bluuebunny/crossref_metadata_2025 huggingface dataset:
Different
a
alleles of
Ustilago maydis
are necessary for maintenance of filamentous growth but not for meiosis
Authors: Banuett Flora , Herskowitz Ira
Published: May 2006

Two loci (the incompatibility or mating type loci),
a
and
b
, govern the life cycle of
Ustilago maydis
, a fungal pathogen of corn.
U. maydis
diploids heterozygous at both
a
and
b
(
a
[unk]
b
[unk]) form mycelial colonies (Fuz
+
phenotype), induce tumors, and produce teliospores able to undergo meiosis. We report here the isolation and characterization of nonmycelial (Fuz
-
) derivatives. These Fuz
-
diploids have allowed us to examine the requirement of
a
[unk] and
b
[unk] in maintenance of filamentous growth and tumor-inducing ability. The Fuz
-
diploids are of four classes: two are inferred to be homozygous for
b
(
a
[unk]
b
=); the other two are shown to be homozygous for
a
(
a
=
b
[unk]). These observations confirm the requirement for
b
[unk] and reveal the requirement for
a
[unk] in filamentous growth.
U. maydis
is thus like other basidiomycetes that have two loci governing growth of the dikaryotic filament. The mating behavior of the Fuz
-
diploids indicates that heterozygosity at
a
or
b
does not block mating. Although both
a
=
b
[unk] and
a
[unk]
b
= diploids are nonmycelial, they differ in that
a
[unk]
b
= diploids are nonpathogenic, whereas
a
=
b
[unk] diploids are pathogenic and produce teliospores able to undergo meiosis. These findings substantiate previous, more limited observations. They demonstrate that ability to induce tumors and produce normal teliospores requires different alleles at
b
but not at
a
.
Mass spectrometric charting of bovine posterior/intermediate pituitary peptides.
Authors: Feistner G J , Højrup P , Evans C J , Barofsky D F , Faull K F , Roepstorff P
Published: May 2006

The feasibility for charting neuropeptides in neuroendocrine tissues on the basis of the universal property and inherent specificity of their molecular weights was explored. As a model, a comprehensive MS analysis of extractable peptides from bovine posterior/intermediate pituitary was performed. Two suitable MS techniques--namely, plasma-desorption time-of-flight and fast atom bombardment MS--were evaluated, and each method could identify more than 20 peptides, including N-terminally acetylated and C-terminally amidated species. In toto these peptides account for almost the entire lengths of propressophysin, prooxyphysin, and proopiomelanocortin. Some of the experimentally determined molecular weights did not match any known peptides. Three of these species were identified as acidic joining peptide (4-24) [proopiomelanocortin(83-103)], C-terminal glycopeptide(22-39) [propressophysin(130-147)], and glycosylated C-terminal glycopeptide(1-19) [propressophysin(109-127)] by conventional sequence analysis.
Induction of an antioxidant protein of Saccharomyces cerevisiae by O2, Fe3+, or 2-mercaptoethanol.
Authors: Kim I H , Kim K , Rhee S G
Published: May 2006

A soluble 27-kDa protein from Saccharomyces cerevisiae specifically prevents the inactivation of various enzymes caused by a nonenzymatic Fe3+/O2/thiol mixed-function oxidation system but not by mixed-function oxidation systems in which the thiol component is replaced by another electron donor-e.g., ascorbate. In this report, using a 125I-labeled monospecific antibody against the 27-kDa protein, we measured changes in the 27-kDa protector protein in response to changes in oxidative stress and heat shock. With a shift from an anaerobic (95% N2/5% CO2) to a hyperaerobic (95% O2/5% CO2) atmosphere, a 3-fold increase was observed. This increase was prevented by cycloheximide, indicating that the induction requires new protein synthesis. The antioxidant protein synthesis was also significantly enhanced by the addition of either 2-mercaptoethanol or Fe3+ to the growth medium. Radioimmunoassay results also show that the antioxidant protein is an abundant protein, as it constitutes 0.7% of total soluble protein from yeast grown aerobically. Immunoblotting experiments revealed that rat tissues also contain a 27-kDa protein that can be specifically recognized by antibodies against the yeast protein. These results suggest that in vivo induction in yeast of the 27-kDa protein may represent an adaptive response that evolved to protect cells against damage caused by thiol-dependent mixed-function oxidation systems, and the antioxidant protein is conserved in mammalian tissues. A heat shock applied to yeast did not cause any significant increases in the concentration of the 27-kDa protein.
20S small nuclear ribonucleoprotein U5 shows a surprisingly complex protein composition.
Authors: Bach M , Winkelmann G , Lührmann R
Published: May 2006

U5 small nuclear ribonucleoprotein (snRNP), purified from HeLa nuclear extracts (splicing extracts), shows a complex protein composition. In addition to the snRNP proteins B', B, D, D', E, F, and G, which are present in each of the major snRNPs U1, U2, U4/U6, and U5, U5 snRNP contains a number of unique proteins characterized by apparent molecular masses of 40, 52, 100, 102, 116, and 200 (mostly a double band) kDa. The latter set of proteins may be regarded as U5-specific for the following reasons. They are not only eluted specifically, together with snRNP particles, from anti-2,2,7-trimethylguanosine immunoaffinity columns by 7-methylguanosine, they also cofractionate with U5 snRNP during chromatography and, most importantly, in glycerol gradient centrifugation. These U5 snRNP particles show a high sedimentation constant of about 20S. U5 snRNPs that lack the U5-specific proteins are also found in nuclear extracts but have (in comparison) a lower sedimentation value of only 8-10S. Autoimmune sera from patients with systemic lupus erythematosus were identified that, on immunoblots with purified U5 snRNP proteins, reacted selectively with the 100- or 200-kDa proteins. This indicates that at least the high molecular mass U5-specific proteins are structurally distinct and not derived one from the other by proteolytic degradation. The existence of so many unique proteins in the U5 snRNP suggests that this snRNP particle may exert its function during splicing mainly by virtue of its protein components.
Evolutionary conserved modules associated with zinc fingers in Xenopus laevis.
Authors: Knöchel W , Pöting A , Köster M , el Baradi T , Nietfeld W , Bouwmeester T , Pieler T
Published: May 2006

Many DNA-binding proteins that are involved in the differential regulation of gene expression are composed of multiple discrete modules. Association of the homeobox-encoded helix-turn-helix DNA-binding motif with conserved modules, such as the paired box or the POU domain, has led to the definition of structurally and functionally related subfamilies of regulatory proteins. The zinc finger, which is the second major nucleic acid-binding motif characterized to date, defines large multigene families in higher eukaryotes; we have isolated more than 100 Xenopus finger protein-encoding cDNAs and in this study we show that at least 10 of these clones share extensive sequence homologies in a region of more than 200 amino acids in the N-terminal nonfinger portion of the predicted proteins, which is connected to variable finger clusters. We refer to this element as a finger-associated boxes (FAX) domain. Cross-hybridization with human genomic DNA indicates that the finger-associated boxes domain is evolutionary conserved. Northern blot analysis shows that the corresponding genes are differentially expressed in the course of early Xenopus embryogenesis.
Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase.
Authors: Mustelin T , Coggeshall K M , Altman A
Published: May 2006

T lymphocytes express a tyrosine protein kinase (TPK; protein-tyrosine kinase; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112), pp56lck that is encoded by the lck protooncogene. This TPK was recently found to be associated with the intracellular domain of the T-cell surface glycoproteins, CD4 and CD8, suggesting that it plays an important role in T-cell development and activation. We have studied the regulation of pp56lck and found that this kinase can be rapidly activated by an endogenous mechanism present in T-lymphocyte membranes. This activation was sensitive to sodium orthovanadate and O-phosphotyrosine, consistent with the involvement of a phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphatase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) in pp56lck activation. Based on a recent report demonstrating that CD45, the leukocyte common antigen, is a membrane-bound PTPase, we analyzed its role in pp56lck activation. CD45 was found to be the major (greater than 90%) PTPase in membranes of the murine T-lymphoma line BW5147. Moreover, activation of pp56lck was undetectable in a mutant BW5147 line lacking CD45 expression (and the associated PTPase activity). In contrast, activation of pp56lck was readily detected in the wild-type lymphoma line. More important, when immunoprecipitated CD45 was added to pp56lck, the TPK activity of the latter increased greater than 2-fold within minutes. This effect of CD45 was completely blocked by sodium orthovanadate. These findings indicate an important role for the CD45 PTPase in pp56lck activation. This role could be mediated by direct dephosphorylation of a regulatory tyrosine residue in pp56lck.
Comparative 13C and 31P NMR assessment of altered metabolism during graded reductions in coronary flow in intact rat hearts.
Authors: Weiss R G , Chacko V P , Glickson J D , Gerstenblith G
Published: May 2006

13C NMR spectroscopy may offer a unique ability to characterize the metabolic response to graded reduction in coronary flow since it allows repeated, nondestructive identification of products of intermediary metabolism in the same heart. The sensitivity of 13C parameters of glucose metabolism was compared with changes in levels of phosphocreatine, ATP, and pH as determined by 31P NMR in the intact, beating rat heart model during graded reductions in coronary flow. Experiments were performed during 60 min of perfusion with [1-13C]glucose (5 mM) at normal flow (15 ml/min) and at the reduced flow rates of 5 and 2 ml/min. During flow at 5 ml/min, isovolumic developed pressure fell to 51 +/- 4% of control. Although phosphocreatine, ATP, and pH were not changed, [3-13C]lactate was increased (1.46 +/- 0.12 mumol/g of wet weight vs. 0.63 +/- 0.08 during normal flow). In addition, the time to 50% maximum enrichment of [2-13C]glutamate was prolonged (17 +/- 1 min vs. 9 +/- 1 min during normal flow), indicating that glucose-supported flux through the tricarboxylic acid (TCA) cycle was decreased. The relative anaplerotic contribution to citrate synthase-supported TCA flux was increased from 6% to 35%. These 13C metabolic changes could not be reproduced by reduced [1-13C]glucose delivery in the absence of ischemia, although similar reduced TCA flux indices were reproduced in additional hearts when workload was reduced by low calcium (0.7 mM) perfusion. Therefore, the information provided by 13C NMR spectroscopy can be a more sensitive indicator of flow-induced alterations in cardiac metabolism than that provided by the much more commonly used 31P NMR technique.
Mismatch-specific 3'----5' exonuclease associated with the mitochondrial DNA polymerase from Drosophila embryos.
Authors: Kaguni L S , Olson M W
Published: May 2006

The mitochondrial DNA polymerase from Drosophila embryos lacks dNTP turnover activity. However, a potent 3'----5' exonuclease activity can be detected by a specific assay in which the exonuclease excises mispaired nucleotides at the 3' termini of primed synthetic and natural DNA templates. The excision of a mispaired nucleotide occurs at a significantly greater rate than excision of a correctly paired nucleotide and, under conditions of DNA synthesis, hydrolysis of a mispaired terminal nucleotide occurs prior to primer extension. The 3'----5' exonuclease copurifies quantitatively with DNA polymerase gamma and cosediments with the nearly homogeneous enzyme under native conditions. These results suggest that the 3'----5' exonuclease provides a proofreading function to enhance the fidelity of DNA synthesis during Drosophila mitochondrial DNA replication.
Covalently bound phosphate residues in bovine milk xanthine oxidase and in glucose oxidase from Aspergillus niger: a reevaluation.
Authors: Johnson J L , London R E , Rajagopalan K V
Published: May 2006

The reported presence of covalently bound phosphate residues in flavoproteins has significant implications with regard to the catalytic mechanisms and structural stability of the specific enzymes themselves and in terms of general cellular metabolic regulation. These considerations have led to a reevaluation of the presence of covalently bound phosphorus in the flavoproteins xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22) and glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4). Milk xanthine oxidase purified by a procedure that includes anion-exchange chromatography is shown to contain three phosphate residues. All three are noncovalently associated with the protein, two with the FAD cofactor, and one with the molybdenum cofactor. Results of chemical analysis and 31P NMR spectroscopy indicate that enzyme purified by this method contains no phosphoserine residues. Xanthine oxidase preparations purified by chromatography on calcium phosphate gel in place of DEAE-Sephadex yielded higher phosphate-to-protein ratios, which could be reduced to the expected values by additional purification on a folate affinity column. Highly active, highly purified preparations of glucose oxidase are shown to contain only the two phosphate residues of the FAD cofactor. The covalently bound bridging phosphate reported by others may arise in aged or degraded preparations of the enzyme but appears not to be a constituent of functional glucose oxidase. These results suggest that the presence of covalent phosphate residues in other flavoproteins should be rigorously reevaluated as well.
Cis- and trans-acting elements involved in the regulation of the erythroid promoter of the human porphobilinogen deaminase gene.
Authors: Mignotte V , Eleouet J F , Raich N , Romeo P H
Published: May 2006

Two cis-acting sequences, recognized by two erythroid-specific trans-acting factors, are involved in the regulation of the erythroid promoter of the human gene coding for porphobilinogen deaminase (PBGD). The first region, located at -70, binds the erythroid factor NF-E1, and point mutations within this region abolish the induction of transcription of this promoter during murine erythroleukemia (MEL) cell differentiation. The second region, located at -160, binds the erythroid-specific factor NF-E2 and the ubiquitous factor AP1. Using UV cross-linking, we show that NF-E2 has a higher molecular weight than AP1, demonstrating that NF-E2 is not an erythroid-specific degradation product of AP1. By point mutagenesis of the NF-E2/AP1 binding site, we define mutations that abolish binding of either NF-E2 alone or AP1 and NF-E2 together. Regulation of transcription of the PBGD erythroid promoter is abolished by those mutations, suggesting that NF-E2 but not AP1 is necessary for correct regulation of this promoter in erythroid cells.
K+/H+-antiporter nigericin arrests DNA synthesis in Ehrlich ascites carcinoma cells.
Authors: Margolis L B , Novikova I Y u , Rozovskaya I A , Skulachev V P
Published: May 2006

Acidification of the cytoplasm of Ehrlich ascites carcinoma cells to pH 6.3 arrests DNA synthesis in these cells. Such an effect can be achieved by incubating the cells at pH 6.2 or by adding low concentrations of the K+/H+ antiporter, the antibiotic nigericin, at neutral pH. Glucose and anaerobiosis potentiate the nigericin effect. The inhibition of DNA synthesis by nigericin occurs without any significant decrease in the ATP concentration and in the mitochondrial membrane potential. The DNA synthesis inhibition is caused neither by a decrease in the intracellular [K+] nor by an increase in the intracellular [Na+] accompanying the nigericin effect (at least at low concentrations of the antibiotic). Nigericin should thus be regarded as a type of a cytostatic primarily affecting intracellular pH.
Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor.
Authors: Poulter L , Earnest J P , Stroud R M , Burlingame A L
Published: May 2006

Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the alpha subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the gamma subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up approximately equal to 22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide.
T cells specific for alpha-beta interface regions of hemoglobin recognize the isolated subunit but not the tetramer and indicate presentation without processing.
Authors: Atassi M Z , Yoshioka M , Bixler G S
Published: May 2006

Processing of a protein antigen into fragments is believed to be a prerequisite for its presentation by the antigen-presenting cell to the T cell. This model would predict that, in oligomeric proteins, T cells prepared with specificity for regions that are buried within subunit association surfaces should recognize the respective regions in vitro equally well on the isolated subunit or on the oligomer. Three hemoglobin (Hb) alpha-chain synthetic peptides, corresponding to areas that are situated either completely [alpha-(31-45)] or partially [alpha-(41-45) and alpha-(81-95)] within the interface between the alpha and beta subunits of Hb, and a fourth peptide representing a completely exposed area in tetrameric Hb were used as immunogens in SJL/J (H-2s) mice. Peptide-primed T cells were passaged in vitro with the respective peptide to obtain peptide-specific T-lymphocyte lines. T-cell clones were isolated from these lines by limiting dilution. T-cell lines and clones that were specific for buried regions in the subunit association surfaces recognized the free peptide and the isolated subunit but not the Hb tetramer. On the other hand, T cells with specificity against regions that are not involved in subunit interaction and are completely exposed in the tetramer recognized the peptide, the isolated subunit, and the oligomeric protein equally well. The responses of the T-cell lines and clones were major histocompatibility complex-restricted. Since the same x-irradiated antigen-presenting cells were employed, the results could not be attributed to differences or defects in Hb processing. The findings indicate that in vitro the native (unprocessed and undissociated) oligomeric protein was the trigger of major histocompatibility complex-restricted T-cell responses.
S-phase-specific regulation by deletion mutants of the human thymidine kinase promoter.
Authors: Lipson K E , Chen S T , Koniecki J , Ku D H , Baserga R
Published: May 2006

The levels of thymidine kinase (TK; EC 2.7.1.21) mRNA were determined in nine established cell lines derived from TK-ts13, a temperature-sensitive mutant cell line that arrests in late G1 phase of the cell cycle at the restrictive temperature. The derivative cell lines carried either a cDNA or a minigene of human TK under the control of TK promoters of different lengths. A tenth cell line carried a human TK cDNA under the control of a simian virus 40 promoter. Two different assays were used to determine the S-phase-specific regulation of human TK mRNA levels in quiescent cells stimulated to proliferate. Results from these two assays indicated that (i) the first two introns of the human TK gene had no effect on the S-phase-specific regulation of TK mRNA levels, although the presence of introns increased the amount of TK mRNA; (ii) similar amounts of TK mRNA were present in cells containing constructs with an 83-base-pair (bp) promoter as with other TK promoters comprising up to approximately 4000 bp of 5' flanking sequence; (iii) a 456-bp promoter was fully S-phase-regulated, whereas the 83-bp promoter was only partially regulated; (iv) a 63-bp promoter was much less regulated than an 83-bp promoter; and (v) the crucial element in the 20-bp fragment comprising bp -83 to -64 has been localized, by site-directed mutagenesis, to the CCAAT element at -70.
A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is recognized by an evolutionarily conserved nuclear protein.
Authors: Staiger D , Kaulen H , Schell J
Published: May 2006

In the chalcone synthase gene of Antirrhinum majus (snapdragon), 150 base pairs of the 5' flanking region contain cis-acting signals for UV light-induced expression. A nuclear factor, designated CG-1, specifically recognizes a hexameric motif with internal dyad symmetry, CACGTG, located within this light-responsive sequence. Binding of CG-1 is influenced by C-methylation of the CpG dinucleotide in the recognition sequence. CG-1 is a factor found in a variety of dicotyledonous plant species including Nicotiana tabacum, A. majus, Petunia hybrida, Arabidopsis thaliana, and Glycine max. CACGTG motifs contained within trans-acting factor recognition sites in various other plant promoters can interact with CG-1. In addition, the binding site of the human adenovirus major late transcription factor USF can compete for CG-1 binding to the chalcone synthase promoter. This suggests an evolutionary conservation of trans-acting factor recognition sites involved in divergent mechanisms of gene control.
The human beta-globin locus activation region alters the developmental fate of a human fetal globin gene in transgenic mice.
Authors: Enver T , Ebens A J , Forrester W C , Stamatoyannopoulos G
Published: May 2006

We linked a 3.3-kilobase fragment containing the entire A gamma-globin gene together with 1.3 kilobases of 5' flanking and 0.37 kilobase of 3' flanking DNA to a 2.5-kilobase fragment containing four of the developmentally stable hypersensitive sites normally located in the 5' region of the human beta-globin locus. This construct was injected into fertilized mouse eggs, and its expression was analyzed in the primitive and definitive erythroid cells, as well as the brain of 14-day embryos. All six transgenic individuals that contained intact copies of the construct expressed the transgene in an erythroid-specific fashion. Expression was observed in both primitive and definitive erythroid cells. This is in marked contrast to previous transgenic mice experiments using the same A gamma-globin gene fragment in isolation, where expression was restricted to primitive erythroid cells. Our results show that the region containing the developmentally stable globin locus hypersensitive sites changes the developmental stage specificity of a human fetal globin gene in transgenic mice. These observations imply that sequences additional to those used here are involved in the developmental control of fetal globin gene expression in vivo. The ability to express fetal globin in adult erythroid cells allows one to consider using fetal globin genes for gene therapy of sickle cell disease.
Salmonella typhimurium phoP virulence gene is a transcriptional regulator.
Authors: Groisman E A , Chiao E , Lipps C J , Heffron F
Published: May 2006

Salmonella typhimurium is a facultative intracellular pathogen capable of surviving within host phagocytic cells. Salmonella strains carrying phoP mutations are avirulent, unable to survive in macrophages, and extremely sensitive to peptides having antimicrobial activity such as the host-derived defensins. We present here the DNA sequence of the phoP gene and show that the deduced amino acid sequence of phoP has extensive homology with the Escherichia coli transcriptional regulators PhoB and OmpR, which control the expression of loci in response to different environmental stimuli. The psiD locus, which is regulated by phosphate availability, was found to be under the control of the phoP gene product. Sequences homologous to phoP were found in several Gram-negative species and in the yeast Saccharomyces cerevisiae.
Protection of macaques against simian AIDS by immunization with a recombinant vaccinia virus expressing the envelope glycoproteins of simian type D retrovirus.
Authors: Hu S L , Zarling J M , Chinn J , Travis B M , Moran P A , Sias J , Kuller L , Morton W R , Heidecker G , Benveniste R E
Published: May 2006

Simian AIDS (SAIDS) is an endemic disease of macaques that shares many characteristics with AIDS in humans. SAIDS is etiologically linked to infection by a type D retrovirus, SAIDS retrovirus (SRV). Immunization with an inactivated whole-virus vaccine was shown to protect macaques against infection by SRV serotype 1. To identify the antigen(s) responsible for eliciting protective immunity, we have constructed a recombinant vaccinia virus (v-senv5) that expresses the envelope glycoproteins of SRV serotype 2 (SRV-2/W). Pig-tailed macaques (Macaca nemestrina) immunized with v-senv5 showed lymphoproliferative responses to purified SRV-2/W. They also generated antibodies that neutralized SRV-2/W infectivity in vitro and mediated antibody-dependent cellular cytotoxicity against SRV-2-infected cells. Four v-senv5-immunized animals, together with four control animals, were challenged intravenously with 5 x 10(3) tissue culture infectious doses of SRV-2/W. As early as 2 weeks after challenge, three of four control animals became viremic, and two of these three animals also seroconverted. The animal that was viremic but remained antibody negative died of symptoms of SRV infection 6 1/2 weeks after challenge. In contrast, all four v-senv5-immunized animals remained healthy, virus-free, and seropositive against only the immunizing envelope antigens. These results indicate that immunization with a recombinant vaccinia virus expressing the envelope antigens of SRV-2/W protects primates from infection by a retrovirus that causes immunodeficiency diseases.
Expression of an intrinsic growth strategy by mammalian retinal neurons.
Authors: Montague P R , Friedlander M J
Published: May 2006

Postnatal cat retinal ganglion cells (RGCs) were retrogradely labeled with fluorescent microspheres, dissociated from the retina using a peeling procedure, and monitored in cell culture with a time-lapse video microscopy system. The spatial patterns formed by the growing neurites were analyzed using conventional and fractal measures (Hausdorff dimension, H) of their extent and complexity. The results presented were obtained from the arborizations formed by the neurites of 48 labeled and isolated ganglion cells growing separate from each other and separate from a feeder layer of astrocytes. Cells were obtained from animals when the RGCs were postmitotic and after dendritic differentiation in vivo at age 0-1 week (4/48), 2-5 weeks (35/48), or 6-8 weeks (9/48). By 48 hr after plating, the number of surviving labeled RGCs was reduced to 22-28% of its initial value. After removal of all processes and isolation in vitro, these RGCs expressed neurite patterns strikingly similar to those seen in the intact retina, although the RGCs had been deprived of potential cues from the intact retina and target tissue. Self crossings of the growing neurites were rare (less than 0.5%, 20 cells, n = 2500 neurites). Calculation of the Hausdorff dimension, a metric for the space-filling capacity of the neurite patterns, revealed that after 3-day culture 77% (n = 56) of the RGCs achieved relatively uniform coverage of territory (1.6 less than H less than 1.9). This coverage was independent of the number of interbranchpoint segments and/or the total neurite length of a particular neurite pattern. A sample of dendritic arbors from RGCs in intact retina yielded similar values for the Hausdorff dimension (H = 1.73, SD = 0.12, n = 18, range 1.54-1.94). These results reveal that a mammalian central nervous system neuron, for at least 8 postnatal weeks, has the intrinsic capacity for reexpression of in vivo structure characteristic of that cell type in the absence of interaction with neighboring neurons, afferent input, and target tissue. These neurons exhibit stereotyped growth resulting in uniform coverage of a restricted territory by the strategic selection of the length, location, and orientation of interbranchpoint segments.
Retinal transplant-mediated learning in a conditioned suppression task in rats.
Authors: Coffey P J , Lund R D , Rawlins J N
Published: May 2006

Retinae were transplanted from embryonic rats to a location over the midbrain of unilaterally anucleated newborn rats. At maturity, the rats were trained to "lever-press" for a food reward. By using a Pavlovian conditioned suppression paradigm, it was found that light delivered to the transplant could be recognized by the host rat as a conditioned stimulus, resulting in a reduced rate of lever pressing.
Reentrant signaling among simulated neuronal groups leads to coherency in their oscillatory activity.
Authors: Sporns O , Gally J A , Reeke G N , Edelman G M
Published: May 2006

Recent experiments have revealed tightly synchronized oscillatory discharges in local assemblies of cortical neurons as well as phase coherency of oscillations at distant cortical sites. These findings are consistent with the theory of neuronal group selection, a population theory of brain function that is based on the properties of local groups of neurons. A set of computer simulations shows that cooperative interactions within and among neuronal groups can generate the observed phenomena. In the simulations, oscillations within neuronal groups are generated through local excitatory and inhibitory interactions. Different groups in general oscillate in an uncorrelated fashion. Coherency of the oscillatory activity of different neuronal groups depends crucially on reciprocal reentrant signaling and can reflect the spatial continuity of a stimulus. Separated or discontinuous features of a given stimulus can be transiently associated in a temporally coherent pattern through reentrant signaling between groups in networks responding to different aspects of that stimulus. A simulation of reentrant activity between arrays of neuronal groups selective for oriented lines and pattern motion displays cross-correlations between groups that are responsive to different parts of a stimulus contour if these parts move together. Such coherency among neuronal groups might be used in the discrimination of a stimulus from other stationary or differentially moving elements in a visual scene.
Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.
Authors: Lee S H , Eki T , Hurwitz J
Published: May 2006

Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase delta that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase delta on a DNA singly primed phi X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase delta, in contrast to DNA polymerase alpha, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase alpha and delta are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I.
Fruit flies with additional expression of the elongation factor EF-1 alpha live longer.
Authors: Shepherd J C , Walldorf U , Hug P , Gehring W J
Published: May 2006

In Drosophila melanogaster, the decrease in protein synthesis that accompanies aging is preceded by a decrease in elongation factor EF-1 alpha protein and mRNA. Here we show that Drosophila transformed with a P-element vector containing an EF-1 alpha gene under control of hsp70 regulatory sequences have a longer life-span than control flies.
Horizontal cell gap junctions: single-channel conductance and modulation by dopamine.
Authors: McHahon D G , Knapp A G , Dowling J E
Published: May 2006

Horizontal cells form an electrically coupled network for the transmission of inhibitory signals in the outer retina. In teleosts, horizontal cell coupling is modulated by the neurotransmitter dopamine. Using voltage-clamped pairs of teleost horizontal cells, we have examined the effects of dopamine on the conductance and gating properties of the cell-to-cell channels that mediate electrical synaptic transmission. Variance analysis of the junctional current noise showed that dopamine substantially reduced the open probability of gap junction channels, from 0.75 to 0.14. Direct observation of unitary junctional gating events in poorly coupled cell pairs indicated that these channels have a unitary conductance of 50-60 pS. The elementary conductance of channels in cell pairs treated with dopamine (48.7 +/- 6.6 pS) was statistically indistinguishable from channels in untreated cells (53.2 +/- 7.2 pS). Uncoupling with octanol also yielded a similar unitary conductance (61.1 +/- 11.1 pS). Our results suggest that dopamine reduces the open probability of gap junctional channels by decreasing their open duration.
Presence of the hypermodified nucleotide N6-(delta 2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA.
Authors: Wilson R K , Roe B A
Published: May 2006

The overall structure of transfer RNA is optimized for its various functions by a series of unique post-transcriptional nucleotide modifications. Since many of these modifications are conserved from prokaryotes through higher eukaryotes, it has been proposed that most modified nucleotides serve to optimize the ability of the tRNA to accurately interact with other components of the protein synthesizing machinery. When a cloned synthetic Escherichia coli tRNAPhe gene was transfected into a bacterial host that carried a defective phenylalanine tRNA-synthetase gene, tRNAPhe was overexpressed by 11-fold. As a result of this overexpression, an undermodified tRNAPhe species was produced that lacked only N6-(delta 2-isopentenyl)-2-methylthioadenosine (ms2i6A), a hypermodified nucleotide found immediately 3' to the anticodon of all major E. coli tRNAs that read UNN codons. To investigate the role of ms2i6A in E. coli tRNA, we compared the aminoacylation kinetics and in vitro codon-reading properties of the ms2i6A-lacking and normal fully modified tRNAPhe species. The results of these experiments indicate that while ms2i6A is not required for normal aminoacylation of tRNAPhe, its presence stabilizes codon-anticodon interaction and thereby prevents misreading of the genetic code.
ATP stimulates Ca2+ uptake and increases the free Ca2+ concentration in isolated rat liver nuclei.
Authors: Nicotera P , McConkey D J , Jones D P , Orrenius S
Published: May 2006

Addition of ATP to a highly purified fraction of rat liver nuclei incubated with submicromolar concentrations of Ca2+ and trace amounts of 45Ca2+ resulted in the rapid accumulation of 45Ca2+ in the nuclei. This was associated with an increase in intranuclear free Ca2+ concentration as measured with the fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-a mino-5'- methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (fura-2). Inhibitors of microsomal and mitochondrial Ca2+ translocases had no effect on nuclear Ca2+ sequestration, indicating that it was distinct from previously known intracellular Ca2+-transporting systems. Ca2+ uptake and the associated increase in intranuclear free Ca2+ concentration were prevented by calmidazolium, a potent calmodulin antagonist. Partial characterization of the ATP-stimulated nuclear Ca2+ uptake showed that maximal rates of Ca2+ uptake and increase in intranuclear free Ca2+ level occurred at concentrations of Ca2+ normally present in the cytosol of mammalian cells. Together, these results show that a distinct, ATP- and calmodulin-dependent Ca2+ uptake system exists in liver nuclei. This system may play an important role in the regulation of intranuclear Ca2+-dependent processes.
Replacement of aspartic residues 85, 96, 115, or 212 affects the quantum yield and kinetics of proton release and uptake by bacteriorhodopsin.
Authors: Marinetti T , Subramaniam S , Mogi T , Marti T , Khorana H G
Published: May 2006

Recently, a number of aspartic acid mutants of bacteriorhodopsin have been shown to be defective in steady-state proton transport. Here we report time-resolved measurements of light-induced proton release and uptake for these mutants. Proton transfers between the protein and the aqueous phase were directly monitored by measuring changes in the bulk conductivity of a micellar solution of bacteriorhodopsin. For the Asp-96----Asn mutant, proton uptake was slowed by greater than 1 order of magnitude with no observable effect on the release step. For Asp-85----Asn, H+ uptake occurred with normal kinetics, but the yield was significantly lower compared with either the Asp-96----Asn mutant or wild type, especially at pH 6. Substitution of glutamate for Asp-85 or Asp-96 had smaller but detectable effects on the kinetics and quantum yield of proton movements. Both asparagine and glutamate substitutions of aspartates at positions 115 and 212 lowered the proton quantum yields. Of these, only the Asp-115----Asn mutant showed an effect on the proton release step, and only the Asp-212----Glu mutation decreased the proton uptake rate. These experiments imply an obligatory role for Asp-96 in H+ uptake in the normal operation of the bacteriorhodopsin proton pump. The results also indicate that the amino acid substitutions affect the kinetics of either H+ release or H+ uptake, but not both. This implies that the two steps occur independently of each other after initiation of the photocycle.
Different gamma delta T-cell receptors are expressed on thymocytes at different stages of development.
Authors: Ito K , Bonneville M , Takagaki Y , Nakanishi N , Kanagawa O , Krecko E G , Tonegawa S
Published: May 2006

We have analyzed the structural diversity of the murine gamma delta T-cell receptor (TCR) heterodimer expressed on CD4- CD8- thymocyte populations and on TCR gamma delta-expressing hybridomas derived from thymocytes of fetal, newborn, and adult mice. We found that CD4- CD8- thymocytes derived from mice of different pre- and postnatal age preferentially express a gamma delta TCR encoded by different subsets of gamma and delta gene segments. This age-dependent differential expression of gamma delta TCR on thymocytes seems to be accomplished in part by a specific control of rearranged gamma genes operating at the level of transcription and/or RNA stability. We discuss the implications of these findings with respect to the recognition roles of the gamma delta TCR.
Fate of aerosolized recombinant DNA-produced alpha 1-antitrypsin: use of the epithelial surface of the lower respiratory tract to administer proteins of therapeutic importance.
Authors: Hubbard R C , Casolaro M A , Mitchell M , Sellers S E , Arabia F , Matthay M A , Crystal R G
Published: May 2006

To evaluate the possibility of administering therapeutic proteins via the respiratory route, we administered an aerosol of recombinant DNA-produced human alpha 1-antitrypsin (rAAT) to anesthetized sheep and measured levels of the protein in epithelial lining fluid (ELF), lung lymph, blood, and urine. Using a nebulizer that generated aerosol droplets with a mass median aerodynamic diameter of 2.7 micron (55% of droplets were less than 3 micron, a particle size optimal for deposition on the alveolar epithelium), in vitro studies demonstrated that the aerosolized rAAT remained intact and fully functional as an inhibitor of neutrophil elastase. When aerosolized to sheep, the 45-kDa rAAT molecule diffused across the alveolar epithelium, as evidenced by its presence in lung lymph and in blood. Comparison of ELF, lymph, blood, and urine rAAT levels demonstrated that the process was concentration dependent, with highest levels in ELF and in descending concentrations with approximately 10-fold concentration differences in each consecutive compartment, respectively. Importantly, evaluation with aerosolized 125I-labeled rAAT demonstrated that the rAAT molecules that reached the lung lymph and the systemic circulation remained intact as a 45-kDa protein. These results demonstrate the feasibility of using aerosolization to the pulmonary epithelial surface to administer sizeable proteins of therapeutic interest, thus circumventing the necessity of the traditional parenteral modes of administration of such molecules.
Voltage-gated channels formed in lipid bilayers by a positively charged segment of the Na-channel polypeptide.
Authors: Tosteson M T , Auld D S , Tosteson D C
Published: May 2006

The Na-channel polypeptide is responsible for the voltage-gated and time-dependent ionic permeability changes that give rise to the action potential in the membranes of nerve cells. We have synthesized a 22-amino acid peptide with a sequence identical to that of the segment named S4, repeat IV of the primary structure of the Na channel. We have found that this peptide induces a voltage- and time-dependent conductance in bilayers formed by a mixture of phosphatidyl-ethanolamine and phosphatidylserine. This conductance is activated when the cis side is made positive, with an apparent gating charge of 3. The results are consistent with the idea that this segment plays a role in determining the voltage sensitivity of the Na channel.
Nucleotide-specific cleavage and minor-groove interaction of DNA with esperamicin antitumor antibiotics.
Authors: Sugiura Y , Uesawa Y , Takahashi Y , Kuwahara J , Golik J , Doyle T W
Published: May 2006

The cleavage of DNA by esperamicin is greatly accelerated in the presence of thiol compounds. Oxygen and active oxygen-radical scavengers have no significant influence upon DNA strand breakage by esperamicin. The preferential cutting sites of esperamicin are at thymidylate residues, and the frequency of bases attacked (T greater than C greater than A greater than G) is different from that of calicheamicin (C much greater than T greater than A = G), neocarzinostatin (T greater than A greater than C greater than G), or bleomycin (C greater than T greater than A greater than G). Esperamicin preferentially attacks at T and C bases in oligopyrimidine sequences such as 5'-CTC-3', 5'-TTC-3', and 5'-TTT-3'. In contrast to the preferred sites of cleavage by bleomycin, 5'-GT-3' and 5'-GC-3', the preferred sites of esperamicin-mediated DNA degradation are 5'-TG-3' and 5'-CG-3' sequences. The nucleotide-specific cleavage mode of esperamicin is significantly affected by pretreatment of DNA with netropsin and distamycin A, suggesting that interaction of esperamicin occurs through the minor groove of B-DNA. This is further supported by the asymmetric cleavage pattern to the 3' side on the opposite strand of the DNA. The roles of the fucose-anthranilate moiety and the trisaccharide side chain of esperamicin in DNA binding and base recognition are discussed.
Refined apoprotein structure of rat intestinal fatty acid binding protein produced in Escherichia coli.
Authors: Sacchettini J C , Gordon J I , Banaszak L J
Published: May 2006

Rat intestinal fatty acid binding protein (I-FABP) is a member of a family of cytoplasmic hydrophobic ligand-binding proteins. To gain insights about the contribution of bound fatty acid to I-FABP's conformation and mechanism of ligand binding, we have determined the structure of Escherichia coli-derived rat apo-I-FABP to 1.96-A resolution and compared it to the recently refined structure of I-FABP with bound palmitate. Both apo- and holo-I-FABP are composed primarily of anti-parallel beta-strands which form two nearly orthogonal beta-sheets ("beta-clam"). The overall structures of the apo- and holo-I-FABP are nearly identical, with a root mean square (rms) difference of 0.37 A between C alpha atoms, 0.38 A between all main-chain atoms, and 0.94 A between all side-chain atoms. However, rms differences of greater than 1.3 A were noted for the side chains of Ile-23, Lys-27, Arg-56, Leu-72, Ala-73, and Asp-74. The space occupied by bound ligand in the core of the holoprotein is occupied in the apo-protein by ordered solvent molecules. This results in an increase in the total number of internal ordered solvent molecules from 7 in the holoprotein to 13 in apo-I-FABP. This finding, together with observed differences in the side-chain orientations of two residues (Arg-56 and Lys-27) situated over a potential opening to the cores of the apo- and holoproteins, suggests that solvent molecules play a critical role in ligand binding. Moreover, the data indicate that the beta-clam structure is stable even in the absence of bound ligand.
Hydroxyl radical "footprinting" of RNA: application to pre-mRNA splicing complexes.
Authors: Wang X D , Padgett R A
Published: May 2006

We present an adaptation of the hydroxyl radical DNA "footprinting" technique that permits high-resolution mapping of protected regions of RNA. Hydroxyl radical cleaves RNA independently of base sequence and secondary structure of the RNAs examined and allows resolution of protected regions at the single nucleotide level. By using this technique, we show that several regions of the 3' splice site of mRNA precursors are protected during the formation of splicing-specific ribonucleoprotein complexes in an in vitro splicing system. These regions include the 3' intron/exon junction and a portion of the adjacent exon, the polypyrimidine tract, and the site of branch formation. These protections appear to be due to splicing specific complexes since their formation is sensitive to point mutations at crucial residues and requires ATP and incubation. The formation of these protected regions is independent of the presence of a 5' splice site.
Clostridial glycine reductase: protein C, the acetyl group acceptor, catalyzes the arsenate-dependent decomposition of acetyl phosphate.
Authors: Stadtman T C
Published: May 2006

The highly purified protein C component of clostridial glycine reductase is required in addition to seleno-protein A and protein B for the conversion of glycine to acetate and ammonia in the presence of arsenate. As shown by Arkowitz and Abeles [Arkowitz, R. A. & Abeles, R. H. (1989) Biochemistry 28, 4639-4644], the products are ammonia and acetyl phosphate in the presence of phosphate. The protein C component alone catalyzes an arsenate-dependent decomposition of acetyl phosphate, showing that it serves as the acetyl group acceptor in the overall reaction. A thiol-reducing agent and Mg2+ are required for catalysis of the arsenolysis reaction by protein C. Alkylation or heating at 60 degrees C completely abolishes the ability of protein C to catalyze the arsenolysis reaction and to participate as an essential component in the overall glycine reductase reaction.