bluuebunny/crossref_metadata_2025 huggingface dataset:
Unusual mechanism of hydrocarbon formation in the housefly: cytochrome P450 converts aldehyde to the sex pheromone component (Z)-9-tricosene and CO2.
Authors: Reed J R , Vanderwel D , Choi S , Pomonis J G , Reitz R C , Blomquist G J
Published: May 2006

An unusual mechanism for hydrocarbon biosynthesis is proposed from work examining the formation of (Z)-9-tricosene (Z9-23:Hy), the major sex pheromone component of the female housefly, Musca domestica. Incubation of (Z)-15-[1-14C]- and (Z)-15-[15,16-3H2]tetracosenoic acid (24:1 fatty acid) with microsomes from houseflies gave equal amounts of [3H]Z9-23:Hy and 14CO2. The formation of CO2 and not CO, as reported for hydrocarbon formation in plants, animals, and microorganisms [Dennis, M. & Kolattukudy, P. E. (1992) Proc. Natl. Acad. Sci. USA 89, 5306-5310], was verified by trapping agents and by radio-GLC analysis. Incubation of (Z)-15-[15,16-3H2]tetracosenoyl-CoA with microsomal preparations in the presence of NADPH and O2 gave almost equal amounts of (Z)-15-3H2]tetrasosenal (24:1 aldehyde) and Z9-23:Hy. Addition of increasing amounts of hydroxylamine (aldehyde trapping agent) caused a decrease in hydrocarbon formation with a concomitant increase in oxime (aldehyde derivative) formation. The 24:1 aldehyde was efficiently converted to (Z)-9-tricosene only in the presence of both NADPH and O2. Bubbling carbon monoxide (20:80 CO/O2) or including an antibody against housefly cytochrome P450 reductase inhibited the formation Z9-23:Hy from 24:1 aldehyde. These data demonstrate an unusual mechanism for hydrocarbon formation in insects in which the acyl-CoA is reduced to the corresponding aldehyde and then carbon-1 is removed as CO2. The requirement for NADPH and O2 and the inhibition by CO and the antibody to cytochrome P450 reductase strongly implicate the participation of a cytochrome P450 in this reaction.
Modulation by gangliosides of the lamellar-inverted micelle (hexagonal II) phase transition in mixtures containing phosphatidylethanolamine and dioleoylglycerol.
Authors: Perillo M A , Scarsdale N J , Yu R K , Maggio B
Published: May 2006

We studied the effect of gangliosides GD1a and GM1 on the lamellar-to-hexagonal II phase transition of mixtures of dioleoylphosphatidylethanolamine/dioleoylphosphatidyl choline, 3:1, and of transphosphatidylated phosphatidylethanolamine with dioleoylglycerol by high-sensitivity differential scanning calorimetry, 31P-NMR, and pyrene fluorescence of a phosphatidylcholine probe. Gangliosides had a dual effect. Below 1 mol % ganglioside the hexagonal II phase transition was affected but still occurred at lower temperature than in the absence of gangliosides. The presence of between 1 and 2 mol % gangliosides increased the temperature for formation of the hexagonal II phase and progressively decreased its cooperativity. Above 3 mol % gangliosides totally inhibited the formation of both the temperature-induced and composition-induced hexagonal phase, probably by opposing the geometric distortions necessary for the inverted micellar structures.
Negative regulation of mitosis in fission yeast by catalytically inactive pyp1 and pyp2 mutants.
Authors: Hannig G , Ottilie S , Erikson R L
Published: May 2006

The Schizosaccharomyces pombe genes pyp1+ and pyp2+ encode protein tyrosine phosphatases (PTPases) that act as negative regulators of mitosis upstream of the wee1+/mik1+ pathway. Here we provide evidence that pyp1+ and pyp2+ function independently of cdr1+(nim1+) in the inhibition of mitosis and that the wee1 kinase is not a direct substrate of either PTPase. In a pyp1::ura4 cdc25-22 genetic background, overexpression of either the N-terminal domain of pyp1+ or a catalytically inactive mutant, pyp1C470S, causes cell cycle arrest. This phenotype reverses the suppression of a cdc25 temperature-sensitive mutation at 35 degrees C caused by a pyp1 disruption. Furthermore, pyp1C470S and a catalytically inactive mutant of pyp2, pyp2C630S, induce mitotic delay as do their wild-type counterparts. Analysis of pyp1+ and pyp2+ further reveals that in vitro PTPase activity of pyp1 and pyp2, as well as their biological activity, is dependent on the presence of N-terminal sequences that are not normally considered part of PTPase catalytic domains.
Non-Hebbian properties of long-term potentiation enable high-capacity encoding of temporal sequences.
Authors: Granger R , Whitson J , Larson J , Lynch G
Published: May 2006

A hypothesis commonly found in biological and computational studies of synaptic plasticity embodies a version of the 1949 postulate of Hebb that coactivity of pre- and postsynaptic elements results in increased efficacy of their synaptic contacts. This general proposal presaged the identification of the first and still only known long-lasting synaptic plasticity mechanism, long-term potentiation (LTP). Yet the detailed physiology of LTP induction and expression differs in many specifics from Hebb's rule. Incorporation of these physiological LTP constraints into a simple non-Hebbian network model enabled development of "sequence detectors" that respond preferentially to the sequences on which they were trained. The network was found to have unexpected capacity (e.g., 50 x 10(6) random sequences in a network of 10(5) cells), which scales linearly with network size, thereby addressing the question of memory capacity in brain circuitry of realistic size.
Km mutants of the Chlorella monosaccharide/H+ cotransporter randomly generated by PCR.
Authors: Will A , Caspari T , Tanner W
Published: May 2006

The HUP1 gene codes for the monosaccharide/H+ cotransporter protein of Chlorella kessleri. The gene is functionally expressed in Schizosaccharomyces pombe. This heterologous system has been used to screen for Km mutants of the Chlorella symporter. Since S. pombe transformed with HUP1 cDNA showed a 1000-fold increase in sensitivity toward the toxic sugar analogue 2-deoxyglucose, we screened for transformants with a decreased 2-deoxyglucose sensitivity. The transformants were produced with HUP1 cDNA randomly mutagenized by PCR. From 73 transformants with decreased 2-deoxyglucose sensitivity, four mutants with increased Km values for D-glucose were obtained. The amino acid exchanges responsible for the increased Km values are located in the center of the putative transmembrane helices V (Q179E), VII (Q298R), and XI (V433L/N436Y). Q179N and Q299N had previously been shown by directed mutagenesis to affect the Km value of the transporter for D-glucose. The drastic mutational changes Q298R and N436Y gave rise to very high Km values; however, the corresponding conservative amino acid changes Q298N or N436Q obtained by directed mutagenesis also result in Km values increased by a factor of 10 or 20, respectively. The data therefore support the proposal that at least helices V, VII, and XI may line the sugar translocation path and determine its specificity. These results are discussed in relation to other sugar transporters and to the interaction of the yeast hexokinase B with D-glucose as known from published crystal structures.
Transfection of an Fc gamma receptor cDNA induces T cells to become phagocytic.
Authors: Hunter S , Kamoun M , Schreiber A D
Published: May 2006

The human receptor Fc gamma RIIA for the Fc portion of IgG (Fc gamma) was expressed in a human T-cell line and conferred on these cells the ability to perform IgG antibody-stimulated phagocytosis. Crosslinking Fc gamma RIIA with anti-Fc gamma RII monoclonal antibody also induced tyrosine phosphorylation of multiple proteins including Fc gamma RIIA, ZAP-70, p72SYK, and phospholipase C gamma 1 subunit and an increase in intracellular Ca2+ concentration. The T cell receptor-associated zeta-chain was not tyrosine-phosphorylated after crosslinking of Fc gamma RIIA, suggesting that the Fc gamma RIIA-mediated signals were independent of CD3. Fc gamma RIIA-mediated signal transduction was defective in a transfected mutant T-cell line exhibiting reduced expression of the tyrosine kinases LCK and FYN. These studies indicate that certain T cells can assume phagocytic properties after transfection of cDNA encoding an Fc gamma receptor with the capability of inducing a phagocytic signal.
Human adhalin is alternatively spliced and the gene is located on chromosome 17q21.
Authors: McNally E M , Yoshida M , Mizuno Y , Ozawa E , Kunkel L M
Published: May 2006

Mutations in the dystrophin gene cause the X chromosome-linked, recessive Duchenne and Becker muscular dystrophies. Dystrophin, a large cytoskeletal protein, copurifies with a complex of dystrophin-associated proteins which serve to anchor dystrophin to the sarcolemma. One of these associated proteins, adhalin, has been implicated as a candidate for severe childhood autosomal recessive muscular dystrophy (SCARMD) due to absence of anti-adhalin staining in muscle biopsy samples taken from SCARMD patients. Furthermore, the Duchenne-like dystrophic phenotype seen in the SCARMD families was shown to be tightly linked to chromosome 13 markers. To determine the genetic mutation responsible for autosomal dystrophy, we characterized the human adhalin gene. Contrary to our expectation, human adhalin was mapped to chromosome 17q21, excluding adhalin as the gene causing chromosome 13-associated SCARMD. Additionally, a splice form of adhalin message was found that predicts a 35-kDa nontransmembrane adhalin. The expression of both adhalin splice forms is exclusively restricted to striated muscle, unlike other components of the dystrophin-glycoprotein complex.
Asymptotic theory for gene mapping.
Authors: Kong A , Wright F
Published: May 2006

In genetic linkage mapping, the precision of the location estimate depends on both the number of individuals and the number of markers. Convergence rates and limit laws are established for several situations in which the number of markers depends on the sample size. Practical implications of the results are discussed.
Protection of retroviral DNA from autointegration: involvement of a cellular factor.
Authors: Lee M S , Craigie R
Published: May 2006

An essential step in the retrovirus life cycle is integration of a DNA copy of the viral genome into a host chromosome. After reverse transcription, there can be a delay of many hours before the viral DNA is integrated. It is important for the retrovirus to ensure that the viral DNA does not integrate into itself during this period; such autointegration is a suicidal process that would result in destruction of the viral genome. Understanding of the mechanism that blocks autointegration of the viral DNA may lead to insights into how to inhibit viral replication by inducing the viral DNA to autointegrate. Evidence is presented in this report that viral nucleoprotein complexes isolated from cells infected with Moloney murine leukemia virus exhibit a barrier to autointegration. The barrier can be disrupted by high salt treatment and, subsequently, restored by addition of factors provided by a host cell extract. Our data indicate an involvement of host machinery in protecting retroviral DNA from autointegration.
Cloning and sequencing of parafusin, a calcium-dependent exocytosis-related phosphoglycoprotein.
Authors: Subramanian S V , Wyroba E , Andersen A P , Satir B H
Published: May 2006

A cDNA for parafusin, an evolutionarily conserved phosphoglycoprotein involved in exocytosis, has been cloned and sequenced from a unicellular eukaryote, Paramecium tetraurelia. A Paramecium cDNA library was screened with an oligonucleotide probe synthesized to an internal amino acid sequence of isolated parafusin. The insert was 3 kb long with an open reading frame of 1.75 kb. Data base searches of the deduced amino acid sequence showed that Paramecium parafusin had a 50.7% sequence identity to rabbit muscle phosphoglucomutase, although no detectable phosphoglucomutase activity has been detected in isolated parafusin. The deduced parafusin amino acid sequence had four inserts and two deletions, which might confer on the protein specific functions in signal transduction events related to exocytosis. Furthermore, searches for potential phosphorylation sites showed the presence of a protein kinase C site (KDFSFR) specific to parafusin. Southern blot analysis with probes specific for parafusin and phosphoglucomutase suggested that these proteins were products of different genes. We propose that parafusin and phosphoglucomutase are members of a superfamily that conserve homologies important for the tertiary structure of the molecules.
A distinct segment of the sigma 32 polypeptide is involved in DnaK-mediated negative control of the heat shock response in Escherichia coli.
Authors: Nagai H , Yuzawa H , Kanemori M , Yura T
Published: May 2006

Induction of heat shock proteins in Escherichia coli is caused by a transient increase in the cellular level of sigma 32 (the rpoH gene product), a protein required for transcription of heat shock genes. Both increased synthesis and stabilization of sigma 32 contribute to the increase in sigma 32. We previously showed that heat-induced translation of sigma 32-beta-galactosidase fusion protein encoded by an rpoH-lacZ gene fusion was mediated by an mRNA secondary structure formed between two 5'-proximal segments (A and B) of rpoH coding sequence spanning some 200 nt. We now report that a portion of the sigma 32 polypeptide that corresponds to further downstream (designated region C) is involved in the DnaK-mediated negative control resulting in the shutoff of heat-induced synthesis and degradation of fusion protein. Gene fusions carrying the 5' half (433 nt) or more of the rpoH coding sequence exhibited normal shutoff of synthesis, and the fusion proteins produced were very unstable, like authentic sigma 32; both the shutoff of synthesis and the instability of protein were markedly affected by the dnaK and dnaJ mutations. In contrast, gene fusions carrying < or = 364 nt (lacking region C) and a fusion carrying most of the rpoH sequence but with a frameshift mutation specifically affecting region C exhibited little or no shutoff and produced stable proteins. These results indicate that a distinct segment of sigma 32 plays a critical role in the negative feedback control of sigma 32. The control may be exerted during or after completion of sigma 32 synthesis mediated by interaction between nascent or mature sigma 32 and DnaK/DnaJ proteins.
Single versus parallel pathways of protein folding and fractional formation of structure in the transition state.
Authors: Fersht A R , Itzhaki L S , elMasry N F , Matthews J M , Otzen D E
Published: May 2006

Protein engineering and kinetic experiments indicate that some regions of proteins have partially formed structure in the transition state for protein folding. A crucial question is whether there is a genuine single transition state that has interactions that are weakened in those regions or there are parallel pathways involving many transition states, some with the interactions fully formed and others with the structural elements fully unfolded. We describe a kinetic test to distinguish between these possibilities. The kinetics rule out those mechanisms that involve a mixture of fully formed or fully unfolded structures for regions of the barley chymotrypsin inhibitor 2 and barnase, and so those regions are genuinely only partially folded in the transition state. The implications for modeling of protein folding pathways are discussed.
Lymphocyte antigen receptor activation of a focal adhesion kinase-related tyrosine kinase substrate.
Authors: Kanner S B , Aruffo A , Chan P Y
Published: May 2006

One of the earliest responses of T and B lymphocytes to stimulation through their antigen receptors is the activation of protein tyrosine kinases and the tyrosine phosphorylation of multiple cellular substrates. Here we describe a tyrosine kinase substrate, fakB, a putative homologue of the focal adhesion kinase pp125FAK. Tyrosine phosphorylation of fakB was rapidly augmented in human T and B cells following antigen receptor cross-linking with antibody, while pp125FAK was nonresponsive. Costimulation of the T-cell antigen receptor (TCR/CD3) with either the CD2 or CD4 costimulatory receptors induced synergistic fakB tyrosine phosphorylation in normal human T cells. Engagement of TCR/CD3 induced the stable association of fakB with ZAP-70, the TCR/CD3 sigma-chain-associated tyrosine kinase involved in antigen receptor-induced T-cell activation. In addition, preformed complexes of fakB and ZAP-70 were observed in T-cell leukemia lines. Phosphorylation of fakB on serine, threonine, and tyrosine residues was observed both in vivo and in vitro, where a functional increase of in vitro kinase activity was observed following TCR/CD3 stimulation. fakB is thus a focal adhesion kinase-related tyrosine kinase substrate that is differentially regulated from that of pp125FAK and likely plays a role in antigen-induced lymphocyte signaling.
Topological analysis of the human beta 2-adrenergic receptor expressed in Escherichia coli.
Authors: Lacatena R M , Cellini A , Scavizzi F , Tocchini-Valentini G P
Published: May 2006

We have investigated the topology of the human beta 2-adrenergic receptor expressed in Escherichia coli, using the genetic method described by Beckwith and coworkers. We found that fusions with alkaline phosphatase beyond a certain point on the human beta 2-adrenergic receptor sequence were assembled into the bacterial membrane with the same topology as the human beta 2-adrenergic receptor in the mammalian membrane. The pattern that might have been expected on the basis of the topology of the human beta 2-adrenergic receptor in mammalian membranes was not reflected in the levels of alkaline phosphatase activity of the fusions occurring between the N-terminal region and positions close to the second external domain. Our data suggest that the correct positioning of the N terminus of the receptor depends on the presence of its C-terminal portions.
Generic affinities among crocodilians as revealed by DNA fingerprinting with a Bkm-derived probe.
Authors: Aggarwal R K , Majumdar K C , Lang J W , Singh L
Published: May 2006

Genetic fingerprint profiles have been successfully used for establishing biological relationships, in linkage analysis, and in studies of population structure but have not so far been used for ascertaining phylogenetic relationships among related groups of species and genera. This is largely because these profiles are thought to evolve too rapidly to be informative over large time intervals. However, we show here that among the Crocodilia, whose phylogeny is a debated issue, these profiles can provide phylogenetically useful information. By using the probe Bkm-2(8), DNA fingerprints with distinct bands distributed in the size range 0.5-23.0 kb were obtained for individuals of 18 species belonging to seven of the eight genera of crocodilians. These genetic profiles showed individual-, species-, and restriction enzyme-specific patterns. In addition, striking differences were observed in the copy number of Bkm-related sequences in genomes of different crocodilian species. The qualitative data from DNA fingerprint profiles, and quantitative data on copy number variation in Bkm-related sequences, suggest that these genera belong to two distinct groups, one of which includes Alligator, Paleosuchus, and Caiman; the other includes Crocodylus, Osteolaemus, Tomistoma, and Gavialis. A close relationship between Tomistoma and Gavialis is also suggested by these results.
Role of the 75-kDa tumor necrosis factor receptor: inhibition of early hematopoiesis.
Authors: Jacobsen F W , Rothe M , Rusten L , Goeddel D V , Smeland E B , Veiby O P , Slørdal L , Jacobsen S E
Published: May 2006

Biological effects of tumor necrosis factor alpha (TNF-alpha) are mediated through two cell surface receptors, the 55-kDa TNF receptor and the 75-kDa TNF receptor. The present study investigated the relative roles of the two TNF receptors in normal hematopoiesis. Using agonists (antibodies) specific for the 55- and 75-kDa TNF receptors, we demonstrate differential roles of the two TNF receptors in hematopoiesis in that only the 55-kDa TNF receptor mediates antiproliferative effects of TNF-alpha on mature Lin- hematopoietic progenitor cells responding to granulocyte colony-stimulating factor or interleukin 3 alone. In contrast, the 75-kDa TNF receptor is essential in mediating inhibition of primitive Lin-Sca-1+ high-proliferative-potential colony-forming cells and inhibition of the total number of proliferative clones of individually cultured Lin-Sca-1+Rh123lo and Lin-Sca-1+Rh123hi cells.
The crystal structure of allosteric chorismate mutase at 2.2-A resolution.
Authors: Xue Y , Lipscomb W N , Graf R , Schnappauf G , Braus G
Published: May 2006

The crystal structure of an allosteric chorismate mutase, the Thr-226-->Ile mutant, from yeast Saccharomyces cerevisiae has been determined to 2.2-A resolution by using the multiple isomorphous replacement method. Solvent-flattening and electron-density modification were applied for phase improvement. The current crystallographic R factor is 0.196. The final model includes 504 of the 512 residues and 97 water molecules. In addition, two tryptophan molecules were identified in the interface between monomers. The overall structure is completely different from the reported structure of chorismate mutase from Bacillus subtilis. This structure showed 71% helices with essentially no beta-sheet structures.
Localization of the long form of beta-1,4-galactosyltransferase to the plasma membrane and Golgi complex of 3T3 and F9 cells by immunofluorescence confocal microscopy.
Authors: Youakim A , Dubois D H , Shur B D
Published: May 2006

beta-1,4-Galactosyltransferase (GalTase) is localized to two subcellular compartments, the Golgi complex, where it participates in cellular glycosylation, and the plasma membrane, where it functions as a receptor for oligosaccharide ligands on opposing cells or in the extracellular matrix. The gene for GalTase encodes two nearly identical proteins that differ only in their N-terminal cytoplasmic domains: both short and long GalTases share an 11-aa cytoplasmic tail, but long GalTase has an additional 13-aa sequence on its cytoplasmic domain. In this study, we investigated the subcellular distribution of endogenous long GalTase in untransfected F9 and 3T3 cells by using confocal microscopy and antibodies specific for the 13-aa sequence unique to long GalTase. Long GalTase was found in the Golgi complex as expected; long GalTase was also found on the plasma membrane in cell-type-specific distributions. In 3T3 cells, long GalTase was evident on the basal surface of cells possessing a migratory phenotype, being concentrated at the leading and trailing edges; nonmigratory cells had little detectable surface immunoreactivity. In F9 cells, long GalTase was localized on the plasma membrane, being concentrated at the apical aspect of intercellular junctions. These results demonstrate that in 3T3 and F9 cells, long GalTase is present on the cell surface in addition to the Golgi complex. The pattern of surface expression shows cell-type specificity that is consistent with GalTase function in cellular interactions.
The DNA-binding domain of the MotA transcription factor from bacteriophage T4 shows structural similarity to the TATA-binding protein.
Authors: Finnin M S , Hoffman D W , White S W
Published: May 2006

The bacteriophage T4 middle-mode transcription factor MotA consists of two domains of approximately equal size. The C-terminal domain has been shown to contain the DNA-binding elements of the molecule, and the N-terminal domain appears to interact with RNA polymerase. A 12.5-kDa fragment of the C-terminal domain (MotCF), comprising residues 105-211 of MotA, was found to be suitable for structural studies by NMR. The 1H and 15N assignments have been made for MotCF by using two-dimensional homonuclear and heteronuclear experiments. A secondary structure has been determined which consists of a six-stranded antiparallel beta-pleated sheet with three alpha-helical segments. The secondary structure of MotCF has a clear similarity to one half of the eukaryotic TATA-binding protein (TBP), which is an intramolecular dimer. Therefore, MotCF may be related to a monomeric ancestral protein of TBP. TBP binds its target DNA in the minor groove by specific interactions with hydrophobic and aromatic residues on the exposed sheet surface of the protein. Similar residues are also present on the beta-sheet surface of MotCF, suggesting that it too binds DNA in the minor groove.
Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.
Authors: Farese R V , Standaert M L , Yamada K , Huang L C , Zhang C , Cooper D R , Wang Z , Yang Y , Suzuki S , Toyota T
Published: May 2006

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.
A system for tissue-specific gene targeting: transgenic mice susceptible to subgroup A avian leukosis virus-based retroviral vectors.
Authors: Federspiel M J , Bates P , Young J A , Varmus H E , Hughes S H
Published: May 2006

Avian leukosis viruses (ALVs) have been used extensively as genetic vectors in avian systems, but their utility in mammals or mammalian cell lines is compromised by inefficient viral entry. We have overcome this limitation by generating transgenic mice that express the receptor for the subgroup A ALV under the control of the chicken alpha sk-actin promoter. The skeletal muscles of these transgenic animals are susceptible to efficient infection by subgroup A ALV. Because infection is restricted to cell lineages that express the transgene, the method has utility for studies of development and oncogenesis and will provide models for tissue-specific gene therapy.
Monozygotic twinning: an evolutionary hypothesis.
Authors: Gleeson S K , Clark A B , Dugatkin L A
Published: May 2006

Monozygotic twinning is rare within populations yet taxonomically widespread. We explore the evolution of monozygotic twinning by modeling an allele in a newly formed offspring that causes it to undergo mitosis and separation to form one or more clones (twins), potentially in conflict with the parents' best interest. The success of this twinning allele in our haploid models depends on the balance of the benefit of increased frequency in the clutch and the cost of reduced survival resulting from limited parental resources. The trait reaches high frequency in a broad range of plausible conditions but also fails to spread or is kept at low frequency in others when the survival cost is high (e.g., in small clutch sizes). Interestingly, there are two reasonable conditions that predict high frequency of the trait but low visibility: random parental abortion and selection for low penetrance. Thus our models suggest reasons why monozygotic twinning might be rare, or alternatively, be common yet appear rare. In addition, we discuss the implications for sex-linked twinning, dizygotic twinning, and twinning by gametes.
A pathway for the biosynthesis of straight and branched, odd- and even-length, medium-chain fatty acids in plants.
Authors: Kroumova A B , Xie Z , Wagner G J
Published: May 2006

Pathways and enzymes of fatty acid synthase-mediated, long-even-chain (generally C16-C20) fatty acid synthesis are well studied, and general metabolism involved in short-chain (C4-C7) fatty acid biosynthesis is also understood. In contrast, mechanisms of medium-chain (C8-C14) fatty acid synthesis are unclear. Recent work suggests involvement of chain-elongation-terminating thioesterases in medium-chain fatty acid formation in oilseeds and animals. We have shown that iso- and anteiso-branched and straight, odd- and even-length, short-chain fatty acids esterified in plant-trichome-gland-produced sucrose esters are synthesized by using carbon skeletons provided by modified branched-chain amino acid metabolism/catabolism. The principal enzymes involved are those catalyzing leucine biosynthesis in all organisms and those leading to short-chain alcohols in mutant yeasts and alkyl acids in Clostridium species (products often serving as mammalian pheromones). Here we provide evidence that C10-C12 straight medium-chain and C10-C12 branched medium-chain acyl acids of tomato, C6-C8 straight-chain acyl acids of Petunia, and C6 and C8 branched acyl acids of Nicotiana glutinosa are formed by alpha-ketoacid elongation without participation of fatty acid synthase-mediated reactions or -independent thioesterases. This different metabolism suggests greater integration of amino acid and fatty acid metabolism than previously considered and provides other avenues to study and manipulate not only straight even-length but also odd- and even-length straight and branched medium-chain fatty acid biosynthesis.
Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing.
Authors: Kim U , Wang Y , Sanford T , Zeng Y , Nishikura K
Published: May 2006

We have cloned human cDNA encoding double-stranded RNA adenosine deaminase (DRADA). DRADA is a ubiquitous nuclear enzyme that converts multiple adenosines to inosines in double-helical RNA substrates without apparent sequence specificity. The A --> I conversion activity of the protein encoded by the cloned cDNA was confirmed by recombinant expression in insect cells. Use of the cloned DNA as a molecular probe documented sequence conservation across mammals and detected a single transcript of 7 kb in RNA of all human tissues analyzed. The deduced primary structure of human DRADA revealed a bipartite nuclear localization signal, three repeats of a double-stranded RNA binding motif, and the presence of sequences conserved in the catalytic center of other deaminases, including a cytidine deaminase involved in the RNA editing of apolipoprotein B. These structural properties are consistent with the enzymatic signature of DRADA, and strengthen the hypothesis that DRADA carries out the RNA editing of transcripts encoding glutamate-gated ion channels in brain.
Certain HLA-DR5 and -DR6 major histocompatibility complex class II alleles are associated with a CD8 lymphocytic host response to human immunodeficiency virus type 1 characterized by low lymphocyte viral strain heterogeneity and slow disease progression.
Authors: Itescu S , Rose S , Dwyer E , Winchester R
Published: May 2006

Either of two structurally related major histocompatibility complex class II alleles, DRB1*1102, which encodes a DR5 specificity, or DRB1*1301, which encodes a DR6 specificity, was found in 67% of individuals responding to human immunodeficiency virus type 1 (HIV-1) infection with a syndrome characterized by persistent circulating and diffusely infiltrative CD8 lymphocytosis (DILS), slow progression to opportunistic infections, and delayed CD4 T-cell depletion. These alleles were present in only 28% of ethnically matched HIV-positive controls (P = 0.001). The frequency of DRB1*1301 was increased in both Blacks and Caucasians with this syndrome, while that of DRBI*1102 was increased only in Blacks, where 80% had either of these alleles. To investigate whether the host response associated with these alleles influences the evolutionary divergence of the HIV-1 genome, sequencing of the envelope V3 loop was performed. This revealed a significantly diminished lymphocyte viral heterogeneity compared with random HIV+ controls matched for CD4 T-cell levels. These results suggest that the immunogenetics of the host influence the nature of the immune response to HIV-1, which may lead to constrained evolution of HIV-1 gene products. Of possible relevance, the alpha-helical third diversity region common to both the DRB1*1102 and DRB1*1301 allelic products was noted to have homology with the C-terminal region of the HIV-1 envelope V3 loop at six of nine consecutive residues. This suggests the possibility that these alleles may bias the anti-HIV T-cell receptor repertoire through a mimicry mechanism.
Targeting of DNA polymerase to the adenovirus origin of DNA replication by interaction with nuclear factor I.
Authors: Armentero M T , Horwitz M , Mermod N
Published: May 2006

Efficient initiation by the DNA polymerase of adenovirus type 2 requires nuclear factor I (NFI), a cellular sequence-specific transcription factor. Three functions of NFI--dimerization, DNA binding, and activation of DNA replication--are colocalized within the N-terminal portion of the protein. To define more precisely the role of NFI in viral DNA replication, a series of site-directed mutations within the N-terminal domain have been generated, thus allowing the separation of all three functions contained within this region. Impairment of the dimerization function prevents sequence-specific DNA binding and in turn abolishes the NFI-mediated activation of DNA replication. NFI DNA-binding activity, although necessary, is not sufficient to activate the initiation of adenovirus replication. A distinct class of NFI mutations that abolish the recruitment of the viral DNA polymerase to the origin also prevent the activation of replication. Thus, a direct interaction of NFI with the viral DNA polymerase complex is required to form a stable and active preinitiation complex on the origin and is responsible for the activation of replication by NFI.
Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations.
Authors: Martinez M A , Vartanian J P , Wain-Hobson S
Published: May 2006

The finding of G-->A hypermutated retroviral genomes in which up to 40% of guanines may be substituted by adenines was proposed to result from the depletion of the intracellular dCTP concentration and suggested a means to hypermutagenize nucleic acids. Using a RNA/reverse transcriptase ratio of approximately 1:30, comparable to that within the retroviral replication complex, G-->A hypermutants were produced in a simple in vitro reaction using highly biased dNTP concentrations--i.e., a low ratio of [dCTP]/[dTTP]. Up to 38% of G residues could be substituted, the proportion being inversely proportional to the concentration of dCTP. As G-->A hypermutation resulted from elongation beyond multiple rG.dT mismatches, U-->C hypermutants resulting from multiple rU.dG mismatches were sought, and found, during cDNA synthesis using low [dATP] and high [dGTP]. Mixed G-->A and U-->C hypermutants could also be produced under conditions of low [dCTP] plus low [dATP] and high [dTTP] plus high [dGTP]. Hypermutagenesis should allow jumping through, and subsequent exploration of, sequence space to a greater degree than heretofore and, in conjunction with genetic screening, might be of use in the search of proteins or ribozymes with novel or enhanced properties.
Fialuridine accumulates in DNA of dogs, monkeys, and rats following long-term oral administration.
Authors: Richardson F C , Engelhardt J A , Bowsher R R
Published: May 2006

Accumulation of the antiviral nucleoside analogue fialuridine (FIAU; 1-(2'-deoxy-2'-fluoro-beta-D-arab-inofuranosyl-5-iodouracil) in genomic DNA was examined with a modified version of a recently developed RIA for FIAU. DNA was obtained from tissues of dogs administered FIAU at 0, 1, 2, or 3 mg/kg of body weight per day for 90 days, monkeys administered FIAU at 0 or 25 mg/kg per day for 30 days, and rats administered FIAU at 0, 255, or 510 mg/kg per day for 70 days. FIAU incorporation was observed in all species. In the rat, FIAU was incorporated into DNA of all tissues examined, with highest concentrations in the liver followed by jejunum, spleen, and heart. FIAU was also incorporated into sperm DNA. Incorporation rates were as high as 11,000 pmol of FIAU per mumol of thymidine or 1 FIAU molecule per 90 thymidine molecules. In dogs and rats, the extent of incorporation was dose-dependent. Across species, FIAU concentrations in DNA were not singly dependent on the total dose administered but also may have been dependent on the duration of exposure. These studies show that FIAU accumulates to high concentrations in genomic DNA of liver as well as other tissues during chronic oral administration and suggest that net accumulation of FIAU in DNA may be a critical step in FIAU-induced toxicity.
Oligodeoxynucleotides antisense to mRNA encoding protein kinase A, protein kinase C, and beta-adrenergic receptor kinase reveal distinctive cell-type-specific roles in agonist-induced desensitization.
Authors: Shih M , Malbon C C
Published: May 2006

The roles of three protein kinases, cyclic AMP-dependent protein kinase (protein kinase A), protein kinase C, and beta-adrenergic receptor kinase (beta ARK), implicated in agonist-induced desensitization of guanine nucleotide-binding protein (G-protein)-coupled receptors were explored in four different cell lines after 48 hr of incubation with oligodeoxynucleotides antisense to the mRNA encoding each kinase. Desensitization of beta 2-adrenergic receptors was analyzed in cell types in which the activities of the endogenous complement of protein kinases A and C and beta ARK were distinctly different. Protein kinase A was necessary for desensitization of rat osteosarcoma cells (ROS 17/2.8), whereas the contribution of beta ARK to desensitization was insignificant. In Chinese hamster ovary cells that stably express beta 2-adrenergic receptors and in smooth muscle cells (DDT1MF-2), oligodeoxynucleotides antisense to beta ARK mRNA nearly abolished desensitization, whereas oligodeoxynucleotides antisense to protein kinase A mRNA attenuated desensitization to a lesser extent. In human epidermoid carcinoma cells (A-431), oligodeoxynucleotides antisense to either protein kinase A mRNA or beta ARK mRNA attenuated agonist-induced desensitization, providing a third scenario in which two kinases constitute the basis for agonist-induced desensitization. In sharp contrast, oligodeoxynucleotides antisense to protein kinase C mRNA were found to enhance rather than attenuate desensitization in DDT1MF-2 and A-431 cell lines, demonstrating counterregulation between prominent protein kinases in desensitization. Using antisense oligodeoxynucleotides to "knock out" target protein kinases in vivo, we reveal distinctive cell-type-specific roles of protein kinase A, protein kinase C, and beta ARK in agonist-induced desensitization.
Human immunodeficiency virus type 1 (HIV-1) inhibition, DNA-binding, RNA-binding, and ribosome inactivation activities in the N-terminal segments of the plant anti-HIV protein GAP31.
Authors: Lee-Huang S , Kung H F , Huang P L , Bourinbaiar A S , Morell J L , Brown J H , Huang P L , Tsai W P , Chen A Y , Huang H I
Published: May 2006

GAP31 (gelonium anti-HIV protein of 31 kDa) is an anti-HIV protein which we have identified and purified from a medicinal plant, Gelonium multiflorum. It is capable of inhibiting HIV-1 infection and replication. GAP31 also exhibits DNA topoisomerase inhibitor activity and RNA N-glycosidase activity. The ability of GAP31 to interrupt both DNA and RNA functions may be related to its multiple antiviral actions. To define the roles of these activities in the anti-HIV action of GAP31, a series of peptides corresponding to the N-terminal segment of GAP31 were synthesized and assayed for the aforementioned activities of the parent molecule. A 33-aa segment (KGATYITYVNFLNELRVKTKPEGNSHGIPSLRK) designated as K10-K42 is the shortest peptide necessary and sufficient for HIV-1 inhibition, DNA and RNA binding, and ribosome inactivation. The peptides were 2-5 orders of magnitude less active than GAP31. Truncation of 19 aa from the C terminus of K10-K42 resulted in the loss of all of these activities. On the other hand, deletion of N-terminal residues to give E23-K42 did not alter ribosome-inactivation activity but eliminated the other activities. These findings permit identification of a 7-aa sequence, KGATYIT, at the N terminus of K10-K42 that is critical for DNA binding and RNA binding, whereas a 9-aa sequence, SHGIPSLRK, at the C terminus is important to ribosome inactivation. Both regions contribute to anti-HIV activity. Histidine at position 35 is critical for all of these activities. The disparity of sequence requirements for inhibition of HIV infection and replication and for ribosome-inactivation activity suggests that the anti-HIV activity of most ribosome-inactivating proteins may not be the result of N-glycosidase activity alone. Mapping the minimal domain of GAP31 offers insights into the rational design of molecular mimetics of anti-HIV drugs.
Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs.
Authors: Gardner P R , Nguyen D D , White C W
Published: May 2006

The effect of hyperoxia on activity of the superoxide-sensitive citric acid cycle enzyme aconitase was measured in cultured human epithelial-like A549 cells and in rat lungs. Rapid and progressive loss of > 80% of the aconitase activity in A549 cells was seen during a 24-hr exposure to a PO2 of 600 mmHg (1 mmHg = 133 Pa). Inhibition of mitochondrial respiratory capacity correlated with loss of aconitase activity in A549 cells exposed to hyperoxia, and this effect could be mimicked by fluoroacetate (or fluorocitrate), a metabolic poison of aconitase. Exposure of rats to an atmospheric PO2 of 760 mmHg or 635 mmHg for 24 hr caused respective 73% and 61% decreases in total lung aconitase activity. We propose that early inactivation of aconitase and inhibition of the energy-producing and biosynthetic reactions of the citric acid cycle contribute to the sequelae of lung damage and edema seen during exposure to hyperoxia.
Transposition of the LINE-like retrotransposon TART to Drosophila chromosome termini.
Authors: Sheen F M , Levis R W
Published: May 2006

TART, a telomere-associated DNA element from Drosophila, is shown in this paper to have structural homology to LINE (long interspersed element)-like retrotransposons and to transpose to broken chromosome ends. TART DNA was detected by in situ hybridization in 7 of 10 independent additions of DNA to a chromosome end. We found evidence that a TART element had transposed to the chromosome end in each of two additions that were examined in detail. From the DNA sequence of a TART element that recently transposed, we infer that TART encodes two proteins having significant sequence similarity to the putative proteins of many LINEs. These results support the hypothesis that TART elements preferentially retrotranspose to the termini of chromosomes as part of the essential process by which Drosophila telomeres are maintained.
Scallop striated and smooth muscle myosin heavy-chain isoforms are produced by alternative RNA splicing from a single gene.
Authors: Nyitray L , Jancsó A , Ochiai Y , Gráf L , Szent-Györgyi A G
Published: May 2006

We report here that the catch and striated adductor muscle myosin heavy-chain (MHC) isoforms of scallop (Argopecten irradians, previously Aequipecten irradians) are generated by alternative RNA splicing from a single gene. Scallop catch muscle cDNA and genomic DNA were amplified by PCR using primers based on the previously sequenced scallop striated muscle MHC cDNA. Mapping of the exon/intron borders and sequencing of a full-length catch muscle MHC in overlapping fragments revealed that the 24-kb gene encodes the MHC polypeptide in 27 exons and that four sets of tandem exon pairs are alternatively spliced into a striated and a catch MHC isoform. An additional alternative exon was identified in catch cDNA and is apparently spliced into a minor MHC isoform. The striated muscle-specific isoform is not expressed in other tissues, whereas the catch-type isoforms were also detected in various smooth muscles, but not in the striated one. Of the alternative exons, exons 5 and 6 encode part of the ATP-binding region and the 25-kDa/50-kDa proteolytic junction; exon 13 encodes part of one of the actin-binding regions and extends to the active site; exon 20 encodes the middle of the rod hinge region; exon 26 in the striated-specific sequence starts with the stop codon, whereas the catch-specific exon codes for an additional 10 residues. Differences between the alternative exons presumably determine the lower ATPase activity of smooth muscle myosin, contribute to the different structure of the striated and smooth muscle thick filaments, and may also be important for the molecular mechanism of the catch phenomenon.
The inositol high-polyphosphate series blocks synaptic transmission by preventing vesicular fusion: a squid giant synapse study.
Authors: Llinás R , Sugimori M , Lang E J , Morita M , Fukuda M , Niinobe M , Mikoshiba K
Published: May 2006

Presynaptic injection of inositol 1,3,4,5-tetraphosphate, inositol 1,3,4,5,6-pentakisphosphate, or inositol 1,2,3,4,5,6-hexakisphosphate--which we denote here the inositol high-polyphosphate series (IHPS)--is shown to block synaptic transmission when injected into the preterminal of the squid giant synapse. This effect is not produced by injection of inositol 1,4,5-trisphosphate. The synaptic block is characterized by a time course in the order of 15-45 min, depending on the injection site in the preterminal fiber; the fastest block occurs when the injection is made at the terminal release site. Presynaptic voltage clamp during transmitter release demonstrates that IHPS block did not modify the presynaptic inward, calcium current. Analysis of synaptic noise at the postsynaptic axon shows that both the evoked and spontaneous transmitter release are blocked by the IHPS. Tetanic stimulation of the presynaptic fiber at frequencies of 100 Hz indicates that block is accompanied by gradual reduction of the postsynaptic response, demonstrating that the block interferes with vesicular fusion rather than with vesicular docking. These results, in combination with the recently demonstrated observation that the IHPS bind the C2B domain in synaptotagmin [Fukada, M., Aruga, J., Niinobe, M., Aimoto, S. & Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211], suggest that IHPS elements are involved in vesicle fusion and exocytosis. In addition, a scheme is proposed in which synaptotagmin triggers transmitter release directly by promoting the fusion of synaptic vesicles with the presynaptic plasmalemma, in agreement with the very rapid nature of transmitter release in chemical synapses.