bluuebunny/crossref_metadata_2025 huggingface dataset:
Deletion of a B-cell-specific enhancer affects transfected, but not endogenous, immunoglobulin heavy-chain gene expression.
Authors: Zaller D M , Eckhardt L A
Published: May 2006

A transcriptional enhancer has recently been identified between the variable and constant region coding segments of an assembled immunoglobulin heavy-chain gene. This enhancer is required for efficient expression of such genes when transfected into myeloma cells. A class switch rearrangement in the mouse myeloma cell line 9.9.2.1 has resulted in deletion of this enhancer, and yet this cell line continues to produce heavy chains at a high level. Cell line 9.9.2.1 is a gamma 2a-producing class-switch variant derived from the gamma 2b-producing myeloma cell line, MPC11. We demonstrate that despite the high level of heavy-chain production in 9.9.2.1, the cloned 9.9.2.1 heavy-chain gene is not expressed efficiently when transfected into myeloma cells. Efficient expression after transfection can be obtained only if the deleted enhancer is reinserted into the gene. The implication of this finding for the role of this enhancer in establishing and/or maintaining immunoglobulin heavy-chain gene expression is discussed.
Inositol trisphosphate-induced calcium release and contraction in vascular smooth muscle.
Authors: Somlyo A V , Bond M , Somlyo A P , Scarpa A
Published: May 2006

Inositol 1,4,5-trisphosphate (InsP3) caused Ca release and tension development in rabbit main pulmonary artery smooth muscle permeabilized with saponin or digitonin. Both of these responses to single additions of InsP3 (0.5-30 microM) were repeatable and occurred in the presence of 0.0-1.9 mM free Mg2+. Sustained contractions were induced by InsP3. The amount of Ca released by InsP3, measured with a Ca2+-selective electrode, was also estimated to be sufficient to stimulate contraction in intact smooth muscle. Ca release was not influenced by inhibitors of mitochondrial oxidative phosphorylation. The uptake of Ca2+ from the medium into the InsP3-sensitive pool was ATP-dependent. The present results support the hypothesis that, in smooth muscle, InsP3 is the messenger, or one of the messengers, involved in transmitter-induced (pharmacomechanical) Ca release from the sarcoplasmic reticulum, which is the intracellular Ca store identified previously as the source of Ca released by norepinephrine in main pulmonary artery.
Remarkable homology among the internal repeats of erythroid and nonerythroid spectrin.
Authors: Birkenmeier C S , Bodine D M , Repasky E A , Helfman D M , Hughes S H , Barker J E
Published: May 2006

A cDNA clone for nonerythroid alpha-spectrin was identified by direct immunological screening of a chicken smooth muscle cDNA library. A library prepared in the expression plasmids pUC8 and pUC9 was screened with an antiserum specific for chicken alpha-spectrin. Blots of poly(A)+ RNA from various tissues of chicken and mouse show that the cDNA hybridizes to an 8-kilobase mRNA. The cDNA hybridizes to a single-copy sequence on Southern blots of chicken genomic DNA. The complete nucleic acid sequence of the clone has a single 1419-base open reading frame. The derived amino acid sequence is organized into two partial and three complete 106-amino-acid repeats that show homology to the repeats described for human erythroid alpha- and beta-spectrin. Immunological and biochemical data indicate that chicken nonerythroid and human erythroid alpha-spectrin are two of the more widely diverged members of the spectrin family of proteins. In this respect, the degree of homology found between them was unexpected. Our data suggest a common evolutionary origin for these two alpha-spectrins and allow some predictions concerning spectrin gene structure.
Model of alpha 2-macroglobulin structure and function.
Authors: Feldman S R , Gonias S L , Pizzo S V
Published: May 2006

A model of alpha 2-macroglobulin is presented that is compatible with previous structural, functional, and phylogenetic studies of the protein. The model of the molecule resembles a hollow cylinder and is comprised of two identical functional halves with three C2 axes of symmetry and no mirror planes. The "trap mechanism" of this proteinase inhibitor is effected by slight movement of two trap arms per "half-molecule." Evidence for this model is obtained from the study of the structure and proteinase binding of the molecule. By using this model, predictions are made concerning proteinase binding ratios, receptor recognition, and "slow-to-fast" conformational change of the molecule.
Azotobacter vinelandii nifD-
and
nifE
-encoded polypeptides share structural homology
Authors: Dean Dennis R. , Brigle Kevin E.
Published: May 2006

The
Azotobacter vinelandii nifE
gene was isolated and its complete nucleotide sequence was determined. The amino acid sequences deduced from the
A. vinelandii nifE
and
nifD
gene sequences were compared and found to share striking primary sequence homology. This homology implies a functional and possibly an evolutionary relationship between these two gene products. The structural homology is discussed with regard to the potential FeMo cofactor binding properties of these polypeptides and the possible role of a
nifEN
product complex as a surrogate MoFe protein.
Demonstration of a stimulatory protein for virus-specified DNA polymerase in phorbol ester-treated Epstein-Barr virus-carrying cells.
Authors: Chiou J F , Li J K , Cheng Y C
Published: May 2006

A heat-labile Epstein-Barr virus-specific DNA polymerase stimulatory protein having a molecular mass of 45 kDa was purified from phorbol 12-myristate 13-acetate-treated P3HR-1 cells by column chromatography. The virus DNA polymerase stimulatory protein was precipitated by sera from patients with nasopharyngeal carcinoma but not by sera from healthy donors. The interaction of the stimulatory protein with DNA polymerase was stoichiometric. Furthermore, this protein stimulated Epstein-Barr virus DNA polymerase but not herpes simplex virus type 1 or type 2 or human DNA polymerase alpha. The stimulatory protein did not alter the Km value of dTTP or DNA but did increase the Vmax of DNA polymerase. Salt concentrations between 100 mM and 150 mM KCl were optimal for this protein-induced stimulation of Epstein-Barr virus DNA polymerase activity. The presence of the stimulatory protein in the reaction mixture enhanced the sensitivity of virus DNA polymerase to phosphonoformate.
Pellet microfossils: Possible evidence for metazoan life in Early Proterozoic time
Authors: Robbins Eleanora Iberall , Porter Karen Glaus , Haberyan Kurt A.
Published: May 2006

Microfossils resembling fecal pellets occur in acid-resistant residues and thin sections of Middle Cambrian to Early Proterozoic shale. The cylindrical microfossils average 50 × 110 μm and are the size and shape of fecal pellets produced by microscopic animals today. Pellets occur in dark gray and black rocks that were deposited in the facies that also preserves sulfide minerals and that represent environments analogous to those that preserve fecal pellets today. Rocks containing pellets and algal microfossils range in age from 0.53 to 1.9 gigayears (Gyr) and include Burgess Shale, Greyson and Newland Formations, Rove Formation, and Gunflint Iron-Formation. Similar rock types of Archean age, ranging from 2.68 to 3.8 Gyr, were barren of pellets. If the Proterozoic microfossils are fossilized fecal pellets, they provide evidence of metazoan life and a complex food chain at 1.9 Gyr ago. This occurrence predates macroscopic metazoan body fossils in the Ediacaran System at 0.67 Gyr, animal trace fossils from 0.9 to 1.3 Gyr, and fossils of unicellular eukaryotic plankton at 1.4 Gyr.
Nucleotide sequence and predicted amino acid sequence of a protein encoded in a small herpes simplex virus DNA fragment capable of trans-inducing alpha genes.
Authors: Pellett P E , McKnight J L , Jenkins F J , Roizman B
Published: May 2006

The five alpha genes of herpes simplex virus 1 are the first set of genes to be expressed after infection. Previous studies have shown that alpha genes resident in eukaryotic cells are induced by infection with herpes simplex virus 1 or 2 but not by other herpesviruses and indicate that the alpha trans-inducing factor was a structural component of the virion. This factor induces genes linked to a bona fide promoter and containing at the 5' end a small sequence derived from the promoter-regulatory domains of alpha genes. We report the sequence of a small DNA fragment shown previously to be capable of expressing the alpha trans-inducing factor in transient expression systems. The only gene encoded in its entirety in this fragment is predicted to specify a 479 amino acid protein with a Mr of 53,053. The precise termini of the 1.74-kilobase mRNA specifying this protein were determined in our 5' and 3' S1 nuclease protection studies.
Enkephalins have a direct positive inotropic effect on cultured cardiac myocytes.
Authors: Laurent S , Marsh J D , Smith T W
Published: May 2006

Enkephalins have peripheral vascular effects, and enkephalinergic innervation of the heart has been reported. To determine whether enkephalins have direct effects on myocardium, we studied the effects of [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin on amplitude of contraction (measured with an optical-video system) in spontaneously beating monolayer cultures of chicken embryo ventricular cells, a preparation devoid of intact neural elements. [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin as well as [Met5]- and [Leu5]enkephalin increased contractility in a concentration-dependent manner. The enkephalin-induced maximal contractile effects were 28% and 30% above control, with EC50 values of 0.53 and 0.17 microM for [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin, respectively. The positive inotropic effect was antagonized by naloxone but not by propranolol, phentolamine, diphenhydramine, or cimetidine. Naloxone alone had no effect on contractility at a concentration (0.1 microM) that blocked positive inotropic effects of [D-Ala2, Met5]enkephalinamide and [D-Ala2, D-Leu5]enkephalin. To demonstrate the presence of opiate receptors, we studied [3H]naloxone binding in homogenates of cultured chicken embryo ventricular cells. Analysis of binding curves under equilibrium conditions indicated that [3H]naloxone bound specifically to membranes of cultured heart cells with KD = 18.5 +/- 5.4 nM and Bmax = 46.8 +/- 11.7 fmol/mg of protein. We conclude that enkephalins exert a direct positive inotropic effect on cultured heart cells, increasing contractile state via specific opiate receptors.
Cloning and sequence analysis of a cDNA encoding porcine mitochondrial aspartate aminotransferase precursor.
Authors: Joh T , Nomiyama H , Maeda S , Shimada K , Morino Y
Published: May 2006

The primary structure of pig mitochondrial aspartate aminotransferase (mAspATase; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) precursor was deduced from its cDNA sequence. A library of cDNA clones was constructed from pig liver poly(A)+ RNA by applying the vector/primer method of Okayama and Berg [Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170]. The library was screened for pig mAspATase sequences by using a mixture of eight oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known amino acid sequence of pig mAspATase residues 196-201. Two recombinant plasmids containing inserts of about 2500 and 2600 base pairs were selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the pig mAspATase precursor consists of the mature enzyme of 401 amino acid residues and an amino-terminal segment of 29 amino acid residues called the "presequence" that contains four basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The sequence of this 29-amino acid mAspATase precursor segment was compared with the presequences of other mitochondrial enzymes.
Molecular structure of the human cytoplasmic beta-actin gene: interspecies homology of sequences in the introns.
Authors: Nakajima-Iijima S , Hamada H , Reddy P , Kakunaga T
Published: May 2006

A recombinant phage that carries the cytoplasmic beta-actin gene was isolated from a human DNA library. The nucleotide sequence of this gene was determined. The amino acid sequence deduced from the nucleotide sequence matches perfectly that of beta-actin from human fibroblasts. The gene contains five introns. A large intron was found in the 5' untranslated region six nucleotides upstream from the ATG initiation codon. Four introns were found within the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. In contrast to the human cardiac muscle actin gene, the aorta-type smooth muscle actin gene, and the stomach-type smooth muscle actin gene, the beta-actin gene lacks the codon for cysteine between the ATG initiation codon and the codon for the NH2-terminal amino acid of the mature protein. Hybridization of genomic DNA with DNA fragments derived from intron I in the 5' untranslated region and from intron III strongly suggests the presence of a single beta-actin gene in the human genome. The DNA sequences of the coding region, of the 3' untranslated region, and of the sequence block between the "CCAAT" box and "TATA" box in the 5' flanking DNA of the human beta-actin gene are highly homologous to the corresponding sequences of the rat and chicken beta-actin genes. Unexpectedly, the sequence of intron III of the human beta-actin gene shows considerable homology to that of the rat beta-actin gene.
Transplants of Schwann cell cultures promote axonal regeneration in the adult mammalian brain.
Authors: Kromer L F , Cornbrooks C J
Published: May 2006

Transplantation of embryonic brain tissue or mature peripheral nerves into the adult mammalian central nervous system promotes axonal regrowth from axotomized central nervous system neurons; however, the cellular origin and molecular nature of the factors promoting axonal growth in vivo are unknown. To further characterize cellular environments that facilitate regeneration of central nervous system axons, we developed a methodology whereby cultured cell preparations can be transplanted into the brain of mature mammals. For this procedure, lesions are produced in the septal-hippocampal system of adult rats, and selected regions from collagen-supported Schwann cell/neuron cultures (consisting of Schwann cells, extracellular matrix, and degenerating neuronal processes and myelin but devoid of neuronal perikarya and fibroblasts) are positioned within the intracephalic cavity so that they bridge the lesion gap (approximately 3 mm) separating the septum and hippocampus. At various time up to 3 weeks after transplantation, specimens were prepared for acetylcholinesterase histochemistry and the immunocytochemical localization of laminin (an extracellular matrix protein) and C-4 (a Schwann cell membrane antigen). All specimens (from uninjured controls and from animals with either acellular collagen or mature Schwann cell/extracellular matrix transplants) contained laminin immunoreactivity associated with the meninges, choroid plexus, ependyma, and cerebral blood vessels. All animals with transplants showed prominent laminin staining on astrocytic processes along the intracephalic cavity, but only the Schwann cell/extracellular matrix transplants exhibited dense laminin and C-4 immunoreactivity within the cellular portion of the transplants. Regeneration of acetylcholinesterase-positive septal fibers occurred only in animals containing Schwann cell/extracellular matrix transplants. By 6 days after transplantation, acetylcholinesterase-positive fibers were observed both on laminin-positive cellular tissue strands connecting the septum and the Schwann cell/extracellular matrix transplants and on the initial portions of the transplants. By day 14, acetylcholinesterase-positive fibers traversed the entire lesion cavity in intimate association with the laminin- and C-4-positive cellular layer of the transplants and reinnervated the host hippocampus. However, cholinergic fibers were not associated with all laminin-containing processes along the lesion cavity nor did they grow along acellular collagen transplants. These results indicate the presence of factors in transplants of cultured Schwann cells and their associated extracellular matrix that promote rapid regeneration of central nervous system cholinergic axons in vivo.
Morphology and distribution of tyrosine hydroxylase-like immunoreactive neurons in the cat retina.
Authors: Oyster C W , Takahashi E S , Cilluffo M , Brecha N C
Published: May 2006

In cat retina, antisera directed against tyrosine hydroxylase (tyrosine 3-monooxygenase; EC 1.14.16.2) labeled three morphologically distinct classes of neurons; amacrine cells, displaced amacrine cells, and interplexiform cells. These three cell populations differed not only in their morphologies but also in their numbers, densities, and retinal distributions. Insofar as tyrosine hydroxylase-like immunoreactivity is associated with dopaminergic neurons, these observations suggest that the dopamine system in cat retina is morphologically heterogeneous and that the cat retina contains functional subdivisions based on the different distributions of specific cell populations.
Chloride/formate exchange with formic acid recycling: a mechanism of active chloride transport across epithelial membranes.
Authors: Karniski L P , Aronson P S
Published: May 2006

The pathways for transport of Cl- and formate in microvillus membrane vesicles isolated from rabbit renal cortex were evaluated. An outward formate gradient stimulated the uptake of Cl-, and an outward Cl- gradient stimulated the uptake of formate, indicating Cl-/formate exchange. In addition, an inside alkaline pH gradient induced the accumulation of formate, consistent with nonionic diffusion of formic acid. Although an inward Na+ gradient also stimulated uphill formate accumulation, suggesting Na+/formate cotransport, this effect was abolished when ionophores were used to prevent the generation of a transmembrane pH gradient, indicating an indirect coupling of formic acid transport to Na+/H+ exchange. An inside alkaline pH gradient only minimally stimulated the uptake of 82Br-, used as tracer for Cl-, confirming the absence of appreciable Cl-/OH- exchange. However, the same pH gradient in the presence of a physiologic formate concentration (0.2 mM) markedly stimulated 82Br- influx. These data suggest that Cl-/formate exchange with recycling of formic acid by nonionic diffusion is a potential mechanism for active Cl- absorption across the luminal membrane in the proximal tubule and perhaps in other epithelia.
Sequence of a second human asialoglycoprotein receptor: conservation of two receptor genes during evolution.
Authors: Spiess M , Lodish H F
Published: May 2006

The asialoglycoprotein (ASGP) receptor isolated from human liver and from the human hepatoma cell line HepG2 migrates on NaDodSO4 gel electrophoresis as a single species of 45,000 daltons. Recently, we isolated a cDNA clone encoding this receptor (H1) from a HepG2 lambda gt11 library. From the same library, we have isolated and sequenced a clone encoding a second ASGP receptor, H2, with a protein sequence homology of 58% to H1. There are two subspecies of H2 that differ only by the presence of a five-amino acid insertion in the COOH-terminal extracytoplasmic domain. Comparison with the available sequences of the two rat ASGP receptors R1 and R2 indicates that H1 is more homologous to R1 than to H2, and H2 is more similar to R2 than to H1. Thus, the two receptor genes evolved before the separation of rat and man. As judged by RNA blot hybridization of HepG2 RNA using RNA transcribed in vitro from cDNA clones of the human receptors as standards, H1 and H2 mRNA are present in equimolar amounts, each 0.005-0.01% of the total mRNA. This finding raises the question of whether the three ASGP receptor proteins are functional as heterodimers or whether they might serve different functions in the cell.
Chloride uptake into cultured airway epithelial cells from cystic fibrosis patients and normal individuals.
Authors: Stutts M J , Cotton C U , Yankaskas J R , Cheng E , Knowles M R , Gatzy J T , Boucher R C
Published: May 2006

The chloride permeability of airway and sweat ductal epithelium of cystic fibrosis (CF) patients is decreased. This abnormality could represent an intrinsic characteristic of the epithelial cell or the response to a tonic extrinsic stimulus, in vivo. We cultured airway epithelial cells derived from CF and non-CF individuals under identical conditions that were free from donor-specific factors. Differences in the characteristics of cells that multiplied under these circumstances are unlikely to reflect the effects of extrinsic modulation present in the host. After 8-12 days in culture, the cells of CF and non-CF patients were similar in morphology and intracellular electrolyte content, but the CF cultures took up chloride at a reduced rate. The difference could not be attributed to a higher intracellular potential in CF cells or to the presence of a stilbene anion-sensitive chloride-chloride exchange in non-CF cells. We conclude that epithelial cells from CF patients grown in the absence of extracellular factors of the host express reduced cellular chloride permeability, a defect similar to that found in vivo and in freshly excised nasal epithelium.
Polyneuropathy with monoclonal gammopathy: glycolipids are frequently antigens for IgM paraproteins.
Authors: Ilyas A A , Quarles R H , Dalakas M C , Brady R O
Published: May 2006

Immunoglobulins from patients with paraproteinemic polyneuropathy were screened for reactivity with nerve and brain glycolipids by ELISA and/or a thin-layer-chromatogram-overlay technique. The myelin-associated glycoprotein (MAG) has been shown to be an antigen in many neuropathy patients with IgM gammopathy, but this study focused on seven neuropathy patients in which the IgM paraproteins had been shown not to react with this glycoprotein. Five of these seven had IgM that reacted with components in the acidic glycolipid fraction of human sciatic nerve, and three of these IgMs also reacted with components in the acidic glycolipid fraction of human brain. Little or no reactivity with glycolipids was detected for two patients with neuropathy and IgG gammopathy or for two with neuropathy and IgA gammopathy. The results suggest that neuropathy-patient IgM paraproteins not reactive with MAG often react with acidic glycolipids and thus define a subset of paraproteinemic neuropathies. Since the IgM paraproteins that react with MAG also react with acidic glycolipids of nerve, glycolipid antigens appear to be quite common among the IgM paraproteinemic neuropathies.
Quantitative assessment of the role of O6-methylguanine in the initiation of carcinogenesis by methylating agents.
Authors: Doniger J , Day R S , DiPaolo J A
Published: May 2006

Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syrian hamster embryo cells (HEC) treated with three different methylating agents: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Each induced transformation in a dose-dependent manner. On a molar basis, MNNG was approximately equal to 100- and 500-fold more effective than MNU and MMS, respectively. For each carcinogen the induction and repair of O6- and N7-methylguanine (O6- and N7-MeGua) relative to total guanine content was compared. At concentrations that induced equivalent transformation frequencies, the induction of O6-MeGua was the same for all three carcinogens, but N7-MeGua induction was 30-fold higher with MMS than with MNNG or MNU. The capacity to repair methylation lesions in HEC is limited because only between 50% and 70% of both O6- and N7-MeGua lesions were removed from the DNA within 24 hr after treatment, independent of methylating carcinogen. No consistent effect on either the rate of DNA replication or the size distribution of nascent strands correlated with O6-MeGua induction. These data support the hypothesis that O6-MeGua is the critical lesion for initiation of carcinogenesis by methylating agents. The frequency of transformation relative to O6-MeGua induction is 40- to 750-fold more than that of mutation. Based on the quantitative data for induction of O6-MeGua and transformation, the target size for initiation of carcinogenesis was calculated as a minimum of 10(4) nucleotides. This suggests that one of many genes can initiate carcinogenesis or that initiation is not the result of a single base mutation.
Two separate functions of class II (Ia) molecules: T-cell stimulation and B-cell excitation.
Authors: Corley R B , LoCascio N J , Ovnic M , Haughton G
Published: May 2006

We have evaluated the role of major histocompatibility complex-encoded class II (Ia) molecules as transmembrane signaling receptors in the T helper cell-dependent activation of B lymphocytes. For these studies, we utilized the murine B-cell lymphoma CH12, which expresses both I-A and I-E class II molecules. In addition, CH12 cells carry IgM of known antigen specificity and require both specific antigen and Ia-restricted T-cell help for the induction of antibody secretion. In this respect, they resemble normal resting B cells. We have studied the ability of antigen-specific or alloreactive T helper cells reactive with either the I-A or the I-E molecules on CH12 to be activated and their ability to stimulate antibody production by CH12. The results show that, although CH12 cells present antigen to T helper cells that interact with either the I-A or the I-E molecules, CH12 cells are stimulated to secrete antibody only by T helper cells reactive with their I-E molecules. Our data demonstrate that class II molecules are transducers of signals for B-cell excitation in addition to serving a restricting function for helper T-cell stimulation. Moreover, the data demonstrate that these two functions, T-cell stimulation and B-cell excitation, are discrete and need not be expressed by the same Ia molecule.
Electroconvulsive shock increases preproenkephalin messenger RNA abundance in rat hypothalamus.
Authors: Yoshikawa K , Hong J S , Sabol S L
Published: May 2006

Daily administration of electroconvulsive shock (ECS) to rats for 10 days increased the content of [Met5]enkephalin in the hypothalamus and the striatum by 64% and 45%, respectively. The effect of ECS on the relative abundance of mRNA coding for the enkephalin precursor preproenkephalin was investigated. Analysis by cell-free translation of polyadenylylated RNA and immunoprecipitation of preproenkephalin revealed ECS-elicited increases of 79% and 14% in preproenkephalin mRNA activity in the hypothalamus and striatum, respectively. ECS treatment did not affect the general translational activity of total polyadenylylated RNA from these brain regions. A 32P-labeled probe prepared from a rat preproenkephalin cDNA clone hybridized with an apparently single species of polyadenylylated RNA of approximately equal to 1450 nucleotides from both hypothalamus and striatum. Dot-blot hybridization of polyadenylylated RNA with the rat probe indicated that ECS elicits a 76% increase in the preproenkephalin mRNA abundance in the hypothalamus and no significant change in the striatum. These results suggest that ECS treatment leads to enhanced biosynthesis of the enkephalin precursor in hypothalamic neurons.
Rapid transient induction of phenylalanine ammonia-lyase mRNA in elicitor-treated bean cells
Authors: Edwards Keith , Cramer Carole L. , Bolwell G. Paul , Dixon Richard A. , Schuch Wolfgang , Lamb Chris J.
Published: May 2006

DNAs complementary to a size-selected fraction of poly(A)
+
RNA present in elicitor-treated cells of bean (
Phaseolus vulgaris
L.) were inserted into pAT153 and used to transform
Escherichia coli
strain C600. Five clones were identified by hybrid-selected translation and cross-hybridization that contained sequences complementary to mRNA encoding phenylalanine ammonia-lyase (EC 4.3.1.5), which catalyzes the first reaction of phenylpropanoid biosynthesis. The longest insert contained a single open reading frame of 1520 base pairs together with 223 base pairs of 3′ untranslated sequence. RNA blot hybridization showed that elicitor caused a rapid, marked but transient increase in phenylalanine ammonia-lyase mRNA that was closely correlated with changes in translatable mRNA activity
in vitro
and enzyme synthesis
in vivo
. Blot hybridization of newly synthesized mRNA purified by organomercurial affinity chromatography following
in vivo
pulse-labeling with 4-thiouridine indicates that elicitor caused a rapid stimulation of phenylalanine ammonia-lyase mRNA synthesis as an early in the defense response leading to accumulation of phenylpropanoid-derived phytoalexins.
Mapping the cysteine residues and actin-binding regions of villin by using antisera to the amino and carboxyl termini of the molecule.
Authors: Matsudaira P , Jakes R , Cameron L , Atherton E
Published: May 2006

Peptide antisera specific for either the amino- or carboxyl-terminal regions of villin were used to locate the position of cysteine residues in immunoblots of villin cleaved with 2-nitro-5-thiocyanobenzoic acid. Maps constructed from the cleavage pattern suggest that villin contains six cysteine residues, two located in its amino-terminal peptide of Mr 44,000, and four located in the carboxyl-terminal peptide of Mr 51,000. Gel overlays of the partial cleavage fragments with 125I-labeled actin identified a calcium-dependent actin-binding region located within the amino-terminal peptide of Mr 32,000 of villin. The peptide antibody method used, called cleavage mapping, should be a convenient technique for mapping residues and ligand binding sites in proteins.
Structure of glycolate oxidase from spinach
Authors: Lindqvist Ylva , Brändén Carl-Ivar
Published: May 2006

A high-resolution structure determination of glycolate oxidase from spinach is reported. X-ray data were collected on films at the synchrotron radiation source in Daresbury, England. The structure was solved by using two heavy-atom derivatives and a solvent-flattening procedure developed by B.-C. Wang. The subunit structure is essentially a structure of the eight-stranded α/β-barrel type first described for triosephosphate isomerase. In addition, there are 70 residues at the NH
2
terminus and 45 residues between strand four and helix four of the barrel, which are arranged in a helical domain outside the COOH end of the parallel strands of the barrel. The active site is in a cleft between these domains with the coenzyme FMN essentially bound to the barrel and a substrate analogue, thioglycolate, bound to the helical domain. The molecule is octameric with 422 symmetry and has a 15- to 20-Å-wide hole in the middle.
Prevention of a plant disease by specific inhibition of fungal polyamine biosynthesis.
Authors: Rajam M V , Weinstein L H , Galston A W
Published: May 2006

DL-alpha-Difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17), strongly retards the growth of several species of phytopathogenic fungi in vitro. Such inhibition can be completely reversed by putrescine or spermidine, confirming the essentiality of polyamines for growth of fungal hyphae. We now show that DFMO can protect bean plants (Phaseolus vulgaris Linnaeus cv. Pinto) against infection by uredospores of the bean rust fungus, Uromyces phaseoli Linnaeus, race O. Unifoliolate leaves of 10-day-old greenhouse-grown seedlings were sprayed with 400 microliter per leaf of DFMO at various concentrations in 0.01% Tween 20 at pH 7.0 before or after inoculation with uredospores of Uromyces. After 16 hr in darkness in dew chambers to facilitate spore germination, plants were transferred to the greenhouse, arranged randomly, and examined for local lesions 7 days later. All concentrations of DFMO 0.50 mM or higher gave complete protection against the pathogen; at lower concentrations, postinoculation treatments with DFMO were generally more effective than preinoculation. The appearance of lesions on plants treated with lower concentrations of DFMO was retarded 2-6 days. DFMO also confers protection on unsprayed parts of treated plants, indicating the translocation of some protective effect from sprayed areas. DL-alpha-Difluoromethylarginine, an analogous inhibitor of arginine decarboxylase (EC 4.1.1.19), which is the rate-limiting enzyme in an alternative pathway for polyamine biosynthesis in higher plants, confers no protection even at 5 mM. This emphasizes ornithine decarboxylase as the biochemical locus of choice for the prevention of plant diseases by inhibiting polyamine metabolism.
Organization of pigment proteins in the photosystem II complex of the cyanobacterium Anacystis nidulans R2.
Authors: Pakrasi H B , Riethman H C , Sherman L A
Published: May 2006

Two chlorophyll-protein complexes associated with photosystem II (PSII) of the cyanobacterium Anacystis nidulans R2 have been detected. The larger of the two complexes, CPVI-1, contained a 71-kDa and a 42-kDa protein. The 71-kDa protein was determined to be the anchor protein of the phycobilisomes (the light-harvesting complex of A. nidulans PSII), since it was recognized by an antibody raised against a similar protein from another cyanobacterium. The second complex, CPVI-4, contained a previously unobserved 36-kDa chlorophyll-binding protein. Additionally, two other PSII chlorophyll-protein bands were characterized. CPVI-2 contained a 52-kDa band that was recognized by an antibody raised against the presumptive PSII reaction center protein of Chlamydomonas reinhardtii. It gave rise to a fluorescence emission peak (77K) at 695 nm, indicating that this chlorophyll-protein complex may harbor the reaction center of PSII. Finally, CPVI-3 was found to have a 45-kDa protein and to be immunologically related to the presumptive immediate-antenna protein of the C. reinhardtii PSII.
DNA amplification patterns in maize endosperm nuclei during kernel development
Authors: Kowles Richard V. , Phillips Ronald L.
Published: May 2006

Increased DNA levels in centrally located endosperm nuclei are shown to be related to endosperm development in
Zea mays
. Mitotic activity sharply decreases in endosperm cells 10-12 days after pollination. At this time nuclear size and DNA content per nucleus (where C = haploid content) sharply increase until peak levels are reached at about 14-18 days after pollination. Mean DNA content per endosperm nucleus in strain A188 was shown by Feulgen cytophotometry to increase to about 90C by this peak stage, with the pattern being remarkably consistent over four consecutive growing seasons. Some individual nuclei achieved levels of >200C. Most other strains compared during one growing season averaged even higher peak levels of DNA per nucleus than did A188. Individual nuclei in those strains reached levels as high as 690C. A decrease in DNA level was observed in older endosperms with most strains. Endosperm mutant strains did not show a significant reduction in DNA. Opaque-2 mutants in several backgrounds achieved higher levels of DNA per nucleus. DNA levels from F
1
endosperms did not indicate heterosis. Regardless of differences in DNA content, the pattern of DNA increasing as development proceeds followed by a DNA decrease was observed for most strains. Cytological studies reveal much variation in chromatin strandedness, a maximum of three nucleoli, a maximum of three nucleolar organizer regions, and ≈30 diffuse chromatin masses in older endosperm tissue. A form of DNA amplification, perhaps polytenization, appears to be occurring during endosperm development.
Diversity among Purkinje cells in the monkey cerebellum.
Authors: Ingram V M , Ogren M P , Chatot C L , Gossels J M , Owens B B
Published: May 2006

A monoclonal antibody (B1) produced against rat embryonic forebrain membranes shows specific and striking immunohistochemical staining of Purkinje cells in the monkey cerebellum in a pattern of broad parasagittal alternating bands of cells either possessing or lacking the B1 antigen. In addition, the neurons of the deep cerebellar nuclei and some neurons of the motor cortex and of the spinal cord also contain the B1 antigen. Neurons with the B1 antigen were also seen in the somatosensory cortex, the vestibular and cochlear nuclei, and the retina.
Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions.
Authors: Symington L S , Kolodner R
Published: May 2006

An enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions was partially purified approximately 500- to 1000-fold by DEAE-cellulose chromatography, gel filtration on Sephacryl S300, and chromatography on single-stranded DNA-cellulose. The partially purified enzyme did not have any detectable nuclease activity when tested with single-stranded or double-stranded bacteriophage T7 substrate DNA and did not have detectable endonuclease activity when tested with bacteriophage M13 viral DNA or plasmid pBR322 covalently closed circular DNA. Analysis of the products of the cruciform cleavage reaction by electrophoresis on polyacrylamide gels under denaturing conditions revealed that the cruciform structure was cleaved at either of two sites present in the stem of the cruciform and was not cleaved at the end of the stem. The cruciform cleavage enzyme was able to cleave the Holliday junction present in bacteriophage G4 figure-8 molecules. Eighty percent of these Holliday junctions were cleaved in the proper orientation to generate intact chromosomes during genetic recombination.
Sustained release of epidermal growth factor accelerates wound repair.
Authors: Buckley A , Davidson J M , Kamerath C D , Wolt T B , Woodward S C
Published: May 2006

Epidermal growth factor (EGF) is a potent mitogen in vitro, but its biological role is less clear. The vulnerary effects of EGF were evaluated in a model of wound repair, the polyvinyl alcohol sponge implanted subcutaneously in rats. EGF was purified to homogeneity by reverse-phase HPLC and quantified by receptor binding assay and amino acid analysis. Preliminary data showed moderate promotion of granulation tissue formation by daily injections of 10 micrograms of EGF. To test the hypothesis that long-term exposure to EGF is required for complete cellular response, the factor was incorporated into pellets releasing 10 or 20 micrograms of biologically active EGF per day, and the pellets were embedded within the sponges. Slow release of EGF caused a dramatic increase in the extent and organization of the granulation tissue at day 7, a doubling in the DNA content, and 33% increases in protein content and wet weight, as compared with placebo controls. Although collagen content was also increased by almost 50%, the relative rate of collagen synthesis remained the same, suggesting that the morphological and biochemical increase in collagen resulted from increased numbers of fibroblasts rather than a specific stimulation of collagen synthesis. These results indicate that the local sustained presence of EGF accelerates the process of wound repair, specifically neovascularization, organization by fibroblasts, and accumulation of collagen.
Use of anti-idiotypic antibodies to identify a receptor for the T-cell I-J determinant.
Authors: Zupko K , Waltenbaugh C , Diamond B
Published: May 2006

In order to identify the molecule(s) interacting with the I-J determinant on suppressor T cells, we have generated two anti-idiotypic sera: one to monoclonal anti-I-Jd antibody and one to monoclonal anti-I-Jk antibody. These antisera specifically block suppressor T-cell function in a genetically restricted manner and have no effect on helper T-cell activation. Both recognize a marker on primary monocytes and B cells but not on T cells. A myeloma cell line bearing this marker has been identified. Therefore, these antisera may recognize a molecule on cells interacting with suppressor T cells that is involved in mediating suppressor T-cell activity. The relationship between the T-cell I-J determinant and the molecule identified by the anti-idiotype may be similar to the relationship between the receptor on helper T cells and Ia molecules.
On the nature of the 5-lipoxygenase reaction in human leukocytes: characterization of a membrane-associated stimulatory factor.
Authors: Rouzer C A , Shimizu T , Samuelsson B
Published: May 2006

When 10,000 X g supernatants of human leukocyte homogenates were subjected to centrifugation at 100,000 X g for 75 min, the activity of 5-lipoxygenase decreased by 30-60%, even though no enzyme was detectable in the resuspended 100,000 X g pellet. Recombination of the 100,000 X g supernatant and pellet resulted in a restoration of the lost enzymatic activity, indicating the presence of a 5-lipoxygenase stimulatory factor in the microsomal membrane preparation. Dialysis of human leukocyte supernatants resulted in an apparent decrease in 5-lipoxygenase activity, but only in samples that contained the membrane-associated stimulatory factor, suggesting that the factor required a small molecular weight component for optimal function. The 5-lipoxygenase stimulatory activity was highly unstable to washing of the 100,000 X g pellet or to incubation (16-20 hr) at 4 degrees C. In contrast, the activity was remarkably stable to heat (100 degrees C for 40 min). The responses of the 12- and 15-lipoxygenases in human leukocyte homogenates to the membrane-associated factor and to dialysis were notably different from that of the 5-lipoxygenase. These results demonstrate, therefore, that the 5-lipoxygenase is unique among the human lipoxygenases, not only in its requirement for Ca2+ and ATP but also in its regulation by a membrane-associated stimulatory factor. The mechanism of action of this regulatory factor is of obvious interest for the understanding of the control of leukotriene and lipoxin biosynthesis.
Amplification of DNA sequences in human multidrug-resistant KB carcinoma cells.
Authors: Fojo A T , Whang-Peng J , Gottesman M M , Pastan I
Published: May 2006

Four KB carcinoma cell lines selected independently for resistance to either colchicine, adriamycin, or vinblastine were studied. All cell lines showed high levels of resistance to the selecting drug and cross-resistance to the other drugs and to actinomycin D. Double-minute chromosomes could be identified on chromosomal spreads of these multidrug-resistant KB cell lines. Amplification of specific DNA sequences was demonstrated by using the technique of in-gel renaturation. All the cell lines share common amplified sequences. There are also amplified sequences that are specific for each cell line. A revertant cell line that has reacquired drug sensitivity has lost its amplified sequences. Specific probes obtained by cloning amplified sequences from the cell line selected in vinblastine recognize amplified sequences in all the resistant lines. The presence of common amplified sequences in these cell lines is strong evidence for the importance of these regions in multiple drug resistance.
Experimental diabetic neuropathy: impairment of slow transport with changes in axon cross-sectional area.
Authors: Medori R , Autilio-Gambetti L , Monaco S , Gambetti P
Published: May 2006

Analysis of slow axonal transport in the sciatic and primary visual systems of rats with streptozotocin-induced diabetes of 4-6 weeks duration showed impairment of the transport of neurofilament subunits, tubulin, actin, and a 30- and a 60-kDa polypeptide in both systems. The degree of impairment was not uniform. Transport of polypeptide constituents of the slow component b, such as the 30- and 60-kDa polypeptides, appeared to be more severely affected than the transport of constituents of the slow component a, such as neurofilaments. Morphometric analysis of sciatic axons revealed a proximal increase and a distal decrease of axonal cross-sectional area. It is proposed that impairment of axoplasmic transport and changes of axonal size are related. Transport impairment results in a larger number of neurofilaments, microtubules, and other polypeptides in the proximal region of the axon, which increases in size, whereas fewer neurofilaments, microtubules, and other polypeptides reach the distal axons that show a size decrease. Such changes in axonal transport and area are likely to occur in other diabetic animal models and in human diabetes.
Bacterial expression of the acquired immunodeficiency syndrome retrovirus p24 gag protein and its use as a diagnostic reagent.
Authors: Dowbenko D J , Bell J R , Benton C V , Groopman J E , Nguyen H , Vetterlein D , Capon D J , Lasky L A
Published: May 2006

A retrovirus [lymphoadenopathy-associated virus, human T-cell leukemia virus type III, acquired immunodeficiency syndrome (AIDS)-related virus] suspected of causing AIDS has been isolated recently. The detection of exposure to this retrovirus in donors of various blood products is important to prevent transmission of the disease from these donors to recipients. In the majority of cases, the detection of antibodies directed against either the viral core protein, a Mr approximately equal to 24,000 protein termed p24 gag, or the viral envelope antigen is proof of previous viral infection. Thus, we have expressed the p24 gag antigen in Escherichia coli in order to produce a diagnostic reagent for the detection of virus exposure. The bacterially synthesized antigen reacts with human and rabbit antisera directed against the native p24 gag protein in both electrophoretic transfer blot assay and ELISA. In addition, the use of bacterially produced antigens for ELISAs gave results that were comparable to those obtained by using antigens isolated from the virus.
Loss of the normal N-ras allele in a mouse thymic lymphoma induced by a chemical carcinogen.
Authors: Guerrero I , Villasante A , Corces V , Pellicer A
Published: May 2006

Young mice injected with the carcinogen N-nitroso-N-methylurea develop thymic lymphomas 2-4 months later. We previously have shown that these tumors frequently contain an activated N-ras gene that can transform rodent fibroblasts in vitro. We report here the intron/exon structure of such an activated N-ras gene and the sequence of its four coding exons. A single nucleotide change is responsible for the transforming alteration, a C----A transversion in the first base of codon 61. Through the use of synthetic oligonucleotides as hybridization probes, we show that this tumor lacks the normal allele of the N-ras gene. The implications of this finding for oncogene dominance are discussed.
Lens-specific expression and developmental regulation of the bacterial chloramphenicol acetyltransferase gene driven by the murine alpha A-crystallin promoter in transgenic mice.
Authors: Overbeek P A , Chepelinsky A B , Khillan J S , Piatigorsky J , Westphal H
Published: May 2006

Two lines of transgenic mice with one to two copies of a DNA fragment containing nucleotides -364 to +45 of the murine alpha A-crystallin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene expressed the CAT gene only in their eye lenses. Both CAT activity and alpha A-crystallin were first detected in eyes at approximately 12.5 days of embryonic development, suggesting that the alpha A-CAT fusion gene and the endogenous alpha A-crystallin gene are co-regulated during lens development in the transgenic mice. These experiments show that the murine alpha A-crystallin gene contains a short, cis-acting, tissue-specific regulatory sequence at its 5' end that can target the expression of the bacterial CAT gene, and probably foreign eukaryotic genes, specifically to the ocular lens.