealed greater amounts of 70-kDa heat shock protein mRNA in both control and heated variant cells. The greater levels of 70-kDa heat shock protein synthesized in the variant cells presumably are a reflection of altered levels of its messenger mRNA. In addition, we found that translational control plays a role in the elevated expression of heat shock proteins in heat-shocked HA-1 cells and their heat-resistant variants. The association of the heat-resistant phenotype with increased levels of a 70-kDa heat shock protein suggests strongly that this gene product plays a role in protecting cells from damage inflicted by elevated temperatures.
Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase.
Authors: Schook W J , Puszkin S
Published: May 2006

A protein kinase activity was observed in coated vesicles, prepared from bovine brain, that had clathrin-associated protein 2 (CAP2, also known as clathrin light chain 2) as its principal substrate. Coated vesicles were purified by sucrose density gradient centrifugation followed by Sephacryl S-1000 column chromatography, and all buffers utilized in these procedures contained a mixture of proteolysis inhibitors to maintain CAP2 kinase activity. Incubation of vesicles with [gamma-32P]ATP in the presence of 7 microM polylysine resulted in an overall increase in the incorporation of phosphate. NaDodSO4/PAGE revealed that the principal recipient of this additional phosphate was CAP2 (Mr 33,000), the faster-migrating component of the clathrin coat-associated proteins, whereas CAP1 (Mr 36,000) was not phosphorylated. A number of other proteins, in the Mr 140,000 and 100,000 regions, were phosphorylated to a lesser extent. Polyarginine and polyethylenimine also supported CAP2 phosphorylation, but arginine and lysine were ineffective. The phosphorylated protein was identified as CAP2 because addition of exogenous CAPs resulted in increased incorporation of label into Mr 33,000 polypeptides and because heat treatment of labeled vesicles followed by ultracentrifugation resulted in recovery of labeled Mr 33,000 protein in the supernatant. Phosphorylation of CAP2 may play a regulatory role in clathrin coat/coated vesicle functions.
Catabolite-resistant sporulation (crsA) mutations in the Bacillus subtilis RNA polymerase sigma 43 gene (rpoD) can suppress and be suppressed by mutations in spo0 genes.
Authors: Kawamura F , Wang L F , Doi R H
Published: May 2006

The catabolite-resistant sporulation (crsA) mutation is able to overcome the repressive effect of glucose on sporulation in Bacillus subtilis. Three chromosomal crsA mutations, crsA1, crsA4, and crsA47, were transferred by the "gene conversion" process to B. subtilis plasmid pRPD11, which consists of the entire wild-type rpoD coding sequence, encoding the major sigma 43 factor of B. subtilis RNA polymerase, and pUB110. By DNA sequence analysis we showed that all three crsA mutations were identical two-base changes, CCT (proline) to TTT (phenylalanine), within the rpoD coding sequence. Furthermore, the crsA47 mutation restored spo0J and spo0K sporulation to wild-type levels and partially improved the sporulation efficiencies of spo0B, spo0D, and spo0F. Extragenic suppressors (scr) of crsA47 included mutations in spo0A, spo0D, spo0F, and spo0K plus other mutations that have not been specifically identified. Thus sigma 43 appears to be involved in catabolite repression by glucose, to interact either directly or indirectly with spo0 gene products, and to play an important role in the initiation of spore development in B. subtilis.
Class II major histocompatibility complex molecules regulate the development of the T4+T8- inducer phenotype of cultured human thymocytes.
Authors: Blue M L , Daley J F , Levine H , Schlossman S F
Published: May 2006

We demonstrate that a variety of Ia+ cells has the ability to promote the development of human T4+T8- thymocytes in vitro. Prolonged thymocyte culture in the absence of Ia+ accessory cells results in a predominantly T8+T4- cell population. The generation of T4+ cells in the presence of irradiated Ia+ cells could be suppressed up to 70% by a monoclonal antibody directed against a nonpolymorphic epitope on HLA-DR. Using two-color fluorescence sorting techniques, we were able to identify the activated T4+T8+ thymocyte as the cell that interacts with Ia and gives rise to the T4+T8- cell subset. These results directly and specifically implicate class II major histocompatibility complex molecules in the differentiative pathway of the human thymocyte.
Hyperglycemia-induced teratogenesis is mediated by a functional deficiency of arachidonic acid.
Authors: Goldman A S , Baker L , Piddington R , Marx B , Herold R , Egler J
Published: May 2006

Congenital malformations now represent the largest single cause of mortality in the infant of the diabetic mother. The mechanism by which diabetes exerts its teratogenic effects is not known. This study evaluated whether arachidonic acid might be involved, a possibility raised by the role of arachidonic acid in palatal elevation and fusion, processes analogous to neural tube folding and fusion. This hypothesis was tested in two animal models of diabetic embryopathy, the in vivo pregnant diabetic rat and the in vitro hyperglycemic mouse embryo culture. The subcutaneous injection of arachidonic acid (200-400 mg/kg per day) into pregnant diabetic rats during the period of organ differentiation (days 6-12) did not alter the maternal glucose concentration, the maternal weight gain, or the weight of the embryos. However, the incidence of neural tube fusion defects was reduced from 11% to 3.8% (P less than 0.005), the frequency of cleft palate was reduced from 11% to 4% (P less than 0.005), and the incidence of micrognathia was reduced from 7% to 0.8% (P less than 0.001). The addition of arachidonic acid to B10.A mouse embryos in culture also resulted in a reversal of hyperglycemia-induced teratogenesis. The teratogenic effect of D-glucose (8 mg/ml) in the medium resulted in normal neural tube fusion in only 32% of the embryos (P less than 0.006 when compared to controls). Arachidonic acid supplementation (1 or 10 micrograms/ml) produced a rate of neural tube fusion (67%) that was not significantly different from that observed in controls. The evidence presented indicates that arachidonic acid supplementation exerts a significant protective effect against the teratogenic action of hyperglycemia in both in vivo (rat) and in vitro (mouse) animal models. These data therefore suggest that the mechanism mediating the teratogenic effect of an increased glucose concentration involves a functional deficiency of arachidonic acid at a critical stage of organogenesis.
Analysis of the transcript encoding the latent Epstein-Barr virus nuclear antigen I: a potentially polycistronic message generated by long-range splicing of several exons.
Authors: Speck S H , Strominger J L
Published: May 2006

The Epstein-Barr virus nuclear antigen (EBNA I) present in latently infected cells is encoded in a 2-kilobase exon contained in the BamHI K viral genomic fragment. This exon is, however, found within a 3.7-kilobase mRNA transcript. The origin of the remaining 1.7 kilobases is unknown, although it is not derived from adjacent Epstein-Barr virus DNA sequences. A 1.1-kilobase cDNA clone generated by primer extension using an oligonucleotide complimentary to a sequence 245 base pairs 3' to the putative initiation codon for EBNA I in the BamHI K fragment has been isolated. This clone contains seven exons (from the BamHI W, Y, U, E, and K viral genomic fragments), which are spread over approximately 70 kilobases of the viral genome. However, this clone does not appear to contain the complete 5' end of the transcript. In addition to the open reading frame in the exon encoding EBNA I, three other open reading frames are found in this transcript that potentially encode other viral antigens present in latently infected cells.