bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermodynamics and structure of a DNA tetraplex: a spectroscopic and calorimetric study of the tetramolecular complexes of d(TG3T) and d(TG3T2G3T).
Authors: Jin R , Gaffney B L , Wang C , Jones R A , Breslauer K J
Published: May 2006

We report a combined thermodynamic and structural characterization of a DNA tetraplex. Using spectroscopic and calorimetric techniques, we demonstrate that d(TG3T) and d(TG3T2G3T), in the presence of K+, form stable tetramolecular complexes. From differential scanning calorimetry measurements, we obtain the following thermodynamic profiles for formation of each tetraplex at 25 degrees C: delta G degrees = -6.9 kcal/mol of tetraplex (or -2.3 kcal/mol of tetrad; 1 cal = 4.184 J), delta H degrees = -62.6 kcal/mol of tetraplex (or -20.9 kcal/mol of tetrad), and delta S degrees = -186.9 cal.K-1.mol-1 of tetraplex (or -62.3 cal.K-1.mol-1 of tetrad) for the d(TG3T) tetraplex; and delta G degrees = -20.2 kcal/mol of tetraplex (or -3.4 kcal/mol of tetrad), delta H degrees = -123.2 kcal/mol of tetraplex (or -20.5 kcal/mol of tetrad), and delta S degrees = -346.0 cal.K-1.mol-1 of tetraplex (or -57.7 cal.K-1.mol-1 of tetrad) for the d(TG3T2G3T) tetraplex. These data demonstrate that at 25 degrees C a G-tetrad can exhibit considerable stability, comparable to or even exceeding that of most Watson-Crick nearest-neighbor interactions, with this stability resulting from a very favorable enthalpy of formation. Temperature-dependent CD measurements reveal that the melting temperatures of both tetraplexes exhibit unusually low salt dependences. This unexpected behavior may reflect a diminished charge density due to bound K+ ions. For each complex, the Na+ and K+ forms exhibit drastically different isothermal and temperature-dependent CD profiles, with the K+ forms of each tetraplex melting more sharply and at a higher temperature than the Na+ forms. Using one- and two-dimensional NMR techniques, we show that the strands in the tetramolecular complex of d(TG3T), K+ are all parallel and that the guanine glycosidic conformations are all anti.
Centromere DNA mutations induce a mitotic delay in Saccharomyces cerevisiae.
Authors: Spencer F , Hieter P
Published: May 2006

Cytological observations of animal cell mitoses have shown that the onset of anaphase is delayed when chromosome attachment to the spindle is spontaneously retarded or experimentally interrupted. This report demonstrates that a centromere DNA (CEN) mutation carried on a single chromosome can induce a cell cycle delay observed as retarded mitosis in the yeast Saccharomyces cerevisiae. A 31-base-pair deletion within centromere DNA element II (CDEII delta 31) that causes chromosome missegregation in only 1% of cell division elicited a dramatic mitotic delay phenotype. Other CEN DNA mutations, including mutations in centromere DNA elements I and III, similarly delayed mitosis. Single division pedigree analysis of strains containing the CDEII delta 31 CEN mutation indicated that most (and possibly all) cells experienced delay in each cell cycle and that the delay was not due to increased chromosome copy number. Furthermore, a synchronous population of cells containing the CDEII delta 31 mutation underwent DNA synthesis on schedule with wild-type kinetics, but subsequently exhibited late chromosomal separation and concomitant late cell separation. We speculate that this delay in cell cycle progression before the onset of anaphase provides a mechanism for the stabilization of chromosomes with defective kinetochore structure. Further, we suggest that the delay may be mediated by surveillance at a cell cycle checkpoint that monitors the completion of chromosomal attachment to the spindle.
Retinoid X receptor RXR alpha binds to and trans-activates the hepatitis B virus enhancer.
Authors: Huan B , Siddiqui A
Published: May 2006

A retinoid X receptor (RXR) response element was located within the functionally defined hepatitis B virus (HBV) enhancer element. A short segment of the enhancer that contains this region has been shown with genetic analysis to play a key role in the regulation of enhancer function and to represent a major determinant of liver-specific activity. Both the full-length protein and the DNA-binding domain of the liver-specific receptor RXR alpha bound to the putative retinoic acid response element in the HBV enhancer. In vivo, an HBV enhancer-reporter gene construct responds to induction with retinoic acid when cotransfected with an RXR alpha expression vector. A single-base transition (G----A) in the HBV retinoic acid response element leads to a dramatic reduction both in the in vitro binding activity of RXR alpha and the in vivo activity of the HBV enhancer. Thus, retinoic acid and the RXR alpha are implicated as being significant determinants in the liver-specific regulation of HBV gene expression and the resultant disease pathogenesis.
Growth arrest induced by wild-type p53 protein blocks cells prior to or near the restriction point in late G1 phase.
Authors: Lin D , Shields M T , Ullrich S J , Appella E , Mercer W E
Published: May 2006

Conditional expression of wild-type (wt) p53 protein in a glioblastoma tumor cell line has been shown to be growth inhibitory. We have now more precisely localized the position in the cell cycle where growth arrest occurs. We show that growth arrest occurs prior to or near the restriction point in late G1 phase of the cell cycle. The effect of wt p53 protein on the expression of four immediate-early genes (c-FOS, c-JUN, JUN-B, and c-MYC), one delayed-early gene (ornithine decarboxylase), and two late-G1/S-phase genes (B-MYB and DNA polymerase alpha) was also examined. Of this subset of growth response genes, only the expression of B-MYB and DNA polymerase alpha was significantly repressed. The possibility that decreased expression of B-MYB may be an important component of growth arrest mediated by wt p53 protein is discussed.
Identification and prevalence of a genetic defect that causes leukocyte adhesion deficiency in Holstein cattle.
Authors: Shuster D E , Kehrli M E , Ackermann M R , Gilbert R O
Published: May 2006

Two point mutations were identified within the gene encoding bovine CD18 in a Holstein calf afflicted with leukocyte adhesion deficiency (LAD). One mutation causes an aspartic acid to glycine substitution at amino acid 128 (D128G) in the highly conserved extracellular region of this adhesion glycoprotein, a region where several mutations have been found to cause human LAD. The other mutation is silent. Twenty calves with clinical symptoms of LAD were tested, and all were homozygous for the D128G allele. In addition, two calves homozygous for the D128G allele were identified during widespread DNA testing, and both were subsequently found to exhibit symptoms of LAD. The carrier frequency for the D128G allele among Holstein cattle in the United States is approximately 15% among bulls and 6% among cows. This mutation is also prevalent among Holstein cattle throughout the world, placing this disorder among the most common genetic diseases known in animal agriculture. All cattle with the mutant allele are related to one bull, who through the use of artificial insemination sired many calves in the 1950s and 1960s. The organization of the dairy industry and the diagnostic test described herein will enable nearly complete eradication of bovine LAD within 1 year. These results also demonstrate that bovine LAD is genetically homologous and phenotypically similar to human LAD, thus providing a useful animal model for studies of LAD and beta 2 integrin function.
Expression cloning of a cDNA encoding UDP-GlcNAc:Gal beta 1-3-GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen.
Authors: Bierhuizen M F , Fukuda M
Published: May 2006

A cDNA encoding UDP-GlcNAc:Gal beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase (EC 2.4.1.102), which forms critical branches in O-glycans, has been isolated by an expression cloning approach using Chinese hamster ovary (CHO) cells. Increased activity of this enzyme and the concomitant occurrence of the O-glycan core 2 structure [Gal beta 1-3(GlcNAc beta 1-6)GalNAc] has been observed in a variety of biological processes, such as T-cell activation and immunodeficiency due to the Wiskott-Aldrich syndrome and AIDS. Since CHO cells do not express this enzyme, CHO cell lines were established to stably express polyoma large tumor (T) antigen, which enables transient expression cloning. Because the antibody used was found to detect most efficiently the oligosaccharide products attached to leukosialin, the CHO cells were also stably transfected with leukosialin cDNA. By using this particular CHO cell line, a cDNA that encodes a protein determining the formation of the core 2 structure was isolated from an HL-60 cDNA library. The cDNA sequence predicts a protein with type II membrane topology, as has been found for all other mammalian glycosyltransferases cloned to date. The expression of the presumed catalytic domain as a fusion protein with the IgG binding domain of protein A enabled us to demonstrate unequivocally that the cDNA encodes the core 2 beta-1,6-N-acetylglucosaminyltransferase, the enzyme responsible for the formation of Gal beta 1-3(GlcNAc beta 1-6)GalNAc structures. No activity with this enzyme was detected toward the acceptors for other beta 1-6GlcNAc transferases.
Expression patterns of gamma-aminobutyric acid type A receptor subunit mRNAs in primary cultures of granule neurons and astrocytes from neonatal rat cerebella.
Authors: Bovolin P , Santi M R , Puia G , Costa E , Grayson D
Published: May 2006

Using a competitive polymerase chain reaction assay, we have quantitated the absolute amounts of mRNA encoding 14 distinct subunits of the gamma-aminobutyric acid type A (GABAA) receptor in primary cultures of rat cerebellar granule neurons and cerebellar astrocytes. We found that the total amount of GABAA receptor subunit mRNA in astrocytes was 2 orders of magnitude lower than in neuronal cells. Furthermore, granule cell cultures expressed all 14 different GABAA subunit mRNAs, while the astroglial cultures contained detectable amounts of all the subunits expressed by granule cells except the alpha 6 and the gamma 2L subunits. Of the alpha subunit family members, the alpha 1, alpha 5, and alpha 6 mRNAs were prominent in granule cells, while the alpha 1 and alpha 2 mRNAs were abundant in astrocytes. Of the beta receptor subunit mRNAs, the beta 1 and beta 3 mRNAs were abundantly expressed in both cultures. The gamma 2S and gamma 2L mRNAs constituted the great majority of gamma subunit mRNAs in neurons, while the gamma 1 subunit mRNA was the most abundant gamma subunit mRNA in astrocytes. When various allosteric modulators of GABAA receptors were tested electrophysiologically, methyl 6,7-dimethoxy-4-ethyl-beta-carboline- 3-carboxylate (DMCM) was the only one to modulate chloride currents elicited by GABA in a significantly different manner in granule cells (negative modulation) compared with astrocytes (positive modulation). The latter effect was previously observed in transiently expressed recombinant GABAA receptors containing a gamma 1 instead of a gamma 2 subunit. Our quantitative mRNA results suggest that an important molecular determinant responsible for the DMCM-positive modulatory effect on astroglial native GABAA receptors is the presence of the gamma 1 subunit in the receptor assembly.
Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor.
Authors: Beck von Bodman S , Hayman G T , Farrand S K
Published: May 2006

The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gall tumors. The opines also induce Ti plasmid-encoded genes required by the bacteria for opine catabolism. We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B. The gene, accR, is closely linked to the agrocinopine catabolic locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR. Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac. Analysis of lacZ fusions in conjugal transfer and opine catabolic structural genes demonstrated that the accR-encoded function is a transcriptional repressor. accR can encode a 28-kDa protein. This protein is related to a class of repressor proteins that includes LacR, GutR, DeoR, FucR, and GlpR that regulate sugar catabolic systems in several bacterial genera.
A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major seed storage protein of soybean
Authors: Scott M. Paul , Jung Rudolf , Muntz Klaus , Nielsen Niels C.
Published: May 2006

The assembly of 11S globulin seed storage proteins in plants is regulated in part by the activity of a protease that cleaves between asparagine and glycine residues. Post-translational cleavage of subunit precursors into acidic and basic polypeptides is associated with the ability of subunits in trimers to aggregate into hexamers
in vitro
. An activity is present in extracts from immature soybean seeds that specifically cleaves immature 11S seed storage proteins of soybean and
Vicia faba
into the polypeptides of the mature proteins. Sequence microanalysis has been used to demonstrate that proglycinin and prolegumin are cut at the legitimate site when proteins synthesized
in vitro
are used as substrates. A single amino acid change in the cleavage site renders the substrate uncleavable. The protease responsible for this activity also hydrolyzes a synthetic octapeptide whose sequence reproduces four amino acids on either side of the glycinin subunit G4 cleavage site. This assay permitted the purification and characterization of the protease. It is a glycosylated enzyme with an acidic pH optimum and a molecular mass of about 45 kDa in solution.
Pituitary follicular cells secrete an inhibitor of aortic endothelial cell growth: identification as leukemia inhibitory factor.
Authors: Ferrara N , Winer J , Henzel W J
Published: May 2006

Medium conditioned by bovine pituitary follicular cells paradoxically inhibits the growth of adult bovine aortic endothelial (ABAE) cells at dilutions that are instead mitogenic to adrenal cortex capillary endothelial (ACCE) cells, suggesting that follicular cells secrete a growth inhibitor with a selectivity for ABAE cells. The ABAE cell inhibitory activity was purified to apparent homogeneity by a combination of size-exclusion chromatography, ion-exchange chromatography, and two reversed-phase steps on a C4 column. Microsequencing of the purified material revealed a single NH2-terminal amino acid sequence, identical to that of leukemia inhibitory factor (LIF), a glycoprotein originally identified by its ability to inhibit the growth of MT1 mouse leukemia cells and subsequently found to have numerous effects. Recombinant human LIF inhibited the growth of ABAE cells as effectively as transforming growth factor beta (TGF beta 1). However, it failed to inhibit markedly the growth of ACCE cells, whereas TGF beta 1 dramatically inhibited their growth. Recombinant human LIF also failed to induce a significant angiogenic response in the chicken chorioallantoic membrane, indicating that, unlike TGF beta, LIF probably does not induce the release of direct-acting angiogenic factors from inflammatory cells. The presence of LIF in follicular cells may relate to the peculiar vascular organization of the pituitary gland, where no arteries reach the pars distalis and all of the blood supply to this area is by capillaries.
Complementation of a red-light-indifferent cyanobacterial mutant.
Authors: Chiang G G , Schaefer M R , Grossman A R
Published: May 2006

Many cyanobacteria alter their phycobilisome composition in response to changes in light wavelength in a process termed complementary chromatic adaptation. Mutant strains FdR1 and FdR2 of the filamentous cyanobacterium Fremyella diplosiphon are characterized by aberrant chromatic adaptation. Instead of adjusting to different wavelengths of light, FdR1 and FdR2 behave as if they are always in green light; they do not respond to red light. We have previously reported complementation of FdR1 by conjugal transfer of a wild-type genomic library. The complementing DNA has now been localized by genetic analysis to a region on the rescued genomic subclone that contains a gene designated rcaC. This region of DNA is also able to complement FdR2. Southern blot analysis of genomic DNA from FdR1 and FdR2 indicates that these strains harbor DNA insertions within the rcaC sequence that may have resulted from the activity of transposable genetic elements. The predicted amino acid sequence of RcaC shares strong identity to response regulators of bacterial two-component regulatory systems. This relationship is discussed in the context of the signal-transduction pathway mediating regulation of genes encoding phycobilisome polypeptides during chromatic adaptation.
Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V.
Authors: Doi T , Recktenwald A , Karaki Y , Kikuchi M , Morikawa K , Ikehara M , Inaoka T , Hori N , Ohtsuka E
Published: May 2006

T4 endonuclease V [endodeoxyribonuclease (pyrimidine dimer); deoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] initiates repair of damaged DNA by hydrolysis of the N-glycosyl bond at the 5' side of a pyrimidine photodimer in double-stranded DNA. To study one of the active sites of T4 endonuclease V, systematic site-directed mutagenesis was performed on the synthetic T4 endonuclease V gene, in parallel with three-dimensional structure analysis by x-ray crystallography. The mutant proteins were evaluated for DNA glycosylase activity using an oligonucleotide duplex (14-mer) containing a single thymidine dimer as a substrate. Replacement of either Glu-23 with glutamine or asparatic acid or Arg-3 with glutamine completely abolished DNA glycosylase activity. Mutation of Arg-3 to lysine or of Arg-26 to glutamine or lysine in a basic amino acid cluster caused serious defects in DNA glycosylase activity, which are reflected in the increases in Km and decreases in kcat of DNA glycosylase activity. On the other hand, substitutions of lysine for Arg-22 or of glutamine for Arg-117 or Lys-121 resulted in increases in the Km value. The completely inactive mutant proteins, E23Q and R3Q, in which glutamine was substituted for Glu-23 and Arg-3, respectively, were further investigated by CD spectroscopy for their ability to bind the oligonucleotide substrate. It was found that the E23Q protein retained specific substrate-binding ability, whereas the R3Q protein did not. These results indicate that Glu-23 plays an important role in catalysis of the DNA glycosylase reaction, and that Arg-3 is a crucial residue for substrate binding. In addition, Arg-22, Arg-26, Arg-117, and Lys-121 in the basic amino acid cluster also participate in substrate binding. We conclude that the basic amino acid cluster in T4 endonuclease V is an essential structure for DNA glycosylase activity.
Increased expression of beta-amyloid precursor protein during neuronal differentiation is not accompanied by secretory cleavage.
Authors: Hung A Y , Koo E H , Haass C , Selkoe D J
Published: May 2006

Despite increasing evidence for a pathogenetic role for the beta-amyloid precursor protein (beta APP) in Alzheimer disease, the physiological function of the protein remains unclear. The expression of the neural-specific isoform containing 695 amino acids, beta APP695, is consistent with a role for the protein in neuronal development. In this study, we analyzed the expression of beta APP during the retinoic acid-induced neuronal differentiation of P19 murine embryonal carcinoma cells. Northern blot and RNase protection analyses show a selective increase in beta APP695 expression, concomitant with the morphologic differentiation of P19-derived neurons. Moreover, the time course of increase observed for the beta APP695 mRNA is paralleled by other neuronal-specific transcripts. A similar increase in beta APP695 is observed at the protein level. Furthermore, we show that levels of beta APP695 protein progressively increase during the in vitro differentiation of primary hippocampal neurons. The finding that beta APP695 increases selectively and progressively during neuronal differentiation in two different cell culture systems suggests that this isoform has an important cellular function during this process in the brain. Unlike beta APP in most peripheral cell types, the increased levels of beta APP found in terminally differentiated neuronal cells are not processed in significant amounts by secretory cleavage. Thus, differentiation of neurons is accompanied by increased beta APP695 expression and membrane retention of the protein as intact, full-length molecules that could serve as potential substrates for amyloidogenesis.
A phylogenetic analysis of the myxobacteria: basis for their classification.
Authors: Shimkets L , Woese C R
Published: May 2006

The primary sequence and secondary structural features of the 16S rRNA were compared for 12 different myxobacteria representing all the known cultivated genera. Analysis of these data show the myxobacteria to form a monophyletic grouping consisting of three distinct families, which lies within the delta subdivision of the purple bacterial phylum. The composition of the families is consistent with differences in cell and spore morphology, cell behavior, and pigment and secondary metabolite production but is not correlated with the morphological complexity of the fruiting bodies. The Nannocystis exedens lineage has evolved at an unusually rapid pace and its rRNA shows numerous primary and secondary structural idiosyncrasies.
Hypoxia and N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, but not nerve growth factor, induce Na+ channels and hypertrophy in chromaffin-like arterial chemoreceptors.
Authors: Stea A , Jackson A , Nurse C A
Published: May 2006

Chronic hypoxia sensitizes the ventilatory reflex in mammals and causes enlargement of the carotid body, a peripheral arterial chemosensory organ. To investigate possible underlying mechanisms, in the absence of circulatory changes, we exposed cultures of dissociated rat carotid body containing the oxygen sensors (i.e., chromaffin-like glomus cells) to chronic hypoxia (6% O2) over a period of 2 weeks. After a delay of a few days, the Na+ current density in hypoxia-treated glomus cells increased significantly, reaching values up to 6 times that seen in normoxic (20% O2) controls. In addition the whole-cell capacitance, an indicator of cell size, was also significantly larger (3-4 times control) in glomus cells exposed to chronic hypoxia. Both effects were mimicked qualitatively by chronic treatment of normoxic cultures with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, but not nerve growth factor, which is known to induce similar changes in the chromaffin cell line PC12. Thus, the physiological and morphological effects of chronic hypoxia on the carotid body in vivo may be due in part to a cAMP-mediated stimulation of Na+ channel expression and hypertrophy in the chemosensory glomus cells.
Engagement of the T-cell receptor during positive selection in the thymus down-regulates RAG-1 expression.
Authors: Brändle D , Müller C , Rülicke T , Hengartner H , Pircher H
Published: May 2006

We have examined the expression of the recombination activating gene RAG-1 by in situ hybridization to thymi from mice bearing transgenes for the T-cell receptor (TCR) alpha chain, TCR beta chain, or both TCR alpha and beta chains. RAG-1 transcription was found in the thymic cortex of transgenic mice carrying a single TCR alpha- or TCR beta-chain transgene, comparable to normal mice. However, RAG-1 transcription was strikingly reduced in the thymic cortex from transgenic mice carrying both TCR alpha- and beta-chain genes and expressing major histocompatibility complex (MHC) class I (H-2b) molecules necessary for positive selection of the transgenic TCR. In contrast, thymi of transgenic mice also carrying both TCR alpha- and beta-chain genes but expressing MHC molecules (H-2d) that did not positively select the transgenic TCR displayed high levels of RAG-1 transcription. The low thymic RAG-1 expression coincided with high transgenic TCR alpha-chain surface expression and with inhibition of endogenous TCR alpha-chain rearrangement. Our findings suggest that binding of the TCR to self MHC molecules during positive selection down-regulates RAG-1 transcription in cortical thymocytes and thereby prevents further TCR alpha-chain rearrangements.
Somatostatin receptors are expressed by immature cerebellar granule cells: evidence for a direct inhibitory effect of somatostatin on neuroblast activity.
Authors: Gonzalez B , Leroux P , Lamacz M , Bodenant C , Balazs R , Vaudry H
Published: May 2006

Somatostatin and somatostatin receptors are transiently expressed in the immature rat cerebellar cortex but virtually undetectable in the cerebellum of adults. Although somatostatin binding sites have been visualized during the postnatal period in the external granule cell layer, the type of cell that expresses somatostatin receptors has never been identified; thus, the potential function of somatostatin in the developing cerebellum remains unknown. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the presence of somatostatin receptors and the effect of somatostatin on intracellular messengers on cerebellar neuroblasts in primary culture. Autoradiographic labeling revealed the occurrence of a high density of binding sites for radioiodinated Tyr-[D-Trp8]somatostatin-(1-14) on 1-day-old cultured immature granule cells. Saturation and competition studies showed the existence of a single class of high-affinity binding sites (Kd = 0.133 +/- 0.013 nM, Bmax = 3038 +/- 217 sites per cell). Somatostatin induced a dose-dependent inhibition of forskolin-evoked cAMP formation (ED50 = 10 nM), and this effect was prevented by preincubation of cultured immature granule cells with pertussis toxin. Somatostatin also caused a marked reduction of intracellular calcium concentration. These results show the presence of functionally active somatostatin receptors on immature granule cells. Our data suggest the possible involvement of somatostatin in the regulation of proliferation and/or migration of neuroblasts during the development of the cerebellar cortex.
Dendritic field size and morphology of midget and parasol ganglion cells of the human retina.
Authors: Dacey D M , Petersen M R
Published: May 2006

The visual system of the macaque monkey has provided a useful model for understanding the neural basis of human vision, yet, there are few detailed comparisons of neural populations other than photoreceptors for the two species. Using intracellular staining in an in vitro preparation of the isolated and intact human retina, we have characterized the relationship of dendritic field size to retinal eccentricity for the two major ganglion cell classes, the midget and the parasol cells. We report three findings. (i) The difference in dendritic field diameter between the parasol and midget cells increases from a ratio of approximately 3:1 in the retinal periphery to approximately 10:1 at 3 degrees eccentricity, suggesting that human midget cells may outnumber parasol cells by as much as 30:1 in the central retina. (ii) The dendritic fields of human ON-center parasol and midget cells are 30-50% larger in diameter than their OFF-center counterparts, suggesting a distinct asymmetry in the human ON-OFF visual pathways. (iii) The dendritic fields of parasol cells, but not midget cells, are larger in humans than in macaques. The difference increases from approximately 20% in the retinal periphery to approximately 90% at 5 degrees eccentricity. This result predicts that the human parasol cells should show a lower resolving ability and an increased sensitivity to luminance contrast than their equivalents in the macaque.
Quinolone binding to DNA is mediated by magnesium ions.
Authors: Palù G , Valisena S , Ciarrocchi G , Gatto B , Palumbo M
Published: May 2006

The binding of plasmid DNA to norfloxacin, a quinolone antibacterial agent, was investigated by fluorescence, electrophoretic DNA unwinding, and affinity chromatography techniques. The amount of quinolone bound to DNA was modulated by the concentration of Mg2+. No interaction was evident in the absence of Mg2+ or in the presence of an excess of Mg2+, whereas maximum binding was observed at a Mg2+ concentration of 1-2 mM. The experimental data can be fitted to the formation of three types of Mg adducts: a binary adduct with norfloxacin and Mg2+, a binary adduct with DNA and Mg2+, and a ternary adduct with quinolone, plasmid, and Mg2+. We propose a model for the ternary complex, in which Mg acts as a bridge between the phosphate groups of the nucleic acid and the carbonyl and carboxyl moieties of norfloxacin. Additional stabilization may arise from stacking interactions between the condensed rings of the drug and DNA bases (especially guanine and adenine), which may account for the preference exhibited by quinolones for single-stranded and purine-rich regions of nucleic acids. Other possible biochemical pathways of drug action are suggested by the observation that norfloxacin binds Mg2+ under conditions that are close to physiological.
A broad molecular phylogeny of ciliates: identification of major evolutionary trends and radiations within the phylum.
Authors: Baroin-Tourancheau A , Delgado P , Perasso R , Adoutte A
Published: May 2006

The cellular architecture of ciliates is one of the most complex known within eukaryotes. Detailed systematic schemes have thus been constructed through extensive comparative morphological and ultrastructural analysis of the ciliature and of its internal cytoskeletal derivatives (the infraciliature), as well as of the architecture of the oral apparatus. In recent years, a consensus was reached in which the phylum was divided in eight classes as defined by Lynn and Corliss [Lynn, D. H. & Corliss, J. O. (1991) in Microscopic Anatomy of Invertebrates: Protozoa (Wiley-Liss, New York), Vol. 1, pp. 333-467]. By comparing partial sequences of the large subunit rRNA molecule, and by using both distance-matrix and maximum-parsimony-tree construction methods (checked by boot-strapping), we examine the phylogenetic relationships of 22 species belonging to seven of these eight classes. At low taxonomic levels, the traditional grouping of the species is generally confirmed. At higher taxonomic levels, the branching pattern of these seven classes is resolved in several deeply separated major branches. Surprisingly, the first emerging one contains the heterotrichs and is strongly associated with a karyorelictid but deeply separated from hypotrichs. The litostomes, the oligohymenophorans, and the hypotrichs separate later in a bush-like topology hindering the resolution of their order of diversification. These results show a much more ancient origin of heterotrichs than was classically assumed, indicating that asymmetric, abundantly ciliated oral apparatuses do not correspond to "highly evolved" traits as previously thought. They also suggest the occurrence of a major radiative explosion in the evolutionary history of the ciliates, yielding five of the eight classes of the phylum. These classes appear to differ essentially according to the cytoskeletal architecture used to shape and sustain the cellular cortex (a process of essential adaptative and morphogenetic importance in ciliates).
Growing tips of type I collagen fibrils formed in vitro are near-paraboloidal in shape, implying a reciprocal relationship between accretion and diameter.
Authors: Holmes D F , Chapman J A , Prockop D J , Kadler K E
Published: May 2006

Collagen fibrils generated in vitro at 37 degrees C by enzymic removal of C-terminal propeptides from type I pC-collagen (an intermediate in the normal processing of type I procollagen to collagen containing the C-terminal propeptides but not the N-terminal propeptides) display shape polarity, with one tip fine tapered and the other coarse tapered. Mass measurements by scanning transmission electron microscopy show that the mass per unit length along both kinds of tip increases roughly linearly over distances of approximately 100 D periods from the fibril end [D (axial periodicity) = 67 nm]. The fine tips of fibrils of widely differing lengths exhibit near-identical mass distributions, the mass in all cases increasing at the rate of approximately 17 molecules per D period, irrespective of fibril length. Coarse tips display less regular behavior. These results show that (i) the shape of a fine tip is not conical but resembles more closely a paraboloid of revolution, and (ii) for this shape to be maintained throughout growth, accretion (rate of mass uptake per unit area) cannot everywhere be the same on the surface of the tip but must decrease as the diameter increases. To a first approximation, accretion alpha (diameter)-1.
Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative Rev trans-activator.
Authors: Bevec D , Dobrovnik M , Hauber J , Böhnlein E
Published: May 2006

Human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome (AIDS). Currently, no satisfactory treatment for this viral disease is available. Somatic gene therapy has been proposed as an alternative to conventional therapies. Several antiviral gene therapy approaches including ribozymes, antisense inhibition, and RNA-decoy strategies, as well as dominant-negative mutants of HIV-1 proteins (Gag, Tat, and Rev) have been suggested. To prove the concept of trans-dominant inhibition of HIV-1 replication, we transduced CEM cells with a retroviral vector encoding a dominant-negative rev gene. Amplification of integrase-specific proviral sequences from high molecular weight DNA indicated successful HIV-1 human T-lymphotropic virus type IIIB (HTLV-IIIB) infection of all cells. In contrast to CEM cells and CEM cells expressing the rev wild-type (wt) gene, infection of two CEM-RevM10 clones with HIV-1 did not result in the release of significant levels of p24 Gag antigen as measured by antigen capture assay, indicating a block in HIV-1 replication due to the presence of the trans-dominant Rev protein. Furthermore, the parental CEM cells as well as CEM cells expressing the Rev wt protein were effectively killed in the course of the HIV-1 infection, whereas all CEM cells expressing the RevM10 protein were unaffected in their growth rate.
Unsaturation of fatty acids in membrane lipids enhances tolerance of the cyanobacterium Synechocystis PCC6803 to low-temperature photoinhibition.
Authors: Gombos Z , Wada H , Murata N
Published: May 2006

Effect of the unsaturation of fatty acids in the glycerolipids of thylakoid membranes on low-temperature photoinhibition of photosynthesis was studied by mutation and transformation of the cyanobacterium Synechocystis PCC6803. When grown at 34 degrees C, the wild type contained mono-, di-, and triunsaturated lipids; a mutant, designated Fad6, contained mono- and diunsaturated lipids; and a transformant of Fad6, with a disrupted gene for desaturation and designated Fad6/desA::Kmr, contained only monounsaturated lipids. Fad6/desA::Kmr was the most susceptible among these strains to low-temperature photoinhibition of photosynthesis, whereas Fad6 and the wild type were apparently indistinguishable in terms of sensitivity to photoinhibition. This result suggests that the presence of diunsaturated fatty acids is important in protecting against low-temperature photoinhibition. The photoinhibition at room temperature, although much less significant than that at low temperature, was also affected by the unsaturation of fatty acids. By contrast, the photosynthetic transport of electrons, measured at various temperatures, was not affected by changes in extent of fatty acid unsaturation.
Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.
Authors: Alpuche Aranda C M , Swanson J A , Loomis W P , Miller S I
Published: May 2006

Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification.
Retention of habituation in PC12 cells.
Authors: Cheever L , Koshland D E
Published: May 2006

The time course of habituation and recovery of neurotransmitter release was measured in neuronally differentiated PC12 cells stimulated with either acetylcholine or ATP. The release of norepinephrine in response to either stimulant declined exponentially with repeated presentation of that stimulant. When the stimulus was withheld, the cells' ability to respond recovered to initial levels with an exponential time course. The rate of response recovery depended on the stimulant used and, in the case of stimulation with acetylcholine, on the number of previous stimuli. After habituation and recovery, or partial recovery, of norepinephrine release, the response to a second series of repetitive stimuli declined more rapidly than in the naive case. This increase in habituation rate was dependent on the number of previous stimuli and, in the case of stimulation with acetylcholine, was stable with time for at least 90 min after stimulation. These phenomena are analogous to characteristics of short- and long-term memories of habituative learning observed in behavioral studies. Kinetic equations based on a putative reversible stimulation-dependent inactivation of the cellular response mechanism were used to analyze the rates of habituation and response recovery.
Mouse cortical collecting duct cells show nonselective cation channel activity and express a gene related to the cGMP-gated rod photoreceptor channel.
Authors: Ahmad I , Korbmacher C , Segal A S , Cheung P , Boulpaep E L , Barnstable C J
Published: May 2006

Apical nonselective cation channels with an average single-channel conductance of 34 +/- 2.3 pS were found in M-1 mouse cortical collecting duct cells. Channel activity is increased by depolarization and abolished by cytoplasmic calcium removal. Cytoplasmic application of 0.1 mM cGMP decreases channel open probability by 27%. cDNAs corresponding to approximately 40% of the coding region of the photoreceptor channel were isolated by the polymerase chain reaction from M-1 cells and a rat kidney cDNA library. The rat kidney-derived sequence differs by a single base, and the M-1-cell-derived sequence differs by only two bases, from the photoreceptor sequence. A second clone from M-1 cells differs by 20 out of 426 bases from the photoreceptor sequence. In all three clones, the deduced amino acid sequence is identical to that of the rat photoreceptor channel. Northern blot analysis of poly(A)+ RNA from M-1 cells reveals the presence of a 3.2-kilobase band hybridizing with a retinal cGMP-gated cation channel probe. The results suggest the expression in M-1 cells of more than one gene coding for nonselective cation channels or channel subunits, one of which is identical to the cGMP-gated cation channel gene of rod photoreceptors.
Molecular cloning of a somatostatin-28 receptor and comparison of its expression pattern with that of a somatostatin-14 receptor in rat brain.
Authors: Meyerhof W , Wulfsen I , Schönrock C , Fehr S , Richter D
Published: May 2006

The tetradecapeptide somatotropin-release inhibiting factor somatostatin-14 regulates the release of peptide hormones and also functions as neurotransmitter. The octacosapeptide somatostatin-28, the N-terminally extended form of somatostatin-14, shows similar biological activities yet with different potencies. Both peptides most likely function through distinct receptors. Here we report on the molecular and functional characterization of a somatostatin-28 receptor (SSR-28) cloned from a rat brain cDNA library. The nucleotide sequence contains an open reading frame for a protein of 428 amino acid residues with a predicted molecular mass of 47 kDa. Binding assays using radiolabeled somatostatin-14 and membranes from COS cells transfected with the cloned cDNA show that this receptor, SSR-28, has a higher binding affinity for somatostatin-28 (IC50 = 0.24 nM) than for somatostatin-14 (IC50 = 0.89 nM). RNA blot analysis reveals a 4.4-kilobase mRNA in rat cerebellum and at significantly lower abundance in other brain regions. In situ hybridization indicates that SSR-28 mRNA is present in the granular and Purkinje cell layers of the cerebellum and in the large cells of the hypoglossal nucleus of the brain stem. Signals for SSR-28 mRNA do not overlap with those of a previously cloned rat receptor that preferentially binds somatostatin-14 (SSR-14). SSR-14 mRNA is found in the medial cerebellar nucleus, horizontal limb of the diagonal band, various hypothalamic nuclei, and in layers IV and V of the cortex. In the rat cerebellum, SSR-14 and SSR-28 mRNAs are developmentally regulated; the levels of the former are highest around birth and levels of the latter are highest at the adult stage.
Uptake of exogenous free cholesterol induces upregulation of tissue factor expression in human monocyte-derived macrophages.
Authors: Lesnik P , Rouis M , Skarlatos S , Kruth H S , Chapman M J
Published: May 2006

Lipid-laden macrophages present as foam cells may contribute to the hyperthrombotic state of human atherosclerotic lesions by the production of tissue factor (TF). We investigated the effect of exogenous nonlipoprotein cholesterol on the expression of TF by human monocyte-derived macrophages in culture. Nonlipoprotein cholesterol at 50 micrograms/ml increased TF activity 4-fold; TF induction was dose- and time-dependent. Expression of TF activity was positively correlated with the free cholesterol content of monocyte-derived macrophages, was increased upon inhibition of cholesterol esterification, and reflected de novo synthesis of TF protein. TF expression in cholesterol-loaded macrophages remained sensitive to stimulation (approximately 12-fold) by bacterial lipopolysaccharide, indicating that intracellular free cholesterol and lipopolysaccharide act by distinct mechanisms in inducing TF procoagulant activity. Our results suggest that loading human monocyte-derived macrophages with free cholesterol induces upregulation of TF expression, thereby contributing to thrombus formation at sites of plaque rupture.
Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family.
Authors: Aymerich S , Steinmetz M
Published: May 2006

Induction of the Bacillus subtilis sacB gene and sacPA operon and Escherichia coli bgl operon is mediated by structurally homologous antiterminators encoded by the sacY, sacT, and bglG genes, respectively. When activated, these proteins prevent early transcription termination at terminators located in the leader regions of the three operons. BglG was previously shown to bind in vitro to an imperfectly palindromic 29-nucleotide RNA sequence located upstream of the terminator and partially overlapping with it [Houman, F., Diaz-Torres, M.R. & Wright, A. (1990) Cell 62, 1153-1163]. Similar motifs, here termed ribonucleic antiterminators (RATs), strongly conserved in sequence and in position, are found in the leader of both sacB and sacPA. Mutations were created in sacB RAT and tested in B. subtilis; this showed that sacB RAT is the target for SacY-mediated induction of sacB and that a stem-loop structure in the mRNA is required for regulatory function. Mutations increasing the similarity of the sacB RAT with those of sacPA or bgl rendered sacB inducible by SacT or BglG, respectively; most of these changes did not strongly affect induction by SacY, suggesting that the nucleotides at these variable positions act as negative specificity determinants.
Parental imprinting studied by allele-specific primer extension after PCR: paternal X chromosome-linked genes are transcribed prior to preferential paternal X chromosome inactivation.
Authors: Singer-Sam J , Chapman V , LeBon J M , Riggs A D
Published: May 2006

The preferential inactivation of the paternal X chromosome in extraembryonic cells during early mouse development is an example of parental imprinting, but it has not been studied at the transcriptional level because standard methods of measuring RNA levels do not allow detection of allele-specific RNAs in individual early embryos. We sought to determine whether the paternal allele of the X chromosome-linked gene for 3-phosphoglycerate kinase 1 (Pgk-1), which is located very near the center of X chromosome inactivation, is transcribed prior to differentiation of extraembryonic lineages. Previous reports indicated that in heterozygous embryos there is a delay in the appearance of the phosphoglycerate kinase 1 allozyme encoded by the paternal X chromosome until 2 days after the appearance of the corresponding maternal allozyme. We report results obtained by use of a reverse transcription/PCR-based method which allows the quantitative measurement of allele-specific RNA. The assay is sensitive enough for the quantitative analysis in single embryos of allele-specific transcripts differing by only one nucleotide. We have used this assay to analyze mouse embryos heterozygous at the Pgk-1 and Hprt [hypoxanthine (guanine) phosphoribosyltransferase] loci, and we find that individual 8-cell and blastocyst embryos express both Hprt and Pgk-1 paternal transcripts, as do pooled 2- to 4-cell embryos. These results are discussed in view of the apparent temporal delay in paternal expression of the Pgk-1 gene at the enzyme level.