bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatitis B viral DNA in liver and serum of asymptomatic carriers.
Authors: Kam W , Rall L B , Smuckler E A , Schmid R , Rutter W J
Published: May 2006

Cloned DNA probes were used to test for hepatitis B virus (HBV) DNA in the liver and serum of 14 asymptomatic hepatitis B surface antigen (HBsAg) carriers and two former carriers. The results were compared with serological markers of HBV infection and liver histopathology. Three groups of carriers were distinguished. In group I, HBV DNA was present in both liver and serum. Hepatitis B e antigen (HBeAg), a marker of viral replication, was uniformly positive in serum. In one individual in this group, viral DNA was also integrated into liver genomic DNA. In group II, lower levels of nonintegrated HBV DNA were detected in the liver, but no HBV DNA was found in the serum. HBeAg was negative and with one exception there were antibodies to HBeAg. Integrated viral DNA was present in each case. In group III, there was no detectable nonintegrated viral DNA in serum or liver, but one individual had integrated sequences. All carriers lacked antibodies to HBsAg and had antibodies to hepatitis B core antigen and demonstrated nonspecific histological abnormalities in the liver. These findings indicate significant quantitative and qualitative differences among asymptomatic HBsAg carriers and suggest that their infectivity may be highly variable.
The cAMP-binding domains of the regulatory subunit of cAMP-dependent protein kinase and the catabolite gene activator protein are homologous.
Authors: Weber I T , Takio K , Titani K , Steitz T A
Published: May 2006

Comparison of the recently determined amino acid sequences of the regulatory subunit of cAMP-dependent protein kinase (RII) from bovine cardiac muscle and the Escherichia coli catabolite gene activator protein (CAP) shows significant homology. This homology extends over most of the amino-terminal domain in CAP and is particularly good for the region of the beta-roll structure. The RII sequence contains two adjacent and internally homologous regions, both of which have high resemblance to the cAMP-binding domain in CAP. This suggests that the protein kinase regulatory subunit contains two cAMP-binding domains in the carboxyl-terminal region, each having a beta-roll structure similar to that in CAP. The cAMP molecule is expected to bind to the RII within a pocket formed by residues from the beta-roll, as is the case with CAP. One cAMP molecule would interact with residues from about 163 to 220, and the other cAMP would interact with amino acids in the stretch 285-350 of the RII protein kinase sequence. As the carboxyl-terminal domain of CAP shows homologies to the DNA-binding domains of other regulatory proteins, the protein appears to be of modular construction: a DNA-binding domain joined to a cAMP-binding domain.
Cytochalasins inhibit arachidonic acid metabolism in thrombin-stimulated platelets.
Authors: Siess W , Lapetina E G , Cuatrecasas P
Published: May 2006

Low concentrations (0.5-1 microM) of cytochalasins inhibit the thrombin-stimulated polymerization of monomeric actin to filamentous actin in platelets. Similar concentrations of cytochalasin B inhibit the formation and metabolism of arachidonic acid in horse platelets stimulated by low concentrations of thrombin (0.1-0.5 unit/ml). However, the release of serotonin is not inhibited by cytochalasin B. Cytochalasins B and D (0.5-1 microM) markedly reduce, in thrombin-stimulated human or horse platelets, the metabolism of the liberated arachidonic acid by cyclooxygenase activity to thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid and the conversion of arachidonic acid by lipoxygenase activity to 12-hydroxy-5,8,10,14-icosatetraenoic acid. The generation of arachidonic acid from platelet phospholipids and the formation of phosphatidic acid are much less affected by cytochalasin B or D. Cytochalasins do not directly inhibit platelet cyclooxygenase, lipoxygenase, phospholipase A2, or phosphatidyl-inositol-specific phospholipase C. In addition, the metabolism of exogenously added arachidonic acid by intact platelets is not inhibited by cytochalasins B and D. The results indicate that polymerization of actin in platelets stimulated by thrombin may be required for the effective metabolism of arachidonic acid released from platelet phospholipids.
Excessive mineralization with growth plate closure in rats on chronic warfarin treatment.
Authors: Price P A , Williamson M K , Haba T , Dell R B , Jee W S
Published: May 2006

Rats maintained for 8 months on a level of warfarin sufficient to decrease the vitamin K-dependent protein of bone (bone Gla protein) to 2% of normal have an excessive mineralization disorder characterized by complete fusion of the proximal tibial growth plate and cessation of longitudinal growth. The general features of this abnormality resemble the fetal warfarin syndrome in humans, a disorder also characterized by excessive mineralization of the growth plate. These excessive mineralization disorders may be caused by the decreased levels of bone Gla protein, a protein that potently inhibits mineralization in vitro.
alpha-Amylase biosynthesis: signal sequence prevents normal conversion of the unprocessed precursor molecule to the biologically active form.
Authors: Miyata S , Akazawa T
Published: May 2006

Recently, we showed that completion of the polypeptide chains on the polysomes isolated from germinating rice seed scutellum in a cell-free translation system can direct the synthesis of (i) unprocessed polypeptide containing the signal sequence (precursor 1), (ii) signal sequence-cleaved but nonglycosylated polypeptide (precursor 2), and (iii) the fully processed and glycosylated form of alpha-amylase molecules. The two precursors as well as the mature form of alpha-amylase can thus be produced in almost the same condition. Here, the binding affinity of the enzyme molecule to the substrate analogue beta-cyclodextrin was used as a probe to compare the conformations of the three distinctly different polypeptide chains produced on the polysomes. It was found that the mature secretory form and the nonglycosylated precursor form (precursor 2) specifically bind to beta-cyclodextrin immobilized on an epoxy-activated Sepharose 6B column but the form that has an attached signal sequence (precursor 1) does not. The results provide evidence that the NH2-terminal signal sequence prevents acquisition of beta-cyclodextrin-binding activity, indicating that, in rice seed alpha-amylase, the signal sequence impairs conversion of the unprocessed polypeptide to the enzymically active configuration.
HLA antigen structural gene mutants selected with an allospecific monoclonal antibody.
Authors: Pious D , Krangel M S , Dixon L L , Parham P , Strominger J L
Published: May 2006

The HLA-A2 antigen-specific monoclonal antibody BB7.2 and complement were used to immunoselect mutants from an ethyl methanesulfonate-mutagenized human B lymphoid cell line, T5-1. Surviving colonies were screened by radioimmune binding with BB7.2 and with a monospecific HLA-A2 alloantiserum, Stewart, and HLA antigens of selected clones were immunoprecipitated and studied by isoelectric focusing. Several classes of mutants could be distinguished: mutants that expressed no HLA-A2 heavy chain; mutants that expressed an HLA-A2 heavy chain that was unable to associate with beta 2-microglobulin (beta 2m) and was not expressed at the cell surface; mutants with reduced HLA-A2 heavy chain-beta 2m association and cell surface expression of HLA-A2 dimer with or without heavy chain charge alterations; mutants with normal HLA-A2 heavy chain-beta 2m association and normal quantitative cell surface HLA-A2 expression but with HLA-A2 heavy chain charge alterations; and mutants with as yet incompletely defined lesions. Mutants with altered cell surface HLA antigens were not found in previous selections with alloantisera and should be useful for epitope mapping and structure-function studies of HLA molecules.
Quantitative karyotyping of human chromosomes by dual beam flow cytometry.
Authors: Langlois R G , Yu L C , Gray J W , Carrano A V
Published: May 2006

Dual beam flow cytometry of chromosomes stained with Hoechst 33258 and chromomycin A3 has been proposed as a method for quantitative classification of human chromosomes (bivariate flow karyotyping). In this paper we investigate the sources and magnitudes of variability in the mean fluorescence intensities of each chromosome group resolved in bivariate flow karyotypes and study the impact of this variability on chromosome classification. Replicate bivariate flow karyotypes of chromosomes isolated from lymphocytes from 10 individuals demonstrated that person-to-person variability was significantly greater than run-to-run variability. The total variability was sufficiently small that it did not interfere with classification of normal chromosome types except chromosomes 9 through 12 and chromosomes 14 and 15. Furthermore, the variability was generally smaller than 1/600th of the mitotic genome, so that one-band rearrangements should be detectable in bivariate flow karyotypes.
Interferon induces morphologic reversion with elimination of extrachromosomal viral genomes in bovine papillomavirus-transformed mouse cells.
Authors: Turek L P , Byrne J C , Lowy D R , Dvoretzky I , Friedman R M , Howley P M
Published: May 2006

The effect of mouse L-cell interferon on bovine papillomavirus type 1 (BPV-1) transformation of murine cells was examined. Mouse interferon reduced the level of BPV-1-induced transformation of mouse C127 cells by 95%. Long-term treatment of established BPV-1-transformed mouse cell clones with mouse L-cell interferon led to a decrease in the average number of the plasmid viral genomes present in these cells to 1/3 to 1/8. Although revertant lines could not be isolated from these lines in the absence of treatment with interferon, flat revertants were easily selected from two independent clonal transformed lines carried for 60 generations in the continued presence of 200 units of interferon per ml. These flat revertants had the biological characteristics of nontransformed C127 cells and could be retransformed by BPV-1. Southern blot hybridization failed to detect BPV-1 DNA in any of eight independent revertant lines examined under conditions that could detect 0.2 copies per cell. We conclude that interferon treatment has resulted in a selective reduction of the amount of extrachromosomal BPV-1 DNA in transformed cells and has cured some treated cells completely of their viral DNA.
Identification of thymidine-5′-aldehyde at DNA strand breaks induced by neocarzinostatin chromophore
Authors: Kappen Lizzy S. , Goldberg Irving H. , Liesch Jerrold M.
Published: May 2006

Snake venom phosphodiesterase or endonuclease S1 digestion of neocarzinostatin chromophore-treated DNA, labeled in its thymidine residues, liberates an unusual labeled nucleoside from the 5′ end of a drug-induced break. This substance, isolated by reverse-phase HPLC, possesses carbons from both the thymine and the deoxyribose moieties of thymidine in the DNA but, unlike thymidine, is readily degraded at pH 12 to thymine and a sugar fragment. The altered nucleoside was shown to contain a carbonyl group by its reduction with NaBH
4
to form a substance that has the chromatographic properties of thymidine and by its reaction with various hydrazines to form the respective hydrazone derivatives; the carbonyl exists as the 5′ aldehyde as shown by its mild chemical oxidation to the carboxylic acid with simultaneous loss of the 5′
3
H. Mass spectral analysis showed a fragmentation pattern compatible with the structure thymidine-5′-aldehyde. These data indicate that the nonprotein chromophore of neocarzinostatin, in the presence of a reducing substance (2-mercaptoethanol) and molecular oxygen, selectively oxidizes the 5′ carbon of nucleosides in DNA to the aldehyde, resulting in a strand break and a DNA fragment bearing nucleoside-5′-aldehyde at its 5′ end.
Cytochrome P-450-dependent oxygenation of arachidonic acid to hydroxyicosatetraenoic acids.
Authors: Capdevila J , Marnett L J , Chacos N , Prough R A , Estabrook R W
Published: May 2006

Arachidonic acid is oxidized by a NADPH-dependent oxygenase of rat liver microsomes to a number of oxygen-containing products, which can be resolved by HPLC. Several of these products have been purified and characterized. They exhibit an absorbance in the UV region of the spectrum that has a maximum at approximately 235 nm, indicative of the presence of a conjugated diene function. Mass spectral analysis of the trimethylsilyl ether derivatives of the methyl esters of the hydrogenated and nonhydrogenated metabolites shows that they are the 9-, 11-, 12-, and 15-monohydroxy derivatives of arachidonic acid, the hydroxyicosatetraenoic acids (HETEs). Their UV absorbance and chromatographic properties suggest that these products possess cis,transdiene geometry characteristic of HETEs isolated from other mammalian sources. THe isolation of these isomeric HETEs suggests that cytochrome P-450 may play a role in the oxidative metabolism of arachidonic acid to physiologically and pharmacologically important hydroxylated unsaturated fatty acids.
Overlap between ampC and frd operons on the Escherichia coli chromosome.
Authors: Grundström T , Jaurin B
Published: May 2006

The promoter for the Escherichia coli ampC beta-lactamase gene is shown to be located within the last gene of the fumarate reductase (frd) operon. Evidence is presented that the ampC attenuator serves as the terminator for transcription of this preceding operon. The nucleotide sequence was determined for two proteins that were shown to be encoded by a DNA segment preceding the ampC gene. The two proteins consisted of 131 and 119 triplets and had molecular weights of 15,000 and 13,100, respectively. The twelve COOH-terminal amino acids of the 13,100 molecular weight protein were found to overlap the ampC promoter. Accordingly, a G.C insertion in the promoter gave both increased transcription of ampC and a frameshift in this overlapping gene, resulting in readthrough proteins. Thus, we describe a type of very compact genetic organization of operons in prokaryotes.
Analysis of cytoskeletal proteins and Ca
2+
-dependent regulation of structure in intestinal brush borders from rachitic chicks
Authors: Howe Christine L. , Keller Thomas C. S. , Mooseker Mark S. , Wasserman Robert H.
Published: May 2006

We have investigated several structural aspects of the intestinal epithelial brush border from rachitic chicks. At both the light and electron microscope levels, rachitic brush borders are indistinguishable from controls. Although several of the prominent periodic acid-Schiff-positive proteins of the brush border membrane have slightly slower mobilities on sodium dodecyl sulfate/polyacrylamide gels than do corresponding proteins from control brush borders, the major components of the microvillus core, including subunits of 105, 95, and 68 kilodaltons, actin, and calmodulin, are not detectably different. As assayed by a
125
I-labeled calmodulin gel overlay technique, the same calmodulin-binding proteins are present in rachitic and control brush borders. Two proteins, the 105-kilodalton subunit of the microvillus core and an approximately 30-kilodalton membrane protein, bind calmodulin in a calcium-independent manner. Four cytoskeletal proteins (250, 190, 180, and 150 kilodaltons) and one membrane protein (35 kilodaltons) bind calmodulin only in the presence of calcium. Calcium-dependent solation of microvillus core proteins and calcium-dependent phosphorylation of the 20-kilodalton light chain of brush border myosin both occur as in controls. Our results show that rachintic chicks have brush borders that are quite similar to controls with respect to their ultrastructural organization, constituent contractile proteins, and calcium-dependent regulation of contractility and microvillus core structure. Therefore, the decreased absorption of calcium by intestinal epithelial cells in rachitic chicks is probably not due to gross structural or chemical differences in the brush border cytoskeleton.
Clonidine p-isothiocyanate, an affinity label for alpha 2-adrenergic receptors on human platelets.
Authors: Atlas D , Steer M L
Published: May 2006

Exposure of intact human platelets or platelet membranes to the clonidine analog clonidine p-isothiocyanate (clonidine-NCS), followed by extensive washing, results in the loss of [3H]yohimbine binding to platelet alpha 2-receptors. In addition, exposure of intact platelets to clonidine-NCS, followed by extensive washing, results in the loss of of epinephrine-induced inhibition of adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in frozen--thawed platelets and in purified platelet membranes. This effect is dependent on time and concentration (t 1/2 at 30 degrees C is less than 15 min; half-maximal effect occurs with clonidine-NCS at less than 10 microM). Clonidine-NCS appears to interact by irreversibly blocking the platelet alpha 2-receptors because (i) it abolishes alpha 2-receptor effects of adenylate cyclase activity (i.e., epinephrine-induced inhibition of basal and prostaglandin E1-stimulated activity) while not altering other cyclase activity (basal, prostaglandin E1-stimulated, and NaF-stimulated) and (ii) its effect on both [3H]yohimbine binding and epinephrine-induced inhibition of adenylate cyclase can be specifically prevented by alpha-agonists [(-)-epinephrine and clonidine] and alpha-antagonists (yohimbine and phentolamine). These observations indicate that clonidine-NCS is an effective affinity label for platelet alpha 2-receptors.
GTP is not required for calmodulin stimulation of bovine brain adenylate cyclase.
Authors: Heideman W , Wierman B M , Storm D R
Published: May 2006

The importance of guanyl nucleotides for calmodulin stimulation of bovine cerebral cortex adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was examined by using a partially purified calmodulin-sensitive adenylate cyclase that was resolved from calmodulin-insensitive forms of the enzyme. By using 5'-adenylyl imidodiphosphate as a substrate, in the absence of an ATP-regenerating system, it was determined that GTP is not required for calmodulin stimulation of the enzyme. Maximal activation by 5'-guanylyl imidodiphosphate (p[NH]ppG) was 5.3-fold, whereas the combination of p[NH]ppG and calmodulin stimulated the enzyme 27-fold. Although GDP inhibited p[NH]ppG stimulation of the calmodulin-sensitive adenylate cyclase, it did not affect calmodulin stimulation. In addition, calmodulin did not alter the kinetics for activation of the enzyme by p[NH]ppG. It is concluded that GTP is not required for calmodulin stimulation of brain adenylate cyclase and that calmodulin regulation of this enzyme is probably not due to effects of calmodulin on the affinity of the guanyl nucleotide regulatory complex for guanyl nucleotides.
Synthesis of thyroid hormone metabolites by photolysis of thyroxine and thyroxine analogs in the near UV.
Authors: van der Walt B , Cahnmann H J
Published: May 2006

Photolysis of thyroxine and its analogs in the near UV permitted synthesis in good yield of picogram to gram quantities of thyroid hormone metabolites. Preparation of the same metabolites by classical chemical synthesis requires multistep procedures. Specifically labeled metabolites of high specific activity (e.g., those carrying the label in the nonphenolic ring) were obtained by photolysis of appropriately labeled thyroxine or 3',3',5'-triiodothyronine (reverse triiodothyronine). Some of these labeled metabolites, which are required for metabolic studies (3-iodothyronine and 3,3'-diiodothyronine, labeled in the nonphenolic ring), had not previously been obtained by other methods. Irradiation of thyroxine and reverse triiodothyronine in 150 mM methanolic ammonium hydroxide with greater than 340-nm light caused removal of one iodine atom from the phenolic ring with formation of 3,5,3'-triiodothyronine and 3,3'-diiodothyronine, respectively. Irradiation with higher-energy light (greater than 300 nm) led to stepwise removal of additional iodine atoms. Those in the phenolic ring were removed preferentially, so that 3,5-diiodothyronine and 3-iodothyronine, respectively, were formed. The iodine atoms in the nonphenolic ring were lost more slowly. Tetraiodothyroacetic acid followed a similar photodeiodination pattern. Photolysis with light in the near UV is a simple method for the synthesis of thyroid hormone metabolites.
Sex determination in Drosophila melanogaster: analysis of transformer-2, a sex-transforming locus.
Authors: Belote J M , Baker B S
Published: May 2006

The transformer-2 (tra-2) locus is one of a set of regulatory loci that control sex determination in Drosophila melanogaster. Temperature-shift experiments with temperature-sensitive tra-2 mutants demonstrate that within single cell lineages tra-2+ function is required at several times, and probably continuously, during development for the occurrence of a series of determinative decisions necessary for female sexual differentiation. Analysis of the effects of tra-2 in the genital disc demonstrates that the tra-2+ function is necessary in females both to prevent male sexual differentiation and to permit female differentiation. These and other results support the model that the tra-2+ and tra+ loci act to control the expression of the bifunctional doublesex (dsx) locus.
Reciprocal modulation of agonist and antagonist binding to muscarinic cholinergic receptor by guanine nucleotide.
Authors: Burgisser E , De Lean A , Lefkowitz R J
Published: May 2006

The ability of guanine nucleotide to decrease the binding affinity of agonists but not antagonists has been documented in a number of hormone and neurotransmitter receptor systems. By contrast, recent reports indicate that both agonist and antagonist binding to the muscarinic cholinergic receptors appear to be regulated in a reciprocal fashion by guanine nucleotide. We document two forms of the muscarinic cholinergic receptor in frog heart, which are present in approximately equal proportions and which display high-agonist/low antagonist and low-agonist/high-antagonist affinities, respectively. Guanine nucleotide appears to convert the former type of site into the latter type. These observations can be interpreted in terms of a model for two interconvertible forms of the muscarinic cholinergic receptor reciprocally favored by agonists and antagonists. This model has implications both for the understanding of neurotransmitter-receptor interactions generally and for the nature of the biological effects of receptor antagonists.
Heat shock induces rapid dephosphorylation of a ribosomal protein in Drosophila.
Authors: Glover C V
Published: May 2006

Ribosomes isolated from Drosophila melanogaster tissue culture cells labeled in vivo with 32Pi contain a single, heavily phosphorylated, ribosomal protein. As much as 40% of this protein is phosphorylated in cells cultured at 25 degrees C. The molecular weight and other characteristics of this protein suggest possible homology with ribosomal protein S6. Following a shift-up to 37 degrees C, the protein is specifically and quantitatively dephosphorylated. The kinetics of this dephosphorylation are rapid with a half-time on the order of a few minutes. These kinetics closely parallel the heat shock-induced breakdown of the preexisting polysome population.
Organization of human histone genes.
Authors: Sierra F , Lichtler A , Marashi F , Rickles R , Van Dyke T , Clark S , Wells J , Stein G , Stein J
Published: May 2006

We describe the isolation and initial characterization of seven independent lambda Charon 4A recombinant phages which contain human histone genomic sequences (designated lambda HHG). Restriction maps of these clones and localization of the genes coding for histones H2A, H2B, H3, and H4 are presented. The presence of histone encoding regions in the lambda HHG clones was demonstrated by several independent criteria including hybridization with specific DNA probes, hybrid selection/in vitro translation, and hybridization of lambda HHG DNAs to reserve Southern blots containing cytoplasmic RNAs from G1-, S-, and arabinofuranosylcytosine (cytosine arabinoside)-treated S-phase cells. In addition, the lambda HHG DNAs were shown to protect in vivo labeled H4 mRNAs from S1 nuclease digestion. Based on the analysis of the lambda HHG clones, human histone genes appear to be clustered in the genome. However, gene clusters do not seem to be present in identical tandem repeats. The lambda HHG clones described in this report fall into at least three distinct types of arrangement. One of these arrangements contains two coding regions for each of the histones H3 and H4. The arrangement of histone genes in the human genome, therefore, appears to be different from that in the sea urchin and Drosophila genomes in which each of the five histone-encoding regions (H1, H2A, H2B, H3, and H4) is present only once in each tandemly repeated cluster. At least one clone, lambda HHG 41, contains, in addition to the histone genes, a region that hybridizes with a cytoplasmic RNA approximately 330 nucleotides in length. This RNA is not similar in size to known histone-encoding RNAs and is present in the cytoplasm of HeLa cells predominantly in the G1 phase of the cell cycle.
Self-normalization of highly transformed 3T3 cells through maximized contact interaction.
Authors: Rubin H , Chu B M
Published: May 2006

Nontransformed and moderately and highly transformed BALB/c 3T3 cells maintained on small coverslips in a large volume of medium multiplied to 2, 3, and 4 times higher population density, respectively, than they did in conventional cultures. Deprivation of Mg2+ caused highly transformed cells on coverslips to assume the appearance of nontransformed cells, decrease their rate of multiplication, and stop further growth at a much lower saturation density than the same cells in physiological Mg2+. The latter cells reached a saturation density of 10(6)/cm2 and their rate of DNA synthesis decreased progressively with increased crowding. At saturation density, cells in physiological Mg2+ took on an appearance and arrangement similar to normal fibroblasts. They developed a high requirement for serum to initiate DNA synthesis. When transferred at low density, they flattened out on a plastic surface and maintained the appearance of nontransformed cells for approximately 1 day. Onset of DNA synthesis and multiplication in the transferred cells was delayed for periods characteristic of quiescent nontransformed cells stimulated by fresh medium or transfer. Cells from crowded coverslips were approximately 1/10th as efficient at colony formation when suspended in agar as cells from uncrowded coverslips. They also had a significantly lower Mg2+ content. The crowded cells returned to their transformed morphological and growth behavior 2 to 3 days after transfer at low density. We conclude that a very high degree of crowding causes highly transformed cells to revert to the phenotype of nontransformed cells. Other treatments such as deprivation of Mg2+ or inorganic orthophosphate can achieve similar results. It appears that a balanced reduction in rates of metabolism and multiplication can restore the normal phenotype to transformed cells, implying that they differ only quantitatively from nontransformed cells. The putative role of Mg2+ in the regulation of multiplication and in transformation of animal cells is discussed.
Potential mechanism of emphysema: alpha 1-proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity.
Authors: Carp H , Miller F , Hoidal J R , Janoff A
Published: May 2006

The elastase inhibitory capacity per mg of alpha 1-proteinase inhibitor (alpha 1 PI) was measured in the bronchoalveolar lavage (BAL) fluid from 26 healthy smokers and 24 nonsmokers. Activity was decreased by 40% in smokers' BAL fluid compared to nonsmokers. This effect was demonstrable by using human neutrophil elastase as well as porcine pancreatic elastase as test enzyme (elastase, EC 3.4.21.11) and was reproducible when selected individuals in each group underwent lavage on repeated occasions. In contrast, the functional activity of alpha 1-antichymotrypsin was not decreased in smokers' BAL fluid. Crossed antigen-antibody electrophoresis confirmed that inactivation of alpha 1 PI was responsible for the decrease in the elastase inhibitory capacity of smokers' BAL fluid. alpha 1 PI purified from smokers' BAL fluids contained methionine sulfoxide (4 mol/mol of inactive alpha 1 PI), whereas alpha 1 PI from nonsmokers' BAL fluid did not. Smokers' alpha 1 PI was indistinguishable from nonsmokers' alpha 1 PI on the basis of electrophoretic mobility, molecular weight, and immunoreactivity. Thus, oxidation of methionine residues in lung alpha 1 PI is associated with cigarette smoking. Because chemical oxidation of alpha 1 PI in vitro causes loss of its elastase inhibitory activity, the present observations suggest that methionine oxidation may also be the cause of decreased functional activity of lung alpha 1 PI in smokers. Oxidative inactivation of alpha 1 PI in the lungs of cigarette smokers may play a role in the development of pulmonary emphysema in this group.
Post-translational modification and processing of Escherichia coli prolipoprotein in vitro.
Authors: Tokunaga M , Tokunaga H , Wu H C
Published: May 2006

Escherichia coli strain MM18 cells containing malE-lacZ hybrid protein was reported to accumulate prolipoprotein when they were induced with maltose [Ito, K., Bassford, P. J. & Beckwith, J. (1981) Cell 24, 707-717]. We have shown that the prolipoprotein accumulated in maltose-induced MM18 cells is not modified, lacking covalently linked glyceride. When the cell envelope of MM18 containing unmodified prolipoprotein was incubated in the presence of detergent with [2-3H]glycerol-labeled cell envelope of strain JE5505 lacking murein lipoprotein, incorporation of [2-3H]glycerol radioactivity into both prolipoprotein and processed mature lipoprotein was observed. Likewise, when [3H]-palmitate-labeled JE5505 cell envelope was incubated with the MM18 cell envelope containing unmodified prolipoprotein in the presence of detergent, [3H]palmitate radioactivity was incorporated into prolipoprotein by ester linkage and into mature lipoprotein by both ester and amide linkages. These results indicate that our in vitro system contains activities of prolipoprotein modification and processing enzymes, including glyceryltransferase, O-acyltransferase, signal peptidase, and N-acyltransferase. The signal peptidase activity in our in vitro system was completely inhibited by globomycin. At pH 5.0, glyceryltransferase was inactive. Signal peptidase was active at pH 5.0, provided that prolipoprotein had been modified by glyceryltransferase (O-acyl-transferase) during a prior incubation at pH 9.1. These results strongly suggest that the modification of prolipoprotein by glyceryltransferase (and O-acyltransferase) precedes, and may in fact be a prerequisite for, the processing of prolipoprotein by signal peptidase.
Inheritance of DNA methylation in microinjected eggs of Xenopus laevis.
Authors: Harland R M
Published: May 2006

Plasmid DNA methylated at Hpa II sites was injected into eggs of Xenopus laevis. The methylated DNA replicates extrachromosomally, and the methylated state of the H pa II sites is inherited through replication. Unmethylated sites remain unmethylated in progeny molecules. To test whether replication is necessary for new methylation to occur, DNA methylated on one strand only was injected and unreplicated DNA was selected for analysis. Methylation was copied onto a previously unmethylated strand in the absence of replication but less efficiently than in its presence. These experiments show that the individual methylated site contains enough information for inheritance of the methylated state and that inheritance of methylation does not require integration of the DNA into the chromosome.
Altered epidermal growth factor (EGF)-stimulated protein kinase activity in variant A431 cells with altered growth responses to EGF
Authors: Buss Janice E. , Kudlow Jeffrey E. , Lazar Cheri S. , Gill Gordon N.
Published: May 2006

To determine the role of epidermal growth factor (EGF)-stimulated protein kinase in the biological effects caused by EGF, tyrosine-specific kinase activity has been quantitated in A431 human epidermoid carcinoma cells and six variant cell lines. Because EGF inhibited proliferation of A431 cells, variants resistant to this inhibition were selected by treatment with mutagen and maintenance for 1 month in 0.1 μM EGF. After cloning and growth for 6-20 generations without EGF, the resistance of the variants to the growth-inhibitory effect of EGF was confirmed. Whereas EGF increased cellular phosphotyrosine content ≈10-fold in parental A431 cells, EGF caused smaller or undetectable increases in the six variant cell lines. Solubilized membranes from the six variants displayed diminished EGF-stimulated phosphorylation of the EGF receptor and of antibodies to p60
src
(the product of the Rous sarcoma virus transforming gene), which act as an exogenous substrate. The decrease in EGF-stimulated tyrosine-specific protein kinase activity varied from ≈40% (clone 16) to ≈8% (clone 18) of parental A431 activity. Phosphorylated EGF receptors from parental and variant cells migrated identically on sodium dodecyl sulfate/polyacrylamide gels. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity, so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in the six variant cell lines with reductions in both activities to as low as 10%. These results suggest that this tyrosine-specific protein kinase activity mediates the growth-inhibitory effect of EGF on A431 cells and that both EGF binding and kinase activities reside in the same or tightly associated molecules.
Pluripotential hemopoietic stem cells in adult mouse brain.
Authors: Bartlett P F
Published: May 2006

Single cell suspensions of adult mouse brain were shown to contain large numbers of pluripotential hemopoietic stem cells as detected by the ability to form hemopoietic colonies in the spleens of irradiated hosts. These colony forming unit, spleen (CFU-s) cells derived from brain gave rise to colonies identical in morphology and histology to those of bone marrow-derived CFU-s. The average number of CFU-s obtained per 10(5) dissociated adult brain cells was 14, whereas other adult tissues such as lung, kidney, heart, and thymus contained insignificant CFU-s levels when tested. As the level of CFU-s in adult blood is less than 1 per 10(6) nucleated cells, blood contamination does not contribute to the high levels found in adult brain. Individual spleen colonies isolated from irradiated CBA (H-2k) recipients injected with (BALB/c x CBA)F1 (H-2d x H-2k) brain cells were shown by immunofluorescence to contain cells bearing surface H-2d molecules, thus indicating that the colonies arose from the brain cell inoculum and were not endogenously derived. The surface phenotype of brain- and bone marrow-derived CFU-s was found to differ in that brain CFU-s could be inhibited by prior incubation with a monoclonal antibrain antibody B2A2, whereas bone marrow CFU-s were not. Further differences were found between brain and bone marrow CFU-s in the congenitally anemic Wf/Wf mice. These mice were shown to have a very few CFU-s in the adult bone marrow, whereas the brain contained normal adult levels. The large number of hemopoietic stem cells in the brain may indicate an essential requirement for the continual generation of cells such as microglia or phagocytic cells, without the disruption of the blood-brain barrier.
Prothoracic glands of the saturniid moth
Samia cynthia ricini
possess a circadian clock controlling gut purge timing
Authors: Mizoguchi Akira , Ishizaki Hironori
Published: May 2006

In the moth
Samia cynthia ricini
, timing of the release of prothoracicotropic hormone is controlled by a circadian clock in the cephalic organ (brain); while this hormone release is necessary for gut purge, the final timing of this event is controlled by a circadian clock in the prothoracic glands that gates release of ecdysone. The photoreceptor of the prothoracic gland clock(s) is extraocular and evidently in the glands themselves. Demonstration that the clock and its photoreceptor are in the prothoracic glands is based on a mixture of localized illuminations and the transplantation of glands from one larva to another.
Initial localization of the memory trace for a basic form of learning.
Authors: McCormick D A , Clark G A , Lavond D G , Thompson R F
Published: May 2006

Electrophysiological recording of neuronal unit activity during paired training trials from various regions of the ipsilateral cerebellum in rabbits well trained in the classically conditioned eyelid/nictitating membrane response have revealed both stimulus-evoked responses and responses that form an amplitude/temporal model of the learned behavioral response. Ablation of the ipsilateral, lateral cerebellum completely and permanently abolished the behavioral conditioned response in well-trained animals but had no effect at all on the unconditioned reflex response. In marked contrast, conditioned responses were easily trained in the eye contralateral to the cerebellar lesion. We suggest that at least part of the essential neuronal plasticity that codes the learned response may be localized to the cerebellum.
Human cytoplasmic superoxide dismutase cDNA clone: a probe for studying the molecular biology of Down syndrome.
Authors: Lieman-Hurwitz J , Dafni N , Lavie V , Groner Y
Published: May 2006

The gene locus for human cytoplasmic superoxide dismutase (SOD-1; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is located in or near a region of chromosome 21 known to be involved in Down syndrome. To approach the molecular biology of this genetic disease we have constructed a SOD-1 cDNA clone. Poly(A)-containing RNA enriched for human SOD-1 mRNA was isolated, used to synthesize double-stranded cDNA, and inserted into the endonuclease Pst I site of the plasmid pBR322. The chimeric molecules were used to transform Escherichia coli. Two clones containing SOD-1 cDNA inserts were identified by their ability to hybridize specifically with mRNA coding for SOD-1. Each of these clones carries a 650-base-pair insert, as was determined by restriction enzyme digestion and electron microscopic heteroduplex analysis. Hybridization of labeled cloned cDNA to RNA blots revealed two distinct SOD-1 mRNA classes of 500 and 700 nucleotides. The data suggest that both are polyadenylylated and are coded by chromosome 21.
Abnormal oxidative metabolism of estradiol in women with breast cancer.
Authors: Schneider J , Kinne D , Fracchia A , Pierce V , Anderson K E , Bradlow H L , Fishman J
Published: May 2006

The three dominant oxidative biotransformations of estradiol were examined in 10 normal women and 33 females with breast cancer by using a recently devised radiometric method. Estradiol tracers, labeled with 3H specifically in the 17 alpha, C-2, or 16 alpha position, were used to measure both the rate and extent of 17 beta-ol oxidation (the initial metabolic step) and the subsequent 2- and 16 alpha-oxidative reactions. The mean +/- SEM values for the extent of extradiol metabolism at these three specific sites for the extent of estradiol metabolism at these three specific sites were 76.9 +/- 5.3%, 31.1 +/- 4.0%, and 9.3 +/- 0.8%, respectively in normal subjects. Corresponding data in patients with breast cancer--i.e., 73.0 +/- 4.2%, 32.7 +/- 2.7%, and 14.9 +/- 1.5%--revealed a significantly greater extent of 16 alpha-hydroxylation in the latter population. Because the 16 alpha-hydroxylated compounds (including estriol) are themselves potent estrogens, these changes may have important hyperestrogenic consequences that could have a bearing on the etiology of the disease.
Demethylation and expression of murine mammary tumor proviruses in mouse thymoma cell lines.
Authors: Mermod J J , Bourgeois S , Defer N , Crépin M
Published: May 2006

Murine mammary tumor virus (MMTV) expression is analyzed in a T-lymphoid cell line (T1M1) sensitive to the killing effect of glucocorticoids and in two of its variants, one resistant (T1M1r) and one supersensitive (T1M1ss) to glucocorticoid-induced lymphocytolysis. In the T1M1 line, MMTV is expressed and induced approximately 10-fold by short treatment with dexamethasone. Southern blot analyses of restriction enzyme digests of DNA from T1M1 cells reveal three proviruses similar to those of normal C57BL mouse tissue. In the T1M1ss line, which has retained functional glucocorticoid receptors, MMTV mRNA is inducible by glucocorticoids, while induction is reduced in the T1M1r line defective in glucocorticoid receptors. Moreover, the T1M1r line expresses a strikingly elevated basal level of MMTV mRNA in the absence of hormone. No rearrangements or superinfection have occurred in the variants, but all the regions containing 5'-long terminal repeats are demethylated in the T1M1r variant although other sites of the provirus remain methylated. Because this variant was selected by prolonged treatment with dexamethasone, these observations raise the possibility that the continuous transcription of MMTV that occurred during this selection can result in glucocorticoid-induced demethylation of long-terminal-repeat sequences.
Immunocytochemical localization of prosomatostatin fragments in maturing and mature secretory granules of pancreatic and gastrointestinal D cells.
Authors: Ravazzola M , Benoit R , Ling N , Guillemin R , Orci L
Published: May 2006

Pancreatic and gastrointestinal D cells were examined by immunocytochemistry using antisera against somatostatin-28 (SS28) and its NH2-terminal fragment SS28-(1-12), followed by the staphylococcal protein A-gold (pAg) complex. In pancreatic and gastric D cells incubated with antiserum against SS28-(1-12) the gold particles produced intense staining of the mature secretory granules but weaker staining of the immature granules associated with the Golgi area, whereas after SS28 antiserum treatment the particles accumulated selectively over the population of immature secretory granules. In intestinal D cells not only SS28-(1-12) but also SS28 antiserum produced an intense gold staining over the mature delta granules. These observations show that the relative amounts of immunoreactive sites related to SS28 and its cleavage product SS28-(1-12) in maturing and mature secretory granules are different in pancreatic, gastric, and intestinal D cells.
Directed mutagenesis method for analysis of mutagen specificity: application to ultraviolet-induced mutagenesis.
Authors: Livneh Z
Published: May 2006

A directed mutagenesis method has been developed for the analysis of mutagen specificity. The method is based on the construction of a plasmid damaged by the mutagen at a specific segment within a given marker gene, followed by screening for mutant plasmids and nucleotide sequence analysis of the damaged segment. By using this method plasmid pXf3 has been specifically damaged by UV radiation at the BamHI-Sph I segment in the tetracycline resistance (tet) gene(s) and used to transform SOS-induced Escherichia coli. Fourteen ampicillin-resistant, tetracycline-sensitive mutants of pXf3 were isolated and subjected to sequence analysis. The data revealed the induction of transitions, a transversion, a frameshift mutation, and deletions. The single base changes all were located within runs of pyrimidines, and the deletions mapped between direct repeats of polypyrimidine tracts. In addition, mutant plasmids were found with no mutation within the damaged segment. In these cases it is likely that "untargeted mutagenesis" in other portions of the tet gene(s) is responsible for the mutant phenotype. The method can be applied to any mutagen that reacts with DNA, and it also can be used for genetic analysis as a means to mutate specific segments of DNA.
Sequence specificity in aflatoxin B1--DNA interactions.
Authors: Muench K F , Misra R P , Humayun M Z
Published: May 2006

The activated form of aflatoxin B1 (AFB1) causes covalent modification primarily of guanine residues, leading to alkali-labile sites in DNA. A simple extension of the Maxam-Gilbert procedure for sequence analysis permits the identification of alkali-labile sites induced by AFB1 and determination of the frequency of alkali-labile AFB1 modifications at particular sites on a DNA fragment of known sequence. Using this strategy, we have investigated the influence of flanking nucleotide sequences on AFB1 modification in a number of DNA fragments of known sequence. Our results show that certain guanine residues in double-stranded DNA are preferentially attacked by AFB1 over others in a manner predictable from a knowledge of vicinal nucleotide sequences. The observed in vitro sequence specificity is independent of a number of tested parameters and is likely to occur in vivo.
Control of UV induction of recA protein.
Authors: Salles B , Paoletti C
Published: May 2006

The basal level of recA protein in Escherichia coli K-12 was estimated by an immunoradiometric assay; it is approximately equal to 1,200 molecules per wild-type bacteria in midexponential phase of growth, slightly more in an excision-deficient (uvrA) strain, and markedly more in recF mutants. Kinetics of induction after UV irradiation showed a rapid increase of recA protein content, which reached a peak level after 60-90 min (20- to 55-fold amplification) and then decreased by dilution of the protein in the growing population. In order to obtain an identical extent of induction of recA protein, a 10-fold higher UV dose was necessary in a wild-type strain compared to the uvrA mutant strain. In the uvrA strain, the presence of one or only very few pyrimidine dimers on DNA was accompanied by a measurable increase of the constitutive level of recA protein; however, the unexcised dimers were unable to permanently induce the formation of recA protein. The derepressed promoter of recA gene is one of the strongest in E. coli. Its sequence displays many similarities with that of the strongest early promoters of T5 phage. Mutants (umuC uvrB and recF uvrB) unable to carry out W-reactivation produced high levels of recA protein after UV irradiation. The data suggested that the recF and umuC genes negatively control the regulation of recA protein level.
Relationship between promoter structure and template specificities exhibited by the bacteriophage T3 and T7 RNA polymerases.
Authors: Bailey J N , Klement J F , McAllister W T
Published: May 2006

To explore the basis for the template specificities of the bacteriophage T3 and T7 RNA polymerases (EC 2.7.7.6), we determined the nucleotide sequences of six promoters recognized by the T3 RNA polymerase and compared them with the previously determined promoter sequences recognized by the bacteriophage T7 RNA polymerase. Recombinant plasmids containing random Hpa II and Taq I fragments of T3 DNA were screened for T3 promoter activity in vitro in a transcription assay using purified T3 RNA polymerase. Five promoters for the T3 RNA polymerase were identified in this manner and their sequences were determined; the sequence of an additional promoter was determined directly from a genomic DNA fragment. In five of the T3 promoters an identical 16-base-pair sequence (A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A) extends from -12 to +4 (initiation occurring with GTP at +1); this sequence is preceded by a 6-base-pair A + T region. The remaining promoter contains an inserted C at position -1 and an A at the +1 position. The sequence of the 5' end of the RNA transcript from the latter promoter confirms that transcription is initiated with ATP at the +1 position. Previously, late T3 or T7 transcripts had not been found to initiate with ATP. The highly conserved T3 promoter sequence was compared to the T7 promoter consensus sequence. The fundamental difference between the two kinds of phage promoters is the occurrence of G-A at positions -11 and -10 in the T7 promoter, whereas there is a single C at position -10 in the T3 promoter.