bluuebunny/crossref_metadata_2025 huggingface dataset:
ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle.
Authors: Siemankowski R F , Wiseman M O , White H D
Published: May 2006

The rate constant for dissociation of ADP from actomyosin subfragment 1 (S1) has been measured in this laboratory and elsewhere for a variety of vertebrate muscle types. We have made the following observations: (i) In solution, the dissociation of ADP from actomyosin-S1 limits the rate of dissociation of actomyosin-S1-ADP by ATP and, presumably, also limits the rate of crossbridge detachment in contracting muscle. (ii) For muscle types in which the rate of ADP dissociation from actomyosin-S1 is slow enough to measure using stopped-flow methods, the rate constants are nearly the same as the theoretical value for the minimum allowable rate constant for dissociation of an attached crossbridge. Therefore, ADP dissociation is sufficiently slow to be the molecular step that limits the maximum shortening velocity of these muscles. (iii) Variation with muscle type of the rate constant for ADP dissociation may be a general phylogenetic mechanism for regulating shortening velocity.
N-nitrosodialkylamines do not function as substrates for liver monoamine oxidase.
Authors: Edmondson D E , Bruice T C
Published: May 2006

Since reports in the literature [Rowland, I.R., Lake, B. G. & Gangolli, S. D. (1980) Mutat. Res. 72, 63-72] have implicated hepatic monoamine oxidase (EC 1.4.3.4) in the activation of N-nitrosodialkylamines to mutagens, it is of basic pharmacological and biochemical interest to determine if these compounds could serve as substrates for this enzyme. The dialkylnitrosamines N-nitrosodiethylamine, N-nitrosodimethylamine, and N-nitrosodibenzylamine were tested and found not to be substrates, competitive inhibitors, or irreversible inhibitors of liver monoamine oxidase. Thus, any role for monoamine oxidase participation in the mutagenic activation of these compounds must be subsequent to an initial conversion of these compounds to their respective secondary amines.
Variances and covariances of identity coefficients of a multigene family.
Authors: Ohta T
Published: May 2006

By using a model of gene conversion for treating concerted evolution of multigene families, a theory for calculating the variances and covariances of identity coefficients has been developed. Six coefficients on triple identity and 15 coefficients on quadruple identity are needed. The variances and covariances are obtained from the quadruple identity coefficients and divided into within-population and between-population components. The former represents the variation of identity coefficients among individuals within a population, whereas the latter represents the magnitude of fluctuation of population averages. Monte Carlo simulations confirmed the theoretical predictions at equilibrium.
Complete nucleotide sequences of three VH genes in Caiman, a phylogenetically ancient reptile: evolutionary diversification in coding segments and variation in the structure and organization of recombination elements.
Authors: Litman G W , Murphy K , Berger L , Litman R , Hinds K , Erickson B W
Published: May 2006

Complete nucleotide sequences are described for three caiman (Caiman crocodylus crocodylus) immunoglobulin VH genes (C3, E1, and G4) that hybridize with a murine VH probe. The E1 and G4 genes are physically linked (intergenic distance, approximately equal to 6.5 kilobases) in the same transcriptional orientation but are not directly contiguous with the C3 gene. When the coding segments, including both framework and complementarity-determining regions, of these genes and the murine probe sequences are compared by metric analysis, it is apparent that the caiman genes are only slightly more related to each other than to the mammalian sequence, consistent with significant preservation of nucleotide sequence over an extended period of phylogenetic time. Based on the presence of transcriptionally critical 5' sequences and the absence of terminator codons, frameshift mutations, or other recognizable alterations, the genes do not appear to be pseudogenes. The E1 gene, however, is distinguished from other VH genes because (i) the spacer region within the 3' recombination signal sequence is 12 base pairs, typical of VK genes but not of VH genes, which possess 22- to 23-base-pair spacers and (ii) a near-perfect VH recombination signal sequence is present within the intervening sequence that splits the segment encoding the leader. These studies establish VH gene multiplicity in a species that arose prior to mammalian radiation and provide a description of differences in the configuration and location of recombination elements associated with an otherwise potentially functional gene.
Biochemical rationale and myocardial tissue data on the effective therapy of cardiomyopathy with coenzyme Q10.
Authors: Folkers K , Vadhanavikit S , Mortensen S A
Published: May 2006

The tissue levels of coenzyme Q10 (CoQ10) in endomyocardial biopsy samples and blood from 43 patients with cardiomyopathy were determined by steps of extraction, purification, and HPLC. The biopsy samples were obtained from the patients after a routine heart catheterization. Six patients were of class I, 18 of class II, 11 of class III, and 8 of class IV (classified according to guidelines of the New York Heart Association). True control biopsies of healthy hearts are not available for ethical reasons, but the data of the four classes by severity of disease may be justifiably compared. Patients of class IV had lower (P less than 0.01) levels of CoQ10 than those of class I. Patients of classes III and IV had a lower (P less than 0.0001) level than those of classes I and II. Biopsy samples were obtained from five patients after treatment with CoQ10 for 2-8 months. The increases of CoQ10 levels ranged from 20% to 85%; the mean value was higher (P less than 0.02) than before treatment. Blood deficiencies also increase with severity of disease, but not as markedly as for the biopsies. These data reveal a myocardial deficiency of CoQ10, which is higher with increasing severity of disease and is reduced by therapy. This biochemistry correlates with the effective treatment of cardiomyopathy with CoQ10.
"Retroposon" insertion into the cellular oncogene c-myc in canine transmissible venereal tumor.
Authors: Katzir N , Rechavi G , Cohen J B , Unger T , Simoni F , Segal S , Cohen D , Givol D
Published: May 2006

We examined by Southern blotting the state of the cellular oncogene c-myc in the dog transmissible venereal tumor. The tumor DNA contains a 16.8-kilobase pair (kbp) rearranged c-myc fragment in addition to the normal 15-kbp and 7.5-kbp fragments. We compared the structure of the cloned rearranged c-myc (re-myc) with that of a cloned normal c-myc and found that the rearrangement was due to the insertion of a 1.8-kbp DNA upstream to the first exon of c-myc. The inserted DNA is flanked by 10-base-pair direct repeats and contains a dA-rich tail, suggesting its origin from mRNA. Partial sequence of the inserted element showed 62% homology with the primate interdispersed Kpn I repetitive element. These results provide an example for the behavior of repetitive DNA sequences like the Kpn I family, as movable elements that can transpose nearby to oncogenes or other structural genes and perhaps affect their activity.
Accurate initiation of rat ribosomal RNA gene transcription using a fractionated nuclear extract from normal liver and a hepatoma.
Authors: Kurl R N , Jacob S T
Published: May 2006

Nuclear extracts obtained from normal rat liver and from Morris hepatoma 3924A were fractionated by DEAE-Sephadex chromatography. The fraction eluted with 175 mM (NH4)2SO4 (DE-B), which contains greater than 90% of RNA polymerase I activity, supported accurate transcription of cloned rat rDNA. A similar fraction obtained from the cytosol had all of the factors required for rDNA transcription. However, its transcriptional activity was at most one-sixth that of the corresponding nuclear fraction, as determined by the amount of protein needed to produce a similar quantity of the transcript. Unfractionated nuclear or cytosol preparations did not yield an accurate transcript. Optimal KCl and magnesium concentrations for rDNA transcription were 60 mM and 5-7.5 mM, respectively. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-B greater than whole cell extract derived from rat mammary adenocarcinoma cells much greater than normal liver fraction DE-B. The hepatoma preparation produced at least 10 times the amount of transcript produced by the corresponding liver nuclear preparation. Transcriptional activity was proportional to the levels of RNA polymerase I and to the rate of rRNA synthesis in these tissues.
Polyadenylylation of sea urchin histone RNA sequences in transfected COS cells.
Authors: Nordstrom J L , Hall S L , Kessler M M
Published: May 2006

The region of pSV2neo that encompasses the simian virus 40 early polyadenylylation signal was replaced with a DNA fragment that spans the 3' end of a sea urchin (Psammechinus miliaris) histone H2A gene. This clone, pMK2.H2A(3'), was used to transfect COS cells. RNA analysis revealed that transcripts from pMK2.H2A(3') were polyadenylylated at a site 85 nucleotides downstream from the expected 3' end of mature H2A mRNA. Nucleotide sequencing showed that the site of poly(A) addition was located 10 nucleotides downstream from a cluster of four A-A-U-A-A-A sequences. The lower accumulation of MK2.H2A(3') mRNA, which was 5-10% that of SV2neo mRNA, suggests that the H2A polyadenylylation signal is relatively inefficient. The relationship of the above findings to the 3' end processing of other histone mRNAs is discussed.
Efficient cloning of single-copy genes using specialized cosmid vectors: isolation of mutant dihydrofolate reductase genes.
Authors: Urlaub G , Carothers A M , Chasin L A
Published: May 2006

A method for the efficient cloning of single-copy genes from restriction digests of mammalian DNA is described. The method is illustrated by the cloning of several mutant genes as well as the wild-type gene for Chinese hamster dihydrofolate reductase (DHFR; 7,8-dihydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). This gene is isolated within a 41-kilobase Bgl I fragment by using cosmid (plasmids containing a cohesive-end site) vectors that have been constructed especially for this purpose. Two cosmids are used: one contains a short region from the 5' flanking region of the dhfr gene, and the other contains a short region from the 3' flanking region. These two regions contain the Bgl I sites that bound the dhfr gene. Bgl I leaves staggered ends that are different depending on the DNA sequence within the enzyme binding site. When these cosmids are cut with Bgl I and hybridized with total Bgl I-cut genomic DNA, they preferentially associate with the fragment bearing the dhfr gene, since it has the same Bgl I ends. An approximately 500-fold enrichment for the dhfr gene in cosmid libraries from Chinese hamster ovary cells was achieved by using this method coupled with a single-step size fractionation. As a result, only several hundred cosmid colonies need to be screened in order to clone a dhfr gene from a particular mutant Chinese hamster ovary cell. This method should facilitate the repetitive cloning of any gene or gene fragment.
Repetitive A epitope (type 3 chain A) defined by blood group A1-specific monoclonal antibody TH-1: chemical basis of qualitative A1 and A2 distinction.
Authors: Clausen H , Levery S B , Nudelman E , Tsuchiya S , Hakomori S
Published: May 2006

The IgG2a monoclonal antibody TH-1, which reacts specifically with blood group A1 but with neither A2 nor O erythrocytes, has been established. The antibody reacted only with A1 erythrocytes in hemagglutination and antibody absorption assays; it did not react with A2 erythrocytes, even after trypsin or sialidase treatment. This antibody detected, on TLC immunostaining, a series of glycolipids from A1 erythrocytes but virtually none or very weak bands from A2 erythrocytes. It did not react with type 1 or type 2 chain A, or with globo-A. The simplest reactive component was isolated from a previously assigned Ab fraction by HPTLC of acetylated compounds. The structure of the reactive component was characterized by 1H NMR spectroscopy, methylation analysis, and enzymatic degradation, as shown below: (Formula: see text). The structure is essentially a repetitive A epitope attached to type 2 chain and is hereby called type 3 chain A. The determinant can be carried on extended and/or branched structures, but it was not detectable in glycoproteins. The structure was characteristic of A1 erythrocytes and present in only trace amounts in A2 erythrocytes. The precursor H (Fuc alpha 1----2Gal beta 1----3GalNAc alpha 1----3[Fuc alpha 1----2]Gal beta 1----4GlcNAc beta 1----R; type 3 chain H) was present in greater quantity in A2 erythrocytes than in A1 erythrocytes, but it was absent in both O and B erythrocytes. The A1 transferase apparently can transfer alpha-GalNAc to type 3 chain H, while the A2 transferase may not have this ability.
Group actions and higher signatures
Authors: Weinberger Shmuel
Published: May 2006

Let π be a nontrivial finite group and
M
be a closed manifold. An interesting question is whether or not
M
has the
R
-homology type of a manifold admitting a free π action. Here this problem is studied for actions that are “homologically trivial.” If π
1
M
is nontrivial these questions are intimately related to the Novikov higher signature conjecture, but the results are new even in the simply connected case.
Purification and characterization of polynucleotide phosphorylase from cucumber
Authors: Khan Zulfaqar Alam , Fraenkel-Conrat H.
Published: May 2006

Polynucleotide phosphorylase (polyribonucleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8) activity has been found in many prokaryotes and studied in detail since 1955. Such enzymes have been detected also in plants. We now describe the purification of polynucleotide phosphorylase from cucumber cotyledons and leaves. This enzyme is a complex of three subunits, possibly not identical, of about
M
r
50,000. Its enzymatic properties are similar to those of the tobacco enzyme. Unlike the prokaryotic enzymes, the plant enzyme shows activity in the absence of primer but is to various extents stimulated by various ribopolynucleotides or RNAs. RNA-dependent RNA polymerase, not previously shown to exist in non-virus-infected cucumber, has been found to be present at a low level and was separated from the much greater amount of polynucleotide phosphorylase, although some of the physical properties of the two enzymes are rather similar.
Mapping the location of psoralen crosslinks on RNA by mung bean nuclease sensitivity of RNA.DNA hybrids.
Authors: Hui C F , Cantor C R
Published: May 2006

An indirect high resolution method has been developed for finding the location of intrastrand crosslinks in RNA. An end-labeled DNA strand that overlaps the approximate crosslink position is hybridized to the RNA and then treated with mung bean nuclease. The resulting digest is analyzed on a sequencing-type gel. The method was tested with the major psoralen crosslink seen in the 16S rRNA of inactivated Escherichia coli 30S ribosomal subunits. This crosslink was previously mapped between residues 930 +/- 25 and a region close to the 3' end by electron microscopy. The new indirect method reveals that the crosslink occurs between residues 919 and 923 and residues 1530 and 1534. When these results are examined in the light of existing consensus secondary structure models for the 16S rRNA, it appears that the Shine-Dalgarno sequence is located close to the peptidyl tRNA binding site.
Treatment of mice with 5-azacytidine efficiently activates silent retroviral genomes in different tissues.
Authors: Jaenisch R , Schnieke A , Harbers K
Published: May 2006

The drug 5-azacytidine was injected into mice to activate silent retroviral genomes. The Mov-7 and Mov-10 substrains of mice were used, each of which carries a Moloney murine leukemia provirus with mutations in the coding regions at nonidentical positions. These proviral genomes are highly methylated and are not expressed in the animal. A single injection of the drug into postnatal mice induced transcription of the endogenous defective proviral genomes in thymus, spleen, and liver at 3 days after treatment. No viral transcription was detected in the brain of drug-exposed animals. When postnatal Mov-7/Mov-10 F1 mice were treated with the drug, infectious virus was generated efficiently and resulted in virus spread and viremia in all animals by 3 weeks of age. In contrast, infectious virus was not generated in F1 mice that had been treated during gestation with up to sublethal doses of the drug. Our results demonstrate that injection of 5-azacytidine can be used to efficiently and reproducibly activate silent genes in different cell populations of postnatal mice.
Linear empirical Bayes estimation of means and variances.
Authors: Robbins H
Published: May 2006

Linear empirical Bayes estimators have been obtained for means and variances that apply to random sampling from populations whose structure is unknown or only partly known.
Calmodulin accelerates the rate of polymerization of human platelet actin and alters the structural characteristics of actin filaments.
Authors: Piazza G A , Wallace R W
Published: May 2006

Calmodulin stimulated the rate of Mg2+-induced polymerization of human platelet actin. The stimulatory effect was due to an increase in the nucleation phase of the reaction; there was no effect on the steady-state viscosity. The calmodulin antagonist trifluoperazine blocked the stimulatory effect of calmodulin. Addition of EGTA to the reaction mixture also stimulated the rate of actin polymerization; however, the effect of calmodulin on actin polymerization is not due to Ca2+ chelation, as is presumed to be the case for EGTA. Electron microscopy revealed structural differences in the filaments prepared in the presence of calmodulin as compared to those prepared with trifluoperazine. In the presence of calmodulin, the filaments were thicker, suggesting that they consisted of multiple actin polymers. In addition, numerous projections were present perpendicular to the filaments, as well as localized areas of filament bundling. It was not possible to demonstrate a direct interaction between calmodulin and actin, which raises the possibility that the calmodulin effect may be indirect through a calmodulin-binding protein or calmodulin-dependent enzyme. Regardless of whether calmodulin is acting directly or indirectly, these results provide evidence that calmodulin may play a regulatory role in either the polymerization of actin or in determining the structural characteristics of actin filaments.
Structure of proteins with single-site mutations: a minimum perturbation approach.
Authors: Shih H H , Brady J , Karplus M
Published: May 2006

A large number of mutant proteins with single amino acid substitutions are now being produced. The ability to predict the structural changes expected from such mutations would aid greatly in the efficient utilization of the mutagenic techniques and in the interpretation of the changes in stability and function that result. A minimum perturbation approach is suggested as a first step in such structural predictions and is tested by application to a recently isolated variant of the hemagglutinin glycoprotein. The agreement between the predicted structure and that inferred from the x-ray refinement is encouraging and provides support for the proposed modeling procedure.
Sex-dependent expression of mouse testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha): cDNA cloning and pretranslational regulation.
Authors: Harada N , Negishi M
Published: May 2006

By using both double-colony hybridization and an in situ immunostaining assay for transformants, 39 cDNA clones (clone p-16 alpha) encoding mouse liver microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) were isolated from a cDNA library constructed in the cloning vector pUC-9 with poly(A)+ RNA immunoenriched from total liver polysomes of male 129/J mice. mRNA selected by hybridization with clone p-16 alpha translated the P-450(16) alpha apoprotein in vitro. Total cellular proteins, which were prepared from immunopositive transformant Escherichia coli cells, were conjugated with Sepharose 4B. Antibody purified with the Sepharose 4B conjugate from mixed antiserum to P-450(16) alpha and P-450(15) alpha specifically inhibited testosterone 16 alpha-hydroxylase activity in microsomes. The cDNA insert of one recombinant plasmid (clone P-16 alpha-1) was 1.75 kilobases in size and contained one or more internal restriction sites for HindIII, BamHI, Bgl I, Pst I, Alu I, HinpI, and Rsa I. 32P-labeled clone p-16 alpha-1 hybridized with a single mRNA (2000 bases) that was 10 times more concentrated in liver cells from male 129/J mice than in female mice. This result was consistent with the finding that poly(A)+ RNA from male mice translated 10 times as much P-450(16) alpha in vitro as did the poly(A)+ RNA from females. Thus, the predominant expression of testosterone 16 alpha-hydroxylase in male 129/J mice is regulated pretranslationally, presumably at the transcriptional level of the P-450(16) alpha gene.
Identification and chromosomal distribution of 5S rRNA genes in Neurospora crassa.
Authors: Metzenberg R L , Stevens J N , Selker E U , Morzycka-Wroblewska E
Published: May 2006

The 5S rRNA genes of Neurospora crassa, unlike those of most organisms, are not tandemly arranged, and they are found imbedded in a variety of unique sequences. The 5S rRNA regions of most of the genes are of one type, alpha; however, several other "isotypes" (beta, gamma, delta, zeta, and eta) are also found. We asked whether Neurospora 5S rRNA genes are dispersed on a chromosomal scale and whether genes of different isotypes are spatially segregated. We identified, by DNA sequencing, 5S rRNA genes in 22 5S DNA clones, and we mapped these genes by conventional crosses by using restriction fragment length polymorphisms in their flanking sequences as genetic markers. The results show that the 5S rRNA genes are distributed on at least six of the seven chromosomes. Their location does not appear to be completely random. Some of them are closely linked. One of the chromosomes carries a disproportionate number of 5S rRNA genes of the most common structural type, alpha; another chromosome carries three of the four mapped beta 5S rRNA genes. None of the 5S rRNA genes studied maps close to the nucleolus organizer, the site of the genes that code for the three larger rRNAs.
Spectrum and chemical composition of the remarkable planetary nebula NGC 6537
Authors: Feibelman Walter , Aller Lawrence H. , Keyes C. D. , Czyzak S. J.
Published: May 2006

Observations with the image tube scanner at the Shane 3-m telescope are combined with data obtained with the International Ultraviolet Explorer to assess the spectrum of the remarkable high-excitation planetary nebula NGC 6537. We have analyzed the spectrum of this nitrogen-rich object with the aid of the theoretical nebular models. The models permit one to estimate the fraction of unobservable ions of abundant elements. On the scale log N(H) = 12, the logarithmic abundance values for He, C, N, and O are as follows: [Table: see text] The abundances of Ne S, Cl, and Ar appear to be essentially solar to within a factor of 2. Our interpretation is that the progenitor of NGC 6537 had a chemical composition not differing greatly from that of the Sun. In the course of its prenebular evolution, C and probably O were converted to N and much H was converted to helium.
Two nonidentical forms of subunit V are functional in yeast cytochrome c oxidase.
Authors: Cumsky M G , Ko C , Trueblood C E , Poyton R O
Published: May 2006

In Saccharomyces cerevisiae, the inner mitochondrial membrane protein cytochrome c oxidase is composed of nine polypeptide subunits. Six of these subunits (IV, V, VI, VII, VIIa, VIII) are encoded by the nuclear genome, and the remaining three (I, II, III) are encoded by mitochondrial DNA. We report here the existence of two nonidentical subunit V polypeptides, which are encoded by separate genes within the yeast genome. One gene, COX5a, encodes the polypeptide Va, normally found in preparations of holocytochrome c oxidase. The other gene, COX5b, encodes the polypeptide Vb, which cross-reacts with anti-subunit Va antiserum and restores respiratory competency and cytochrome oxidase activity in transformants of cox5a structural gene mutants. This polypeptide also copurifies with the holoenzyme prepared from these transformants. We have found that COX5b is expressed in vegetatively growing yeast cells, and that the Vb polypeptide can be detected in mitochondria from strain JM28, a cox5a mutant. This mutant has 15%-20% residual cytochrome oxidase activity, and it respires at 10%-15% the wild-type rate. By disrupting the COX5b gene in this strain, we show that this residual activity is directly attributable to the presence of a chromosomal copy of the COX5b gene. Taken together, these results suggest that Va or Vb can function as cytochrome oxidase subunits in yeast and that Vb may be used under some specific, as yet undefined, physiological conditions.
Characterization of the ColE1 primer-RNA1 complex: analysis of a domain of ColE1 RNA1 necessary for its interaction with primer RNA.
Authors: Tamm J , Polisky B
Published: May 2006

RNA1 is a small, plasmid-encoded transcript involved in the replication control of the plasmid ColE1. RNA1 blocks replication by preventing processing of the primer RNA necessary for the initiation of replication. It has been proposed that inhibition by RNA1 involves a direct interaction between RNA1 and primer RNA. Here we describe an in vitro system that allows the detection and characterization of the RNA1-primer complex. Using this system, we have demonstrated that the association between ColE1 RNA1 and primer results in the formation of an RNA X RNA hybrid between RNA1 and the 5' end of primer RNA. Furthermore, analysis of mutant RNA1 molecules containing sequence alterations in the 5' single-stranded region of the molecule has revealed that this portion of RNA1 is necessary for the formation of the RNA1-primer complex. These experiments indicate that, in addition to the three single-stranded loops of RNA1, the 5' single-stranded region is a functional domain of the molecule.
Expression in Escherichia coli of a fusion protein product containing a region of the adenovirus DNA polymerase.
Authors: Rekosh D , Lindenbaum J , Brewster J , Mertz L M , Hurwitz J , Prestine L
Published: May 2006

The bulk of an open reading frame extending from map coordinates 23.3 to 14.2 in region E2b of the adenoviral genome has been cloned and expressed from a chimeric plasmid in Escherichia coli. The cloning strategy used created a fusion protein of 124,000 daltons, which contained greater than 98% adenovirus-encoded sequences. Antiserum raised against this protein reacted with the authentic 140,000-dalton adenovirus DNA polymerase. Another serum raised against a synthetic hexapeptide whose sequence corresponded to the predicted carboxyl terminus of adenovirus-encoded DNA polymerase also reacted with the fusion protein and authentic adenovirus DNA polymerase. These results demonstrate that the cloned region of DNA encodes the adenovirus DNA polymerase.
Fluctuation of the Ca
2+
-sequestering activity of permeabilized sea urchin embryos during the cell cycle
Authors: Suprynowicz Frank A. , Mazia Daniel
Published: May 2006

We have followed the sequestration of Ca
2+
by intracellular compartments in sea urchin embryos through the first cell cycles. To gain biochemical access to these compartments, the embryos were permeabilized by brief exposure to an intense electric field. Sequestration was determined as the retention of tracer,
45
Ca, after filtration of aliquots on Millipore filters. The permeabilized cells sequester Ca
2+
at a constant rate for at least 20 min, with the following characteristics: (
i
) ATP is required. (
ii
) Sequestration occurs at Ca
2+
levels corresponding to those estimated
in vivo
. (
iii
) The Ca
2+
concentration dependence of sequestration and its insensitivity to mitochondrial poisons imply that the activity derives from a single, nonmitochondrial transport system. The Ca
2+
-sequestering activities of embryos that are permeabiized at successive stages of the first cell cycle (one-cell stage) progressively increase to 5 times the initial level. The rate of sequestration is maximal during telophase and, in some populations of zygotes, is nearly as great throughout prophase. Over the course of the second cell cycle (two-cell stage), the activity undergoes a 2-fold oscillation that bears the same temporal relationship to mitosis as the previous fluctuation.
Negative control for the initiation of meiosis in
Schizosaccharomyces pombe
Authors: Iino Yuichi , Yamamoto Masayuki
Published: May 2006

Temperature-sensitive mutants of the
pat1
gene of
Schizosaccharomyces pombe
are able to produce poorly viable spores from the haploid state without the requirement for nitrogen starvation if exposed to the restrictive temperature. Three lines of evidence strongly suggest that this gene codes for a factor whose physiological role is inhibition of initiation of meiosis. First,
pat1
haploids arrested in G
1
phase undergo apparent premeiotic DNA synthesis at the restrictive temperature. Second, the
pat1
gene was found to exert its function at a specified stage of the meiotic pathway by comparison with other meiotic mutants. Third, meiotic recombination and chromosome segregation take place quite normally in
pat1
diploids at the restrictive temperature, resulting in the production of highly viable spores. A negative control mechanism for meiosis is proposed.
Natural killer cell recognition of target cells expressing different antigens of vesicular stomatitis virus.
Authors: Moller J R , Rager-Zisman B , Quan P C , Schattner A , Panush D , Rose J K , Bloom B R
Published: May 2006

Natural killer (NK) cells have the capability of lysing virus-infected, transformed, and embryonal cells, yet the nature of the target structure(s) recognized remains unclear. The availability of well-characterized temperature-sensitive (ts) mutants of vesicular stomatitis virus, defective in expression of individual viral-encoded polypeptides at the nonpermissive temperature (39 degrees C), offered an approach to elucidating NK-cell recognition of virus-infected cells. Target cells were infected with ts mutants in three functions: the viral surface glycoprotein (G protein; ts 045); the matrix (M) protein (ts G31, ts G33), and the polymerase (ts G11). Cells infected with wild-type virus and all ts mutants at the permissive temperature (31 degrees C) were killed by murine spleen cells. Similar to results on cytotoxic T lymphocytes, target cells infected by ts 045 defective in expression of G protein at 39 degrees C were not killed by NK cells. Unexpectedly, cells infected at 39 degrees C with the M-protein mutants also were not killed, although G protein was expressed at the cell surface. Target binding studies indicated that conjugates were not formed by cells infected with the ts mutants at the nonpermissive temperature. That expression of G protein was not sufficient for NK cell-mediated cytotoxicity was established in experiments in which a plasmid (pSVGL) containing the gene for vesicular stomatitis virus G protein was transfected into COS cells. Although G antigen was expressed on the plasma membrane, the cells were not lysed. These results suggest either that recognition of virus-infected cells depends on an appropriate conformation imparted to the viral G protein by association with the M protein or that NK cells can recognize alterations in the structure of the cell membrane induced by insertion of viral M and G molecules.
Metabolically activated steviol, the aglycone of stevioside, is mutagenic.
Authors: Pezzuto J M , Compadre C M , Swanson S M , Nanayakkara D , Kinghorn A D
Published: May 2006

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a noncaloric sugar substitute in Japan. Consistent with reports in the literature, we have found that stevioside is not mutagenic as judged by utilization of Salmonella typhimurium strain TM677, either in the presence or in the absence of a metabolic activating system. Similar negative results were obtained with several structurally related sweet-tasting glycosides. However, steviol, the aglycone of stevioside, was found to be highly mutagenic when evaluated in the presence of a 9000 X g supernatant fraction derived from the livers of Aroclor 1254-pretreated rats. Expression of mutagenic activity was dependent on both pretreatment of the rats with Aroclor 1254 and addition of NADPH; unmetabolized steviol was not active. The structurally related species, isosteviol, was not active regardless of metabolic activation. Similarly, chemical reduction of the unsaturated bond linking the carbon-16 and -17 positions of steviol resulted in the generation of two isomeric products, dihydrosteviol A and B, that were not mutagenic. In addition, ent-kaurenoic acid was found to be inactive. It is therefore clear that a metabolite of an integral component of stevioside is mutagenic; structural features of requisite importance for the expression of mutagenic activity include a hydroxy group at position 13 and an unsaturated bond joining the carbon atoms at positions 16 and 17. A potential metabolite of steviol, steviol-16 alpha,17-epoxide, was synthesized chemically and found to be ineffective as a direct-acting mutagen. Thus, although stevioside itself appears innocuous, it would seem prudent to expeditiously and unequivocally establish the human metabolic disposition of this substance.
Expression of Plasmodium falciparum surface antigens in Escherichia coli.
Authors: Ardeshir F , Flint J E , Reese R T
Published: May 2006

The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity.
Isolation of stable preinitiation, initiation, and elongation complexes from RNA polymerase II-directed transcription.
Authors: Safer B , Yang L , Tolunay H E , Anderson W F
Published: May 2006

Distinct RNA polymerase II transcription preinitiation, initiation, and elongation complexes can be formed in vitro on cloned adenovirus 2 DNA sequences containing the major late promoter. These transcription complexes are stable and can be rapidly isolated by gel filtration of HeLa whole cell extracts. In the absence of exogenous nucleotides and under appropriate salt conditions, a stable but transcriptionally incomplete preinitiation complex is formed. When this complex is incubated in the presence of adenosine or deoxyadenosine triphosphates, the beta-gamma phosphodiester bond is hydrolyzed, and RNA polymerase II joins the complex, thereby converting it into a stable initiation complex capable of forming (but prior to the formation of) the first phosphodiester bond. When this complex is isolated and incubated in the presence of all four nucleoside triphosphates, it is converted into an elongation complex that then permits the synthesis of phosphodiester bonds and the correct run-off transcript. A limiting transcription component is sequestered in the preinitiation complex. This factor is released upon elongation and can reassociate with new DNA templates during subsequent rounds of initiation. Therefore, class II genes do not appear to form activated transcription units stable for multiple rounds of transcription; rather, their transcriptional activity may be controlled in part by regulating the association of transcription factors at each initiation event.
Incorporation of cellular and plasma fibronectins into smooth muscle cell extracellular matrix in vitro.
Authors: Millis A J , Hoyle M , Mann D M , Brennan M J
Published: May 2006

Fibronectins isolated from the conditioned medium produced by cultures of undifferentiated (monolayer) and differentiated (nodular) swine vascular smooth muscle cells are similar but not identical. In general, the nodular-cell fibronectin has a smaller molecular mass than monolayer-cell fibronectin and appears to lack the COOH-terminal interchain disulfide linkage. We studied the incorporation of cellular and plasma fibronectins into the cell layer. Smooth muscle cells bound 2.5 times more monolayer-cell fibronectin than nodular-cell fibronectin. Polypeptide fragments of human plasma fibronectin were used as a model system to investigate fibronectin incorporation into the cell layer. Only intact molecules were incorporated into the cell layer and subsequently organized into fibers. Polypeptide fragments of molecular mass 205 kDa and 185 kDa were not incorporated even though they retained the collagen-, cell-, and heparin-binding regions. Incorporation appears to require an activity associated with either the NH2-terminal or COOH-terminal domains. We propose that fibronectin activity is lost during differentiation of smooth muscle cells.
Silkmoth chorion gene families contain patchwork patterns of sequence homology.
Authors: Eickbush T H , Burke W D
Published: May 2006

The late chorion locus of Bombyx mori, containing paired members from two multigene families, has been analyzed in detail. The 15 gene pairs, irregularly spaced over 140 kilobases, exhibit an identical structure and a high overall sequence homology, while the flanking DNA (intergene regions) varies considerably. Segments of DNA of 150-300 base pairs from a reference gene pair were used as probes in a series of DNA hybridization experiments. It was found that the sequence variants within the locus are in a "patchwork" arrangement. Each gene pair contains a unique pattern of regions or "patches" that are highly homologous to the reference gene pair, interspersed with regions exhibiting less homology. We suggest that phenotypic selection is unable to account for the observed patchwork patterns; rather, sequence exchange between genes must be postulated. The nature of the DNA flanking the gene pairs would suggest that most of this sequence exchange is by means of gene conversion rather than unequal crossover events.
mRNA precursor splicing in vivo: sequence requirements determined by deletion analysis of an intervening sequence.
Authors: van Santen V L , Spritz R A
Published: May 2006

To define the extent of intervening sequence required for splicing higher eukaryotic mRNA precursors in vivo, we constructed deletions within the second intervening sequence of the human G gamma-globin gene that progressively approach the donor or acceptor splice sites. Most of the intervening sequence can be deleted with no effect on splicing. At the donor splice site, 6 bases of intervening sequence are sufficient for accurate and efficient splicing. At the acceptor splice site, 20 bases are sufficient for accurate and efficient splicing, and 16 bases are sufficient for accurate splicing but at a reduced level. However, 15 bases are insufficient for splicing at a significant level. The effect of deletions ending near the acceptor splice site is independent of whether an A-G dinucleotide is introduced into the acceptor splice site region by the deletion.
Involvement of I-J epitopes in the self- and allo-recognition sites of T cells: blocking of syngeneic and allogeneic mixed lymphocyte reaction-responder cells by monoclonal anti-I-J antibodies.
Authors: Uracz W , Abe R , Tada T
Published: May 2006

Monoclonal anti-I-Jk antibodies (mAbs) were found to inhibit syngeneic and allogeneic mixed lymphocyte reactions by blocking the responder T cells but not the stimulator cells. Only the responses of H-2k and H-2a strains were inhibited. Three different anti-I-Jk mAbs (1G8, 4B11, and KN34) showed different inhibitory patterns in allogeneic mixed lymphocyte reactions of individual H-2k strains, depending on the H-2 and immunoglobulin heavy chain variable region (IgVH) genes possessed by the stimulator strains. The results indicated that I-J epitopes are involved in the self- and allo-recognition sites of T cells, which are clonally distributed and used to recognize Ia plus IgVH-linked products.
NMR imaging shows water distribution and transport in plant root systems
in situ
Authors: Bottomley Paul A. , Rogers Hugo H. , Foster Thomas H.
Published: May 2006

Images of
Vicia faba
L. root systems at 0.6-mm resolution, undisturbed and within the soil medium in which they were grown, have been obtained by using a 1.5-Tesla proton (
1
H) NMR medical imaging research system. Images of root systems in seven soil media (Cahaba soil, vermiculite, sand, perlite, fritted clay, potting soil, and peatlite) exhibited variable but useful quality owing to a diversity of magnetic properties of the soils. Root structure and pathology in the form of partial decay of hypogeal cotyledons were easily discernible. Water transport in roots with light-stressed foliage was demonstrated by using water doped with a paramagnetic NMR contrast agent, and the process of plant-wilt and recovery was monitored
in situ
. Images of germinated seeds within soil media indicated that dynamic observations of germination and growth are possible. The results suggest that NMR imaging can be an effective noninvasive tool for studying plants
in situ
.
The chicken delta 1-crystallin gene promoter: binding of transcription factor(s) to the upstream G+C-rich region is necessary for promoter function in vitro.
Authors: Das G C , Piatigorsky J
Published: May 2006

There are two linked delta-crystallin genes in the chicken (5' delta 1-delta 2 3'). Only the delta 1 gene has been shown definitively to be active in the lens. Transcription of deletion mutants, reported here, shows that the sequences necessary for the functioning of the delta 1 promoter in a HeLa cell extract are located upstream from the RNA initiation site, between nucleotide positions -121 and -38. This region includes a number of G+C-rich motifs, including one hexanucleotide sequence, CCGCCC, that is repeated six times in the simian virus 40 (SV40) promoter. Competition experiments with purified fragments from the delta 1-crystallin gene promoter showed that binding of transcription factor(s) from the HeLa cell extract to this G+C-rich region is required for promoter activity in vitro. Further, competition experiments using three different fragments from the SV40 promoter suggest that the transcription factor(s) is similar to Sp1, which stimulates transcription by binding to the G+C-rich 21-base-pair repeats of the SV40 promoter, and differs from that which interacts with the SV40 enhancer region.
Human peripheral monocytes express putative receptors for neuroexcitatory amino acids.
Authors: Malone J D , Richards M , Kahn A J
Published: May 2006

Human peripheral mononuclear cells responded chemotactically to 4-carboxyl-L-glutamic acid. The maximal chemotactic response occurred at 0.1 nM. No chemotactic response was found with neutrophils or fetal bovine fibroblasts. Glutamic acid, a neuroexcitatory dicarboxylic amino acid and the parent compound of 4-carboxyglutamic acid, did not stimulate chemotaxis in any of the cells tested. However, it functioned as an antagonist to 4-carboxyglutamic acid (ED50 approximately 2 pM; ED100 approximately 10 pM). In contrast to the lack of response to glutamic acid, its dicarboxylic cyclic analogue, kainic acid, excited a chemotactic response in mononuclear cells. The data suggest that mononuclear phagocytes have receptors for dicarboxylic neuroexcitatory amino acids, and we speculate that 4-carboxyglutamic acid, a tricarboxylic acid, may have a previously unrecognized role as a neuroexcitatory amino acid.
Human seminal alpha-inhibins: detection in human pituitary, hypothalamus, and serum by immunoreactivity.
Authors: Ramasharma K , Li C H
Published: May 2006

An antiserum generated in rabbits against human seminal alpha-inhibin-52 has been used to develop a sensitive radioimmunoassay for the detection of alpha-inhibins. The alpha-inhibin-52 antiserum reacts with alpha-inhibin-92 and alpha-inhibin-31 with equal avidity. These peptides were found to be present in human pituitary, hypothalamus, and serum. In exclusion chromatography on Sephadex G-100, the immunoreactive material eluted in a large molecular size region. Immunoblot analysis of column-derived fractions of these extracts revealed the presence of alpha-inhibin-92. The mean concentrations of immunoreactive alpha-inhibin were found to be 7.2 ng/ml in normal adult male serum, 70.3 ng/g (wet weight) of pituitary, and 12.9 ng/g (wet weight) of hypothalamus. This communication reports on the evidence for the existence of gonadal peptides in the brain.
Human Cu/Zn superoxide dismutase gene family: molecular structure and characterization of four Cu/Zn superoxide dismutase-related pseudogenes.
Authors: Danciger E , Dafni N , Bernstein Y , Laver-Rudich Z , Neer A , Groner Y
Published: May 2006

Four distinct human Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1)-related sequences were isolated from genomic DNA libraries. Genomic blots, heteroduplex analyses, and DNA sequencing showed that they are processed pseudogenes not residing on chromosome 21. Three of them originated from the 0.7-kilobase SOD1 mRNA, while the fourth was derived from the 0.9-kilobase mRNA species. Comparison between the coding sequences of the functional gene and two of the processed genes suggested that they integrated into the genome about 25 million years ago.
Insulin stimulates the generation of an adipocyte phosphoprotein that is isolated with a monoclonal antibody against the regulatory subunit of bovine heart cAMP-dependent protein kinase.
Authors: Lawrence J C , Hiken J F , Inkster M , Scott C W , Mumby M C
Published: May 2006

Incubating 32P-labeled fat cells with insulin increased by as much as 80-fold the amount of 32Pi in a soluble species of apparent Mr 62,000. This species, designated isp62, was specifically immunoprecipitated from cellular extracts with a monoclonal antibody against the type II regulatory subunit (RII) of cAMP-dependent protein kinase. Fat-cell RII, purified from extracts with cAMP-Sepharose or labeled with 8-azido [32P]cAMP, had an apparent Mr 51,000. Peptide mapping indicated that isp62 and adipocyte RII were different proteins. When cells were metabolically labeled with [35S]methionine, insulin stimulated the appearance of 35S-labeled isp62, indicating that the hormonal effect involves generation of the protein. The insulin-induced increase in isp62 could be observed within 1 min, occurred with physiological concentrations of the hormone, and was rapidly reversible. The increase in isp62 was unaffected by cycloheximide, indicating that insulin stimulates the posttranslational processing of a precursor, rather than de novo synthesis of the protein.