bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation of renal beta-intercalated cells to alpha-intercalated and principal cells in culture.
Authors: Fejes-Tóth G , Náray-Fejes-Tóth A
Published: May 2006

The renal collecting duct is a heterogenous epithelium consisting of intercalated cells (ICC) and principal cells (PC). The origin of this cellular heterogeneity is not clear. To test the hypothesis that the two cell types might originate from one another, pure populations of ICC (beta subtype) and PC were isolated by fluorescence-activated cell sorting and grown on permeable supports. After the monolayers reached confluence, the expression of ICC- and PC-specific functions and antigens was monitored. Cultures of sorted beta-ICC, in addition to expressing ICC-specific functions (such as an electrogenic H+ secretion) and antigens, progressively acquired PC functions (amiloride-sensitive Na+ transport and K+ secretion). On day 6, cultures of sorted beta-ICC exhibited a lumen-negative transepithelial potential difference of 83 +/- 4 mV and a short circuit current of 107 +/- 15 microA/cm2 and created a lumen-to-bath K+ concentration ratio of approximately 10. The percentage of cells staining with two PC-specific antibodies was 53% and 65%. On the other hand, cultures of sorted PC failed to acquire ICC-specific functions while maintaining PC characteristics. To rule out preferential proliferation of a few contaminating PC as an explanation of these results, we have generated a continuous collecting duct cell line (M-1) originating from mice transgenic for the early region of simian virus 40. Cell lines cloned from M-1 cells exhibit both PC and ICC functions and show mutually exclusive heterogenous expression of PC and ICC antigens, demonstrating a common origin of the two cell types. These data indicate that while beta-ICC in culture can give rise to both alpha-ICC and PC, PC cannot convert to ICC, which raises the possibility that beta-ICC is the stem cell in the renal collecting duct. Differentiation of ICC to PC may explain the cellular heterogeneity in the cortical collecting duct.
Sodium action potentials in the dendrites of cerebellar Purkinje cells.
Authors: Regehr W G , Konnerth A , Armstrong C M
Published: May 2006

We report here that in cerebellar Purkinje cells from which the axon has been removed, positive voltage steps applied to the voltage-clamped soma produce spikes of active current. The spikes are inward, are all-or-none, have a duration of approximately 1 ms, and are reversibly eliminated by tetrodotoxin, a Na channel poison. From cell to cell, the amplitude of the spikes ranges from 4 to 20 nA. Spike latency decreases as the depolarizing step is made larger. These spikes clearly arise at a site where the voltage is not controlled, remote from the soma. From these facts we conclude that Purkinje cell dendrites contain a sufficient density of Na channels to generate action potentials. Activation by either parallel fiber or climbing fiber synapses produces similar spikes, suggesting that normal input elicits Na action potentials in the dendrites. These findings greatly alter current views of how dendrites in these cells respond to synaptic input.
Influence of calorie restriction on oncogene expression and DNA synthesis during liver regeneration.
Authors: Himeno Y , Engelman R W , Good R A
Published: May 2006

Controlling calorie intake (CCI) extends healthful life-span by a mechanism that may involve reduced rates of cell division without detriment to inducible cellular responses. To test whether inducible cellular proliferation is preserved by CCI and whether the mRNA expression levels of oncogenes activated by cell division can be reduced by CCI, we evaluated the effect of dietary energy on the hepatocellular proliferative burst and on oncogene and growth factor mRNA expression induced by partial hepatectomy. Eighty Fischer 344 rats were separated into two dietary groups and were fed semipurified diets for 10 weeks that differed only in calories by 40%. Mean hepatic levels of [3H]thymidine incorporation were greater among CCI animals at 18, 24, 28, and 36 hr after partial hepatectomy. The expression of c-fos and c-Ki-ras mRNAs, activated during hepatic regeneration, was reduced by CCI. Peak expression of c-fos among ad libitum fed controls to levels 4-6 times greater than prehepatectomy levels was not detected among CCI animals. Protracted elevated expression of c-Ki-ras among ad libitum fed animals was foreshortened by CCI. These findings demonstrate that inducible cellular proliferative responses are preserved by CCI and that the mRNA expression levels of c-fos and c-Ki-ras activated during cell division are reduced by controlling dietary energy. Preserved inducible cellular responses and lowered oncogene expression during cell division may be attributes of the healthful protective effect of CCI.
Oxoammonium cation intermediate in the nitroxide-catalyzed dismutation of superoxide.
Authors: Krishna M C , Grahame D A , Samuni A , Mitchell J B , Russo A
Published: May 2006

Dismutation of superoxide has been shown previously to be catalyzed by stable nitroxide compounds. In the present study, the mechanism of superoxide (.O2-) dismutation by various five-membered ring and six-membered ring nitroxides was studied by electron paramagnetic resonance spectrometry, UV-visible spectrophotometry, cyclic voltammetry, and bulk electrolysis. Electron paramagnetic resonance signals from the carbocyclic nitroxide derivatives (piperidinyl, pyrrolidinyl, and pyrrolinyl) were unchanged when exposed to enzymatically generated .O2-, whereas, in the presence of .O2- and reducing agents such as NADH and NADPH, the nitroxides underwent reduction to their respective hydroxylamines. The reaction of 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (Tempol-H) with .O2- was measured and, in agreement with earlier reports on related compounds, the rate was found to be too slow to be consistent with a mechanism of .O2- dismutation involving the hydroxylamine as an intermediate. Voltammetric analyses of the carbocyclic nitroxide derivatives revealed a reversible one-electron redox couple at positive potentials. In contrast, oxazolidine derivatives were irreversibly oxidized. At negative potentials, all of the nitroxides studied exhibited a broad, irreversible reductive wave. The rate of .O2- dismutation correlated with the reversible midpoint redox potential. Bulk electrolysis at positive potentials was found to generate a metastable oxidized form of the nitroxide. The results indicate that the dismutation of .O2- is catalyzed by the oxoammonium/nitroxide redox couple for carbocyclic nitroxide derivatives. In addition to the one-electron mitochondrial reduction pathway, the present results suggest the possibility that cellular bioreduction by a two-electron pathway may occur subsequent to oxidation of stable nitroxides. Furthermore, the cellular destruction of persistent spin adduct nitroxides might also be facilitated by a primary univalent oxidation.
Coevolution of genes and languages revisited.
Authors: Cavalli-Sforza L L , Minch E , Mountain J L
Published: May 2006

In an earlier paper it was shown that linguistic families of languages spoken by a set of 38 populations associate rather strongly with an evolutionary tree of the same populations derived from genetic data. While the correlation was clearly high, there was no evaluation of statistical significance; no such test was available at the time. This gap has now been filled by adapting to this aim a procedure based on the consistency index, and the level of significance is found to be much stronger than 10(-3). Possible reasons for coevolution of strictly genetic characters and the strictly cultural linguistic system are discussed briefly. Results of this global analysis are compared with those obtained in independent local analysis.
Sequence and molecular characterization of human monocyte/neutrophil elastase inhibitor.
Authors: Remold-O'Donnell E , Chin J , Alberts M
Published: May 2006

cDNA encoding human monocyte/neutrophil elastase inhibitor (EI), a M(r) approximately 42,000 protein with serpin-like functional properties, has been sequenced. The 1316-base-pair sequence was obtained from overlapping clones and amplified DNA from libraries of monocyte-like and neutrophil-like cells. Hybridization with EI cDNA identified three EI mRNA species of 1.5, 1.9, and 2.6 kilobases in U937 monocyte-like cells and no hybridizing mRNA in lymphoblastoid cells lacking detectable EI. The cDNA open reading frame encodes a 379-amino acid protein, of which 167 residues were confirmed by tryptic peptides. Although EI may function extracellularly as well as intracellularly, its deduced sequence lacks a typical cleavable N-terminal signal sequence. Sequence analysis established that EI is a member of the serpin superfamily. EI has greatest homology (50.1% identity of amino acids) with plasminogen activator inhibitor 2, also a monocyte protein, and ovalbumin and gene Y, which were previously grouped as an ancient branch of the serpin superfamily. The extent of EI identity with the functionally related serpin alpha 1 antitrypsin is only 30.1%. Sequence alignment indicates that the reactive center P1 residue is Cys-344, consistent with abrogation of elastase inhibitory activity by iodoacetamide and making EI a naturally occurring Cys-serpin. The cleavable bond, Cys-Met, suggests an oxidation-sensitive molecule capable of inhibiting more than one serine protease. Oxidation sensitivity would limit the place of action of EI to the immediate vicinity of carrier cells. The molecular structure will help clarify the likely role of EI in regulating protease action and preventing tissue damage by phagocytic cells.
Cloned human neuropeptide Y receptor couples to two different second messenger systems.
Authors: Herzog H , Hort Y J , Ball H J , Hayes G , Shine J , Selbie L A
Published: May 2006

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system and exhibits a diverse range of important physiological activities, including effects on psychomotor activity, food intake, regulation of central endocrine secretion, and potent vasoactive effects on the cardiovascular system. Two major subtypes of NPY receptor (Y1 and Y2) have been defined by pharmacological criteria. We report here the molecular cloning of a cDNA sequence encoding a human NPY receptor and the corrected sequence for a rat homologue. Analysis of this sequence confirms that the receptor is a member of the G protein-coupled receptor superfamily. When expressed in Chinese hamster ovary (CHO) or human embryonic kidney (293) cells, the receptor exhibits the characteristic ligand specificity of a Y1 type of NPY receptor. In the 293 cell line, the receptor is coupled to a pertussis toxin-sensitive G protein that mediates the inhibition of cyclic AMP accumulation. In the CHO cell line, the receptor is coupled not to the inhibition of adenylate cyclase but rather to the elevation of intracellular calcium. These results demonstrate that second messenger coupling of the NPY-Y1 receptor is cell type specific, depending on the specific repertoire of G proteins and effector systems present in any cell type.
Expression of the cholecystokinin gene in pediatric tumors.
Authors: Friedman J M , Vitale M , Maimon J , Israel M A , Horowitz M E , Schneider B S
Published: May 2006

We have examined a wide range of cultured human tumor cell lines and found that a specific subset of tumors expresses the cholecystokinin (CCK) gene. All neuroepitheliomas (eight) and Ewing sarcoma (eight) cell lines that were tested express CCK RNA. In addition, two of six rhabdomyosarcoma cell lines also express the CCK gene, suggesting that rhabdomyosarcomas are probably heterogenous and that a subset may be similar to Ewing sarcoma and neuroepithelioma. Very few of the positive tumors express completely processed immunoreactive CCK. However, we have used a radioimmunoassay that detects the CCK precursor to demonstrate synthesis of CCK precursor-like peptides by all of the Ewing sarcoma and neuroepithelioma lines that were tested and by the rhabdomyosarcoma cell line that expresses CCK mRNA. These data demonstrate a consistent association of CCK gene expression with a specific group of human neoplasms. The data also add credence to the theory that Ewing sarcoma and neuroepithelioma are derived from the same transformed cell type. Finally, our results suggest that CCK gene expression may serve as a marker to distinguish these tumors, which are considered to be small-round-cell tumors of childhood, from other pediatric tumors.
Inhibition of assimilatory nitrate reductase activity in soil by glutamine and ammonium analogs.
Authors: McCarty G W , Bremner J M
Published: May 2006

Recent work in our laboratory indicated that the inhibitory effect of ammonium (NH4+) on assimilatory nitrate reductase (ANR) activity in soil is not due to NH4+ per se but to glutamine formed by microbial assimilation of NH4+. To test this conclusion, we studied the effects of eight analogs of L-glutamine (L-glutamic acid gamma-methyl ester, L-glutamic acid gamma-hydrazide, L-glutamic acid gamma-hydroxamate, L-glutamic acid gamma-ethyl ester, L-glutamic acid dimethyl ester, L-asparagine, L-aspartic acid beta-methyl ester, and L-aspartic acid beta-hydroxamate) and two analogs of ammonium (hydroxylamine and methylamine) on ANR activity in soil slurries. The studies with the L-glutamine analogs showed that all except L-glutamic acid dimethyl ester inhibited ANR activity in soil. The sharp contrast observed between the strong inhibitory effect of L-glutamic acid gamma-methyl ester on ANR activity and the complete lack of an inhibitory effect with the corresponding dimethyl ester suggests that only the free-acid form of glutamine effectively inhibits ANR activity. The studies with hydroxylamine and methylamine showed that both of these ammonium analogs inhibited ANR activity in soil and that this inhibition was dependent upon glutamine synthetase activity. This dependence indicates that inhibition of ANR activity by hydroxylamine and methylamine was due to formation of the glutamine analogs L-glutamic acid gamma-hydroxamate and L-glutamic acid gamma-methylamide, respectively. These observations support the conclusion that the inhibitory effect of NH4+ on ANR activity in soil is due to glutamine formed by microbial assimilation of NH4+.
Identification of the major soluble cuticular glycoprotein of lymphatic filarial nematode parasites (gp29) as a secretory homolog of glutathione peroxidase.
Authors: Cookson E , Blaxter M L , Selkirk M E
Published: May 2006

We have cloned and identified the major cuticular glycoprotein (gp29) of lymphatic filarial nematode parasites as a homolog of the antioxidant enzyme glutathione peroxidase. The derived amino acid sequence predicted a protein of 25.8 kDa, with an amino-terminal hydrophobic signal peptide and two sites for N-linked glycosylation, consistent with the documented properties of gp29. Transcription of a full-length cDNA in an SP65 vector and subsequent translation of the RNA in reticulocyte lysates in vitro generated a protein of 27 kDa, which was glycosylated upon the addition of pancreatic microsomal membranes. A postulated role for this secreted enzyme could be inhibition of the oxidative burst of leukocytes and neutralization of secondary products of lipid peroxidation, thus providing one explanation for the resistance of these parasites to immune effector mechanisms and their persistence in the mammalian host.
Specific binding of the diphtheria tox regulatory element DtxR to the tox operator requires divalent heavy metal ions and a 9-base-pair interrupted palindromic sequence.
Authors: Tao X , Boyd J , Murphy J R
Published: May 2006

The structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages; however, the regulation of tox expression is controlled by a Corynebacterium diphtheriae-encoded regulatory element, dtxR. The molecular cloning and sequence analysis of dtxR was recently described. Previous studies have suggested that DtxR-mediated regulation of the diphtheria tox operator involves the formation of an iron-repressor complex, which specifically binds to the tox operator. We have expressed and purified DtxR from recombinant Escherichia coli. Immunoblot analysis shows DtxR to be a single M(r) 28,000 protein band in both recombinant E. coli and the C7(-) and C7hm723(-) strains of C. diphtheriae. In addition, we demonstrate that the binding of DtxR to a diphtheria tox promoter/operator probe requires the addition of Mn2+ to the reaction mixture; however, binding may be blocked by addition of the chelator 2,2'-dipyridyl, anti-DtxR antiserum, and excess unlabeled probe to the reaction mixture. Deletion of one of the 9-base-pair inverted repeat sequences from the tox operator results in a loss of DtxR binding. The results presented here demonstrate that regulation of diphtheria toxin expression by DtxR requires direct interaction between this regulatory factor and the tox operator in the presence of a divalent heavy metal ion.
Nonconservative recombination in Escherichia coli.
Authors: Takahashi N K , Yamamoto K , Kitamura Y , Luo S Q , Yoshikura H , Kobayashi I
Published: May 2006

Homologous recombination between two duplex DNA molecules might result in two duplex DNA molecules (conservative) or, alternatively, it might result in only one recombinant duplex DNA molecule (nonconservative). Here we present evidence that the mode of homologous recombination is nonconservative in an Escherichia coli strain with an active RecF pathway (a recBC sbcBC mutant). We employed plasmid substrates that enable us to recover both recombination products. These plasmids carry two mutant alleles of neo gene in direct orientation, two drug-resistance marker genes, and two compatible replication origins. After their transfer to the cells followed by immediate selection for the recombination to neo+, we could recover only one recombination product. A double-strand break at the region of homology increased this nonconservative recombination. If a nonconservative exchange should leave an end, this end may stimulate another exchange. Such "successive half crossing-over events" can explain several recombination-related phenomena in E. coli, including the origin of plasmid linear multimers and of transcribable, nonreplicated recombination products, and also in yeast and mammalian cells.
Multiple mechanisms mediate glucose repression of the yeast GAL1 gene.
Authors: Lamphier M S , Ptashne M
Published: May 2006

Several mechanisms contribute to the glucose repression of the GAL1 gene in Saccharomyces cerevisiae. We show that one mechanism involves the transcriptional down-regulation of the GAL4 gene and a second requires the GAL80 gene. We also examine the contribution of cis-acting negative elements in the GAL1 promoter to glucose repression. In an otherwise wild-type strain disruption of any one of these three mechanisms alleviates repression of GAL1 only 2- to 4-fold. However, in the absence of the other two mechanisms the transcriptional down-regulation of GAL4 is sufficient to repress GAL1 expression 40- to 60-fold and the GAL80-dependent mechanism is sufficient to repress GAL1 expression 20- to 30-fold. These first two mechanisms constitute a functionally redundant system of repression and both must be disrupted in order to abolish glucose repression of GAL1. In contrast, negative elements in the GAL1 promoter are effective in repressing GAL1 expression 2- to 4-fold in glucose medium only when at least one of the other two mechanisms of repression is present. Thus, glucose repression of GAL1 is mediated primarily by the first two mechanisms, whereas the third mechanism supplements repression severalfold.
Developmental failure of chimeric embryos expressing high levels of H-2Dd transplantation antigens.
Authors: Jaffe L , Robertson E J , Bikoff E K
Published: May 2006

The absence of expression of class I products of the major histocompatibility complex at early stages of development is thought to play a key role in maternal tolerance of the fetal allograft. To test this, we developed a strategy that would allow us to describe the consequences of overexpression of the H-2Dd transplantation antigen in the developing embryo. A construct containing the H-2Dd gene under control of the human beta-actin promoter was transfected into pluripotent embryonic stem (ES) cells. Particularly in this case, since overexpression of major histocompatibility complex class I gene products may profoundly affect embryonic development, an important advantage of the ES cell system is the ability to analyze gene expression and study effects on cell growth and differentiation in vitro. ES cells do not constitutively express beta 2-microglobulin. Consistent with this, H-2Dd H chains expressed by ES cell transformants were not associated with beta 2-microglobulin or transported to the cell surface. Significant levels of beta 2-microglobulin and H-2Dd membrane glycoproteins were expressed following differentiation in vitro. H-2Dd-transfected ES cells gave rise to a wide range of differentiated cell types, and there was no evidence to suggest that expression of the introduced H-2Dd gene affects the differentiation abilities of ES cells in vitro. When introduced into blastocysts, H-2Dd-transfected ES cells extensively contribute to embryonic and extraembryonic tissues, but this results in the failure of chimeric conceptuses at midgestation. Considering that transgenic chimeras cannot be rescued by transfer into syngeneic foster females, it seems likely that nonimmunological mechanisms are responsible for these prenatal lethalities.
Estimating substitution rates from molecular data using the coalescent.
Authors: Lundstrom R , Tavaré S , Ward R H
Published: May 2006

A coalescent model is used to estimate the rate at which neutral substitutions occur in a DNA sequence, without the necessity for an independent estimate of divergence times. Given a random sample of molecular sequences from a finite population, the distribution of the time to a common ancestor can be obtained from the coalescent model. With this principle, summary statistics are developed that use the distribution of molecular diversity within the sample to estimate the relative magnitude of nucleotide substitution rates. If, in addition, the effective population size is known, absolute substitution rates can also be estimated. These techniques are illustrated by estimating the transition rates that underlie the evolution of the first 360 nucleotides of the mitochondrial control region in an Amerindian tribal population.
Cloning of a cuticular antigen that contains multiple tandem repeats from the filarial parasite Dirofilaria immitis.
Authors: Poole C B , Grandea A G , Maina C V , Jenkins R E , Selkirk M E , McReynolds L A
Published: May 2006

An unusual antigen composed of tandemly repeated protein units was cloned from the filarial parasite Dirofilaria immitis. The antigen was initially identified by screening a lambda gt11 cDNA library with serum from dogs immunized with irradiated D. immitis third-stage larvae. DNA sequence analysis of the cDNA clone, Di5, revealed a continuous open reading frame composed of two 399-base-pair repeats arranged in tandem. Southern blot analysis of genomic D. immitis DNA showed that the gene coding for Di5 is composed of a tandem array of 25-50 copies of this same 399-base-pair repeat. Antiserum raised against recombinant Di5 protein detected a protein "ladder," from about 14 to greater than 200 kDa with steps approximately 15 kDa apart, on immunoblots of D. immitis extract. Metabolic labeling of adult parasites with [35S]methionine showed that Di5 is synthesized as a large precursor that is subsequently cleaved to produce the ladder-like array. These results suggest that the characteristic ladder is created by proteolytic cleavage of the precursor at the same site in each monomer. The Di5 antigen was localized to the cuticle and hypodermis of adult D. immitis by immunoelectron microscopy. Both male and female parasites were found to release Di5 when cultured in vitro. DNA hybridization analysis demonstrated that Di5 is a member of a gene family present in many filarial parasites that infect both animal and human populations.
Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid.
Authors: Lin L L , Lin A Y , Knopf J L
Published: May 2006

Cytosolic phospholipase A2 (cPLA2) binds to natural membrane vesicles in a Ca(2+)-dependent fashion, resulting in the selective release of arachidonic acid, thus implicating cPLA2 in the hormonally regulated production of eicosanoids. Here we report that the treatment of Chinese hamster ovary (CHO) cells overexpressing cPLA2 with ATP or thrombin resulted in an increased release of arachidonic acid as compared with parental CHO cells, demonstrating the hormonal coupling of cPLA2. In contrast, CHO cells overexpressing a secreted form of mammalian PLA2 (sPLA2-II) failed to show any increased hormonal responsiveness. Interestingly, we have noted that the activation of cPLA2 with a wide variety of agents stimulates the phosphorylation of cPLA2 on serine residues. Pretreatment of cells with staurosporin blocked the ATP-mediated phosphorylation of cPLA2 and strongly inhibited the activation of the enzyme. Increased cPLA2 activity was also observed in lysates prepared from ATP-treated cells and was sensitive to phosphatase treatment. These results suggest that in addition to Ca2+, the phosphorylation of cPLA2 plays an important role in the agonist-induced activation of cPLA2.
Complexity of sea urchin embryo nuclear proteins that contain basic domains.
Authors: Harrington M G , Coffman J A , Calzone F J , Hood L E , Britten R J , Davidson E H
Published: May 2006

We describe a quantitative two-dimensional gel electrophoretic analysis of nuclear extract from 24-hr sea urchin embryos. The extract was fractionated by using a weak cation-exchange resin, and eight known DNA-binding proteins were shown to be entirely included in a salt eluate that releases proteins containing basic domains. This fraction and a lower-salt fraction containing the majority of the protein species were mapped two-dimensionally by using new algorithms that permit reproducible spot identification, storage of intensity and map-position data, and subtractive comparison of one pattern with respect to another. By reference to a previously characterized DNA-binding factor, spot intensity could be interpreted in terms of the number of molecules per embryo nucleus. A map was constructed displaying all nuclear proteins containing basic domains that are present within the concentration range per nucleus of a set of known DNA-binding factors of the sea urchin embryo. The map includes 265 spots that fulfill both of these criteria, probably representing about 100 different protein species.
A microsomal endoprotease that specifically cleaves isoprenylated peptides.
Authors: Ma Y T , Rando R R
Published: May 2006

A microsomal enzymatic activity is described that can specifically cleave the tetrapeptide N-acetyl-S-farnesyl-L-Cys-L-Val-L-Ile-L-Ser between the isoprenylated cysteine residue and the valine residue. Km and Vmax values are measured as 5.8 microM and 251 pmol/min per mg of protein, respectively. Proteolytic cleavage of the substrate is stereospecific because the substitution of a farnesylated D-cysteine residue for the L-amino acid leads to the abolition of substrate activity. A free carboxyl-terminal group is also required for substrate activity because methyl esterification renders the substrate inert. The tripeptide N-acetyl-S-farnesyl-L-Cys-L-Val-L-Ile and the dipeptide N-acetyl-S-farnesyl-L-Cys-L-Val are also hydrolyzed by the protease. Again, stereospecificity is observed at the isoprenylated residue. Hydrolysis of the farnesylated tetrapeptide is not inhibited by a 5-fold excess of the nonfarnesylated tetrapeptide, suggesting that isoprenylation is important for substrate activity. This activity is probably the same as the proteolytic activity proposed to cleave isoprenylated proteins terminating in a Cys-Ali-Ali-Xaa motif, where Ali refers to aliphatic amino acid. These proteins include the ras family of G proteins and the heterotrimeric G proteins. Proteolytic maturation of these essential isoprenylated signal-transducing elements is a key step in their activation.
Murine Hox-1.11 homeobox gene structure and expression.
Authors: Tan D P , Ferrante J , Nazarali A , Shao X , Kozak C A , Guo V , Nirenberg M
Published: May 2006

The Hox-1.11 gene encodes a protein 372 amino acid residues long that contains a conserved pentapeptide, a homeodomain, and an acidic region. The amino acid sequence of the homeodomain of Hox-1.11 is identical to that of Hox-2.8, and the N-terminal and C-terminal regions of Hox-1.11 are similar to those of human HOX2H, which is the equivalent of murine Hox-2.8. The Hox-1.11 gene was shown to reside on murine chromosome 6, which contains the Hox-1 cluster of homeobox genes. One species of Hox-1.11 poly(A)+ RNA approximately 1.7 kb long was detected in mouse embryos, which is most abundant in 12-day-old embryos and progressively decreases during further embryonic development. The most anterior expression of Hox-1.11 poly(A)+ RNA in 12- to 14-day-old mouse embryos was shown by in situ hybridization to be in the mid and posterior hindbrain. Hox-1.11 poly(A)+ RNA also is expressed in the VII and VIII cranial ganglia, spinal cord, spinal ganglia, larynx, lungs, vertebrae, sternum, and intestine.
Disruption of a binding site for hepatocyte nuclear factor 4 results in hemophilia B Leyden.
Authors: Reijnen M J , Sladek F M , Bertina R M , Reitsma P H
Published: May 2006

Hemophilia B Leyden is an X chromosome-linked bleeding disorder characterized by very low plasma levels of blood coagulation factor IX (fIX) during childhood. After puberty, plasma fIX levels gradually rise to a maximum of 60% of normal, probably under the influence of testosterone. Single point mutations in the fIX promoter region of hemophilia B Leyden patients have been reported at -20, -6, -5, +8 and +13. In addition, one promoter mutation (G----C at -26) has been detected that abolishes fIX expression throughout life (M. Ludwig, personal communication). We examined how one of the hemophilia B Leyden mutations (T----A at -20) and the G----C mutation at -26 interfere with fIX gene transcription. We report that the wild-type promoter of the human fIX gene contains a binding site (at nucleotides -34 to -10) for hepatocyte nuclear factor 4 (HNF-4), a member of the steroid hormone receptor superfamily of transcription factors. The binding of HNF-4 is disrupted by both the T----A mutation at -20 and the G----C mutation at -26. Whereas HNF-4 transactivates the wild-type promoter sequence in liver (HepG2) and non-liver (HeLa) cell types quite well, it transactivates the -20 mutated promoter to only a limited extent and the -26 mutated promoter not at all. These data suggest that HNF-4 is a major factor controlling fIX expression in the normal individual and that its inability to bind efficiently to the -20 T----A and the -26 G----C mutated promoter sequence results in hemophilia. Further, the severity of the hemophilia phenotype appears to be directly related to the degree of disruption of HNF-4 binding and transactivation.
MT-III, a brain-specific member of the metallothionein gene family.
Authors: Palmiter R D , Findley S D , Whitmore T E , Durnam D M
Published: May 2006

A third member of the metallothionein (MT) gene family, designated MT-III, was cloned by virtue of its homology to a human protein that was shown previously to inhibit neuronal survival in culture and to be deficient in the brains of people with Alzheimer disease. Human and mouse MT-IIIs have two insertions relative to all other known mammalian MTs: a threonine after the fourth amino acid and a block of six amino acids near the carboxyl terminus. The genes encoding MT-III resemble all other mammalian MT genes in their small size and exon/intron organization. The MT-III genes are closely linked to the other functional MT genes on human chromosome 16 and mouse chromosome 8. Mouse MT-III gene expression appears to be restricted to brain; in addition, it fails to respond to zinc, cadmium, dexamethasone, or bacterial endotoxin in vivo, thereby distinguishing MT-III from other known MTs.
Developmental expression of surface antigen genes in the parasitic ciliate Ichthyophthirius multifiliis.
Authors: Clark T G , McGraw R A , Dickerson H W
Published: May 2006

A 1.2-kilobase (kb) cDNA encoding a major surface antigen of the holotrich ciliate Ichthyophthirius multifiliis (an obligate parasite of fish) has been isolated and used as a probe to examine the expression of immobilization antigen (i-antigen) genes in this system. The cDNA encodes a predicted protein of 394 amino acids with a tandemly repeated structure characteristic of the i-antigens of the related free-living ciliates Paramecium and Tetrahymena. As shown by Northern hybridization analysis with both total and poly(A)+ RNAs, the 1.2-kb cDNA recognizes distinct transcripts of 1.6 and 1.9 kb which are differentially expressed through the parasite life cycle. During the transition from the host-associated trophozoite stage to the infective tomite stage, steady-state levels of the 1.9-kb RNA undergo a marked increase of greater than or equal to 50-fold, while the 1.6-kb transcript increases only slightly. The absolute amounts of RNA encoding the i-antigen have been quantitated and were found to reach extremely high levels equivalent to approximately 6% of the poly(A)+ RNA of I. multifiliis tomites. Southern hybridization analysis with I. multifiliis genomic DNA suggests that at least two genes encode the i-antigen transcripts. In experiments to examine the effects of temperature on the expression of I. multifiliis i-antigen genes, levels of the 1.6- and 1.9-kb transcripts were found to remain relatively constant in cells maintained at different temperature extremes. These studies indicate that genes encoding i-antigens of I. multifiliis are developmentally regulated, and they suggest the existence of alternative mechanisms for the control of surface antigen expression in ciliates.
Three-dimensional scroll waves organize Dictyostelium slugs.
Authors: Siegert F , Weijer C J
Published: May 2006

To test the hypothesis that periodic signals and chemotaxis direct later morphogenesis in Dictyostelium discoideum, we investigated cell behavior and cell movement in slugs. Trails of neutral red-stained prestalk and anterior-like cells were recorded by high-resolution digital image processing. Neutral red-stained anterior-like cells in the prespore zone of slugs move straight forward in the direction of slug migration and, furthermore, show coherent periodic cell movement. In contrast, cells in the prestalk zone move along completely different trajectories. Prestalk cells move perpendicular to the direction of slug migration; that is, they rotate around the tip. The cell movement data show that the chemotactic signal in the slug propagates as a three-dimensional scroll wave in the prestalk zone and as a planar wave in the prespore zone. The different behavior of prestalk and prespore cells is most likely caused by a difference in the oscillatory properties of the two cell types. We provide evidence that the slug stage of Dictyostelium behaves like an excitable system and that a (twisted) scroll wave organizes slug formation and migration.
Long-chain fatty acids activate calcium channels in ventricular myocytes.
Authors: Huang J M , Xian H , Bacaner M
Published: May 2006

Nonesterified fatty acids accumulate at sites of tissue injury and necrosis. In cardiac tissue the concentrations of oleic acid, arachidonic acid, leukotrienes, and other fatty acids increase greatly during ischemia due to receptor or nonreceptor-mediated activation of phospholipases and/or diminished reacylation. In ischemic myocardium, the time course of increase in fatty acids and tissue calcium closely parallels irreversible cardiac damage. We postulated that fatty acids released from membrane phospholipids may be involved in the increase of intracellular calcium. We report here that low concentrations (3-30 microM) of each long-chain unsaturated (oleic, linoleic, linolenic, and arachidonic) and saturated (palmitic, stearic, and arachidic) fatty acid tested induced multifold increases in voltage-dependent calcium currents (ICa) in cardiac myocytes. In contrast, neither short-chain fatty acids (less than 12 carbons) or fatty acid esters (oleic and palmitic methyl esters) had any effect on ICa, indicating that activation of calcium channels depended on chain length and required a free carboxyl group. Inhibition of protein kinases C and A, G proteins, eicosanoid production, or nonenzymatic oxidation did not block the fatty acid-induced increase in ICa. Thus, long-chain fatty acids appear to directly activate ICa, possibly by acting at some lipid sites near the channels or directly on the channel protein itself. We suggest that the combined effects of fatty acids released during ischemia on ICa may contribute to ischemia-induced pathogenic events on the heart that involve calcium, such as arrhythmias, conduction disturbances, and myocardial damage due to cytotoxic calcium overload.
N-methyl-D-aspartate receptor-mediated neuroprotection in cerebellar granule cells requires new RNA and protein synthesis.
Authors: Marini A M , Paul S M
Published: May 2006

Cerebellar granule cells are susceptible to the excitotoxin glutamate, which acts at N-methyl-D-aspartate (NMDA) receptors, as well as the neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+), the active cytotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Paradoxically, preincubation of cultured cerebellar granule cells with low concentrations of NMDA or glutamate markedly antagonizes the neurotoxicity resulting from subsequent exposure to toxic concentrations of either MPP+ or glutamate. The neuroprotective effects of NMDA and glutamate against MPP+ toxicity are observed at agonist concentrations as low as 1 microM, are blocked by specific NMDA receptor antagonists, and require at least 30 min to develop fully. Moreover, NMDA receptor-mediated neuroprotection is prevented by the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cycloheximide. Thus, in cerebellar granule cells activation of NMDA receptors by glutamate can result in either neurotoxicity or neuroprotection, depending on the apparent degree of receptor stimulation. NMDA receptor-mediated neuroprotection requires new RNA and protein synthesis and therefore appears to be mediated by the expression of a neuroprotective protein(s). These data demonstrate the presence of an active NMDA receptor-mediated and transcriptionally directed neuroprotective mechanism in cerebellar granule cells.
Coinfection with multiple strains of the Epstein-Barr virus in human immunodeficiency virus-associated hairy leukoplakia.
Authors: Walling D M , Edmiston S N , Sixbey J W , Abdel-Hamid M , Resnick L , Raab-Traub N
Published: May 2006

Epstein-Barr virus DNA was analyzed from specimens of hairy leukoplakia, an oral lesion that occurs in patients infected with the human immunodeficiency virus. The simultaneous presence of both type 1 and type 2 Epstein-Barr virus was demonstrated by Southern blot analysis and polymerase chain reaction assay. Restriction fragment length polymorphisms in the BamHI WYH region and in clones of the EcoRI C region suggested the presence of multiple strains of type 1 and type 2 viruses. The demonstration of multiple variably sized BamHI H fragments on Southern blot analysis and cloning of the EBNA-2 gene coding region also suggested the presence of multiple viral strains or variants coinfecting hairy leukoplakia. Recombination of the viral genome in and around the EBNA-2 gene apparently generated viral variants that replicated efficiently, one of which appeared to increase in abundance in a lesion over time. These data indicate that hairy leukoplakia involves coinfection with multiple strains of replicating Epstein-Barr virus and the endogenous generation of viral variants, some of which have mutations of the EBNA-2 gene.
Competitive inhibition of nitric oxide synthase prevents the cortical hyperemia associated with peripheral nerve stimulation.
Authors: Northington F J , Matherne G P , Berne R M
Published: May 2006

With combined microdialysis and hydrogen clearance techniques for simultaneous local delivery of drugs and blood-flow measurement in the rat hindlimb sensory-motor cortex, we examined the role of nitric oxide in cerebral blood-flow regulation during sciatic nerve stimulation. Infusion of 1 mM nitric oxide synthase antagonist, N eta-nitro-L-arginine methyl ester (L-NAME), blocked the cortical blood-flow response to sciatic nerve stimulation (152 +/- 43 ml.min-1.100 g-1 of tissue in controls and 73 +/- 11 ml.min-1.100 g-1 in the presence of L-NAME; P less than 0.05). Addition of 10 mM L-arginine to the dialysate containing L-NAME partially restored the hyperemic response to nerve stimulation (125 ml.min-1.100 g-1). L-NAME also produced a decrease in baseline cerebral blood flow when compared with the control (66 +/- 14 ml.min-1.100 g-1 vs. 93 +/- 25 ml.min-1.100 g-1). We conclude that nitric oxide from activated neurons participates in the local regulation of cortical blood flow in response to sciatic nerve stimulation and also in the maintenance of basal cortical blood flow.
NADPH-dependent beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms.
Authors: Smeland T E , Nada M , Cuebas D , Schulz H
Published: May 2006

The mitochondrial metabolism of 5-enoyl-CoAs, which are formed during the beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms, was studied with mitochondrial extracts and purified enzymes of beta-oxidation. Metabolites were identified spectrophotometrically and by high performance liquid chromatography. 5-cis-Octenoyl-CoA, a putative metabolite of linolenic acid, was efficiently dehydrogenated by medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cis-octadienoyl-CoA, which was isomerized to 3,5-octadienoyl-CoA either by mitochondrial delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) or by peroxisomal trifunctional enzyme. Further isomerization of 3,5-octadienoyl-CoA to 2-trans-4-trans-octadienoyl-CoA in the presence of soluble extracts of either rat liver or rat heart mitochondria was observed and attributed to a delta 3,5,delta 2,4-dienoyl-CoA isomerase. Qualitatively similar results were obtained with 2-trans-5-trans-octadienoyl-CoA formed by dehydrogenation of 5-trans-octenoyl-CoA. 2-trans-4-trans-Octadienoyl-CoA was a substrate for NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34). A soluble extract of rat liver mitochondria catalyzed the isomerization of 2-trans-5-cis-octadienoyl-CoA to 2-trans-4-trans-octadienoyl-CoA, which upon addition of NADPH, NAD+, and CoA was chain-shortened to hexanoyl-CoA, butyryl-CoA, and acetyl-CoA. Thus we conclude that odd-numbered double bonds, like even-numbered double bonds, can be reductively removed during the beta-oxidation of polyunsaturated fatty acids.
Five subtypes of type A gamma-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies.
Authors: Fritschy J M , Benke D , Mertens S , Oertel W H , Bachi T , Möhler H
Published: May 2006

The extraordinary structural diversity of subunits forming type A gamma-aminobutyric acid (GABAA) receptors in the brain is expected to give rise to different modes of GABAergic synaptic inhibition and different profiles of modulatory drugs effective in anxiolytic, hypnotic, and antiepileptic therapy. To identify receptor subtypes in situ, the most prevalent subunits were visualized by double and triple immunofluorescence staining in rat brain, using polyclonal antibodies to the alpha 1, alpha 3, and gamma 2 subunits and a monoclonal antibody to locate both the beta 2 and the beta 3 subunit. At both cellular and subcellular levels five distinct patterns of subunit colocalization were identified: I, alpha 1 beta 2,3 gamma 2; II, alpha 3 beta 2,3 gamma 2; III, alpha 1 alpha 3 beta 2,3 gamma 2; IV, alpha 3 gamma 2; and V, alpha 1 alpha 3 gamma 2. As analyzed by confocal laser microscopy, different subunits displayed the same local variations of staining intensity ("hot spots") along the plasma membrane. The covisualized subunits appear therefore to be coassembled in receptor subtypes. Most neurons expressed only a single major receptor subtype with no apparent distinction between synaptic and extrasynaptic sites. However, in some neurons, most notably in Purkinje cells, the subunit composition varied between the soma and the dendrites, pointing to the existence of receptor heterogeneity within single neurons. Furthermore, different populations of neurons may be characterized by particular receptor subtypes. Cells displaying alpha 1-subunit immunoreactivity were mostly identified as GABAergic, whereas monoaminergic neurons displayed intense alpha 3-subunit immunoreactivity but virtually no alpha 1-subunit immunoreactivity. The allocation of defined GABAA receptor subtypes to identified neurons opens the way for a functional analysis of receptor heterogeneity.
Copper deficiency reversibly impairs DNA synthesis in activated T lymphocytes by limiting interleukin 2 activity.
Authors: Bala S , Failla M L
Published: May 2006

The essentiality of adequate copper (Cu) nutriture for normal T-cell function in laboratory and domestic animals is well established. However, specific biochemical roles of Cu in the maturation and activation of T cells have not been defined. Previous work showed that when cultures of splenic mononuclear cells (MNCs) from Cu-deficient rats were exposed to T-cell mitogens, DNA synthesis was markedly reduced despite normal up-regulation of interleukin 2 (IL-2) receptors, transferrin receptors, and class II major histocompatibility complex molecules. In the present study, IL-2 activity in PHA-treated cultures of MNCs from Cu-deficient rats was 40-50% that of controls as determined by bioassay. Addition of rat IL-2 to phytohemagglutinin-treated cultures of MNCs from Cu-deficient rats increased blastogenic activity to control levels, demonstrating that Cu deficiency does not inhibit transition of quiescent cells to the competence phase of the activation process. Moreover, supplementation of MNC cultures from Cu-deficient rats with physiological levels of Cu enhanced IL-2 activity and DNA synthesis in response to phytohemagglutinin. These data indicate that IL-2 activity in cultures of activated splenic T lymphocytes from Cu-deficient rats is insufficient for optimal blastogenesis.
Presence of a member of the Tc1-like transposon family from nematodes and Drosophila within the vasotocin gene of a primitive vertebrate, the Pacific hagfish Eptatretus stouti.
Authors: Heierhorst J , Lederis K , Richter D
Published: May 2006

Molecular cloning of the vasotocin gene of a cyclostome, the Pacific hagfish Eptatretus stouti, reveals, in contrast to other known members of the vertebrate vasopressin/oxytocin hormone gene family, an unusual exon-intron organization. Although the location of three exons and two introns is conserved, an additional intron is present 5' of the coding region of the hagfish gene. The third intron, which is greater than 14 kilobase pairs in size, contains on the opposite DNA strand to that encoding vasotocin an open reading frame exhibiting striking similarity to the putative transposase of Tc1-like nonretroviral mobile genetic DNA elements, so far reported only from nematodes and Drosophila. The hagfish element, called Tes1, is flanked by inverted terminal repeats representing an example of the existence of a typical inverted terminal-repeat transposon within vertebrates. The presence of Tc1-like elements in nematodes, Drosophila, and cyclostomes indicates that these genetic elements have a much broader phylogenetic distribution than hitherto expected.