bluuebunny/crossref_metadata_2025 huggingface dataset:
A ligand-specific action of chelated copper on hypothalamic neurons: stimulation of the release of luteinizing hormone-releasing hormone from median eminence explants.
Authors: Barnea A , Colombani-Vidal M
Published: May 2006

We have previously shown that chelated copper stimulates the release of luteinizing hormone-releasing hormone (LHRH) from isolated hypothalamic granules. In this study, we wished to ascertain if chelated copper acts on hypothalamic neurons to stimulate LHRH release and, if so, what is the ligand specificity of this interaction. An in vitro system of explants of the median eminence area (MEA) was established and characterized. MEA explants were exposed for 15 min to 50 microM copper, and then they were incubated for 75 min in copper-free medium. Copper led to a transient increase in the rate of LHRH release; the maximal rate was attained 15 min after transfer of the MEA to copper-free medium. In addition, we found that copper complexed to histidine (Cu-His), but not ionic copper, stimulated LHRH release, the magnitude of which was dependent on the dose of Cu-His. The chelator specificity for Cu complex action was such that Cu-His stimulated LHRH release 4.9-fold and Cu-Cys stimulated release 2.5-fold, whereas neither Cu-Thr, Cu-Gly-His-Lys, Cu-bovine serum albumin, nor ceruloplasmin stimulated LHRH release. Based on these results and those of others indicating that the concentration of copper in hypothalamic axonal terminals is 1-2 orders of magnitude greater than plasma, we propose that copper released in the vicinity of the LHRH neurons interacts with specific sites on the LHRH axonal terminals, which leads to release of the peptide.
Atrial natriuretic factor elicits an endothelium-independent relaxation and activates particulate guanylate cyclase in vascular smooth muscle.
Authors: Winquist R J , Faison E P , Waldman S A , Schwartz K , Murad F , Rapoport R M
Published: May 2006

A 26 amino acid synthetic peptide fragment of atrial natriuretic factor (ANF) relaxed isolated rabbit aortic segments in which the endothelium was either intact or functionally destroyed. The relaxations were temporally associated with increases in levels of cGMP with no change in the levels of cAMP. The ANF-induced increases in cGMP were also observed in aortic segments pretreated with calcium-free buffer or the cGMP phosphodiesterase inhibitor M&B 22,948. Qualitatively similar results were obtained for sodium nitroprusside. ANF selectively activated particulate guanylate cyclase, having no effect on the soluble form of the enzyme. Thus, the direct (endothelium-independent) vasodilator effect of ANF may be mediated via increased tissue levels of cGMP. ANF appears to increase vascular cGMP levels by activation of particulate guanylate cyclase.
Soldier formation regulated by a primer pheromone from the soldier frontal gland in a higher termite,
Nasutitermes lujae
Authors: Lefeuve Patrick , Bordereau Christian
Published: May 2006

In the nasute higher termite
Nasutitermes lujae
, the differentiation of new soldiers is suppressed or delayed by the soldiers themselves. Experimental data strongly suggest that this inhibitory effect results from the action of a primer pheromone secreted by the frontal gland of soldiers. The pheromone must be contacted directly. Thus, the frontal gland of termite soldiers assumes a new role in addition to alarm and defense, although this extension cannot be generalized to all termite species. The soldier inhibitory pheromone is evidently only one of multiple factors contributing soldier regulation in termite societies.
High-affinity monoclonal antibodies for aflatoxins and their application to solid-phase immunoassays.
Authors: Groopman J D , Trudel L J , Donahue P R , Marshak-Rothstein A , Wogan G N
Published: May 2006

Monoclonal antibodies specific for aflatoxin B1, aflatoxin B2, aflatoxin M1, and the major aflatoxin-DNA adducts were obtained following fusion of mouse SP-2 myeloma cells with spleen cells of mice immunized with aflatoxin B1 covalently bound to bovine gamma globulin. The aflatoxin-modified protein used to immunize mice was produced chemically by activating aflatoxin B1 to a 2,3-epoxide derivative, which then covalently bound to the protein. One of the monoclonal antibodies isolated (2B11) was found to be a high-affinity IgM antibody with an affinity constant for aflatoxin B1, aflatoxin B2, and aflatoxin M1 of about 1 X 10(9) liters per mol. In a competitive radioimmunoassay using [3H]aflatoxin B1, 3 pmol (1 ng) of aflatoxin B1, aflatoxin B2, or aflatoxin M1 caused 50% inhibition with this antibody. The antibody also had significant cross-reactivity for the major aflatoxin-DNA adducts: 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 and 2,3-dihydro-2-(N5-formyl-2',5', 6'-triamino-4'oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1. The antibody was also covalently bound to Sepharose-4B and used in a column-based solid-phase immunosorbent assay system. Aflatoxins added in vitro to phosphate buffer, human urine, human serum, or human milk at levels expected to be obtained in human samples acquired from environmentally exposed individuals were quantitatively recovered by applying the mixture to this antibody affinity column purification system. Preliminary studies using urine samples from rats injected with radiolabeled aflatoxin B1 have also indicated that aflatoxin metabolites can be isolated by these methods. Furthermore, we have found that the monoclonal antibody affinity columns can be regenerated for multiple use. Therefore, the monoclonal antibodies and their application to affinity chromatography represents a useful and rapid technique to purify environmentally occurring levels of this carcinogen and some of its metabolites for quantitative measurements.
Clusters of HLA class II beta restriction fragments describe allelic series.
Authors: Cohen D , Le Gall I , Marcadet A , Font M P , Lalouel J M , Dausset J
Published: May 2006

Eighty-eight HLA haplotypes have been investigated for the presence or absence of 52 restriction fragments generated by four restriction enzymes (EcoRI, EcoRV, HindIII, BamHI), and detected by a DQ beta cDNA probe. Correlation analysis showed several sets of positively associated fragments forming 11 clusters. They constitute three different allelic series. The first coincides with DR alleles, the second with DQ alleles, and within the third, one cluster coincides with DRw53 (MT). As shown by comparative hybridization, most fragments belonging to the DR- as well as the DQ-related series correspond to DQ beta genes. In contrast, the MT-related series corresponds to DR beta genes. The evolutionary significance of these restriction fragment clusters is discussed.
Proteolysis and the domain organization of myosin subfragment 1.
Authors: Mornet D , Ue K , Morales M F
Published: May 2006

Because the proteolytic cleavage of a folded polypeptide depends not only on the specificity of the protease but on the nature of the folding, we investigated the cleavage of (chymotryptically produced) subfragment 1 (designated "S-1") or "head" segment of myosin by seven proteases with different specificities. All seven produced approximately the same three fragments of S-1--namely, fragments (from the NH2 terminus) of 27, 50, and 20 kilodaltons, suggesting that in intact S-1 these fragments are distinct domains. The same proteases were used to hydrolyze the MgADP complex of S-1. All failed to do so except trypsin, which, as found earlier [Hozumi, T. (1983) Biochemistry 22, 799-804], makes two additional cleavages. This result suggests that the conformational change induced by MgADP opens up only a small stretch of polypeptide chain, which stretch happens to be vulnerable to trypsin.
Exposure of nondividing populations of primary human fibroblasts to UV (254 nm) radiation induces a transient enhancement in capacity to repair potentially lethal cellular damage.
Authors: Tyrrell R M
Published: May 2006

Nondividing (arrested) populations of primary human fibroblasts from normal individuals exposed to an initial dose (1.5 or 3 J X m-2) of far-UV (254 nm) radiation and then incubated in medium containing low (0.5%) serum develop enhanced resistance to inactivation of cloning efficiency by a second (challenge) dose of UV. The resistance develops within 2-4 days, after which there is a decline. Resistance develops to a higher degree and more rapidly (1-2 days) in cells derived from patients with the variant form of xeroderma pigmentosum. Excision-deficient cells from xeroderma pigmentosum complementation group A individuals also develop UV resistance after a lower (0.2 J X m-2) exposure to UV. Enhanced UV resistance does not develop in UV-irradiated cell populations incubated with the protein synthesis inhibitor cycloheximide (5 microM). These observations are consistent with the interpretation that exposure of human fibroblasts to low doses of UV induces synthesis of a protein involved in a metabolic pathway that transiently enhances the capacity of cells to repair potentially lethal damage resulting from a subsequent dose of UV.
Cell growth and net Na+ flux are inhibited by a protein produced by kidney epithelial cells in culture.
Authors: Walsh-Reitz M M , Toback F G , Holley R W
Published: May 2006

Proliferation of confluent kidney epithelial cell cultures (BSC-1 line) is inhibited by a protein (Mr approximately equal to 24,000) that is secreted by the cells. The mechanism of action of this growth inhibitor was sought by studying its effect on net Na+ flux because increased availability of Na+ in the culture medium had been shown to stimulate cell growth. The increase in cell Na+ content observed during stimulation of the growth after a medium change was attenuated in the presence of the purified inhibitor. Inhibition of both cell Na+ accumulation and growth in the presence of the protein was reversed completely by addition of NaCl to the medium. These results suggest that control of net Na+ flux and growth in kidney epithelial cells could be mediated, at least in part, by a secreted cellular protein.
Unusual alleles of recB and recC stimulate excision of inverted repeat transposons Tn10 and Tn5.
Authors: Lundblad V , Taylor A F , Smith G R , Kleckner N
Published: May 2006

Precise and nearly precise excision of transposon Tn10 occur by host-mediated processes unrelated to transposition. Both types of excision involve interactions between short (9 or 24 base-pair) direct repeat sequences at or near the termini of the transposon and are stimulated by the large (1,329-base-pair) inverted repeats that form the ends of Tn10. We describe here three mutations of Escherichia coli K-12, designated texA, that enhance excision of Tn10 and of the structurally analogous transposon Tn5. Genetic mapping and complementation analysis show that these mutations are unusual alleles of the recB and recC genes that alter but do not abolish RecBC function. As Tn10 excision normally does not depend on RecA or RecBC functions, texA mutations appear to provide another pathway for excision that depends on altered RecBC function; for one texA allele, excision has become dependent on RecA function as well. The available evidence suggests that texA mutations alter the stimulatory interaction between the inverted repeats of Tn10.
Products of a reciprocal chromosome translocation involving the c-myc gene in a murine plasmacytoma.
Authors: Stanton L W , Yang J Q , Eckhardt L A , Harris L J , Birshtein B K , Marcu K B
Published: May 2006

The structures of the rearranged c-myc gene products derived from a rcp T(12;15) have been investigated in the MPC-11 (IgG2b kappa) plasmacytoma. The rcp T(12;15) in MPC-11 is a reciprocal exchange between the c-myc gene on chromosome 15 and the immunoglobulin gamma 2a switch region (S gamma 2a) on chromosome 12. The c-myc gene is broken within a 5'-nontranslated exon, thereby separating the promoter region of the normal c-myc gene from its protein coding sequences. This reciprocal rearrangement results in the loss of 11 base pairs of c-myc sequence and 300 base pairs of S gamma 2a sequence at the point of recombination. Sequences that represent the promoter region of the normal c-myc gene are present in the 5'-myc reciprocal fragment. A comparison of the nucleotide sequences at the recombination site of a number of c-myc rearrangements reveals a common feature that may have mechanistic importance for these translocation events.
Activation of factor IX bound to cultured bovine aortic endothelial cells.
Authors: Stern D M , Drillings M , Kisiel W , Nawroth P , Nossel H L , LaGamma K S
Published: May 2006

Previous studies have shown that factor IX and its activated form, factor IXa, bind to cultured vascular endothelial cells and that cell-bound factor IXa retains its procoagulant activity. The present studies provide evidence that factor IX bound to cultured bovine aortic endothelial cells can be activated. Factor IX activation was assessed by finding cleavage of the factor IX molecule on NaDodSO4/polyacrylamide gel electrophoresis and by the generation of procoagulant activity as assessed by thrombin-treated factor VIII-dependent generation of factor Xa activity. Cell-bound factor IX (0.8 micrograms per 4 X 10(8) cells per ml) could be activated by factor XIa (5 micrograms/ml) or by factor VIIa (0.1 micrograms/ml) without exogenous tissue factor when endothelial cells were treated with phorbol ester and acquired tissue factor-like procoagulant activity. Regardless of how factor IX was activated, the cell-bound factor IXa required thrombin-treated factor VIII and calcium, but not exogenous phospholipid, to activate factor X. In further experiments, factor X bound to endothelial cells specifically and reversibly with a dependence on calcium and with a lower affinity (half-maximal at 480 nM) than factor IX. At saturation, 9.1 X 10(6) factor X molecules were bound per cell. After activation of factor X by factor IXa, approximately 50% of the factor Xa formed could be eluted from the cells by 10 mM EDTA, suggesting that the factor Xa was cell associated. These observations indicate that endothelial cells can bind and promote the activation of factors IX and X in the absence of platelets or exogenous phospholipid.
Quantitative investigation of hepatic genomic response to hormonal and pathophysiological stimuli by multivariate analysis of two-dimensional mRNA activity profiles.
Authors: Carr F E , Bingham C , Oppenheimer J H , Kistner C , Mariash C N
Published: May 2006

We have applied techniques of multivariate analysis to the characterization and comparison of the effects of various pathophysiological and hormonal stimuli on the expression of the rat hepatic genome at a pretranslational level. In vitro translated products were resolved by two-dimensional gel electrophoresis. We analyzed 10 pathophysiological states brought about by variation in thyroidal status, starvation, administration of high carbohydrate diet, and the production of experimentally induced diabetes mellitus. Each state differed significantly from every other state in the two-dimensional electrophoretic profiles. The set consisting of the minimal number of products necessary for maintaining the distinctive patterns was identified. The analysis also defined those clusters of products that behaved in a coordinate fashion in response to the various stimuli. Lastly, the similarity and dissimilarity of hepatic mRNA activity profiles to each other could be geometrically represented in three-dimensional space. Our finding that the hepatic mRNA activity profile could distinguish reliably between closely related hormonal and pathophysiological stimuli indicates the specificity of hepatic genomic expression. A systematic analysis of such profiles may be useful as an overall index of the biologic response at the hepatocellular level.
Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus.
Authors: Bellofatto V , Shapiro L , Hodgson D A
Published: May 2006

A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance. A fragment of DNA containing the neomycin phosphotransferase II (NPT II) gene from Tn5, lacking its promoter region but retaining its translation initiation signal, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS50L sequence while retaining its ability to transpose. This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10. Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments. Transcriptional regulation by exogenous cysteine of NPT II gene expression was demonstrated in a cysteine auxotroph generated by Tn5-VB32 insertional inactivation. NPT II synthesis, measured by agar plate assays of kanamycin resistance and by immunoprecipitation of the NPT II protein, was repressed in the presence of cysteine and derepressed in its absence. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences.
Adaptive response in mammalian cells: crossreactivity of different pretreatments on cytotoxicity as contrasted to mutagenicity.
Authors: Laval F , Laval J
Published: May 2006

Pretreatment of H4 (rat hepatoma) cells for 48 hr with low nontoxic doses of alkylating agents [methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and N-methyl-N-nitrosourea] renders the cells more resistant to the toxic effect of these compounds. Crossreactivity for survival is also observed with the different alkylating agents tested. Pretreatment with MNNG enables the cells to be less mutated than control cultures during a subsequent challenge with high doses of this compound. However, pretreatment with MMS does not modify the mutation frequency of cells challenged with either MMS or MNNG. The adaptive response to mutagenesis is correlated with a faster and more efficient removal of O6-methylguanine in MNNG-pretreated cells as compared to control cultures, whereas the disappearance of this lesion is not modified in MMS-pretreated cells. As MMS produces less methylation at the O6 position of guanine and more methylation at the N7 position in comparison to MNNG, the results suggest that: (i) N7-methylguanine is not implicated in the adaptive response and (ii) adaptation to mutagenesis can be correlated with the amount of O6-methylguanine induced during the pretreatment. The effect of pretreatment on other O-alkylated derivatives is not known.
Isolation and characterization of a mouse cDNA clone that expresses mast-cell growth-factor activity in monkey cells.
Authors: Yokota T , Lee F , Rennick D , Hall C , Arai N , Mosmann T , Nabel G , Cantor H , Arai K
Published: May 2006

A cDNA sequence coding for mouse mast-cell growth-factor (MCGF) has been cloned from a cDNA library prepared from mRNA derived from a concanavalin A-activated mouse T-cell clone. Cloning was achieved using the pcD vector that permits expression of cDNA inserts in mammalian cells. The DNA sequence codes for a polypeptide of 166 amino acid residues including a putative signal peptide. The supernatant fluid obtained after transfection of COS-7 monkey cells with the pcD-MCGF plasmid had mouse MCGF activity. The MCGF amino acid sequence between amino acids 33 and 41, deduced from the nucleotide sequence of its cDNA, is identical to the NH2-terminal sequence of the mouse lymphokine, interleukin 3, suggesting that MCGF is related to interleukin 3.
Molecular cloning of hormone-responsive genes from the yeast Saccharomyces cerevisiae.
Authors: Stetler G L , Thorner J
Published: May 2006

A method for identifying yeast genes whose transcription is differentially regulated was developed. The technique is based on incorporation of the analog 4-thiouridine into nascent RNAs, which allows their purification. The purified RNAs are used to prepare cDNA copies for screening of genomic DNA libraries by hybridization. Using this procedure, several cloned yeast DNA segments were found whose transcription in MATa haploids in vivo is apparently modulated in dramatic fashion within 10-15 min after exposure to the mating pheromone, alpha factor. Subsequent analysis indicated that these sequences fall into three major classes: (i) genes expressed in vegetatively growing cells that are no longer transcribed after alpha-factor administration ("turn-off" genes); (ii) genes whose expression is increased 10- to 20-fold after exposure of the MATa cells to alpha factor ("turn-up" genes); and (iii) genes that are expressed only after alpha-factor treatment ("turn-on" genes). The first class may encode products required for cell cycle progression; the third class may code for products uniquely involved in the mating process.
Neurotrophic factor for central neurons.
Authors: Müller H W , Beckh S , Seifert W
Published: May 2006

A serum-free culture of dissociated neurons from embryonic rat hippocampus has been established as a rapid and quantitative in vitro test system for neurotrophic signals in the mammalian brain. By means of this cell culture bioassay, a novel low molecular weight neurotrophic factor (NTF) could be identified. NTF is essential for in vitro brain neuron development, promoting survival and neurite outgrowth. The diffusible factor is synthesized and secreted into serum-free defined medium by cultured astrocytes from rat cerebral hemispheres. The number of viable neurons responding to NTF by neurite outgrowth is dependent on the concentration of the factor. Fractionation of astroglial conditioned medium by gel filtration on columns of Sephadex G-10 recovered biological activity of NTF in a single sharp peak corresponding to an apparent molecular weight of approximately equal to 500. NTF is stable to heat and cold and resistant to trypsin and pronase. Unlike nerve growth factor, NTF has no apparent effect on the neurite outgrowth of peripheral neurons. NTF-like activity is present in situ in the mammalian brain, in certain other nonneural tissues, and in C6 and B12 glioma cell conditioned media.
In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon.
Authors: Groisman E A , Castilho B A , Casadaban M J
Published: May 2006

A mini-Mu bacteriophage containing a high copy number plasmid replicon was constructed to clone genes in vivo. A chloramphenicol resistance gene for independent selection and the lacZYA operon to form gene fusions were also incorporated into this phage. This mini-Mu element can transpose at a high frequency when derepressed, and it can be complemented by a helper Mu prophage for lytic growth. DNA sequences that are flanked by two copies of this mini-Mu can be packaged along with them. After infection, homologous recombination can occur between the mini-Mu sequences, resulting in the formation of plasmids carrying the transduced sequences. lac operon fusions can be formed with promoters and translation initiation sites on the cloned sequences in the resulting plasmids. The utility of this system was demonstrated by cloning genes from eight different Escherichia coli operons and by identifying lac fusions to the regulated araBAD operon among clones selected for the nearby leu operon.
Molecular cloning of the mouse ouabain-resistance gene.
Authors: Levenson R , Racaniello V , Albritton L , Housman D
Published: May 2006

DNA prepared from ouabain-resistant mouse cells was able to transform ouabain-sensitive CV-1 cells to ouabain resistance after DNA-mediated gene transfer. The murine DNA fragment responsible for ouabain resistance was detected on the background of CV-1 DNA by virtue of a repetitive DNA sequence element that reacts positively with a mouse repeat DNA clone. CV-1 DNA is nonreactive with this probe. Southern analysis of several independently derived ouabain-resistant transformants indicates that the mouse ouaR gene is located on a 6.5-kilobase EcoRI restriction fragment. The 6.5-kilobase DNA fragment was initially isolated from a lambda phage library made from a ouabain-resistant secondary transformant and subsequently was subcloned in the plasmid vector pAT153. This plasmid was able to transform wild-type CV-1 cells to ouabain resistance at a frequency of about 10 cells per ng of DNA.
pH-sensitive liposomes: acid-induced liposome fusion.
Authors: Connor J , Yatvin M B , Huang L
Published: May 2006

Sonicated unilamellar liposomes containing phosphatidylethanolamine and palmitoylhomocysteine fuse rapidly when the medium pH is lowered from 7 to 5. Liposome fusion was demonstrated by (i) mixing of the liposomal lipids as shown by resonance energy transfer, (ii) gel filtration, and (iii) electron microscopy. The pH-sensitive fusion of liposomes was observed only when palmitoylhomocysteine (greater than or equal to 20 mol%) was present in the liposomes. The presence of phosphatidyl-ethanolamine in the liposomes greatly enhanced fusion whereas the presence of phosphatidylcholine inhibited fusion. During fusion of liposomes containing phosphatidylethanolamine and palmitoylhomocysteine (8:2, mol/mol), almost all of the encapsulated calcein was released. Inclusion of cholesterol (40 mol%) in the liposomes substantially decreased leakage without impairing fusion.
Murine leukemia virus sequences are encoded in the murine major histocompatibility complex.
Authors: Meruelo D , Kornreich R , Rossomando A , Pampeno C , Mellor A L , Weiss E H , Flavell R A , Pellicer A
Published: May 2006

The studies reported here localize murine leukemia viral sequences to the TL region of the major histocompatibility complex, H-2. We examined a battery of 38 cosmids, isolated from two large genomic libraries constructed from C57BL/10 spleen DNA, that define 25 class I gene sequences. The viral probes used hybridized with only four cosmids, containing overlapping mouse sequences, that define four class I gene-related sequences in a region of 90 kilobases of DNA. The data show that two distinct viral envelope sequences are contained in the cluster. One of these sequences is situated with its 3' end next to the 3' end of a class I sequence. The other sequence, which does not contain the entire viral envelope, is proximal to the 3' end of a different class I sequence. Hybridization of the viral probes with the H-2 cosmid clones does not appear to be due to homology between viral and H-2 sequences. Rather, the viral sequences detected appear to be linked to or inserted amid class I genes. These findings may be significant in understanding molecular mechanisms involved in the generation of H-2 class I gene diversity.
Characterization of leukemogenic virus produced by a new line of Friend erythroleukemia virus-transformed cells.
Authors: Friend C , Pogo B G , Holland J G
Published: May 2006

A new cell line designated SQ-A was established from the spleen of a leukemic DBA/2J mouse inoculated with the anemic strain of Friend erythroleukemia virus (FLV-A). The cells are similar in morphology, growth pattern, and tumorigenicity to our prototype erythroleukemia line 5-86 but are more sensitive to the cytotoxic effects of inducers of differentiation. The virus produced by SQ-A cells induces erythroleukemia associated with anemia in adult mice but has little activity when assayed on XC cells. It was characterized to determine what factors influence its leukemogenic potential. As compared to the attenuated virus from cultures of 5-86 and G-2 cells, the subunits of the RNA from the virions of SQ-A cells are the same size, and the amount of reverse transcriptase activity and RNase H present in the purified virions of the three lines are similar. However, differences are observed in levels of endonuclease and protein kinase. Both enzymes are increased in SQ-A virions. The activity of protein kinase in SQ-A virions is about 5 times higher than that in the attenuated virions. The number of polypeptides and their phosphorylation patterns also distinguish the virions of SQ-A. Whereas 5-86 virions contain seven proteins, three of which are phosphorylated in vitro, SQ-A virions contain eight proteins, all of which are phosphorylated. The extra protein in SQ-A virions has a molecular weight of 25,000 and is not glycosylated.
Dynorphin converting enzyme with unusual specificity from rat brain.
Authors: Devi L , Goldstein A
Published: May 2006

A rat brain membrane extract was shown to convert synthetic dynorphin B-29 ("leumorphin") to dynorphin B [dynorphin B-29-(1-13), "rimorphin"]. This represents a "single arginine cleavage" at Thr-Arg at positions 13 and 14 of the substrate. The product was identified by immunoprecipitation with a highly specific dynorphin B antiserum and by coelution with radiolabeled dynorphin B on reversed-phase high-performance liquid chromatography. The converting activity exhibits a pH optimum of 8. It is inhibited by a thiol protease inhibitor but not by inhibitors of cathepsin B or of serine proteases. It is inhibited by dynorphin A but not by various dynorphin A fragments. These results suggest that the converting activity is due to a novel thiol protease distinct from any known protease believed to function in the processing of biologically active peptides.
Extensive and widespread homologies between mitochondrial DNA and chloroplast DNA in plants
Authors: Stern David B. , Palmer Jeffrey D.
Published: May 2006

We used hybridization techniques to demonstrate that numerous sequence homologies exist between cloned mung bean and spinach chloroplast DNA (ctDNA) restriction fragments and mtDNAs from corn, mung bean, spinach, and pea. The strongest cross-homologies are between clones derived from the ctDNA inverted repeat and mtDNA from corn and pea, although all the ctDNA clones tested hybridized to at least one mtDNA restriction fragment. Known chloroplast genes showing strong mtDNA homologies include those for the large subunit of ribulosebisphosphate carboxylase, which hybridizes to corn mtDNA, and the β subunit of the chloroplast ATPase, which hybridizes to mung bean mtDNA. Certain of these homologies were confirmed by using cloned spinach mtDNA restriction fragments as probes in reciprocal hybridizations to ctDNA. Several of these ctDNA-homologous mtDNA sequences were shown to be much more closely related to ctDNA from the same species than to that of a distantly related species. We interpret these differential homologies as evidence for relatively recent DNA sequence transfer events, suggesting that transpostion between the two genomes is an ongoing evolutionary process.
Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically.
Authors: Abdel-Meguid S S , Grindley N D , Templeton N S , Steitz T A
Published: May 2006

The 20,500-dalton gamma delta resolvase monomer can be cleaved by chymotrypsin into a 5000-dalton COOH-terminal fragment and a 15,500-dalton NH2-terminal fragment that have been purified. Two crystal forms of the large fragment have been obtained, one of which is isomorphous with crystals of the native protein, showing that the large fragment makes the protein-protein contacts in the crystal and that the small fragment is segmentally disordered relative to the large fragment. Nuclease protection demonstrates that the small fragment binds specifically to all three DNA binding sites protected by resolvase. However, unlike native resolvase, which binds to all three complete sites with equal affinity, the small fragment binds to each of the six half sites with a different affinity. It has not been possible to demonstrate specific DNA binding of the larger fragment. Thus, resolvase has a modular construction analogous to that found for some repressors and activators; its COOH-terminal domain recognizes specific sequences in the DNA and its NH2-terminal domain mediates protein-protein interactions and probably has the enzymatic activity.
NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation.
Authors: Bos T J , Davis A R , Nayak D P
Published: May 2006

Influenza virus neuraminidase (NA), unlike the majority of integral membrane proteins, does not contain a cleavable signal sequence. It contains an NH2-terminal hydrophobic domain that functions as an anchor. We have investigated the signal function for translocation of this NH2-terminal hydrophobic domain of NA by constructing chimeric cDNA clones in which the DNA coding for the first 40 NH2-terminal hydrophobic amino acids of NA was joined to the DNA coding for the signal-minus hemagglutinin (HA) of influenza virus. The chimeric HA (N4OH) containing the NH2 terminus of NA was expressed in CV1 cells by using a simian virus 40 late-expression vector. The chimeric HA is synthesized, translocated into the rough endoplasmic reticulum, and glycosylated, whereas HA lacking the signal sequence is present only in small amounts and is unglycosylated. These results clearly show that the NH2 terminus of NA, in addition to its anchor function, also provides the signal function in translocation. However, the acquisition of complex oligosaccharides and the transport of N4OH to the cell surface are greatly retarded. To determine if the presence of two anchor sequences, one provided by NA at the NH2 terminus and the other provided by HA at the COOH terminus of N4OH, was responsible for the slow transport, the NH2 terminus of NA was fused to an "anchorless" HA. The resulting chimeric HA (N4OH482) contains the hydrophobic domain of NA at the NH2 terminus but lacks the HA anchor at the COOH terminus. N4OH482 was synthesized and glycosylated; however, as with N4OH, the acquisition of complex oligosaccharides and the migration to the cell surface are greatly retarded. Immunofluorescence data also support that, compared to the native HA, only a small amount of chimeric HA proteins is transported to the cell surface. Thus, the hydrophobic NH2 terminus of NA, although capable of providing the signal function in translocation across the rough endoplasmic reticulum, interferes with the transport of the chimeric HA to the cell surface.
Temporal expression of late histone messenger RNA in the sea urchin Lytechinus pictus.
Authors: Knowles J A , Childs G J
Published: May 2006

Sea urchin histones are encoded by several multigene families. The temporal expression of one of these families, the late histones, has been studied during the early development of Lytechinus pictus. Using a nuclease S1 assay, we detected about 10,000 transcripts encoding both late H3 and H4 proteins in the unfertilized egg. This suggests that the late genes were active at some point during oogenesis. The number of late gene transcripts begins to increase 6.5 hr after fertilization (64-cell stage), indicating that these genes probably become reactivated 4.5-6.5 hr after fertilization. The maximum rate of accumulation of transcripts (4600 molecules per min per embryo) occurs 9-14 hr after fertilization (from blastula stage to hatching). The number of transcripts peaks 21 hr after fertilization (onset of gastrulation) when the embryo has accumulated 1.8 X 10(6) copies of each late mRNA (a 164-fold increase). A 5.5-fold increase in the relative rate of transcription, between 7 and 15 hr after fertilization, is partly responsible for the accumulation of these gene products. The relative synthesis of early histone message, which is encoded by a different family, decreases 18-fold during this time. Synthesis of the late transcripts continues at the higher rate after accumulation has ceased (24 hr after fertilization). The number of late transcripts begins to decrease 48 hr after fertilization, reaching about 10,000 copies at 72 hr.
Ty-mediated gene expression of the LYS2 and HIS4 genes of Saccharomyces cerevisiae is controlled by the same SPT genes.
Authors: Simchen G , Winston F , Styles C A , Fink G R
Published: May 2006

Five Ty insertion mutations were isolated at the LYS2 locus of Saccharomyces cerevisiae. Genetic and physical analyses show that four Ty insertions are in the 5' noncoding region of LYS2 and one is within the structural gene. Three of these Ty elements have been cloned and characterized. The Ty mutations differ from each other in restriction pattern, phenotypic effects on LYS2, reversion frequency, and the nature of reversion events. Spt2 and spt3 mutations, known to suppress Ty insertions and their solo delta derivatives at HIS4, can also suppress at least one of the Ty insertions (Ty61) at LYS2 and can also suppress the Lys- phenotype of a solo delta derivative of another Ty insertion (Ty128) at LYS2. These results demonstrate that spt mutations can suppress Ty and delta mutations at both HIS4 and LYS2, suggesting that they are general for their effects on Ty and delta elements.
Isolation of hybridomas expressing a specific heavy chain variable region gene segment by using a screening technique that detects mRNA sequences in whole cell lysates.
Authors: Manser T , Gefter M L
Published: May 2006

A technique is described that allows single hybridoma cell colonies to be assayed for the productive rearrangement of a single immunoglobulin variable region (V) gene segment by utilizing expression of V mRNA for analysis. Hybridomas growing in microwell tissue culture plates are lysed in situ, cellular RNA is directly transferred to nitrocellulose by filtration, and specific immunoglobulin mRNA is detected by hybridization of the filter with a DNA probe. The method is simple and sensitive. A single species of mRNA can be detected in a lysate of 1000 cells; 5000 hybridoma colonies can be easily screened per day. The technique has been successfully used to isolate cell lines from nonimmune mice expressing a particular heavy chain variable region (VH) gene segment.
Restricted association of V and J-C gene segments for mouse lambda chains.
Authors: Reilly E B , Blomberg B , Imanishi-Kari T , Tonegawa S , Eisen H N
Published: May 2006

The frequencies of diverse rearrangements of variable (V)lambda to joining (J)lambda gene segments were examined by Southern blot hybridization in 30 murine B-cell lines, each producing an immunoglobulin lambda light chain of known subtype (lambda 1, lambda 2, or lambda 3). For 11 out of 12 lambda 1 chains, the rearrangement was V lambda 1----J lambda 1; for 9 out of 9 lambda 2 chains, it was V lambda 2----J lambda 2; and for 8 out of 9 lambda 3 chains, it was V lambda 1----J lambda 3. Similar results were obtained by considering the partial or complete sequences at the amino acid or cDNA level of 44 other lambda chains (24 previously described): for 43 of these chains the rearranged V-J gene segments were evidently V lambda 1-J lambda 1 for 28 lambda 1 chains, V lambda 2-J lambda 2 for 10 lambda 2 chains, and V lambda 1-J lambda 3 for 5 lambda 3 chains. Of the combined total of 74 chains there were 3 with unusual V lambda rearrangements, all involving the V lambda 2 gene segment: for 2 of these unusual chains, the encoding segments were V lambda 2-J lambda 1-C lambda 1 and for one they were V lambda 2-J lambda 3-C lambda 3. Thus, the results for all 74 lambda chains show that, in contrast to the apparently unrestricted V kappa----J kappa rearrangements for kappa chains, for each of the 3 murine lambda-chain subtypes V-J recombination is severely restricted: the V lambda gene segment expressed in lambda 1 and lambda 3 chains was nearly always V lambda 1 (95% and 93%, respectively), whereas in lambda 2 chains it was without exception V lambda 2 (19 out of 19 chains). Therefore V lambda-J lambda combinatorial variation is not a significant source of amino acid sequence diversity of lambda chains of inbred mice. If the order of the lambda gene segments is 5' V lambda 2-J lambda 2C lambda 2J lambda 4C lambda 4-V lambda 1-J lambda 3C lambda 3J lambda 1C lambda 1 3', as suggested previously and by the present findings, it appears that (i) when a V lambda gene segment rearranges in a developing B cell it ordinarily recombines with a J lambda gene segment in the nearest downstream (3') cluster of J lambda C lambda segments, and (ii) V lambda rearrangement to the upstream (5') cluster is very rare and possibly may not take place at all.
Characterization of monoclonal antibodies to serum galactosyltransferase.
Authors: Podolsky D K , Isselbacher K J
Published: May 2006

Monoclonal IgG and IgM antibodies (mAbs) directed against serum galactosyltransferase (GalTase) activities were prepared and characterized for their relative specificity for GalTase isoenzymes I and II (GalTase I and GalTase II). After immunization of mice with purified GalTase, 7 of 1680 fusion products screened were positive for anti-GalTase activity in a solid-phase assay; of these 7, 2 were found to bind GalTase I in a somewhat selective manner while 1 (C6) was relatively specific for GalTase II. The Ka for anti-GalTase mAbs ranged from 2.7 X 10(7) to 1.1 X 10(8) M-1. Enzymatically active GalTase could be recovered from an affinity column of C6 coupled to Sepharose 4B following application of a cell extract from a human colon cancer cell line, confirming that the mAb is directed against GalTase. A sandwich RIA was developed to measure soluble GalTase II in serum by using a combination of two isoenzyme nonspecific mAbs (F5 and V10) coated on a solid-phase support followed by the addition of antigen and GalTase II-specific 125I-labeled C6. This assay was specific for GalTase II with a sensitivity of approximately 10 ng/ml. Evaluation of 240 sera demonstrated higher levels of GalTase II in patients with gastrointestinal cancer (48 ng/ml). However, in contrast to previous results with a radiochemical assay, some normal sera contained GalTase II (mean, 14 ng/ml) and some patients with liver disease had elevated levels (mean, 23 ng/ml). These studies demonstrate the production of moderate-affinity antibodies directed to serum GalTase isoenzymes and the development of an RIA useful in the study of GalTase.
Carboxypeptidase B-like converting enzyme activity in secretory granules of rat pituitary.
Authors: Hook V Y , Loh Y P
Published: May 2006

Recent amino acid sequence data suggest that trypsin-like and carboxypeptidase B-like activities are required for the processing of pituitary prohormones--e.g., pro-opiocortin (pro-adrenocorticotropin/lipotropin) and provasopressin in secretory granules. In this study the existence of a carboxypeptidase B activity in purified secretory granules from anterior, intermediate, and neural lobes of rat pituitary has been examined. A carboxypeptidase B activity that cleaved the COOH-terminal -Lys-Lys-Arg residues from the adrenocorticotropin fragment ACTH-(1-17) (a potential hormone product liberated from pro-opiocortin by a trypsin-like enzyme) was detected in anterior and intermediate lobe granules. A similar carboxypeptidase B activity was also present in purified secretory granules from rat pituitary neural lobes that cleaved the -Lys-Arg residues from [Arg8]vasopressin-Gly-Lys-Arg, a potential product cleaved from provasopressin. Secretory granule carboxypeptidase(s) from the three lobes of the pituitary was shown to cleave 125I-[Met]enkephalin-Arg6 to form 125I-[Met]enkephalin as well. 125I-[Met]Enkephalin was used as a model substrate for the quantitative assay of pituitary carboxypeptidase activity. The carboxypeptidase B in secretory granules from all three lobes was shown to be active at pH 5.5, but not at pH 7.4. Inhibition by the zinc metallocarboxypeptidase inhibitors guanidinopropylsuccinic acid, aminomercaptosuccinic acid, benzylsuccinic acid, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and the potato carboxypeptidase B inhibitor, and inhibition by the metal chelators EDTA and 1,10-phenanthroline demonstrate metal ion dependence of the pituitary granule carboxypeptidase activities. However, Co2+ stimulated the secretory granule carboxypeptidase B activities. Thiol protease inhibitors such as Cu2+ and p-chloromercuriphenylsulfonic acid also inhibited the activity. Thus, the secretory granule carboxypeptidase B-like activities in all three lobes of the pituitary appear to be similar thiol-metallopeptidases that differ from other carboxypeptidase activities previously described and may play an exclusive role in hormone biosynthesis in the pituitary.
Organization of collagen types I and V in the embryonic chicken cornea: monoclonal antibody studies.
Authors: Fitch J M , Gross J , Mayne R , Johnson-Wint B , Linsenmayer T F
Published: May 2006

To determine whether type V collagen is antigenically masked in situ by its fibrillar organization, two different methods were used to perturb selectively the structure of collagen fibrils in sections of embryonic chicken corneas. The experimentally modified tissues were probed by immunohistochemical procedures with monoclonal antibodies against types V and I. A lathyritic agent was used to block crosslinking of newly synthesized collagen. This results in reversible temperature-sensitive alterations in fibrillar packing, such that freshly formed collagen fibrils retain their aggregated state at 37 degrees C but become dissociated upon cooling. Type V-specific immunofluorescence remained masked at 37 degrees C but was revealed at 0 degree C. The effect of temperature was partially reversible, indicating that type V collagen is normally unavailable for antibody binding because of its fibrillar arrangement. In sections of normal corneas, treatment with corneal collagenase, which degrades type I collagen, but not type V, also unmasked the latter. This implicates type I collagen as the masking agent. We propose that collagen types I and V are incorporated together in heterotypic fibrils.
Methylation of the hypoxanthine phosphoribosyltransferase locus on the human X chromosome: implications for X-chromosome inactivation.
Authors: Wolf S F , Jolly D J , Lunnen K D , Friedmann T , Migeon B R
Published: May 2006

To explore the role of DNA methylation in maintaining dosage compensation of X chromosome-linked genes and in regulating the transcriptional activity of "housekeeping" genes, we characterized DNA methylation of active, inactive, and derepressed alleles at the locus for hypoxanthine phosphoribosyltransferase (HPRT) on the human X chromosome. The methylation of Hpa II and Hha I sites in HPRT alleles on the active X chromosome was the same in all tissues. The consensus pattern includes hypomethylation of 5' clustered sites and extensive methylation of the 3' sequence. The striking feature of methylation of inactive X-chromosome alleles is nonuniformity and less extensive hypomethylation of the 5' cluster. Analysis of HPRT alleles reactivated in response to 5-azacytidine showed at least partial restoration of the consensus pattern. These observations indicate that methylation of housekeeping genes on the X chromosome is the same as that of autosomal ones and that the overall pattern and methylation of multiple sites within a cluster may cooperate to facilitate transcription. Furthermore, the fidelity of methylation of the active allele and the extensive drift in methylation of the inactive allele suggest that mechanisms involved in X-chromosome dosage compensation may be directed at the active rather than inactive X chromosome.