bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophages and smooth muscle cells express lipoprotein lipase in human and rabbit atherosclerotic lesions.
Authors: Ylä-Herttuala S , Lipton B A , Rosenfeld M E , Goldberg I J , Steinberg D , Witztum J L
Published: May 2006

Lipoprotein lipase (LPL; EC 3.1.1.34) may promote atherogenesis by producing remnant lipoproteins on the endothelial surface and by acting on lipoproteins in the artery wall. In vitro, smooth muscle cells and macrophages synthesize LPL, but in human carotid lesions only a few smooth muscle cells were reported to contain LPL protein. Endothelial cells do not synthesize LPL in vitro, but in normal arteries intense immunostaining for LPL is present on the endothelium. We used Northern blot analysis, in situ hybridization, and immunocytochemistry of human and rabbit arteries to determine cellular distribution and the site of the synthesis of LPL in atherosclerotic lesions. Northern blot analysis showed that LPL mRNA was detectable in macrophage-derived foam cells isolated from arterial lesions of "ballooned" cholesterol-fed rabbits. In situ hybridization studies of atherosclerotic lesions with an antisense riboprobe showed a strong hybridization signal for LPL mRNA in some, but not all, lesion macrophages, which were mostly located in the subendothelial and edge areas of the lesions. Also, some smooth muscle cells in lesion areas also expressed LPL mRNA. Immunocytochemistry of frozen sections of rabbit lesions with a monoclonal antibody to human milk LPL showed intense staining for LPL protein in macrophage-rich intimal lesions. The results suggest that lesion macrophages and macrophage-derived foam cells express LPL mRNA and protein. Some smooth muscle cells in the lesion areas also synthesize LPL. These data are consistent with an important role for LPL in atherogenesis.
Factor-binding element in the human c-myc promoter involved in transcriptional regulation by transforming growth factor beta 1 and by the retinoblastoma gene product.
Authors: Pietenpol J A , Münger K , Howley P M , Stein R W , Moses H L
Published: May 2006

Previous studies have shown that transforming growth factor beta 1 (TGF-beta 1) inhibition of keratinocyte proliferation involves suppression of c-myc transcription, and indirect evidence has suggested that the retinoblastoma gene product (pRB) may be involved in this process. In this study, transient expression of pRB in skin keratinocytes was shown to repress transcription of the human c-myc promoter as effectively as TGF-beta 1. The same c-myc promoter region was required for regulation by both TGF-beta 1 and pRB. These sequences, termed the TGF-beta control element (TCE), lie between positions -86 and -63 relative to the P1 transcription start site. Oligonucleotides containing the TCE bound to several nuclear factors in mobility-shift assays using extracts from cells with or without normal pRB. Binding of some factors was inhibited by TGF-beta 1 treatment of TGF-beta-sensitive but not TGF-beta-insensitive cells. These data indicate that pRB can suppress c-myc transcription and suggest the involvement of cellular factors in addition to pRB in the TGF-beta 1 pathway for the inhibition of c-myc transcription and growth inhibition.
Thyrotropin-releasing hormone induces opposite effects on Ca2+ channel currents in pituitary cells by two pathways.
Authors: Gollasch M , Haller H , Schultz G , Hescheler J
Published: May 2006

Thyrotropin-releasing hormone (TRH) stimulates pituitary secretion by steps involving a cytosolic Ca2+ rise. We examined various pathways of Ca2+ elevation in pituitary GH3 cells. By using the patch clamp technique in the whole-cell configuration and Ba2+ as divalent charge carrier through Ca2+ channels, TRH (1 microM) reversibly reduced the current by about 55%. This hormonal effect was prevented by infusing guanine 5'-[beta-thio]diphosphate (GDP[beta S]) intracellularly but not by pretreating the cell with pertussis toxin (PT). Since PT-insensitive guanine nucleotide-binding regulatory (G) proteins are known to mediate a hormone-stimulated inositol trisphosphate-mediated Ca2+ release from intracellular stores, we assume that the inhibitory effect of TRH on Ba2+ currents through Ca2+ channels is caused by the increased intracellular Ca2+. To prevent a Ca(2+)-release-dependent inhibition of Ca2+ channels, we preincubated GH3 cells in a medium free of divalent charge carriers and measured the Na+ current through Ca2+ channels. When fura-2 was used as indicator for the cytosolic Ca2+, TRH induced a release from intracellular stores only once and had no effect on the intracellular Ca2+ concentration during further applications. In line with this observation, TRH initially reduced the Na+ current through Ca2+ channels but stimulated it during subsequent applications. The stimulation was sensitive to GDP[beta S] and was abolished by pretreatment with PT, suggesting that the stimulatory action of TRH is mediated by a G protein different from the one that functionally couples the receptor to phosphatidylinositol 4,5-bisphosphate hydrolysis. In conclusion, the present data suggest that TRH increases the intracellular Ca2+ concentration by two interacting pathways, that release from intracellular stores causes a secondary blockage of Ca2+ channels, and that, especially with empty intracellular Ca2+ stores, Ca2+ channels are stimulated by a PT-sensitive G protein.
Structural requirements for the carbohydrate ligand of E-selectin.
Authors: Tyrrell D , James P , Rao N , Foxall C , Abbas S , Dasgupta F , Nashed M , Hasegawa A , Kiso M , Asa D
Published: May 2006

The acute inflammatory response requires that circulating leukocytes adhere to, and then migrate through, the vascular wall at the site of injury or infection. Several receptors have been implicated in this adhesion and migration process, including the selectins, a family of carbohydrate-binding proteins. The ligand for one of these proteins, E-selectin (LECAM-2, ELAM-1) has been described by several groups to contain a polylactosamine structure bearing a terminal sialic acid residue and at least one fucose residue. We report here a more detailed investigation into the minimum structural requirements for carbohydrate recognition by E-selectin. Using both direct binding and inhibition studies we demonstrate that the sialyl Lewisx tetrasaccharides Sia(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, and Sia(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]Glc are the smallest oligosaccharides recognized by the lectin. In addition, an oligosaccharide containing the sialyl Lewisa epitope is also recognized, but less avidly. We propose a structural model of functional groups necessary for recognition by E-selectin, based on these data and additional experiments on modifications of sialic acid and the reducing terminal saccharide.
Compartmentalized expression of a gene under the control of sporulation transcription factor sigma E in Bacillus subtilis.
Authors: Driks A , Losick R
Published: May 2006

Immunoelectron microscopy was used to visualize the expression of a gene under the control of developmental transcription factor sigma E during spore formation in Bacillus subtilis. sigma E is generated by cleavage of an inactive proprotein (pro-sigma E) shortly after the formation of the sporulation septum, which partitions the sporangium into mother-cell and forespore compartments. Specific antibodies and gold-conjugated secondary antibodies were used to localize beta-galactosidase in thin sections of sporangia from cells bearing a lacZ transcriptional fusion to a gene (spoIID) under the direct control of sigma E. Transcription of spoIID was found to be induced shortly after the formation of the sporulation septum and was largely confined to the mother cell. Cell-type-specific transcription of genes under the control of sigma E could be responsible for establishing the mother-cell line of gene expression.
DNA binding by the archaeal histone HMf results in positive supercoiling.
Authors: Musgrave D R , Sandman K M , Reeve J N
Published: May 2006

HMf, a histone from the hyperthermophilic archaeon Methanothermus fervidus binds double-stranded DNA molecules in vitro, forming compact structures that visibly resemble eukaryal nucleosomes. We show here that HMf binding increases the helical periodicity of DNA molecules to approximately 11 base pairs (bp) per turn and that DNA molecules in these nucleosome-like structures are constrained in positive toroidal supercoils. Based on the mass of HMf needed to cause a change in linking number (delta Lk), the maximum delta Lk introduced into circular DNA molecules of known sizes, and electron microscopy, we estimate that each HMf-DNA structure contains between 90 and 150 bp of DNA wrapped in 1.5 positive toroidal supercoils around a core of four HMf molecules. A model and pathway for the formation of these structures in vitro are presented and the possible role of positive toroidal wrapping of the M. fervidus genome in vivo is discussed.
Disulfide cross-linking studies of the transmembrane regions of the aspartate sensory receptor of Escherichia coli.
Authors: Lynch B A , Koshland D E
Published: May 2006

The Escherichia coli aspartate receptor, a dimer of identical subunits, has two transmembrane regions (TM1, residues 7-30; TM2, residues 189-212) of 24 residues each. To study the relative placement and orientation of the regions, cysteine residues were introduced individually into the center of each: at positions 17, 18, and 19 in TM1; and at positions 198, 199, 200, and 201 in TM2. Based on the patterns of disulfide cross-linking observed between subunits in the mutant receptors, there appears to be close contact between the TM1 and TM1' regions at the dimer interface but no such direct interaction between the TM2 and TM2' regions. The cross-linking results are consistent with an alpha-helical structure extending across the transmembrane region up through at least residue 36, which lies on the periplasmic side of TM1. The ability of an 18-18' cross-linked dimer to transmit an aspartate-induced transmembrane signal is also supportive of such an extended helix. The changes in relative rates of disulfide cross-linking provide experimental evidence of a conformational change transmitted through the transmembrane domain during signaling. Once formed, disulfides between the transmembrane regions are unusually resistant to reduction by low molecular weight thiols in the presence of denaturants like SDS. These targeted disulfide cross-links can be used to reveal structural and dynamic aspects of protein function.
T lymphocytes inhibit the vascular response to injury.
Authors: Hansson G K , Holm J , Holm S , Fotev Z , Hedrich H J , Fingerle J
Published: May 2006

The proliferation of vascular smooth muscle cells is controlled by specific growth factors and cytokines acting in paracrine networks. Macrophage products such as the platelet-derived growth factor and interleukin 1 promote smooth muscle proliferation and are released in the arterial wall during atherosclerosis and repair processes. T lymphocytes are also present in vascular tissue, but their role in vascular growth control in vivo has been unclear. We now demonstrate that rats in which T lymphocytes have been eliminated by a monoclonal antibody develop larger proliferative arterial lesions after balloon-catheter injury. Larger lesions also develop in athymic rnu/rnu rats that lack T lymphocytes, when compared with rnu/+ littermates with normal T-cell levels. Finally, injection of the lymphokine interferon gamma inhibits smooth muscle proliferation and results in smaller lesions compared with controls injected with buffer alone. These results indicate that T lymphocytes modulate smooth muscle proliferation during vascular repair. We propose that T lymphocytes may play an important, immunologically nonspecific role in tissue repair processes.
Deletion of the c-kit protooncogene in the human developmental defect piebald trait.
Authors: Fleischman R A , Saltman D L , Stastny V , Zneimer S
Published: May 2006

The protooncogene c-kit is critical for development of hematopoietic stem cells, germ cells, and melanoblasts in the mouse. Homozygous mutations of this gene in the mouse cause anemia, infertility, and albinism, whereas heterozygous mutant mice usually exhibit only a white forehead blaze and depigmentation of the ventral body, tail, and feet. The heterozygous mouse phenotype is very similar to human piebald trait, which is characterized by a congenital white hair forelock and ventral and extremity depigmentation. To investigate the possibility that alterations in the human c-kit gene may be a cause of piebald trait, DNA from seven unrelated affected individuals was examined by Southern blot analysis. One subject, although cytogenetically normal, has a heterozygous deletion of the c-kit protooncogene. This deletion encompasses the entire coding region for c-kit and also involves the closely linked gene for platelet-derived growth factor receptor alpha. Fluorescence in situ hybridization of genomic c-kit probes to metaphase chromosomes independently confirmed the deletion in this case. These findings provide molecular evidence mapping piebald trait to the c-kit locus on chromosome 4. Although we cannot exclude the involvement of other closely linked genes, the demonstration of a genomic c-kit deletion in one subject with piebald trait and the marked concordance of the human and mouse phenotypes provide strong evidence for the role of c-kit in the development of human melanocytes and in the pathogenesis of piebald trait.
De novo chromosome formation in rodent cells.
Authors: Praznovszky T , Keresö J , Tubak V , Cserpán I , Fátyol K , Hadlaczky G
Published: May 2006

A hybrid cell line was produced by the fusion of an EC3/7 mouse cell with a Chinese hamster ovary cell. The EC3/7 cell carries a dicentric chromosome with a functional marker centromere. This marker centromere contains human, lambda, and bacterial vector DNA sequences and a dominant selectable gene (aminoglycoside 3'-phosphotransferase type II; neo). In the hybrid, the marker centromere separated from the dicentric chromosome and formed a full-sized chromosome (lambda neo). The newly formed chromosome is stable, even under nonselective culture conditions. This functional chromosome, which is the result of an amplification process, is composed of seven large, different-sized amplicons. Each amplicon contains multiple copies of human, lambda, neo, and mouse telomeric DNA sequences. Individual amplicons are separated from each other by mouse major satellite DNA sequences. The marker centromere was localized to a terminal amplicon by anticentromere immunostaining. The number of amplicons in the newly formed chromosome is remarkably consistent. This finding suggests that the length of the newly formed chromosome is highly constrained.
Substance P-mediated slow excitatory postsynaptic potential elicited in dorsal horn neurons in vivo by noxious stimulation.
Authors: De Koninck Y , Henry J L
Published: May 2006

The original proposal that substance P is involved in the regulation of nociceptive information at the first sensory synapse in the spinal cord has been substantiated by a wide range of evidence, but definitive support has been lacking, due primarily to the lack of evidence that a specific nociceptive response in the dorsal horn can be blocked by a substance P antagonist. Here, we present evidence that CP-96,345, a specific substance P (NK-1) receptor antagonist, selectively blocks a slow, prolonged excitatory postsynaptic potential following noxious cutaneous stimulation or a train of intense electrical stimuli to sensory nerves but does not affect the response to innocuous input or the brief response to single electrical stimuli to C fibers. These results indicate the specific involvement of substance P in the mediation of a prolonged after-excitation to noxious stimulation. This may have important implications for the etiology and treatment of chronic pain and for plastic changes in nociceptive pathways.
A test of Fisher's theory of dominance.
Authors: Orr H A
Published: May 2006

One of the first patterns noticed by geneticists was that mutations are almost always recessive to their wild-type alleles. Several explanations of this striking pattern have been offered. The two most influential are Fisher's theory--which argues that dominance results from natural selection against recurring deleterious mutations--and Wright's theory--which argues that dominance results from the physiology of gene action. The debate over which of these theories is correct represents one of the most protracted controversies in the history of evolutionary biology. Here I test Fisher's theory by assessing the dominance of mutations in an organism that is typically haploid, the alga Chlamydomonas reinhardtii. The results show that mutations are recessive just as often among haploid as among diploid species. This result falsifies Fisher's theory of dominance and provides strong support for the alternative physiological theory.
How does RNase H recognize a DNA.RNA hybrid?
Authors: Nakamura H , Oda Y , Iwai S , Inoue H , Ohtsuka E , Kanaya S , Kimura S , Katsuda C , Katayanagi K , Morikawa K
Published: May 2006

The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA.RNA hybrid Escherichia coli RNase H complex. Site-directed mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out. A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively. In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme. The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model. Moreover, the specific cleavage of the RNA strand in DNA.RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes. A mechanism of enzymatic action is proposed.
Continuous microspectrophotometric measurement of DNA polymerase activity: application to the Klenow fragment of Escherichia coli DNA polymerase I and human immunodeficiency virus type 1 reverse transcriptase.
Authors: Baillon J G , Nashed N T , Sayer J M , Jerina D M
Published: May 2006

Progress of DNA- and/or RNA-directed DNA polymerization reactions can be measured continuously using circular dichroism (CD) or ultraviolet (UV) spectroscopy. In the presence of the Klenow fragment of Escherichia coli DNA polymerase I, a CD change of -0.27 +/- 0.06 millidegree at 248 nm and a UV change of -2.7 +/- 0.3 milliabsorbance units at 275 nm occur upon incorporation of 120 pmol of dTMP in a reaction volume of 120 microliters (1 microM dTMP incorporation) into a synthetic template-primer, p(dA)40-60.p(dT)20. The transcription of poly(A).p(dT)12-18 by reverse transcriptases can also be monitored using these methods. Kinetic parameters for the polymerization reaction catalyzed by the Klenow fragment were determined from initial velocity measurements using CD or UV assays and were in close agreement with those measured by the standard single point radiochemical filtration assay. The generality of optical techniques for the measurement of DNA polymerase activity was shown by the use of a partially self-complementary hairpin-shaped oligonucleotide substrate for the Klenow fragment. Addition of a single nucleotide residue under steady-state conditions to this 35-mer at a concentration of 1.5-3 microM gave an easily measurable absorbance decrease at 275 nm, and the absorbance changes upon sequential addition of nucleotide units were additive.
Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA.
Authors: Gegner J A , Dahlquist F W
Published: May 2006

An essential step in the signal transduction pathway of Escherichia coli is the control of the protein kinase activity of CheA by the chemotaxis receptor proteins. This control requires the participation of the CheW protein. Although the biochemical nature of the coupling between the receptors and the kinase is unknown, it is likely that CheW interacts with the receptors and with CheA. In this communication, we report direct measurement of a physical interaction between CheW and CheA. We utilized the equilibrium column chromatography method of Hummel and Dreyer to show that CheW and CheA exhibit reversible binding with the stoichiometry of two CheW monomers per CheA dimer. CheW was found to exist as monomers and CheA was found to exist as dimers by equilibrium analytical ultracentrifugation. The dissociation constant for the CheW-CheA interaction (in 160 mM KCl/5 mM MgCl2, pH 7.4 at 4 degrees C) was determined to be in the physiologically relevant range of 17 microM. No evidence for cooperativity in the association of CheW with CheA was found.
Polarized expression of cytokines in cell conjugates of helper T cells and splenic B cells.
Authors: Kupfer A , Mosmann T R , Kupfer H
Published: May 2006

We describe the intracellular localization, by double immunofluorescence microscopy, of four cytokines that were produced during the prolonged interaction of cloned helper T cells with resting splenic B cells. When two rabbit immunoglobulin-specific helper-T-cell clones were mixed, either separately or together, with splenic B cells in the presence of the antigen rabbit anti-mouse immunoglobulin antibodies, stable T-cell-B-cell conjugates were seen up to 29 hr later. Microscopic observations of these cells revealed that interferon gamma and interleukin 2, inside one of the T-cell clones, and interleukins 4 and 5, inside the other T-cell clone, were concentrated very close to the T-cell-B-cell contact area. The cytokines were not seen in the T cells prior to their interaction with the B cells and their production was strictly antigen-specific. These studies show, at the single-cell level, that helper-T-cell clones can remain bound to splenic B cells long enough for the T cells to produce cytokines, which are synthesized near the bound B cells. We propose that the polarized synthesis of the cytokines may result in their directed secretion toward the bound B cells. By locally secreting the cytokines, which are not antigen-specific, at the contacting T-cell-B-cell membranes, where T- and B-cell surface receptors are engaged and clustered, the helper T cells can induce selective and specific B-cell responses.
Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: correlation with susceptibility to hepatocarcinogenesis.
Authors: Buchmann A , Bauer-Hofmann R , Mahr J , Drinkwater N R , Luz A , Schwarz M
Published: May 2006

The frequency and pattern of mutations at codon 61 of the c-Ha-ras gene have been analyzed in 195 liver tumors and 132 precancerous liver lesions from various rodent strains with differing susceptibility to hepatocarcinogenesis. By using the polymerase chain reaction and allele-specific oligonucleotide hybridization, C----A transversions at the first base and A----T transversions or A----G transitions at the second base of c-Ha-ras codon 61 were detected in 20-60% of spontaneous or carcinogen-induced liver tumors of the C3H/He, CBA, CF1, and B6C3F1 mouse strains, which are highly susceptible to hepatocarcinogenesis. No such mutations, however, could be found in any of the 31 liver tumors of the insensitive C57BL/6J and BALB/c mouse strains or in any of the 35 liver tumors of the comparatively resistant Wistar rat. Further analyses of c-Ha-ras codon 12 mutations in liver tumors from the three insensitive rodent strains also failed to give any positive results. In early precancerous liver lesions, c-Ha-ras codon 61 mutations were found in 13-14% of lesions of the sensitive C3H/He and B6C3F1 mouse strains but not in any of the 34 lesions of the insensitive C57BL/6J mouse. Taken together, our results indicate a close correlation between the mutational activation of the c-Ha-ras gene in liver tumors of the different rodent strains and their susceptibility to hepatocarcinogenesis, whereby the mutations appear to provide a selective growth advantage, leading to a clonal expansion of the mutated liver cell population, only in livers of sensitive but not of insensitive strains.
Evidence for an involvement of the brain cholecystokinin B receptor in anxiety.
Authors: Singh L , Lewis A S , Field M J , Hughes J , Woodruff G N
Published: May 2006

The effect of neuropeptide cholecystokinin (CCK) receptor agonists and antagonists was examined in the rat elevated X-maze model of anxiety. The selective CCK-B receptor antagonists CI-988 (PD 134308) and L-365,260 produced anxiolytic-like effects, whereas MK-329, a CCK-A receptor antagonist, was respectively less potent by factors of 313 and 200. The intracerebroventricular administration of the nonselective CCK receptor agonist caerulein or the selective CCK-B receptor agonist pentagastrin increased dose dependently the level of anxiety. CI-988 dose dependently antagonized the anxiogenic response to pentagastrin but not that induced by pentylenetetrazol. These results strongly suggest that activation of the brain CCK-B receptor induces anxiety and that selective antagonists of this receptor represent a separate class of anxiolytic agents.
Anti-reovirus receptor antibody accelerates expression of the optic nerve oligodendrocyte developmental program.
Authors: Cohen J A , Williams W V , Geller H M , Greene M I
Published: May 2006

Previous studies showed that the cell-surface receptor for reovirus serotype 3 (Reo3R) appears at an early stage of oligodendrocyte differentiation and that anti-Reo3R antibodies and Reo3R-binding peptides induce galactocerebroside expression by developing oligodendrocytes. In the present studies, anti-Reo3R antibodies are shown to stimulate additional features of the program of oligodendrocyte development, including the loss of the A2B5 marker and expression of myelin basic protein. In anti-Reo3R antibody-treated cultures, galactocerebroside was expressed by cells having the morphology of immature oligodendrocyte precursors. Reo3R binding did not appear directly to inhibit or stimulate proliferation of glial progenitor cells or to affect their lineage commitment. Cell-surface structures utilized as a receptor by reovirus type 3 appear to play a role in the regulation of the initiation or rate of execution of the oligodendrocyte developmental program.
Surface immunoglobulin crosslinking activates a tyrosine kinase pathway in B cells that is independent of protein kinase C.
Authors: Brunswick M , Samelson L E , Mond J J
Published: May 2006

It has been found that the principal biochemical pathway activated in B cells stimulated by antigen- or anti-immunoglobulin-mediated crosslinking of surface immunoglobulin is that resulting in hydrolysis of phosphatidylinositol bisphosphate with generation of diacylglycerol and inositol trisphosphate. Recent evidence suggests that surface immunoglobulin-mediated B-cell activation can proceed without detectable increases in the concentration of either diacylglycerol or intracellular Ca2+ concentration, implicating involvement of other non-protein-kinase-C/Ca2(+)-dependent signal-transduction pathways. Therefore, we sought evidence for activation of a signaling pathway that is associated with growth regulation in other cell types--i.e., the protein-tyrosine kinases. We now show that crosslinking of membrane immunoglobulin by mitogenic antibodies leads to rapid tyrosine phosphorylation of several cellular substrates, consistent with the induction of a tyrosine kinase activity. This increase in tyrosine phosphorylation is weakly (if at all) stimulated by other B-cell mitogens, including phorbol esters and ionophores, and does not require the presence of detectable protein kinase C. Furthermore, inhibition of anti-immunoglobulin-stimulated phosphatidylinositol bisphosphate hydrolysis does not inhibit activation of this tyrosine kinase-dependent pathway. These findings suggest that occupancy of the membrane immunoglobulin receptor may induce multiple pathways of activation.
Evidence for separate pathways within the tecto-geniculate projection in the tree shrew.
Authors: Diamond I T , Conley M , Fitzpatrick D , Raczkowski D
Published: May 2006

Two layers (3 and 6) in the dorsal lateral geniculate nucleus (GLd) of the tree shrew (Tupaia belangeri) receive projections from the superficial layers of the superior colliculus. The goal of this study was to determine whether the same or different cells in the superior colliculus give rise to the projections to layers 3 and 6 by following individual axons labeled with biocytin from the superior colliculus to the GLd. The results show that the terminal fields differ in the two layers--those in layer 3 are restricted to a line of projection, whereas those in layer 6 are elongated along the dimension orthogonal to a line of projection. Another important difference between axons that project to GLd layers 3 and 6 is that those that project to layer 6 give off collaterals to the posterior pretectal nucleus, whereas at least some axons that project to layer 3 send a collateral to the ventral lateral geniculate nucleus (GLv). These results suggest that the superior colliculus exerts separate influences on these two GLd layers, both of which project to separate targets above layer IV in the striate cortex. The biocytin method has proved useful by showing the dendritic trees of the superior colliculus cells of origin, the pathways taken by the axons (including the presence of collaterals), and the terminal fields both within and outside the GLd.
Binding protein BiP is required for translocation of secretory proteins into the endoplasmic reticulum in Saccharomyces cerevisiae.
Authors: Nguyen T H , Law D T , Williams D B
Published: May 2006

The endoplasmic reticulum of mammalian cells contains a heat shock protein of approximately 70 kDa (hsp70) termed binding protein BiP that is thought to promote the folding and subunit assembly of newly synthesized proteins. To study BiP function, we placed the BiP-encoding gene from Saccharomyces cerevisiae under the control of a regulated promoter and examined the effects of BiP depletion. Reduction of BiP protein to about 15% of normal levels led to a profound reduction in secretion of alpha factor and invertase. At the same time, unglycosylated precursors of these proteins accumulated intracellularly. The predominant form of the invertase precursor had undergone signal sequence cleavage but accumulated as a soluble species in the cytosol. In contrast, the alpha-factor precursor was exclusively in the signal-uncleaved form. It sedimented with microsomal membranes and was exposed at the cytoplasmic face in a protease-resistant form. These findings suggest that, in yeast, BiP function is required for translocation of soluble proteins into the endoplasmic reticulum at a stage beyond the initial nascent chain-membrane association.
Complementation of a yeast cell cycle mutant by an alfalfa cDNA encoding a protein kinase homologous to p34cdc2.
Authors: Hirt H , Páy A , Györgyey J , Bakó L , Németh K , Bögre L , Schweyen R J , Heberle-Bors E , Dudits D
Published: May 2006

The cdc2 protein kinase plays a central role in control of the eukaryotic cell cycle of animals and yeasts. We have isolated a cDNA clone (cdc2Ms) from alfalfa (Medicago sativa L.) that is homologous to the yeast cdc2/CDC28 genes. The encoded protein is 64% identical to the yeast and mammalian counterparts and shows all the prominent structural features known from these organisms. Antibody raised against a 16-amino acid synthetic peptide with crossreactivity against p34 proteins recognized a 34-kilodalton protein in extracts of alfalfa cells. When transferred into a fission yeast, the plant cdc2 homolog can complement a temperature-sensitive cdc2 mutant. Northern analysis revealed higher transcript levels in shoots and suspension cultures than in roots. In addition to the dominant transcript of 1.4 kilobases detected in the poly(A)+fraction, 2.5- and 1.2-kilobase transcripts were detected in total RNA preparations from shoots or somatic embryos. Suspension cultures that were induced to form somatic embryos by an auxin (2,4-dichlorophenoxyacetic acid) showed fluctuations in transcription pattern during the induction period and embryogenesis.
Characteristics of erythroleukemia cells selected for vincristine resistance that have accelerated inducer-mediated differentiation.
Authors: Richon V M , Weich N , Leng L , Kiyokawa H , Ngo L , Rifkind R A , Marks P A
Published: May 2006

The induction of murine erythroleukemia cells (MELC; DS19/Sc9) to terminal differentiation by hexamethylenebisacetamide (HMBA) is characterized by a latent period of 10-12 hr before onset of commitment to terminal-cell division and increased transcription of globin genes. MELC variants, derived from this parental cell line, selected for resistance to vincristine (VC), can be induced to differentiate with little or no latent period. This study shows that accelerated HMBA-induced commitment is characteristic of MELC with a low level (2- to 5-fold) of VC resistance in four independently derived cell lines. Both resistance to VC and accelerated differentiation are stable phenotypes for at least 50 passages (approximately 5 months) in the absence of VC. Low-level VC-resistant MELC do not display increased levels of P-glycoprotein or mdr1, mdr2, and mdr3 mRNAs, nor do they exhibit cross-resistance to colchicine or doxorubicin. These cells do show (i) increased level of protein kinase C activity, (ii) reduced accumulation of [3H]VC, and (iii) restoration of VC sensitivity in the presence of verapamil. MELC selected for higher levels of VC resistance (approximately 500-fold) do express high levels of P-glycoprotein and the mdr3 gene. During HMBA-induced differentiation, DS19/Sc9 decrease [3H]VC accumulation, but P-glycoprotein content does not change. A VC-transport-associated protein, also critical for the process of induced differentiation, may be constitutively present in VC-resistant MELC, accounting for their enhanced sensitivity to inducer. This protein accumulates by exposure of VC-sensitive cells to HMBA, contributing to their differentiation and decreased level of VC accumulation.
High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine.
Authors: Wall R J , Pursel V G , Shamay A , McKnight R A , Pittius C W , Hennighausen L
Published: May 2006

The whey acidic protein (WAP) is a major milk protein in mice, rats, and rabbits but has not been found in milk of livestock including swine. To determine whether mammary gland regulatory elements from the WAP gene function across species boundaries and whether it is possible to qualitatively alter milk protein composition, we introduced the mouse WAP gene into the genome of swine. Three lines of transgenic swine were analyzed, and mouse WAP was detected in milk from all lactating females at concentrations of about 1 g/liter; these levels are similar to those found in mouse milk. Expression of the corresponding RNA was specific to the mammary gland. Our results suggest that the molecular basis of mammary-specific gene expression is conserved between swine and mouse. In addition the WAP gene must share, with other milk protein genes, elements that target gene expression to the mammary gland. Mouse WAP accounted for about 3% of the total milk proteins in transgenic pigs, thus demonstrating that it is possible to produce high levels of a foreign protein in milk of farm animals. that it is possible to produce high levels of a foreign protein in
Chloroplast DNA evidence for a North American origin of the Hawaiian silversword alliance (Asteraceae).
Authors: Baldwin B G , Kyhos D W , Dvorak J , Carr G D
Published: May 2006

Chloroplast DNA restriction-site comparisons were made among 24 species of the Hawaiian silversword alliance (Argyroxiphium, Dubautia, and Wilkesia) and 7 species of North American perennial tarweeds in Adenothamnus, Madia, Raillardella, and Raillardiopsis (Asteraceae-Madiinae). These data and results from intergeneric hybridization indicated surprisingly close genetic affinity of the monophyletic Hawaiian group to two diploid species of montane perennial herbs in California, Madia bolanderi and Raillardiopsis muirii. Of 117 restriction-site mutations shared among a subset of two or more accessions, more than one-fifth (25 mutations) separated the silversword alliance, M. bolanderi, and Raillardiopsis from Adenothamnus and Raillardella. An additional 10 mutations distinguished the silversword alliance, M. bolanderi, and R. muirii from Adenothamnus, Raillardella, and Raillardiopsis scabrida. Phylogenetic analyses of these data and production of vigorous hybrids of the combinations Dubautia laevigata x R. muirii and (Dubautia knudsenii x Dubautia laxa) x M. bolanderi reinforce and refine Carlquist's hypothesis [Carlquist, S. (1959) Aliso 4, 171-236] that the Hawaiian silversword alliance arose from American tarweeds. Ultimate origin of silversword alliance chloroplast DNA from within the Californian-endemic paraphyletic genus Raillardiopsis was supported with high bootstrap confidence. Geologic considerations and the distribution of sporophytic self-incompatibility among these species demonstrate that the tarweed ancestor of the silverswords overcame (i) a dispersal barrier of at least 3900 km of open ocean and (ii) the breeding barrier of self-incompatibility.
Rabies virus nucleoprotein expressed in and purified from insect cells is efficacious as a vaccine.
Authors: Fu Z F , Dietzschold B , Schumacher C L , Wunner W H , Ertl H C , Koprowski H
Published: May 2006

A cDNA copy of the RNA gene that encodes the nucleoprotein N of rabies virus Evelyn-Rokitnicki-Abelseth strain was cloned into baculovirus. The recombinant baculovirus expressed the N protein abundantly in Spodoptera frugiperda cells. The N protein was extracted from infected Spodoptera frugiperda cells and purified to near homogeneity by affinity chromatography. The purified N protein reacted with 31 of 32 monoclonal antibodies that recognize native rabies virus ribonucleoprotein. Like the ribonucleoprotein, the purified N protein was a major antigen capable of inducing virus-specific helper T cells. Priming of mice with the purified N protein prior to a booster inoculation with inactivated Evelyn-Rokitnicki-Abelseth virus vaccine resulted in a 20-fold increase in the production of virus-neutralizing antibodies. After immunization with the purified N protein, mice developed a strong anti-ribonucleoprotein antibody response and were protected against a lethal challenge of rabies virus. These data indicate that the N protein expressed in insect cells is antigenically and immunogenically comparable to the authentic rabies virus ribonucleoprotein and therefore represents a potential source of an effective and economical vaccine for large-scale immunization of humans and animals against rabies.
Dipoles localized at helix termini of proteins stabilize charges.
Authors: Aqvist J , Luecke H , Quiocho F A , Warshel A
Published: May 2006

The charge-stabilization effect associated with alpha-helices in proteins has been reexamined by microscopic calculations without any a priori assumptions about the dielectric constant of the protein. The calculations reproduce the observed charge stabilization effect of a helix in two well-defined test cases: a histidine residue situated at the C-terminal end of a helix in barnase and the sulfate ligand located near the N-terminal end of a helix of the sulfate-binding protein. They also show that the effective dielectric constant for helix-charge interactions is much larger than previously assumed and that the stabilizing effect of the helix is not associated with the helix macrodipole but rather with a few localized dipoles confined mostly to the first turn of the helix. It is predicted that mutations at one end of the helix should have very small effects on the stabilization of charges at the opposite terminus. It is pointed out that the relatively short-ranged effect of the helix is essentially similar to other cases in which localized dipoles play key roles in electrostatic stabilization.
Identification and purification to near homogeneity of the vitamin K-dependent carboxylase.
Authors: Wu S M , Morris D P , Stafford D W
Published: May 2006

Vitamin K-dependent carboxylase catalyzes the modification of specific glutamic acids to gamma-carboxyglutamic acid in several blood-coagulation proteins. This modification is required for the blood-clotting activity of these proteins and has thus been the subject of intense investigation. We have now identified the bovine vitamin K-dependent carboxylase and purified it to near homogeneity by an affinity procedure that uses the 59-amino acid peptide FIXQ/S (residues -18 to 41 of factor IX with mutations Arg----Gln at residue -4 and Arg----Ser at residue -1). The carboxylase as purified has a molecular weight of 94,000. It is also the major protein that can be cross-linked to iodinated FIXQ/S and is the only protein whose cross-linking is prevented by a synthetic factor IX propeptide. The degree of purification is about 7000-fold with reference to ammonium sulfate-fractionated microsomal protein from liver.
Curved DNA without A-A: experimental estimation of all 16 DNA wedge angles.
Authors: Bolshoy A , McNamara P , Harrington R E , Trifonov E N
Published: May 2006

The principal sequence feature responsible for intrinsic DNA curvature is generally assumed to be runs of adenines. However, according to the wedge model of DNA curvature, each dinucleotide step is associated with a characteristic deflection of the local helix axis. Thus, an important test of a more general view of sequence-dependent DNA curvature is whether sequence elements other than A-A cause the DNA axis to deflect. To address this question, we have applied the wedge model to a large body of experimental data. The axial path of DNA can be described at each step by three Eulerian angles: the helical twist, the deflection angle (wedge angle), and the direction of the deflection. Circularization and gel electrophoretic mobility data on 54 synthetic DNA fragments, both from other laboratories and from our own, were used to compare the theoretical predictions of the wedge model with experiment. By minimizing misfit between calculated and observed DNA curvature, we have found that the stacks AG/CT, CG/CG, GA/TC, and GC/GC, in addition to AA/TT, have large wedge values. We have also synthesized seven sequences without AA/TT elements but with these other wedges correctly phased to cause appreciable predicted curvature. All appear curved as demonstrated by anomalous gel mobilities. The full set of 16 roll and tilt wedge angles is estimated and, together with the known 10 helical twists, these allow prediction of the general sequence-dependent trajectory of the DNA axis.
The c-fms gene complements the mitogenic defect in mast cells derived from mutant W mice but not mi (microphthalmia) mice.
Authors: Dubreuil P , Forrester L , Rottapel R , Reedijk M , Fujita J , Bernstein A
Published: May 2006

Mutations at three loci in the mouse--W, Steel Sl), and microphthalmia (mi)--can lead to a deficiency in melanocytes and mast cells. As well, W and Sl mutants can be anemic and sterile, whereas mi mice are osteopetrotic due to a monocyte/macrophage defect. Recent data have shown that the c-kit receptor tyrosine kinase is the gene product of the W locus, whereas Sl encodes the ligand for this growth factor receptor. We show here that ectopic expression of c-fms, a gene that encodes a macrophage growth factor receptor that is closely related to the c-kit receptor, complements mutations at the W locus in an in vitro mast cell/fibroblast coculture system but is unable to reverse the inability of mi/mi mast cells to survive under these conditions. Furthermore, mast cells expressing the c-fms receptor survive on a monolayer of fibroblasts homozygous for the Sl mutation. These results suggest that ligand binding to the c-kit or c-fms receptor activates identical or overlapping signal transduction pathways. Furthermore, they suggest that mi encodes a protein necessary for transducing signals mediated by way of either the c-kit or c-fms receptor.
Chemiluminescence detection of proteins from single cells.
Authors: Gillespie P G , Hudspeth A J
Published: May 2006

The analysis of proteins from single cells requires techniques of supreme sensitivity. Although radiochemical procedures are capable of detecting small amounts of electrophoretically separated proteins, their sensitivity falls short of that required for routine detection of minor components of single cells. Utilizing the avidin-biotin interaction and the alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD), we have developed an alternative, chemiluminescence-based method for protein detection whose sensitivity exceeds that of other methods. Applying this method to a purified protein, we could detect as little as 63 fg (0.9 amol) of biotinylated bovine serum albumin. The sensitivity of the method was demonstrated by the detection of proteins from individual photoreceptor outer segments, including proteins constituting approximately 1% of the total. Chemiluminescence detection also proved extremely sensitive for immunoblotting: a comparison of five methods for detection of antibody-antigen interactions showed that the AMPPD technique was more sensitive than detection with a colorimetric alkaline phosphatase substrate, 125I-labeled protein A, 125I-labeled anti-mouse IgG, or colloidal gold-conjugated anti-mouse IgG.
The reaction of hydroxylamine with bacteriorhodopsin studied with mutants that have altered photocycles: selective reactivity of different photointermediates.
Authors: Subramaniam S , Marti T , Rösselet S J , Rothschild K J , Khorana H G
Published: May 2006

The reaction of the retinylidene Schiff base in bacteriorhodopsin (bR) to the water-soluble reagent hydroxylamine is enhanced by greater than 2 orders of magnitude under illumination. We have used this reaction as a probe for changes in Schiff base reactivity during the photocycle of wild-type bR and mutants defective in proton transport. We report here that under illumination at pH 6, the D85N mutant has a 20-fold lower rate and the D212N mutant has a greater than 4-fold higher rate for the light-dependent reaction with hydroxylamine compared with wild-type bR. In contrast, the reactivities of wild-type bR and the D96N and T46V mutants are similar. It has been previously shown that the D96N and T46V replacements have no significant effect on the kinetics of "M" formation but have dramatic effects on rate of the decay of M. We therefore conclude that the hydroxylamine reaction occurs before formation of the M intermediate. Most likely it occurs at the "L" stage of the cycle and reflects increased water accessibility to the Schiff base due to a light-driven change in protein conformation.