bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+- and phospholipid-independent activation of protein kinase C by selective oxidative modification of the regulatory domain.
Authors: Gopalakrishna R , Anderson W B
Published: May 2006

The susceptibility of purified protein kinase C to oxidative inactivation by H2O2 was found to be increased by Ca2+ either alone at a high (5 mM) concentration or at a low (approximately 50 microM) concentration along with phosphatidylserine and diacylglycerol and by tumor-promoting phorbol esters even in the absence of Ca2+. This suggested that the membrane-bound and/or catalytically active form of protein kinase C is relatively more susceptible to oxidative inactivation. Although both the regulatory and catalytic domains of protein kinase C were susceptible to oxidative inactivation, a selective modification of the regulatory domain was obtained under mild oxidative conditions by protecting the catalytic site with ATP/Mg2+. Under these conditions there was a loss of both phorbol ester binding and Ca2+/phospholipid-stimulated kinase activity. However, this modified form of enzyme exhibited an increase in Ca2+/phospholipid-independent kinase activity. This suggests that selective oxidative modification of the regulatory domain may negate the requirement for Ca2+ and lipids for activation. Treatment of intact C6 glioma or B16 melanoma cells with H2O2 resulted in a time- and temperature-dependent decrease in Ca2+/phospholipid-dependent protein kinase C activity along with a concomitant transient increase in an oxidatively modified isoform of protein kinase C that exhibited activity in the absence of Ca2+ and phospholipids. Since protein kinase C can initially be activated by mild oxidative modification and subsequently inactivated by further oxidation, this dual activation-inactivation of protein kinase C in response to H2O2 suggests an effective on/off signal mechanism to influence cellular events.
Conservation of the human telomere sequence (TTAGGG)n among vertebrates.
Authors: Meyne J , Ratliff R L , Moyzis R K
Published: May 2006

To determine the evolutionary origin of the human telomere sequence (TTAGGG)n, biotinylated oligodeoxynucleotides of this sequence were hybridized to metaphase spreads from 91 different species, including representative orders of bony fish, reptiles, amphibians, birds, and mammals. Under stringent hybridization conditions, fluorescent signals were detected at the telomeres of all chromosomes, in all 91 species. The conservation of the (TTAGGG)n sequence and its telomeric location, in species thought to share a common ancestor over 400 million years ago, strongly suggest that this sequence is the functional vertebrate telomere.
Targeted correction of a major histocompatibility class II E alpha gene by DNA microinjected into mouse eggs.
Authors: Brinster R L , Braun R E , Lo D , Avarbock M R , Oram F , Palmiter R D
Published: May 2006

DNA molecules containing the 5' end of a functional major histocompatibility class II E alpha gene were injected into mouse eggs bearing E alpha genes with 630-base-pair (bp) deletions encompassing the promoter and first exon. The deletion was corrected by homologous recombination in 1 of about 500 transgenic mice that incorporated the injected DNA. The corrected E alpha gene was transmitted to progeny, which were bred to homozygosity. Southern blot analysis, polymerase chain reaction amplification of the DNA spanning the deletion, and sequence analysis revealed that the corrected allele resembles the wild-type E alpha gene. At sites of single-base-pair polymorphisms, there was apparently random conversion to either the donor or recipient sequence. In addition, many point mutations were introduced. mRNAs were produced from the corrected allele in a tissue-specific manner, but their sizes were different from the wild-type allele, and they did not produce detectable E alpha protein. This experiment demonstrates the feasibility of targeting foreign DNA to a gene that is completely inactive in fertilized mouse eggs.
Labeling monoclonal antibodies and F(ab')2 fragments with the alpha-particle-emitting nuclide astatine-211: preservation of immunoreactivity and in vivo localizing capacity.
Authors: Zalutsky M R , Garg P K , Friedman H S , Bigner D D
Published: May 2006

alpha-Particles such as those emitted by 211At may be advantageous for radioimmunotherapy since they are radiation of high linear energy transfer, depositing high energy over a short distance. Here we describe a strategy for labeling monoclonal antibodies and F(ab')2 fragments with 211At by means of the bifunctional reagent N-succinimidyl 3-(trimethylstannyl)benzoate. An intact antibody, 81C6, and the F(ab')2 fragment of Me1-14 (both reactive with human gliomas) were labeled with 211At in high yield and with a specific activity of up to 4 mCi/mg in a time frame compatible with the 7.2-hr half-life of 211At. Quantitative in vivo binding assays demonstrated that radioastatination was accomplished with maintenance of high specific binding and affinity. Comparison of the biodistribution of 211At-labeled Me1-14 F(ab')2 to that of a nonspecific antibody fragment labeled with 211At and 131I in athymic mice bearing D-54 MG human glioma xenografts demonstrated selective and specific targeting of 211At-labeled antibody in this human tumor model.
Functional expression of the amiloride-sensitive sodium channel in Xenopus oocytes.
Authors: George A L , Staub O , Geering K , Rossier B C , Kleyman T R , Kraehenbuhl J P
Published: May 2006

Expression of the amiloride-sensitive sodium channel was examined in Xenopus oocytes that were microinjected with A6 cell mRNA. Amiloride-inhibitable 22Na flux could be measured in intact oocytes 2-3 days after injection with 25 ng of poly(A)+ RNA isolated from aldosterone-treated A6 cells. The rate of 22Na uptake was approximately 15-fold greater in oocytes microinjected with 25 ng of poly(A)+ RNA than in water-injected control oocytes. An increase in 22Na uptake by mRNA-injected oocytes occurred whether the mRNA was isolated from A6 cells grown on a porous or nonporous support. In the presence of 4 mM NaCl, amiloride caused dose-dependent inhibition of 22Na uptake in mRNA-injected oocytes, which was half-maximal at 6 x 10(-8) M. Both 1 microM amiloride and 0.1 microM benzamil inhibited 22Na uptake in mRNA-injected oocytes by greater than 95%, whereas less than 50% inhibition occurred with 1 microM 5-(N-ethyl-N-isopropyl)amiloride. When A6 cell mRNA was size fractionated by sucrose density-gradient centrifugation, amiloride-sensitive 22Na uptake was expressed predominantly by oocytes injected with mRNA from two contiguous fractions.
Maize nuclear background regulates the synthesis of a 22-kDa polypeptide in
Zea luxurians
mitochondria
Authors: Cooper Pam , Newton Kathleen J.
Published: May 2006

When cytoplasm of the teosinte
Zea luxurians
is introduced into certain maize inbred nuclear backgrounds, the pattern of protein synthesis in the teosinte mitochondria is altered. Teosinte mitochondria purified from plants possessing a maize A619 nuclear background (
Z.l.
—A619 plants) synthesize a novel 22-kDa polypeptide that is associated exclusively with the membrane fraction of the organelle. Mitochondria from plants possessing a W23 nuclear background do not synthesize this protein. The F
1
hybrids
Z.l.
—A619 × W23 and
Z.l.
—W23 × A619 do not synthesize the protein. However, synthesis of the polypeptide was observed in 14 of 21 individual progeny from the backcross of the F
1
hybrid
Z.l.
—A619 × W23 to the pollen parent A619. These data suggest that a single nuclear gene controls the synthesis of the 22-kDa protein in mitochondria, with the recessive allele of the gene allowing expression of the polypeptide. Mitochondria from the F
1
hybrid
Z.l.
—A619 × Mo17 synthesize the 22-kDa protein, whereas mitochondria from
Z.l.
—A619 × B73 do not. Data from these outcrosses demonstrate that other maize lines also possess nuclear genes capable of regulating the synthesis of the 22-kDa
Zea luxurians
mitochondrial protein.
Cell-specific induction of mouse Cyp1a1 mRNA during development.
Authors: Dey A , Westphal H , Nebert D W
Published: May 2006

The dioxin-inducible cytochrome P(1)450 (Cyp1a1 gene) and P(3)450 (Cyp1a2 gene) enzymes have been implicated in the metabolism of numerous polycyclic hydrocarbons and arylamines, respectively. The prototypic inducer 3-methylcholanthrene was given to the pregnant mouse, and the intrauterine response was examined with the use of in situ hybridization. During the early postimplantation stage, inducible Cyp1a1 mRNA is detected in specific cell types in the extraembryonic tissues only. This selective expression along with the lack of detectable constitutive Cyp1a1 and constitutive or inducible Cyp1a2 gene transcripts between 5.3 and 14.5 days of gestation suggest that (i) these two genes appear to play no endogenous role during differentiation and (ii) the metabolic activity of the inducible Cyp1a1 enzyme may be important to the embryo and fetus from the standpoint of protection against toxic foodstuff and other environmental chemicals.
Low-affinity IgE receptor (CD23) function on mouse B cells: role in IgE-dependent antigen focusing.
Authors: Kehry M R , Yamashita L C
Published: May 2006

B-cell surface immunoglobulin very efficiently focuses specific protein antigens for presentation to T cells. We have demonstrated a similar role in antigen focusing for the low-affinity Fc epsilon receptor (Fc epsilon RII) on mouse B lymphocytes. B cells treated with an IgE monoclonal antibody to 2,4,6-trinitrophenyl (TNP) (IgE-B cells) were 100-fold more effective than were untreated B cells in presenting low concentrations of TNP-antigen to T cells. Blocking the binding of IgE to Fc epsilon RII on IgE-B cells with a monoclonal antibody to Fc epsilon RII completely eliminated this increased effectiveness. Preformed complexes of IgE anti-TNP and TNP-antigen were more effectively presented (approximately 100-fold) than TNP-antigen in the presence of nonspecific IgE. In contrast, complexes of IgG1 anti-TNP and TNP-antigen, capable of binding to Fc gamma receptors on B cells, were presented less effectively than TNP-antigen in the presence of nonspecific IgG1. Antigens focused by means of Fc epsilon RII or by means of B-cell surface immunoglobulin receptors were presented at comparably low concentrations. For several reasons, Fc epsilon RII on B lymphocytes seems to be particularly effective in efficiently focusing IgE-antigen complexes.
Distribution of D2 dopamine receptor mRNA in rat brain.
Authors: Meador-Woodruff J H , Mansour A , Bunzow J R , Van Tol H H , Watson S J , Civelli O
Published: May 2006

The distribution of mRNA coding for the D2 dopamine receptor was studied in the rat brain by in situ hybridization. A cDNA probe corresponding to the putative third cytosolic loop and sixth and seventh transmembrane domains of the rat D2 receptor was used to generate an 35S-labeled riboprobe to hybridize to D2 receptor mRNA. D2 mRNA was found both in dopamine projection fields and in regions associated with dopamine-containing cell bodies, suggesting both postsynaptic and presynaptic autoreceptor localization. Highest concentrations of D2 mRNA were found in neostriatum, olfactory tubercle, substantia nigra, ventral tegmental area, and the nucleus accumbens. This distribution is consistent with those reported with D2 receptor autoradiography.
Elevated levels of an exocrine pancreatic secretory protein in Alzheimer disease brain.
Authors: Ozturk M , de la Monte S M , Gross J , Wands J R
Published: May 2006

We identified by a monoclonal antibody-based immunoradiometric assay high concentrations of an exocrine pancreatic protein called pancreatic thread protein (PTP) in several areas of Alzheimer disease (AD) brain. The saline-extractable soluble immunoreactivity shares at least three epitopes in common with the native pancreatic form of the protein; the Mr varies from approximately 17,000 to 20,000. Quantitative measurements of PTP immunoreactivity in various regions of several AD brains revealed levels varying from 12 to 295 ng/g of tissue (mean, 116 ng/g) compared with 1-11 ng/g of tissue (mean, 5 ng/g) found in comparable areas of control brains. Immunocytochemistry performed with the anti-PTP monoclonal antibodies demonstrate PTP immunoreactivity within large pyramidal neurons--many of which contain neurofibrillary tangles in both AD and Down syndrome. Less accumulation was observed in astrocytes, and some PTP immunoreactivity was found extracellularly. The highest number of labeled cells in AD and Down syndrome was seen in the hippocampal formation. Fewer positive-staining cells were noted in normal and disease control brains. We conclude, therefore, that an exocrine pancreatic protein is present in the central nervous system of normal individuals at low levels; in AD brain concentrations of this protein are much higher.
"Frizzy" aggregation genes of the gliding bacterium Myxococcus xanthus show sequence similarities to the chemotaxis genes of enteric bacteria.
Authors: McBride M J , Weinberg R A , Zusman D R
Published: May 2006

The frz genes of Myxococcus xanthus are necessary for proper aggregation of cells to form fruiting bodies. Mutations in the frz genes affect the frequency with which individual cells reverse their direction of movement. We have subcloned and determined the nucleotide sequence of three of the frz genes. From the sequence we predict three open reading frames corresponding to frzA, frzB, and frzCD. The putative FrzA protein (17,094 Da) exhibits 28.1% amino acid identity with the CheW protein of Salmonella typhimurium. The putative FrzCD protein (43,571 Da) contains a region of about 250 amino acids which is similar to the C-terminal portions of the methyl-accepting chemotaxis receptor proteins of the enteric bacteria. FrzCD also contains a region with potentially significant similarity to the DNA-binding region of the Bacillus subtilis sigma 43. The putative FrzB protein (12,066 Da) shares no significant identity with known chemotaxis proteins. The sequence similarities between the putative Frz proteins and the chemotaxis proteins of the enteric bacteria strongly support the hypothesis that the frz genes define a system of signal transduction analogous to the enterobacterial chemotaxis systems.
Retroregulation of the synthesis of ribosomal proteins L14 and L24 by feedback repressor S8 in Escherichia coli.
Authors: Mattheakis L , Vu L , Sor F , Nomura M
Published: May 2006

Previous studies on regulation of the spc operon containing genes for ribosomal proteins have shown that S8, encoded by the fifth gene of the operon in Escherichia coli, is a translational repressor and regulates the synthesis of the third gene product (L5) and distal gene products by acting at a site near the L5 mRNA translation initiation site. We have now shown that S8 also regulates the synthesis of the first and second gene products (L14 and L24) of the operon by acting at the same mRNA target site--that is, the site located distal to sites coding for L14 and L24--and that mRNA degradation is involved in this retroregulation. It was shown that single base substitutions in the target site, which abolish repression of the synthesis of L5 and L5-distal gene products by S8, also cause derepression of L14-L24 synthesis. Inhibition of L14-L24 synthesis by S8 was also shown by overproducing S8 in trans from a plasmid carrying the S8 gene under lac promoter/operator control. A strain carrying temperature-sensitive mutations in genes for polynucleotide phosphorylase and RNase II was found upon shift to nonpermissive temperature to show higher differential synthesis rates of L14-L24 (and L5) relative to those of several L5-distal spc operon gene products. We suggest that repression of distal ribosomal protein synthesis by S8 triggers nucleolytic cleavage of spc operon mRNA, followed by mRNA degradation by these 3'- to 5'- exonucleases, which is then responsible for inhibition of L14-L24 synthesis.
Correlation between oxytocin neuronal sensitivity and oxytocin receptor binding: an electrophysiological and autoradiographical study comparing rat and guinea pig hippocampus.
Authors: Raggenbass M , Tribollet E , Dubois-Dauphin M , Dreifuss J J
Published: May 2006

In transverse hippocampal slices from rat and guinea pig brains, we obtained unitary extracellular recordings from nonpyramidal neurones located in or near the stratum pyramidale in the CA1 field and in the transition region between the CA1 and the subiculum. In rats, these neurones responded to oxytocin at 50-1000 nM by a reversible increase in firing rate. The oxytocin-induced excitation was suppressed by a synthetic structural analogue that acts as a potent, selective antioxytocic on peripheral receptors. Nonpyramidal neurones were also excited by carbachol at 0.5-10 microM. The effect of this compound was postsynaptic and was blocked by the muscarinic antagonist atropine. In guinea pigs, by contrast, nonpyramidal neurones were unaffected by oxytocin, although they were excited by carbachol. Light microscopic autoradiography, carried out using a radioiodinated selective antioxytocic as a ligand, revealed labeling in the subiculum and in the CA1 area of the hippocampus of rats, whereas no oxytocin-binding sites were detected in the hippocampus of guinea pigs. Our results indicate (i) that a hippocampal action of oxytocin is species-dependent and (ii) that a positive correlation exists between neuronal responsiveness to oxytocin and the presence in the hippocampus of high-affinity binding sites for this peptide.
Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies.
Authors: Basu A K , Loechler E L , Leadon S A , Essigmann J M
Published: May 2006

The mutational specificity and genetic requirements for mutagenesis by 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), one of the principal DNA lesions induced by oxidation and ionizing radiation, has been investigated in Escherichia coli. Thymine glycol was positioned at a unique site in the single-stranded genome of a bacteriophage M13mp19 derivative. Replication of the genome in E. coli yielded targeted mutations at a frequency of 0.3%; the mutations were exclusively T----C. Mutagenesis was independent of SOS and nth (nth encodes endonuclease III, a thymine glycol repair enzyme). The adduct was not detectably mutagenic in duplex DNA. A chemical rationalization for the mutation observed for thymine glycol was developed by applying molecular modeling and molecular mechanical calculations to the same DNA sequence studied in vivo. Modeling suggested that the 5R,6S isomer of cis-thymine glycol, when not base paired, was displaced laterally by approximately 0.5 A toward the major groove in comparison to the position that thymine would otherwise occupy. This perturbation of DNA structure should increase the likelihood of a guanine.thymine glycol wobble base pair during replication, which would explain the mutational specificity of the base observed in the genetic experiments.
A member of the Hsp70 family is localized in mitochondria and resembles Escherichia coli DnaK.
Authors: Leustek T , Dalie B , Amir-Shapira D , Brot N , Weissbach H
Published: May 2006

A 71-kDa protein (P71) with properties similar to those of the Escherichia coli heat shock protein DnaK has been found in extracts of HeLa cells. P71 was copurified by ATP-agarose affinity chromatography with three additional proteins of the Hsp70 family. Of these proteins, only P71 crossreacted strongly with antiserum raised against purified DnaK, and both DnaK and P71 could be phosphorylated in vitro with [gamma-32]ATP in a reaction that was markedly stimulated by Ca2+. In HeLa cells, P71 was found to be concentrated in mitochondria. A protein similar to P71 was also found in calf liver and yeast mitochondria.
ATP-sensitive K+ channels in a plasma membrane H+-ATPase mutant of the yeast Saccharomyces cerevisiae.
Authors: Ramirez J A , Vacata V , McCusker J H , Haber J E , Mortimer R K , Owen W G , Lecar H
Published: May 2006

A mutant in the plasma membrane H+-ATPase gene of the yeast Saccharomyces cerevisiae with a reduced H+-ATPase activity, when examined at the single-channel level with the patch-clamp technique, was found to exhibit K+ channels activated by intracellular application of ATP. In the parent strain, the same channel, identified by its conductance and selectivity, is not activated by ATP. This activity in the mutant is blocked by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. ADP and the ATP analog adenosine 5'-[gamma-[35S]thio]triphosphate do not activate the channel. These findings suggest a tight physical coupling between the plasma membrane ATPase and the K+ channel.
Familiar neighbors enhance breeding success in birds.
Authors: Beletsky L D , Orians G H
Published: May 2006

We tested the hypothesis that long-term familiarity with neighbors is advantageous by determining whether male red-winged blackbirds (Agelaius phoeniceus) breeding adjacent to familiar neighbors have better reproductive success than other males. Using data gathered during a 10-yr study of breeding success, we found that males with familiar neighbors fledged, on average, significantly more offspring annually than males without familiar neighbors. We also found that the same males, breeding in different years on the same territories, had significantly larger harems in the years they had familiar neighbors. Improved reproductive success was due to the males' abilities to attract more females to nest in their territories. Alternative hypotheses to explain the positive relationship between familiar neighbors and breeding success were not supported by our data. Relatively high reproductive success for breeders with long-term neighbors may provide a basis for the evolution of cooperative behavior in this and other species.
Higher-order structure of Saccharomyces cerevisiae chromatin.
Authors: Lowary P T , Widom J
Published: May 2006

We have developed a method for partially purifying chromatin from Saccharomyces cerevisiae (baker's yeast) to a level suitable for studies of its higher-order folding. This has required the use of yeast strains that are free of the ubiquitous yeast "killer" virus. Results from dynamic light scattering, electron microscopy, and x-ray diffraction show that the yeast chromatin undergoes a cation-dependent folding into 30-nm filaments that resemble those characteristic of higher-cell chromatin; moreover, the packing of nucleosomes within the yeast 30-nm filaments is similar to that of higher cells. These results imply that yeast has a protein or protein domain that serves the role of the histone H 1 found in higher cells; physical and genetic studies of the yeast activity could help elucidate the structure and function of H 1. Images of the yeast 30-nm filaments can be used to test crossed-linker models for 30-nm filament structure.
Effect of 3' flanking neighbors on kinetics of pairing of dCTP or dTTP opposite O6-methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used.
Authors: Singer B , Chavez F , Goodman M F , Essigmann J M , Dosanjh M K
Published: May 2006

O6-Methylguanine (m6G) was incorporated site-specifically into two 25-base oligonucleotides differing only in the nucleotide on the 3' side of the modified base. Templates were primed with oligonucleotides terminating one or two bases prior to the site at which incorporation kinetics were to be investigated. Escherichia coli DNA polymerase I (Klenow fragment) was used to determine the apparent Km and relative Vmax of incorporation of either dCTP or dTTP opposite m6G or G. These data were used to calculate the relative frequency of incorporation opposite the m6G or the unmodified G. When the sequence was 3'-Cm6G-5', there was a 6- to 7-fold preference for formation of a m6G.T pair compared with m6G.C. The m6G.T frequency, based on Vmax/Km, was at least 50-fold greater than that of a G.T pair at the same site. Changing the sequence to 3'-Tm6G-5' had a marked effect on both Km and Vmax of pairs containing m6G and on the incorporation frequency of T opposite m6G, which was then only slightly favored over m6G.C. When replication was started directly opposite m6G, the kinetics appeared unaffected. These data indicate that the frequency of incorporation of C or T opposite m6G in a DNA template is dependent on the flanking neighbors and that a change of even a single base at the 3' position can have a major effect on mutagenic efficiency. Replication using Drosophila Pol alpha gave the same values for relative frequencies. Pairing of either C or T with m6G on the primer terminus did not significantly inhibit extension of the next normal base pair, in contrast to terminal mismatches of unmodified bases. It is concluded that, in the absence of repair, m6G can exhibit widely differing mutation frequencies which, in these experiments, can be as high as 85% of the replicated base. This variation in frequency of changed pairing could contribute to the occurrence of mutational "'hot spots" after replication of damaged DNA.
Triton,... electron,... cosmon,...: An infinite regression?
Authors: Dehmelt Hans
Published: May 2006

I propose an elementary particle model in which the simplest near-Dirac particles triton, proton, and electron are members of the three top layers of a bottomless stack. Each particle is a composite of three particles from the next layer below in an infinite regression approaching Dirac point particles. The cosmon, an immensely heavy lower layer subquark, is
the
elementary particle. The world-atom, a tightly bound cosmon/anticosmon pair of zero relativistic total mass, arose from the nothing state in a quantum jump. Rapid decay of the pair launched the big bang and created the universe.
Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae.
Authors: Dowson C G , Hutchison A , Brannigan J A , George R C , Hansman D , Liñares J , Tomasz A , Smith J M , Spratt B G
Published: May 2006

Resistance to penicillin in clinical isolates of Streptococcus pneumoniae has occurred by the development of altered penicillin-binding proteins (PBPs) that have greatly decreased affinity for the antibiotic. We have investigated the origins of penicillin-resistant strains by comparing the sequences of the transpeptidase domain of PBP2B from 6 penicillin-sensitive and 14 penicillin-resistant strains. In addition we have sequenced part of the amylomaltase gene from 2 of the sensitive and 6 of the resistant strains. The sequences of the amylomaltase gene of all of the strains and of the PBP2B gene of the penicillin-sensitive strain show that S. pneumoniae is genetically very uniform. In contrast the PBP2B genes of the penicillin-resistant strains show approximately equal to 14% sequence divergence from those of the penicillin-sensitive strains and the development of penicillin resistance has involved the replacement, presumably by transformation, of the original PBP2B gene by a homologous gene from an unknown source. This genetic event has occurred on at least two occasions, involving different sources, to produce the two classes of altered PBP2B genes found in penicillin-resistant strains of S. pneumoniae. There is considerable variation among the PBP2B genes of the resistant strains that may have arisen by secondary transformation events accompanied by mismatch repair subsequent to their original introductions into S. pneumoniae.
Molecular resemblance of an AIDS-associated lymphoma and endemic Burkitt lymphomas: implications for their pathogenesis.
Authors: Haluska F G , Russo G , Kant J , Andreef M , Croce C M
Published: May 2006

Non-Hodgkin lymphoma is a common feature of AIDS. Approximately 30-40% of these tumors exhibit clinical features suggestive of endemic Burkitt lymphoma: they are aggressive malignancies that occur in association with Epstein-Barr virus infection, they arise in the setting of immunosuppression, and they carry t(8;14) translocations without detectable rearrangement of the MYC oncogene. To understand the molecular basis of these parallels, we analyzed a case of Epstein-Barr-positive AIDS-associated undifferentiated lymphoma. Southern blots show that the tumor exhibits immunoglobulin joining segment rearrangement but no rearrangement of the MYC oncogene. Cloning of the rearranged joining segment allowed the isolation of recombinant clones encompassing the translocation breakpoint, and sequencing of the translocation junction disclosed that the breakpoint is situated 7 base pairs from the chromosome 14 site involved in a previously described endemic Burkitt lymphoma translocation. Furthermore, the breakpoint is situated far from MYC on chromosome 8, a constant finding in endemic Burkitt lymphomas. That the molecular architecture of the translocation in this case is strikingly similar to previously analyzed translocations from endemic Burkitt lymphomas strongly suggests that common molecular mechanisms must be operative in the pathogenesis of these tumors.
Enzymatic conversion of all-trans-beta-carotene to retinal by a cytosolic enzyme from rabbit and rat intestinal mucosa.
Authors: Lakshman M R , Mychkovsky I , Attlesey M
Published: May 2006

Enzymatic conversion of all-trans-beta-carotene to retinal by a partially purified enzyme from rabbit and rat intestinal mucosa was demonstrated. The enzymatic product was characterized based on the following evidence: (i) The product gave rise to its O-ethyloxime by treatment with O-ethylhydroxylamine with an absorption maximum at 363 nm in ethanol characteristic of authentic retinal O-ethyloxime. High-pressure liquid chromatography (HPLC) of this derivative yielded a sharp peak with a retention time of 7.99 min corresponding to the authentic compound. The enzyme blank and boiled enzyme blank failed to show any significant HPLC peaks corresponding to retinal O-ethyloxime, retinal, or retinol. (ii) The mass spectrum of the O-ethyloxime of the enzymatic product was identical to that of authentic retinal O-ethyloxime (m/z 327: 45%, M+. and m/z 282: 100%, M--ethoxy). (iii) The specific activity of the enzymatically formed [14C]retinal O-ethyloxime remained constant even after repeated crystallization. (iv) The enzymatic product exhibited an absorption maximum at 370 nm in light petroleum characteristic of authentic retinal. Furthermore, it was reduced by horse liver alcohol dehydrogenase to retinol with an absorption maximum at 326 nm in light petroleum. This retinol was enzymatically esterified to retinyl palmitate by rat pancreatic esterase with a retention time of 10 min on HPLC corresponding to authentic retinyl palmitate. Thus, the enzymatic product of beta-carotene cleavage by the partially purified intestinal enzyme was unequivocally confirmed to be retinal.
Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation.
Authors: Dürrenberger F , Crameri A , Hohn B , Koukolíková-Nicola Z
Published: May 2006

We show that upon induction of Agrobacterium tumefaciens, free linear double-stranded T-DNA molecules as well as the previously described T-strands are generated from the Ti plasmid. A majority of these molecules are bound to a protein. We show that this protein is the product of the virulence gene virD2. This protein was found to be attached to the 5' terminus of processed T-DNA at the right border and to the rest of the Ti plasmid at the left border. The protein remnant after Pronase digestion rendered the right end of the double-stranded T-DNA resistant to 5'----3' exonucleolytic attack in vitro. The protein-DNA association was resistant to SDS, mercaptoethanol, mild alkali, piperidine, and hydroxylamine, indicating that it involves a covalent linkage. The possible involvement of this T-DNA-protein complex in replication, transduction to the plant, nuclear targeting, and integration into the plant nuclear DNA is discussed.
Complementation of adenovirus virus-associated RNA I gene deletion by expression of a mutant eukaryotic translation initiation factor.
Authors: Davies M V , Furtado M , Hershey J W , Thimmappaya B , Kaufman R J
Published: May 2006

Adenovirus VA RNAs (virus-associated RNAs) are small polymerase III transcripts that are required for efficient initiation of mRNA translation late in adenovirus infection. VAI RNA prevents double-stranded RNA (dsRNA) activation of the interferon-induced protein kinase (DAI kinase). Activation of this kinase results in phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) and correlates with inhibition of translation initiation. In this report we show growth complementation of adenoviruses harboring deletions in the VAI gene in cell lines expressing a serine-to-alanine mutant of eIF-2 alpha. This serine-to-alanine mutant is resistant to phosphorylation by DAI kinase. These results directly show that the primary function of VAI RNA in the lytic adenovirus infection is the inhibition of eIF-2 alpha phosphorylation by DAI kinase and identify eIF-2 alpha as the target that mediates the effects of DAI kinase activation. Cells that express a mutant eIF-2 alpha will enable the isolation of specific host-range mutants for other types of viruses that are defective in the ability to inhibit DAI kinase.
Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein.
Authors: Liu G , Topping T B , Randall L L
Published: May 2006

It has been shown that folding of precursor maltose-binding protein of Escherichia coli in vitro is retarded by the leader peptide. We now present evidence that this modulation of folding plays a role during the export of maltose-binding protein in vivo. Maltose-binding protein synthesized in vivo without a leader sequence did not engage the cellular export apparatus. However, the requirement for the leader in at least one step, that of binding the export factor SecB, could be overcome by an amino acid substitution in the mature portion of maltose-binding protein. This substitution retarded the folding of the polypeptide even in the absence of a leader. Investigations using purified proteins in vitro demonstrated that SecB would stably bind to species of maltose-binding protein devoid of a leader when the folding of the binding proteins was sufficiently slow. Thus, we conclude that one of the roles of the leader is to retard folding and expose the binding site for SecB.
A mechanism for the Hebb and the anti-Hebb processes underlying learning and memory.
Authors: Lisman J
Published: May 2006

In a previous paper, a model was presented showing how the group of Ca2+/calmodulin-dependent protein kinase II molecules contained within a postsynaptic density could stably store a graded synaptic weight. This paper completes the model by showing how bidirectional control of synaptic weight could be achieved. It is proposed that the quantitative level of the activity-dependent rise in postsynaptic Ca2+ determines whether the synaptic weight will increase or decrease. It is further proposed that reduction of synaptic weight is governed by protein phosphatase 1, an enzyme indirectly controlled by Ca2+ through reactions involving phosphatase inhibitor 1, cAMP-dependent protein kinase, calcineurin, and adenylate cyclase. Modeling of this biochemical system shows that it can function as an analog computer that can store a synaptic weight and modify it in accord with the Hebb and anti-Hebb learning rules.
Urinary 8-hydroxy-2'-deoxyguanosine as a biological marker of in vivo oxidative DNA damage.
Authors: Shigenaga M K , Gimeno C J , Ames B N
Published: May 2006

DNA is subject to constant oxidative damage from endogenous oxidants. The oxidized DNA is continuously repaired and the oxidized bases are excreted in the urine. A simple routine analytical procedure is described for urinary 8-hydroxy-2'-deoxyguanosine, an oxidative DNA damage adduct, as an indicator of oxidative damage in humans and rodents. This adduct was purified from human urine and characterized. The described assay employs a series of solid-phase extraction steps that separate 8-hydroxy-2'-deoxyguanosine from other urinary constituents, followed by analysis by gradient reversed-phase HPLC coupled to a dual-electrode high-efficiency electrochemical detection system. Analysis of urine from three species by this method indicates that mice excrete approximately 3.3-fold more 8-hydroxy-2'-deoxyguanosine than humans (582 vs. 178 residues per cell per day), a result that supports the proposal that oxidative damage to DNA increases in proportion to species-specific basal metabolic rates.
Quantitation of mRNA by the polymerase chain reaction.
Authors: Wang A M , Doyle M V , Mark D F
Published: May 2006

A method for the quantitation of specific mRNA species by the polymerase chain reaction (PCR) has been developed by using a synthetic RNA as an internal standard. The specific target mRNA and the internal standard are coamplified in one reaction in which the same primers are used. The amount of mRNA is then quantitated by extrapolating against the standard curve generated with the internal standard. The synthetic internal standard RNA consists of a linear array of the sequences of upstream primers of multiple target genes followed by the complementary sequences to their downstream primers in the same order. This quantitative PCR method provides a rapid and reliable way to quantify the amount of a specific mRNA in a sample of less than 0.1 ng of total RNA. In addition, the same internal standard RNA is used, with appropriate primer pairs, to quantitate multiple different mRNA species.
Hippocampal specialization of food-storing birds.
Authors: Krebs J R , Sherry D F , Healy S D , Perry V H , Vaccarino A L
Published: May 2006

In a study of 52 individuals belonging to 35 species or subspecies of passerine birds it was shown that the volume of the hippocampal complex relative to brain and body size is significantly larger in species that store food than in species that do not. Retrieval of stored food relies on an accurate and long-lasting spatial memory, and hippocampal damage disrupts memory for storage sites. The results suggest, therefore, that food-storing species of passerines have an enlarged hippocampal complex as a specialization associated with the use of a specialized memory capacity. Other life-history variables were examined and found not to be correlated with hippocampal volume.
Juvenile hormone bisepoxide biosynthesis in vitro by the ring gland of Drosophila melanogaster: a putative juvenile hormone in the higher Diptera.
Authors: Richard D S , Applebaum S W , Sliter T J , Baker F C , Schooley D A , Reuter C C , Henrich V C , Gilbert L I
Published: May 2006

The in vitro production of juvenile hormone (JH) was investigated by using isolated ring glands from third instar Drosophila melanogaster. A JH-like molecule is secreted that comigrates with a synthetic sample of methyl 6,7;10,11-bisepoxy-3,7,11-trimethyl-(2E)-dodecenoate (JHB3) during TLC, liquid chromatography, and GC analysis. Purified product from farnesoic acid-stimulated ring glands was analyzed by electron impact GC/MS and gave a mass spectrum identical to synthetic JHB3. Additional structure confirmation was obtained following conversion of product from unstimulated biosynthesis to a derivative that comigrated on liquid chromatography with the derivative prepared from synthetic JHB3. Physiological studies revealed that JHB3 is produced solely by the corpus allatum portion of the ring gland in vitro. Isolated ring glands from other cyclorrhaphous dipteran larvae also produce JHB3 almost exclusively in vitro. Corpora allata from mosquito larvae, however, produce only JH III, indicating that JHB3 production may be restricted to the higher Diptera. Topically applied synthetic JHB3 caused developmental responses in newly formed D. melanogaster white puparia similar to those obtained with JH III. The data suggest that JHB3 is a fly juvenile hormone.
Two putative protein-tyrosine kinases identified by application of the polymerase chain reaction.
Authors: Wilks A F
Published: May 2006

The pivotal role that protein-tyrosine kinases (PTKs) play in the growth regulation of eukaryotic cells is manifest in the frequent appearance of members of the PTK family as growth factor receptors or as the transforming agents of acutely transforming retroviruses. A feature common to all members of the PTK family is a highly conserved catalytic domain which is characteristic of the group as a whole and whose activity appears to be tightly regulated within the cell by other domains of the PTK. Degenerate oligonucleotide probes corresponding to two invariant amino acid sequence motifs within the catalytic domains of all PTK family members were synthesized and employed in the polymerase chain reaction (PCR) to amplify cDNA sequences between them. An M13 PCR library was produced in this way from cDNA prepared against mRNA from the murine hemopoietic cell line FDC-P1. The PCR library was then screened by DNA sequencing for PTK-related sequences. Two sequences were identified that, on the basis of sequence comparison with known PTKs, may encode representatives of a new class of PTK.
Regulation of pheromone biosynthesis by a brain hormone in two moth species
Authors: Tang Juliet D. , Charlton Ralph E. , Jurenka Russell A. , Wolf Walter A. , Phelan P. Larry , Sreng Leam , Roelofs Wendell L.
Published: May 2006

Experiments were performed to characterize the action of a brain hormone on pheromone biosynthesis in female redbanded leafroller and cabbage looper moths. Results showed that the two species differed in their respective control mechanisms. In the cabbage looper, pheromone titer from decapitated females that received either saline or brain extract injections was not significantly different from control females, suggesting that pheromone biosynthesis was not dependent on the presence of the brain hormone. In contrast, with redbanded leafroller females, studies using radiolabeled acetate incorporation as well as incorporation of deuterium-labeled hexadecanoic acid showed that (
i
) the brain hormone was required for pheromone biosynthesis, (
ii
) the brain hormone regulated pheromone biosynthesis by activating synthesis of octadecanoyl and hexadecanoyl intermediates, and (
iii
) the brain hormone did not control other enzymes in the pathway. Regulation of fatty acid synthetase was unlikely since assays of the enzyme from decapitated and normal females showed no differences in the amount or distribution of the 18- and 16-carbon acyl end products. These results in conjunction with those from organ cultures of the pheromone gland suggest that the brain hormone acts by increasing the substrate supply for fatty acid synthesis.
A detailed structural model of cytotactin: protein homologies, alternative RNA splicing, and binding regions.
Authors: Jones F S , Hoffman S , Cunningham B A , Edelman G M
Published: May 2006

A combination of cDNA sequencing of the complete coding region, protein comparisons, binding site mapping, and electron microscopic imaging has permitted the formulation of a structural model of cytotactin. Cytotactin is a large extracellular matrix glycoprotein that displays a restricted tissue distribution during development. Although there appears to be a single cytotactin gene, multiple cytotactin polypeptides and mRNAs are detected in a variety of tissues. We report here the sequences and relationships of cDNAs that encode the complete amino acid sequences of two cytotactin polypeptides in chicken brain. The translated cDNA sequences agree with those obtained by direct analysis of cytotactin and fragments of the molecule. All regions of the polypeptides appear to be identical except for a 273 amino acid segment found in the larger but not in the smaller. At their amino termini, both polypeptides contain a cysteine-rich segment that probably includes those residues that link monomers into hexamers. This segment is followed by 13 epidermal growth factor-like (EGFL) repeats and then 8 consecutive segments that each resemble the type III repeats found in fibronectin. At their carboxyl termini, the polypeptides are similar to the beta and gamma chains of fibrinogen, including a calcium-binding segment. The additional sequence in the large polypeptide is inserted after the fifth type III repeat and includes three additional type III repeats. On RNA transfer blot analyses, cytotactin cDNA probes detected a 6.4-kilobase (kb) component in both brain and gizzard and larger mRNAs in both tissues, but those in gizzard were larger by about 1 kb than those in brain. A probe specific to the insert did not hybridize to the 6.4-kb mRNA in either tissue but detected the larger mRNAs in both tissues. At least a portion of the insert is thus present in both tissues, but there may be additional inserts in the gizzard mRNAs. The proposed model of cytotactin specifies the orientation of the polypeptides, the localization of interchain disulfide bonds, the structural elements constituting the thin and thick segments (EGFL repeats and type III repeats, respectively), the terminal fibrinogen-like nodular region, and the relative location of the cell-binding region.
Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells.
Authors: Tsujimoto Y
Published: May 2006

The biological activity of the human BCL-2 gene product was analyzed in an Epstein-Barr virus (EBV)-infected human lymphoblastoid B-cell line transfected with BCL-2 sequences driven by the simian virus 40 promoter and enhancer. Overproduction of the BCL-2 protein conferred a selective growth advantage to the EBV-infected B cells as selective growth advantage to the EBV-infected B cells as compared with control transfectants in low-serum medium and also after seeding at limiting dilution but did not render the cells tumorigenic in athymic nude mice. This growth enhancement was also seen in cells transfected with the BCL-2 gene with its own promoter juxtaposed to the immunoglobulin heavy chain gene enhancer, which represents the translocated form of the BCL-2 gene observed in follicular lymphomas with the t(14;18) translocation. The growth advantage of EBV-infected B cells overproducing the BCL-2 protein is neither due to the enhanced growth factor production nor due to an enhanced sensitivity of the BCL-2 transfectants to interleukins 1 or 6, although both lymphokines are known to stimulate proliferation of EBV-infected B-cell lines. The growth advantage of EBV-infected B cells by overproduction of the BCL-2 protein suggests the direct involvement of the BCL-2 gene product in the pathogenesis of follicular lymphoma.
cDNA sequences of two inducible T-cell genes.
Authors: Kwon B S , Weissman S M
Published: May 2006

We have previously described a set of human T-lymphocyte-specific cDNA clones isolated by a modified differential screening procedure. Apparent full-length cDNAs containing the sequences of 14 of the 16 initial isolates were sequenced and were found to represent five different species of mRNA; three of the five species were identical to previously reported cDNA sequences of preproenkephalin, T-cell-replacing factor, and a serine esterase, respectively. The other two species, 4-1BB and L2G25B, were inducible sequences found in mRNA from both a cytolytic T-lymphocyte and a helper T-lymphocyte clone and were not previously described in T-cell mRNA; these mRNA sequences encode peptides of 256 and 92 amino acids, respectively. Both peptides contain putative leader sequences. The protein encoded by 4-1BB also has a potential membrane anchor segment and other features also seen in known receptor proteins.