bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibody that defines a unique human T-cell leukemia antigen
Authors: Seon Ben K. , Negoro Shigeru , Barcos Maurice P.
Published: May 2006

We have generated and characterized a hybridoma monoclonal antibody, termed SN1, that defines a unique human T-cell leukemia antigen. This antibody was generated by using a human leukemia antigen preparation isolated from cell membranes of MOLT-4, a leukemia T-cell line derived from a patient with T-cell-type acute lymphoblastic leukemia (T-ALL). SN1 was characterized by a sensitive microscale radioimmunoassay using a variety of cultured and uncultured human cells. In selected cases, the cell specimens were further tested by immunoperoxidase staining and an immunofluorescence staining test. The results of the radioimmunoassay were in agreement with those of the two other tests. Among the various cultured malignant and nonmalignant cell lines, SN1 reacted only with leukemia T-cell lines derived from patients with T-ALL; it reacted with all six T-ALL cell lines tested—i.e., JM, CCRF-CEM, CCRF-H-SB2, RPMI 8402, PEER, and MOLT-4. In the case of uncultured cell specimens derived from cancer patients, SN1 reacted with four of four cases of T-ALL but did not react with specimens derived from 41 patients with other types of cancer. SN1 did not react with any normal human cell specimens tested, both cultured and uncultured. These specimens include normal lymphoblastoid cell lines, thymocytes, bone marrow cells, spleen cells, lymph node cells, peripheral blood mononuclear cells, lymphocytes containing B and T cells, purified T cells, monocytes, granulocytes, erythrocytes, and platelets. Furthermore, SN1 did not react with phytohemagglutinin-activated T cells nor with concanavalin A-activated T cells. The results show that monoclonal antibody SN1 defines a type of human leukemia antigen that is expressed on the cell surface of T-cell-type ALL cells. The results further show the usefulness of SN1 in the diagnosis of cancer patients and suggest its therapeutic potential. We designate this antigen TALLA, a T-cell ALL antigen.
Influence of diet on vascular lesions in autoimmune-prone B/W mice.
Authors: Fernandes G , Alonso D R , Tanaka T , Thaler H T , Yunis E J , Good R A
Published: May 2006

Autoimmune-prone B/W mice, which are known to develop severe glomerulonephritis and vasculitis, also are found to develop arteritis and proliferative and fatty-proliferative lesions of the aorta and its branches as well as renal inflammatory lesions. High intake of saturated fat in the diet enhances the development of these atherosclerotic and autoimmune lesions significantly in female mice, whereas restriction of dietary calories and fat inhibits their development. Ad lib feeding of laboratory chow, high in fiber and low in fat, does not foster development of vascular lesions but does permit the development of autoimmune renal disease.
Synthesis of aspartate transcarbamoylase in Escherichia coli: transcriptional regulation of the pyrB-pyrI operon.
Authors: Navre M , Schachman H K
Published: May 2006

The first committed reaction in pyrimidine biosynthesis in Escherichia coli and Salmonella typhimurium is catalyzed by the allosteric enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), the product of the pyrB-pyrI operon. Regulation of the pyrimidine pathway is achieved in part by changes in the enzyme's catalytic activity as a function of the concentration of substrates and other metabolites as well as by variations in enzyme synthesis in response to changes in cellular levels of pyrimidine nucleotides. Although there is substantial evidence that UTP concentration has a marked influence on expression of the pyrB-pyrI operon, the mechanism of this control is not known. We have cloned the operon and determined the nucleotide sequence of the region preceding the first structural gene (pyrB). These studies show two regions sharing considerable homology with the consensus sequence of E. coli promoters, a segment that can code for a 44-amino-acid leader peptide, and a sequence very similar to that of the attenuator of the trp operon. RNA transcripts from several bacterial strains were studied by S1 nuclease mapping. Under conditions leading to extensive enzyme synthesis there was a large production of transcript whose 5' end correlated with the putative promoter closer to the structural genes. At low levels of operon expression there was little transcript in the extracts and both promoters appeared to serve as initiation sites. The results are interpreted in terms of transcriptional control of the pyrB-pyrI operon according to an attenuation model that differs in novel ways from the mechanisms proposed for the regulation of amino acid biosynthesis.
Structure of a Bacillus subtilis bacteriophage SPO1 gene encoding RNA polymerase sigma factor.
Authors: Costanzo M , Pero J
Published: May 2006

Gene 28 of Bacillus subtilis bacteriophage SPO1 codes for a regulatory protein, a sigma factor known as sigma gp28, that binds to the bacterial core RNA polymerase to direct the recognition of phage middle gene promoters. middle promoters exhibit distinctive and conserved nucleotide sequences in two regions centered about 10 and 35 base pairs upstream from the start point of mRNA synthesis. Here we report the cloning of gene 28 and its complete nucleotide sequence. We infer that sigma gp28 is a 25,707-dalton protein of 220 amino acids. Neither the nucleotide sequence of gene 28 nor the inferred amino acid sequence of sigma gp28 exhibits extensive homology to the gene or protein sequence of Escherichia coli sigma factor.
Unwinding associated with synapsis of DNA molecules by recA protein.
Authors: Wu A M , Bianchi M , DasGupta C , Radding C M
Published: May 2006

In the presence of adenosine 5'-[gamma-thio]triphosphate, a nonhydrolyzable analog of ATP, Escherichia coli recA protein extensively unwinds duplex DNA in a reaction that is strongly stimulated by either homologous or heterologous single-stranded DNA [Cunningham, R.P., Shibata, T., DasGupta, C. & Radding, C.M. (1979) Nature (London) 281, 191-195]. In the presence of ATP and homologous circular single-stranded DNA, recA protein also unwinds circular duplex DNA that is nicked at a heterologous site. When DNA ligase seals this nick, the product is a highly negatively superhelical molecule that can be relaxed by E. coli topoisomerase I. This unwinding requires a high degree of homology since phi X174 single-stranded DNA does not serve as a cofactor in the unwinding of G4 DNA, even though these molecules are 70% homologous. Like synapsis itself, and unlike strand exchange which follows synapsis, unwinding is sensitive to inhibition by ADP. Because recA protein unwinds duplex DNA when neither the single-stranded DNA nor the duplex DNA has a free end in the region of homology, unwinding can be initiated or mediated by a synaptic structure that differs from that of a simple D loop. The paired circular single strand in the synaptic structure behaves like one strand of an under-wound helix because E. coli topoisomerase I can interwind it with its complement.
Regulation of differentiated cell-specific functions.
Authors: Savageau M A
Published: May 2006

The demand theory of gene regulation predicts that regulated cell-specific functions in high demand (i.e., high level of gene expression frequently required) are under the influence of a positive regulatory element whereas those in low demand (i.e., high level of gene expression not frequently required) are under the influence of a negative regulatory element. Furthermore, during differentiation, when the demand regimen for cell-specific functions changes, a switch in the regulatory mechanism itself is predicted. For the case in which a function is regulated in both demand regimens, the mode of regulation will switch from positive (high demand) to negative (low demand) or vice versa. These predictions are compared with published experimental evidence and found to be in good agreement.
Bacterio-opsin mutants of
Halobacterium halobium
Authors: Betlach Mary , Pfeifer Felicitas , Friedman James , Boyer Herbert W.
Published: May 2006

The bacterio-opsin (
bop
) gene of
Halobacterium halobium
R1 has been cloned with about 40 kilobases of flanking genomic sequence. The 40-kilobase segment is derived from the (G+C)-rich fraction of the chromosome and is not homologous to the major (pHH1) or minor endogenous covalently closed circular DNA species of
H. halobium
. A 5.1-kilobase
Pst
I fragment containing the
bop
gene was subcloned in pBR322 and a partial restriction map was determined. Defined restriction fragments of this clone were used as probes to analyze the defects associated with the
bop
gene in 12 bacterio-opsin mutants. Eleven out of 12 of the mutants examined had inserts ranging from 350 to 3,000 base pairs either in the
bop
gene or up to 1,400 base pairs upstream. The positions of the inserts were localized to four regions in the 5.1-kilobase genomic fragment: within the gene (one mutant), in a region that overlaps the 5′ end of the gene (seven mutants), and in two different upstream regions (three mutants). Two revertants of the mutant with the most distal insert had an additional insert in the same region. The polar effects of these inserts are discussed in terms of inactivation of a regulatory gene or disruption of part of a coordinately expressed operon. Given the defined nature of the
bop
mRNA—i.e., it has a 5′ leader sequence of three ribonucleotides—these observations indicate that the
bop
mRNA might be processed from a large mRNA transcript.
Human T-cell leukemia-lymphoma virus (HTLV): cloning of an integrated defective provirus and flanking cellular sequences.
Authors: Manzari V , Wong-Staal F , Franchini G , Colombini S , Gelmann E P , Oroszlan S , Staal S , Gallo R C
Published: May 2006

Human T-cell leukemia-lymphoma virus (HTLV) is the first unequivocal human retrovirus. Seroepidemiological and virus isolation studies indicate that HTLV is etiologically associated with a subtype of adult T-cell malignancy. We have molecularly cloned approximately 1 kilobase of sequences derived from the 5' and 3' termini of the HTLV genome. Use of these clones as probes allowed isolation of a 9.8-kilobase EcoRI fragment from a genomic DNA library of an HTLV-infected neoplastic T-cell line (CR). Analysis of this clone revealed the presence of cellular sequences flanking approximately 5 kilobases of viral sequences including one long terminal repeat sequence. The 5' and 3' clones, as well as subclones derived from different regions of the genomic clone, were used as probes to compare integrated proviruses and viral RNA expression in different HLTV-infected neoplastic T cell lines. The results indicate that the infected cells are of clonal origin with respect to the virus integration sites and they express multiple viral mRNA species including a 35S RNA.
Senescent cell antigen is immunologically related to band 3.
Authors: Kay M M , Goodman S R , Sorensen K , Whitfield C F , Wong P , Zaki L , Rudloff V
Published: May 2006

IgG autoantibodies in human serum selectively bind to a glycopeptide antigen that appears on senescent and damaged cells in situ. We identified the membrane protein from which the senescent cell antigen is derived by using a phagocytosis-inhibition assay and immunoautoradiographic gel staining and electroblotting techniques. Results of the phagocytosis-inhibition assay revealed that only the purified transmembrane glycoprotein designated "band 3" and senescent cell antigen inhibited the phagocytosis of erythrocytes induced by IgG eluted from senescent erythrocytes. Purified spectrin, syndein, band 4.1, actin, glycophorin A, and intact or desialylated sialoglycoprotein periodic acid/Schiff (PAS) staining bands 1-4 containing glycophorins A, B, and C did not inhibit phagocytosis. Specific antibodies against the senescent cell antigen and erythrocyte band 3 were used to identify the membrane protein from which the senescent cell antigen is derived. Band 3-related polypeptides (MrS approximately equal to 60,000, 42,000, and 18-26,000) were identified in erythrocyte ghosts prepared in the presence of diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and EDTA by immunoautoradiography with antiband 3. Antibodies to senescent cell antigen reacted with band 3 and the same lower Mr band 3-related polypeptides. Thus, the senescent cell antigen is immunologically related to band 3.
Bacterial and phage mutations that reveal helix-unwinding activities required for bacteriophage T4 DNA replication.
Authors: Gauss P , Doherty D H , Gold L
Published: May 2006

An Escherichia coli strain with a mutation in the optA gene restricts the growth of bacteriophage T4 strains partially defective in gene 43 (DNA polymerase) or missing gene dda (DNA-dependent ATPase). The mutations in the dda gene inactivate a DNA-dependent ATPase that has been shown to have DNA helicase activity in vitro. We show that the restriction of phage growth after infection of the optA bacterium is the result of a block in DNA replication. We infer that the block arises from a defect in DNA unwinding.
Allergen challenge of lung tissue from asthmatics elicits bronchial contraction that correlates with the release of leukotrienes C4, D4, and E4.
Authors: Dahlén S E , Hansson G , Hedqvist P , Björck T , Granström E , Dahlén B
Published: May 2006

The leukotrienes C4, D4, and E4, previously referred to as slow reacting substance of anaphylaxis, elicited long-lasting contractions of bronchi isolated from two birch pollen-sensitive asthmatics. The leukotrienes were 1,000 times more potent on a molar basis than was histamine or prostaglandin F2 alpha. Moreover, allergen released leukotrienes C4, D4, and E4 from the lung tissue of the asthmatics in amounts that appeared to correlate well to the anaphylactic bronchial contraction. Irrespectively of whether the lung was stimulated with specific allergen, the ionophore A23187 or 14C-labeled arachidonic acid, 15-hydroxyicosatetraenoic acid, and other lipoxygenase-derived monohydroxy acids were the major metabolites of arachidonic acid in the lung, and thromboxane A2 and prostaglandin I2 were the predominant cyclooxygenase products identified. However, cyclooxygenase inhibition with indomethacin had no effect on the contraction response to antigen in the bronchi, whereas, in the presence of U-60257, an inhibitor of leukotriene biosynthesis, the allergen neither released leukotrienes from the lung nor caused bronchial contraction. These findings indicate that leukotrienes C4, D4, and E4 are major mediators of allergic bronchoconstriction in man.
Inhibition of the growth and differentiation of erythroid precursor cells by an endotoxin-induced mediator from peritoneal macrophages.
Authors: Sassa S , Kawakami M , Cerami A
Published: May 2006

Conditioned medium from cultures of mouse macrophages incubated with endotoxin in a serum-free medium contains an inhibitor of the growth and differentiation of erythroid precursor cells of mouse Friend virus-transformed erythroleukemia cells. Endotoxin itself has no inhibitory effect. The endotoxin-induced macrophage mediator inhibits the growth and differentiation of dimethyl sulfoxide-, hexamethylenebisacetamide-, butyric acid-, and hypoxanthine-induced cells but has no effect on hemin-induced cells. The conditioned medium has its maximal inhibitory effect on committed erythroid precursor cells, a decreased effect on uncommitted stem cells, and no effect on fully differentiated erythroid cells. These results demonstrate that endotoxin stimulation of macrophages leads to the production of a humoral factor(s) which is critical for the growth and differentiation of erythroid precursor cells.
Perfect palindromic lac operator DNA sequence exists as a stable cruciform structure in supercoiled DNA in vitro but not in vivo.
Authors: Sinden R R , Broyles S S , Pettijohn D E
Published: May 2006

A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation greater than 90% of the sequence is in cruciforms. Results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.
Cells injected with guanosine 5'-[alpha, beta-methylene]triphosphate, an alpha, beta-nonhydrolyzable analog of GTP, show anomalous patterns of tubulin polymerization affecting cell translocation, intracellular movement, and the organization of Golgi elements.
Authors: Wehland J , Sandoval I V
Published: May 2006

Injection of the alpha, beta-nonhydrolyzable GTP analog, guanosine 5'-[alpha, beta-methylene]triphosphate (pp[CH2]pG) into PtK2, A549, and Swiss 3T3 cells produced dramatic changes in the normal pattern of long radiating microtubules displayed by the cells before injection. Injection of pp[CH2]pG into cells growing in normal medium resulted in the formation of microtubule bundles resistant to depolymerization by Colcemid and calcium. Cells injected with pp[CH2]pG after incubation with Colcemid for 2 hr showed polymerization of tubulin into long wavy ribbons within 2 hr after injection. Removal of Colcemid 1 hr after the injection of pp[CH2]pG resulted in assembly of tubulin into short single randomly oriented microtubules. All cells injected with pp[CH2]pG showed impeded translocation and restriction or absence of intracellular movement. pp[CH2]pG also prevented the fragmentation of Golgi elements in A549 cells treated with Colcemid. Cells first treated with Colcemid and then injected with pp[CH2]pG failed to reassemble the Golgi elements after the removal of Colcemid. Cells in intimate membrane contact with cells injected with pp[CH2]pG showed similar changes in microtubule polymerization, cell movement, and organization of Golgi elements.
Photoaffinity label for the alpha 1-adrenergic receptor: synthesis and effects on membrane and affinity-purified receptors.
Authors: Hess H J , Graham R M , Homcy C J
Published: May 2006

An azide analog, 2-[4-(4-azidobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (CP59,430), of the highly selective alpha 1-adrenergic receptor antagonist prazosin was synthesized and its effects on rat hepatic membrane and affinity-purified alpha 1-adrenergic receptor preparations were examined. CP59,430 behaved as a competitive antagonist before photolysis. When the membrane or purified preparations pretreated with CP59,430 were irradiated with UV light, CP59,430 behaved as a noncompetitive antagonist. Labeling of membrane alpha 1-adrenergic receptors was irreversible; repeated dialysis or washing could not reverse the photolysis-induced inactivation by CP59,430, whereas dialysis completely reversed the antagonism by the same concentration of the label prior to photolysis. Additionally, photolabeling of purified receptors was resistant to Sephadex G-50 chromatography, whereas in the absence of photolysis the same concentration of CP59,430 or prazosin (10 microM) could be readily removed by this procedure. CP59,430 appears to label specifically only alpha 1-adrenergic receptors because prazosin protected the membrane and purified receptors from photolysis-induced inactivation by CP59,430. Furthermore, specific [3H]dihydroalprenolol and [3H]yohimbine binding to membrane beta- and alpha 2-adrenergic receptors, respectively, was unchanged by CP59,430 at 1 microM, a concentration that decreased specific [3H]prazosin binding to alpha 1-adrenergic receptors by 72%. In additional studies, the photolysis-induced receptor inactivation by CP59,430 remained unchanged in the presence of the scavenger p-aminobenzoic acid. It is likely, therefore, that receptor labeling by CP59,430 occurs via a true photoaffinity mechanism. CP59,430, which specifically and irreversibly labels the alpha 1-adrenergic receptor after photolysis, should thus be uniquely valuable for the molecular characterization of this receptor.
Genes for catecholamine biosynthesis: cloning by expression and identification of the cDNA for rat dopamine beta-hydroxylase.
Authors: O'Malley K L , Mauron A , Raese J , Barchas J D , Kedes L
Published: May 2006

mRNA for dopamine beta-hydroxylase [3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1] has been partially purified from poly(A)+ mRNA isolated from a rat pheochromocytoma cell line. Shared antigenic determinants between tyrosine hydroxylase and dopamine beta-hydroxylase allowed us to obtain enriched fractions of dopamine beta-hydroxylase mRNA by immunoprecipitating translated mRNA products with tyrosine hydroxylase antisera. The enriched dopamine beta-hydroxylase mRNA was used to synthesize the corresponding cDNAs, which were then cloned in the Pst I site of pBR322. Recombinant colonies were characterized by an in situ colony immunoassay and hybrid-selected translation. In vitro translation of the mRNA selected from one recombinant clone produced a protein of 75,000 daltons that comigrated with authentic dopamine beta-hydroxylase. Partial proteolysis of both authentic dopamine beta-hydroxylase and the protein encoded by the recombinant clone produced identical peptide patterns.
"Silent" nucleotide substitution in a beta+-thalassemia globin gene activates splice site in coding sequence RNA.
Authors: Goldsmith M E , Humphries R K , Ley T , Cline A , Kantor J A , Nienhuis A W
Published: May 2006

A beta+-thalassemia globin gene was isolated from the genome of a Black individual by molecular cloning. DNA sequence analysis revealed only a single difference between this gene and the normal human beta-globin gene--adenine is substituted for thymine in the third position of codon 24. Codon 24 in both the normal gene (GGT) and the beta+-thalassemia gene (GGA) encodes glycine. The function of this beta+-thalassemia gene was compared to the function of the normal human beta-globin gene in monkey kidney cells by using plasmid expression vectors. The codon 24 substitution activates a 5' splice site that involves the guanine-thymine dinucleotide present in codon 25, 16 nucleotides upstream from the normal exon 1-intron I boundary. The splice, involving the abnormal 5' site in codon 25, is completed with the normal 3' splice site at the end of intron I. This splicing abnormality leads to a 75% decrease in the accumulation of normally processed beta-globin mRNA, thereby causing the beta+-thalassemia phenotype.
Retrograde transport of gamma-amino[3H]butyric acid reveals specific interlaminar connections in the striate cortex of monkey.
Authors: Somogyi P , Cowey A , Kisvárday Z F , Freund T F , Szentágothai J
Published: May 2006

Several lines of evidence suggest that gamma-aminobutyric acid is an inhibitory neurotransmitter in the cerebral cortex. To study the intracortical projection of neurons that selectively accumulate this amino acid, we injected radioactive gamma-aminobutyric acid into the upper layers of the striate cortex of monkeys along tracks at an oblique angle to the pia. Sections from the injected area were then processed by a combination of autoradiography and Golgi impregnation to reveal the distribution of labeled neurons and their morphological characteristics. Labeled neurons always occurred around the injection site in each layer. In addition, a consistent radial pattern of perikaryal labeling was observed in layers IVc-VI below the injection track in layers I-IVa. The closer the injection track was to the pia the deeper the peak density of labeled cells appeared. After injection in layers IVa and the lower part of III, the highest number of labeled neurons was in layer IVc; after injection in the upper part of layer III, most labeled neurons were in layer V; and, after injection in layers I and II, the proportion of labeled neurons increased in the lower part of layer V and in layer VI. All these neurons in the infragranular layers are presumably labeled by retrograde axonal transport via the labeled fiber bundles that extended from upper to lower layers. Thirty-four Golgi-stained neurons of various types were also examined for retrograde labeling. Two were labeled, and both were aspiny stellate cells in layer V. The arrangement of these putative GABAergic neurones, with axons that ascend from lower to upper layers in a regular pattern and arborize locally, would enable them to mediate inhibition within cortical columns and between neighboring columns.
Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes.
Authors: Kirkegaard K , Buc H , Spassky A , Wang J C
Published: May 2006

A method based on the differential rate of cytosine methylation in single- and double-stranded nucleic acids by dimethyl sulfate [Peattie, D.A. & Gilbert, W. (1980) Proc. Natl. Acad. Sci. USA 77, 4679-4682] has been developed for probing unpaired cytosines in DNA and DNA-protein complexes at the sequence level. Application of the method to the complexes between Escherichia coli RNA polymerase (EC 2.7.7.6) and three related promoters, lac UV5, trp, and a hybrid promoter tac resulting from the fusion of the two, reveals distinct differences in the way RNA polymerase unpairs DNA in these promoters. No single-stranded region is detectable in the complex with the trp promoter. For the lac UV5 promoter, the cytosines at positions -6, -4, -2, and -1 are in an unpaired region. The same cytosines in the tac promoter, which is homologous in sequence to lac UV5 in this region, are also found to be single stranded. For the pair of promoters lac UV5 and tac, the cytosine methylation reaction has also been used to demonstrate the steep temperature dependence of opening of base pairs by RNA polymerase. One striking feature is that the midpoint of this transition for the tac promoter is 3 degrees C lower than the corresponding value for lac UV5, even though the sequence of the unpaired region in the two promoters is identical.
Identification of high-affinity phorbol ester receptor in cytosol of EL4 thymoma cells: requirement for calcium, magnesium, and phospholipids.
Authors: Sando J J , Young M C
Published: May 2006

A specific high-affinity phorbol ester binding component has been identified in the cytosol of an EL4 mouse thymoma line by using conditions similar to those for demonstrating activity of a calcium/phospholipid-dependent protein kinase. Specific binding is absolutely dependent on acidic phospholipids (maximal binding at 96 micrograms of phosphatidylserine per ml or 200 micrograms of phosphatidylinositol per ml) and is greatly enhanced by addition of calcium (0.5 mM) and magnesium (75 mM). Scatchard analysis of the cytosolic binding component indicated a Kd of 4.2 +/- 2.5 nM with 1.8 +/- 0.6 x 10(4) sites per cell compared to a Kd of 11.9 +/- 4.4 nM and 4.1 +/- 1.0 x 10(4) sites per cell for the membrane receptor. Consistent with the existence of at least two phorbol ester binding sites in EL4 cells was the observation of curvilinear Scatchard plots for binding to whole homogenates. The cytosolic binding showed the same order of potency for binding phorbol ester analogs as has been observed for intact cells. These results further support the similarity between phorbol ester receptors and the calcium/phospholipid-dependent protein kinase and suggest that the cytosolic receptor may be involved in subsequent phorbol ester effects in EL4 cells including loss of the kinase activity from the cytosol and production of T-cell growth factor.
IgA1 proteases of Haemophilus influenzae: cloning and characterization in Escherichia coli K-12.
Authors: Bricker J , Mulks M H , Plaut A G , Moxon E R , Wright A
Published: May 2006

Haemophilus influenzae is one of several bacterial pathogens known to release IgA1 proteases into the extracellular environment. Each H. influenzae isolate produces one of at least three distinct types of these enzymes that differ in the specific peptide bond they cleave in the hinge region of human IgA1. We have isolated the gene specifying type 1 IgA1 protease from a total genomic library of H. influenzae, subcloned it into plasmid vectors, and introduced these vectors into Escherichia coli K-12. The enzyme synthesized by E. coli was active and had the same specificity as that of the H. influenzae donor. Unlike that of the donor, E. coli protease activity accumulated in the periplasm rather than being transported extracellularly. The position of the protease gene in H. influenzae DNA and its direction of transcription was approximated by deletion mapping. Tn5 insertions, and examination of the polypeptides synthesized by minicells. A 1-kilobase probe excised from the IgA1 protease gene hybridized with DNA restriction fragments of all H. influenzae serogroups but not with DNA of a nonpathogenic H. parainfluenzae species known to be IgA1 protease negative.
Polymorphic human somatostatin gene is located on chromosome 3.
Authors: Naylor S L , Sakaguchi A Y , Shen L P , Bell G I , Rutter W J , Shows T B
Published: May 2006

Somatostatin is a 14-amino-acid neuropeptide and hormone that inhibits the secretion of several peptide hormones. The human gene for somatostatin SST has been cloned, and the sequence has been determined. This clone was used as a probe in chromosome mapping studies to detect the human somatostatin sequence in human-rodent hybrids. Southern blot analysis of 41 hybrids, including some containing translocations of human chromosomes, placed SST in the q21 leads to qter region of chromosome 3. Human DNAs from unrelated individuals were screened for restriction fragment polymorphisms detectable by the somatostatin gene probe. Two polymorphisms were found: (i) an EcoRI variant located at the 3' end of the gene, found in Caucasian, U.S. Black, and Asian populations with a frequency of approximately 0.10 and (ii) a BamHI variant in the intron, which occurs in Caucasians at a frequency of 0.13.
Estradiol-activated alpha-fetoprotein suppresses the uterotropic response to estrogens.
Authors: Mizejewski G J , Vonnegut M , Jacobson H I
Published: May 2006

The binding of estrogen to alpha-fetoprotein (AFP) in the plasma cannot account for the impaired estrogen response seen in immature rodents because estradiol (E2) doses that far exceed the total body burden of AFP will stimulate only modest uterine growth. We investigated this phenomenon in immature female mice by determining their uterine weights 23 hr after intraperitoneal injection of estrogens or AFP or both. Administration of either 0.5 micrograms of E2 or 10 ng of moxestrol (MOX) approximately doubled the uterine weight. Giving 1 microgram of AFP 1 hr before injection of either estrogen did not alter that response. Combining the E2 and AFP just prior to injection resulted in decreased uterine growth (34% inhibition). Preincubating the estrogens with purified AFP (0.1-50 micrograms) did not affect the growth response to moxestrol but markedly decreased the response to E2. This was not due to sequestering of hormone because maximal reduction of the E2 response (ca. 65% inhibition) required only 1.0 microgram of AFP (AFP/E2 molar ratio, 1:130), and higher AFP doses inhibited less. About 40% of the growth elicited by injection of either 0.5 micrograms of E2 or 10 ng of MOX was inhibited when these doses were preceded by injection of the preincubated AFP/E2 mixture but not when preceded by either of the components. In each experiment, the mitotic index of luminal epithelium was affected to the same degree as uterine weight. AFP and E2 incubated for 1 hr thus produce a potent inhibitor of estrogen-stimulated mitotic activity and growth. This inhibitor might act upon estrogen-responsive cells at specific sites at which competition by an inactive component of AFP can block the process.
Pleiotropic activities of human interferons are mediated by multiple response pathways.
Authors: Forti R L , Mitchell W M , Hubbard W C , Workman R J , Forbes J T
Published: May 2006

Among the pleiotropic effects of human interferon are the inhibition of viral replication, the activation of natural killer cells, and the inhibition of cellular growth. Oxyphenbutazone, a nonsteroidal antiinflammatory agent, is a potent inhibitor of the antiviral activity of human alpha and beta interferons as determined by cytopathic effect and vesicular stomatitis virus synthesis and release in human foreskin fibroblasts. The inhibition of interferon activity is dose dependent with maximal inhibition at 25-50 microM and minimal inhibition at 1 microM. In contrast, oxyphenbutazone at concentrations as high as 100 microM has no effect on the activation of natural killer cells by human interferon. Similarly, oxyphenbutazone has no inhibitory effect on interferon-induced antigrowth activity in the human breast carcinoma cell line MDA-MB-231. This cell line is sensitive to oxyphenbutazone inhibition of interferon-induced antiviral activity in vitro. In another human cell line, the vulvar carcinoma A431, oxyphenbutazone apparently augments the antigrowth activity of interferon. Although oxyphenbutazone inhibits the fatty acid cyclooxygenase enzyme in these systems, other inhibitors of cyclooxygenase fail to inactivate the antiviral activity of human interferon. Thus, oxyphenbutazone appears to inhibit the interferon antiviral cascade at a site distinct from prostaglandin biosynthesis. Moreover, the failure to inhibit natural killer cell activation or cellular antigrowth effects of human interferon suggests a pathway different from that associated with the antiviral effect of human interferon.
Inhibition of growth of pancreatic carcinomas in animal models by analogs of hypothalamic hormones.
Authors: Redding T W , Schally A V
Published: May 2006

Using animal models of acinar and ductal pancreatic cancer, we investigated the effect of analogs of hypothalamic hormones on tumor growth. In Wistar/Lewis rats bearing the acinar pancreatic tumor DNCP-322, chronic administration of [L-5-Br-Trp8]somatostatin-14 significantly decreased tumor weights and volume. Somatostatin-28 and the cyclic hexapeptide analog of somatostatin cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) failed to influence the growth of this tumor. The agonistic analog of luteinizing hormone-releasing hormone [D-Trp6]LH-RH also significantly decreased tumor weight and volume in this model and reduced testosterone levels and the weights of the ventral prostate and tests. In Syrian hamsters bearing ductal type of pancreatic carcinoma, chronic administration of [L-5-Br-Trp8]somatostatin diminished tumor weights and volume. The percentage change in tumor volume was significantly decreased when compared to control animals. In one experiment, cyclic hexapeptide of somatostatin also inhibited growth of this tumor. [D-Trp6]LH-RH, given twice daily or injected in the form of microcapsules for constant controlled release, significantly decreased tumor weight and volume and suppressed serum testosterone levels. Hamsters castrated 4 days after transplantation of the pancreatic tumors showed a significant decrease in weight and volume of these tumors. This suggests that pancreatic cancers may, at least in part, be sex hormone sensitive. [D-Trp6]LH-RH may decrease the growth of pancreatic carcinomas by suppressing androgens. Somatostatin analogs reduce the growth of pancreatic ductal and acinar cancers, probably by inhibiting the release or stimulatory action of gastrointestinal hormones on tumor cells (or both). Inhibition of animal models of pancreatic tumors by chronic administration of somatostatin analogs and [D-Trp6]LH-RH suggests that these compounds should be considered for the development of a new hormonal therapy for cancer of the pancreas.
Reconstitution of a functional synaptosomal membrane possessing the protein constituents involved in acetylcholine translocation.
Authors: Israël M , Lesbats B , Morel N , Manaranche R , Gulik-Krzywicki T , Dedieu J C
Published: May 2006

Reconstitution of a functional presynaptic membrane possessing calcium-dependent acetylcholine release properties has been achieved. The proteoliposomal membrane obtained gains its acetylcholine-releasing capabilities from presynaptic membrane proteins. At the peak of acetylcholine release, intramembrane particles became more numerous in one of the proteoliposomal membrane faces. This phenomenon resembles the intramembrane particle rearrangements found in stimulated synaptosomes. No visible structures capable of releasing acetylcholine as a result of the calcium influx were found inside the proteoliposomes. This supports the view that the release of free cytosolic acetylcholine from stimulated nerve terminals can be directly attributed to presynaptic membrane proteins. These proteins were extracted in a functional form from the synaptosomal membrane.
Streptococcal L-forms isolated from Drosophila paulistorum semispecies cause sterility in male progeny.
Authors: Somerson N L , Ehrman L , Kocka J P , Gottlieb F J
Published: May 2006

The Drosophila paulistorum complex contains six semispecies that do not normally interbreed. In the laboratory, crosses between semispecies produce fertile daughters and sterile sons. Microbial endosymbionts have been observed in all D. paulistorum flies that display this male sterility. Streptococcal L-forms have been isolated from the Andean-Brazilian (Mesitas) and Transitional (Santa Marta) semispecies and cultured in artificial medium. Transfer of these L-forms from their native hosts into reciprocal semispecies resulted in sterile male progeny. When L-forms were inoculated into the semispecies from which they had been isolated, most of the male progeny were fertile. Control streptococcal L-forms did not show this sterility pattern.
Synthesis of peptides related to the prosegment of mouse submaxillary gland renin precursor: an approach to renin inhibitors.
Authors: Evin G , Devin J , Castro B , Menard J , Corvol P
Published: May 2006

The complete sequence of the structural gene coding for mouse submaxillary gland renin was recently reported and the amino acid sequence of preprorenin was deduced. This sequence includes a 45-amino acid peptide that corresponds to the prosegment of the renin precursor. To investigate whether peptides related to the renin prosegment are able to inhibit renin activity, we have synthesized four peptides having the following structures: Arg-Ile-Pro-OMe, butyloxycarbonyl(Boc)-Leu-Lys-Lys-Met-Pro-OMe, Boc-Arg-Ile-Pro-Leu-Lys-Lys-Met-Pro-OMe, and Boc-Glu-Arg-Ile-Pro-Leu-Lys-Lys-Met-Pro-OMe (corresponding to amino acids 12-14, 15-19, 12-19, and 11-19, respectively, of the renin prosegment). All four peptides were found to inhibit the activity of pure mouse submaxillary renin on a porcine synthetic tetra-decapeptide in vitro, and the most potent inhibitors exhibited IC50 values in the micromolar range. Enzymatic kinetic studies carried out using peptide 15-19 showed an uncompetitive or a mixed type of inhibition with a Ki value of 2.3 X 10(-6) M at 37 degrees C in 0.5 M citrate/phosphate buffer (pH 6.0).
DNA sequences required for the in vitro replication of adenovirus DNA.
Authors: Guggenheimer R A , Stillman B W , Nagata K , Tamanoi F , Hurwitz J
Published: May 2006

Initiation of adenovirus (Ad) DNA replication occurs on viral DNA containing a 55-kilodalton (kDa) protein at the 5' terminus of each viral DNA strand and on plasmid DNAs containing the origin of Ad replication but lacking the 55-kDa terminal protein (TP). Initiation of replication proceeds via the synthesis of a covalent complex between an 80-kDa precursor to the TP (pTP) and the 5'-terminal deoxynucleotide, dCMP. Formation of the covalent pTP-dCMP initiation complex with Ad DNA as the template requires the viral-encoded pTP and DNA polymerase and, in the presence of the Ad DNA binding protein, is dependent upon a 47-kDa host protein, nuclear factor I. Initiation of replication with recombinant plasmid templates requires the aforementioned proteins and an additional host protein, factor pL. Deletion mutants of the Ad DNA replication origin contained within the 6.6-kilobase plasmid pLA1 were used to analyze the nucleotide sequences required for the formation and subsequent elongation of the pTP-dCMP initiation complex. The existence of two domains within the first 50 base pairs of the Ad genome, both of which are required for the efficient use of recombinant DNA molecules as templates in an in vitro DNA replication system, was demonstrated. The first domain, consisting of a 10-base-pair "core" sequence located at nucleotide positions 9-18, has been identified tentatively as a binding site for the pTP [ Rijinders , A. W. M., van Bergen, B. G. M., van der Vliet , P. C. & Sussenbach , J. S. (1983) Nucleic Acids Res. 11, 8777-8789]. The second domain, consisting of a 32-base-pair region spanning nucleotides 17-48, was shown to be essential for the binding of nuclear factor I.
Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR.
Authors: Miller V L , Mekalanos J J
Published: May 2006

We have cloned a positive regulatory gene ( toxR ) from Vibrio cholerae that controls cholera toxin transcription. This was done by first constructing a genetic fusion consisting of the lacZ gene fused to the promoter of the cholera toxin operon ctxAB . This operon fusion was used to screen a V. cholerae genomic library for genes that could activate the ctx promoter in Escherichia coli. This method allowed the identification of a gene, toxR , that increases ctx expression by more than 100-fold. Complementation analysis indicated that certain hypotoxinogenic mutants of V. cholerae 569B probably have mutations in the toxR gene. Southern blot analysis suggests that all V. cholerae, including nontoxinogenic strains, have the toxR gene. Moreover, nontoxinogenic strains not only lack the structural genes for cholera toxin but also sequences associated with the larger 7-kilobase ctx genetic element.
Genes required for initiation and resolution steps of mating-type switching in fission yeast.
Authors: Egel R , Beach D H , Klar A J
Published: May 2006

The fission yeast Schizosaccharomyces pombe switches mating type by transposition of a copy of DNA derived from either of the two storage cassettes, mat2 -P and mat3 -M, into the expression locus, mat1 . The recombinational event of switching is initiated by a double-stranded DNA break present in approximately 20% of the molecules at mat1 . Fifty-three mutants defective in switching of mating type have been isolated previously, and each has been assigned to 1 of 10 linkage groups. One group consists of cis-acting mutations at mat1 , which reduce the amount of the DNA double-strand cut. The remaining nine groups are mutations in genes that are unlinked to the mating-type locus and are studied here. Three ( swi1 , -3, -7) are required for formation of the double-strand cut, whereas the others are not. Mutants of three genes ( swi4 , -8, -9) undergo high-frequency rearrangement of the mating-type locus indicative of errors of resolution of recombinational intermediates. The remaining three ( swi2 , -5, -6) have normal levels of cut, do not make errors of resolution, and possibly are required either for efficient utilization of the cut or determining the directionality of switching. The data suggest that the switching process can be dissected into genetically distinguishable steps.
Human endothelial cells in culture produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with thrombin.
Authors: Prescott S M , Zimmerman G A , McIntyre T M
Published: May 2006

Cultured human endothelial cells produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) when stimulated with human thrombin. The response to thrombin is dose dependent, with a half-maximal effect at 0.17 unit/ml. The product is identified as PAF by the incorporation of radiolabeled precursors, its behavior in chromatographic systems, the recovery of biological activity, and the effect of treatment with phospholipase A2. Incorporation of [3H]acetate into PAF is maximal by 5 min and decreases thereafter. Endothelial cells produce 0.10-0.17 nmol of PAF per 10(6) cells in a 5-min exposure to thrombin, as judged by the amount of neutrophil-aggregating activity. The production of this potent agonist for platelet activation and neutrophil chemotaxis by endothelial cells suggests that it may play a role in the maintenance of vascular integrity and perhaps in pathological events such as thrombosis and atherogenesis.
Isolation of cloned cDNA encoding mammalian ornithine decarboxylase.
Authors: Kahana C , Nathans D
Published: May 2006

During the growth of mammalian cells the level of ornithine decarboxylase ( OrnDCase ; L-ornithine carboxy-lyase, EC 4.1.1.17), the first enzyme in polyamine biosynthesis, undergoes rapid changes. As an initial step in the study of possible genetic mechanisms involved in these changes, we have isolated cDNA clones encoding OrnDCase . To obtain RNA enriched for OrnDCase messenger, mouse myeloma cells that overproduce OrnDCase were selected in the presence of the OrnDCase inhibitor, difluoromethylornithine. A pBR322 cDNA library was prepared from poly(A)+ RNA isolated from difluoromethylornithine-resistant cells, and the library was probed with [32P]cDNA representing mRNA sequences from resistant or parental (sensitive) cells. All clones hybridizing preferentially to the resistant cell probe shared nucleotide sequences. A representative clone containing 1.1 kilobases of cDNA was shown to encode OrnDCase sequences by in vitro translation of hybrid-selected mRNA followed by precipitation of the translation products with anti- OrnDCase antiserum. Using this cDNA clone as a probe, we found that mouse DNA yielded several restriction fragments that react with the OrnDCase cDNA. In the difluoromethylornithine-resistant myeloma cells, one of these DNA segments is amplified and the level of OrnDCase mRNA is greatly increased compared with that in parental plasmacytoma cells. The level of OrnDCase mRNA is also increased in cultured 3T3 cells stimulated with serum and in mouse kidneys after administration of androgen, indicating that OrnDCase gene transcription and/or mRNA stability are regulated during cell growth.
Correlation of lac operator DNA imino proton exchange kinetics with its function.
Authors: Cheung S , Arndt K , Lu P
Published: May 2006

The kinetics for imino hydrogen exchange, at individual base pairs in the DNA sequence corresponding to the lactose operon operator of Escherichia coli, has been examined by NMR saturation recovery measurements as a function of temperature. Three 17-base-pair subsections of the lac operator DNA were chemically synthesized for these studies. The results support our previous observations in the 36-base-pair complete lac operator DNA fragment that has been used in our previous NMR studies. The results indicate faster opening kinetics at a GTG/CAC that is also the site of operator mutations leading to the highest level of constitutive beta-galactosidase synthesis. The GTG/CAC sequence occurs frequently and often symmetrically in prokaryotic and eukaryotic DNA sites where one anticipates specific protein interaction for gene regulation or recombination.