bluuebunny/crossref_metadata_2025 huggingface dataset:
N,N'-dimethylthiourea dioxide formation from N,N'-dimethylthiourea reflects hydrogen peroxide concentrations in simple biological systems.
Authors: Curtis W E , Muldrow M E , Parker N B , Barkley R , Linas S L , Repine J E
Published: May 2006

We hypothesized that measurement of a specific product from reaction of N,N'-dimethylthiourea (Me2TU) and H2O2 would provide a good indication of the H2O2 scavenging and protection seen after addition of Me2TU to biological systems. We found that addition of H2O2 to Me2TU yielded a single stable product, Me2TU dioxide. Me2TU dioxide formation correlated with Me2TU consumption as a function of added H2O2 concentration and was prevented by simultaneous addition of catalase (but not boiled catalase), superoxide dismutase, dimethyl sulfoxide, mannitol, or sodium benzoate. Me2TU dioxide formation, Me2TU consumption, and H2O2 concentration increases occurred in mixtures containing phorbol 12-myristate 13-acetate (PMA) and normal human neutrophils but not in mixtures containing PMA and neutrophils from patients with chronic granulomatous disease or in mixtures containing PMA and normal neutrophils and catalase. Me2TU dioxide formation also occurred in isolated rat lungs perfused with Me2TU and H2O2 but not in lungs perfused with Me2TU and elastase, histamine, or oleic acid. In contrast, Me2TU dioxide formation did not occur after exposure of Me2TU to 60Co-generated hydroxyl radical or hypochlorous acid in the presence of catalase. The results indicate that reaction of Me2TU with H2O2 selectively forms Me2TU dioxide and that measuring Me2TU dioxide formation from Me2TU may be useful for assessing the presence and significance of H2O2 in biological systems.
Doxorubicin cardiotoxicity may be caused by its metabolite, doxorubicinol.
Authors: Olson R D , Mushlin P S , Brenner D E , Fleischer S , Cusack B J , Chang B K , Boucek R J
Published: May 2006

Doxorubicin (former generic name, adriamycin), a highly effective anticancer drug, produces cardiotoxicity, which limits its therapeutic potential. The mechanism of this cardiotoxicity has remained elusive. Our data suggest that this toxicity could involve doxorubicinol, the primary circulating metabolite of doxorubicin. Doxorubicinol was markedly more potent than doxorubicin at compromising both systolic and diastolic cardiac function. Similarly, doxorubicinol was much more potent than doxorubicin at inhibiting the calcium pump of sarcoplasmic reticulum [ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38], the Na+/K+ pump of sarcolemma [ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37], and the F0F1 proton pump of mitochondria [ATP phosphohydrolase (H+-transporting, EC 3.6.1.34]. Our finding that this highly toxic metabolite was produced by cardiac tissue exposed to doxorubicin suggests that doxorubicinol could accumulate in the heart and contribute significantly to the chronic cumulative cardiotoxicity of doxorubicin therapy. Our observation that doxorubicin was more potent than doxorubicinol in inhibiting tumor cell growth in vitro suggests that the cardiotoxicity of doxorubicin is dissociable from its anticancer activity.
Roles for chloride ion and fibrinogen in the activation of [Glu1]plasminogen in human plasma.
Authors: Gaffney P J , Urano T , de Serrano V S , Mahmoud-Alexandroni M , Metzger A R , Castellino F J
Published: May 2006

Using two-dimensional immunoelectrophoresis and an antibody to alpha 2-antiplasmin, we assessed the plasmin generated in serum under different conditions as the plasmin-alpha 2-antiplasmin complex. Activation in serum of human [Glu1]plasminogen ([Glu1]Pg) by recombinant tissue plasminogen activator was inhibited by the normal serum levels of Cl- and was enhanced by physiological levels of fibrinogen in the presence or absence of Cl-. These results agree with the recognized ability of Cl- to induce a conformation in [Glu1]Pg less favorable for its activation than the conformation that results without Cl-. The enhancing effect of fibrinogen surpassed the inhibitory effect of Cl- over a wide range of recombinant tissue plasminogen activator concentrations in physiological serum. Lesser inhibition by Cl- was seen in a purified clot-lysis system, suggesting that [Glu1]Pg conformation when attached to soluble fibrin matrix was less affected by the anion. The data regarding the roles of circulating fibrinogen and Cl- in controlling the plasma level of activated [Glu1]Pg have important implications in thrombolytic therapy with recombinant tissue plasminogen activator.
Production of a monoclonal antibody against the Snow Mountain agent of gastroenteritis by in vitro immunization of murine spleen cells.
Authors: Treanor J , Dolin R , Madore H P
Published: May 2006

The Snow Mountain agent (SMA) is a 27- to 32-nm noncultivatable virus that causes acute gastroenteritis in humans. SMA is morphologically similar to but immunologically distinct from the Norwalk agent. SMA has been partially purified from the stool of experimentally infected volunteers and contains a single structural protein of Mr 62,000 as well as one or more non-virion-associated soluble proteins. Further characterization of this important human pathogen and other Norwalk-like viruses has been hindered by the lack of reagents with which to study them. To further characterize SMA, we developed a monoclonal antibody to SMA using in vitro immunization--a technique that permitted use of small quantities of antigen for immunization. The monoclonal antibody, SM-4, was specific for SMA and did not react with the Norwalk or Hawaii agents. In addition, SM-4 reacted with purified virion but not with the soluble protein. SM-4 also blocked the ability of labeled postinfection human IgG to bind to purified virion. Finally, both SM-4 and human postinfection sera specifically recognized the Mr 62,000 virion-associated protein. Thus, SM-4 is directed against an epitope present on the SMA structural protein that is not shared by the Norwalk or Hawaii agents and that is not present on the soluble protein. The availability of a monoclonal antibody against SMA should facilitate further purification and characterization of this agent. The techniques utilized in these studies provide a method for the production of additional monoclonal antibodies to this group of viruses and also should be useful for the study of other occult viral agents.
Selective elimination of idiotype-binding cells in vivo by a drug-idiotype conjugate demonstrates the functional significance of these cells in immune regulation.
Authors: Abu-Hadid M M , Bankert R B , Mayers G L
Published: May 2006

A receptor-specific cytotoxic drug delivery system has been used to eliminate idiotype-binding cells in vivo to ascertain the possible functional significance of these cells in regulating the humoral immune response to dextran. Protein M104E, a mouse myeloma protein that binds dextran, expresses a private idiotope that is present on a significant proportion of the normal dextran-specific antibody repertoire. Immunocompetent cells that bind and internalize M104E idiotype-bearing molecules were eliminated by the intravenous administration of a single dose of cytosine arabinonucleoside conjugated to purified M104E protein. The administration of this cytotoxic drug-idiotype conjugate had a profound effect upon the expression of the M104E idiotype in euthymic but not in athymic BALB/c mice following immunization with dextran. In euthymic mice, the depletion of the idiotype-binding cells resulted in a marked elevation in the level of M104E idiotype present in the immune sera. Moreover, treated but not control mice developed idiotype-positive molecules that did not bind dextran. These results demonstrate the functional significance of idiotype-binding cells in the regulation of individual clonotypes during an immune response.
Insulin-like growth factor II stimulates production of inositol trisphosphate in proximal tubular basolateral membranes from canine kidney.
Authors: Rogers S A , Hammerman M R
Published: May 2006

To determine whether insulin-like growth factor II (IGF-II) activates phospholipase C in the basolateral membrane of the renal proximal tubular cell, we incubated basolateral membranes isolated from canine kidney with rat IGF-II (rIGF-II) and measured levels of inositol trisphosphate (Ins-P3) in suspensions and of diacylglycerol extractable from the membranes. Incubation with rIGF-II increased levels of Ins-P3 and diacylglycerol in a concentration-dependent manner. Significant enhancement of Ins-P3 levels and extractable diacylglycerol occurred in suspensions incubated with as little as 10(-10) M rIGF-II. Elevated levels of Ins-P3 were measured after as little as 5 sec of incubation. Increases were no longer detectable after 45 sec of incubation, due to dephosphorylation of Ins-P3 in membrane suspensions. Incubation with either insulin or insulin-like growth factor I did not affect the level of Ins-P3. IGF-II-stimulated increases in Ins-P3 did not occur when basolateral membranes were suspended in the absence of free calcium. Increases were demonstrable in basolateral membrane suspensions in 0.1, 0.2, or 0.3 microM calcium, but not in 1.0 microM calcium. Inclusion of guanosine 5'-[gamma-thio]triphosphate in incubation mixtures did not increase levels of Ins-P3, nor did it enhance the action of rIGF-II in this regard. However, inclusion of guanosine 5'-[beta-thio]diphosphate inhibited rIGF-II stimulation of Ins-P3 production. In contrast to findings with basolateral membrane suspensions, incubation with rIGF-II did not increase levels of Ins-P3 in suspensions of isolated brush-border membranes. Our data are consistent with IGF-II-mediated activation of phospholipase C in isolated proximal tubular basolateral membranes. Such an action could reflect the mechanism by which the IGF-II "signal" is transmitted across the basolateral membrane of the renal proximal tubular cell and by which the actions of this peptide are mediated in renal and non-renal cells.
Cooperative DNA binding of heterologous proteins: evidence for contact between the cyclic AMP receptor protein and RNA polymerase.
Authors: Ren Y L , Garges S , Adhya S , Krakow J S
Published: May 2006

Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. In the absence of cGMP, CRP*598 shows a more open conformation than CRP, as indicated by its sensitivity to proteolytic attack and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated subunit crosslinking. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 can activate lacP+-directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection ("foot-printing") indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the [32P]lacP+ fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding. In contrast, the weakly active unliganded CRP*598 can be shifted to a functional state not only by cAMP but also by cGMP and RNA polymerase.
Trans-activation of the human immunodeficiency virus long terminal repeat sequences, expressed in an adenovirus vector, by the adenovirus E1A 13S protein.
Authors: Rice A P , Mathews M B
Published: May 2006

The human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) sequences were inserted into adenovirus in place of the E1 region. The HIV-1 LTR contained in this recombinant adenovirus responds to trans-activation by tatIII in a HeLa cell line constitutively expressing that HIV-1 gene product. In addition, the HIV-1 LTR is activated by the adenovirus E1A 13S, but not 12S or 9S, gene product when it is supplied in trans by a coinfecting wild-type adenovirus. The Rous sarcoma virus LTR, in a similar recombinant adenovirus, is insensitive to tatIII but is also trans-activated by the E1A 13S protein. The action of the 13S E1A and tatIII proteins are additive for the HIV-1 LTR in the context of adenovirus and they appear to act at the transcriptional level. As in HeLa cells, the adenovirus-borne HIV-1 LTR is inactive in the absence of a trans-activator in H9 and Jurkat cells, two human leukemic T-cell lines. This suggests that recombinant adenoviruses have diagnostic potential for the detection of trans-activators of the HIV-1 LTR that are present in circulating human lymphocytes.
Structural analysis of the 5' flanking region of the beta-globin gene in African sickle cell anemia patients: further evidence for three origins of the sickle cell mutation in Africa.
Authors: Chebloune Y , Pagnier J , Trabuchet G , Faure C , Verdier G , Labie D , Nigon V
Published: May 2006

Haplotype analysis of the beta-globin gene cluster shows two regions of DNA characterized by nonrandom association of restriction site polymorphisms. These regions are separated by a variable segment containing the repeated sequences (ATTTT)n and (AT)xTy, which might be involved in recombinational events. Studies of haplotypes linked to the sickle cell gene in Africa provide strong argument for three origins of the mutation: Benin, Senegal, and the Central African Republic. Nevertheless, the haplotype determination does not give any information about the variable segment and does not totally exclude the possibility of recombination leading to different haplotypes linked to the mutation. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT)n and (AT)xTy repeats. We found three additional structures for (AT)xTy correlating with the geographic origin of the patients. Ten other nucleotide positions, 5' and 3' to the (AT)xTy copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5' flanking region of the beta-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.
Additional evidence from x-ray powder diffraction patterns that icosahedral quasi-crystals of intermetallic compounds are twinned cubic crystals
Authors: Pauling Linus
Published: May 2006

Analysis of the measured values of
Q
for the weak peaks (small maxima, usually considered to be background fluctuations, “noise”) on the x-ray powder diffraction curves for 17 rapidly quenched alloys leads directly to the conclusion that they are formed by an 820-atom or 1012-atom primitive cubic structure that by icosahedral twinning produces the so-called icosahedral quasi-crystals.
Specificity of tumor necrosis factor toxicity for human mammary carcinomas relative to normal mammary epithelium and correlation with response to doxorubicin.
Authors: Dollbaum C , Creasey A A , Dairkee S H , Hiller A J , Rudolph A R , Lin L , Vitt C , Smith H S
Published: May 2006

By using a unique short-term culture system capable of growing both normal and malignant breast epithelial tissue, human recombinant tumor necrosis factor (TNF) showed preferential cytotoxicity to malignant cells as compared to the corresponding nonmalignant cells. Most of the malignant specimens were sensitive to TNF with 13 of 18 specimens showing 90% inhibition of clonal growth (ID90) by less than 500 units of TNF per ml of culture fluid. In contrast, all 13 nonmalignant specimens tested clustered at the resistant end of the TNF response spectrum, with ID90 values being greater than 5000 units of TNF per ml of culture fluid. This differential sensitivity to TNF was seen in three cases in which malignant and nonmalignant breast epithelial tissues from the same patient were studied. To investigate the mechanism of resistance to TNF by normal cells, the presence of receptors for TNF was determined. Five of six cultures showed specific binding of 125I-labeled TNF and there was no relationship between the degree of resistance and the degree of specific binding. Simultaneous comparison of tumor responsiveness to doxorubicin and TNF revealed a positive correlation in ID90 values; these results may have important implications for the clinical use of TNF in cancer patients heavily pretreated with doxorubicin.
Characterization of specific pancreatic polypeptide receptors on basolateral membranes of the canine small intestine.
Authors: Gilbert W R , Frank B H , Gavin J R , Gingerich R L
Published: May 2006

We have identified specific binding sites for pancreatic polypeptide (PP) on the mucosal lining of canine small intestine. The present study was undertaken to further characterize these binding sites (receptors) on purified intestinal membranes and to establish their location on the brush border or basolateral surface of the intestinal enterocyte. Basolateral and brush border membranes were prepared by sorbitol density centrifugation. PP receptors were localized predominantly to the vascular surface, and thus binding of PP 125I-labeled on Tyr-27 to the basolateral preparation was used to evaluate receptor characteristics. Binding of PP was calcium, time, temperature, and pH dependent. Maximum specific binding of labeled PP occurred after an 8-hr incubation at 4 degrees C with 5 mM calcium at pH 6.8. Data analysis by Scatchard plot showed high- and low-affinity binding sites with relative affinities of 1.5 x 10(-9) M and 2.6 x 10(-8) M and with corresponding binding capacities of 0.23 pmol/mg and 0.84 pmol/mg of protein, respectively. This receptor was specific for PP since peptide YY and neuropeptide Y, peptides of the PP family, cross-reacted by less than 3%, as judged from comparisons of half-maximal displacement of label. Structurally dissimilar peptides, insulin and glucagon, did not compete for binding. Specific 125I-labeled PP binding was localized primarily to basolateral membranes (9.8 +/- 0.8%) with little binding by brush border membranes (0.8 +/- 0.2%). Thus, we have identified highly specific receptors for PP, located predominantly on the vascular surface of the small intestinal mucosa. These data suggest that the mucosal lining of the small intestine is a target tissue for PP and that PP participates in the hormonal regulation of fuel metabolism and substrate transport in the small intestinal mucosa.
Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC.
Authors: Keener J , Kustu S
Published: May 2006

The NTRC protein (ntrC product) of enteric bacteria activates transcription of nitrogen-regulated genes by a holoenzyme form of RNA polymerase that contains the ntrA product (sigma 54) as sigma factor. Although unmodified NTRC will bind to DNA, it must be phosphorylated to activate transcription. Both phosphorylation and dephosphorylation of NTRC occur in the presence of the NTRB protein (ntrB product). We here demonstrate rigorously that it is the NTRB protein that is a protein kinase by showing that NTRB can phosphorylate itself, whereas NTRC cannot. Phosphorylated NTRC (NTRC-P) is capable of autodephosphorylation with a first-order rate constant of 0.14-0.19 min-1 (t 1/2 of 5.0-3.6 min) at 37 degrees C. In addition, there is regulated dephosphorylation of NTRC-P. By contrast to the autophosphatase activity, regulated dephosphorylation requires three components in addition to NTRC-P: the PII regulatory protein, NTRB, and ATP. NTRC is phosphorylated within its amino-terminal domain, which is conserved in one partner of a number of two-component regulatory systems in a wide variety of eubacteria. A purified amino-terminal fragment of NTRC (approximately equal to 12.5 kDa) is sufficient for recognition by NTRB and is autodephosphorylated at the same rate as the native protein.
Molecular evidence for somatic recombination in the ribosomal DNA of Tetrahymena thermophila.
Authors: Løvlie A , Haller B L , Orias E
Published: May 2006

The ribosomal DNA (rDNA) in Tetrahymena thermophila is a 21-kilobase-pair palindromic DNA molecule that replicates autonomously in the macronucleus and is maintained at the level of about 10,000 copies per macronucleus. The rDNA of inbred strain C3 outreplicates the rDNA of inbred strain B in most B/C3 heterozygous macronuclei, generating macronuclei containing exclusively C3 rDNA sequences. In 1% or less of the B/C3 heterozygous macronuclei, however, rDNA sequences derived from both B and C3 strains persist in the macronucleus (co-maintainers). We report here that long-term culture of co-maintainers has yielded recombinant rDNA molecules combining sequences from both parental inbred strains. The genetic structure of such molecules also gives us virtual certainty that the differential replication of C3 rDNA with respect to B rDNA is due to the DNA sequence difference previously reported in domain 2 of the rDNA replication regions of the two strains.
Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia.
Authors: Vulliamy T J , D'Urso M , Battistuzzi G , Estrada M , Foulkes N S , Martini G , Calabro V , Poggi V , Giordano R , Town M
Published: May 2006

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. We have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A we have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. In one of the commonest, G6PD Mediterranean, which is associated with favism among other clinical manifestations, a single amino acid replacement was found (serine----phenylalanine): it must be responsible for the decreased stability and the reduced catalytic efficiency of this enzyme. Single point mutations were also found in G6PD Metaponto (Southern Italy) and in G6PD Ilesha (Nigeria), which are asymptomatic, and in G6PD Chatham, which was observed in an Indian boy with neonatal jaundice. In G6PD "Matera," which is now known to be the same as G6PD A-, two separate point mutations were found, one of which is the same as in G6PD A. In G6PD Santiago, a de novo mutation (glycine----arginine) is associated with severe chronic hemolytic anemia. The mutations observed show a striking predominance of C----T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.
Alterations of amino acid repeats in the Escherichia coli hemolysin affect cytolytic activity and secretion.
Authors: Felmlee T , Welch R A
Published: May 2006

The primary structure of the Escherichia coli hemolysin polypeptide (HlyA) is used to predict intramolecular structures involved in the secretion and cytolytic activity of the molecule. The C-terminal region of HlyA contains a repeated, 8-amino acid chain represented by the consensus sequence Leu-Xaa-Gly-Gly-Xaa-Gly-Asn-Asp. Three in vitro derived mutations of hlyA are described that encode molecules missing various portions of the C-terminal region, including the repeat region. The wild-type and mutated HlyA molecules were analyzed for the ability to be secreted and to lyse erythrocytes. Hemolytic activity absolutely requires the presence of the repeats. The ability of the mutated HlyA molecules to initiate membrane translocation and be secreted required the presence of the C terminus and, to a degree, the repeated amino acid octets.
Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-β-1,3-glucanase
Authors: Shinshi H. , Wenzler H. , Neuhaus J.-M. , Felix G. , Hofsteenge J. , Meins F.
Published: May 2006

Tobacco glucan endo-1,3-β-glucosidase (β-1,3-glucanase; 1,3-β-D-glucan glucanohydrolase; EC 3.2.1.39) exhibits complex hormonal and developmental regulation and is induced when plants are infected with pathogens. We determined the primary structure of this enzyme from the nucleotide sequence of five partial cDNA clones and the amino acid sequence of five peptides covering a total of 70 residues. β-1,3-Glucanase is produced as a 359-residue preproenzyme with an N-terminal hydrophobic signal peptide of 21 residues and a C-terminal extension of 22 residues containing a putative N-glycosylation site. The results of pulse-chase experiments with tunicamycin provide evidence that the first step in processing is loss of the signal peptide and addition of an oligosaccharide side chain. The glycosylated intermediate is further processed with the loss of the oligosaccharide side chain and C-terminal extension to give the mature enzyme. Heterogeneity in the sequences of cDNA clones and of mature protein and in Southern blot analysis of restriction endonuclease fragments indicates that tobacco β-1,3-glucanase is encoded by a small gene family. Two or three members of this family appear to have their evolutionary origin in each of the progenitors of tobacco,
Nicotiana sylvestris
and
Nicotiana tomentosiformis
.
Glucocorticoids facilitate the stable transformation of embryonal rat fibroblasts by a polyomavirus large tumor antigen-deficient mutant.
Authors: Martens I , Nilsson M , Magnusson G , Linder S
Published: May 2006

The addition of glucocorticoids to the growth medium could substitute for the expression of the polyomavirus large tumor antigen in the transformation of rat fibroblasts in vitro. After transfection with a large tumor antigen-deficient mutant of polyomavirus, pbc1051, high-frequency permanent transformation was observed, if the cells were grown in medium containing dexamethasone. Growth of pbc1051-transfected rat fibroblasts was strictly dependent on the presence of glucocorticoids during the initial phase of transformation. In the second phase, the growth of pbc1051-transfected cells was stimulated by dexamethasone, but the hormone was not essential for growth. After approximately 10 weeks in culture, pbc1051-transfected cells had progressed to hormone independent growth. Rat embryo cells transfected with wild-type polyomavirus DNA had the second phase in which growth was stimulated by glucocorticoid, and after this phase growth was steroid independent. Addition of glucocorticoids to rat fibroblasts transfected with a plasmid encoding only the middle-sized tumor antigen resulted in only a weak stimulation of growth. In contrast, embryo cells transfected with a plasmid containing the human homologue of the cellular T24 Ha-ras gene linked to murine sarcoma virus and simian virus 40 enhancers could be efficiently established as cell lines in medium supplemented with glucocorticoids. The data suggest that, in the transformation of primary rodent cells by polyomavirus, the activity of large tumor antigen can be substituted for by stimulating normal cellular functions with dexamethasone.
Structure and expression of Hox-2.2, a murine homeobox-containing gene.
Authors: Schughart K , Utset M F , Awgulewitsch A , Ruddle F H
Published: May 2006

The Hox-2.2 gene is one of a cluster of homeobox-containing genes on mouse chromosome 11. A cDNA clone containing the Hox-2.2 homeobox has been isolated from an adult spinal cord library. Our analysis of the Hox-2.2 cDNA and genomic clones indicates that there are at least two oxons and one intron. The largest open reading frame includes the homeobox and codes for a 224 amino acid protein of molecular weight 25,312. Comparisons of the predicted Hox-2.2 protein with other homeodomain-containing proteins revealed four regions of sequence similiarity: an N-terminal octapeptide, a hexapeptide upstream of the homeodomain, the homeodomain, and a glutamic acid-rich region at the C terminus. Possible functions of these regions are discussed. The Hox-2.2 gene is expressed in 13.5-day embryos in the developing hindbrain and spinal cord. The expression patterns of Hox-2.2 and Hox-2.1 in 13.5-day embryos are compared.
A repressor heterodimer binds to a chimeric operator.
Authors: Hollis M , Valenzuela D , Pioli D , Wharton R , Ptashne M
Published: May 2006

Replacement of the solvent-exposed residues of the DNA recognition helix of the 434 repressor with the corresponding residues of the P22 repressor generates a hybrid protein, 434R[alpha 3(P22R)], which binds specifically to P22 operators. We show here that a new DNA-binding specificity is generated by combining 434 and 434R[alpha 3(P22R)] repressor monomers to form a heterodimer. The heterodimer specifically recognizes a chimeric P22/434 operator that lacks two-fold rotational symmetry.
Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.
Authors: Kole H K , Abdel-Ghany M , Racker E
Published: May 2006

Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.
Chemical oscillations in closed macromolecular systems.
Authors: Di Cera E , Phillipson P E , Wyman J
Published: May 2006

A cycle of irreversible, first-order, autocatalytic reactions among different states of a polyfunctional macromolecule, subject to the conservation of mass, can display stable chemical oscillations. This introduces a class of nonlinear dynamic models for energy transduction in closed macromolecular systems.
Epitope mapping of human factor VIII inhibitor antibodies by deletion analysis of factor VIII fragments expressed in Escherichia coli.
Authors: Scandella D , DeGraaf Mahoney S , Mattingly M , Roeder D , Timmons L , Fulcher C A
Published: May 2006

Epitopes for antibodies that inhibit factor VIII procoagulant protein were analyzed by deletion mapping of factor VIII protein fragments expressed in Escherichia coli. A human factor VIII cDNA clone was used to generate E. coli expression vectors encoding fragments containing the 80-kDa factor VIII light chain (A3, C1, and C2 domains) and the 44-kDa carboxyl-terminal half of the factor VIII heavy chain (A2 domain). A series of deletions of each fragment was constructed and tested by immunoblotting for the binding of alloantibody and autoantibody inhibitors. Analysis of derivatives of the 80-kDa fragment showed that six inhibitors recognized a major epitope(s) within the carboxyl-terminal 17.3 kDa of factor VIII. These inhibitors also recognized weaker epitopes nearby and one inhibitor recognized epitopes scattered throughout the 80-kDa fragment. Deletions within the heavy chain fragment revealed one epitope-containing region confined to the amino-terminal 18.3 kDa recognized by six inhibitors. Bacterially produced factor VIII fragments containing the major epitopes were capable of neutralizing inhibitors in vitro but fragments containing weaker or no epitopes did not. These data suggest a potential therapeutic use of factor VIII fragments for neutralization of inhibitor antibodies.
Importance of monoamine oxidase A in the bioactivation of neurotoxic analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
Authors: Heikkila R E , Kindt M V , Sonsalla P K , Giovanni A , Youngster S K , McKeown K A , Singer T P
Published: May 2006

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent dopaminergic neurotoxin that causes biochemical, pharmacological, and pathological deficits in experimental animals similar to those seen in human parkinsonian patients. All of the deficits can be prevented by treating mice with selective inhibitors of monoamine oxidase B (MAO-B), including deprenyl, prior to MPTP administration. We now report that the dopaminergic neurotoxicity of two potent MPTP analogs, namely the 2'-methyl and 2'-ethyl derivatives (2'-MeMPTP and 2'-EtMPTP), cannot be prevented by deprenyl pretreatment. However, the neurotoxicity of these two analogs can be prevented by pretreatment with a combination of deprenyl and the selective MAO-A inhibitor clorgyline at doses that are sufficient to almost completely inhibit both MAO-B and MAO-A activities. Moreover, the neurotoxicity of 2'-EtMPTP (but not of 2'-MeMPTP and MPTP) can be significantly attenuated by clorgyline alone. There was a parallel between the capacity of the MAO inhibitors to decrease the brain content of the pyridinium species after administration of the tetrahydropyridines and the capacity of the MAO inhibitors to protect against the neurotoxic action of the tetrahydropyridines. The data support the conclusion that both 2'-MeMPTP and 2'-EtMPTP are bioactivated to pyridinium species to a significant extent by MAO-A. Further, it appears that the formation of the pyridinium species plays an important role in the neurotoxic process.
Transsynaptic impulse activity regulates postsynaptic density molecules in developing and adult rat superior cervical ganglion.
Authors: Wu K , Black I B
Published: May 2006

Ganglionic postsynaptic density protein (PSDp) was used to monitor the influence of transsynaptic impulse activity on synaptic structure in the developing and adult rat superior cervical sympathetic ganglion (SCG). Since transsynaptic activity is known to regulate ontogeny of postsynaptic transmitter enzymes, we initially studied the developing ganglion. Denervation in neonates prevented normal development, decreasing calmodulin binding to the ganglionic PSDp by 71% after 4 weeks. During this period, denervation elicited only a 42% decrease in total protein of the synaptic membrane fraction, suggesting that innervation regulates development of various synaptic components differentially. Effects of denervation were extremely rapid, resulting in a 44% decrease in calmodulin binding within 1 day, consistent with regulation by a signaling process such as impulse activity. The effect of impulse activity was examined more directly in adults by treatment with the agents reserpine or phenoxybenzamine, which elicit reflex increases in sympathetic transmission. Administration of reserpine resulted in a progressive 90% increase in calmodulin binding to the PSDp over 4 weeks. Phenoxybenzamine also elicited an increase, mimicking the effects of reserpine. Neither agent altered total protein of the synaptic membrane fraction, suggesting that impulse activity regulates specific synaptic components. Finally, ganglionic denervation in adults decreased PSDp binding within 12 hr, consistent with acute effects of impulse reduction. Our results suggest that transsynaptic impulse activity plays an important role in regulation of specific molecular components of the synapse.
Maintenance by glycyl-L-glutamine in vivo of molecular forms of acetylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.
Authors: Koelle G B , Skau K A , Thampi N S , Hymel D M , Han M S
Published: May 2006

The 24-hr intracarotid infusion of plasma-treated glycyl-L-glutamine (3 microM) produced significant enhancement of the monomeric G1 and tetrameric G4 forms of acetylcholinesterase of the cat superior cervical ganglion 48 hr after denervation, in comparison with denervated, noninfused controls. No significant effect of glycyl-L-glutamine could be demonstrated 4 or 6 days after denervation. These findings are consistent with the conclusion, drawn from a previous in vitro study, that glycyl-L-glutamine acts at a stage prior to the aggregation of the G1 form into higher polymers to maintain the acetylcholinesterase content of denervated ganglia. It is proposed that the dipeptide may regulate the transcription of the DNA for acetylcholinesterase to its corresponding mRNA.
Phosphorylation of Alzheimer disease amyloid precursor peptide by protein kinase C and Ca2+/calmodulin-dependent protein kinase II.
Authors: Gandy S , Czernik A J , Greengard P
Published: May 2006

The amino acid sequence of the Alzheimer disease amyloid precursor (ADAP) has been deduced from the corresponding cDNA, and hydropathy analysis of the sequence suggests a receptor-like structure with a single transmembrane domain. The putative cytoplasmic domain of ADAP contains potential sites for serine and threonine phosphorylation. In the present study, synthetic peptides derived from this domain were used as model substrates for various purified protein kinases. Protein kinase C rapidly catalyzed the phosphorylation of a peptide corresponding to amino acid residues 645-661 of ADAP [ADAP peptide(645-661)] on Ser-655. Ca2+/calmodulin-dependent protein kinase II phosphorylated ADAP peptide (645-661) on Thr-654 and Ser-655. This peptide was virtually ineffective as a substrate for cAMP-dependent protein kinase, cGMP-dependent protein kinase, casein kinase II, or insulin receptor protein-tyrosine kinase. When a homogenate of rat cerebral cortex was used as the source of protein kinase, phosphorylation of ADAP peptide(645-661) was stimulated by calcium/phosphatidylserine/diolein to a level 4.6-fold above the basal level of phosphorylation, consistent with a prominent stimulation by protein kinase C. Using rat cerebral cortex synaptosomes prelabeled with 32Pi, a 32P-labeled phosphoprotein of approximately equal to 135 kDa was immunoprecipitated by using antisera prepared against ADAP peptide(597-624), consistent with the possibility that the holoform of ADAP in rat brain is a phosphoprotein. Based on analogy with the effect of phosphorylation by protein kinase C of juxtamembrane residues in the cytoplasmic domain of the epidermal growth factor receptor and the interleukin 2 receptor, phosphorylation of ADAP may target it for internalization.
Structure of an alternating-B DNA helix and its relationship to A-tract DNA.
Authors: Yoon C , Privé G G , Goodsell D S , Dickerson R E
Published: May 2006

The crystal structure of the synthetic DNA dodecamer CGCATATATGCG has been solved at 2.2-A resolution. Its central 6 base pairs adopt the alternating-B-DNA helix structure proposed nearly a decade ago. This alternating poly(AT) structure contrasts with the four known examples of what can be termed a poly(A) subfamily of B-DNA structures: CGCAAAAAAGCG, CGCAAATTTGCG, CGCGAATTCGCG, and CGCGAATTbrCGCG, their defining characteristic being a succession of two or more adenines along one strand, in a region of 4 or more A.T base pairs. All five helices show a characteristically narrow minor groove in their AT centers, but the mean propeller twist at A.T base pairs is lower in the alternating poly(AT) helix than in the poly(A) subfamily of helices. Three general principles emerge from x-ray analyses of B-DNA oligonucleotides: (i) GC and mixed-sequence B-DNA have a wide minor groove, whereas the minor groove is narrow in heteropolymer or homopolymer AT sequences. (ii) G.C base pairs have low propeller twist; A.T pairs can adopt a high propeller twist but need not do so. A high propeller twist can be stabilized by cross-strand hydrogen bonds in the major or minor groove, examples being the minor groove bonds seen in CCAAGATTGG and the major groove bonds that can accompany AA sequences in the poly(A) family. (iii) Homopolymer poly(A) tracts may be stiffer than are alternating AT or general-sequence DNA because of these cross-strand major groove hydrogen bonds. Poly(A) tracts appear internally unbent, but bends may occur at junctions with mixed-sequence DNA because of differences in propeller twist, base pair inclination, and base stacking on the two sides of the junction. Bending occurs most easily via base roll, favoring compression of the broad major groove.
Capture and characterization of 5-aza-2'-deoxycytidine-treated C3H/10T1/2 cells prior to transformation.
Authors: Rainier S , Feinberg A P
Published: May 2006

A model system was developed to capture phenotypically normal cells committed to transformation to address two fundamental questions in cancer biology: (i) what are the earliest events in transformation; and (ii) what is the role of DNA methylation in carcinogenesis? Individual C3H/10T1/2 cells were treated with 5-aza-2'-deoxycytidine, which causes hypomethylation of DNA. Cells were grown to subconfluence, and individual microcolonies were trypsinized into two fractions. One fraction was cryopreserved, and the other was replated and maintained in culture. Ten percent of these replated colonies became morphologically transformed after 4-6 weeks. The cryopreserved ancestral cells of both transformed and nontransformed microcolonies were then cultured and compared to each other and to the transformants for phenotypic properties of cellular transformation. Pretransformed and nonpretransformed ancestral cells were initially morphologically indistinguishable, their early growth curves did not differ significantly, and they did not form colonies in soft agar. On continued growth in culture, however, the pretransformants displayed all of the phenotypic characteristics of transformation. Furthermore, transformation occurred in a given pretransformant in most or all of the cells of that clone. Thus, cells committed to transformation could be isolated in this way prior to phenotypic transformation. Studies of these pretransformed cells will permit examination of the earliest events in carcinogenesis and the role of DNA methylation in transformation.
Genetic instability in Drosophila melanogaster: P-element mutagenesis by gene conversion.
Authors: Geyer P K , Richardson K L , Corces V G , Green M M
Published: May 2006

We report the molecular characterization of several P element-induced mutations and their revertants at the yellow (y) locus of Drosophila melanogaster. One of the mutants analyzed, y76d28, results from the insertion of a P element into the 5'-transcribed, untranslated portion of the y gene. Sequence analysis of several revertants of y76d28 shows that P excision occurs imprecisely. These events result in insertion of additional ATG codons in the y locus mRNA but are without phenotypic effect. In addition, we describe the molecular structure of P-associated mutations induced in a near wild-type revertant of y76d28 that carries an internally deleted 0.4-kilobase P element in the 5' noncoding region. Sequence analysis of two of these mutants demonstrates that they arose as a result of the integration of a larger P element at the exact location as in the parental stock without the 8-base-pair additional duplication associated with P insertions. The phenotype of these y alleles is dependent on the size and orientation of the integrated P element. We infer that P-element replacement in these mutants has occurred by a recombination/gene conversion mechanism.
Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition.
Authors: Gibbs J S , Chiou H C , Bastow K F , Cheng Y C , Coen D M
Published: May 2006

Herpes simplex virus (HSV) encodes a DNA polymerase that is similar in several respects to the replicative mammalian DNA polymerase alpha. Recently, these and other DNA polymerases have been shown to share several regions of protein sequence similarity. Despite these similarities, antiviral drugs that mimic natural polymerase substrates specifically inhibit herpesvirus DNA polymerases. To study amino acids involved in substrate and drug recognition, we have characterized and mapped altered drug sensitivity markers of nine HSV pol mutants and sequenced the relevant portions of these mutants. The mutations were found to occur within four relatively small regions. One such region, which we designate region A, has sequence similarity only to DNA polymerases that are sensitive to certain antiviral drugs. The other three regions contain sequences that are similar among various DNA polymerases. The multiple mutations occurring within two of these regions make it likely that the regions interact directly with drugs and substrates. Our results lead us to favor a model in which protein folding allows interactions among the four regions to form the substrate and drug binding sites.
Structure of the medium-chain acyl-CoA dehydrogenase from pig liver mitochondria at 3-A resolution.
Authors: Kim J J , Wu J
Published: May 2006

The three-dimensional structure of the medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) from pig liver mitochondria has been determined to 3.0-A resolution by the x-ray diffraction method. The enzyme is a tetramer of four identical 43-kDa subunits and contains one equivalent of flavin adenine dinucleotide (FAD) per subunit. The polypeptide is folded into three domains. The N-terminal and the C-terminal domains are composed mainly of alpha-helices, and the middle domain is packed with orthogonal beta-sheets. The FAD has an extended conformation: the flavin ring lies between the N-terminal and the beta-sheet domains, and the adenine moiety is found at the junction between the C-terminal and the beta-sheet domains of one subunit and the C-terminal domain of a neighboring subunit. The polypeptide chain folding near the FAD binding site is different from those observed in other flavoproteins, such as glutathione reductase and glycolate oxidase.