bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2.
Authors: Mulligan M S , Desrochers P E , Chinnaiyan A M , Gibbs D F , Varani J , Johnson K J , Weiss S J
Published: May 2006

The pulmonary tree is exposed to neutrophil-derived serine proteinases and matrix metalloproteinases in inflammatory lung diseases, but the degree to which these enzymes participate in tissue injury remains undefined, as does the therapeutic utility of antiproteinase-based interventions. To address these issues, an in vivo rat model was examined in which the intrapulmonary deposition of immune complexes initiates a neutrophil-mediated acute alveolitis. In vitro studies demonstrated that rat neutrophils can release neutrophil elastase and cathepsin G as well as a neutrophil progelatinase, which was subsequently activated by either chlorinated oxidants or serine proteinases. Based on structural homologies that exist between rat and human neutrophil proteinases, rat neutrophil elastase and cathepsin G activities could be specifically regulated in vitro by recombinant human secretory leukoproteinase inhibitor, and rat neutrophil gelatinase activity proved sensitive to inhibition by recombinant human tissue inhibitor of metalloproteinases 2. When either of the recombinant antiproteinases were instilled intratracheally, in vivo lung damage as assessed by increased permeability or hemorrhage was significantly reduced. Furthermore, the coadministration of the serine and matrix metalloproteinase inhibitors almost completely prevented pulmonary damage while effecting only a modest decrease in neutrophil influx. These data support a critical role for neutrophil-derived proteinases in acute lung damage in vivo and identify recombinant human secretory leukoproteinase and recombinant human tissue inhibitor of metalloproteinases 2 as potentially efficacious interventions in inflammatory disease states.
The relationship between synthetic and editing functions of the active site of an aminoacyl-tRNA synthetase.
Authors: Kim H Y , Ghosh G , Schulman L H , Brunie S , Jakubowski H
Published: May 2006

We have analyzed, by site-directed mutagenesis, the molecular basis of the editing function and its relation to the synthetic function of Escherichia coli methionyl-tRNA synthetase. The data obtained fit a model of the active site that partitions an amino acid substrate between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions direct the cognate substrate methionine through the synthetic pathway and prevent it from entering the editing pathway. Two hydrophobic interactions are proposed: between the side chain of Trp-305 and a methyl group of methionine and between the benzene ring of Tyr-15 and the beta- and gamma-CH2 groups of the substrate. An essential hydrogen bond forms between the OH of Tyr-15 and an electron pair of the sulfur atom of methionine. Consistent with these functions, side chains of Trp-305 and Tyr-15 are localized on opposite sides of the cavity forming a putative methionine binding pocket that is observed in the three-dimensional crystallographic structure of methionyl-tRNA synthetase. Enzymes W305A, Y15A, and Y15F have diminished ability to discriminate against homocysteine in the synthetic reaction, compared to the wild-type enzyme. At the same time, mutant enzymes have lost the ability to discriminate against methionine in the editing reaction and edited Met-AMP to a similar extent as Hcy-AMP. Interactions of residues Arg-233 and Asp-52 of methionyl-tRNA synthetase with the carboxyl and amino groups, respectively, of the substrate, which are essential for the synthetic function, were also essential for the editing function of the enzyme. Deacylation of Met-tRNA to S-methylhomocysteine thiolactone catalyzed by W305A, Y15A, and Y15F mutant enzymes was only slightly impaired relative to the wild-type enzyme. However, enzymes R233Q, R233A, and D52A did not deacylate Met-tRNA. The model also explains why the noncognate homocysteine is edited by methionyl-tRNA synthetase.
Transcriptional regulation of HLA-A and -B: differential binding of members of the Rel and IRF families of transcription factors.
Authors: Girdlestone J , Isamat M , Gewert D , Milstein C
Published: May 2006

HLA-A and -B transplantation antigens can be expressed differentially at the basal level and in response to interferons (IFNs). To determine which DNA control elements and nuclear factors are responsible for these differences, HLA-A and -B upstream regulatory regions were used in expression and mobility-shift analyses. The HLA-A enhancer was found to contain two Rel (KBF/NF-kappa B) binding motifs, while the HLA-B enhancer has only one and is transactivated less well by overexpression of the NF-kappa B p65 subunit. On the other hand, the HLA-B IFN response element mediates a much stronger induction by IFNs and has a higher affinity for IRF-1 and -2, which are transcription factors implicated in the regulation of major histocompatibility complex class I genes. These results suggest a molecular basis for the way in which HLA-A and -B loci have adapted to be differentially expressed and to respond to different sets of cytokine signals.
Calcium permeability of the N-methyl-D-aspartate receptor channel in hippocampal neurons in culture.
Authors: Jahr C E , Stevens C F
Published: May 2006

We have developed a quantitative description for calcium permeability of the N-methyl-D-aspartate receptor channel to permit predictions of the reversal potential and calcium influx at different voltages for different extracellular calcium concentrations. Increasing the external calcium concentration markedly shifted the reversal potential to positive values and simultaneously decreased the single-channel conductance at potentials negative to the reversal potential. Very simple quantitative descriptions of calcium permeation and channel block by calcium ions accurately characterize our data and permit the prediction of reversal potentials and magnitudes of calcium influx for a wide range of conditions.
Induction of erythroid-specific gene expression in lymphoid cells.
Authors: Chiba T , Nagata Y , Kishi A , Sakamaki K , Miyajima A , Yamamoto M , Engel J D , Todokoro K
Published: May 2006

Erythropoietin (Epo) is a cytokine which specifically regulates differentiation and proliferation of erythroid progenitor cells. We report here that Epo receptor expressed in interleukin 3-dependent lymphoid Ba/F3 cells transmits both differentiation and growth signals. Epo stimulation of these cells leads to activation of transcription and/or translation of the erythroid-specific transcription factors GATA-1 and SCL, followed by the accumulation of both alpha- and beta-globin chains. These results suggest that expression and activation of the Epo receptor regulates erythroid-specific gene expression and might play a role in determining a cell lineage in vivo and that GATA-1 and SCL may exert their effects after Epo binds to its receptor. It was further found that chimeric receptors composed of extracellular domains of Epo receptor and cytoplasmic domains of interleukin 2 or interleukin 3 receptors could also induce erythroid-specific gene expression in Ba/F3 cells. Taking these data together with previous observations, we conclude that interaction of the extracellular domains of the Epo receptor with other membrane components is essential for transmission of both the erythroid differentiation and the growth signals.
Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase.
Authors: Mitchell J A , Akarasereenont P , Thiemermann C , Flower R J , Vane J R
Published: May 2006

Constitutive cyclooxygenase (COX-1; prostaglandin-endoperoxide synthase, EC 1.14.99.1) is present in cells under physiological conditions, whereas COX-2 is induced by some cytokines, mitogens, and endotoxin presumably in pathological conditions, such as inflammation. Therefore, we have assessed the relative inhibitory effects of some nonsteroidal antiinflammatory drugs on the activities of COX-1 (in bovine aortic endothelial cells) and COX-2 (in endotoxin-activated J774.2 macrophages) in intact cells, broken cells, and purified enzyme preparations (COX-1 in sheep seminal vesicles; COX-2 in sheep placenta). Similar potencies of aspirin, indomethacin, and ibuprofen against the broken cell and purified enzyme preparations indicated no influence of species. Aspirin, indomethacin, and ibuprofen were more potent inhibitors of COX-1 than COX-2 in all models used. The relative potencies of aspirin and indomethacin varied only slightly between models, although the IC50 values were different. Ibuprofen was more potent as an inhibitor of COX-2 in intact cells than in either broken cells or purified enzymes. Sodium salicylate was a weak inhibitor of both COX isoforms in intact cells and was inactive against COX in either broken cells or purified enzyme preparations. Diclofenac, BW 755C, acetaminophen, and naproxen were approximately equipotent inhibitors of COX-1 and COX-2 in intact cells. BF 389, an experimental drug currently being tested in humans, was the most potent and most selective inhibitor of COX-2 in intact cells. Thus, there are clear pharmacological differences between the two enzymes. The use of such models of COX-1 and COX-2 activity will lead to the identification of selective inhibitors of COX-2 with presumably less side effects than present therapies. Some inhibitors had higher activity in intact cells than against purified enzymes, suggesting that pure enzyme preparations may not be predictive of therapeutic action.
Fast events in protein folding initiated by nanosecond laser photolysis.
Authors: Jones C M , Henry E R , Hu Y , Chan C K , Luck S D , Bhuyan A , Roder H , Hofrichter J , Eaton W A
Published: May 2006

Initiation of protein folding by light can dramatically improve the time resolution of kinetic studies. Here we present an example of an optically triggered folding reaction by using nanosecond photodissociation of the heme-carbon monoxide complex of reduced cytochrome c. The optical trigger is based on the observation that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide to the covalently attached heme group in the unfolded state. Photodissociation of the carbon monoxide thus triggers the folding reaction. We used time-resolved absorption spectroscopy to monitor binding at the heme. Before folding begins we observe transient binding of both nonnative and native ligands from the unfolded polypeptide on a microsecond time scale. Kinetic modeling suggests that the intramolecular binding of methionine-65 and -80 is faster than that of histidine-26 and -33, even though the histidines are closer to the heme. This optical trigger should provide a powerful method for studying chain collapse and secondary structure formation in cytochrome c without any limitations in time resolution.
Expression and phosphorylation of the Listeria monocytogenes ActA protein in mammalian cells.
Authors: Brundage R A , Smith G A , Camilli A , Theriot J A , Portnoy D A
Published: May 2006

Movement of Listeria monocytogenes within infected eukaryotic cells provides a simple model system to study the mechanism of actin-based motility in nonmuscle cells. The actA gene of L. monocytogenes is required to induce the polymerization of host actin filaments [Kocks, C., Gouin, E., Tabouret, M., Berche, P., Ohayon, H. & Cossart, P. (1990) Cell 68, 521-531; Domann, E., Wehland, J., Rohde, M., Pistor, S., Hartl, M., Goebel, W., Leimeister-Wachter, M., Wuenscher, M. & Chakraborty, T. (1992) EMBO J. 11, 1981-1990]. In this study, an in-frame deletion mutation within the actA gene was constructed and introduced into the L. monocytogenes chromosome by allelic exchange. This mutation resulted in a decrease (3 orders of magnitude) in virulence for mice. In tissue culture cells, the actA mutant was absolutely defective for the nucleation of actin filaments and consequently was impaired in cell-to-cell spread. Antiserum raised to a synthetic peptide encompassing the proline-rich repeat (DFPPPPTDEEL) of ActA was used to characterize the expression of the ActA protein. The ActA protein derived from extracellular bacteria migrated as a 97-kDa polypeptide upon SDS/PAGE, whereas the protein from infected cells migrated as three distinct polypeptides, one that comigrated with the 97-kDa extracellular form and two slightly larger species. Treatment of infected cells with okadaic acid resulted in decreased amounts of all forms of ActA and the appearance of a larger species of ActA. Phosphatase treatment of ActA immunoprecipitated from intracellular bacteria resulted in conversion of the larger two species to the 97-kDa form. Labeling of infected cells with 32Pi followed by immunoprecipitation showed that the largest molecular form of ActA was phosphorylated. Taken together, these data indicate that ActA is phosphorylated during intracellular growth. The significance of the intracellular modification of ActA is not known, but we speculate that it may modulate the intracellular activity of ActA.
Highly localized tracks of human immunodeficiency virus type 1 Nef in the nucleus of cells of a human CD4+ T-cell line.
Authors: Murti K G , Brown P S , Ratner L , Garcia J V
Published: May 2006

A human T-cell line constitutively expressing the nef gene from the human immunodeficiency virus type 1 SF2 isolate was used to examine the distribution of the Nef protein in the nucleus. High-resolution immunogold labeling/electron microscopic studies with polyclonal anti-Nef antibodies on nef+ and nef- cells revealed that a small fraction of Nef is in the nucleus and it is localized in specific curvilinear tracks that extend between the nuclear envelope and the nucleoplasm. An examination of the sequence of the SF2 nef gene revealed a putative nuclear targeting sequence that was previously found in several other eukaryotic nucleoplasmic proteins. The nuclear localization of Nef suggests a potential nuclear function for this protein. The presence of Nef in distinct nuclear tracks suggests that Nef is transported along a specific pathway that extends from the nuclear envelope into the nucleoplasm. A previous study [Meier, U. T. & Blobel, G. (1992) Cell 70, 127-138] has shown that the nucleolar protein of rat liver cells (Nopp140) shuttles from the nucleolus to the nuclear envelope on distinct tracks. The present study has suggested that the transport of a nucleoplasmic protein may also occur on distinct nuclear pathways.
Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase.
Authors: Pillai R , Kytle K , Reyes A , Colicelli J
Published: May 2006

Saccharomyces cerevisiae strain PP5 has a phosphodiesterase (PDE) deficiency that results in heat-shock sensitivity due to the intracellular accumulation of cAMP. This strain also carries the cam mutation, which confers permeability to cAMP and, as shown here, to other compounds. Expression of rat type IV PDE in these cells caused them to revert to heat-shock resistance. Treatment of the transformed PP5 cells with rolipram, an antidepressant in humans and a potent inhibitor of type IV PDEs, reinstated sensitivity to heat shock. The biochemical properties of deletion mutants of this PDE were determined, and an active enzyme of minimum length was created. Reversion to heat-shock resistance was then used to select for PDE mutants refractory to the inhibitory effects of rolipram. Four mutants (A1, A2, A3, and A5) were isolated. Each carries a single point mutation; two have mutations in the same codon. Each mutant showed distinct properties, based on analysis of their substrate kinetics and IC50 values for a variety of inhibitors. Mutant A5 had a reduced activity for substrate, mutants A1 and A3 showed no change in substrate kinetics, and mutant A2 displayed an increase in activity. For most mutants, the drug resistance was confined to the class of drug used in the selection. This study shows that it is possible to recreate in yeast cells the susceptibility of mammalian enzymes to pharmacological agents. Our study also demonstrates that such systems can be used to select rare mutants useful in the analysis of drug-protein interactions.
Two functionally distinct forms of the photosystem II reaction-center protein D1 in the cyanobacterium Synechococcus sp. PCC 7942.
Authors: Clarke A K , Hurry V M , Gustafsson P , Oquist G
Published: May 2006

The cyanobacterium Synechococcus sp. PCC 7942 possesses a small psbA multigene family that codes for two distinct forms of the photosystem II reaction-center protein D1 (D1:1 and D1:2). We showed previously that the normally predominant D1 form (D1:1) was rapidly replaced with the alternative D1:2 when cells adapted to a photon irradiance of 50 mumol.m-2.s-1 are shifted to 500 mumol.m-2.s-1 and that this interchange was readily reversible once cells were allowed to recover under the original growth conditions. By using the psbA inactivation mutants R2S2C3 and R2K1 (which synthesize only D1:1 and D1:2, respectively), we showed that this interchange between D1 forms was essential for limiting the degree of photoinhibition as well as enabling a rapid recovery of photosynthesis. In this report, we have extended these findings by examining whether any intrinsic functional differences exist between the two D1 forms that may afford increased resistance to photoinhibition. Initial studies on the rate of D1 degradation at three photon irradiances (50, 200, and 500 mumol.m-2.s-1) showed that the rates of degradation for both D1 forms increase with increasing photon flux density but that there was no significant difference between D1:1 and D1:2. Analysis of light-response curves for oxygen evolution for the mutants R2S2C3 and R2K1 revealed that cells with photosystem II reaction centers containing D1:2 have a higher apparent quantum yield (approximately 25%) than cells possessing D1:1. Further studies using chlorophyll a fluorescence measurements confirmed that R2K1 has a higher photochemical yield than R2S2C3; that is, a more efficient conversion of excitation energy from photon absorption into photochemistry. We believe that the higher photochemical efficiency of reaction centers containing D1:2 is causally related to the preferential induction of D1:2 at high light and thus may be an integral component of the protection mechanism within Synechococcus sp. PCC 7942 against photoinhibition.
A pathway for disulfide bond formation in vivo.
Authors: Bardwell J C , Lee J O , Jander G , Martin N , Belin D , Beckwith J
Published: May 2006

Protein disulfide bond formation in Escherichia coli requires the periplasmic protein DsbA. We describe here mutations in the gene for a second protein, DsbB, which is also necessary for disulfide bond formation. Evidence suggests that DsbB may act by reoxidizing DsbA, thereby regenerating its ability to donate its disulfide bond to target proteins. We propose that DsbB, an integral membrane protein, may be involved in transducing redox potential across the cytoplasmic membrane.
Microsatellite mapping of the gene causing weaver disease in cattle will allow the study of an associated quantitative trait locus.
Authors: Georges M , Dietz A B , Mishra A , Nielsen D , Sargeant L S , Sorensen A , Steele M R , Zhao X , Leipold H , Womack J E
Published: May 2006

A genetic disease in cattle, progressive degenerative myeloencephalopathy (weaver disease), is associated with increased milk production. This association could result from population stratification, from a pleiotropic effect of a single gene, or from linkage disequilibrium between the gene causing weaver disease and a quantitative trait locus (QTL) for milk production. To test these hypotheses, we performed an extensive linkage study in a bovine pedigree segregating for the weaver condition and identified a microsatellite locus (TGLA116) closely linked to the weaver gene (zmax, 8.15; theta, 0.03). TGLA116 and, by extension, the weaver locus were assigned to bovine synteny group 13. This microsatellite can be used to identify weaver carriers, to select against this genetic defect, and to study the effect of the corresponding chromosomal region on milk production in Brown Swiss and other breeds of cattle.
Toward an understanding of the molecular mechanisms of physiological cell death.
Authors: Vaux D L
Published: May 2006

Cell death is a normal physiological process. Morphological studies have shown that cells that die by physiological mechanisms often undergo characteristic changes termed "apoptosis" or "programmed cell death." Recent work has begun to unravel the molecular mechanisms of these deaths and has shown that one of the primary cell-death pathways is conserved throughout much of evolution. In the nematode Caenorhabditis elegans programmed cell deaths are mediated by a mechanism controlled by the ced-9 gene; in mammals apoptosis can often be inhibited by expression of the bcl-2 gene. The ability of the human BCL2 gene to prevent cell deaths in C. elegans strongly suggests that bcl-2 and ced-9 are homologous genes. Although the process of cell death controlled by bcl-2 can occur in many cell types, there appears to be more than one physiological cell-death mechanism. Targets of cytotoxic T cells and cells deprived of growth factor both exhibit changes characteristic of apoptosis, such as DNA degradation. However, bcl-2 expression protects cells from factor withdrawal but fails to prevent cytotoxic T-cell killing. DNA degradation is, thus, not specific for any one cell-death mechanism. The ability of bcl-2 to protect cells from a wide variety of pathological, as well as physiological, stimuli indicates that many triggers can serve to activate the same suicide pathway, even some thought to cause necrosis, and not physiological cell death.
Expression of a constitutively active erythropoietin receptor in primary hematopoietic progenitors abrogates erythropoietin dependence and enhances erythroid colony-forming unit, erythroid burst-forming unit, and granulocyte/macrophage progenitor growth.
Authors: Pharr P N , Hankins D , Hofbauer A , Lodish H F , Longmore G D
Published: May 2006

We tested the ability of a constitutively activated erythropoietin receptor [EpoR(R129C)] to alter the growth requirements of primary hematopoietic precursors that terminally differentiate in culture. Two recombinant retroviruses expressing EpoR(R129C), spleen focus-forming virus (SFFVc-EpoR) and myeloproliferative sarcoma virus (MPSVcEpoR), were used to infect fetal liver cells that served as a source of hematopoietic progenitors. Methylcellulose cultures were incubated in the absence of any added growth factors or in combination with selected growth factors. EpoR(R129C) completely abrogated the Epo requirement of erythroid colony-forming units to form erythrocytes after 2-5 days in culture and did not interfere with the differentiation program of these cells. In the absence of added growth factors EpoR(R129C) did not enhance erythroid burst-forming unit development. In contrast to experiments in heterologous cell lines, EpoR(R129C) did not render progenitor cells independent of interleukin 3 or granulocyte/macrophage colony-stimulating factor (GM-CSF). However, when progenitors were cultured with added steel factor, but not with interleukin 3 or GM-CSF, EpoR(R129C) augmented the growth and differentiation of erythroid bursts, mixed erythroid/myeloid, and granulocyte/macrophage (GM) colonies. Furthermore, both viruses were capable of expressing EpoR(R129C) in erythroid, mixed erythroid/myeloid, and GM colonies. Thus an aberrantly expressed and constitutively activated EpoR can stimulate proliferation of some GM progenitors. The ability of EpoR(R129C) to abrogate the Epo requirement of primary hematopoietic cells, but not the requirement for other cytokines, is consistent with the induction of erythroblastosis in vivo.
Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae.
Authors: Miran S G , Lawson J E , Reed L J
Published: May 2006

The gene encoding the pyruvate dehydrogenase (PDH) beta subunit (E1 beta) of the PDH complex from Saccharomyces cerevisiae has been cloned, sequenced, disrupted, and expressed. Two overlapping DNA fragments were generated from a yeast genomic DNA library by the polymerase chain reaction with synthetic oligonucleotide primers based on amino acid sequences of the yeast and human E1 beta subunits. The DNA fragments were subcloned and sequenced. The composite sequence has an open reading frame of 1098 nucleotides encoding a putative presequence of 33 amino acid residues and a mature protein of 333 residues with a calculated M(r) = 36,486. Yeast and human E1 beta exhibit 62% sequence identity. The size of the mRNA is approximately 1.5 kilobases. Hybridization analysis showed that the E1 beta gene (PDH beta 1) is localized to chromosome II. Disruption of PDH beta 1 is not lethal under vegetative growth conditions. The null mutant transformed with PDH beta 1 on a unit-copy plasmid produced mature E1 beta and a functional PDH complex.
A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens.
Authors: Wistow G J , Shaughnessy M P , Lee D C , Hodin J , Zelenka P S
Published: May 2006

A discrete 10-kDa polypeptide (10K) is expressed from early stages in the embryonic chicken lens. Since this has potential as a marker for lens cell development, chicken 10K and its homologues from mouse and human lenses were identified by protein sequencing and cloning. Surprisingly, lens 10K proteins appear to be identical to a lymphokine, macrophage migration inhibitory factor (MIF), originally identified in activated human T cells. Using microdissection and PCR techniques, we find that expression of 10K/MIF is strongly correlated with cell differentiation in the developing chicken lens. Northern blot analysis shows that 10K/MIF is widely expressed in mouse tissues. These results suggest that proteins with MIF activity may have roles beyond the immune system, perhaps as intercellular messengers or part of the machinery of differentiation itself. Indeed, partial sequence of other small lens proteins identifies another MIF-related protein (MRP8) in calf lens. The relatively abundant expression of MIF in lens may have clinical significance, with the possibility of involvement in ocular inflammations that may follow damage to the lens.
Pathway of proton transfer in bacterial reaction centers: second-site mutation Asn-M44-->Asp restores electron and proton transfer in reaction centers from the photosynthetically deficient Asp-L213-->Asn mutant of Rhodobacter sphaeroides.
Authors: Rongey S H , Paddock M L , Feher G , Okamura M Y
Published: May 2006

Site-directed mutagenesis of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides has shown Asp-213 of the L subunit (Asp-L213) to be important for photosynthetic viability. Replacement of Asp-L213 with Asn resulted in a photosynthetically deficient mutant, due to the 10(4)-fold slower rate for the proton-coupled electron transfer reaction QA-QB- + 2H+-->QAQBH2 (k(2)AB). The detrimental effect of Asn-L213 is surprising since RCs from Rhodopseudomonas viridis, Rhodospirillum rubrum, and Chloroflexus aurantiacus have Asn at the homologous position. However, RCs from these bacteria have an Asp located near QB (the secondary quinone acceptor) at the position homologous to Asn-M44 in Rb. sphaeroides which might function in place of Asp-L213. To test this conjecture a "viridis-like" structure was introduced into Rb. sphaeroides by replacing Asp-L213 with Asn and Asn-M44 with Asp. The RCs from this double mutant displayed near-native rates for the electron transfer reaction k(2)AB and restored photosynthetic competence. The rates for the first electron transfer reaction QA-QB-->QAQB- (k(1)AB) and charge recombination D+QAQB--->DQAQB (kBD) were also restored to near-native values. These results indicate that Asp at either the L213 or the M44 site near QB can provide a pathway for rapid proton transfer and explain why Asp-L213 need not be conserved in different photosynthetic bacteria. To test further the effect of Asp at M44 on electron and proton transfer to QB a mutant containing Asp at both L213 and M44 was constructed. The RCs from this mutant (Asn-M44-->Asp) exhibited faster proton-coupled electron transfer to QB-. The increased rate of proton-coupled electron transfer (k(2)AB) in the presence of negatively charged Asp residues near QB suggests the role of an Asp near QB as (i) a proton donor group in the proton transfer chain and/or (ii) a negatively charged residue stabilizing proton transfer to reduced QB.
Evidence for functional interaction between elongation factor Tu and 16S ribosomal RNA.
Authors: Powers T , Noller H F
Published: May 2006

Translation of the genetic code requires the accurate selection of elongation factor (EF)-Tu.GTP.tRNA ternary complexes at the ribosomal acceptor site, or A site. Several independent lines of evidence have implicated the universally conserved 530 loop of 16S rRNA in this process; yet its precise role has not been identified. Using an allele-specific chemical probing strategy, we have examined the functional defect caused by a dominant lethal G-->A substitution at position 530. We find that mutant ribosomes are impaired in EF-Tu-dependent binding of aminoacyl-tRNA in vitro; in contrast, nonenzymatic binding of tRNA to the A and P sites is unaffected, indicating that the defect involves an EF-Tu-related function rather than tRNA-ribosome interactions per se. In vivo, the mutant ribosomes are found in polysomes at low levels and contain reduced amounts of A-site-bound tRNA, but normal levels of P-site tRNA, in agreement with the in vitro results; thus the dominant lethal phenotype of mutations at G530 can be explained by impaired interaction of mutant ribosomes with ternary complex. These results provide evidence for a newly defined function of 16S rRNA--namely, modulation of EF-Tu activity during translation.
Productivity, consumers, and the structure of a river food chain.
Authors: Wootton J T , Power M E
Published: May 2006

We tested models of food chain dynamics in experimentally manipulated channels within a natural river. As light levels increased, primary productivity and the biomass of algae and primary predators increased, but the biomass of grazers remained relatively constant. In the presence of a fourth trophic level, algae and primary predators decreased, but grazers increased. These results match predictions of food chain models based on classical predator-prey theory and suggest that simple models of multitrophic level interactions are sometimes sufficient to predict the responses of natural communities to changes in environmental productivity and predators.
Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase.
Authors: Quadt R , Kao C C , Browning K S , Hershberger R P , Ahlquist P
Published: May 2006

The association of host proteins with viral RNA replication proteins has been reported for a number of (+)-strand RNA viruses. However, little is known about the identity or function of these host proteins in viral replication. In this paper we report the characterization of a host protein associated with the RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley. A host protein was specifically and proportionally enriched with BMV RdRp activity through several purification steps. This RdRp-associated host protein reacted with an antiserum prepared against wheat germ eukaryotic translation initiation factor 3 (eIF-3). The RdRp-associated host protein, the p41 subunit of wheat germ eIF-3, and an antigenically related protein from rabbit reticulocyte lysates were all found to bind with high affinity and specificity to BMV-encoded protein 2a, which is involved in viral RNA replication. Moreover, addition of wheat germ eIF-3 or the p41 subunit from wheat germ to BMV RdRp gave a specific and reproducible 3-fold stimulation of (-)-strand RNA synthesis in vivo. These results suggest that the barley analog of eIF-3 subunit p41, or a closely related protein, associates with BMV RdRp in vivo and is involved in BMV RNA replication. This observation and the established role of translation factors in bacteriophage Q beta RdRp suggest that association with translation factors may be a general feature of RNA replication by (+)-strand RNA viruses.
A Drosophila photoreceptor cell-specific protein, calphotin, binds calcium and contains a leucine zipper.
Authors: Ballinger D G , Xue N , Harshman K D
Published: May 2006

The calphotin protein, encoded by the calphotin (cap) gene, is expressed in the soma and axons of all Drosophila photoreceptor cells. It is expressed early in photo-receptor cell development, at the time when cell-type decisions are being made. Expression of calphotin is not altered by the glass mutation, which blocks photoreceptor cell development. The calphotin protein binds calcium and contains a long C-terminal leucine zipper. Potential implications of these properties are discussed.
Gene 5.5 protein of bacteriophage T7 inhibits the nucleoid protein H-NS of Escherichia coli.
Authors: Liu Q , Richardson C C
Published: May 2006

Gene 5.5 of coliphage T7 is one of the most highly expressed genes during T7 infection. Gene 5.5 protein, purified from cells overexpressing the cloned gene, purifies with the nucleoid protein H-NS of Escherichia coli during three chromatographic steps. A fusion protein of gene 5.5 protein and maltose binding protein also purifies with H-NS. The fusion protein binds to the DNA-H-NS complex and abolishes H-NS-mediated inhibition of transcription by Escherichia coli and T7 RNA polymerases in vitro. Expression of gene 5.5 also relieves the repression of the Escherichia coli proU promoter by H-NS in vivo. The change of leucine to proline at residue 30 of gene 5.5 protein abolishes the interaction between gene 5.5 protein and H-NS.
Functionally distinct roles for glycosylation of alpha and beta integrin chains in cell-matrix interactions.
Authors: Chammas R , Veiga S S , Travassos L R , Brentani R R
Published: May 2006

Laminin interaction with gp120/140, a B16-F10 laminin-binding protein immunologically related to alpha 6 beta 1 integrin, has been shown to be dependent on oligosaccharides from both ligand and receptor. Lectin analysis of gp120/140 led to the conclusion that this integrin is a sialoglycoprotein bearing mainly complex antennary structures. By means of exoglycosidase treatment, it was possible to identify alpha-galactosyl residues on the integrin alpha chain as the laminin-binding determinants. These residues are involved in cell adhesion to laminin. On the other hand, beta-chain complex antennary structures, whose synthesis could be inhibited by swainsonine, were associated with cell spreading rather than cell adhesion. Thus, it was possible to modulate integrin-mediated cell adhesion and spreading through changes in the glycosylation state of integrin alpha and beta chains.
Lysophosphatidic acid is a chemoattractant for Dictyostelium discoideum amoebae.
Authors: Jalink K , Moolenaar W H , Van Duijn B
Published: May 2006

The naturally occurring phospholipid lysophosphatidic acid (LPA) can induce a number of physiological responses in vertebrate cells, including platelet aggregation, smooth muscle contraction, and fibroblast proliferation. LPA is thought to activate a specific G-protein-coupled receptor, thereby triggering classic second messenger pathways such as stimulation of phospholipase C and inhibition of adenylate cyclase. Here we report that 1-oleoyl-LPA, at submicromolar concentrations, evokes a chemotactic response in amoebae of the cellular slime mold Dictyostelium discoideum. LPA-induced chemotaxis is specific in that other lysophospholipids, phosphatidic acid, and monoacylglycerol have no effect. We show that the response to LPA is not secondary to the accumulation of extracellular cAMP, a well-established chemoattractant for nutrient-starved D. discoideum. Compared with cAMP-induced chemotaxis, LPA-induced chemotaxis has a somewhat lower efficiency and is not accompanied by the characteristic cellular elongation and orientation along the gradient. These results indicate that LPA has a previously unsuspected role as a chemoattractant for D. discoideum and imply that its biological function as a "first messenger" is not restricted to vertebrate cells.
Kinship bioassay on hypervariable loci in blacks and Caucasians.
Authors: Morton N E , Collins A , Balazs I
Published: May 2006

Four hypervariable loci were examined in DNA samples of American Blacks and Caucasians. Genetic diversity, measured by mean kinship within race, is 0.004 for a sliding window equal to twice the radius of coalescence of the autoradiographic bands. Kinship increases with the width of a window or bin, but it is an order of magnitude less than for blood groups and isozymes. This must reflect the greater mutability of hypervariable loci and absence of divergent selection. Low kinship implies that matching probabilities do not depend critically on race. Complete matching between pairs of loci shows deviations from independence, presumably because of inadvertent inclusion of replicated samples or related individuals. Multilocus matching probabilities were corrected for this (possibly spurious) dependence; the correction is negligible. When the complexities of coalescence, kinship, and dependent matching are allowed for, the probability calculations used in forensic identification are reliable. Recent counterarguments without theoretical basis or empirical support are discussed.
Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell.
Authors: Byk T , Bar-Yaacov M , Doza Y N , Minke B , Selinger Z
Published: May 2006

Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to the active form of metarhodopsin. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. On the other hand, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the rdgC phosphatase. We propose that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells.
Glucose-dependent insulinotropic peptide: structure of the precursor and tissue-specific expression in rat.
Authors: Tseng C C , Jarboe L A , Landau S B , Williams E K , Wolfe M M
Published: May 2006

Glucose-dependent insulinotropic peptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that stimulates insulin secretion from pancreatic beta cells in the presence of glucose. Approximately 7.8 x 10(5) recombinant clones of a neonatal rat intestinal cDNA library were screened by using plaque hybridization, and three clones were identified and sequenced with the dideoxynucleotide chain-termination method. The translated amino acid sequence deduced from the nucleotide sequence of the cDNA indicated that rat GIP was derived by proteolytic processing of a 144-amino acid precursor polypeptide. The mature peptide is flanked by a 43-amino acid NH2-terminal peptide that contains a 21-amino acid signal peptide and by a 59-amino acid COOH-terminal peptide. Analysis of the nucleotide and amino acid sequence of rat GIP revealed only two substitutions from the known human GIP peptide. The use of high-stringency RNA blot-hybridization analysis of total RNA extracted from various organs demonstrated expression of the GIP gene in the duodenum and jejunum and, to a lesser extent, in the ileum. In addition, expression of the GIP gene was observed in the submandibular salivary gland both by RNA analysis and RIA. In response to duodenal perfusion of a 20% Lipomul meal for 60 min, duodenal mucosal GIP mRNA concentrations increased by 42.8% and 48.2% at 30 and 60 min, respectively.
Trans-activation of glutathione transferase P gene during chemical hepatocarcinogenesis of the rat.
Authors: Morimura S , Suzuki T , Hochi S , Yuki A , Nomura K , Kitagawa T , Nagatsu I , Imagawa M , Muramatsu M
Published: May 2006

Glutathione transferase P (GST-P; glutathione transferase, EC 2.5.1.18) is known to be specifically expressed at high levels in precancerous lesions and in hepatocellular carcinomas from a very early phase of chemically induced hepatocarcinogenesis in the rat. The almost invariable occurrence of this phenotype in these lesions strongly suggests a mechanism by which GST-P gene is activated together with a crucial transforming gene of liver cells. To distinguish the two alternative possibilities--either the GST-P gene is coactivated with a closely located transforming gene by a cis mechanism or it is activated in trans by a common trans-acting factor--we carried out carcinogenesis experiments using transgenic rats harboring the bacterial chloramphenicol acetyltransferase reporter gene ligated to the upstream regulatory sequence of the GST-P gene. In each of three independent lines tested, liver foci and nodules produced by chemical carcinogens (Solt-Farber procedure) were found to express high levels of chloramphenicol acetyltransferase activity, indicating clearly that the GST-P gene is activated by a trans mechanism during hepatocarcinogenesis.
Atomic force microscopy of long DNA: imaging in air and under water.
Authors: Lyubchenko Y , Shlyakhtenko L , Harrington R , Oden P , Lindsay S
Published: May 2006

We have obtained striking atomic force microscopy images of the intact lambda bacteriophage genome and of several lambda restriction fragments both in air and under water. The DNA is unstained and the images are stable under continuous scanning for up to 30 min. Measured contour lengths of fully imaged restriction fragments and intact lambda DNA are accurate to within a few percent. The key to this development is the use of a process for binding unmodified double-stranded DNA to chemically treated mica surfaces. This procedure leads to strong DNA attachment and yields high-quality images that are stable under repeated scanning, even with the sample submerged in water. This allows normal hydration conditions to be maintained during scanning and in addition leads to a general improvement of image quality. Both the lateral resolution and the contrast increase by a factor of approximately 3 under water.
Schwannoma-derived growth factor must be transported into the nucleus to exert its mitogenic activity.
Authors: Kimura H
Published: May 2006

Schwannoma-derived growth factor (SDGF) is a mitogen and neurotrophic protein which belongs to the epidermal growth factor (EGF) family. There are two basic amino acid clusters in the SDGF molecule which are homologous to the nuclear targeting signal of the simian virus 40-encoded large tumor antigen. Mutational analysis of these clusters showed that they function as nuclear targeting signals, and a gel retardation assay showed that SDGF binds to A+T-rich DNA sequences. Both the wild-type SDGF and a mutant defective in the nuclear targeting signals activate the immediate early genes NGFI-A and c-fos. The wild-type SDGF is a mitogen for Swiss mouse 3T3 fibroblasts, but the mutant defective in the nuclear targeting signals is not mitogenic. Moreover, wild-type SDGF potentiates [3H]thymidine incorporation in NIH mouse 3T3 cells bearing an EGF receptor defective in the kinase domain, whereas the mutant SDGF does not stimulate DNA synthesis. These results suggest that transport into the nucleus is required for SDGF to induce a mitogenic response.
Repression of the herpes simplex virus 1 alpha 4 gene by its gene product occurs within the context of the viral genome and is associated with all three identified cognate sites.
Authors: Michael N , Roizman B
Published: May 2006

The infected cell protein 4 (ICP-4), the major regulatory protein encoded by the a4 gene of the herpes simplex virus 1, binds two sites (alpha 4-1 proximal, alpha 4-1 distal) at the 5'-untranscribed domain and at the transcription initiation site (alpha 4-2) of the alpha 4 gene. Chimeric genes consisting of the 5'-untranscribed and transcribed noncoding domains of the alpha 4 gene fused to the coding sequences of the thymidine kinase gene were mutagenized to abolish binding of ICP-4 by substitution of bases, including the guanines whose methylation interferes with binding of the protein, and recombined into the viral genome. The cytoplasmic RNAs extracted from infected cells treated with cycloheximide, from untreated infected cells maintained for 4 or 8 hr, and from cells infected first with a virus deleted in the alpha 22 gene and 3 hr later with the test viruses were tested in RNase protection assay for amounts of the chimeric gene RNA relative to amounts of alpha 22 gene RNA. We report the following: (i) Mutation of the alpha 4-2 binding site resulted in a 5-to 6-fold higher accumulation of chimeric gene RNA at 4 hr and as much as 15-fold higher accumulation by 8 hr after infection. (ii) Mutations of alpha 4-1 sites by themselves had no effect on RNA accumulation. However, mutagenesis of all three sites significantly increased mRNA amounts above the levels seen in cells infected with alpha 4-2 site mutants. (iii) The mutations have no effect on accumulation of alpha 4 mRNA in the absence of ICP-4 synthesis and, therefore, the mutations had no effect on RNA stability or transcription rate. (iv) Accumulation of alpha 4 mRNA relative to that of alpha 22 mRNA is highest in the presence of cycloheximide and decreases with time after infection. We conclude that ICP-4 autoregulates the transcription of its own gene in infected cells and that binding of ICP-4 to three sites in its promoter is additive in its effects on this process.