bluuebunny/crossref_metadata_2025 huggingface dataset:
Rhodopsin mutations in autosomal dominant retinitis pigmentosa.
Authors: Sung C H , Davenport C M , Hennessey J C , Maumenee I H , Jacobson S G , Heckenlively J R , Nowakowski R , Fishman G , Gouras P , Nathans J
Published: May 2006

DNA samples from 161 unrelated patients with autosomal dominant retinitis pigmentosa were screened for point mutations in the rhodopsin gene by using the polymerase chain reaction and denaturing gradient gel electrophoresis. Thirty-nine patients were found to carry 1 of 13 different point mutations at 12 amino acid positions. The presence or absence of the mutations correlated with the presence or absence of retinitis pigmentosa in 174 out of 179 individuals tested in 17 families. The mutations were absent from 118 control subjects with normal vision.
Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coli.
Authors: Akimaru J , Matsuyama S , Tokuda H , Mizushima S
Published: May 2006

Reconstitution of the translocation machinery for secretory proteins from purified constituents was performed. SecY was solubilized from SecY/SecE-overproducing Escherichia coli cells and purified by chromatography on ion-exchange and size-exclusion columns. Proteoliposomes active in protein translocation were reconstituted from the purified preparations of SecY and SecE. The reconstituted translocation activity was SecA- and ATP-dependent. Although the purified preparations of SecY and SecE were still contaminated with minute amounts of other proteins, the elution profiles of SecY and SecE on column chromatographies coincided with the elution profiles of reconstituted translocation activity, indicating that SecY and SecE are the indispensable components in these preparations. We conclude that SecY, SecE, and SecA are essential components of the protein secretion machinery and that translocation activity can be reconstituted from only these three proteins and phospholipids.
Platelet-derived growth factor (PDGF) and PDGF receptor are induced in mesangial proliferative nephritis in the rat.
Authors: Iida H , Seifert R , Alpers C E , Gronwald R G , Phillips P E , Pritzl P , Gordon K , Gown A M , Ross R , Bowen-Pope D F
Published: May 2006

We investigated whether platelet-derived growth factor (PDGF), or its receptor (PDGF-R), was upregulated in a rat model of mesangial proliferative glomerulonephritis. A marked increase in both PDGF A- and B-chain mRNA could be demonstrated in glomerular RNA by Northern blot analysis 3 and 5 days after disease induction, corresponding to the time of mesangial cell proliferation. PDGF-R beta-subunit mRNA and protein were also increased in glomeruli in mesangial proliferative nephritis, being maximal at day 5. The principal cells expressing PDGF B-chain appeared by immunostaining to be a subpopulation of mesangial cells; in contrast, the majority of the mesangial cells expressed the PDGF-R beta-subunit protein. Both complement depletion and platelet depletion significantly reduced cell proliferation and expression of both PDGF and PDGF-R. Thus, in mesangial proliferative nephritis there is a platelet- and complement-mediated induction of PDGF A and B chain and PDGF-R beta-subunit gene transcription and protein synthesis. The finding that the majority of PDGF is produced by the mesangial cell supports the role of PDGF as an autocrine growth factor in glomerulonephritis.
Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ.
Authors: Clinton M , Graeve L , el-Dorry H , Rodriguez-Boulan E , Horecker B L
Published: May 2006

To test the hypothesis that prothymosin and parathymosin contain amino acid sequences that cause them to be targeted to the cell nucleus, expression vectors were constructed containing a simian virus 40 promoter and cDNAs that would code for chimeric proteins composed of truncated human growth hormone (hGH) linked to the NH2 terminus of prothymosin or parathymosin. The truncated hGH lacked the signal peptide sequence required for its secretion. After transfection of these constructs into HeLa S3 cells, which do not normally synthesize hGH, the use of indirect immunofluorescence staining to follow the localization of the hGH chimeras demonstrated that both prothymosin and parathymosin caused targeting to the cell nucleus. Controls with a construct coding for native hGH only, and one coding for the truncated hGH lacking the signal peptide, revealed secretion into culture medium and staining in the endoplasmic reticulum and Golgi apparatus in the first case, and diffuse staining throughout the cytoplasm in the second. The results provide direct evidence, with proteins synthesized in situ, for the presence of nuclear localization signals in both prothymosin and parathymosin.
Avian osteoclasts as estrogen target cells.
Authors: Oursler M J , Osdoby P , Pyfferoen J , Riggs B L , Spelsberg T C
Published: May 2006

Although in vivo effects of 17 beta-estradiol (estrogen) on bone turnover have been shown to occur mainly through influences on osteoclast-mediated bone resorption, the mechanism by which estrogen reduces bone resorption is unclear. To approach this question, we have examined authentic osteoclasts for evidence of a direct osteoclast response to estrogen in vitro. Highly purified (greater than (90%) viable avian osteoclasts from birds maintained on a low calcium diet were obtained using an osteoclast-specific monoclonal antibody coupled to magnetic beads. Isolated cells were either analyzed directly for estrogen receptor (ER) levels or cultured to assess the biological effects of estrogen. Northern blot analysis revealed a 5.2-kilobase mRNA that hybridized with a cDNA to human ER mRNA in the osteoclasts. An anti-human ER antibody recognized proteins of 66 kDa and 140 kDa in osteoclast extracts by Western blot analysis. The 66-kDa size is in close agreement with the reported size of the human ER. Nuclear binding of estrogen to intact viable osteoclasts was steroid-specific and saturable, with 5662 +/- 1420 molecules bound per nucleus (mean +/- SEM). In vitro estrogen responses in osteoclasts included a dose-dependent decrease in resorption as well as an increase in nuclear protooncogene mRNA levels. These observations indicate that osteoclasts are capable of directly responding to estrogen in vivo.
Inhibition of N- and L-type Ca2+ channels by the spider venom toxin omega-Aga-IIIA.
Authors: Mintz I M , Venema V J , Adams M E , Bean B P
Published: May 2006

omega-Aga-IIIA, an 8.5-kDa peptide toxin isolated from the venom of Agelenopsis aperta, was found to be a highly potent inhibitor of Ca channels in cardiac muscle and in peripheral and central neurons of rats and frogs. Cardiac L-type Ca channels were completely (Kd approximately 0.6 nM) blocked by omega-Aga-IIIA. In sensory neurons, the toxin inhibited most high-threshold Ca current but not T-type Ca current. omega-Aga-IIIA blocked with similar potency (Kd approximately 1.5 nM) both omega-conotoxin GVIA-sensitive and dihydropyridine-sensitive current components but left a fraction (approximately 35%) of high-threshold current that was also resistant to omega-conotoxin and dihydropyridines. The toxin blocks N- and L-type channels with equal potency and therefore may identify a high-affinity binding site common to these two Ca channel types.
Biological transport processes and space dimension.
Authors: Nadler W , Stein D L
Published: May 2006

We discuss the generic time behavior of reaction-diffusion processes capable of modeling various types of biological transport processes, such as ligand migration in proteins and gating fluctuations in ion channel proteins. The main observable in these two cases, the fraction of unbound ligands and the probability of finding the channel in the closed state, respectively, exhibits an algebraic t-1/2 decay at intermediate times, followed by an exponential cutoff. We provide a simple framework for understanding these observations and explain their ubiquity by showing that these qualitative results are independent of space dimension. We also derive an experimental criterion to distinguish between a one-dimensional process and one whose effective dimension is higher.
Chemically induced premature mitosis: differential response in rodent and human cells and the relationship to cyclin B synthesis and p34cdc2/cyclin B complex formation.
Authors: Steinmann K E , Belinsky G S , Lee D , Schlegel R
Published: May 2006

Normal eukaryotic cells do not initiate mitosis until DNA replication has been completed. This requirement can be bypassed by exposing cells to certain chemicals. We report here that chemically induced premature mitosis is not readily achieved in all mammalian species. Although hamster cells underwent premature mitosis following treatment with caffeine, the protein phosphatase inhibitor okadaic acid, and the protein kinase inhibitors 2-aminopurine and 6-dimethyl-aminopurine, the mouse and human cells examined in this study displayed little or no response to any of these compounds. Differences in cell permeability or metabolism could not account for the species specificity of these drugs, because other biochemical and mitosis-promoting activities were apparent in human cells. Cell-type specificity can be explained, however, by the timing of cyclin B synthesis and p34cdc2/cyclin B complex formation during the cell cycle. Synthesis of cyclin B and formation of a p34cdc2/cyclin B complex, both of which are required for initiation of mitosis, were prevalent in hamster cells arrested in S phase but were absent or barely detectable in arrested human cells. In hamster cells, the hyperphosphorylated form of p34cdc2 was complexed with cyclin B and underwent tyrosine dephosphorylation during caffeine-induced premature mitosis. These findings indicate that the onset of mitosis is regulated somewhat differently among mammalian cell types and that these differences affect the vulnerability of cells to drug-induced mitotic aberrations and cytogenetic damage.
Differential effects of plant sterols on water permeability and on acyl chain ordering of soybean phosphatidylcholine bilayers.
Authors: Schuler I , Milon A , Nakatani Y , Ourisson G , Albrecht A M , Benveniste P , Hartman M A
Published: May 2006

To gain some insight into the structural and functional roles of sterols in higher plant cells, various plant sterols have been incorporated into soybean phosphatidylcholine (PtdCho) bilayers and tested for their ability to regulate water permeability and acyl chain ordering. Sitosterol was the most efficient sterol in reducing the water permeability of these vesicles and stigmasterol appeared to have no significant effect. Vesicles containing 24zeta-methylcholesterol exhibited an intermediate behavior, similar to that of vesicles containing cholesterol. Cycloartenol, the first cyclic biosynthetic precursor of plant sterols, reduced the water permeability in a very effective way. Of two unusual plant sterols, 24-methylpollinastanol and 14alpha,24zeta-dimethylcholest-8-en-3beta-ol, the former was found to be functionally equivalent to sitosterol and the latter was found to be relatively inefficient. 2H NMR experiments have been performed with oriented bilayers consisting of soybean PtdCho with sitosterol, stigmasterol, or 24-methylpollinastanol. The results provided clear evidence that sitosterol and 24zeta-methylpollinastanol exhibit a high efficiency to order PtdCho acyl chains that closely parallels their ability to reduce water permeability. By contrast, stigmasterol shows a low efficiency for both functions. These results show that sitosterol and stigmasterol, two major 24-ethylsterols differing only by the absence or presence of the Delta22 double bond in the side chain, probably play different roles in regulating plant membrane properties; they also may explain why 9beta,19-cyclopropylsterols behave as good surrogates of sitosterol.
Mutants of Agrobacterium tumefaciens with elevated vir gene expression.
Authors: Pazour G J , Ta C N , Das A
Published: May 2006

Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone. To identify the critical functional domains of virA and virG, a mutational approach was used. Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine. Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed. Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains. By plasmid reconstruction and other procedures, all mutations mapped to the virA locus. These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression. DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG.
Expression of RNase P RNA in Saccharomyces cerevisiae is controlled by an unusual RNA polymerase III promoter.
Authors: Lee J Y , Evans C F , Engelke D R
Published: May 2006

The RNA subunit of Saccharomyces cerevisiae nuclear RNase P is encoded by a single-copy, essential gene, RPR1. The 369-nucleotide mature form of the RNA has an apparent precursor with an 84-nucleotide 5' leader and approximately 33 nucleotides of additional 3' sequence. Analysis of RPR1 transcription in a strain with a temperature-sensitive lesion in RNA polymerase III shows that the gene is transcribed in vivo by RNA polymerase III. Examination of potential promoter regions using both progressive upstream deletions and point mutations indicates that at least two sequences contained within the 5' leader region are essential for expression in vivo, while sequences farther upstream influence efficiency. The required leader elements resemble tRNA gene-like A-box and B-box internal promoters in sequence and spacing. As in the tRNA genes, transcription factor TFIIIC binds to this region in vitro and binding is severely reduced by either A-box or B-box point mutations that impair expression in vivo. It thus appears that the yeast RNase P RNA gene has adopted a promoter strategy that places an RNA polymerase III "internal" promoter upstream of the mature structural domain to help drive transcription.
Evidence for the identity of human scatter factor and human hepatocyte growth factor.
Authors: Weidner K M , Arakaki N , Hartmann G , Vandekerckhove J , Weingart S , Rieder H , Fonatsch C , Tsubouchi H , Hishida T , Daikuhara Y
Published: May 2006

Scatter factor (SF), a secretory protein of fibroblasts, dissociates and increases the motility of epithelial cells and may be involved in cell migration processes during embryogenesis and tumor progression. Hepatocyte growth factor (HGF), a protein isolated from serum of patients with liver failure, is a potent mitogen for hepatocytes and is thought to play a role in liver regeneration. Here we present structural and functional evidence that human SF and human HGF (and also the human lung fibroblast-derived mitogen) are identical proteins encoded by a single gene, since (i) no major difference could be found by protein sequencing, by cDNA analysis, and by immunological comparison and (ii) SF in fact acts as a hepatocyte growth factor--i.e., stimulates DNA synthesis of activity--i.e., dissociates and induces invasiveness of various epithelial cells. The human SF/HGF gene was localized to chromosome bands 7q11.2-21. These results have important consequences for further studies on the involvement of SF/HGF as a modulator of cellular growth and motility in embryonal, malignant, and regenerative processes.
Oxidative modification by low levels of HOOH can transform myoglobin to an oxidase.
Authors: Osawa Y , Korzekwa K
Published: May 2006

It is generally thought that the oxidative modification of hemoproteins leads to their inactivation. In the current study, however, a transiently activated form of myoglobin was shown to be formed when the prosthetic heme group became covalently bound to the polypeptide during the reaction of myoglobin with low levels of HOOH. In the presence of an enzymatic metmyoglobin reducing system containing diaphorase and methylene blue with excess NADH, this HOOH-altered myoglobin catalyzed NADH oxidation and oxygen consumption; the overall stoichiometry indicated a two-electron reduction of oxygen to HOOH. This reaction was not catalyzed by iron released from heme, as desferrioxamine had no effect on the activity. Stoichiometric amounts of HOOH were sufficient to produce the activated oxidase state of myoglobin, whereas larger amounts of HOOH lead to heme destruction, iron release, and inactivation of the oxidase activity. The alteration of myoglobin to an enzyme that can form toxic oxygen metabolites may have pathological importance, especially in myocardial injury caused by ischemia and reperfusion, where myoglobin is present in large amounts and HOOH is formed. Furthermore, the oxidase form may be involved in the mechanism of destruction of the heme seen with oxidative treatment of myoglobin.
The low-affinity p75 nerve growth factor (NGF) receptor mediates NGF-induced tyrosine phosphorylation.
Authors: Berg M M , Sternberg D W , Hempstead B L , Chao M V
Published: May 2006

Protein tyrosine phosphorylation is a potential mechanism for initial signaling in PC12 cells during differentiation in response to nerve growth factor (NGF). NGF-induced tyrosine phosphorylation has been found to be initiated by the trk protooncogene, which participates in the formation of high-affinity NGF binding sites. In contrast to transfection of wild-type low-affinity p75 NGF receptors, transfection of p75NGFR with mutations in the cytoplasmic domain resulted in an inability of NGF to elicit tyrosine phosphorylation of intracellular substrates, indicating that p75NGFR is involved in initiating phosphorylation events by NGF. Even though the p75NGFR receptor does not possess any inherent tyrosine kinase activity, these experiments demonstrate that the p75NGFR has a potential role in NGF-induced tyrosine phosphorylation.
Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.
Authors: Donson J , Kearney C M , Hilf M E , Dawson W O
Published: May 2006

Tobacco mosaic virus (TMV) produces large quantities of RNA and protein on infection of plant cells. This and other features, attributable to its autonomous replication, make TMV an attractive candidate for expression of foreign sequences in plants. However, previous attempts to construct expression vectors based on plant RNA viruses, such as TMV, have been unsuccessful in obtaining systemic and stable movement of foreign genes to uninoculated leaves in whole plants. A hybrid viral RNA (TB2) was constructed, containing sequences from two tobamoviruses (TMV-U1 and odontoglossum ringspot virus). Two bacterial sequences inserted independently into TB2 moved systemically in Nicotiana benthamiana, although they differed in their stability on serial passage. Systemic expression of the bacterial protein neomycin phosphotransferase was demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene sequences were encapsidated by the odontoglossum ringspot virus coat protein, facilitating their transmission and amplification on passaging to subsequent plants. The vector TB2 provides a rapid means of expressing genes and gene variants in plants.
Inhibition of proteolysis protects hippocampal neurons from ischemia.
Authors: Lee K S , Frank S , Vanderklish P , Arai A , Lynch G
Published: May 2006

Intense proteolysis of cytoskeletal proteins occurs in brain within minutes of transient ischemia, possibly because of the activation of calcium-sensitive proteases (calpains). This proteolytic event precedes overt signs of neuronal degeneration, is most pronounced in regions of selective neuronal vulnerability, and could have significant consequences for the integrity of cellular function. The present studies demonstrate that (i) the early phase of enhanced proteolysis is a direct response to hypoxia rather than other actions of ischemia, (ii) it is possible to pharmacologically inhibit the in vivo proteolytic response to ischemia, (iii) inhibition of proteolysis is associated with a marked reduction in the extent of neuronal death, and (iv) protected neurons exhibit normal-appearing electrophysiological responses and retain their capacity for expressing long-term potentiation, a form of physiological plasticity thought to be involved in memory function. These observations indicate that calcium-activated proteolysis is an important component of the post-ischemic neurodegenerative response and that targeting this response may be a viable therapeutic strategy for preserving both the structure and function of vulnerable neurons.
Stimulation of high-affinity adenosine A2 receptors decreases the affinity of dopamine D2 receptors in rat striatal membranes.
Authors: Ferre S , von Euler G , Johansson B , Fredholm B B , Fuxe K
Published: May 2006

Since high-affinity adenosine A2 receptors (A2a) are localized exclusively in dopamine-rich regions in the central nervous system and mediate inhibition of locomotor activity, we have examined the effect of A2a receptor activation on D1 and D2 receptor binding in membrane preparations of the rat striatum. The A2a agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) increased the Kd of the dopamine D2 agonist L-(-)-N-[3H]propylnorapomorphine without affecting the Bmax. The increase in Kd was maximal (40%) at 30 nM CGS 21680. CGS 21680 (30 nM) decreased the dopamine-induced inhibition of [3H]raclopride (a D2 antagonist) binding due to an increase (about 3-fold) in KH and KL, the dissociation constants of high- and low-affinity binding sites. The effects of CGS 21680 were antagonized by the adenosine antagonist 8-phenyltheophylline (10 microM). (-)-N6-(2-Phenylisopropyl)adenosine produced an effect similar to that of CGS 21680, provided the concentration used was high enough to stimulate A2a receptors (300 nM). GTP (50 microM) also decreased the dopamine-induced inhibition of [3H]raclopride binding but, in contrast to CGS 21680, GTP decreased the proportion of D2 receptors in the high-affinity state. CGS 21680 (30 nM) did not affect the Kd or Bmax of [3H]raclopride and failed to affect ligand binding to D1 receptors. Thus, stimulation of A2a receptors potently reduces the affinity of D2 agonist binding sites within the plasma membrane of striatal neurons. This A2a-D2 interaction may underlie the neuroleptic-like actions of adenosine agonists and the enhancing effects of adenosine antagonists, such as caffeine, on locomotor activity.
Critical role for the Val/Gly86 HLA-DR beta dimorphism in autoantigen presentation to human T cells.
Authors: Ong B , Willcox N , Wordsworth P , Beeson D , Vincent A , Altmann D , Lanchbury J S , Harcourt G C , Bell J I , Newsom-Davis J
Published: May 2006

Helper T lymphocytes recognize fragments of foreign (or self) antigens in the peptide-binding clefts of major histocompatibility complex class II molecules; their activation is a crucial step in the induction of many immune and autoimmune responses. While studying the latter, we raised a T-cell line from the thymus of a myasthenia gravis patient against recombinant alpha subunit of the human acetylcholine receptor, the target of this autoimmune disease. The line responds to the 144-156 region of the human sequence and not to the same region of the electric fish homolog, which differs by only three residues. These CD4+ T cells recognize this epitope only in the context of HLA-DR4 class II molecules, of which the variants with Gly86 are absolutely required. Thus the naturally occurring alternatives Dw14.2 (Gly86) and Dw14.1 (Val86)--which differ only at this one position in the entire antigen-binding region--show an all-or-nothing difference in presenting activity. This dimorphism at position 86 is widespread, occurring in subtypes of DR1, DR2, DR3, DR5, and DR6 alleles as well as DR4. Since other DR4 subtypes with substitutions at positions 70-74 also fail to present this peptide, and glycine residues can be uniquely flexible, we suggest that this replacement at position 86 acts locally or at a distance by altering the conformation of the peptide-binding cleft. Such profound functional consequences for T-cell recognition as we report here may explain this example of conserved major histocompatibility complex diversity.
The aba mutant of Arabidopsis thaliana is impaired in epoxy-carotenoid biosynthesis.
Authors: Rock C D , Zeevaart J A
Published: May 2006

The three mutant alleles of the ABA locus of Arabidopsis thaliana result in plants that are deficient in the plant growth regulator abscisic acid (ABA). We have used 18O2 to label ABA in water-stressed leaves of mutant and wild-type Arabidopsis. Analysis by selected ion monitoring and tandem mass spectrometry of [18O]ABA and its catabolites, phaseic acid and ABA-glucose ester (beta-D-glucopyranosyl abscisate), indicates that the aba genotypes are impaired in ABA biosynthesis and have a small ABA precursor pool of compounds that contain oxygens on the ring, presumably oxygenated carotenoids (xanthophylls). Quantitation of the carotenoids from mutant and wild-type leaves establishes that the aba alleles cause a deficiency of the epoxy-carotenoids violaxanthin and neoxanthin and an accumulation of their biosynthetic precursor, zeaxanthin. These results provide evidence that ABA is synthesized by oxidative cleavage of epoxy-carotenoids (the "indirect pathway"). Furthermore the carotenoid mutant we describe undergoes normal greening. Thus the aba alleles provide an opportunity to study the physiological roles of epoxy-carotenoids in photosynthesis in a higher plant.
cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator.
Authors: Rommens J M , Dho S , Bear C E , Kartner N , Kennedy D , Riordan J R , Tsui L C , Foskett J K
Published: May 2006

A cAMP-inducible chloride permeability has been detected in mouse fibroblast (L cell) lines upon stable integration of a full-length cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR). As indicated by a Cl(-)-indicator dye, the Cl- permeability of the plasma membrane increases by 10- to 30-fold within 2 min after treatment of the cells with forskolin, an activator of adenylyl cyclase. The properties of the conductance are similar to those described in secretory epithelial cells; the whole-cell current-voltage relationship is linear and there is no evidence of voltage-dependent inactivation or activation. In contrast, this cAMP-dependent Cl- flux is undetectable in the untransfected cells or cells harboring defective cDNA constructs, including one with a phenylalanine deletion at amino acid position 508 (delta F508), the most common mutation causing cystic fibrosis. These observations are consistent with the hypothesis that the CFTR is a cAMP-dependent Cl- channel. The availability of a heterologous (nonepithelial) cell type expressing the CFTR offers an excellent system to understand the basic mechanisms underlying this CFTR-associated ion permeability and to study the structure and function of the CFTR.
Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo.
Authors: von Andrian U H , Chambers J D , McEvoy L M , Bargatze R F , Arfors K E , Butcher E C
Published: May 2006

The lectin homing receptor LECAM-1 (LAM-1, Leu8) and the beta 2 integrins, particularly Mac-1 (CD11b/CD18), participate in leukocyte-endothelial cell interactions in inflammation. LECAM-1 is rapidly shed while Mac-1 expression is dramatically increased upon neutrophil activation, suggesting functionally distinct roles for these molecules. Using intravital video microscopy, we have compared the effect of antibodies against LECAM-1 and CD18 on leukocyte interactions with rabbit mesenteric venules. Anti-LECAM-1 monoclonal antibody and its Fab fragments inhibited initial reversible leukocyte rolling along the vascular wall. Anti-CD18 monoclonal antibody had no effect on rolling but prevented subsequent firm attachment of leukocytes to venular endothelium. These results support a two-step model of leukocyte-endothelial cell interactions: reversible rolling mediated in part by LECAM-1 facilitates leukocyte recruitment by the local microenvironment and precedes activation-dependent firm attachment involving beta 2 integrins.
Expression of two different forms of fibroblast growth factor receptor 1 in different mouse tissues and cell lines.
Authors: Bernard O , Li M , Reid H H
Published: May 2006

The fibroblast growth factor receptors (FGFRs) form a multigene family of at least four members, all having extracellular regions consisting of either two or three immunoglobin-like (Ig-like) domains. By RNase protection analysis we have analyzed the expression of FGFR-1 mRNA in various tissues and cell lines and demonstrated that all of the cell lines studied expressed at least two different forms of the FGFR-1 at similar levels. Although muscle and heart express forms having either two [FGFR-1 short (FGFR-1S)] or three [FGFR-1 long (FGFR-1L)] Ig-like domains, the developing brain and adult brain express only mRNA encoding the longer form. The two forms of the receptor were characterized further by stably introducing expression vectors expressing them into Rat-2 fibroblasts and FDC-P1 myeloid cells. Treatment of the transfected Rat-2 cells with acidic FGF (aFGF) or basic FGF (bFGF) resulted in focus formation. The transformed phenotype was observed even without addition of ligand after growth in culture for greater than 2 months. Cross-linking of 125I-labeled bFGF (125I-bFGF) to Rat-2 cells expressing either FGFR-1L or FGFR-1S yielded two similar complexes of 150 and 110 kDa. Although Rat-2 cells expressing FGFR-1L yielded similar complexes with 125I-labeled aFGF (125I-aFGF), only the 150-kDa complex was formed with cells expressing FGFR-1S. The 150-kDa complex was also observed when 125I-aFGF or 125I-bFGF was cross-linked to FDC-P1 cells expressing FGFR-1L. Significantly, these complexes were only observed when heparin was present in the cross-linking reaction. FDC-P1 cells expressing FGFR-1 bound aFGF and bFGF with high affinity but only in the presence of heparin. The factor dependence of these cells could be switched from interleukin 3 to FGF in the presence of heparin.
Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide.
Authors: Bugg B Y , Danks M K , Beck W T , Suttle D P
Published: May 2006

Nuclear extracts from teniposide (VM-26)-resistant sublines of the human leukemic cell line CCRF-CEM have decreased levels of DNA topoisomerase II catalytic activity and decreased capacity to form drug-stabilized covalent protein-DNA complexes. The ATP concentration required for equivalent activity in a DNA-unknotting assay is 2- to 8-fold higher in nuclear extracts from drug-resistant cell lines as compared with the parental line. When adenosine 5'-[beta,gamma-imido]triphosphate is substituted for ATP in complex-formation assays, no significant change is seen with drug-sensitive cells, but a 50-65% reduction is seen with VM-26-resistant cells. Collectively, these results indicate that an alteration in ATP binding may be involved in the resistance phenotype. Therefore, we identified regions of the topoisomerase II sequence that conform to previously identified nucleotide-binding sites. Starting with cDNA as the template we determined the sequence of the topoisomerase II mRNA surrounding these sites by sequencing DNA fragments produced by the polymerase chain reaction. In the region corresponding to the consensus B ATP-binding sequence described by Walker et al. [Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982) EMBO J. 1, 945-951], the cDNA from the two VM-26-resistant sublines contained an altered sequence having a G----A base change. This base substitution results in the replacement of the conserved arginine at position 449 with a glutamine. Hybridization with allele-specific oligonucleotides confirmed the presence of both the normal and the altered sequence in the resistant cell lines, whereas only the normal sequence was found in the sensitive CEM cells. A chemical mismatch cleavage procedure for the detection of mispaired bases in DNA duplexes identified no other alterations in the 5' third of the mRNA coding sequence, which contains the complete ATP-binding domain of topoisomerase II. The presence of mRNA encoding topoisomerase II with Gln449 correlates both with the presence of a topoisomerase II protein whose interaction with ATP is altered and with increased resistance to the cytotoxicity of VM-26.
Characterization of a programmed alteration in an 18S ribosomal gene that accompanies the experimental induction of drug resistance in Schistosoma mansoni.
Authors: Brindley P J , Heath S , Waters A P , McCutchan T F , Sher A
Published: May 2006

Stable resistance to the anthelmintic hycanthone can be produced in the human blood fluke Schistosoma mansoni by exposing immature parasites in mice to the drug. Within a single generation, genomic rearrangements, detected as rRNA-encoding DNA restriction fragment length polymorphisms (RFLPs), accompany the appearance of resistance in this model. One of these RFLPs, an approximately 3.6-kilobase BamHI fragment, was shown previously to associate consistently with resistance in independent generations of the JHU strain of S. mansoni. To characterize the genetic changes responsible for this RFLP, the fragment was cloned and sequenced. A comparison of the cloned fragment with a normal 18S rRNA gene demonstrated that the drug resistance-associated RFLP fragment arises through the addition of 732 base pairs into an 18S rRNA gene, 134 base pairs downstream of the junction of the intergenic spacer and the mature 18S rRNA gene. The mutation is nonrandom, targets one, or a few only, of the 100 or so copies of the ribosomal genes, and may represent the incomplete duplication of the gene since the inserted element is identical in sequence to the region contiguous to it. The sequence spanning the junction of the insertion and the original 18S rRNA gene was used as a specific primer for the BamHI RFLP in PCR experiments. The analysis conclusively demonstrated that the mutation is induced rather than selected by the drug since the junctional sequence was not detectable in the drug-sensitive parent population of schistosomes. In addition, analysis of four, independently derived, resistant lines indicated that the same region of the gene was mutated each time. Together, these data demonstrate that reproducible changes are induced during the acquisition of resistance in schistosomes and suggest that the resistant phenotype is induced rather than selected from preexisting forms.
Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein.
Authors: Stuehr D J , Cho H J , Kwon N S , Weise M F , Nathan C F
Published: May 2006

A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3- per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 microM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of approximately 250 kDa but could be dissociated into inactive monomers of approximately 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.
Insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integrin alpha 5 beta 1, independent of cell shape.
Authors: Schwartz M A , Lechene C , Ingber D E
Published: May 2006

Growth of anchorage-dependent cells requires both soluble mitogens and insoluble extracellular matrix molecules such as fibronectin. Soluble growth factors activate chemical signaling pathways and stimulate proliferation by binding to transmembrane receptors. Insoluble fibronectin also binds to cell-surface receptors; however, it is thought to act primarily via effects on the cytoskeleton and cell shape. We recently demonstrated that cell spreading on surface-adsorbed fibronectin activates the Na/H antiporter and that inhibition of this chemical-signaling pathway suppresses growth. We now show that insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integrin alpha 5 beta 1, independent of effects on cell shape. These results show that an extracellular matrix receptor can behave similarly to a growth factor receptor to activate a signaling pathway implicated in growth control.
cAMP-stimulated phosphorylation of an axonemal polypeptide that copurifies with the 22S dynein arm regulates microtubule translocation velocity and swimming speed in Paramecium.
Authors: Hamasaki T , Barkalow K , Richmond J , Satir P
Published: May 2006

In Paramecium tetraurelia, cyclic nucleotides are important physiological second messengers that could regulate dynein mechanochemistry by phosphorylation. A 29-kDa polypeptide that is phosphorylated in a cAMP- and Ca(2+)-sensitive manner in permeabilized cells and isolated axonemes is the only significant phosphorylated moiety that consistently copurifies with 22S dynein from paramecium cilia. It is not a component of 14S dynein. This polypeptide can be thiophosphorylated in a cAMP-sensitive manner, and this form of 22S dynein is stable when stored at -70 degrees C. cAMP-mediated thiophosphorylation of the 29-kDa polypeptide significantly increases the velocity with which 22S dynein causes microtubules to glide in vitro. The increase is abolished, together with the thiophosphorylation of the 29-kDa polypeptide, by preincubation with high Ca2+. Pretreatment with high Ca2+ does not alter the thiophosphorylation pattern of, or the velocity of microtubule translocation by, 14S dynein. The same preincubation conditions that permit or abolish the increase in velocity of microtubule translocation by 22S dynein permit or fail to permit swimming speed of permeabilized cells to increase on reactivation even after cAMP is removed. The effect of cAMP on swimming speed can therefore be accounted for by changes in the mechanically coupled 22S dynein activity via phosphorylation or thiophosphorylation of the 29-kDa polypeptide, which could act as a regulatory dynein light chain.
Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice.
Authors: Kohler S W , Provost G S , Fieck A , Kretz P L , Bullock W O , Sorge J A , Putman D L , Short J M
Published: May 2006

Transgenic mice with a lambda shuttle vector containing a lacI target gene were generated for use as a short-term, in vivo mutagenesis assay. The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal). Phage with mutations in the lacI gene form blue plaques, whereas phage with a nonmutated lacI form colorless plaques. Spontaneous background mutant rates using this system range from 0.6 x 10(-5) to 1.7 x 10(-5), depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue. Treatment of the mice with N-ethyl-N-nitrosourea (EtNU), benzo[a]pyrene (B[a]P), or cyclophosphamide caused an induction of mutations over background. Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins. The predominant mutations identified from untreated and treated populations were base substitutions. Although it has been shown by others that 70% of all spontaneous mutations within the lacI gene, when replicated in Escherichia coli, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system. In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the lacI gene. For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B[a]P-induced mutations, in which only transversions were obtained. In addition, B[a]P mutagenesis showed a predominance of mutations (81%) involving cytosines and/or guanines, consistent with its known mode of action. The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B[a]P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.
Expression of M-cadherin, a member of the cadherin multigene family, correlates with differentiation of skeletal muscle cells.
Authors: Donalies M , Cramer M , Ringwald M , Starzinski-Powitz A
Published: May 2006

Cadherins, a multigene family of transmembrane glycoproteins, mediate Ca(2+)-dependent intercellular adhesion. They are thought to be essential for the control of morphogenetic processes, including myogenesis. Here we report the identification and characterization of the cDNA of another member of the cadherin family, M-cadherin (M for muscle), from differentiating muscle cells. The longest open reading frame of the cDNAs isolated contains almost the entire coding region of the mature M-cadherin as determined by sequence homology to the known cadherins. M-cadherin mRNA is present at low levels in myoblasts and is upregulated in myotube-forming cells. In mouse L cells (fibroblasts), M-cadherin mRNA is undetectable. This expression pattern indicates that M-cadherin is part of the myogenic program and may provide a trigger for terminal muscle differentiation.
Fidelity of targeted recombination in human fibroblasts and murine embryonic stem cells.
Authors: Zheng H , Hasty P , Brenneman M A , Grompe M , Gibbs R A , Wilson J H , Bradley A
Published: May 2006

Targeted recombination in murine embryonic stem cells promises to be a powerful tool for introducing specific mutations into target genes to study development in mice and to create animal models of human disease. Gene targeting also holds potential for correcting genetic defects as an approach to human gene therapy. To precisely modify target genes, homologous recombination must proceed with high fidelity. However, several results have suggested that targeted recombination may be highly mutagenic. To test the accuracy of gene targeting we analyzed 44 independent targeted recombinants at the hypoxanthine phosphoribosyltransferase (HPRT) locus in a human fibroblast cell line and in mouse embryonic stem cells. We surveyed 80 kilobases around the sites of recombination by using chemical cleavage of mismatches. Only two mutations were found: a T----G transversion and a thymidine deletion. Thus, gene targeting in mammalian cells can be extremely accurate. These results demonstrate the feasibility of generating precise modifications of mammalian genomes by gene targeting.
Selection of motile nonchemotactic mutants of Escherichia coli by field-flow fractionation.
Authors: Berg H C , Turner L
Published: May 2006

We have developed a chromatographic method for isolating bacterial cells that are motile but nonchemotactic. Separation of strains of different phenotype occurs along a thin horizontal channel between two stirred chambers, the lower one containing a chemical attractant. The channel is bounded above and below by rigid filters, permeable to the attractant but not to the bacteria. The lower part of the channel is occupied by a porous plate comprising a vertical array of capillary tubes. An aliquot of cells is injected at one end of the channel and eluted by continuous flow of cell-free medium. Fluid leaving the other end of the channel is collected in a fraction collector. Cells that respond to the gradient swim to the bottom of the channel where they are retarded by the capillary array. Nonmotile cells sink to the bottom and are trapped in a similar manner. Motile cells that fail to respond to the gradient diffuse across the full height of the channel and, thus, travel through the apparatus at the average velocity of the eluent. When mixed with wild-type cells at a ratio of 1:1000 and subjected to an aspartate gradient, aspartate-blind cells were recovered quantitatively. The enrichment was approximately 200 to 1. The wild-type cells that survived the selection had a poorly motile phenotype.
Lateral redistribution of cytochrome b6/f complexes along thylakoid membranes upon state transitions.
Authors: Vallon O , Bulte L , Dainese P , Olive J , Bassi R , Wollman F A
Published: May 2006

The cytochrome b6/f complex operates in photosynthetic electron transfer either in linear electron flow from photosystem II to photosystem I or in cyclic flow around photosystem I. Using membrane fractionation and immunocytochemistry, we show a change in lateral distribution of cytochrome b6/f complexes along the thylakoid membranes during state transitions. This change is seen in maize as well as in the green algae Chlamydomonas reinhardtii. When either of the two organisms were adapted to state II in vivo, the proportion of cytochrome b6/f complexes found in the photosystem I-enriched stroma lamellae regions was significantly larger than after adaptation to state I. A similar observation was made upon state I to state II transitions done in vitro by illuminating, in the presence of ATP, broken maize chloroplasts prepared from dark-adapted leaves. This reorganization of the electron-transfer chain is concurrent with the change in light-energy distribution between the two photosystems, which requires lateral displacement of light-harvesting complex II. That the changes in lateral distribution of both cytochrome b6/f and light-harvesting II complexes seen upon state transition in vitro similarly required addition of exogenous ATP, suggests that the change in cytochrome b6/f organization also depends on kinase activity. The increased concentration of cytochrome b6/f complexes in the vicinity of photosystem I in state II is discussed in terms of an increase in cyclic electron flow, thus favoring ATP production. Because transition to state II can be triggered in vivo by ATP depletion, we conclude that state transitions should be regarded not only as a light-adaptation mechanism but also as a rerouting of photosynthetic electron flow, enabling photosynthetic organisms to adapt to changes in the cell demand for ATP.