bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol.
Authors: Sugimoto Y , Whitman M , Cantley L C , Erikson R L
Published: May 2006

The ability of purified Rous sarcoma virus transforming gene product, pp60v-src, to phosphorylate phosphatidylinositol and diacylglycerol was investigated. Phosphatidylinositol was phosphorylated to form both mono- and diphosphorylated derivatives. 1,2-Diacylglycerol was phosphorylated to form phosphatidic acid. These activities showed the same thermolability and the same sensitivity to inhibitors as shown by the casein kinase activity of pp60v-src. In addition, when serum-starved chicken embryo fibroblasts transformed by a virus mutant temperature-sensitive for transformation were shifted from the nonpermissive to permissive temperature, an increase of 50-100% in the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid was observed, as compared to uninfected cells.
In vitro mutagenesis and transcriptional analysis of a mouse ribosomal promoter element.
Authors: Skinner J A , Ohrlein A , Grummt I
Published: May 2006

An RNA polymerase I control region essential for initiation of pre-rRNA transcription has been identified by mutagenesis in vitro of mouse rDNA (ribosomal RNA genes) and transcription in a cell-free system derived from Ehrlich ascites cells. Substitution of nucleotides between -35 and -14 by foreign DNA sequences caused a loss of template activity, which indicates that an important promoter element is located within this region. To identify the nucleotides essential for RNA polymerase I function, single and multiple point mutations within this control region were generated and the modified DNAs were assayed for template activity. The phenotypes of mutants in which C-to-T transitions have been introduced at positions -36, -31, -27, -22, -21, and -13 were identical to the wild type. Conversion of G to A at position -15 resulted in a 20% increase of promoter activity, whereas a G-to-A transition at -16 decreased transcription by 95%. Competition experiments between mutant and wild-type DNAs suggest that the guanine at -16, which is evolutionarily highly conserved, interacts with essential components of the transcription apparatus.
Antigenic stimulation regulates the level of expression of interleukin 2 receptor on human T cells.
Authors: Hemler M E , Brenner M B , McLean J M , Strominger J L
Published: May 2006

Antigen-specific, interleukin 2 (IL-2)-dependent human T-cell lines and clones were utilized to study the relationship between IL-2 receptor expression and antigenic stimulation. T cells that had not been exposed to antigen for 2 wk or more expressed a stable low level of the IL-2 receptor. After reexposure to antigen, a 10- to 30-fold increase in the level of the IL-2 receptor was rapidly induced, with the peak level of IL-2 receptor expression occurring at 15-30 hr. This peak preceded the peak in cell proliferation [( 3H]thymidine incorporation), which was at 48-72 hr. Within 2-14 days after peak IL-2 receptor expression, it returned to a low base-line level. The transient elevation in IL-2 receptor level was antigen specific because it occurred in response to specific allogeneic stimulator cells but not after exposure to cells expressing irrelevant HLA allotypes. The levels of other cell-surface proteins, including those related to T-cell activation (HLA-DR, T10, 4F2, A-1A5) as well as T3, which has been proposed to be a component of the T-cell receptor complex for antigen, did not change in response to antigen exposure or deprivation. Because IL-2 was maintained at a consistently high level throughout these experiments, the antigen-induced changes in the IL-2 receptor appear to be independent of changes induced by IL-2 itself. Both cloned T cells and mixed populations containing T4 and T8 subsets showed similar IL-2 receptor responsiveness, indicating that this finding is generalizable to most, if not to all, antigen-responsive T cells.
Transmission of the hepatitis B virus-associated delta agent to the eastern woodchuck.
Authors: Ponzetto A , Cote P J , Popper H , Hoyer B H , London W T , Ford E C , Bonino F , Purcell R H , Gerin J L
Published: May 2006

delta agent of human origin was inoculated into four woodchucks chronically infected with woodchuck hepatitis virus (WHV). The animals developed delta infections with serologic patterns similar to those previously observed in human and chimpanzee infections. delta antigen was detected transiently in serum and liver and was followed by seroconversion to anti-delta antibody. Analogous to the chimpanzee model of delta infection, serum and hepatocyte markers of WHV were suppressed in the woodchuck during acute delta infection. The suppression of WHV DNA in serum was evident only during the time of delta-antigen positivity, while the inhibition of other WHV markers was more protracted. The delta antigen in woodchuck sera circulated as an internal component of a particle similar in size to the human delta particle (36-nm diameter) and was encapsidated by the woodchuck hepatitis virus surface antigen; delta antigen from infected woodchuck and chimpanzee livers had similar biophysical properties. Histologic analysis showed that experimental delta infection is associated with a transient acute hepatitis in woodchucks and loss of hepatocytes carrying WHV antigens. The lesions differed from the conspicuous hepatitis associated with reappearance of WHV replication. Hepatitis B-like viruses, therefore, appear to provide the requisite helper functions for delta replication and the woodchuck represents a useful model for study of the virology and pathology of the delta agent.
Birth of rhesus monkey infant after in vitro fertilization and nonsurgical embryo transfer.
Authors: Bavister B D , Boatman D E , Collins K , Dierschke D J , Eisele S G
Published: May 2006

The birth of a rhesus monkey resulting from in vitro fertilization is reported. Oocytes recovered at laparoscopy from five gonadotropin-stimulated donors were inseminated in vitro with sperm preincubated with caffeine and dibutyryl cyclic AMP. After insemination, oocytes were cultured for 33-46 hr. Twenty-two embryos were transferred nonsurgically into 11 recipient females. One recipient showed signs of implantation but did not carry to term. A second female became pregnant after receiving one 4-cell and one 6-cell embryo fertilized in vitro. The subsequent course of early pregnancy and embryonic and fetal development were characteristic of normal singleton pregnancies. A healthy term male infant was delivered by Cesarean section 176 days after fertilization. This birth has validated our procedures for in vitro fertilization of rhesus monkey gametes and provides an experimental model for studies of early embryonic development in primates.
Multiple opiate receptors in the guinea pig enteric nervous system: unmasking the copresence of receptor subtypes.
Authors: Gintzler A R , Hyde D
Published: May 2006

Experiments were performed in order to obtain physiological evidence for the presence of delta opioid receptors in the guinea pig isolated ileum. The nonequilibrium narcotic antagonists beta-chlornaltrexamine (beta-CNA) and beta-funaltrexamine (beta-FNA) differentially affected inhibitory responses to mu- and delta-specific opiate ligands (q value). These results indicate that these agonists do not act through identical populations of receptor in the ileum despite the similarity of their naloxone Ke values (apparent naloxone dissociation constant determined physiologically). Moreover, elimination of myenteric mu receptors resulted in a 10-fold increase in the naloxone Ke for the highly selective delta agonist Tyr-D-Ser-Gly-Phe-Leu-Thr, suggesting the involvement of a second receptor subtype. It is suggested that this receptor is delta in nature.
Variations in DNA methylation during mouse cell differentiation in vivo and in vitro.
Authors: Razin A , Webb C , Szyf M , Yisraeli J , Rosenthal A , Naveh-Many T , Sciaky-Gallili N , Cedar H
Published: May 2006

Mouse teratocarcinoma cells induced to differentiate in vitro undergo a massive (30%) demethylation of DNA. A similar undermethylation is also observed in the mouse extraembryonic membranes, the yolk sac and placenta. In both cases, the decrease in methyl moieties occurs at a large number of CpG sites spread out over the entire genome, as indicated by a restriction enzyme analysis of several mouse genes including dhfr, beta-major globin, and the H-2K gene family. In contrast to this, the embryo itself appears to undergo methylation de novo during early stages of embryogenesis. Thus, as opposed to somatic cells, events during early mouse development are associated with wide variations in the level of DNA methylation. Although these changes in DNA methylation seem to be an integral part of the differentiation process, its relation to specific gene expression is still unclear.
Phorbol esters potentiate tyrosine phosphorylation of epidermal growth factor receptors in A431 membranes by a calcium-independent mechanism.
Authors: Moon S O , Palfrey H C , King A C
Published: May 2006

Incubation of membranes prepared from A431 cells with either epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulates the transfer of 32phosphate from [gamma-32P]ATP into 8-10 membrane proteins. The major phosphorylated protein migrates on NaDodSO4/polyacrylamide gels with an apparent Mr of 180,000, corresponding to the previously identified EGF receptor. Stimulation of EGF receptor phosphorylation by PMA does not require Ca2+, suggesting that prior activation of protein kinase C is not a prerequisite for phosphate transfer. PMA-enhanced phosphorylation proceeds at 4 degrees C and requires Mn2+, both properties of tyrosine-specific protein kinases. Phospho amino acid analysis of the Mr 180,000 receptor band shows that only tyrosine residues are phosphorylated when A431 membranes are treated with either EGF or PMA. Moreover, proteolysis reveals that these residues are located in the same peptides of the receptor. These results demonstrate that a potent tumor-promoting phorbol ester can mimic a critical early response usually elicited by EGF.
A large interspersed repeat found in mouse DNA contains a long open reading frame that evolves as if it encodes a protein.
Authors: Martin S L , Voliva C F , Burton F H , Edgell M H , Hutchison C A
Published: May 2006

DNA sequence analysis of a region contained within a large, interspersed repetitive family of mice reveals a long open reading frame. This sequence extends 978 base pairs between two stop codons, creating a reading frame that is open for 326 amino acids. The DNA sequence in this region is conserved between three distantly related Mus species, as well as between mouse and monkey, in a manner that is characteristic of regions undergoing selection for protein function.
Expression of immunoglobulin heavy chain at a high level in the absence of a proposed immunoglobulin enhancer element in cis.
Authors: Wabl M R , Burrows P D
Published: May 2006

The major intron between the J and C gene segments of the immunoglobulin heavy (H) chain locus contains an enhancer-like sequence, and it has been proposed that this enhancer is necessary to achieve high levels of H chain expression. We have isolated a subclone of the lymphoid pre-B cell line 18-81 that lacks this enhancer but nevertheless produces mu chain at the level characteristic of pre-B cells. Another subclone with a larger deletion does not produce mu chain, but upon fusion with a myeloma that does not produce any immunoglobulin chain, mu chain is expressed by the homolog from the pre-B subclone. The hybridoma lacks the proposed enhancer element in cis; nevertheless it produces as much mu chain as other plasma cell hybridomas. Therefore, this enhancer element is not obligatory for a high level of H chain production.
Increased free cholesterol in plasma low and very low density lipoproteins in non-insulin-dependent diabetes mellitus: its role in the inhibition of cholesteryl ester transfer.
Authors: Fielding C J , Reaven G M , Liu G , Fielding P E
Published: May 2006

Recombination of low and very low density lipoproteins (VLDL and LDL) from normal subjects with plasma from patients with non-insulin-dependent diabetes mellitus significantly increased the reduced rate of transfer of cholesteryl ester to these lipoproteins, which is characteristic of diabetic plasma, whereas diabetic VLDL and LDL reduced cholesteryl ester transfer rates in normal plasma. VLDL and LDL from diabetic plasma had an increased ratio of free cholesterol to phospholipid compared to normal, and unlike normal VLDL and LDL spontaneously lost free cholesterol to high density lipoprotein. These data suggest that the block to cholesteryl ester transfer to these lipoproteins in non-insulin-dependent diabetes is mediated by their increased free cholesterol content and may be related to the increased risk of these patients for developing atherosclerosis.
DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication.
Authors: DiNardo S , Voelkel K , Sternglanz R
Published: May 2006

A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified. Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity. Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene. Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication. These cells are arrested at medial nuclear division. Analysis of 2-microns plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers. The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.
Dictyostelium transposable element DIRS-1 has 350-base-pair inverted terminal repeats that contain a heat shock promoter.
Authors: Zuker C , Cappello J , Lodish H F , George P , Chung S
Published: May 2006

DIRS-1 is a 4.7-kilobase-pair repetitive and apparently transposable Dictyostelium genetic element that is transcribed during differentiation or after heat shock. The terminal regions of DIRS-1 are inverted repeats of 330 base pairs. The repeats are highly conserved both within a given element as well as between different members of the family (less than 10% divergence). At the distal end of all left repeats is a 32-nucleotide sequence composed almost entirely of A and T residues. In addition to this 32-base A + T sequence, the distal region of all right repeats is extended by a 28-base-pair A + T-rich sequence that is identical in all copies. The sequences flanking each DIRS-1 sequence are completely dissimilar, and there appears to be no duplication of the genomic DNA sequence at the presumed point of DIRS-1 insertion. The terminal repeats can also be found interspersed in the genome independently of the complete element. In addition, the terminal repeats carry a 15-nucleotide sequence that greatly resembles the Drosophila consensus heat shock promoter and may be involved in the transcriptional induction of the DIRS-1 sequences.
Nucleotide sequence of a multiple-copy gene for the B protein of photosystem II of a cyanobacterium
Authors: Mulligan Bernard , Schultes Neil , Chen Lin , Bogorad Lawrence
Published: May 2006

Chloroplast photogene
32
codes for the 32-kilodalton triazine herbicide-binding protein at the B site of electron transport in photosystem II of the photosynthetic apparatus—its product is the B protein and the gene is accordingly designated
ps2B
here. The cyanobacteria
Anacystis nidulans
R2,
Fremyella diplosiphon
, and
Nostoc
sp. MAC each contain several copies of
ps2B
. The sequence of one copy of
ps2B
from
Fremyella, ps2B-1
, has been determined. The longest open reading frame would code for a protein of 360 amino acids. Although the deduced amino acid sequence of
ps2B-1
is highly homologous overall to that of the corresponding spinach protein [Zurawski, G., Bohnert, H. J., Whitfeld, P. R. & Bottomley, W. (1982)
Proc. Natl. Acad. Sci. USA
79, 7699-7703] and, excluding neutral substitutions, the homology is 95% for an internal segment of 309 amino acids, there are a number of nonneutral amino acid substitutions. Most of the differences in net charge and polarity occur in the first 20 amino acids at the amino terminus and in the amino acid composition at the carboxyl terminus. The nucleotide sequences are 76% homologous overall. Conserved sequences resembling prokaryotic “-10” and “-35” regions are found at remarkably similar positions in the spinach and
F. diplosiphon
sequences although the surrounding sequences show only occasional homologies.
Mapping of DNase I-hypersensitive sites in the upstream DNA of human embryonic epsilon-globin gene in K562 leukemia cells.
Authors: Tuan D , London I M
Published: May 2006

We have mapped the DNase I-hypersensitive sites around the epsilon-globin and c-myc genes in two human leukemia cell lines K562 and HL60. In K562 cells in which the epsilon-globin gene is transcribed, six DNase I-hypersensitive sites are found in 6 kilobases (kb) of upstream flanking DNA; in HL60 cells in which the c-myc gene is expressed, two DNase I-hypersensitive sites are observed in 2 kb of upstream DNA. Neither the inactive epsilon-globin gene in HL60 cells nor the inactive c-myc gene in K562 cells displays such upstream DNase I-hypersensitive sites. Our results are consistent with previous studies that have shown DNase I-hypersensitive sites within 1 kb of the 5' end of other expressed genes. In addition, we have found sites displaying even more DNase I sensitivity further upstream of expressed epsilon-globin and c-myc genes. Among the six DNase I-hypersensitive sites of the expressed epsilon-globin gene in K562 cells, the most sensitive site is located about 6 kb upstream of the epsilon-globin gene. When correlated with the DNA sequence upstream of the epsilon-globin gene, this site was found to correspond to a region that contains a stretch of 28 consecutive Ts, three enhancer core-like sequences, and a stretch of consecutive (C-A)15(T-A)6 alternating purine and pyrimidine bases. These findings suggest the possibility that an enhancer element for epsilon-globin gene expression resides within this DNase I-hypersensitive site.
Tumor-promoting phorbol ester stimulates tyrosine phosphorylation in U-937 monocytes.
Authors: Grunberger G , Zick Y , Taylor S I , Gorden P
Published: May 2006

Solubilized lectin-purified extracts from human monocyte-like cells (U-937) and freshly isolated human mononuclear cells preincubated in the presence of phorbol 12-myristate 13-acetate (PMA) stimulated phosphorylation of synthetic tyrosine-containing polymers and of casein. Tyrosine phosphorylation was confirmed by phospho amino acid analysis. PMA stimulated phosphorylation of exogenous substrates in a time- and concentration-dependent manner. This phosphorylation reaction did not require addition of phospholipid, diolein, or calcium. Biologically inactive phorbol compounds did not stimulate phosphorylation in this system. In addition, PMA enhanced phosphorylation of a Mr approximately equal to 140,000 protein as well as several other endogenous proteins in the U-937 extracts. PMA treatment stimulated predominantly phosphorylation on tyrosine residues of the Mr 140,000 protein. Tyrosine phosphorylation, typical of growth-promoting peptides such as insulin or epidermal growth factor, is believed to play a role in regulating normal and disordered cellular growth and proliferation. The demonstration of PMA-stimulated tyrosine phosphorylation might provide a clue to the mechanism of cellular differentiation and proliferation induced by the tumor promoter.
Human autoantibodies against desmosomes: possible causative factors in pemphigus.
Authors: Jones J C , Arnn J , Staehelin L A , Goldman R D
Published: May 2006

Pemphigus is a human disease that causes extensive blistering of the skin. This blistering is related to a loss of epidermal cell cohesion and is accompanied by circulating autoantibodies that stain epidermal cell surfaces, as shown by immunofluorescence microscopy. One of the major components involved in epidermal cell cohesion is the desmosome. The pathological changes that accompany pemphigus led us to determine whether the autoantibodies are specific for desmosomes. Incubation of cultured mouse keratinocytes in medium containing pemphigus antiserum leads to cell separation at cell-cell contact sites, which possess desmosomes. Tissue sections of mouse skin processed for indirect immunofluorescence, using pemphigus antiserum or a rabbit antiserum directed against components of desmosomes, show similar punctate cell-surface staining patterns within the epidermis. Cultured mouse keratinocytes possessing well-defined intermediate filament bundles (tonofilaments) and desmosomes were processed for double indirect immunofluorescence, using a monoclonal antibody directed against mouse skin keratin and either pemphigus antiserum or the desmosome antiserum. The keratinocytes exhibit a complex system of keratin-containing tonofilaments. Tonofilaments in contacting cells are separated by thin dark bands at the cell surface, which correspond precisely to desmosomal plaques seen by phase-contrast microscopy. These bands specifically stain with both pemphigus antiserum and the desmosome antiserum. Double indirect immunofluorescence of the cultured mouse keratinocytes, using pemphigus antiserum and the desmosome antiserum, reveals that the pemphigus autoantibodies stain the same areas of cell-cell contact as the desmosome antibodies. Our evidence supports the idea that pemphigus blisters form, at least in part, from a specific antibody-induced disruption of desmosomes in the epidermis.
Topology of bonding in π-electron systems
Authors: Merrifield R. E. , Simmons H. E.
Published: May 2006

Delocalization in conjugated π-electron systems is shown to be closely related to the topological concept of connectivity. A topological bond order is defined as a quantitative measure of the extent to which the subspace of a pair of adjacent atoms approximates a disconnected component of the molecular topological space. The agreement between this measure and molecular-orbital bond orders in the ordering of bond strengths within molecules is near perfect. There is also excellent quantitative agreement between these two measures of bond strength. Bond orders appear to be primarily determined by molecular topology. A new type of intramolecular equivalence relation between bonds, called “coresiduality,” emerges from the topological analysis. Two bonds are coresidual if the residues remaining when each pair of adjacent atoms is excised from the molecule are isomorphic. Coresidual bonds have equal bond orders. Coresiduality includes, but is more general than, equivalence due to symmetry.
Multiple polymorphic alpha- and beta-tubulin mRNAs are present in sea urchin eggs.
Authors: Alexandraki D , Ruderman J V
Published: May 2006

Multiple alpha- and beta-tubulin RNAs were found in the mature unfertilized eggs of the sea urchin Lytechinus pictus. The alpha-tubulin RNAs were polymorphic in number, size, and relative amounts in the eggs of different females. Five to seven different size classes [1.75-4.2 kilobases (kb)] were detected on RNA gel blots. All egg preparations contained variable amounts of 1.8- and 2.25-kb beta-tubulin RNAs, and a few of them contained an additional 2.9-kb beta-tubulin RNA. The total amount of alpha-tubulin RNA did not always parallel that of beta-tubulin RNA. A portion of all of the various alpha- and beta-tubulin RNAs were polyadenylylated. RNase H digestions ruled out the possibility that some of these RNAs represented a single transcript bearing different lengths of 3' poly(A). One class of alpha-tubulin RNAs (2.4-2.65 kb) was reduced to 2 kb by RNase H, suggesting the presence of internal oligo(A) regions. All of the egg beta-tubulin RNAs sedimented as free ribonucleoprotein particles. Only a small portion of the 1.75- to 3.6-kb alpha-tubulin RNAs, but most of the 4.2-kb alpha-tubulin RNA, were found on polysomes before fertilization. In the 30-min embryo, small amounts of each of the various alpha- and beta-tubulin RNAs were recruited onto polysomes. Thus, each of the multiple polymorphic alpha- and beta-tubulin RNAs in the egg represent translationally competent mRNA.
Eukaryotic transient expression system dependent on transcription factors and regulatory DNA sequences of vaccinia virus.
Authors: Cochran M A , Mackett M , Moss B
Published: May 2006

A transient expression system in which chimeric genes are expressed in cells infected with vaccinia virus was developed. Recombinant plasmids containing the promoter regions of vaccinia virus genes ligated to the coding segment of the prokaryotic chloramphenicol acetyltransferase (CAT) gene were constructed. When the plasmids were introduced into vaccinia virus-infected cells by transfection, the chimeric gene was expressed and significant levels of CAT accumulated. CAT activity was not detected when the same recombinant plasmid was introduced into uninfected cells, nor was activity detected when the vaccinia virus promoter was absent from the plasmid or was replaced by simian virus 40 or Rous sarcoma virus promoters. This specificity indicated that expression is dependent on a cis-acting vaccinia virus promoter region within the recombinant plasmid and diffusible trans-acting transcription factors produced during virus infection. The lack of effect of a simian virus 40 enhancer element inserted upstream of the vaccinia virus promoter region also distinguished this system from systems dependent on RNA polymerase II. Although replication of the recombinant plasmid could not be detected in either uninfected or vaccinia virus-infected cells, an inhibitor of DNA synthesis significantly reduced CAT expression. This result, as well as the kinetics of CAT synthesis, suggests that replication of viral DNA templates can enhance transcription of chimeric genes in recombinant plasmids.
Calcitonin gene-related peptide and its binding sites in the human central nervous system and pituitary.
Authors: Tschopp F A , Henke H , Petermann J B , Tobler P H , Janzer R , Hökfelt T , Lundberg J M , Cuello C , Fischer J A
Published: May 2006

Binding sites for synthetic human 125I-labeled calcitonin gene-related peptide (125I-CGRP) have been demonstrated in membranes of the human nervous system. Binding was high in the cerebellar cortex (1.35 +/- 0.27 fmol/mg of tissue; mean +/- SEM), spinal cord (1.06 +/- 0.27 to 1.27 +/- 0.23 fmol/mg), and nucleus dentatus (1.02 +/- 0.15 fmol/mg), intermediate in the inferior colliculus (0.80 +/- 0.14 fmol/mg) and substantia nigra (0.75 +/- 0.14 fmol/mg), low in the neocortex, globus pallidus, nucleus caudatus, hippocampus, amygdala, superior colliculus, thalamus, and hypothalamus (0.15-0.32 fmol/mg), and negligible in spinal and sympathetic ganglia and pituitary (less than 0.04 fmol/mg). Autoradiography showed distinct 125I-CGRP binding over the molecular and Purkinje cell layers of the cerebellar cortex and over the substantia gelatinosa posterior of the spinal cord. The highest levels of CGRP-like components were recognized in the dorsal part of the spinal cord and the pituitary gland. In the ventral part of the spinal cord as well as in the pituitary and thyroid glands, CGRP values were higher when measured by radioreceptorassay as compared to RIA, indicating that at least two CGRP-like components are present. The predominant CGRP-like peak on HPLC had the retention time of synthetic human CGRP. Immunohistochemistry revealed the presence of a dense plexus of CGRP immunoreactive nerve fibers in the dorsal horn of the spinal cord.
Low-temperature magnetic circular dichroism studies of native laccase: spectroscopic evidence for exogenous ligand bridging at a trinuclear copper active site.
Authors: Allendorf M D , Spira D J , Solomon E I
Published: May 2006

The detailed nature of N-3 binding at the multi-copper active site in native laccase is investigated through a combination of low-temperature magnetic circular dichroism (LTMCD) and absorption spectroscopies. This combination of techniques allows charge-transfer spectral features associated with N-3 binding to the paramagnetic type 2 Cu(II) to be differentiated from those associated with binding to the antiferromagnetically coupled, and therefore diamagnetic, binuclear type 3 Cu(II) site. Earlier absorption titration studies have indicated that N-3 binds with two different binding constants, yielding a high-affinity and a low-affinity form. The studies presented here are interpreted as strong evidence that low-affinity N-3 bridges the paramagnetic type 2 and diamagnetic type 3 binuclear Cu(II) sites in fully oxidized laccase. This assignment is further supported by features in the MCD spectrum whose intensity correlates with an EPR signal associated with uncoupled type 3 Cu(II) sites. In these sites, N-3 has displaced the endogenous bridge, thereby rendering the site paramagnetic and detectable by both LTMCD and EPR spectroscopy. High-affinity N-3 is found to bind to the paramagnetic type 2 Cu(II) site in a limited fraction of the protein molecules that contains reduced type 3 sites. Finally, the possible role of this trinuclear (type 2-type 3) Cu(II) active site in enabling the irreversible reduction of dioxygen to water is considered.
A glycolipid antigen specific to Mycobacterium paratuberculosis: structure and antigenicity.
Authors: Camphausen R T , Jones R L , Brennan P J
Published: May 2006

Mycobacterium paratuberculosis (National Animal Disease Center strain 18 and American Type Culture Collection strain 19698), the causative agent of Johne Disease (bovine paratuberculosis), contains a major immunoreactive glycopeptidolipid (polar GPL-I) that has been isolated and characterized. (formula; see text) Thus, the glycolipid antigen belongs to the polar mycoside C glycopeptidolipid family present in other mycobacterial species. The distal 2,3-di-O-methyl-alpha-L-fucopyranosyl-(1----3)-alpha-L-rhamnopyranosi de unit, the obvious antigen determinant, appears to be characteristic of M. paratuberculosis. The glycolipid can be recognized readily in isolates of M. paratuberculosis by TLC and its presence may be used as a characteristic marker of the infectious agent. The polar glycopeptidolipid was highly reactive in ELISA against serum from an animal hyperimmunized with M. paratuberculosis strain 18, indicating its basic immunogenicity.
Rapid "footprinting" on supercoiled DNA.
Authors: Gralla J D
Published: May 2006

A DNase protection technique is described and applied to the interaction of three lac control proteins with supercoiled lac DNA. The technique uses end-labeled oligonucleotide primers to probe specific DNA regions as an alternative to protocols requiring restriction endonuclease cleavage or blotting. Thus DNA may be probed with high resolution in its native state. It is demonstrated that the introduction of supercoiling into DNA accelerates the rate of lac ps promoter binding by RNA polymerase but does not alter the positions at which polymerase, c-AMP-binding protein, or lac repressor bind to lac DNA.
Covalent modification of the iron protein of nitrogenase from Rhodospirillum rubrum by adenosine diphosphoribosylation of a specific arginine residue.
Authors: Pope M R , Murrell S A , Ludden P W
Published: May 2006

Nitrogenase in Rhodospirillum rubrum is inactivated in vivo by the covalent modification of the Fe protein with a nucleotide. The preparation of two modified peptides derived from proteolytic digestion of the inactive Fe protein is described. The modifying group is shown to be adenosine diphosphoribose, linked through the terminal ribose to a guanidino nitrogen of arginine. The structural features were established by using proton and phosphorus NMR, positive- and negative-ion fast atom bombardment mass spectrometry, and fast atom bombardment/collisionally activated decomposition mass spectrometry. Spectral methods along with chromatographic analysis and sequential degradation established the sequence of the modification site of Fe protein as Gly-Arg(ADR-ribose)-Gly-Val-Ile-Thr. This corresponds to the sequence in the Fe protein from Azotobacter vinelandii for amino acid residues 99 to 104.
Separation of endogenous calmodulin- and cAMP-dependent kinases from microtubule preparations.
Authors: Vallano M L , Goldenring J R , Buckholz T M , Larson R E , DeLorenzo R J
Published: May 2006

Both cAMP- and calmodulin-dependent kinases are proposed regulators of microtubule function by means of their ability to phosphorylate microtubule-associated protein 2(MAP 2). A cAMP-dependent kinase/MAP 2 complex is endogenous to microtubules. In this report, we demonstrate that an endogenous calmodulin-dependent kinase that phosphorylates MAP 2 as a major substrate is also present in microtubules prepared under conditions that preserve kinase activity. This enzyme is identical to a calmodulin-dependent kinase purified previously from rat brain cytosol. A fraction containing calmodulin-dependent kinase and MAP 2 was separated from the cAMP-dependent kinase/MAP 2 complex by gel filtration chromatography of microtubule protein in high ionic strength buffer. All of the recovered calmodulin-dependent kinase activity in microtubules eluted in a single protein peak. The specific activity of the enzyme for MAP 2 was enriched 31-fold in this fraction compared to cytosol. Two-dimensional tryptic phosphopeptide mapping revealed that the endogenous cAMP- and calmodulin-dependent kinases phosphorylated distinct sites on MAP 2. These data demonstrate that both kinases are present in microtubule preparations and that they may differentially regulate MAP 2 function by phosphorylating separate sites on MAP 2.
A second tRNA binding site on elongation factor Tu is induced while the factor is bound to the ribosome.
Authors: Van Noort J M , Kraal B , Bosch L
Published: May 2006

Previously, we reported that the antibiotic kirromycin induces two tRNA-binding sites on the elongation factor Tu. The classical binding site (site I) binds aminoacyl-tRNA and, with much less affinity, deacylated tRNA. The kirromycin-induced site II binds aminoacyl-tRNA, peptidyl-tRNA, and deacylated tRNA with comparable affinities. Accordingly, 3'-oxidized tRNA can be cross-linked in the presence of the antibiotic to two specific sites of EF-Tu: Lys-237 and Lys-208. Here, we report that 3'-oxidized tRNAPhe, bound to a ribosome-poly(U) complex, can also be cross-linked to either one of these two sites. When located in the ribosomal peptidyl site, it cross-links exclusively to Lys-208; when located in the ribosomal aminoacyl site, it cross-links exclusively to Lys-237, irrespective of the presence of kirromycin. Since no cross-linking could be detected in the absence of ribosomes and kirromycin, we conclude that the tRNA-binding site II is induced upon interaction of aminoacyl-tRNA-EF-Tu-GTP with the ribosome-mRNA complex. The results indicate that, on the ribosome, EF-Tu interacts with peptidyl-site-bound peptidyl-tRNA through tRNA-binding site II and with aminoacyl-site-bound aminoacyl-tRNA through tRNA-binding site I.
Preferential orientation of centrioles toward the heart in endothelial cells of major blood vessels is reestablished after reversal of a segment.
Authors: Rogers K A , McKee N H , Kalnins V I
Published: May 2006

The distribution of centrioles was examined in porcine and rabbit vascular endothelial cells fixed in situ and prepared en face for immunofluorescent staining with rabbit sera that specifically stain these organelles. In endothelial cells lining the major blood vessels of the pig, the centrioles are preferentially located on the heart side of the nucleus regardless of the direction of blood flow. A similar distribution is seen in the inferior vena cava of the rabbit but not in the rabbit aorta. In the major vessels of the pig and in the rabbit inferior vena cava, 60%-80% of the endothelial cells have their centrioles located on the side of the nucleus toward the heart, 10%-20% have them on the side away from the heart, and 7%-15% have them in a central position along the side of the nucleus. To determine whether this preferential orientation is reestablished, microvascular surgical techniques were used to reverse a 3-cm segment of the inferior vena cava between the left renal vein and the common iliac veins of the rabbit. Within 1 week of the reversal, some of the centrioles had migrated from the end away from the heart to a more central position. During the following weeks, an increasing number of endothelial cells had their centrioles located on the heart side of the nucleus; after 12 weeks, values similar to those in the nonreversed inferior vena cava were reached in the reversed segment. The demonstration that the preferential orientation of centrioles on the heart side of the nucleus is reestablished after reversal of a segment suggests that the observed polarity is important for normal functioning of vascular endothelium.
Interleukin 3 promotes erythroid burst formation in "serum-free" cultures without detectable erythropoietin.
Authors: Goodman J W , Hall E A , Miller K L , Shinpock S G
Published: May 2006

Erythroid burst-forming units (BFU-E) from mouse bone marrow were grown for 7 days in agar or serum-free methylcellulose cultures in the presence or absence of erythropoietin (Ep) and/or interleukin 3 (IL-3). It was found that IL-3, even in the absence of serum and detectable Ep, was able to stimulate the full development of many erythroid bursts. This IL-3 effect was cell-dose dependent and did not appear to correlate with Ep dose. Spontaneous bursts and those stimulated by Ep only were rare and when seen were very small relative to those produced by IL-3 or IL-3 plus Ep. When addition of IL-3 or Ep to 7-day cultures was delayed, IL-3 but not Ep was shown to maintain BFU-E. No evidence was found by radioimmunoassay that Ep was produced or released in 7-day, "serum-free" cultures of bone marrow nor was Ep activity detected in culture media except those to which it had been added deliberately.
Basement membrane heparan sulfate proteoglycans are concentrated in the laminae rarae and in podocytes of the rat renal glomerulus.
Authors: Stow J L , Sawada H , Farquhar M G
Published: May 2006

A polyclonal antibody was raised against a proteoglycan fraction purified from extracts of isolated rat renal glomeruli. The antibody was characterized by column chromatography and by NaDodSO4/PAGE analysis of immunoprecipitates and was shown to recognize specifically the core protein of a population of heparan sulfate proteoglycans (Mr 130,000) found in the glomerular basement membrane and in other renal basement membranes. The antibody was used to localize the core proteins of this population of heparan sulfate proteoglycans (HSPG) in the glomerulus by immunoelectron microscopic techniques. These HSPG were found to be concentrated in the lamina rarae interna and externa of the glomerular basement membrane (GBM) and precursor forms were detected in the endoplasmic reticulum and Golgi cisternae of glomerular visceral epithelial cells (podocytes).
Analysis of DNA haplotypes suggests a genetic predisposition to trisomy 21 associated with DNA sequences on chromosome 21.
Authors: Antonarakis S E , Kittur S D , Metaxotou C , Watkins P C , Patel A S
Published: May 2006

To test the hypothesis that there is a genetic predisposition to nondisjunction and trisomy 21 associated with DNA sequences on chromosome 21, we used DNA polymorphism haplotypes for chromosomes 21 to examine the distribution of different chromosomes 21 in Down syndrome and control families from the same ethnic group. The chromosomes 21 from 20 Greek families with a Down syndrome child and 27 control Greek families have been examined for DNA polymorphism haplotypes by using four common polymorphic sites adjacent to two closely linked single-copy DNA sequences (namely pW228C and pW236B), which map somewhere near the proximal long arm of chromosome 21. Three haplotypes, +, +---, and - with respective frequencies of 43/108, 24/108, and 23/108, account for the majority of chromosomes 21 in the control families. However, haplotype - was found to be much more commonly associated with chromosomes 21 that underwent nondisjunction in the Down syndrome families (frequency of 21/50; X2 for the two distributions is 9.550; P = 0.023; degrees of freedom, 3). The two populations (control and trisomic families) did not differ in the distribution of haplotypes for two DNA polymorphisms on chromosome 17. The data from this initial study suggest that the chromosome 21, which is marked in Greeks with haplotype - for the four above described polymorphic sites, is found more commonly in chromosomes that participate in nondisjunction than in controls. We propose an increased tendency for nondisjunction due to DNA sequences associated with a subset of chromosomes 21 bearing this haplotype.
Evidence that receptors mediating central synaptic potentials extend beyond the postsynaptic density.
Authors: Faber D S , Funch P G , Korn H
Published: May 2006

Physiological recordings and computer simulations of unitary inhibitory postsynaptic potentials in the Mauthner cell of the goldfish central nervous system have been used to estimate the expected size of the postsynaptic receptor matrix at individual junctions. Simultaneous pre- and postsynaptic recordings were used to determine the kinetic parameters of the quantal responses under normal conditions and in the presence of strychnine, a competitive antagonist of glycine, which is the putative transmitter at these synapses. Calculations indicate that if the postsynaptic density, which has a radius of 0.1 micron, were to accommodate the population of channels estimated to be opened during a quantal response, the glycine binding site density in that region would be unrealistically high. Computer simulation of the quantal responses included transmitter diffusion, transmitter-receptor interactions, and channel activation under conditions including both normal and lowered binding site densities, the latter corresponding to the experimental data obtained with strychnine. The data indicate that the synaptic receptors involved in generating unitary responses are widely distributed to include regions located outside the junctional area, which directly faces the presynaptic release sites. We further suggest that the receptor matrix is surrounded by a restricted diffusional space; this geometrical organization may underlie the finding that response rise times are relatively independent of receptor binding site densities.
Regulation of methionine synthesis in
Escherichia coli
: Effect of
metJ
gene product and
S
-adenosylmethionine on the expression of the
metF
gene
Authors: Shoeman Robert , Redfield Betty , Coleman Timothy , Greene Ronald C. , Smith Albert A. , Brot Nathan , Weissbach Herbert
Published: May 2006

The regulation of the expression of the
Escherichia coli metF
gene, which codes for 5,10-methylenetet-rahydrofolate reductase (EC 1.1.99.15), has been investigated by using a simplified DNA-directed
in vitro
system that measures the formation of the first dipeptide (fMet-Ser) of the gene product. The synthesis of fMet-Ser directed by a plasmid containing the
metF
gene is specifically inhibited by
metJ
protein (repressor protein).
S
-Adenosylmethionine enhances the inhibition by the
metJ
protein of
metF
gene expression. The inhibition by the
metJ
protein is at the level of transcription and the results suggest that
S
-adenosylmethionine is functioning as an allosteric effector.
Altered gene expression during cold acclimation of spinach.
Authors: Guy C L , Niemi K J , Brambl R
Published: May 2006

Exposure of spinach (Spinacia oleracea L.) plants to a constant 5 degrees C induced a greater tolerance to extracellular freezing. The metabolic basis of this cold acclimation response in plants is not understood. In this study we tested the hypothesis that cold acclimation derives from altered gene transcription. We found that exposure of plants to low temperature resulted in a rapid and stable change in the translatable poly(A)+ RNA populations extracted from leaves, as determined by a cell-free in vitro translation assay. The initial appearance of mRNAs for two high molecular weight translation products correlated with an increase in freezing tolerance. Cold acclimation of plants for 8 days resulted in further qualitative changes in mRNA populations. At least four additional mRNAs increased in concentration upon continued exposure of spinach to 5 degrees C, whereas three other mRNAs present in 20 degrees C-grown leaves decreased. We also tested the possibility that the low temperature-induced mRNAs might encode heat shock proteins. We studied heat shock-induced protein synthesis by in vivo labeling techniques and found that spinach synthesized at least eight distinctive heat shock proteins during exposure to 40 degrees C. Most polypeptides induced by exposure to low temperature, however, appeared not to be heat shock proteins. Thus, the change in mRNAs induced by low temperature is a separate response from that induced by high temperature.
DNA sequence and transcript mapping of a soybean gene encoding a small heat shock protein
Authors: Czarnecka Eva , Gurley William B. , Nagao Ronald T. , Mosquera Luis A. , Key Joe L.
Published: May 2006

The DNA sequence of a gene (
Gmhsp17.5-E
) encoding a small heat shock protein of soybean,
Glycine max
, has been determined. Nuclease S1 mapping of the 5′ terminus of the corresponding RNA indicates that the start site for transcription is located 82 bases upstream from the coding region and 24 bases downstream from a “TATA”-like region (-T-T-T-A-A-A-T-A-). The 5′ flanking region of
Gmhsp17.5-E
contains two imperfect dyads that closely resemble regulatory elements present in the promoters of heat-inducible genes of
Drosophila
. One, positioned 18 bases upstream from the TATA-like region, shows 90% homology to the
Drosophila
heat shock consensus sequence. The other overlaps an upstream TATA sequence and is located at position -213. Analysis of the derived amino acid sequence indicates that the protein encoded by
Gmhsp17.5-E
is related structurally to the four small heat shock proteins of
Drosophila
. This relationship is most evident by comparison of hydropathy profiles; they show conservation of several major hydrophilic and hydrophobic regions, which suggests that these proteins have common structural features.
RAS2 of Saccharomyces cerevisiae is required for gluconeogenic growth and proper response to nutrient limitation.
Authors: Tatchell K , Robinson L C , Breitenbach M
Published: May 2006

Saccharomyces cerevisiae contains two genes with remarkable homology to members of the ras oncogene family. These two genes, RAS1 and RAS2, constitute an essential gene family since spores with disruptions of both genes fail to grow. We report here that strains containing RAS2 disruptions have three distinct phenotypes. First, they fail to grow efficiently on nonfermentable carbon sources. Second, they hyperaccumulate the storage carbohydrates glycogen and trehalose. Third, diploid cells homozygous for the RAS2 disruptions sporulate on rich media. Extragenic suppressors have been isolated that suppress the gluconeogenic defect. These suppressors fall into at least three complementation groups, mutations in two of which bypass the normal requirement of RAS for cell viability, allowing cells containing neither RAS gene to grow. The phenotype of the RAS2 mutant and extragenic suppressors implicate RAS with some function in the normal response to nutrient limitation.