bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac.
Authors: Wong P M , Chung S W , Chui D H , Eaves C J
Published: May 2006

We have found that a variety of clonogenic hemopoietic cells can be obtained in a viable state from mouse conceptuses as early as day 7 of gestation when tissues are disaggregated in a crude collagenase solution containing fetal bovine serum. Examination of the time course of colony formation, and the ultimate size and lineages represented in colonies produced in semisolid medium containing methylcellulose, together with analysis of individual erythroid colonies stained with rabbit antisera specific for adult (HbA) and embryonic (HbE) mouse hemoglobins, revealed the presence on days 7 and 8 of gestation (but not later) of erythropoietic progenitors that give rise to mature erythroid colonies containing up to 100 HbE-containing erythroblasts after 4-6 days of growth in culture. These progenitors are highly sensitive to the disaggregation conditions used. Clonogenic progenitors of exclusively HbA-positive erythroblasts can also be detected in the day-7 conceptus. Assays of progenitors from separately disaggregated yolk sacs and embryos from day-8 conceptuses yielded colonies only from yolk sac suspensions, and again these contained either HbE- and HbA-positive erythroblasts or only HbA-positive erythroblasts. These findings demonstrate the very early appearance in the yolk sac of a population of erythroid progenitors with a number of unique properties. Although most of these yield HbE-positive erythroblasts in vitro, some produce erythroblasts containing HbA only. Such a developmental pattern is consistent with the hypothesis that definitive erythropoiesis in the mammalian fetal liver is derived from stem cells that originate in the yolk sac blood islands.
Nonrandom segregation of centromeres following mitotic recombination in Drosophila melanogaster.
Authors: Pimpinelli S , Ripoll P
Published: May 2006

Mitotic recombination is widely used in Drosophila as a technique to study genetic and developmental problems. It has been generally assumed that, following mitotic exchange between homologous chromatids during the G2 stage, the centromeres attached to the chromatids involved in the exchange segregate randomly. As a result, two equally frequent types of segregation, yielding genetically different products, are produced. However, when epidermal or enzymatic cell-marker mutants are used, only one type of segregation gives rise to marked cells. In the present work we test this assumption of random segregation using cytological markers. With cytological markers, larval neuroblast cells resulting from mitotic recombination exhibit recognizably all possible products from mitotic recombination. We find that one type of segregation is favored, in that, after mitotic recombination, the centromeres attached to the chromatids involved in the mitotic exchange preferentially migrate to opposite poles during anaphase. This favored segregation could be the result of exchange between previously oriented chromatids or could be due to the effect of the exchange upon subsequent orientation of homologous chromosomes. In either case, frequencies of mitotic recombination have been overestimated in the past.
Chloramphenicol induces translation of the mRNA for a chloramphenicol-resistance gene in Bacillus subtilis.
Authors: Duvall E J , Lovett P S
Published: May 2006

cat-86 is a plasmid gene specifying chloramphenicol-inducible chloramphenicol acetyltransferase activity in Bacillus subtilis. Inducibility has been suggested to result primarily from activation of the translation of cat-86 mRNA by subinhibitory levels of chloramphenicol. To directly test the involvement of transcription in cat-86 induction, the gene was transcriptionally activated with a strong promoter, resulting in the synthesis of relatively high levels of cat-86 mRNA in uninduced cells. When RNA synthesis was blocked with rifampin (100 micrograms/ml), de novo inducibility of cat-86 by chloramphenicol could be demonstrated for more than 30 min. These results indicate that concurrent transcription is not essential for cat-86 induction. Accordingly, cat-86 is one of only a few inducible bacterial genes in which the primary form of regulation is at the translational level. This form of regulation may apply to other cat genes of Gram-positive origin whose expression is also inducible by chloramphenicol.
Cotransfer of the Ed alpha and Ad beta genes into L cells results in the surface expression of a functional mixed-isotype Ia molecule.
Authors: Malissen B , Shastri N , Pierres M , Hood L
Published: May 2006

Ia molecules play a key role in antigen recognition by T lymphocytes. To analyze the structural features of the individual alpha and beta chains relevant to the assembly of intact Ia molecules, mouse fibroblasts were cotransfected with various combinations of haplotype- and isotype-mismatched Ia alpha/beta gene pairs. Two important points emerged. First, the level of surface expression of a given haplotype-mismatched A alpha A beta pair appears to depend upon the alpha and beta chain alleles involved. Second, transfection with some isotype-mismatched combinations such as Ed alpha Ad beta results in a significant level of surface expression of a stable mixed-isotype dimer, which also appears to be normally expressed at a low level by an Iad-positive B lymphoma. Moreover, a T-cell hybridoma specific for human gamma globulin and restricted by the Ed molecule was found to be efficiently stimulated by the Ed alpha Ad beta-positive transfectant in the presence of antigen. The stimulation was specifically inhibited by monoclonal antibodies directed to either the Ia or the L3T4 molecule. These findings suggest that the estimates of the potential number of Ia molecules available in an animal for restricting T-lymphocyte recognition of antigens must be revised.
Induction of nerve growth factor receptor in Schwann cells after axotomy.
Authors: Taniuchi M , Clark H B , Johnson E M
Published: May 2006

We have discovered that axotomy of sciatic nerve induces Schwann cells distal to the lesion to express de novo, or at greatly increased levels, receptors for nerve growth factor (NGF). Surgical transection of sciatic nerve was performed on adult Sprague-Dawley rats, and, at various times after the operation, the following tissues were dissected for quantitation of NGF receptor: L4 and L5 dorsal root ganglia, sciatic nerve proximal to the transection, sciatic nerve distal to the transection, tibialis anterior muscle, and skin of the dorsum of the foot. The NGF receptor content of these samples was determined by labeling receptor molecules with radioiodinated NGF (125I-NGF) and then specifically immunoprecipitating the 125I-NGF-receptor complexes with 192-IgG, a monoclonal antibody directed against the rat NGF receptor. We observed a time-dependent increase in the amount of radioligand-labeled NGF receptor proteins found in the distal segment of transected sciatic nerve; by 7 days the density of receptor (crosslinked 125I-NGF molecules per mg of homogenate protein) had increased at least 50-fold. The number of receptor molecules in tibialis anterior muscle and dorsal foot skin, two structures denervated by the transection, also increased, with time courses parallel to that of distal sciatic nerve. There was little increase in the density of NGF receptors in the sciatic nerve proximal to the lesion and in the dorsal root ganglia. Immunohistochemical examination of the distal portion of transected sciatic nerve and of the muscle, with 192-IgG as the primary ligand, revealed prominent and exclusive staining of apparently all Schwann cells of the endoneurium, indicating that these peripheral neuroglial cells were expressing NGF receptors. These results show that axonal damage can induce the expression of NGF receptors in the population of sheath cells thought to promote neuronal regeneration. This dramatic increase in NGF receptors may be a mechanism to facilitate the regeneration of NGF-responsive neurons.
Osteoclasts can be induced in fish having an acellular bony skeleton
Authors: Glowacki J. , Cox K. A. , O'Sullivan J. , Wilkie D. , Deftos L. J.
Published: May 2006

Kelp bass (
Paralabrax clathratus
) and leopard sharks (
Triakis semifasciata
) are characterized by an acellular (anosteocytic) bony skeleton and a focally calcified cartilaginous endoskeleton, respectively. These skeletal forms are not considered to function as mineral reservoirs. Previous studies showed that implanted bone particles are resorbed in rats by large multinucleated cells with ultrastructural features (ruffled borders) characteristic of osteoclasts. We tested the ability of fish to resorb bone matrix and to adapt to reduced salinity conditions. Bone particles were implanted in sharks and bass maintained in seawater (34 ppt, 40.5 mg of calcium per dl) or in diluted seawater (26 ppt, 28.5 mg of calcium per dl). Sera and elicited tissues were harvested 4 weeks later. In sharks, bone particles were not resorbed, and multinucleated cells were not evident under either normal or hyposalinity conditions. Shark sera were isoosmolar with the seawater or diluted seawater, with serum chemistries of the hyposalinity group reflecting the 23% reduction in environmental minerals and electrolytes, compared to sharks in normal seawater. In marked contrast, bass adapted to diluted seawater resorbed bone particles and maintained normal serum chemistries. Electron microcopy showed that the bone particles were surrounded by large, foamy multinucleated cells, many with membrane specializations typical of osteoclasts from higher vertebrates, i.e., extensive clear zones apposed to intact bone matrix and active ruffled borders overlying areas of matrix undergoing dissolution. Although osteoclasts had not been described in these fish, this study shows that bass have stem cells that can be stimulated to differentiate into bone-resorbing osteoclasts.
Inositol bis-, tris-, and tetrakis(phosphate)s: analysis in tissues by HPLC.
Authors: Meek J L
Published: May 2006

The concentration of isomers of inositol tris(phosphate) (InsP3) was measured in tissues of intact animals. The method employed involved anion-exchange HPLC with on-line enzymatic hydrolysis of the phosphate esters and detection of the inorganic phosphate formed. All seven organs tested from rats killed by decapitation contained Ins(1,4,5)P3 in concentrations of 13-40 nmol/g; a distribution that bears no resemblance to that reported for its precursor [phosphatidylinositol bis(phosphate)]. A second InsP3 isomer [probably Ins(1,3,4)P3] was also detectable in brain and salivary gland. The content of Ins(1,4,5)P3 in brain and salivary gland from rats killed by decapitation was 10-60 times greater than that from rats killed by focused microwave irradiation to block postmortem metabolism. Inositol bis(phosphate) concentrations also changed dramatically postmortem. A much smaller postmortem change was seen in the content of Ins(1,3,4)P3. Receptor stimulation by muscarinic cholinergic agonists increased the content not only of Ins(1,3,4)P3, but also its recently discovered probable precursor, inositol tetrakis-(phosphate).
Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription.
Authors: Makino S , Stohlman S A , Lai M M
Published: May 2006

Mouse hepatitis virus (MHV), which replicates in cytoplasm of infected cells, contains an identical leader RNA sequence at the 5' end of each of the virus-specific mRNAs. Previous studies suggested that the synthesis of these mRNAs does not involve conventional RNA splicing and may instead require priming by a free leader RNA. In this communication, we demonstrate that, during a mixed infection with two different MHVs, the leader RNA sequences from one virus could be detected on the mRNAs of the coinfecting virus at a high frequency, as if the leader sequence and mRNAs were joined together from two randomly segregating RNA segments. This finding demonstrates that MHV mRNA transcription utilizes independently transcribed leader RNA species that possess the trans-acting property. This study thus provides further evidence in support of the unique model of "leader-primed transcription" for coronavirus mRNA synthesis.
Activation of the transforming potential of p60c-src by a single amino acid change.
Authors: Levy J B , Iba H , Hanafusa H
Published: May 2006

Previous work showed that overexpression of the cellular src (c-src) gene does not cause transformation of chicken cells in culture. However, viral stocks isolated from cells transfected with Rous sarcoma virus DNA containing the c-src gene in place of the viral src gene did occasionally produce foci. Virus obtained from these foci were highly transforming and appeared to arise via spontaneous mutation in the c-src-containing viral populations. The p60 proteins of the transforming mutant src viruses were found to have higher levels of in vitro tyrosine kinase activity than the levels observed with the parental viruses. In this study, we have molecularly cloned the src DNA sequences of two transforming mutant src viruses. When compared to the DNA sequence of the parental c-src viruses, the mutant viruses contain single point mutations that result in single amino acid changes in the src gene products (p60 proteins). Both amino acid changes reside in the tyrosine kinase domain of the protein. The mutation detected in one virus involves replacement of the normal Glu-378 in p60c-src by Gly, whereas the p60 of the other transforming virus has Phe instead of the normal Ile-441. Our data indicate that when p60c-src is expressed at elevated levels in a retroviral context, a single amino acid change in its primary sequence can activate the kinase activity of this protein and cause cellular transformation.
The arachidonic acid cascade is involved in the masculinizing action of testosterone on embryonic external genitalia in mice.
Authors: Gupta C , Goldman A S
Published: May 2006

We have evaluated whether the arachidonic acid cascade may be involved in the folding and fusion of the penis and scrotum in masculine differentiation, a possibility raised by recent observations of the involvement of the arachidonic acid cascade in the analogous embryonic processes of elevation and fusion of the palatal shelves and of folding and fusion of the neural tube. To test this hypothesis, during embryonic masculine differentiation in mice of the B10.A strain, we administered certain agents that produce blockade of masculinization. We report that arachidonic acid can reverse the inhibition of masculine development in male embryos produced by estradiol-17 beta or by cyproterone acetate, an androgen receptor-site blocker, and that such reversal can be prevented by an inhibitor of cyclooxygenase, such as indomethacin. We have also found that agents that block the arachidonic acid cascade at the level of phospholipase A2 (cortisone, phenytoin) or at the level of cyclooxygenase (indomethacin, aspirin) also block masculine differentiation and that such antimasculinization is reversed by arachidonic acid. The masculinization of male embryos is inhibited by indomethacin and aspirin, and the masculinization of female embryos produced by exogenous testosterone is prevented by indomethacin. These findings provide evidence that the mechanism by which testosterone organizes the genitalia involves a role of the arachidonic acid cascade leading to prostaglandins at a critical period of development and that interference with testosterone synthesis or action leads to a teratogenic deficiency of arachidonic acid during this time in the genital anlagen.
Participation of the murine X and Y chromosomes in genetically determined chemosensory identity.
Authors: Yamazaki K , Beauchamp G K , Matsuzaki O , Bard J , Thomas L , Boyse E A
Published: May 2006

The major histocompatibility complex of the mouse imparts to each mouse an odor that reflects its genetic constitution at this region of chromosome 17. Sensory recognition of these differential odors influences reproductive behavior and evokes neuroendocrine responses critical to the maintenance of pregnancy. To determine whether other parts of the mouse genome contribute to individual scent marking, and so may similarly exert a selective force on loci other than the major histocompatibility complex, mice differing genetically only in their X and/or Y chromosomes were tested for individuality of scent in the Y-maze system previously employed to investigate major histocompatibility complex-related scent distinctions. It is shown that the X and Y chromosomes each confer individually of scent related to genotype.
High-resolution analysis of the human HLA-DR polymorphism by hybridization with sequence-specific oligonucleotide probes.
Authors: Angelini G , de Preval C , Gorski J , Mach B
Published: May 2006

The human major histocompatibility complex class II antigens of the HLA-D are highly polymorphic, surface proteins essential in the cellular interactions necessary for an immune response. The analysis of this polymorphism is crucial for (i) histocompatibility matching for transplantation and (ii) understanding the association between HLA-D and certain important diseases. The polymorphism of certain HLA-D haplotypes may escape detection by current methodologies. Analysis at the genomic level of the polymorphism of one of the HLA-D subregions HLA-DR, using oligonucleotide probes specific for the polymorphic regions, is capable of distinguishing single nucleotide differences. The DRw6 haplotype was analyzed in view of the lack of DRw6 specific sera. On the basis of nucleotide sequence analysis, the DRw6 haplotype consists of at least two subtypes. When analyzed with oligonucleotide probes, this split identifies new polymorphic groups that differ from the DRw6 serological subgroups.
Partial primary structure of the T4 antigens of mouse and sheep: assignment of intrachain disulfide bonds.
Authors: Classon B J , Tsagaratos J , McKenzie I F , Walker I D
Published: May 2006

The T4 antigens of mouse and sheep have been purified to homogeneity and partially sequenced using protein and peptide microsequencing techniques. Both mouse and sheep antigens bear distinct homology to human T4, having an amino-terminal segment that is homologous to the K-chain variable region (VK). A surprisingly high degree of sequence divergence was nevertheless evident between the T4 antigens of the three mammalian species, suggesting an unusually rapid rate of evolution that is possibly related to the functional role of T4 in class II major histocompatibility complex antigen recognition. The mouse and sheep T4 antigens contain at least three intrachain disulfide bonds which in all cases connect adjacent cysteine residues. All three intrachain disulfide linkages are situated within the putative extracellular domain, and the amino-terminal disulfide bond probably involves the two cysteine residues homologous to those which form the intrachain bridge within the VK domain of immunoglobulin molecules. The structural relationship of the T4 antigen to other members of the immunoglobulin supergene family is discussed.
Genetic evidence that Acanthamoeba myosin I is a true myosin.
Authors: Hammer J A , Jung G , Korn E D
Published: May 2006

Acanthamoeba castellanii contains two enzymes, myosins IA and IB, that exhibit the catalytic properties of a myosin but possess very unusual physical properties, the most striking of which are their single, low molecular weight heavy chain, their globular shape, and their inability to form bipolar filaments. We have now isolated a putative myosin IB heavy chain gene from Acanthamoeba, using as a heterologous probe a portion of a sarcomeric myosin heavy chain gene from Caenorhabditis elegans. The amoeba genomic clone hybridizes to a 4250-nucleotide RNA species and hybrid-selects an mRNA encoding a 125-kDa polypeptide. This polypeptide comigrates exactly with the heavy chain of purified amoeba myosin IB and is specifically immunoprecipitated with antiserum to myosin IB. We sequenced two restriction enzyme fragments of this gene, and the deduced amino acid sequences show strong homology with the regions of muscle myosins that contain the reactive thiols and the ATP binding site. Our identification of a myosin IB heavy chain gene demonstrates that myosin IB, despite the unusually low molecular weight of its heavy chain, is a true gene product. The sequence results show that, despite its atypical physical properties, myosin IB is clearly related to conventional myosins.
Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21.
Authors: Sigal I S , Gibbs J B , D'Alonzo J S , Scolnick E M
Published: May 2006

To identify the amino acid residues of the Harvey (Ha) ras-encoded protein that are involved in protein-protein interactions, we have created a series of mutant Ha-ras proteins. In particular, amino acid substitutions have been introduced within two regions, residues 32-42 and 61-80, that are conserved among ras proteins from different species. We observed that amino acid substitutions at positions 35, 36, 38, 40, and, to a lesser extent, 39 and 78 reduce the biological potency of Ha-ras protein in both mammalian and Saccharomyces cerevisiae cells, without noticeably affecting the known intrinsic biochemistry of these proteins. The reduction of in vivo activity for these mutant ras proteins correlates with their reduced ability to stimulate yeast adenylate cyclase. The ras-protein-neutralizing antibody Y13-259 binds to six residues: Glu-63, Ser-65, Ala-66, Met-67, Gln-70, and Arg-73. Single substitutions for these residues reduce Y13-259 antibody binding by at least a factor of 1000 but do not significantly affect biological activity. These data are discussed in terms of the model for Ha-ras protein based on the structure of the elongation factor EF-Tu-GDP complex.
Atrial natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process.
Authors: Kurtz A , Della Bruna R , Pfeilschifter J , Taugner R , Bauer C
Published: May 2006

We have examined the effect of a synthetic analogue of human alpha-atrial natriuretic peptide (ANP), APII, on renin release in cultured renal juxtaglomerular cells (JGA cells). Using cell cultures containing 80-90% renal juxtaglomerular cells, we found that ANP (10(-13)-10(-9) M) strongly inhibited renin release from the cells in a dose-dependent fashion (ki, 10 pM) to about 10% of control. Inhibition of renin release by ANP was paralleled by an increase in cellular cGMP levels; while in the presence of the cGMP-phosphodiesterase inhibitor M&B 22948 (1 mM), concentrations of ANP lower by a factor of 100 were required to obtain the same effects on renin release and cGMP levels. The guanylate cyclase inhibitor methylene blue (10 microM), on the other hand, shifted the dose-response curves for renin release and cGMP levels to 100-fold higher concentrations of ANP. Neither the influx of 45Ca into the cells nor the intracellular quin-2 signal, which is a measure for changes of intracellular Ca concentration, was in any way altered by ANP. Our results suggest that ANP inhibits renin release from juxtaglomerular cells by a cGMP-dependent process that does not involve changes in intracellular calcium.
Structures attached to doublet microtubules of cilia: computer modeling of thin-section and negative-stain stereo images.
Authors: Avolio J , Glazzard A N , Holwill M E , Satir P
Published: May 2006

With a single set of positional coordinates for longitudinal and transverse attachment of the inner and outer rows of dynein arms with respect to the doublet microtubules of Tetrahymena ciliary axonemes, a computer model has been constructed at 4-nm resolution that reconciles negative-stain en face stereo images of arm and spoke positions to traditional images of tannic acid/glutaraldehyde-fixed sections. In this model, inner and outer arms correspond in substructure; both repeat with a 24-nm periodicity without stagger between rows, and a pair of arms is in exact alignment with the first spoke (S1) in each doublet spoke group. The model and the supporting micrographs suggest that each arm cycles in three dimensions and that, during cycling, the inner and outer arms move in opposite directions with respect to the center of subfiber A of the doublet (N). Attachment is off-center with respect to subfiber B of the adjacent doublet (N + 1), causing the sliding doublets to skew with respect to one another.
Recombinant interferon alpha can induce rearrangement of T-cell antigen receptor alpha-chain genes and maturation to cytotoxicity in T-lymphocyte clones in vitro.
Authors: Chen L K , Mathieu-Mahul D , Bach F H , Dausset J , Bensussan A , Sasportes M
Published: May 2006

T-cell receptor genes are found in the germ-line configuration as well as in rearranged forms, as detected in T lymphocytes by different patterns in Southern blot analysis. We have recently shown that cytotoxic T-lymphocyte (CTL) "precursor" clones, in which rearrangements are not detected in the gene encoding the T-cell receptor alpha-chain (TCRA), acquire specific lytic function induced by treatment with recombinant interferon alpha or gamma. We now report that, coincident with the acquisition of cytotoxic function by the precursor CTL clone; recombinant interferon alpha appears to induce a rearrangement of TCRA. In addition, in a mature CTL clone (i.e., one already showing lytic function) in which one TCRA allele is rearranged, treatment with recombinant interferon alpha appears to induce a new rearrangement of a TCRA gene.
Intercontinental spread of a genetically distinctive complex of clones of Neisseria meningitidis causing epidemic disease.
Authors: Caugant D A , Frøholm L O , Bøvre K , Holten E , Frasch C E , Mocca L F , Zollinger W D , Selander R K
Published: May 2006

Strains of Neisseria meningitidis responsible for an epidemic of meningococcal disease occurring in Norway since the mid-1970s and for recent increases in the incidence of disease in several other parts of Europe have been identified by multilocus enzyme electrophoresis as members of a distinctive group of 22 closely related clones (the ET-5 complex). Clones of this complex have also colonized South Africa, Chile, Cuba, and Florida, where they have been identified as the causative agents of recent outbreaks of meningococcal disease. There is strong circumstantial evidence that outbreaks of disease occurring in Miami in 1981 and 1982 were caused in large part by bacteria that reached Florida via human immigrants from Cuba.
Factors influencing redox potentials of electron transfer proteins.
Authors: Moore G R , Pettigrew G W , Rogers N K
Published: May 2006

The redox potentials of electron transfer proteins vary over a wide range, even when the type of redox center is the same. Rees [Proc. Natl. Acad. Sci. USA (1985) 82, 3082-3085] proposed that this variation of redox potential partly reflects the different net charges of the proteins, and he presented a linear correlation between these two properties for 36 proteins. A review of the factors that influence protein redox potentials makes it clear that this linear correlation is fortuitous. The key factors influencing redox potentials are the contributions to the Gibbs energy difference between the two redox states, resulting from bonding interactions at the redox center, electrostatic interactions between the redox-center charge and polar groups within the protein and solvent, and redox-state conformational changes. The relative importance of these terms is likely to vary from protein to protein.
Tissue distribution, immunoreactivity, and physical properties of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
Authors: el-Maghrabi M R , Correia J J , Heil P J , Pate T M , Cobb C E , Pilkis S J
Published: May 2006

6-Phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46) activities were determined in various rat tissues, the latter by using a method based on the formation of a phosphorylated enzyme intermediate during the course of catalysis. Both activities from liver, skeletal muscle, lung, kidney, and testis copurified during polyethylene glycol fractionation, anion-exchange and blue Sepharose chromatography, and gel filtration. The Stokes radius of these enzymes and of the liver bifunctional enzyme was 45 A. Extrahepatic tissues had only 10% or less of the kinase activity found in liver. The results indicate that a liver-type bifunctional enzyme is present in most extrahepatic tissues but that it is minimally expressed. However, the ratio of kinase to bisphosphatase activity in most extrahepatic tissues was 4- to 6-fold higher than in liver, whereas heart 6-phosphofructo-2-kinase had no associated bisphosphatase activity, although its Stokes radius was also 45 A. The heart enzyme was not precipitated by an antiserum to the liver enzyme, whereas only a fraction of the kidney and testis activities was precipitated by this antiserum. The data support the existence of a distinct form of extrahepatic 6-phosphofructo-2-kinase, most readily demonstrated in heart, which may not be bifunctional.
Intracellular site of insulin action: mitochondrial Krebs cycle.
Authors: Bessman S P , Mohan C , Zaidise I
Published: May 2006

Effect of insulin on the oxidation of carbons-1,4 and -2,3 of succinate and their incorporation into protein were studied in isolated rat hepatocytes and diaphragm muscle pieces. Oxidation of carbons-2,3 of succinate and their incorporation into hepatocyte protein were stimulated significantly by insulin. Insulin had only a trivial effect on 14CO2 formation from the carboxyl carbons of succinate. These data suggest that insulin affects only those carbons of succinate that are metabolized in the intramitochondrial Krebs cycle.
Isolation of animal cell mutants deficient in plasmalogen biosynthesis and peroxisome assembly.
Authors: Zoeller R A , Raetz C R
Published: May 2006

A rapid autoradiographic screening procedure has been developed for identifying Chinese hamster ovary cell mutants defective in the peroxisomal enzyme dihydroxyacetonephosphate (DHAP) acyltransferase. Ten mutants were found among 60,000 colonies grown from a stock of mutagen-treated cells, and 3 have been characterized with respect to their enzymology and phospholipid biosynthesis. All three contain 3% (or less) of the parental DHAP acyltransferase activity measured at pH 5.5, the optimum for the peroxisomal enzyme. When measured at pH 7.4, all three contained 70-85% of the wild-type activity, but it was sensitive to N-ethylmaleimide. Glycerol-3-phosphate acyltransferase activities were identical in mutant and parent strains. Two other peroxisomal enzymes, alkyl-DHAP synthase and particulate catalase, were also reduced by factors of 5-10 in all three mutants, suggesting that these strains are deficient in some aspect of peroxisome assembly, possibly like cells from patients with Zellweger syndrome. Short-term and long-term labeling with 32Pi revealed that these mutants are grossly deficient in the de novo synthesis and content of plasmalogens. In parental cells the plasmalogen form of phosphatidylethanolamine constitutes 7.1% of the total phospholipid, but it is reduced to 0.7% in the mutants. This decrease is accompanied by a compensatory increase in the diacyl form of phosphatidylethanolamine. The results presented here support the view that there are two DHAP acyltransferases in animal cells and that the peroxisome is essential for the biosynthesis of plasmalogens.
Autonomously replicating RNA in mitochondria of maize plants with S-type cytoplasm
Authors: Finnegan Patrick M. , Brown Gregory G.
Published: May 2006

Mitochondria isolated from maize plants with S-type male-sterile cytoplasms are capable of synthesizing four species of RNA at concentrations of actinomycin D that eliminate all DNA-directed RNA synthesis. No RNA synthesis occurs under the same conditions with mitochondria from plants possessing normal (N) cytoplasm or with other subcellular fractions from plants with S cytoplasm. The actinomycin D-resistant RNA synthesis occurs within the mitochondria since the labeling of these species is unaffected by inclusion of RNase in the incubation medium and since they become completely sensitive to RNase upon lysis of the mitochondria with low concentrations of Triton X-100. Two of the actinomycin D-resistant products are double stranded. These are 2850 and 900 base pairs in length, whereas the remaining two are 2150 and 850 bases. The synthesis of all four RNAs occurs in at least five different accessions of S cytoplasm, suggesting it is a general feature of S mitochondria. The double-stranded RNAs show homology to single-stranded S mitochondrial RNA but not to N mitochondrial RNA. Our observations indicate that the replication of these RNAs occurs independently of mtDNA and that they thus represent a novel type of inheritable element in organelles, an RNA plasmid.
Tumor necrosis factor: a potent effector molecule for tumor cell killing by activated macrophages.
Authors: Urban J L , Shepard H M , Rothstein J L , Sugarman B J , Schreiber H
Published: May 2006

Activated macrophages (aM phi) destroy more effectively cancer cells than normal cells. The mechanism by which macrophages destroy cancer cells is not known. We report here that tumor cells susceptible to aM phi were killed by recombinant (r) tumor necrosis factor type alpha (TNF-alpha), whereas variant tumor cells resistant to aM phi after selection in vitro or in vivo were resistant to killing by rTNF-alpha. The converse selection for rTNF-alpha-resistant variants resulted in cells that were also resistant to killing by aM phi. The sensitivity of macrophage-resistant variants was not changed to other tumoricidal cells or soluble mediators, except that the macrophage-resistant variants were also resistant to the effects of another cytotoxic protein, B-cell lymphotoxin, which is structurally related to rTNF-alpha. Similar results were obtained regardless of whether short-term or long-term cytotoxic effects of aM phi were measured. Finally, it was shown that killing of tumor cells by murine aM phi was completely inhibited with a polyclonal antibody that neutralizes the effects of murine TNF-alpha. These results suggest a major role for TNF-alpha in tumor cell destruction by aM phi in vitro and in vivo.
Bovine cytotoxic T-cell clones specific for cells infected with the protozoan parasite Theileria parva: parasite strain specificity and class I major histocompatibility complex restriction.
Authors: Goddeeris B M , Morrison W I , Teale A J , Bensaid A , Baldwin C L
Published: May 2006

We present information on the specificity of three bovine cytotoxic T-cell clones reactive with lymphoblasts infected with the protozoan parasite Theileria parva. The clones were derived from peripheral blood mononuclear cells of an animal immunized with T. parva (Muguga stock), after five stimulations in vitro with an autologous parasitized cell line. The three clones belonged to the BoT8+ subset of T cells, which is similar to the human CD8+ T-cell subset. On the basis of analysis on a panel of infected target cells originating from cattle of different major histocompatibility complex (MHC) phenotypes, killing by all three clones was found to be restricted to targets bearing the class I MHC specificity KN104, which is defined by alloantiserum KNA104 and monoclonal antibody IL-A4. This class I MHC restriction was confirmed by blocking of target cell lysis with these antibodies and monoclonal antibody w6/32, which reacts with a nonpolymorphic determinant on bovine class I MHC molecules. The three clones were parasite strain specific, in that they did not kill cells of the appropriate MHC type infected with T. parva (Marikebuni stock). These findings, taken together with previous observations that immunization of cattle with T. parva (Muguga) does not provide protection against challenge with T. parva (Marikebuni), suggest that the cytotoxic T cells recognize a cell surface antigen that may be important in induction of immunity to the parasite.
Inducible adhesion of mesenchymal cells to elastic fibers: elastonectin.
Authors: Hornebeck W , Tixier J M , Robert L
Published: May 2006

The addition of highly purified elastic fibers to confluent human skin fibroblast or porcine aorta smooth muscle cell cultures resulted in a time-dependent, strong adhesion of the fibrils to the cell surface. The kinetics of adhesion was studied by video/time-lapse cinematography. After a 0.5-1 hr lag period, adhesion progressed to a maximum amount in 3-6 hr in the described conditions. Adhesion is strongly accelerated by the prior addition of soluble elastin peptides (kappa-elastin) to the cultures. Cycloheximide inhibits this induced adhesion. Adherent elastic fibers can be detached by treatment with elastase and trypsin but not with collagenase. The radioactive proteins adhering to elastic fibers, after a 6-hr incubation of the induced cultures in presence of [35S]methionine, were extracted and analyzed by NaDodSO4/PAGE. The proteins strongly adhering to the elastic fibers had apparent molecular sizes of about 120, 67, 60, and 45 kDa. Only the 120-kDa protein band showed a significant increase of its associated radioactivity in the induced cultures as compared to the noninduced cultures. We propose that the 120-kDa protein is responsible for the induced adhesion of mesenchymal cells to elastic fibers and designate it "elastonectin."
Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts.
Authors: Rauth S , Song K Y , Ayares D , Wallace L , Moore P D , Kucherlapati R
Published: May 2006

We have examined the ability of single-stranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them. We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mp11. The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo. The resulting single-stranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells. We were able to obtain recombinant molecules containing wild-type neo genes in all three systems. Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate. We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction. We have also tested the ability of single-stranded DNA to yield transfectants. When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TK+ colonies. From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.
Calories versus protein in onset of renal disease in NZB x NZW mice.
Authors: Johnson B C , Gajjar A , Kubo C , Good R A
Published: May 2006

Autoimmunity-prone (NZB x NZW)F1 (B/W) female mice are used as a model of human lupus erythematosus. When full-fed, these mice die of glomerulonephritis between 7 and 11 (average 9) months of age. When food intake is restricted to 60% of calories, the onset of this disease is delayed and the mice live greatly prolonged lives free of disease. Since high protein intake is commonly associated with acceleration of kidney damage in humans and experimental animals, the current experiments were designed to employ diets in which protein concentration was as high as possible. The observations demonstrate clearly that with this model of autoimmune disease, total calorie intake (from whatever source) exerts an overriding influence on life span. A higher calorie intake leads to early death and restricted-calorie intake leads to an increased life span. When B/W mice are full-fed, with respect to calories, feeding diets of greatly differing protein composition did not influence life span significantly. By contrast, calorie restriction of diets, even of very high protein content or of lower protein content, greatly prolonged life of B/W mice. Even with exceedingly high protein intake (greater than 83% of the calories) it is not protein per se but the total calorie intake that exerts the greatest influence that determines length of life in mice of this autoimmunity- and glomerulonephritis-prone strain.
Consecutive A X T pairs can adopt a left-handed DNA structure.
Authors: McLean M J , Blaho J A , Kilpatrick M W , Wells R D
Published: May 2006

The capacity of six sequences with different numbers and orientations of A.T pairs flanked by alternating C.G pairs to adopt left-handed structures was evaluated in recombinant plasmids. A series of synthetic oligodeoxynucleotides were cloned into the BamHI site of pRW790, a small plasmid (approximately 2 kilobases) prepared especially for conformational studies of this type. Supercoil relaxation studies by two-dimensional gel electrophoresis on topoisomers of each plasmid revealed the energetics and structures of the left-handed helices. Also, the presence of supercoil-induced altered DNA conformations within the inserts of topoisomer populations of the plasmids was detected by reaction with S1 nuclease followed by restriction mapping of the cleavage sites. We conclude that consecutive T.A base pairs, whether alternating (TATA) or contiguous (TTTT), can adopt a left-handed conformation (presumably Z) when flanked by reasonably short runs of alternating (C-G)n (n = 3-5). Thus, these results substantially broaden the range of DNA sequences that can adopt left-handed Z conformations.
Effect of amino acid substitutions on the catalytic and regulatory properties of aspartate transcarbamoylase.
Authors: Robey E A , Wente S R , Markby D W , Flint A , Yang Y R , Schachman H K
Published: May 2006

Although intensive investigations have been conducted on the allosteric enzyme, aspartate transcarbamoylase, which catalyzes the first committed reaction in the biosynthesis of pyrimidines in Escherichia coli, little is known about the role of individual amino acid residues in catalysis or regulation. Two inactive enzymes produced by random mutagenesis have been characterized previously but the loss of activity is probably attributable to changes in the folding of the chains stemming from the introduction of charged and bulky residues (Asp for Gly-128 and Phe for Ser-52). Site-directed mutagenesis of pyrB, which encodes the catalytic chains of the enzyme, was used to probe the functional roles of several amino acids by making more conservative substitutions. Replacement of Lys-84 by either Gln or Arg leads to virtually inactive enzymes, confirming chemical studies indicating that Lys-84 is essential for catalysis. In contrast, substitution of Gln for Lys-83 has only a slight effect on enzyme activity, whereas chemical modification causes considerable inactivation. Gln-133, which has been shown by x-ray crystallography to reside near the contact region between the catalytic and regulatory chains, was replaced by Ala. This substitution has little effect on catalytic activity but leads to a marked increase in cooperativity. The Gln-83 mutant, in contrast, exhibits much less cooperativity. Since a histidine residue may be involved in catalysis and His-134 has been shown by x-ray diffraction studies to be in close proximity to the site of binding of a bisubstrate analog, His-134 was replaced by Ala, yielding a mutant with only 5% wild-type activity, considerable cooperativity, and lower affinity for aspartate and carbamoylphosphate. All of the mutants, unlike those in which charged or bulky residues replaced small side chains, bind the bisubstrate analog, which promotes the characteristic "swelling" of the enzymes indicative of the allosteric transition.
Spermine-DNA interactions: a theoretical study.
Authors: Feuerstein B G , Pattabiraman N , Marton L J
Published: May 2006

Models for the interaction of spermine and DNA were studied by performing conformational energy calculations on spermine and molecular mechanics calculations on major and minor groove complexes of spermine and oligomers of DNA. Docked into the major groove of B-DNA, spermine stabilizes the complex by maximizing interactions between proton acceptors on the oligomer and proton donors on spermine. This is achieved by bending the major groove of DNA over spermine and altering oligomer sugar puckering and interstrand phosphate distances. By comparison, Liquori's minor groove model appears to be less stable than the major groove model. This evidence favors a preferential binding of spermine to certain sites in DNA, which provides a powerful force for the modification of DNA conformation.
Human parathyroid antigen: characterization and localization with monoclonal antibodies.
Authors: Cance W G , Wells S A , Dilley W G , Welch M J , Otsuka F L , Davie J M
Published: May 2006

A cell surface antigen on human parathyroid cells was identified by monoclonal antibodies. The antigen, called parathyroid antigen (PTA), is found on both of two major polypeptides (190 kDa and 160 kDa) apparently associated exclusively with parathyroid cells. To determine whether PTA was a suitable target for in vivo imaging, 125I-labeled anti-PTA was injected into nude mice bearing human parathyroid xenografts. IgG1 anti-PTA antibody showed excellent radiolocalization of the grafts, whereas IgM anti-PTA, containing the same variable domains as the IgG1 antibody, showed little specific binding. These results suggest that antibody isotype is an important parameter for the in vivo immunoscintigraphy of specific cellular antigens.
Attachment of Con A or extracellular matrix initiates rapid sprouting by cultured leech neurons.
Authors: Chiquet M , Acklin S E
Published: May 2006

The outgrowth of neurites by single identified leech neurons in culture is markedly influenced by the substrate. Extensive sprouting occurs within a few hours on the plant lectin Con A. In contrast, the same neurons grow far more slowly or not at all when plated on vertebrate extracellular matrix proteins, other lectins, or poly(L-lysine). Sprouting on Con A, unlike that on poly(L-lysine), is inhibited by the Con A-specific hapten sugar methyl alpha-D-mannoside. Another substrate, promoting even more extensive sprouting of leech neurons, is cell-free extracellular matrix obtained from leech ganglion capsules. Urea extracts of extracellular matrix retain full neurite-promoting activity when dialyzed and used to coat culture dishes. Soluble growth factors are not required, since sprouting occurs in medium without macromolecules. These results show that sprouting depends not simply on attachment to the substrate but, critically, on its molecular composition; moreover, the pattern of outgrowth is characteristic and distinguishable for each type of neuron.
A synthetic approach to structure-function relationships in the murine epidermal growth factor molecule.
Authors: Heath W F , Merrifield R B
Published: May 2006

Murine epidermal growth factor, a 53-amino acid peptide that is mitogenic for a number of cell types, has been synthesized by the solid-phase method. The synthetic peptide is identical to the natural material in amino acid composition, chromatographic behavior, receptor binding, and stimulation of DNA synthesis. Fragments of the EGF molecule corresponding to residues 42-53, 32-53, and 15-53 were constructed as well as the methionine sulfoxide derivative of EGF, [Met(O)21]EGF-(1-53), and a polymeric form of EGF. [Met(O)21]EGF-(1-53) was slightly less active than EGF in receptor binding and stimulation of DNA synthesis. Polymeric EGF was 1/100th as active as EGF, while EGF-(15-53) was less potent than EGF-(1-53) by a factor of 10(4). EGF-(32-53) was even less active and EGF-(43-53) was inactive.