bluuebunny/crossref_metadata_2025 huggingface dataset:
Segregation patterns of polymorphic restriction sites of the gene encoding the alpha subunit of human chorionic gonadotropin in trophoblastic disease.
Authors: Hoshina M , Boothby M R , Hussa R D , Pattillo R A , Camel H M , Boime I
Published: May 2006

The gene encoding the alpha subunit of human chorionic gonadotropin contains at least two polymorphic sites in its 3' flanking region detected by restriction enzymes HindIII and EcoRI. We used these polymorphic sites as markers of tissue genotype in normal placenta, hydatidiform mole, choriocarcinoma, and peripheral leukocytes. As expected, inheritance patterns of most hydatidiform moles showed only a paternal genetic contribution. However, one uncommon DNA polymorphism pattern, homozygosity for the absence of the EcoRI site and the presence of the HindIII site, predominated in choriocarcinoma. Thus, our results suggest that moles which have this uncommon polymorphism pattern appear particularly likely to develop into choriocarcinoma.
Methyl transfer from methylcobalamin to diaquocobinamide.
Authors: Fanchiang Y T , Bratt G T , Hogenkamp H P
Published: May 2006

The transfer of the methyl group from methylcobalamin to diaquocobinamide in aqueous solution has been demonstrated by proton, carbon-13, and phosphorus-31 nuclear magnetic resonance spectroscopy. The products of this reaction are aquocobalamin and the methylaquocobinamides. Dicyanocobinamide and the cyanoaquocobinamides do not serve as methyl acceptors, while ligands such as pyridine and histidine reduce the rate of the transfer reactions. The methyl transfer is not affected by oxidizing agents such as O2, N2O, and H2O2, suggesting that the reaction does not involve free Co(I) or Co(II) corrinoids. The pH dependence of the rate of the transfer reaction from methylcobalamin to diaquocobinamide demonstrates that methylcobalamin in the "base-on" form and diaquocobinamide are the most effective methyl donor and acceptor, respectively. The most plausible mechanism for the transfer reaction involves the one-electron oxidation of methylcobalamin by diaquocobinamide to a methylcobalamin radical cation and cob(II)inamide. The very unstable methylcobalamin radical cation releases a methyl radical, which reacts with cob(II)inamide to generate the methylaquocobinamides.
A retroviral myc gene induces preneoplastic transformation of lymphocytes in a bursal transplantation assay.
Authors: Neiman P , Wolf C , Enrietto P J , Cooper G M
Published: May 2006

Treatment of chicken embryos with cyclophosphamide results in ablation of bursal lymphocytes. Bursal follicles can be reconstructed by infusion of embryonic bursal cells. Histologic examination of reconstituting bursal follicles showed that the first lymphocytes to appear were large pyrinophilic lymphoblasts that lined up adjacent to the bursal basement membrane and appeared to serve as progenitors for the differentiation of bursal medullary lymphocytes. When these cells were infected with the avian myelocytomatosis virus HB1 bearing a v-myc oncogene they appeared to home to the region of the bursal basement membrane but failed to differentiate. Instead, they formed structures indistinguishable from the preneoplastic transformed follicles that develop during bursal lymphomagenesis induced by lymphoid leukosis viruses. The DNA from these transformed follicles contained the HB1 v-myc gene but lacked the ability to transform NIH/3T3 mouse cells. Therefore these preneoplastic lesions were induced directly by HB1 myc and did not require the expression of Blym-1 or similar oncogenes. Exploitation of this transplantation technique with the chicken bursa will provide a useful method for assessing the stage-specific activity of oncogenes in vivo.
High-frequency meiotic gene conversion between repeated genes on nonhomologous chromosomes in yeast.
Authors: Jinks-Robertson S , Petes T D
Published: May 2006

We have used a genetic system that allows detection of meiotic recombination events between repeated sequences on nonhomologous chromosomes in the yeast Saccharomyces cerevisiae. We have found that recombination between these sequences occurs at a frequency of about 0.5%, and the events observed were nonreciprocal (gene conversions). Surprisingly, the frequency of conversion between the repeated genes on nonhomologous chromosomes observed in this study is similar to that observed between allelic genes. This result is discussed in connection with the role of the synaptonemal complex in meiotic recombination and with the relationship between reciprocal and nonreciprocal recombination.
Elongation in the major direction of tobacco mosaic virus assembly
Authors: Fukuda Megumi , Okada Yoshimi
Published: May 2006

Butler and Lomonossoff [Butler, P. J. G. & Lomonossoff, G. P. (1978)
J. Mol. Biol.
126, 877-882] claim that the elongation in the major direction (3′→5′) proceeds by incorporation of disk protein in tobacco mosaic virus (TMV) assembly. The strongest argument they have for this theory is the periodicity of 50 or 100 nucleotides that they observed in the banding pattern of the protected RNAs during the first few minutes of the assembly reaction. We repeated their experiment using TMV-OM (a common Japanese strain) disk protein and TMV-OM RNA. We observed a banding pattern similar to theirs, but we found the long protected RNA at 6 min to be from the 260-nm intermediate particle rather than from the full-length TMV RNA. We also carried out the assembly reaction between TMV-OM disk protein, as well as cucumber green mottle mosaic virus (CGMMV) protein, and three strains of TMV RNAs. During the course of each assembly reaction, we examined the banding patterns. We demonstrated that the banding pattern of the protected RNA differs depending on what kind of RNA is used, rather than on what kind of aggregational state the protein is in. Specifically, the similar banding pattern observed for CGMMV subunit protein was also observed for TMV disk protein in the assembly reaction with TMV (OM) RNA. We showed previously that the assembly reaction between CGMMV protein and TMV RNA proceeds by incorporation of CGMMV subunit protein. This strongly indicates that the banding pattern of the protected RNA does not arise from the stepwise addition of the 20S disk protein.
Paired helical filaments from Alzheimer disease patients contain cytoskeletal components.
Authors: Perry G , Rizzuto N , Autilio-Gambetti L , Gambetti P
Published: May 2006

Neurofibrillary tangles from Alzheimer disease patients share antigenic determinants with neurofilaments and microtubule-associated proteins, as shown by light microscopy immunocytology. The present study addresses the issue of whether these determinants are located on the paired helical filaments or on other components of the neurofibrillary tangle. Sections from postmortem brains from Alzheimer disease patients were stained by using Bodian's silver method or immunostained by using poly- and monoclonal antibodies to neurofilaments and polyclonal antibodies to microtubules. Bodian's silver stain has an intense affinity for neurofibrillary tangles and has been shown to bind to specific domains of neurofilament subunits. The antibodies to neurofilaments used here immunostain most or all of the neurofibrillary tangles present in the sections whereas the antiserum to microtubule protein immunoreacted with about half of the neurofibrillary tangles. All of the antibodies as well as Bodian's silver stain reacted with the paired helical filaments. The epitopes that we have shown to be present in the paired helical filament, in contrast to the corresponding epitopes present in normal neuronal cytoskeleton, are insoluble in ionic detergent. It is concluded that these epitopes are integral components of the paired helical filaments and that, at least in part, paired helical filaments are derived from altered elements of the normal neuronal cytoskeleton.
Evolutionary amplification of a pseudogene.
Authors: Ghazal P , Clark A J , Bishop J O
Published: May 2006

The family of mouse major urinary protein (MUP) genes has about 35 members, clustered together on chromosome 4. Most of the genes belong to two major subfamilies (group 1 and group 2) each with 12-15 members. Recently we showed that most of the group 1 and group 2 genes are arranged in pairs, each containing a group 1 and a group 2 gene in divergent transcriptional orientation, with 15 kilobases of DNA between the two cap sites. Here we present the nucleotide sequence of the first exon of six group 1 genes and four group 2 genes. The data confirm the close relationship of the genes within each group and the considerable divergence of the two groups from each other. The four group 2 genes all carry the same nonsense mutation in codon 7 of the sequence that specifies the mature protein. Thus, not only do these genes have a common ancestor, but also it seems that their amplification followed the mutation of the ancestor to a pseudogene. Taking into account the 3' flanking regions of the two genes, the overall size of each gene-pair is about 45 kilobases. The sequencing data supports our earlier suggestion that this 45 kilobase domain is the unit of Mup amplification.
Direct demonstration of structural changes in soluble, monomeric Ca2+-ATPase associated with Ca2+ release during the transport cycle.
Authors: Andersen J P , Jørgensen P L , Møller J V
Published: May 2006

The time courses of changes in protein conformation and Ca2+ binding in the phosphorylated state of membrane-bound and soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum have been examined at pH 8.0, 2 degrees C. The transition from ADP-sensitive to ADP-insensitive phosphoenzyme occurs in the soluble monomer as well as in membranous Ca2+-ATPase and is accompanied by an increase in fluorescence from 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine diphosphate bound to the catalytic site and change in tryptic cleavage pattern. A decrease of Ca2+ affinity occurs simultaneously with the fluorescence rise, suggesting a single-step mechanism for energy transfer between the catalytic site and the Ca2+ transport sites. This is in accordance with the tryptic degradation pattern that suggests proximity between the phosphorylation site and Ca2+ transport sites on the peptide. The structural changes occurring in the soluble monomeric Ca2+-ATPase show that a single polypeptide chain is the functional unit in energy transduction.
Inactivation of S-adenosyl-L-homocysteine hydrolase by cAMP results from dissociation of enzyme-bound NAD+.
Authors: Hohman R J , Guitton M C , Veron M
Published: May 2006

S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) is inactivated by cAMP and also by 2'-deoxyadenosine, and in both cases, activity is restored by incubating the inactivated enzyme with NAD+. We have previously presented evidence that, despite these similarities, inactivation by these two ligands proceeds by different mechanisms. We have now used a fluorescence technique to quantitate enzyme-bound NAD+ and NADH on S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum, and we have confirmed that cAMP and 2'-deoxyadenosine inactivate by different mechanisms. Whereas inactivation by 2'-deoxyadenosine is due to reduction of the enzyme-bound NAD+ to NADH, incubation of S-adenosyl-L-homocysteine hydrolase with cAMP results in dissociation of the enzyme-bound NAD+. The dissociation is reversible, and reactivation likely occurs by restoration of the initial NAD+ content. This reversible inactivation by cAMP may be a mechanism of controlling biological methylation reactions by adjusting intracellular concentrations of S-adenosyl-L-homocysteine through action of S-adenosyl-L-homocysteine hydrolase.
Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution.
Authors: Keese P , Symons R H
Published: May 2006

On the basis of sequence homology a model is proposed for five structural and functional domains in viroids. These domains include (i) a conserved central region capable of forming two alternative structures that may regulate two phases of the viroid replication cycle, (ii) a region associated with pathogenicity, (iii) a domain with high sequence variability, (iv and v) two terminal domains that are interchangeable between viroids. That the evolution of viroids has involved RNA rearrangements of domains is supported by the partial duplication of coconut cadang cadang viroid, which arises de novo during each infection. Similar RNA rearrangements have been established for animal viral defective interfering RNAs, which arise by some form of discontinuous transcription. This mechanism could account for the origin of viroids and also RNA viruses, whereby modules of genetic information may have undergone repeated exchange between RNA pathogens and the RNA of their hosts.
A large intracellular pool of inactive Na channel alpha subunits in developing rat brain.
Authors: Schmidt J , Rossie S , Catterall W A
Published: May 2006

An intracellular pool of Na channel alpha subunits has been detected in developing brain cells in vivo and in vitro by phosphorylation with cAMP-dependent protein kinase, immunoprecipitation with specific antiserum, and NaDodSO4 gel electrophoresis or by radioimmunoassay. These alpha subunits are membrane-bound, contain complex carbohydrate chains, and have an apparent molecular weight of 260,000 like mature alpha subunits. In contrast to mature alpha subunits, the intracellular subunits are not covalently attached to a beta 2 subunit, and they do not bind saxitoxin with high affinity. They comprise 67-77% of the total immunoreactive alpha subunit in developing rat brain cells but are not a prominent component in the adult brain. It is proposed that this intracellular pool of alpha subunits forms a ready reserve of preformed subunits for incorporation into the surface membrane during periods of active membrane biogenesis. The results suggest that disulfide linkage of the alpha and beta 2 subunits, insertion into the cell surface membrane, and attainment of a functional conformation are closely related late events in the biogenesis of the Na channel. These processes may regulate the number of functional Na channels in the developing brain.
Increased rate of degradation of c-myc mRNA in interferon-treated Daudi cells.
Authors: Dani C , Mechti N , Piechaczyk M , Lebleu B , Jeanteur P , Blanchard J M
Published: May 2006

The recent observation made in our laboratory that cellular myc (c-myc) mRNA has a very short half-life in a variety of normal and transformed human cells emphasized the potential importance of post-transcriptional regulation of c-myc gene expression. Jonak and Knight [Jonak, G. J. & Knight, E., Jr. (1984) Proc. Natl. Acad. Sci. USA 81, 1747-1750] have reported a selective reduction of c-myc mRNA accumulation in lymphoblastoid Daudi cells treated with human beta interferon. This provided a suitable situation in which to examine a possible action of negative modulators of c-myc expression at the level of mRNA stability. Our results confirm the observation by Jonak and Knight that c-myc mRNA level is depressed in cells treated with beta interferon and extend it to alpha 2 interferon. Furthermore, we now demonstrate that interferon has no effect on c-myc transcription rate in isolated nuclei but rather reduces the half-life of its mRNA. Conversely, we show that it increases the level of HLA-A2 mRNA by stimulating its transcription.
Arginine in the leader peptide is required for both import and proteolytic cleavage of a mitochondrial precursor.
Authors: Horwich A L , Kalousek F , Rosenberg L E
Published: May 2006

Most mitochondrial proteins are encoded in the nucleus and translated in the cytoplasm as larger precursors containing NH2-terminal "leader" peptides, which are strikingly basic in overall amino acid composition. Recent experiments indicate that these leader peptides are both necessary and sufficient to direct post-translational recognition and import of precursors by mitochondria. In this report, we demonstrate a critical role for one or more of the basic arginine residues in the leader peptide of the subunit precursor for the human mitochondrial matrix enzyme, ornithine transcarbamoylase (ornithine carbamoyltransferase, carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3). The distal three of four basic residues, all arginines, in the leader peptide of ornithine transcarbamoylase were replaced at once with charge-neutral glycine residues. The altered ornithine transcarbamoylase precursor failed to be taken up by intact mitochondria in vitro. Moreover, it also failed to be proteolytically cleaved upon incubation with a mitochondrial matrix fraction containing the Zn2+-dependent protease, which normally cleaves the leader peptide.
The regulation of DNA synthesis in quiescent lymphocytes by cytoplasmic inhibitors.
Authors: Gutowski J K , West A , Cohen S
Published: May 2006

We previously have shown that proliferating lymphoid cells contain an extractable cytoplasmic protein that is capable of inducing DNA synthesis in isolated quiescent nuclei (activator of DNA replication, ADR). ADR is present in continuously proliferating (transformed) lymphoblastoid cell lines and in mitogen- or interleukin 2-stimulated human peripheral blood leukocytes (PBL) but is not detectable in extracts from resting (unstimulated) PBL. In the present study, we investigated the possibility that quiescent PBL may contain a factor than can inhibit ADR activity. We found that unstimulated human PBL contain a heat-stable protein greater than or equal to 50,000 Da that is capable of suppressing the induction of DNA synthesis in isolated nuclei by ADR derived from both normal and neoplastic sources. The inhibitor was not detectable in freshly isolated PBL but appeared within the cells after a brief (2-6 hr) culture period. Depletion experiments revealed that the inhibitor was neither derived from nor dependent upon the macrophage component of the PBL cultures. These results suggest that one mechanism by which resting lymphocytes may maintain quiescence may involve the suppression of cytoplasmic mitogenic signals by intracellular inhibitors. In addition, the implications of our findings provide a possible explanation for the loss of growth control in abnormal proliferative states, such as neoplasia.
A 78-kilobase region of mouse chromosome 3 contains salivary and pancreatic amylase genes and a pseudogene.
Authors: Wiebauer K , Gumucio D L , Jones J M , Caldwell R M , Hartle H T , Meisler M H
Published: May 2006

Genetic studies have demonstrated that salivary and pancreatic amylase genes are closely linked in human and mouse. To analyze the arrangement of genes within the amylase cluster, a library of YBR mouse genomic DNA was cloned in the cosmid vector pJB8. Clones containing amylase genes were identified by hybridization with amylase cDNA probes. Salivary and pancreatic amylase genes were isolated on separate cosmid clones, but no overlapping clones were evident from the initial screening. A strategy for the rapid isolation of terminal noncoding fragments from the cosmid clones was developed. By using these terminal fragments for chromosome "walking," a map of 78 kilobases of the amylase gene region was constructed. The salivary and pancreatic amylase genes are present within this region in the same 5'-to-3' orientation, separated by 22 kilobases of genomic DNA. A truncated amylase pseudogene is located 10 kilobases downstream from the pancreatic amylase gene.
Is the 5S RNA a primitive ribosomal sequence?
Authors: Nazar R N , Wong W W
Published: May 2006

A tandemly arranged cluster of 55 RNA-like sequences in the middle of ribosomal 26S to 28S rRNAs from divergent eukaryotic organisms raises the possibility that the larger ribosomal RNAs were built up, at least in part, by gene amplification events and suggests an intriguing evolutionary relationship between the 55 rRNA and the larger rRNA molecules.
Activation of retinoic acid by coenzyme A for the formation of ethyl retinoate.
Authors: Miller D A , DeLuca H F
Published: May 2006

All-trans-retinoic acid is metabolized to a less polar metabolite in rat liver microsomes. This metabolite was proven to be ethyl retinoate by cochromatography on high-performance liquid chromatography, base hydrolysis to all-trans-retinoic acid, and gas chromatography/mass spectral analysis. The formation of ethyl retinoate is a specific enzymatic process; the apparent Km for all-trans-retinoic acid is 9.8 microM. The production of ethyl retinoate is greatly stimulated by the addition of coenzyme A, suggesting the formation of a retinoic acid-coenzyme A intermediate (retinoyl-coenzyme A).
The human Thy-1 gene: structure and chromosomal location.
Authors: Seki T , Spurr N , Obata F , Goyert S , Goodfellow P , Silver J
Published: May 2006

The human Thy-1 gene has been isolated and sequenced and compared to the rat and mouse Thy-1 genes. All three genes are organized in the same way: one exon encoding the majority of the signal peptide, another encoding the transmembrane segment, and a third encoding the remainder of the protein. One major structural difference between the human and rodent Thy-1 glycoproteins is that the former contains two instead of three glycosylation sites. RNA blot analysis of a human T-cell line expressing the T3 complex showed an absence of Thy-1 mRNA, excluding the possibility that Thy-1 represents one of the component chains of T3. The structural gene for human Thy-1 was localized to the long arm of chromosome 11 by nucleic acid hybridization to genomic DNA isolated from somatic cell hybrids.
Calmodulin binding to the cytoskeletal neuronal calmodulin-dependent protein kinase is regulated by autophosphorylation.
Authors: LeVine H , Sahyoun N E , Cuatrecasas P
Published: May 2006

A brain cytoskeletal preparation that is highly enriched in calmodulin-dependent protein kinase facilitated the study of the binding of 125I-labeled calmodulin to the native enzyme. The binding was specific, saturable, Ca2+-dependent, and inhibited by trifluoperazine. Stoichiometric analysis revealed that the ratio of bound calmodulin to the alpha subunit of the protein kinase was about 1:10 (+/-30%), indicating that in the native state not all of the enzyme subunits were accessible to bind calmodulin. The Kd for the binding reaction was 7 X 10(-9) M and was subject to regulation by divalent cations other than Ca2+, decreasing to 1.7 X 10(-9) M in the presence of 7 mM MgCl2. Activation of the protein kinase in the presence of Ca2+ and calmodulin resulted in marked autophosphorylation of the enzyme subunits. The autophosphorylation was accompanied by a 2-fold decrease in the affinity and number of 125I-labeled calmodulin binding sites. This effect was also reflected by an increase in the apparent Km for Ca2+ from 90 to 200 X 10(-9) M. Thus, enzyme autophosphorylation appears to represent a negative feedback signal, rendering the enzyme less sensitive to subsequent stimulation by physiologic increases in the intracellular Ca2+ concentration. These results help to clarify the mode of neuronal intracellular Ca2+ signaling.
Alteration of leukotriene release by macrophages ingesting Toxoplasma gondii.
Authors: Locksley R M , Fankhauser J , Henderson W R
Published: May 2006

Mouse resident peritoneal macrophages incubated with ionophore A23187 or opsonized zymosan released leukotrienes (LT) B4 and C4 (LTB4 and LTC4) and LTC4 and LTD4, respectively. In contrast, incubation with Toxoplasma gondii, an obligate intracellular protozoan, led to the formation of 11-, 12-, and 15-hydroxyicosatetraenoic acids (HETEs), together with an unidentified compound, designated compound X. Each of these compounds incorporated [3H]arachidonic acid from the macrophage during phagocytosis of T. gondii. Compound X migrated immediately prior to 15-HETE by reverse-phase HPLC and was distinct from authentic monoHETE, monohydroperoxyicosatetraenoic acid (mono-HPETE), and dihydroxyicosatetraenoic acid (diHETE) standards. The generation of compound X by macrophages correlated with the extent of phagocytosis of T. gondii and with intracellular survival of the organisms. Prior antibody-coating of T. gondii or activation of macrophages, either of which inhibited survival and replication of ingested organisms, was associated with production of LTD4 but not compound X. Killed organisms also stimulated LTD4 release only. Although T. gondii concentrated arachidonic acid, they did not metabolize the compound to identifiable lipoxygenase products. Preincubation of macrophages with the relative lipoxygenase inhibitors nordihydroguaiaretic acid or 5,8,11,14-icosatetraynoic acid inhibited the formation of compound X. The absence of leukotriene production by macrophages ingesting T. gondii may explain the relative lack of a neutrophil inflammatory response in diseases due to obligate intracellular organisms. Alternatively, compound X may have functional activities that might mediate some of the host responses to cellular parasitism.
Interspecific chloroplast recombination in a
Nicotiana
somatic hybrid
Authors: Medgyesy Peter , Fejes Erzsebet , Maliga Pal
Published: May 2006

Genetic recombination between chloroplasts of two flowering plant species,
Nicotiana tabacum
and
Nicotiana plumbaginifolia
, after somatic cell fusion is described. The parental lines differed in three cytoplasmic genetic markers. The
N. tabacum
mutant SR1-A15 was streptomycin-resistant, defective in chloroplast greening, and lincomycin-sensitive. The
N. plumbaginifolia
mutant LR400 was streptomycin-sensitive, normal green, and lincomycin-resistant. Streptomycin-resistant clones in cell culture are identified by their ability to form a green callus on a selective medium. Streptomycin resistance in the SR1-A15 mutant could not be expressed due to defective chloroplasts. Protoplasts of the two species were fused, and calli grown from the fused population were screened for the expression of streptomycin resistance from the SR1-A15 line as the result of interspecific chloroplast recombination. A somatic hybrid, pt14, expressed a new combination of the cytoplasmic genetic markers. In the pt14 chloroplast genome three
N. tabacum
and four
N. plumbaginifolia
parent specific restriction sites have been identified, indicating that the pt14 chloroplast genome contains at least six recombination sites.
Purification and characterization of a neurite extension factor from bovine brain.
Authors: Kligman D , Marshak D R
Published: May 2006

The extension of neurites by chicken embryo cerebral cortical neurons can be measured quantitatively at low cell density in serum-free, defined medium. An acidic, heat-stable protein fraction from bovine brain has been shown to have neurite extension activity in this assay. We report the use of reversed-phase HPLC to purify a neurite extension factor from this fraction to apparent homogeneity. The protein was characterized by NaDodSO4/PAGE. In the presence of reducing agents, the protein migrated as a single band, with an apparent molecular weight of 6500. In the absence of reducing agents, the protein showed bands at apparent molecular weights of 6500, 21,000-22,000, 30,000, and 40,000. Reduction and S-carboxymethylation of the protein abolished all biological activity and resulted in a shift of the apparent molecular weight to 11,000. The amino acid composition of the purified neurite-extension factor was nearly identical to that of bovine brain S100 beta. The amino acid sequences of peptides derived from trypsin or cyanogen bromide digests of the protein were identical to those found in S100 beta and accounted for 71 of 91 amino acids in the protein. However, three peptides obtained from cyanogen bromide digestion of the nonreduced protein appeared to be disulfide-linked dimers. Our results indicate that a biological activity, neurite extension, which is critical for the development of the nervous system, is associated with a disulfide form of S100 beta.
The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site.
Authors: Senecoff J F , Bruckner R C , Cox M M
Published: May 2006

The minimal size of the recombination site required for efficient FLP recombinase-catalyzed recombination in vitro is no more than 28 base pairs, which includes parts of two 13-base-pair inverted repeats and all of an 8-base-pair spacer. The FLP recombinase cleaves the DNA at the boundaries of the spacer, becomes covalently linked to the spacer DNA via a 3' phosphate, and leaves a free 5' hydroxyl at the other end of the 8-base-pair spacer. The efficiency of recombination is reduced if the size of the spacer in a recombinant site is increased or decreased by 1 base pair, while the spacer in the second site is unaltered. Recombination between two sites with identical 1-base-pair additions or deletions in the spacer, however, is relatively unaffected. This result suggests that pairing of sequences in the spacer region is important in FLP-promoted recombination events. The sequence asymmetry utilized by the recombinase to determine the orientation of the site is located uniquely within the spacer region.
Synthesis of analogs of human platelet membrane glycoprotein IIb-IIIa complex by chicken peripheral blood thrombocytes.
Authors: Kunicki T J , Newman P J
Published: May 2006

Human platelets and their phylogenetic counterparts, avian thrombocytes, play a key role in primary hemostasis. Based upon extensive studies in mammals, platelet cohesion resulting in the formation of the "hemostatic plug" is known to be mediated by the mammalian platelet glycoprotein IIb-IIIa complex in concert with fibrinogen and calcium. The immunological and biochemical technology already developed for the analyses of mammalian platelet glycoproteins has never been applied to avian thrombocytes. By indirect immunofluorescence, we now show that a polyclonal rabbit antibody specific for human glycoproteins IIb plus IIIa and the well-characterized murine monoclonal anti-IIb-IIIa complex antibody, AP2, both crossreact with IIb and IIIa analogs on intact chicken thrombocytes. By two-dimensional polyacrylamide gel electrophoresis, we also demonstrate that chicken thrombocytes will incorporate [35S]methionine into several proteins, including the glycoprotein IIb and IIIa analogs during short-term (4 hr) incubation in vitro. This finding indicates that peripheral blood nucleated thrombocytes of the chicken, unlike their mammalian counterparts, retain the capacity to synthesize protein. The significance of these findings is 2-fold. First, we provide biochemical and immunological evidence that those proteins responsible for platelet cohesion in humans are structurally conserved in cells of analogous function in chickens despite the fact that these species have diverged from a common ancestor more than 200-250 million years ago. Second, we identify chicken thrombocytes as a readily available source of messenger RNA encoding numerous proteins analogous to those already characterized in human platelets, including glycoproteins IIb and IIIa.
Nerve growth factor promotes cholinergic development in brain striatal cultures.
Authors: Martínez H J , Dreyfus C F , Jonakait G M , Black I B
Published: May 2006

We have examined the effect of the trophic protein, nerve growth factor (NGF), on organotypic cultures of fetal rat striatum. Treatment of cultures with NGF for 10-11 days resulted in a 5- to 12-fold increase in the specific activity of the cholinergic enzyme choline acetyltransferase (CAT; EC 2.3.1.6). in a dose-dependent fashion. This effect was not elicited by insulin, ferritin, or cytochrome c, proteins similar in structure or physicochemical properties to NGF. The effect of NGF on CAT activity was specifically blocked by anti-NGF antiserum, whereas treatment with the antiserum alone did not have a significant effect on the enzyme. Immunocytochemical studies of the treated cultures, using a monoclonal antibody directed against CAT, revealed positively stained neurons exhibiting dendritic and axonal processes. NGF did not have an effect on total protein content of the striatal cultures, suggesting a highly specific effect. Moreover, levels of substance P, a peptide localized to other, noncholinergic neurons, were not altered by NGF. Substance P remained unchanged after treatment with NGF for 12 days, whereas CAT activity increased 12-fold in sister cultures. Although the mechanisms of action of NGF on striatal cholinergic interneurons remain to be determined, the marked, specific response of CAT suggests that this well-defined trophic protein may play a critical role in normal brain development.
Nonlinear natural engine: Model for thermodynamic processes in mesoscale systems
Authors: Wheatley John , Buchanan D. S. , Swift G. W. , Migliori A. , Hofler T.
Published: May 2006

To develop intuition on the possible application of concepts from thermodynamic heat engines to mesoscale systems, we have constructed and studied a model thermoacoustic heat engine. The model consists of a chain of coupled nonlinear acoustic vibrators in which the primary thermodynamic medium is argon gas, the secondary thermodynamic medium is constituted by solids bounding the gas, and frequencies are
ca
. 3 × 10
2
Hz. The nonlinear elements are the necks, made flexible by means of an oil-loaded DuPont Kapton film, of Helmholtz resonators. When the primary medium is driven uniformly by an acoustic driver at a frequency somewhat below the low-amplitude resonant frequency and at a high enough driving amplitude, stationary localized or solitary states appear irreversibly on the chain. These are characterized by a higher vibrational amplitude than that in surrounding vibrators, where the amplitude can decrease; by the appearance of deep subharmonics of the drive frequency, corresponding to driven low-frequency vibrations of the Kapton film-oil systems; and by the pumping of heat toward the localized states. Possible implications of these results for mesoscale systems consisting of chains of molecular vibrators are then discussed.
The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor.
Authors: Ralston R , Bishop J M
Published: May 2006

We have observed a modification of the cellular protein kinase pp60c-src, elicited in murine 3T3 fibroblasts by platelet-derived growth factor (PDGF). The modification occurred rapidly after addition of PDGF to the culture medium and was first detected as a reduction in the electrophoretic mobility of a portion of the pp60c-src molecules. A similarly modified form of the viral homologue pp60v-src occurs in vivo in the absence of stimulation by PDGF. The occurrence of modified forms of both pp60c-src and pp60v-src was associated with a novel phosphorylation at tyrosine in the amino-terminal domains of the proteins. The time-course and dose-response for this modification of pp60c-src paralleled PDGF-induced increases in phosphorylation of pp36, a major cellular substrate for several tyrosine-specific protein kinases. In parallel experiments, treatment of cells with PDGF increased the kinase activity of pp60c-src in an immunocomplex assay. These results suggest pp60c-src may play a role in the mitogenic response to PDGF.
Cloning and expression of cDNA for rat heme oxygenase.
Authors: Shibahara S , Müller R , Taguchi H , Yoshida T
Published: May 2006

Two cDNA clones for rat heme oxygenase have been isolated from a rat spleen cDNA library in lambda gt11 by immunological screening using a specific polyclonal antibody. One of these clones has an insert of 1530 nucleotides that contains the entire protein-coding region. To confirm that the isolated cDNA encodes heme oxygenase, we transfected monkey kidney cells (COS-7) with the cDNA carried in a simian virus 40 vector. The heme oxygenase was highly expressed in endoplasmic reticulum of transfected cells. The nucleotide sequence of the cloned cDNA was determined and the primary structure of heme oxygenase was deduced. Heme oxygenase is composed of 289 amino acids and has one hydrophobic segment at its carboxyl terminus, which is probably important for the insertion of heme oxygenase into endoplasmic reticulum. The cloned cDNA was used to analyze the induction of heme oxygenase in rat liver by treatment with CoCl2 or with hemin. RNA blot analysis showed that both CoCl2 and hemin increased the amount of hybridizable mRNA, suggesting that these substances may act at the transcriptional level to increase the amount of heme oxygenase.
Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells.
Authors: Soellner P , Quinlan R A , Franke W W
Published: May 2006

Intermediate-sized filaments (IF) are among the most insoluble intracellular protein polymer structures. We have analyzed the small amounts of soluble vimentin, an IF protein, present in cytosol fractions obtained from lysis of cultured cells [rat RVF-SM cells, simian virus 40-transformed human fibroblasts, and human rhabdomyosarcoma (RD line) cells]. The molecular form of this soluble vimentin was determined by sucrose density gradient centrifugation, using vimentin-specific antibodies for subsequent ELISA and immunoblotting analyses. The majority of the soluble vimentin appeared in a distinct form indistinguishable in its sedimentation behavior from reconstituted tetrameric subunits of purified vimentin arrested at low ionic strength. The tetrameric coiled-coil nature of the soluble form of vimentin was indicated by the digestion pattern with chymotrypsin and by chemical crosslinking with copper-1,10-phenanthroline and dimethylsuberimidate. The competence of this soluble vimentin to assemble into IF at higher salt concentrations was demonstrated by electron microscopy. Pulse-chase experiments showed that the soluble form was not an exclusively posttranslational intermediate. We propose that in the living cell a small pool of a distinct soluble tetrameric form of vimentin exists which may exchange with polymeric IF vimentin.
Primer-dependent eukaryotic RNA polymerase capable of accurate transcription from the adenovirus major late promoter in a reconstituted system.
Authors: Fradkin L G , Leong K , Morrow C D , Berk A J , Dasgupta A
Published: May 2006

A sensitive assay for detection of eukaryotic RNA polymerase II has been developed. This assay depends on the ability of polymerase II to elongate a small RNA primer, oligo(U), hybridized to a single-stranded homopolymeric DNA template, poly(dA). The poly(dA).oligo(U)-dependent RNA polymerase II from calf thymus has been purified approximately 10,000-fold using this assay. The purified enzyme contains four polypeptides of apparent Mr 180,000, 140,000, 24,000, and 16,000 and is fully active in accurate initiation of transcription from the adenovirus major late promoter in the presence of transcription factors from HeLa cells. The poly(dA).oligo(U)-dependent RNA polymerase activity can be detected in crude cell extracts from a variety of tissue culture cells and appears to be largely due to polymerase II, since 90-95% of this activity is inhibited by alpha-amanitin at a concentration of 1 microgram/ml.
Suppression of IgE secretion from hybridoma cells in allotype congenic mice: suppression of allotype 7a by T cells in allotype b mice.
Authors: Maekawa S , de Szalay C , Ovary Z
Published: May 2006

Anti-2,4-dinitrophenyl (DNP) IgE producing hybridoma B 53 when injected subcutaneously is established equally well in syngeneic BALB/c (heavy-chain allotype a) and congenic CB20 (heavy-chain allotype b) mice. However, secretion of anti-DNP IgE monoclonal antibody is greatly suppressed in CB20 mice. B 53 cells taken from the subcutaneous tumors of CB20 mice produce anti-DNP IgE in vivo in BALB/c mice and in vitro. No difference was observed in IgE production between these cells and the controls taken from BALB/c mice. The suppression of IgE production was due to T cells and/or their product(s) of CB20 mice.
Comparison of theoretical and experimental values of the number of metallic orbitals per atom in hypoelectronic and hyperelectronic metals
Authors: Pauling Linus , Kamb Barclay
Published: May 2006

The statistical resonating-valence-bond theory of metals is applied in the purely theoretical calculation of the composition of the Ni-Cu alloy at the foot of the curve of saturation ferromagnetic moment, which marks the boundary between hypoelectronic and hyperelectronic metals and determines the value of the number of metallic orbitals per atom. The results, Ni
44
Cu
56
and 0.722 metallic orbitals, agree with the observed values. This agreement provides strong support of the theory.
p27x-III and p21x-III, proteins encoded by the pX sequence of human T-cell leukemia virus type I.
Authors: Kiyokawa T , Seiki M , Iwashita S , Imagawa K , Shimizu F , Yoshida M
Published: May 2006

Human T-cell leukemia virus type I (HTLV-I) is an etiological agent of adult T-cell leukemia and has a unique sequence, pX, that contains four possible open reading frames, I-IV. p40x was previously identified as the gene product of frame IV (x-lor) and was suggested to mediate transcriptional trans-activation of the viral long terminal repeats. We have identified two pX gene products, p27x-III and p21x-III, encoded by frame III, which mostly overlapped frame IV. These proteins were detected with rabbit antiserum against the synthetic peptide predicted from the 3' end of frame III. p27x-III is phosphorylated in cultured cells, and the phosphoprotein (pp27x-III) is localized in nuclei; some pp27x-III was tightly bound to nuclear components. p27x-III was detected in a number of cell lines that express other viral antigens, including a cell line previously reported to express only p40x as a viral protein. The function(s) of p27x-III and p21x-III is not known, but the tight binding of pp27x-III to nuclear components suggests that it is associated with regulation of viral gene expression.
Fraction of myosin cross-bridges bound to actin in active muscle fibers: estimation by fluorescence anisotropy measurements.
Authors: Burghardt T P , Ajtai K
Published: May 2006

Time-resolved and steady-state fluorescence anisotropy measurements from fluorescence-labeled myosin cross-bridges in single glycerinated skeletal muscle fibers in rigor, relaxed, MgADP-induced, and contracting states have been made in order to estimate the fraction of actin-bound cross-bridges in active muscle. When the plane of polarization of the excitation light is perpendicular to the fiber axis and its propagation vector has a component parallel to this axis, actin-bound cross-bridge states, such as rigor and MgADP-induced, have time-zero and steady-state anisotropies that are substantially lower than has the relaxed state. This difference provides a means of determining the fraction of cross-bridges bound to actin in active isometric fibers, by comparing the fluorescence anisotropy from active fibers with the anisotropy from bound and unbound cross-bridges in static states. By assuming that the active cross-bridges are either bound (in the manner of rigor or MgADP-induced states) or relaxed, we estimate that greater than 80% of the cross-bridges are actin-bound in active isometric fibers.
Human complement component C3: cDNA coding sequence and derived primary structure.
Authors: de Bruijn M H , Fey G H
Published: May 2006

The complete cDNA coding sequence and derived amino acid sequence of human complement component C3 are presented. The encoded precursor molecule contains a signal peptide of 22 amino acid residues, the beta chain (645 residues), and the alpha chain (992 residues). The two chains are joined by four arginine residues not present in the mature protein. Several functionally important sites have been localized, such as the thiolester site, the cleavage site liberating the anaphylatoxin, and two sites of cleavage by the serine protease factor I, as well as a peptide fragment with leukocyte mobilizing activity. At least two carbohydrate attachment sites, one on each chain, have been identified. Human C3 has 79% identity to mouse C3 at the nucleotide level and 77% identity at the amino acid level. The protease alpha 2-macroglobulin and complement component C4 show considerable homology to C3, suggesting that the three proteins have evolved from a common ancestor.
Voltage-dependent sodium and potassium channels in mammalian cultured Schwann cells.
Authors: Shrager P , Chiu S Y , Ritchie J M
Published: May 2006

Cultured Schwann cells from sciatic nerves of newborn rabbits and rats have been examined with patch-clamp techniques. In rabbit cells, single sodium and potassium channels have been detected with single channel conductances of 20 pS and 19 pS, respectively. Single sodium channels have a reversal potential within 15 mV of ENa, are blocked by tetrodotoxin, and have rapid and voltage-independent inactivation kinetics. Single potassium channels show current reversal close to EK and are blocked by 4-aminopyridine. From these results, and from comparisons of single-channel and whole-cell data, we show that these Schwann cells contain voltage-dependent sodium and potassium channels that are similar in most respects to the corresponding channels in mammalian axonal membranes. Cultured rat Schwann cells also have sodium channels, but at a density about 1/10th that of rabbit cells, a result in agreement with saxitoxin binding experiments on axon-free sectioned nerves. Saxitoxin binding to cultured cells suggests that there are up to 25,000 sodium channels in a single rabbit Schwann cell. We speculate that in vivo Schwann cells in myelinated axons might act as a local source for sodium channels at the nodal axolemma.
Interferon regulates c-myc gene expression in Daudi cells at the post-transcriptional level.
Authors: Knight E , Anton E D , Fahey D , Friedland B K , Jonak G J
Published: May 2006

c-myc gene mRNA is reduced by greater than 75% in the human lymphoblastoid cell line Daudi when growth is inhibited by treatment with human interferon beta (IFN-beta). In the present communication, we describe the effect of IFN-beta treatment on transcription of the c-myc gene and on the steady-state level of c-myc mRNA in the cytoplasm of Daudi cells. The results show that, although the rate of c-myc transcription is not significantly different in nuclei isolated either from untreated cells or from those treated with IFN-beta for 3 or 24 hr, the level of c-myc mRNA in the cytoplasm is reduced by 60% within 3 hr of IFN-beta treatment. These results suggest that IFN-beta regulates the c-myc mRNA at a post-transcriptional level. These results are in contrast to the regulation of two IFN-beta-induced genes that under identical conditions are regulated in these cells at the transcriptional level. We have also detected induction of the (2'-5')oligoadenylate synthetase (2-5A synthetase) gene in IFN-beta-treated Daudi cells. Since certain c-myc transcripts have the capacity to form double-stranded RNA regions, we propose that one mechanism by which c-myc could be regulated post-transcriptionally in IFN-beta-treated cells is by activating, through its own double-strandedness, the 2-5A synthetase/RNase L endonuclease system, which would cause selective degradation of the c-myc RNA.
Bioeffective monoclonal antibody against the decapeptide gonadotropin-releasing hormone: reacting determinant and action on ovulation and estrus suppression.
Authors: Talwar G P , Gupta S K , Singh V , Sahal D , Iyer K S , Singh O
Published: May 2006

A monoclonal antibody generated against the decapeptide gonadotropin-releasing hormone (GnRH) was effective in intercepting the bioactivity of the hormone; it blocked ovulation in rats. The antibody reacted optimally with the native hormone. Substitution of amide at the COOH terminus by a carboxyl group decreased immunoreactivity by a factor of 200. The antibody recognized the amino acid sequences 4-6, 7-10, and 4-10 to a variable degree, which suggests that the epitope has a conformation involving the entire molecule, with the NH2- and COOH-terminal regions probably in proximity. The antibody was also competent to suppress the progression of estrus in dogs, an indication that GnRH may play an inductive role in the reproductive function of dogs.