bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of sea urchin glycoprotein mRNAs during embryonic development
Authors: Lau Joseph T. Y. , Lennarz William J.
Published: May 2006

Gastrulation in sea urchin embryos is accompanied by a striking increase in the synthesis of
N
-linked glycoproteins, and inhibitors of this process block gastrulation. In this report, the messages coding for
N
-glycosylatable proteins in the developing embryo of the sea urchin,
Strongylocentrotus purpuratus
, were examined. Total mRNA and mRNA isolated from membranes of the embryos at various stages of development were used to program a cell-free translation/glycosylation system prepared from rabbit reticulocyte lysate supplemented with dog pancreas microsomes. The glycosylated translation products were separated from the nonglycosylated products by concanavalin A-agarose and analyzed by gel electrophoresis. The results indicate that although the RNA derived from the membranes of gastrula-stage embryos contains messages coding for numerous glycoproteins, only trace amounts of glycoprotein messages are associated with membranes at earlier stages of development. mRNAs coding for four glycoproteins of
M
r
s 70,000, 65,000, 51,000, and 30,000 were examined further in total RNA preparations from the developing embryo. The data indicate that the messages coding for the glycoproteins of
M
r
s 65,000 and 51,000 are present also in the unfertilized egg and in the pregastrulation embryo. Because these two messages are not found associated with the membranes until gastrula stage, it is likely that the synthesis of these glycoproteins during gastrulation is regulated at the translational level. The messages coding for glycoproteins of
M
r
s 70,000 and 30,000, on the other hand, are not detectable in the unfertilized egg and may be synthesized
de novo
by the embryos. Thus, the expression of these two glycoproteins during gastrulation is regulated at least in part on the transcriptional level. On the basis of these findings, it appears that different modes of regulation are used for different glycoproteins that are synthesized during gastrulation.
Silkmoth chorion multigene families constitute a superfamily: comparison of C and B family sequences.
Authors: Regier J C , Kafatos F C , Hamodrakas S J
Published: May 2006

We have characterized a new family of silkmoth chorion genes, called C, which is distinct from previously characterized A and B families. The amino acid compositions of 18 purified C proteins have been determined. Three subgroups are recognized on the basis of compositional similarities and may correspond to distinct gene families or subfamilies. The sequences of two overlapping cDNA clones have been determined in their entirety and shown to correspond to a C-specific sequence. Obvious homology is observed between the middle portions of the C sequence and previously characterized B sequences. By contrast, the arms of the C sequence share no significant similarities either with each other or with the corresponding arms of B sequences. Thus, the same tripartite structure originally observed in A and B family sequences is also present in the C family and may have functional significance. Secondary structure prediction of the C sequence is presented and supports this conclusion. The observed homology between C and B family sequences clearly establishes that silkmoth chorion multigene families constitute a superfamily.
Selective constraint in protein polymorphism: Study of the effectively neutral mutation model by using an improved pseudosampling method
Authors: Kimura Motoo , Takahata Naoyuki
Published: May 2006

To investigate the pattern of allelic distribution in enzyme polymorphism, with special reference to the relationship between the mean (
H
) and the variance (

V
H

) of heterozygosity, we used the model of effectively neutral mutations involving multiple alleles in which selective disadvantage of mutant alleles follows a Γ distribution. A simulation method was developed that enables us to study efficiently the process of random drift in a multiallelic genetic system and that saves a great deal of computer time. It is an improved version of the pseudosampling-variable (PSV) method [Kimura, M. (1980)
Proc. Natl. Acad. Sci. USA
77, 522-526] previously used to simulate random drift in a diallelic system. This method will be useful for simulating many models of population genetics that involve behavior of multiple alleles in a finite population. By using this method, it was shown that, as compared with the model of strictly neutral mutations, the present model gives the reduction of both
H
and

V
H

and an excess of rare variant alleles. The results were discussed in the light of recent observations on protein polymorphism with special reference to the functional constraint of proteins involved.
n-Type silicon photoelectrochemistry in methanol: Design of a 10.1% efficient semiconductor/liquid junction solar cell
Authors: Gronet Chris M. , Lewis Nathan S. , Cogan George , Gibbons James
Published: May 2006

n-Type Si electrodes in MeOH solvent with 0.2 M (1-hydroxyethyl)ferrocene, 0.5 mM (1-hydroxyethyl)ferricenium, and 1.0 M LiClO
4
exhibit air mass 2 conversion efficiencies of 10.1% for optical energy into electricity. We observe open-circuit voltages of 0.53 V and short-circuit quantum efficiencies for electron flow of nearly unity. The fill factor of the cell does not decline significantly with increases in light intensity, indicating substantial reduction in efficiency losses in MeOH solvent compared to previous nonaqueous n-Si systems. Matte etch texturing of the Si surface decreases surface reflectivity and increases photocurrent by 50% compared to shiny, polished Si samples. The high values of the open-circuit voltage observed are consistent with the presence of a thin oxide layer, as in a Schottky metal-insulator-semiconductor device, which yields decreased surface recombination and increased values of open-circuit voltage and short-circuit current. The n-Si system was shown to provide sustained photocurrent at air mass 2 levels (20 mA/cm
2
) for charge through the interface of >2,000 C/cm
2
. The n-Si/MeOH system represents a liquid junction cell that has exceeded the 10% barrier for conversion of optical energy into electricity.
Purification of biologically active simian virus 40 small tumor antigen.
Authors: Bikel I , Roberts T M , Bladon M T , Green R , Amann E , Livingston D M
Published: May 2006

The simian virus 40 small tumor antigen (t antigen) gene has been cloned downstream from a hybrid Escherichia coli trp-lac promoter and a suitable ribosome binding site. A bacterial clone (865i) transformed by such a plasmid (pTR865) expresses this gene and, under optimal conditions, can produce greater than or equal to 5% of its total protein as t antigen. Soluble extracts of such a clone were relatively depleted in t antigen, which was found in the initial pellet fraction. The protein was recovered from this fraction in a significantly purified form by extraction with urea-containing buffer. After gel filtration of such t antigen-enriched solutions, highly purified protein was obtained. When either this fraction (freed of urea) or NaDodSO4 gel-purified 865i t antigen (rendered free of detergent) was injected into untransformed rat cells, dissolution of intracellular actin cable networks was observed.
Characterization of the chicken vimentin gene: Single copy gene producing multiple mRNAs
Authors: Zehner Zendra E. , Paterson Bruce M.
Published: May 2006

Genomic clones and cDNA plasmids were isolated for the intermediate filament protein vimentin from chicken. The identity of the various clones was determined both by mRNA selection [Paterson, B. M. & Roberts, B. E. (1981) in
Gene Amplification and Analysis, Structural Analysis or Nucleic Acids
, eds. Chirikjian, J. G. & Papas, T. S. (Elsevier, North Holland), Vol. 2, pp. 418-435] and nucleotide sequence analysis. Restriction analysis, hybridization data, and heteroduplex studies confirmed that all of the genomic isolates contained overlapping fragments of an identical vimentin gene. No evidence for the existence of a second vimentin gene could be found by a Southern analysis either by using coding fragments from the purified vimentin gene or by using cDNA plasmids as probe. Likewise, copy-number experiments verified that the vimentin gene was present only once in the haploid chicken genome. However, in a RNA blot analysis, at least two equally abundant vimentin mRNA species of approximately 2,200 and 2,500 nucleotides in length were detected in all RNAs tested. Sequence analysis revealed that the vimentin gene contained two sets of tandem polyadenylylation sites, 249 and 532 nucleotides downstream from the stop codon for protein synthesis. It is proposed that the larger mRNA species arise because of complete transcription of the 3′-end of the vimentin gene (560 nucleotides of 3′ nontranslated sequence), whereas the smaller mRNA species terminate after the first set of polyadenylylation sites.
In vitro
discrimination of replicases acting on carcinogen-modified polynucleotide templates
Authors: Singer B. , Kuśmierek J. T. , Fraenkel-Conrat H.
Published: May 2006

Three different poly(dC)s with modifications that block the N-3 of deoxycytidine were used as templates for polymer synthesis by
Escherichia coli
DNA polymerase I (EC 2.7.7.7). In contrast to previously reported results with transcriptases, the hydrated form of 3,
N
4
-ethenodeoxycytidine (εdC·H
2
O) did not mispair. Both 3,
N
4
-ethenodeoxycytidine (εdC) and 3-methyldeoxycytidine (m
3
dC) led to dTMP misincorporation: 1/20 εdC and 1/80 m
3
dC. No other misincorporations appeared to be significant in amount. Thus, both qualitatively and quantitatively, replication errors resulting from carcinogen-modified bases are less frequent than errors in transcription of the same deoxypolynucleotides. Replication of comparable ribopolynucleotide templates by cucumber RNA-dependent RNA polymerase (EC 2.7.7.48) was strongly inhibited by εrC·H
2
O and εrC, so that the fidelity of this enzyme could not be assessed. However, both poly(dC) and poly(rC) containing dU or rU led to incorporation of rA. The presence of even small amounts of purines in poly(rC) greatly depressed synthesis, but the complementary base was incorporated. The finding that an RNA replicase can utilize a deoxypolynucleotide template is a further indication that, at least
in vitro
, the specificity of the relationship of enzymes and their natural templates is not absolute.
Conversion of prohistidine decarboxylase to histidine decarboxylase: Peptide chain cleavage by nonhydrolytic serinolysis
Authors: Recsei Paul A. , Huynh Quang K. , Snell Esmond E.
Published: May 2006

Unlabeled prohistidine decarboxylase and prohistidine decarboxylase containing L-[
carboxyl
-
18
O]serine or L-[
hydroxyl
-
18
O]serine were isolated in homogeneous form from mutant 3 of
Lactobacillus
30a grown with the appropriately labeled serine. There was no randomization or redistribution of label during growth, isolation of the protein, or enzymatic hydrolysis and reisolation of the labeled amino acids. These proteins were used to show that during proenzyme activation, in which individual π subunits of the proenzyme are converted to α and β subunits of the active enzyme [Formula: see text] (in which π, α, and β subunits have the partial structures shown and Prv designates a pyruvoyl group), no
18
O from H
2
18
O is incorporated into the newly formed carboxyl terminus (Ser-81) of the β chain, although no labilization of
18
O from proenzyme labeled with L-[
carboxyl
-
18
O]serine occurred when the proenzyme was activated in H
2
16
O by the same procedures. The additional oxygen atom present in the carboxyl group of Ser-81 of the β subunit is transferred from the hydroxyl group of Ser-82 of the proenzyme during the activation reaction. The same result was obtained with wild-type enzyme formed intracellularly. Peptide bond cleavage during activation of the proenzyme thus proceeds by a hitherto unobserved direct or indirect “serinolysis” coupled to α,β-elimination at Ser-82 to yield the pyruvoyl group of the α subunit, rather than by hydrolysis. Possible mechanisms for the reaction are discussed briefly.
Antibodies specific for NADPH-binding region of enzymes possessing dehydrogenase activities.
Authors: Katiyar S S , Porter J W
Published: May 2006

The results reported in this paper show the presence of a population of antibodies in rabbit polyclonal antiserum that recognize an antigenic site at the NADPH-binding region of enzymes possessing dehydrogenase activities. Antisera from rabbits immunized with glucose-6-phosphate dehydrogenase or fatty acid synthetase were found to inactivate the enzyme dihydrofolate reductase. The inhibitory effect of this site-specific antibody is a time- and concentration-dependent reaction. This immunoinactivation is prevented by preincubation of the enzyme with NADPH.
Synthetic peptide with cell attachment activity of fibronectin.
Authors: Pierschbacher M , Hayman E G , Ruoslahti E
Published: May 2006

Four synthetic peptides that together constitute the cell attachment domain of fibronectin [Pierschbacher, M.D., Ruoslahti, E., Sundelin, J., Lind, P. & Peterson, P. (1982) J. Biol. Chem. 257, 9593-9597] were constructed and tested for their ability to induce cell attachment and spreading. One of these peptides, consisting of the 30 amino acid residues nearest the COOH terminus of the domain, contained all of the cell attachment activity of the whole domain. Under suitable conditions the peptide was approximately as active as intact fibronectin on a molar basis. The activity could be demonstrated by binding the peptide to polystyrene directly, or via albumin, or by coupling it to agarose beads. This synthetic peptide will be useful in the elucidation of the molecular details of the attachment of cells to fibronectin and could allow manipulation of the adhesive properties of cell culture surfaces and prosthetic materials.
alpha-Radiation dose at bronchial bifurcations of smokers from indoor exposure to radon progeny.
Authors: Martell E A
Published: May 2006

Synergistic interactions of indoor radon progeny with the cigarette smoking process have been evaluated experimentally. Smoking enhances the air concentration of submicron particles and attached radon decay products. Fractionation in burning cigarettes gives rise to the association of radon progeny with large particles in mainstream cigarette smoke, which are selectively deposited in "hot spots" at bronchial bifurcations. Because smoke tars are resistant to dissolution in lung fluid, attached radon progeny undergo substantial radioactive decay at bifurcations before clearance. Radon progeny inhaled during normal breathing between cigarettes make an even larger contribution to the alpha-radiation dose at bifurcations. Progressive chemical and radiation damage to the epithelium at bifurcations gives rise to prolonged retention of insoluble 210Pb-enriched smoke particles produced by tobacco trichome combustion. The high incidence of lung cancer in cigarette smokers is attributed to the cumulative alpha-radiation dose at bifurcations from indoor radon and thoron progeny--218Po, 214Po, 212Po, and 212Bi--plus that from 210Po in 210Pb-enriched smoke particles. It is estimated that a carcinogenic alpha-radiation dose of 80-100 rads (1 rad = 0.01 J/kg = 0.01 Gy) is delivered to approximately equal to 10(7) cells (approximately equal to 10(6) cells at individual bifurcations) of most smokers who die of lung cancer.
Sharing of biological effect and receptors between guinea pig insulin and platelet-derived growth factor.
Authors: King G L , Kahn C R , Heldin C H
Published: May 2006

Insulins from the hystricomorphs (guinea pig, porcupine, coypu, and casiragua) at high concentration stimulate DNA synthesis in human fibroblasts to a greater level than other mammalian insulins or insulin-like growth factors (IGFs). 125I-Labeled guinea pig insulin binds to a specific receptor and this binding is competed for by hystricomorph insulins but not by porcine insulin or IGFs. Fetal bovine serum also inhibits the binding of 125I-labeled guinea pig insulin and is more potent than fetal bovine plasma, in concordance with their relative potencies for growth stimulation in human fibroblasts. Of several other known growth factors tested, only platelet-derived growth factor (PDGF) inhibits binding of 125I-labeled guinea pig insulin. Four preparations of PDGF that vary in purity and potency for the stimulation of DNA synthesis in human fibroblasts over a 1,000-fold range compete with binding of 125I-labeled guinea pig insulin in proportion to their biological potencies. The purest preparation of PDGF is able to inhibit binding of 125I-labeled guinea pig insulin by 50% at 15 ng/ml (0.25 nM). Biologically, guinea pig insulin, like PDGF, exhibits a synergistic effect with plasma in initiating DNA synthesis in human fibroblasts; this effect is not observed with other mammalian insulins or IGFs. Thus, hystricomorph insulins appear to be mediating their growth-promoting effect through a different receptor and mechanism than other mammalian insulins or IGFs. Further, hystricomorph insulins may be sharing the mechanism of action for their growth effects with PDGF, perhaps suggesting some relationship between these peptides from very different sources.
Localization of urinary lactosylceramide in cytoplasmic vesicles of renal tubular cells in homozygous familial hypercholesterolemia.
Authors: Chatterjee S , Kwiterovich P O , Gupta P , Erozan Y S , Alving C R , Richards R L
Published: May 2006

An average 15-fold increase in lactosylceramide (LacCer) in the sediment of receptor-negative, familial hypercholesterolemic (FH) homozygotes has been reported [Chatterjee, S., Sekerke, C.S. & Kwiterovich, P.O., Jr. (1982) J. Lipid Res. 23, 513-522]. We report here the abnormal urinary excretion of significant numbers of renal tubular cells in eight FH homozygotes. The mean activity of gamma-glutamyltransferase, a marker for renal tubular cells, was twice as high in urinary sediment of FH homozygotes as in normals. Membrane-enclosed cytoplasmic vesicles that stained strongly positive with a fluorescein-labeled antibody against LacCer were found in the renal tubular cells of all homozygotes except two who had undergone a portacaval shunt. These two had normal urinary levels of LacCer, and the cytoplasmic vesicles were vacuolated. In the other six, most of the fluorescent antibody label was intracellular and perinuclear. The cytoplasmic vesicles stained strongly with polychromatic Papanicolaou stain, periodic acid/Schiff reagent, and oil red O. Electron microscopy revealed perinuclear membrane-enclosed lipid and free lipid droplets. When two FH homozygotes, who excreted increased LacCer, underwent plasma exchange, the cytoplasmic vesicles became empty, and the urinary LacCer level decreased into the normal range. We conclude that the increased urinary excretion of LacCer in FH homozygotes occurs in renal tubular cells and that the intracellular locatin of LacCer is within cytoplasmic vesicles. The presence of LacCer within these vesicles can be modulated by treatment with plasma exchange.
Membrane 126-kilodalton phosphoglycoprotein associated with human carcinomas identified by a hybridoma antibody to mammary carcinoma cells.
Authors: Soule H R , Linder E , Edgington T S
Published: May 2006

A large number of hybridomas producing antibodies to the membrane fraction of a human mammary carcinoma cell line have been produced. Among those hybridoma antibodies that bound to exposed surface membrane epitopes on the human mammary carcinoma cell line BT-20, one has been found to bind to all tested breast carcinoma cell lines as well as to a number of other human carcinomas by solid-phase radioimmunoassay. This hybridoma antibody, designated 10-3D2, does not bind to normal mammary epithelium, a variety of control cell lines, or selected normal human tissue homogenates. The 10-3D2 hybridoma antibody identifies by immunoprecipitation a membrane 126-kilodalton protein which is indistinguishable in different tumor cells. It is glycosylated by reference to incorporation of [3H]glucosamine and also is phosphorylated by reference to incorporation of 32P. It is present in a finely granular pattern on the cell surface of mammary carcinoma cells with concentration at the sites of apparent cell-cell contact. This 126-kilodalton phosphoglycoprotein, which we have consistently been unable to identify by immunoprecipitation of intrinsically labeled normal cells, may represent a heretofore unrecognized tumor-associated marker for human mammary carcinomas as well as certain other types of human neoplasms.
Recessive lethal deletion on mouse chromosome 7 affects glucocorticoid receptor binding activities.
Authors: Goldfeld A E , Firestone G L , Shaw P A , Gluecksohn-Waelsch S
Published: May 2006

The hormone binding activity of glucocorticoid receptors is decreased by approximately equal to 75% in the livers of mice homozygous for c14CoS, one of several overlapping radiation-induced deletions on chromosome 7. These deletions have been shown previously to map at and around the albino (c) locus and to act as recessive lethals. They are associated with intractable hypoglycemia, ultrastructural abnormalities in certain liver cell membranes, and deficiencies of specific liver proteins and enzymes that are induced by insulin or glucocorticoids, or both. Scatchard analysis of [3H]dexamethasone binding to receptors in control and mutant liver extracts revealed that the glucocorticoid binding constants were similar; however, the control extracts bound approximately equal to 4 times as much steroid as did mutant extracts. Analysis by DNA-cellulose column chromatography demonstrated that the ability of activated glucocorticoid receptors to form complexes with DNA appears to be unaltered in the mutants; both mutant and control cytosols contain glucocorticoid receptors that elute from DNA-cellulose columns at two different salt concentrations. Sucrose density gradient centrifugation revealed the glucocorticoid receptors in mutant livers to have approximately the same sedimentation coefficient as receptors in control livers. The decrease in glucocorticoid binding activity in mutant liver cell extracts is comparable in degree to that previously reported for insulin binding activity. The gene sequences deleted in the homozygous deletion mutants appear to be instrumental in the regulation rather than the structural determination of both glucocorticoid and insulin receptors.
Primed lymphocyte testing specificity of alloreactive lymphocyte clones for HLA-B locus determinants.
Authors: Zeevi A , Scheffel C , Annen K , Duquesnoy R J
Published: May 2006

We have generated two alloreactive T-lymphocyte clones that recognize the products of the HLA-B locus. The primed lymphocyte testing (PLT) reactivity of clone HJ21 was monospecific for the HLA-B antigen Bw49, a subtype specificity of HLA-Bw21. The PLT specificity of clone RD3 was strongly associated with HLA-Bw21. Several monoclonal antibodies against class I HLA molecules inhibited the PLT responses of these clones whereas class II monoclonal antibodies had no effect. These observations provide experimental evidence that class I products of the HLA-B locus may directly be involved in lymphocyte alloactivation.
Evidence for long-lived mRNA during fruiting body formation in myxococcus xanthus.
Authors: Nelson D R , Zusman D R
Published: May 2006

Half-lives of Myxococcus xanthus mRNA were determined by inhibiting RNA polymerase with rifampin and then measuring the rate of [35S]methionine incorporation into protein. Vegetative cells resuspended in clone fruiting liquid culture showed an average mRNA half-life of approximately 3.5 min. Developmental cells (24 or 48 hr) exhibited biphasic decay curves with apparent mRNA half-lives of approximately 3.5 min and 20-30 min. The more-stable mRNA species accounted for about 30% of all mRNA present in 24-hr cells and as much as 40% of all mRNA of 48-hr cells. Analysis of the 35S-labeled proteins by NaDodSO4/polyacrylamide gel electrophoresis showed that only 5-10 major polypeptides are synthesized after 30 min of rifampin treatment. One of these is protein S, the spore surface coat protein, because immunoprecipitation of the 35S-labeled proteins with antisera specific for protein S showed continued synthesis of protein S in the presence of rifampin. The half-life for its mRNA was calculated to be 15-30 min. RNA from vegetative and developing cells was pulse labeled with 32PO4 followed by rifampin treatment. Analysis of the labeled RNA on 3% NaDodSO4/polyacrylamide gels showed 5-10 long-lived mRNA bands in developing cells. These results show that there are several abundant mRNA species synthesized developmentally that are exceptionally long lived. The fact that the majority of the mRNA species show the shorter half-life suggests that developing cells retain the normal mechanism for mRNA degradation.
Enzymic modification of a tyrosine residue to a stable free radical in ribonucleotide reductase.
Authors: Barlow T , Eliasson R , Platz A , Reichard P , Sjöberg B M
Published: May 2006

Protein B2, a subunit of ribonucleotide reductase from Escherichia coli, contains in its active form a tyrosyl free radical as part of the polypeptide chain and a dimeric iron center that stabilizes the radical. The enzyme depends on this radical for its catalytic activity. Treatment with hydroxyurea scavenges the radical without disturbing the iron center and, thereby, results in an inactive form of the subunit, B2/HU. A second inactive form, apoB2, lacking both the radical and the iron center, is obtained by treatment of B2 with 8-hydroxyquinoline. Here we describe an enzyme activity in extracts from E. coli that transforms the catalytically inactive B2/HU form into the active B2 subunit by regeneration of the tyrosyl radical. This reaction requires the presence of oxygen, dithiothreitol, and Mg2+ and does not proceed through apoB2. Under anaerobic conditions, we obtained evidence for a second activity in the bacterial extract that destroys the free radical and transforms B2 into B2/HU. We suggest that this novel type of protein modification is functionally related to the synthesis of deoxyribonucleotides and DNA.
In vitro bypass of UV-induced lesions by Escherichia coli DNA polymerase I: specificity of nucleotide incorporation.
Authors: Rabkin S D , Moore P D , Strauss B S
Published: May 2006

A variety of DNA polymerases, synthesizing in vitro on an UV-irradiated phi X174 DNA template, terminate synthesis one nucleotide before the 3' pyrimidines of putative dimers on the template. We have devised a system using Escherichia coli DNA polymerase I (Klenow fragment) that can synthesize past at least some of these dimers. The bypass is carried out in a multistep process--first, the incorporation of nucleotides opposite the pyrimidines in the dimer and, then, the addition of nucleotides complementary to the bases distal to the dimer. The insertion of a nucleotide opposite the first (3') pyrimidine of a putative dimer in the presence of Mn2+ occurs in a concentration-dependent fashion with a 3- to 4-fold preference for purine nucleotides over pyrimidine nucleotides. In the presence of Mg2+, insertion is less frequent. Correlation of these results with in vivo mutation data suggests a role for the polymerase in determining the spectrum of base substitution mutagenesis in SOS induced cells.
Biosynthesis of tetrahydrobiopterin by de novo and salvage pathways in adrenal medulla extracts, mammalian cell cultures, and rat brain in vivo.
Authors: Nichol C A , Lee C L , Edelstein M P , Chao J Y , Duch D S
Published: May 2006

Mammalian cells and tissues were found to have two pathways for the biosynthesis of tetrahydrobiopterin (BH4): (i) the conversion of GTP to BH4 by a methotrexate-insensitive de novo pathway, and (ii) the conversion of sepiapterin to BH4 by a pterin salvage pathway dependent on dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity. In a Chinese hamster ovary cell mutant lacking dihydrofolate reductase (DUKX-B11), endogenous formation of BH4 proceeds normally but, unlike the parent cells, these cells or extracts of them do not convert sepiapterin or 7,8-dihydrobiopterin to BH4. KB cells, which do not contain detectable levels of GTP cyclohydrolase or BH4 but do contain dihydrofolate reductase, readily convert sepiapterin to BH4 and this conversion is completely prevented by methotrexate. In supernatant fractions of bovine adrenal medulla, the conversion of sepiapterin to BH4 is completely inhibited by methotrexate. Similarly, this conversion in rat brain in vivo is methotrexate-sensitive. Sepiapterin and 7,8-dihydrobiopterin apparently do not enter the de novo pathway of BH4 biosynthesis and may be derived from labile intermediates which have not yet been characterized.
Tumor promoter 12-O-tetradecanoylphorbol 13-acetate, like epidermal growth factor, stimulates cell proliferation and inhibits differentiation of mouse mammary epithelial cells in culture.
Authors: Taketani Y , Oka T
Published: May 2006

12-O-Tetradecanoylphorbol 13-acetate (TPA) is a potent tumor promoter and shares several biological activities of epidermal growth factor (EGF). Recently we have shown that EGF stimulates DNA synthesis and inhibits milk protein synthesis induced by insulin, cortisol, and prolactin in a primary mouse mammary epithelial cell culture [Taketani, Y. & Oka, T. (1983) FEBS Lett., in press]. Using this system, we examined the biological action of TPA in reference to that of EGF. TPA stimulated cell proliferation and inhibited the synthesis of milk proteins casein and alpha-lactalbumin during a 5-day culture. The ED50 was about 2.5 ng/ml for DNA synthesis and 0.1 ng/ml for milk protein synthesis. Removal of TPA from the medium normalized DNA synthesis completely and casein synthesis partially. The ability of various TPA analogs to stimulate DNA synthesis and inhibit casein synthesis correlated with their potency as tumor promoters. Both TPA and EGF decreased the specific prolactin binding of cultured cells by approximately equal to 50%. These results indicate that TPA, like EGF, switches the developmental course of mammary epithelium from differentiation to proliferation.
YAC-1 variant clones selected for resistance to natural killer cytotoxic factors are also resistant to natural killer cell-mediated cytotoxicity.
Authors: Wright S C , Bonavida B
Published: May 2006

The possible involvement of natural killer cytotoxic factors (NKCF) in the lytic mechanism of natural killer (NK) cell-mediated cytotoxicity (CMC) was investigated by studying the mechanism of NK resistance of variant clones of the YAC-1 cell line. The NK-resistant YAC-1 (YAC-R) clones were generated by prolonged culture of the initially NK-sensitive YAC-1 cell line in the presence of NKCF. The YAC-R clones were resistant to lysis by NKCF as well as lysis by NK cells in a CMC assay. The defect was specific for NK CMC because the YAC-R clones could still be lysed by alloimmune cytotoxic T lymphocytes. Experiments to determine the mechanism of NK resistance of the YAC-R clones indicated that they still possessed the NK recognition structures because they formed a normal number of conjugates with murine spleen cells. In addition, the YAC-R clones, like the parental YAC-1 cell line, were able to stimulate the release of NKCF during co-culture with spleen cells. However, the YAC-R clones, in contrast to YAC-1 cells, were unable to adsorb NKCF from cell-free supernatants of such cultures. Thus, it appears that the YAC-R clones are NK resistant due to a deficiency of NKCF binding sites. The present studies demonstrate that a NK-sensitive target cell must not only be recognized by the NK cell and stimulate release of NKCF but it must also bind NKCF for cell lysis to ultimately result. These findings support our model for the mechanism of NK CMC in which it is proposed that target cell lysis is mediated by NKCF released from the effector cell.
Laws of composition of Bäcklund transformations and the universal form of completely integrable systems in dimensions two and three
Authors: Chudnovsky D. V. , Chudnovsky G. V.
Published: May 2006

Bäcklund transformations are defined as operations on solutions of a Riemann boundary value problem (vector bundles over P
1
) that add apparent singularities. For solutions of difference and differential linear spectral problems, Bäcklund transformations are presented in explicit form through the Christoffel formula and its generalizations. Identities satisfied by iterations of elementary Bäcklund transformations are represented in the form of the law of addition or as the three-dimensional difference equation of Hirota's type. Matrix two-dimensional isospectral deformation equations are imbedded into three-dimensional scalar systems of Kadomtzev-Petviashvili (law of addition) form. Two-dimensional matrix systems correspond to reductions of Kadomtzev-Petviashvili equations with pseudodifferential operators satisfying algebraic equations.
The 3 A resolution structure of a D-galactose-binding protein for transport and chemotaxis in Escherichia coli.
Authors: Vyas N K , Vyas M N , Quiocho F A
Published: May 2006

X-ray diffraction studies of a D-galactose-binding protein essential for transport and chemotaxis in Escherichia coli have yielded a model of the polypeptide chain backbone. An initial polyalanine backbone trace was obtained at 3.2 A resolution by the molecular replacement technique, using a polyalanine search model derived from the refined structure of the L-arabinose-binding protein. Concurrently, a 3 A resolution electron-density map of the D-galactose receptor was determined from multiple isomorphous replacement (MIR) phases. The properly transformed initial polyalanine model superimposed on the MIR electron-density map proved to be an excellent guide in obtaining a final trace. The few changes made in the polyalanine model to improve the fit to the density were confined primarily to the COOH-terminal peptide and some loops connecting the elements of the secondary structure. Despite the lack of significant sequence homology, the overall course of the polypeptide backbone of the D-galactose-binding protein is remarkably similar to that of the L-arabinose-binding protein, the first structure in a series to be solved from this family of binding proteins. Both structures are elongated (axial ratios of 2:1) and composed of two globular domains. For both proteins, the arrangements of the elements of the secondary structure in both domains are identical; both lobes contain a core of beta-pleated sheet with a pair of helices on either side of the plane of the sheet. The four major hydrophobic clusters that stabilize the structure of the L-arabinose-binding protein are also present in the D-galactose-binding protein.
beta-Hydroxyaspartic acid in vitamin K-dependent protein C.
Authors: Drakenberg T , Fernlund P , Roepstorff P , Stenflo J
Published: May 2006

Previous work has shown that the light chain of protein C, an anticoagulant plasma protein, contains an unusual amino acid [Fernlund, P. & Stenflo, J. (1982) J. Biol. Chem. 257, 12170-12179]. To determine the structure of this amino acid a heptapeptide, CMCys-Ile-X-Gly-Leu-Gly-Gly (residues 69-75 in the light chain), was isolated from enzymatic digests of the light chain. According to automatic Edman sequence analysis, 1H NMR spectroscopy, and mass spectrometry the heptapeptide had beta-hydroxyaspartic acid in its third position, which corresponds to position 71 in the light chain of protein C. Analysis of acid and aminopeptidase M hydrolysates of the heptapeptide showed the beta-hydroxyaspartic acid to be the erythro form. Acid hydrolysis of protein C released approximately equal to 1 mol of beta-hydroxyaspartic acid per mol of protein. The function of this amino acid, which, to the best of our knowledge, has not been found previously in proteins, is unknown.
Reversible interconversion between sulfo and desulfo xanthine oxidase in a system containing rhodanese, thiosulfate, and sulfhydryl reagent.
Authors: Nishino T , Usami C , Tsushima K
Published: May 2006

The desulfo form of milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) was reactivated by incubation with rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), thiosulfate, and sulfhydryl reagent; 50% of full activity was recovered. No further reactivation occurred with additional incubation. It was also found that native enzyme in the sulfo form with full activity was inactivated by incubation with the same system, down to half of full activity and no further inactivation occurred. After these incubations the enzyme was found to be a mixture of functional and nonfunctional enzymes based on spectral changes with xanthine, on [14C]oxipurinol equilibration, and on steady-state kinetics. The 35S of [35S]thiosulfate was incorporated into desulfo xanthine oxidase in parallel with an increase in catalytic activity. Most of the 35S was cyanolysable but was protected from cyanolysis by pretreatment with allopurinol. The 35S was released from 35S-labeled reconstituted xanthine oxidase upon incubation with the rhodanese system containing unlabeled thiosulfate. However, catalytic activity remained unchanged, indicating that the sulfur atom was exchanged during the incubation.
Isolation and sequence of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase from the cyanobacterium
Anabaena
7120
Authors: Curtis Stephanie E. , Haselkorn Robert
Published: May 2006

Cloned DNA probes containing genes coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (
rbc
A) of corn and of
Chlamydomonas
were used to identify, by heterologous hybridization, DNA fragments from
Anabaena
7120 carrying the corresponding gene sequence. The same probes were used to isolate, from a recombinant λ library, a 17-kilobase-pair
Eco
RI
Anabaena
DNA fragment containing the coding sequence for the
rbc
A gene. The entire coding sequence, as well as 210 base pairs of 5′ flanking region and 210 base pairs of 3′ flanking region, was determined. Comparison of the nucleotide and amino acid sequences with those of corn, spinach,
Chlamydomonas
, and
Synechococcus rbc
A genes revealed homology of 71-77% at the nucleotide level and 80-85% at the amino acid level. Conservation of sequence is lost immediately outside the coding region on either side. Codon usage in the
Anabaena rbc
A gene is not significantly different from that in the
Anabaena
genes for nitrogenase reductase and nitrogenase β subunit.
Comparison of the cloned H-2Kbm1 variant gene with the H-2Kb gene shows a cluster of seven nucleotide differences.
Authors: Schulze D H , Pease L R , Geier S S , Reyes A A , Sarmiento L A , Wallace R B , Nathenson S G
Published: May 2006

The gene for the H-2K class I antigen of the bm1 variant was cloned and analyzed at the DNA level and compared with the previously cloned parent B6/Kh gene. Sequence determination and comparative restriction endonuclease studies indicate that Kbm1 is derived from the Kb gene. Seven nucleotide changes within a 13-nucleotide stretch distinguish the mutant from the parent gene and result in amino acid differences at positions 152, 155, and 156 in the antigen. The data confirm previously reported changes at amino acid positions 155 and 156 (arginine to tyrosine and leucine to tyrosine, respectively) and extend the altered region to include two nucleotides encoding a glutamate to alanine substitution at amino acid 152, a change not detected by the protein studies because of limitations of the methods used. The DNA sequence encoding this region of the Kbm1 glycoprotein is identical to the DNA sequence of at least one other known class I gene in the mouse, a finding consistent with the hypothesis that the mutation was not a random event but may be the result of a block transfer of information by a copy mechanism analogous to gene conversion. As the sequence analysis of the coding region for the first 273 amino acid residues shows identity between parent and mutant except for the seven nucleotide changes, all variant-parent functional differences must depend only on the cluster of three amino acid differences in the second domain of the Kb glycoprotein.
Dopamine stimulation of phosphatidylcholine (lecithin) biosynthesis in rat brain neurons.
Authors: Leprohon C E , Blusztajn J K , Wurtman R J
Published: May 2006

Rat brain synaptosomes contain enzymes, phosphatidylethanolamine N-methyltransferase(s) (EC 2.1.1.17), that catalyze the methylation of endogenous phosphatidylethanolamine to form its mono-, di-, and trimethyl (i.e., phosphatidylcholine) derivatives. We observe that the activity of these enzymes is enhanced when synaptosomes are incubated with catecholamines: 0.1 mM dopamine increases incorporation of [3H]methyl groups into monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, and phosphatidylcholine by factors of 1.7, 1.3, and 2.1, respectively, and 0.1 mM norepinephrine increases [3H]methyl incorporation into monomethylphosphatidylethanolamine and dimethylphosphatidylethanolamine by factors of 1.6 and 2.1, respectively. Stimulation by dopamine, which is observed at concentrations as low as 1 microM, is blocked by haloperidol.
Acute transsynaptic regulation of tyrosine 3-monooxygenase activity in the rat superior cervical ganglion: evidence for both cholinergic and noncholinergic mechanisms.
Authors: Ip N Y , Perlman R L , Zigmond R E
Published: May 2006

The rate of dopa synthesis in the rat superior cervical ganglion was increased 4- to 6-fold during continuous electrical stimulation of the cervical sympathetic trunk at 10 Hz for 30 min. This increase was only partially blocked by 3 mM hexamethonium and was not significantly affected by 6 microM atropine. In the presence of both hexamethonium and atropine, nerve stimulation still produced a 2- to 4-fold increase in dopa synthesis. Physostigmine increased dopa synthesis in both control and stimulated ganglia. This effect of physostigmine was completely blocked by hexamethonium and atropine. Dopa synthesis was also significantly increased when ganglia were incubated in a medium containing an elevated concentration of K+ (55 mM). This stimulatory effect of high K+ was totally dependent on the presence of Ca2+ in the medium, was decreased by 60% by prior decentralization of the ganglion, and was unaffected by hexamethonium and atropine. The data demonstrate that tyrosine hydroxylase activity is rapidly increased after preganglionic nerve stimulation and suggest that this increase is mediated in part by acetylcholine and in part by a second (noncholinergic) transmitter. The effects of an elevated K+ concentration may be mediated both by the release of a noncholinergic transmitter from the preganglionic nerve terminals and by direct depolarization of the ganglionic neurons.
Flow dichroism evidence for tilting of the bases when DNA is in solution.
Authors: Dougherty A M , Causley G C , Johnson W C
Published: May 2006

Flow linear dichroism studies of DNAs from Clostridium perfringens, Escherichia coli, and Micrococcus luteus are extended into the vacuum UV region to 175 nm. The short-wavelength pi pi region shows a less negative reduced linear dichroism than the well-studied long-wavelength pi pi region. These results are independent of the type of DNA and the concentration of salt over the range 0.001 to 1.0 M. This proves that the bases are not perpendicular to the flow orientation. Assuming that the DNA tends to align along its helix axis, the largest angle that the bases can make with the helix axis is 73 +/- 3 degrees.
Voltage-gated Ca2+ channel in mouse myeloma cells.
Authors: Fukushima Y , Hagiwara S
Published: May 2006

Electrical properties of the cell membrane were studied in the neoplastic lymphocyte, mouse myeloma cell line S194, by using the whole-cell patch clamp technique. Inward Ca2+ currents due to voltage-gated Ca2+ channels were found. The current, which decayed exponentially after reaching a peak, was first activated at about -50 mV and attained its maximum peak amplitude at about -20 mV in a 10 mM Ca2+ solution. Outward current was negligible for the potential range more negative than +30 mV. The channel was permeable to Sr2+ and Ba2+ in addition to Ca2+. Among these species, Sr2+ carried the greatest current. The time constants of the decay of the current depended neither on the species nor on the concentration of charge carrier. The steady-state inactivation was observed at potentials more negative than those at which the inward Ca2+ current was activated. Thus, we concluded that the inactivation of the channel was mainly voltage dependent. For reasons that are not yet understood, the amplitude of the Ca2+ current varied greatly among cells.
Erythroleukemia cells: variants inducible for hemoglobin synthesis without commitment to terminal cell division.
Authors: Marks P A , Chen Z , Banks J , Rifkind R A
Published: May 2006

Murine erythroleukemia cells (MELC) are virus-transformed erythroid precursors that appear to be blocked at an erythroid precursor stage comparable to the erythroid colony-forming unit (CFU-e). These cells are useful in examining factors regulating terminal differentiation. Induced MELC are characterized by a coordinated program of gene expression, including commitment to terminal cell division, accumulation of globin mRNAs and corresponding hemoglobins, and accumulation of several other proteins, including the chromatin-associated protein H1(0). Two cloned variant cell lines, DR10 and R1, have been developed from inducer-sensitive DS19 cells by selection for inducer resistance. DR10 and R1 cells fail to display commitment to terminal cell division when cultured with dimethyl sulfoxide (Me2SO), hexamethylene bisacetamide (HMBA), or butyric acid. Both cell lines are induced by all three agents to accumulate H1(0). DR10 cells are resistant to Me2SO-mediated accumulation of hemoglobin but are sensitive to HMBA- or butyric acid-mediated accumulation. R1 cells are resistant to Me2SO- and HMBA-mediated accumulation of hemoglobin but are sensitive to butyric acid-mediated accumulation. Both DR10 and R1 are commitment-negative MELC variants, displaying variable responses to inducers with respect to other features of terminal erythroid cell differentiation.
Isotype regulation of antibody production: T-cell hybrids can be selectively induced to produce IgG1 and IgG2 subclass-specific suppressive immunoglobulin-binding factors.
Authors: Löwy I , Brezin C , Neauport-Sautes C , Theze J , Fridman W H
Published: May 2006

T2D4, a T-cell hybrid, spontaneously secretes suppressive immunoglobulin factor(s); when incubated with purified monoclonal mouse immunoglobulins, this hybrid produces high levels of immunoglobulin-binding factors specific for the subclass of the inducing immunoglobulin. Thus, we were able to induce the production of IgG1- or IgG2-specific inhibitory factors by the same T2D4 T-cell hybrid. These subclass-specific suppressive factors bind selectively to the IgG1 or IgG2 subclasses and inhibit specifically the secretion of antibodies of the corresponding subclass. Our results favor a model of negative regulation of isotype expression in which a given isotype triggers suppressor mechanism(s) specifically inhibiting its production.
Linkage of beta 2-microglobulin and ly-m11 by molecular cloning and DNA-mediated gene transfer.
Authors: Margulies D H , Parnes J R , Johnson N A , Seidman J G
Published: May 2006

beta 2-Microglobulin (beta 2m) is expressed on the cell surface after introduction of a beta 2mb (C57BL/6N) genomic clone into thymidine kinase-deficient mouse L cells by cotransformation using the calcium phosphate precipitate method. Stable transformant cell lines were identified that express the beta 2mb allele, as determined by reaction of the cells with appropriate monoclonal antibodies and by two-dimensional gel electrophoresis of endogenously labeled immunoprecipitates of cell extracts. These beta 2mb transformants now express ly-m11.2, as detected by an indirect radioimmunoassay. A plasmid subclone of the beta 2mb gene that contains an 8.4-kilobase insert, after introduction into mouse L cells, similarly directs the synthesis of both the beta 2mb and the ly-m11.2 antigens. Thus, the beta 2mb and ly-m11.2 determinants most likely represent sites on the same protein structure.