bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular levels of thymosin immunoreactive peptides are linked to proliferative events: evidence for a nuclear site of action.
Authors: Conteas C N , Mutchnick M G , Palmer K C , Weller F E , Luk G D , Naylor P H , Erdos M R , Goldstein A L , Panneerselvam C , Horecker B L
Published: June 2006

Thymosin alpha 1 (T alpha 1), the N-terminal 28-amino acid fragment of prothymosin alpha (ProT alpha), and ProT alpha, although originally isolated from whole thymus extracts, are also present in nonthymic cells and tissues. We used an ELISA with an antibody raised against T alpha 1 to investigate the relationship between intracellular levels of thymosin immunoreactive peptide(s) (TIP) and cell proliferation in a rat small intestinal IEC-6 cell line. Increasing TIP levels were observed during cell proliferation, which decreased when proliferation was halted by cellular contact inhibition. Serum feeding of cells previously rendered quiescent by serum starvation resulted in a significant increase in TIP within 1 hr. Conversely, serum starvation decreased TIP levels within 1 hr. Peak TIP levels appeared after 3 hr of serum incubation, while maximum [3H]thymidine incorporation was noted after 9 hr, suggesting maximum TIP concentrations in the G1 phase of the proliferative cycle. Immunoelectron microscopy demonstrated an association of TIP with condensed nuclear chromatin. These results support a relation of intracellular TIP levels to IEC-6 cell proliferation and also a nuclear site of action. HPLC analysis of cellular homogenates from proliferating IEC-6 cells revealed a peak of immune reactivity that elutes in the position of T alpha 1.
Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme E1.
Authors: Handley P M , Mueckler M , Siegel N R , Ciechanover A , Schwartz A L
Published: June 2006

The ubiquitin-activating enzyme E1 catalyzes the first step in ubiquitin conjugation. We have cloned and sequenced the cDNA for human E1. This clone predicts a protein of 110,450 Da. Cys-194 lies within a region of identity to active-site Cys-88 of the ubiquitin carrier protein E2, suggesting a potential role for this region in enzymatic function of this protein. In addition, Cys-454 lies within a region of identity to the thiol ester consensus sequence of several proteins involved in thioester formation. Tissue distribution reveals a single 3.5-kilobase E1 message ubiquitous among tissues and cell lines.
Expression, secretion, and lipid-binding characterization of the N-terminal 17% of apolipoprotein B.
Authors: Herscovitz H , Hadzopoulou-Cladaras M , Walsh M T , Cladaras C , Zannis V I , Small D M
Published: June 2006

The N-terminal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pulse-chase experiments showed that the depletion of intracellular apoB-17 follows an apparent first-order kinetics with t1/2 = 51 min. Under conditions of continuous labeling, greater than 60% of the total synthesized apoB-17 was secreted in a soluble form, approximately 98% lipid-poor and approximately 2% lipid-bound. Inclusion of 1.2 mM oleate resulted in 5- and 2.5-fold increases in the amount of labeled apoB-17 in the p less than 1.063 g/ml and 1.063 less than p less than 1.21 g/ml fractions, respectively, which was coordinated with increased secretion of radiolabeled core lipids, triacylglycerols, and cholesteryl esters. Thus under conditions in which lipid pools are enriched a greater fraction of apoB-17 may be secreted on lipoprotein-like particles. The lipid-poor apoB-17 present in p greater than 1.21 g/ml readily associates with exogenously added dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles to form discoidal particles. Discs formed with DMPC/apoB-17, 7:1 (wt/wt), are 239 +/- 43 A in diameter and 61 +/- 4 A thick and contain approximately 2 molecules of apoB-17 and 2250 molecules of DMPC per disc. Based on volume calculations we conclude that apoB-17 forms an annulus about one bilayer high and 10 A thick surrounding the DMPC disc. Circular dichroic spectra of apoB-17 on DMPC discs showed apoB-17 to contain 39% alpha-helix, 36% beta-sheet, 9% beta-turn, and 16% random coil. To be consistent with this model greater than 70% of apoB-17 on DMPC discs must bind to lipid. These data suggest that the N-terminal 17% of apoB-100 can bind lipid and may contribute to some extent to the stabilization of triglyceride-rich lipoproteins.
Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase.
Authors: Cyert M S , Kunisawa R , Kaim D , Thorner J
Published: June 2006

Calcineurin, or phosphoprotein phosphatase type 2B (PP2B), is a calmodulin-regulated phosphoprotein phosphatase. We isolated a gene encoding a yeast PP2B homolog (CNA1) by screening a yeast genomic DNA library in the expression vector lambda gt11, first with 125I-labeled yeast calmodulin and then with a human cDNA encoding the catalytic (or A) subunit of calcineurin. The predicted CNA1 gene product is 54% identical to its mammalian counterpart. Using the polymerase chain reaction (PCR) with oligonucleotide primers based on sequences conserved between CNA1 and mammalian PP2B genes, we isolated a second gene, CNA2. CNA2 is identical to PP2Bw, a partial cDNA clone previously described by others as originating from rabbit brain tissue. Our findings demonstrate that a unicellular eukaryote contains phosphoprotein phosphatases of the 2B class. Haploid cells containing a single cna1 or cna2 null mutation, or both mutations, were viable. MATa cna1 cna2 double mutants were more sensitive than wild-type cells or either single mutant to growth arrest induced by the mating pheromone alpha factor and failed to resume growth during continuous exposure to alpha factor. Thus, calcineurin action antagonizes the mating-pheromone response pathway.
RETRACTED: Accumulation of glycerophosphocholine (GPC) by renal cells: osmotic regulation of GPC:choline phosphodiesterase.
Authors: Zablocki K , Miller S P , Garcia-Perez A , Burg M B
Published: June 2006

Although GPC has long been recognized as a degradation product of phosphatidylcholine, only recently is there wide appreciation of its role as a compatible and counteracting osmolyte that protects cells from osmotic stress. GPC is osmotically regulated in renal cells. Its level varies directly with extracellular osmolality. Cells in the kidney medulla in vivo and in renal epithelial cell cultures (MDCK) accumulate large amounts of GPC when exposed to high concentrations of NaCl and urea. Osmotic regulation of GPC requires choline in the medium, presumably as a precursor for synthesis of GPC. Choline transport into the cells, however, is not osmoregulated. The purpose of the present studies was to use MDCK cell cultures as a defined model to distinguish whether osmotically induced accumulation of GPC results from increased GPC synthesis or decreased GPC disappearance. The rate of incorporation of 14C from [14C]choline into GPC, the steady-state GPC synthesis rate, and the activity of phospholipase A2 (which can catalyze a step in the synthesis of GPC from phosphatidylcholine) are not increased by high NaCl and urea. In fact all are decreased by approximately one-third. Therefore, we find no evidence that high NaCl and urea increases the GPC synthesis rate. On the other hand, the rate coefficient for cellular GPC disappearance and the activity of GPC:choline phosphodiesterase (EC 3.1.4.2), which catalyzes degradation of GPC, are decreased by approximately two-thirds by high NaCl and urea. We conclude that high NaCl and urea increase the level of GPC by inhibiting its enzymatic degradation.
Rolling-circle replication of the plasmid pKYM isolated from a gram-negative bacterium.
Authors: Yasukawa H , Hase T , Sakai A , Masamune Y
Published: June 2006

Plasmid pKYM isolated from a Gram-negative bacterium encodes a Rep protein that is essential for plasmid replication. A comparison of Rep protein from pKYM to Rep proteins encoded by other plasmids shows that it has homology to Rep proteins of the pUB110 plasmid family from Gram-positive bacteria. These plasmids replicate by a rolling-circle mechanism in which a tyrosine residue in the Rep protein acts as the acceptor for the 5' end of the single-strand break introduced by the Rep protein. A Tyr----Phe substitution in the pKYM Rep protein abolishes its activity. Strand-specific single-stranded circular plasmid DNA can be recovered from the cells carrying pKYM and thus we propose that the plasmid pKYM replicates by a rolling-circle mechanism.
Chromostatin, a 20-amino acid peptide derived from chromogranin A, inhibits chromaffin cell secretion.
Authors: Galindo E , Rill A , Bader M F , Aunis D
Published: June 2006

Chromogranin A (CGA) is a ubiquitous 48-kDa secretory protein present in adrenal medulla, anterior pituitary, central and peripheral nervous system, endocrine gut, thyroid, parathyroid, and endocrine pancreas. Recently, we have demonstrated that the protein could be a precursor of bioactive peptides capable of modulating catecholamine secretion from cultured adrenal medullary chromaffin cells. Here we cleaved CGA purified from bovine chromaffin granules with endoproteinase Lys-C, and we isolated and partially sequenced the peptide inhibiting catecholamine secretion from cultured chromaffin cells. A corresponding synthetic peptide composed of the first 20 N-terminal amino acids produced a dose-dependent inhibition in the 10(-9) to 10(-6) M range (with an ID50 of 5 nM) of the catecholamine secretion evoked by carbamoylcholine or by potassium at a depolarizing concentration. This peptide affected secretagogue-induced calcium fluxes but did not alter sodium fluxes. It was found to increase desensitization of cell responses and to modify the kinetics of catecholamine release. Our results indicate that the peptide is extracellularly generated from CGA by a calcium-dependent proteolytic mechanism. We suggest that this peptide, named chromostatin, may be an endocrine modulator of catecholamine-associated responses.
Sequence identity in the nick regions of IncP plasmid transfer origins and T-DNA borders of Agrobacterium Ti plasmids.
Authors: Waters V L , Hirata K H , Pansegrau W , Lanka E , Guiney D G
Published: June 2006

The IncP antibiotic-resistance plasmids transfer to a broad range of bacterial species. The RK2 origin of DNA transfer (oriT) consists of a 250-base-pair segment including the single-stranded cleavage site (nic) needed to generate the DNA strand believed to be transferred. Deletion derivatives and a bank of hydroxylamine-generated oriT mutants were screened for loss of transferability. DNA regions flanking both sides of nic are required for optimal transfer of the oriT clone. Of the chemically induced mutants, critical base-pair changes that dramatically reduced transfer frequency were found in a 10-base-pair region adjacent to nic. Relaxation (nicking) assays performed with these point mutants using protein-DNA complexes reconstituted in vitro revealed a correlation between DNA nicking and transfer frequency. Base-pair changes within the proximal arm of an inverted repeat upstream from the nick site resulted in reduced binding of the essential transfer protein TraJ and correspondingly reduced transfer frequencies. The results support a model of relaxosome formation involving at least two essential proteins: TraI and TraJ. The nick region defined by the point mutants was located in a segment known to be nearly identical in the related plasmid R751. This sequence was also found to be highly conserved in both border junctions of the transfer DNA (T-DNA) of plant tumor-inducing plasmids of Agrobacterium tumefaciens, indicating a relationship between IncP-mediated broad-host-range bacterial conjugation and T-DNA transfer to plants.
Transcription is associated with Z-DNA formation in metabolically active permeabilized mammalian cell nuclei.
Authors: Wittig B , Dorbic T , Rich A
Published: June 2006

Mammalian cells have been encapsulated in agarose microbeads, and from these cells metabolically active permeabilized nuclei were prepared. Previously, we showed that biotin-labeled monoclonal antibodies against Z-DNA can be diffused into the nuclei and, over a specific concentration range, they will bind to Z-DNA within the nucleus in a concentration-independent manner. By using radiolabeled streptavidin, we showed that the amount of Z-DNA antibody bound is related to the torsional strain of the DNA in the nucleus. Relaxation of the DNA results in a decrease of Z-DNA formation, whereas increasing torsional strain through inhibiting topoisomerase I results in increased Z-DNA formation. Here we measure the influence of RNA transcription and DNA replication. Transcription is associated with a substantial increase in the binding of anti-Z-DNA antibodies, paralleling the increased level of RNA synthesized as the level of ribonucleoside triphosphate in the medium is increased. DNA replication yields smaller increases in the binding of Z-DNA antibodies. Stopping RNA transcription with inhibitors results in a large loss of Z-DNA antibody binding, whereas only a small decrease is associated with inhibition of DNA replication.
Three-dimensional structure of human basic fibroblast growth factor, a structural homolog of interleukin 1 beta.
Authors: Zhang J D , Cousens L S , Barr P J , Sprang S R
Published: June 2006

The three-dimensional structure of the 146-residue form of human basic fibroblast growth factor (bFGF), expressed as a recombinant protein in yeast, has been determined by x-ray crystallography to a resolution of 1.8 A. bFGF is composed entirely of beta-sheet structure, comprising a three-fold repeat of a four-stranded antiparallel beta-meander. The topology of bFGF is identical to that of interleukin 1 beta, showing that although the two proteins share only 10% sequence identity, bFGF, interleukin 1, and their homologs comprise a family of structurally related mitogenic factors. Analysis of the three-dimensional structure in light of functional studies of bFGF suggests that the receptor binding site and the positively charged heparin binding site correspond to adjacent but separate loci on the beta-barrel.
The Drosophila homologue of vertebrate myogenic-determination genes encodes a transiently expressed nuclear protein marking primary myogenic cells.
Authors: Paterson B M , Walldorf U , Eldridge J , Dübendorfer A , Frasch M , Gehring W J
Published: June 2006

We have isolated a cDNA clone, called Dmyd for Drosophila myogenic-determination gene, that encodes a protein with structural and functional characteristics similar to the members of the vertebrate MyoD family. Dmyd clone encodes a polypeptide of 332 amino acids with 82% identity to MyoD in the 41 amino acids of the putative helix-loop-helix region and 100% identity in the 13 amino acids of the basic domain proposed to contain the essential recognition code for muscle-specific gene activation. Low-stringency hybridizations indicate that Dmyd is not a member of a multigene family similar to MyoD in vertebrates. Dmyd is a nuclear protein in Drosophila, consistent with its role as a nuclear-gene regulatory factor, and is proposed to be a transiently expressed marker for muscle founder cells. We have used an 8-kilobase promoter fragment from the gene, which contains the first 55 amino acids of the Dmyd protein, joined to lacZ, to follow myogenic precursor cells into muscle fibers with antibodies to beta-galactosidase and to Dmyd. Unlike the myogenic factors in vertebrate muscle cells, Dmyd appears to be expressed at a much lower level in differentiated Drosophila muscles, so Dmyd cannot be followed continuously as a muscle marker. This fact is reflected in the loss of Dmyd RNA expression in 12- to 24-hr embryos, a major period of early myogenesis, as well as in the undetectable level of the nuclear antigen in primary cultures of embryonic and adult Drosophila muscle.
Nucleokinesis: distinct pattern of cell translocation in response to an autocrine motility factor-like substance or fibronectin.
Authors: Klominek J , Sundqvist K G , Robért K H
Published: June 2006

Human lung adenocarcinoma cells develop bipolar shape with prominent pseudopodia (greater than or equal to 200 microns) when cultured in the presence of autocrine motility factor (AMF)-like substance or on fibronectin-coated substrata. AMF was partially purified from a human lung adenocarcinoma cell line and has a peak biological activity at a molecular mass of 67 kDa. Using time-lapse photography, we observed that during AMF- or fibronectin-induced cell translocation, the nuclei of some bipolar cells are transported to the opposite end of the cell, while gross cell shape and position remain unchanged. Following this nuclear movement, which we call "nucleokinesis," the posterior pseudopodium is retracted behind the nucleus. Thus, extension of a pseudopodium followed by nucleokinesis in the same direction and retraction of the cell body behind the nucleus is a normal motile sequence in translocating bipolar cells. This suggests that nucleokinesis is a distinct step in whole-cell translocation of bipolar cells on biological substrata and that pseudopodia can be used as nuclear transport organs. In contrast, adenocarcinoma cells cultured on artificial substrata and in the absence of AMF display a fibroblast-like motility pattern with the nucleus centrally located within the migrating cell.
A molecular mechanism for pyrimidine and purine nucleotide control of aspartate transcarbamoylase.
Authors: Stevens R C , Lipscomb W N
Published: June 2006

CTP (ATP) binding to the T or R state causes reorientation of several key residues and results in a decrease (increase) in the size of the nucleotide binding site and a related decrease (increase) in the extension of the outer parts of the dimer of the regulatory chains, R1 and R6. As a result, CTP pinches the regulatory dimers together by 0.3 A in the R state; ATP pushes the regulatory dimers apart by 0.3 A in the T state. These changes influence key residues in the R1-C1 interface of the R state and the R1-C1 and R1-C4 interfaces of the T state, such that the separation of catalytic trimers (c3 ... c3) is decreased by 0.5 A by CTP in the R state and increased by 0.4 A by ATP in the T state. (Smaller effects on c3 ... c3 are observed when CTP binds to the sterically crowded T state or when ATP binds to the elongated R state). These changes reorient key residues in the active site (e.g., catalytic chain residue Arg-229, a residue involved in aspartate binding). This pattern for action of CTP and ATP in perturbing the regulatory dimer, and consequently both the structure and flexibility in critical parts of the T state or R state, is called the nucleotide perturbation mechanism.
Oldest fossil flowers of hamamelidaceous affinity, from the Late Cretaceous of New Jersey.
Authors: Crepet W L , Nixon K C , Friis E M , Freudenstein J V
Published: June 2006

Exceptionally well-preserved staminate inflorescences, pistillate inflorescences, and detached stamens with important phylogenetic and paleoecological implications have been discovered from the Turonian (ca. 88.5-90.4 million years B.P.) Raritan Formation of New Jersey. The fossils have a combination of floral and pollen characters found in various genera of modern entomophilous and anemophilous Hamamelidaceae and anemophilous Platanus (Platanaceae). The floral characters of the fossils, including a sepal cup, staminal tube, and apparently nectariferous staminodes, indicate that this taxon was probably insect pollinated. The juxtaposition of character complexes in an extinct taxon from disparate modern taxa provides an interesting phylogenetic perspective on the origins of Hamamelidaceae and is a striking example of a fossil that is a mosaic of familial level characters relative to modern taxa. Of even broader interest, however, is the occurrence of staminodal nectaries that have structural characters intermediate between the fossil's functional stamens and modern hamamelidaceous petals. This transitional staminode morphology in the context of the other fossil characters suggests a staminodal origin of petals in the hamamelid-rosid lineage. This hypothesis is supported by the apparent staminode position within the fossil flowers where petals are found in modern genera. The character complex of morphologically transitional staminodes, a staminal tube, and sepal cup can be viewed as prehypanthial, lacking only fusion of the staminal tube to the sepal cup. The appearance of the character complex embodied in these flowers during the late mid-Cretaceous may signal the early stages of the relationship between specialized pollinators, such as bees, and the hamamelid-rosid-asterid lineage of angiosperms, arguably one of the most important events in angiosperm radiation.
Reductive detoxification as a mechanism of fungal resistance to singlet oxygen-generating photosensitizers.
Authors: Daub M E , Leisman G B , Clark R A , Bowden E F
Published: June 2006

Fungi that are resistant or sensitive to the singlet oxygen-generating toxin cercosporin were assayed for their ability to detoxify it by reduction. Cercosporin reduction was assayed microscopically by using bandpass filters to differentiate between fluorescence emission from cercosporin and reduced cercosporin. Hyphae of the resistant Cercospora and Alternaria species emitted a green fluorescence, indicative of reduced cercosporin. Hyphae of nonviable cultures and of cercosporin-sensitive fungi did not reduce cercosporin. Sensitive fungi occasionally reduced cercosporin when incubated with reducing agents that protect against cercosporin toxicity. Cercosporin could not be efficiently photoreduced in the absence of the fungus. Cercospora species were also resistant to eosin Y but were sensitive to rose bengal. Microscopic observation demonstrated that Cercospora species were not capable of reducing rose bengal but were capable of reducing eosin Y. These observations were supported by in vitro electrochemical measurements that revealed the following order with respect to ease of reduction: cercosporin >> eosin Y > rose bengal. The formal redox potential (E 0') of cercosporin at pH 7.5 was found to be -0.14 V vs. the normal hydrogen electrode. We conclude that Cercospora species protect themselves against cercosporin by the reduction and detoxification of the toxin molecule.
Structural basis for the nucleic acid binding cooperativity of bacteriophage T4 gene 32 protein: the (Lys/Arg)3(Ser/Thr)2 (LAST) motif.
Authors: Casas-Finet J R , Fischer K R , Karpel R L
Published: June 2006

To identify the functional residues of the N-terminal B region of bacteriophage T4 gene 32 protein involved in its cooperative binding to single-stranded nucleic acids, a process dependent on homotypic protein-protein interaction, we have studied the interaction of the protein with synthetic peptides containing different portions of this domain. Gel-permeation chromatography showed that a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan)-labeled fluorescent peptide corresponding to the first 17 residues of gene 32 protein formed a complex with whole protein. The fluorescence was blue-shifted 14 nm upon interaction with intact protein, and somewhat less so (7-11 nm) with cleavage products of the protein lacking B domains. The intrinsic tryptophan fluorescence of whole and truncated protein was quenched by this peptide and by the nonderivatized peptide. The peptide bound tightly to truncated protein at both 0.015 and 0.44 M Na+, with a stoichiometry of 1:1. Similar tryptophan quenching or acrylodan blue shifts were obtained with peptides corresponding to residues 1-9 and 3-8, but not residues 1-4, 5-9, or 5-17, indicating that the essential amino acids are contained within positions 3-8, Lys-Arg-Lys-Ser-Thr-Ala. Several other DNA binding proteins contain a LAST motif with documented involvement of these residues in nucleic acid interaction. The amino acid and coding sequence of residues 110-114, a region proposed to be involved in nucleic acid binding, is virtually identical to that of residues 3-7. Based on these observations, we have formulated a model for the cooperative interactions of gene 32 protein with single-stranded nucleic acids.
Rat brain expresses an alternatively spliced form of the dihydropyridine-sensitive L-type calcium channel alpha 2 subunit.
Authors: Kim H L , Kim H , Lee P , King R G , Chin H
Published: June 2006

We have cloned and sequenced a cDNA (rB-alpha 2) encoding an alpha 2 subunit of the voltage-sensitive L-type calcium channel (dihydropyridine receptor) of rat brain. The cDNA (3823 base pairs) encodes a protein of 1091 amino acids with a Mr of 123,822. The deduced amino acid sequence of rB-alpha 2 cDNA is highly similar (95% amino acid identity) to that of rabbit skeletal muscle alpha 2 subunit. The rB-alpha 2 protein is distinct from the previously cloned skeletal muscle alpha 2-subunit protein [Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., Sharp, A. H., Leung, A. T., Campbell, K. P., McKenna, E., Koch, W., Hui, A., Schwartz, A. & Harpold, M. M. (1988) Science 241, 1661-1664] because it contains an insertion of 7 amino acid residues and a deletion of a 19-amino acid segment between putative transmembrane domains 1 and 2. We show that the rB-alpha 2 and skeletal muscle alpha 2-subunit transcripts are the variants produced by alternative splicing of the primary transcript and that they are differentially expressed in brain and skeletal muscle, respectively.
Expression cloning of cDNA encoding a seven-helix receptor from human placenta with affinity for opioid ligands.
Authors: Xie G X , Miyajima A , Goldstein A
Published: June 2006

Here we report the expression cloning of cDNA encoding a putative opioid receptor from a human placenta cDNA library. Placental opioid receptors are of the kappa type. As the dynorphin opioid peptides are kappa-selective, a dynorphin ligand was used in an affinity-enrichment (panning) procedure to select transiently transfected COS-7 cells expressing kappa receptor binding sites. The cloned cDNA encodes a 440-residue protein of the seven-helix guanine nucleotide-binding protein (G-protein)-coupled receptor family. Ligand binding reveals a stereospecific site with typical opioid properties, which binds peptide and nonpeptide opioids with moderate affinity (Kd approximately 100 nM) and which lacks the expected kappa selectivity. The deduced transmembrane domain is 93% identical to the homologous region of the human neuromedin K (neurokinin B) receptor, but the N-terminal and C-terminal sequences have many dissimilarities. The expressed receptor binds opioid ligands but not tachykinins; and under the same conditions, a cloned rat neuromedin K receptor binds tachykinins but not opioids.
Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.
Authors: Uckun F M , Schieven G L , Tuel-Ahlgren L M , Dibirdik I , Myers D E , Ledbetter J A , Song C W
Published: June 2006

Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation.
A receptor tyrosine kinase found in breast carcinoma cells has an extracellular discoidin I-like domain.
Authors: Johnson J D , Edman J C , Rutter W J
Published: June 2006

We have identified a breast carcinoma tyrosine phosphoprotein, discoidin domain receptor (DDR), that defines an unusual class of receptor tyrosine kinases. The DDR cDNA predicts a C-terminal tyrosine kinase domain and an N-terminal domain similar to the Dictyostelium discoideum lectin discoidin I. These domains are connected by an extraordinary hydrophilic proline/glycine-rich domain, which is interrupted by a predicted transmembrane sequence. This extended proline/glycine-rich region may be required for an unusual geometry of interaction with ligand or substrates. Discoidin I domains are also found in other proteins, including coagulation factors V and VIII, and may represent a class of domains that interact with specific cell surface molecules.
Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata.
Authors: Donly B C , Ding Q , Tobe S S , Bendena W G
Published: June 2006

Allatostatins (ASTs) are insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have isolated a cDNA from the cockroach Diploptera punctata that encodes a 41.5-kDa precursor polypeptide containing the AST family of peptides. Translation of the cDNA revealed a 370-amino acid pre-pro-peptide consisting of 13 AST-type peptides and appropriate processing sites for endoproteolytic cleavage and amidation. The 13 potential AST sequences are characterized by the C-terminal AST corestructure Phe-Gly-Leu-NH2, with only one exception. Separating the clustered ASTs in the precursor, three acidic spacer regions are found. Contained within the largest of these are two potentially related peptides that may also be processed. Southern blot analysis revealed the presence of a single copy of the AST gene per haploid genome, as well as the probability that the gene may be present in at least two allelic forms. In situ hybridization indicated the AST-encoding gene is expressed in neurosecretory cells of D. punctata brain.
Rapid application and removal of second messengers to cyclic nucleotide-gated channels from olfactory epithelium.
Authors: Zufall F , Hatt H , Firestein S
Published: June 2006

The last step in the second-messenger cascade mediating vertebrate olfactory transduction is the direct opening of a nonspecific cation channel by cAMP. The kinetic properties of this interaction are critical in determining the time course of the sensory response. To analyze these properties, excised inside-out membrane patches containing either the native channel from salamander olfactory-receptor neurons or a recombinant rat olfactory cyclic nucleotide-gated channel were exposed to short pulses of known concentrations of cAMP or cGMP to mimic a rapid and transient production of second messenger. Channel activity outlasted cyclic nucleotide pulses for several hundred milliseconds. This effect was due to an intrinsic property of the olfactory channel protein because it did not occur with cGMP-activated channels from retinal photoreceptors. Gating kinetics of the olfactory channel were both voltage and agonist dependent. These results demonstrate that the overall slow channel-gating kinetics could account for the difference in time course between the odor-induced changes in cAMP concentration and the subsequent sensory generator current.
An arcane role of DNA in transcription activation.
Authors: Ryu S , Garges S , Adhya S
Published: June 2006

The mechanism by which the cAMP receptor protein (CRP) activates transcription has been investigated using the lac promoter of Escherichia coli. For transcription activation, an interaction between DNA-bound CRP and RNA polymerase is not sufficient. CRP must bind to a site in the same DNA and close to the promoter. CRP action requires an intact spacer DNA to provide a rigid support in building a CRP-RNA polymerase protein bridge or to allow a conformational change in the DNA to be transmitted to the lac promoter using the protein bridge as a structural support.
Intracellular pH and the control of multidrug resistance.
Authors: Simon S , Roy D , Schindler M
Published: June 2006

Many anticancer drugs are classified as either weak bases or molecules whose binding to cellular structures is pH dependent. Accumulation of these drugs within tumor cells should be affected by transmembrane pH gradients. Indeed, development of multidrug resistance (MDR) in tumor cells has been correlated with an alkaline shift of cytosolic pH. To examine the role of pH in drug partitioning, the distribution of two drugs, doxorubicin and daunomycin, was monitored in fibroblasts and myeloma cells. In both cell types the drugs rapidly accumulated within the cells. The highest concentrations were measured in the most acidic compartments--e.g., lysosomes. Modifying the cellular pH in drug-sensitive cells to mimic reported shifts in MDR caused an immediate change in the cellular drug concentration. Drug accumulation was enhanced by acidic shifts and reversed by alkaline shifts. All of these effects were rapid and reversible. These results demonstrate that the alkaline shift observed in MDR is sufficient to prevent the accumulation of chemotherapeutic drugs independent of active drug efflux.
Long-term repopulation of hematolymphoid cells with only a few hemopoietic stem cells in mice.
Authors: Ogata H , Bradley W G , Inaba M , Ogata N , Ikehara S , Good R A
Published: June 2006

A PCR-based assay has been devised for the detection and semiquantitation of cells originating from a few donor hematopoietic stem cells (HSCs) in a background of recipient cells. Upon sequencing a segment of murine Y chromosome contained in the plasmid pY2, oligonucleotide primers were designed for specific amplification of the Y chromosome-restricted segment. The HSCs were isolated from the bone marrow of mice on day 4 following a single i.v. injection of 5-fluorouracil and were readily distinguished from other bone marrow elements by the characteristics of low density, absence of lineage-specific surface markers, lack of expression of transferrin receptor, and a high expression of major histocompatibility complex class I antigen. Injection of as few as four such HSCs was shown to produce donor-derived cells (including lymphoid cells) for at least 8 months after transplantation into syngeneic female recipients. Retransplantation, employing 10(6) bone marrow cells from the initial recipients, also yielded clear evidence of repopulation with detectable levels of male donor cells. On statistical grounds, it is clear that long-term repopulation in vivo may result from even a single HSC having the characteristics defined herein.
The putative actin-binding role of hydrophobic residues Trp546 and Phe547 in chicken gizzard heavy meromyosin.
Authors: Onishi H , Morales M F , Katoh K , Fujiwara K
Published: June 2006

In the course of myosin-catalyzed ATP hydrolysis, certain amino acid residues in myosin interact with counterparts in actin to produce the relational changes that underlie muscle contraction; some of these interactions are ionic, but the stronger interactions are hydrophobic. In an effort to identify myosin residues participating in hydrophobic interactions, myosin (from smooth muscle) fragments with mutations at suspected sites were engineered and compared with wild-type fragments. It was found that the ATPase of doubly mutated (Trp546Ser and Phe547His) fragments was minimally activated by actin and did not decorate actin well to form the regular arrowhead pattern characteristic of myosin binding to actin filaments. Thus, we suggest that Trp546 and Phe547 are important participants in the hydrophobic actin-myosin interaction.
Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor.
Authors: Normant E , Martres M P , Schwartz J C , Gros C
Published: June 2006

The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor. This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain. Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library. Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity. TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases. This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites. The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation.
Climate warming and disease risks in temperate regions – Argulus coregoni and Diplostomum spathaceum as case studies
Authors: Hakalahti T. , Karvonen A. , Valtonen E.T.
Published: June 2006

The link between climate changes and disease risks from various pathogens has been increasingly recognized. The effect of climatic factors on host–parasite population dynamics is particularly evident in northern latitudes where the occurrence and transmission of parasites are strongly regulated by seasonality-driven changes in environmental temperatures. Shortened winter periods would increase growth potential of many parasite populations. The ways in which climate warming could affect life history dynamics of the directly transmitted crustacean ectoparasite Argulus coregoni and complex life cycle trematode Diplostomum spathaceum, which frequently cause problems in northern fish farming, are discussed. Increased problems for fish farming are predicted in terms of increased infection pressure from these parasites in future. This would increase problems associated with infections and increase the use of expensive management protocols with high environmental impact.
'Oh Comrade, What Times Those Were!' History, Capital Punishment and the Urban Square
Authors: Basson Steve
Published: June 2006

From the perspective of traditional Western histories of the urban realm, public squares have been seen to represent a privileged site of urban containment expressive of a community's highest values of individual freedom, social inclusion and cultural refinement. But such views can be misleading. For what is omitted from the scope of these conventional historical visions and their ideal and conforming subjects of public spatial discourse, is an entire array of other and darker narratives that equally speak of personal choice, collective participation and cultural value. Capital punishment reflects such an example, a practice that once comprised an integral part of the political, social and cultural landscape of a Western city's squares and streets. Drawing from Michel Foucault's Discipline and Punish and its implications on how we might begin to re-read the history of the urban square, the paper seeks to explore those practices and modes of rationality that underpinned the once-public spectacle of executions and torture as a vital condition of urban life. In particular, this discussion will question the assumptions of an historical tradition that continues to reduce our understanding of the city and its open spaces of public appearance and action to an idealistic and illusory reality of the urban realm and its narrow framing of collective conduct, necessity and significance.
Dual Loss of ER Export and Endocytic Signals with Altered Melanosome Morphology in thesilverMutation of Pmel17
Authors: Theos Alexander C. , Berson Joanne F. , Theos Sarah C. , Herman Kathryn E. , Harper Dawn C. , Tenza Danièle , Sviderskaya Elena V. , Lamoreux M. Lynn , Bennett Dorothy C. , Raposo Graça , Marks Michael S.
Published: June 2006

Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine–based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.
Stable CpG Hypomethylation of Adipogenic Promoters in Freshly Isolated, Cultured, and Differentiated Mesenchymal Stem Cells from Adipose Tissue
Authors: Noer Agate , Sørensen Anita L. , Boquest Andrew C. , Collas Philippe
Published: June 2006

Mesenchymal stem cells from adipose tissue can differentiate into mesodermal lineages. Differentiation potential, however, varies between clones of adipose stem cells (ASCs), raising the hypothesis that epigenetic differences account for this variability. We report here a bisulfite sequencing analysis of CpG methylation of adipogenic (leptin [LEP], peroxisome proliferator-activated receptor gamma 2 [PPARG2], fatty acid-binding protein 4 [FABP4], and lipoprotein lipase [LPL]) promoters and of nonadipogenic (myogenin [MYOG], CD31, and GAPDH) loci in freshly isolated human ASCs and in cultured ASCs, in relation to gene expression and differentiation potential. Uncultured ASCs display hypomethylated adipogenic promoters, in contrast to myogenic and endothelial loci, which are methylated. Adipogenic promoters exhibit mosaic CpG methylation, on the basis of heterogeneous methylation between cells and of variation in the extent of methylation of a given CpG between donors, and both between and within clonal cell lines. DNA methylation reflects neither transcriptional status nor potential for gene expression upon differentiation. ASC culture preserves hypomethylation of adipogenic promoters; however, between- and within-clone mosaic methylation is detected. Adipogenic differentiation also maintains the overall CpG hypomethylation of LEP, PPARG2, FABP4, and LPL despite demethylation of specific CpGs and transcriptional induction. Furthermore, enhanced methylation at adipogenic loci in primary differentiated cells unrelated to adipogenesis argues for ASC specificity of the hypomethylated state of these loci. Therefore, mosaic hypomethylation of adipogenic promoters may constitute a molecular signature of ASCs, and DNA methylation does not seem to be a determinant of differentiation potential of these cells.
Model–data synthesis in terrestrial carbon observation: methods, data requirements and data uncertainty specifications
Authors: Raupach M. R. , Rayner P. J. , Barrett D. J. , DeFries R. S. , Heimann M. , Ojima D. S. , Quegan S. , Schmullius C. C.
Published: June 2006

Systematic, operational, long‐term observations of the terrestrial carbon cycle (including its interactions with water, energy and nutrient cycles and ecosystem dynamics) are important for the prediction and management of climate, water resources, food resources, biodiversity and desertification. To contribute to these goals, a terrestrial carbon observing system requires the synthesis of several kinds of observation into terrestrial biosphere models encompassing the coupled cycles of carbon, water, energy and nutrients. Relevant observations include atmospheric composition (concentrations of CO2 and other gases); remote sensing; flux and process measurements from intensive study sites; in situ vegetation and soil monitoring; weather, climate and hydrological data; and contemporary and historical data on land use, land use change and disturbance (grazing, harvest, clearing, fire).

A review of model–data synthesis tools for terrestrial carbon observation identifies ‘nonsequential’ and ‘sequential’ approaches as major categories, differing according to whether data are treated all at once or sequentially. The structure underlying both approaches is reviewed, highlighting several basic commonalities in formalism and data requirements.

An essential commonality is that for all model–data synthesis problems, both nonsequential and sequential, data uncertainties are as important as data values themselves and have a comparable role in determining the outcome.

Given the importance of data uncertainties, there is an urgent need for soundly based uncertainty characterizations for the main kinds of data used in terrestrial carbon observation. The first requirement is a specification of the main properties of the error covariance matrix.

As a step towards this goal, semi‐quantitative estimates are made of the main properties of the error covariance matrix for four kinds of data essential for terrestrial carbon observation: remote sensing of land surface properties, atmospheric composition measurements, direct flux measurements, and measurements of carbon stores.
Association between age and clinical characteristics of renal cell carcinoma in adult patients
Authors: JUN CHEN , ZHISHUN XU , XIANZHOU JIANG , QIANG FU , JIN WANG
Published: June 2006

Aim:  To study the association between age and clinical characteristics of renal cell carcinoma in adult patients.

Methods:  Three hundred and ten patients with renal cell carcinoma were classified into three groups: ≤40 years group, 41–59 years group and ≥60 years group. The clinical characteristics of the three groups were compared to define the association.

Results:  The male/female ratio was 1.3/1, 2.0/1, 3.3/1 in the three groups, respectively, and a significant difference appeared when comparing the ≤40 years group to the ≥60 years group (P = 0.010). The respective percentage of incidental renal cell carcinoma was 27.9%, 43.2%, 31.2%, and it was significantly higher in the 41–59 years group than the ≥60 years group (P = 0.047). The incidence of poorly differentiated renal cell carcinoma decreased with age increasing (11.6% vs 5.2% vs 2.7%), and there was significant difference between the ≤40 years group and the ≥60 years group (P = 0.038). In the ≤40 years group, the incidence of lymph node metastasis was 18.6%, which was significantly higher than the other two groups (P < 0.05). The percentage of T3a tumors was lower in ≤40 years group and the percentage of T1−3aN0M0 tumors was significantly higher in the 41–59 years group.

Conclusions:  The percentage of female patients decreases with increasing age. The higher percentage of incidental renal cell carcinoma leads to the higher percentage of T1−3aN0M0 tumors in middle‐aged patients. The higher percentage of poorly differentiated renal cell carcinoma led to the higher incidence of lymph node metastasis and decreased percentage of T3aN0M0 tumors in younger patients.