bluuebunny/crossref_metadata_2025 huggingface dataset:
Narrowband mid-infrared transmission filtering of a single layer dielectric grating
Authors: Foley J. M. , Young S. M. , Phillips J. D.
Published: August 2013

We experimentally demonstrate a simple narrowband transmission filter using a silicon/air grating, exhibiting broadband high-reflectance between 8 and 14 μm and narrow transmission peaks at slightly off-normal incidence. We explain the response in terms of guided modes in a dielectric slab waveguide, with numerical modal analysis corroborating an intuitive slab waveguide picture and the experimental results. The filtered wavelength depends predictably on the grating period, making wavelength selection straightforward, while the length-scale independent operation principle shows promise for enabling similar filtering capabilities at higher frequencies.
Impact of in-plane spread of wave vectors on spin Hall effect of light around Brewster's angle
Authors: Pan Meng-Meng , Li Yan , Ren Jin-Li , Wang Bo , Xiao Yun-Feng , Yang Hong , Gong Qihuang
Published: August 2013

We theoretically and experimentally demonstrate that displacements induced by the spin Hall effect of light are determined by not only the out-plane but also the in-plane spreads of wave vectors when a P-polarized Gaussian beam reflected from an air-glass interface around the Brewster's angle. The decisive impacts of the wave vector spreads are manifested in the dramatic changes of the reflected light intensity distribution when the polarization or the incident angle of the incident beam slightly varies. The experimental displacement data agree well with the theoretical prediction by taking into account of the in-plane spread of wave-vectors.
The effects of storm-induced events on the seasonal dynamics of epilithic algal biomass in subtropical mountain streams
Authors: Tsai Jeng-Wei , Chuang Yi-Li , Wu Zih-Yi , Kuo Mei-Hwa , Lin Hsing-Juh
Published: August 2013

Information concerning the drivers of seasonal variation in algal biomass in subtropical mountain streams is limited. To identify the drivers of biomass dynamics for epilithic algae, a 20-month study was conducted in mountain streams in Taiwan, an area characterised by different levels of riparian vegetation coverage and agricultural activity, in which a process-based model was optimally fit to field data. We found that episodic typhoon-induced floods were the major drivers shaping the seasonal variations in algal biomass. Flow-induced detachment was frequently observed in periods of higher algal biomass. In contrast, an increased flow stimulated algal growth during periods with slower flow rates. Increased temperature stimulated algal growth at sites with an open canopy cover and higher light availability but constrained biomass at sites with dense canopy shading. Overall, scraper biomass exerted less influence on algal biomass than did environmental factors. The effects of grazing were visible only at the pristine, low-stream-order site in winter. The effects of minimal algal biomass required for recovery was comparable to environmental factors only at sites with intermediate canopy cover, moderate discharge, and higher nutrient concentrations. We suggest that agricultural activity and riparian vegetation can affect epilithic algal biomass in subtropical mountain streams.
Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A
Authors: Jiang Daohua , Zhao Yan , Wang Xianping , Fan Junping , Heng Jie , Liu Xuehui , Feng Wei , Kang Xusheng , Huang Bo , Liu Jianfeng , Zhang Xuejun Cai
Published: August 2013

The major facilitator superfamily (MFS) is the largest family of secondary active transporters and is present in all life kingdoms. Detailed structural basis of the substrate transport and energy-coupling mechanisms of these proteins remain to be elucidated. YajR is a putative proton-driven MFS transporter found in many Gram-negative bacteria. Here we report the crystal structure of
Escherichia coli
YajR at 3.15 Å resolution in an outward-facing conformation. In addition to having the 12 canonical transmembrane helices, the YajR structure includes a unique 65-residue C-terminal domain which is independently stable. The structure is unique in illustrating the functional role of “sequence motif A.” This highly conserved element is seen to stabilize the outward conformation of YajR and suggests a general mechanism for the conformational change between the inward and outward states of the MFS transporters.
A novel regulatory mechanism for whey acidic protein gene expression.
Authors: Chen L H , Bissell M J
Published: August 2013

When primary mouse mammary epithelial cells (PMME) are cultured on a basement membrane type matrix, they undergo extensive morphogenesis leading to the formation of 3-dimensional alveoli-like spherical structures surrounding a closed lumen. We show for the first time that cells cultured on basement membrane-type matrix express high levels of whey acidic protein (WAP) mRNA and secrete the protein into the lumen. The expression of WAP appears to be dependent upon the formation of the alveoli-like spheres: prevention of sphere formation by fixation or drying of the matrix abolishes the expression of WAP. Co-culturing PMME on native and fixed basement membrane matrix indicates that the suppression of WAP expression is dominant, thereby revealing the existence of a diffusible inhibitor(s). The inhibitory activity is present in the conditioned medium of PMME cultured on plastic surface and floating collagen gels, substrata that do not form alveoli and do not allow WAP expression. These findings are consistent with the model that the synthesis, or the action, of the WAP inhibitory factor is regulated by the tissue-like multicellular organization of mammary cells. When PMME do not have correct 3-dimensional structures, one (or more) inhibitor is secreted into the medium which suppresses WAP expression by an autocrine or paracrine mechanism. Nuclear run-on experiments suggest that the suppression of WAP expression is posttranscriptional. These results have obvious bearings on the understanding of the mechanisms by which cell-cell and cell-extracellular matrix interaction regulate tissue specific gene expressions.
Multiple actin-based motor genes in Dictyostelium.
Authors: Titus M A , Warrick H M , Spudich J A
Published: August 2013

Dictyostelium cells, devoid of conventional myosin, display a variety of motile activities, consistent with the presence of other molecular motors. The Dictyostelium genome was probed at low stringency with a gene fragment containing the conserved conventional myosin head domain sequences to identify other actin-based motors that may play a role in the observed motility of these mutant cells. One gene (abmA) has been characterized and encodes a polypeptide of approximately 135 kDa with a head region homologous to other myosin head sequences and a tail region that is not predicted to form either an alpha-helical structure of coiled-coil interactions. Comparisons of the amino acid sequences of the tail regions of abmA, Dictyostelium myosin I, and Acanthamoeba myosins IB and IL reveal an area of sequence similarity in the amino terminal half of the tail that may be a membrane-binding domain. The abmA gene, however, does not contain an unusual Gly, Pro, Ala stretch typical of many of the previously described myosin Is. Two additional genes (abmB and abmC) were identified using this approach and also found to contain sequences that encode proteins with typical conserved myosin head sequences. The abm genes may be part of a large family of actin-based motors that play various roles in diverse aspects of cellular motility.
Single-cell analysis of the mitogen-induced calcium responses of normal and protein kinase C-depleted Swiss 3T3 cells.
Authors: Corps A N , Cheek T R , Moreton R B , Berridge M J , Brown K D
Published: August 2013

Single-cell fluorescence image analysis has been used to characterize the mitogen-induced increases in intracellular free [Ca2+] ([Ca2+]i) in control and protein kinase C-depleted Swiss 3T3 cells. More than 80% of the control cells exhibited fast, transient responses to bombesin, vasopressin, or prostaglandin F2 alpha (PGF2 alpha). In contrast, the [Ca2+]i responses induced by platelet-derived growth factor (PDGF) were markedly more heterogeneous, slower, and often biphasic, with fewer cells (60-70%) responding. The peak [Ca2+]i values obtained in response to each mitogen showed substantial variation between cells. Brief pretreatment of the cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) reduced the [Ca2+]i responses to bombesin, but did not affect the responses to PDGF. Long-term pretreatment of the cells with TPA to down-modulate protein kinase C resulted in substantially prolonged [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha, but had no such effect on the responses to PDGF. We conclude that differences between the [Ca2+]i responses to bombesin and PDGF, previously reported using cell populations, reflect differences occurring in individual cells, and that the [Ca2+]i responses to bombesin, vasopressin, and PGF2 alpha (but not PDGF) are subject to feedback inhibition via protein kinase C.
Biological activity of the mammalian RAP genes in yeast.
Authors: Xu H P , Wang Y , Riggs M , Rodgers L , Wigler M
Published: August 2013

We have screened expression libraries for mammalian cDNAs capable of suppressing defects in ras1- Schizosaccharomyces pombe. Both the RAP1A and RAP1B genes were identified in this manner. They suppress defects in cell morphology and sporulation, although not conjugation. In contrast, RAP genes do not suppress phenotypes in the yeast Saccharomyces cerevisiae that are deficient in RAS. Indeed, expression of RAP1A appears to antagonize the activated S. cerevisiae RAS2val19 gene. These results indicate that RAP proteins can interact with RAS targets, sometimes productively, sometimes nonproductively.
Latency correlates with period in a model for signal-induced Ca2+ oscillations based on Ca2(+)-induced Ca2+ release.
Authors: Dupont G , Berridge M J , Goldbeter A
Published: August 2013

Oscillations in cytosolic Ca2+ develop in a variety of cells after an induction phase, called latency, the duration of which depends on the magnitude of external stimulation. Experiments in hepatocytes indicate that the period and latency of Ca2+ oscillations both decrease as the level of the stimulus increases. We analyze the correlation between period and latency in a model recently proposed for signal-induced Ca2+ oscillations. We show that the linear relationship between period and latency observed in the experiments arises naturally in this model as a result of the mechanism of Ca2(+)-induced Ca2+ release on which it is based.
Transcriptional regulation of early growth response genes in FOS-expressing PC-12 cells.
Authors: Ito E , Nomura N , Narayanan R
Published: August 2013

Deregulated c-fos expression in the rat pheochromocytoma cell line, PC-12, causes pronounced downregulation of nerve growth factor (NGF)-induced c-fos and c-jun activation, accompanied by a block in NGF-induced differentiation of PC-12 cells. The FOS-expressing PC-12 cells were exposed to diverse agents such as NGF, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), dibutyryl cyclic adenosine 3',5' monophosphate (db cAMP), and Ca-ionophore; and the expression of egr-1, c-fos, c-jun, jun-B, and jun-D was analyzed. Pronounced downregulation of c-fos, c-jun, and--to a lesser extent--jun-B was observed on treatment with NGF, bFGF, db cAMP, and Ca-ionophore, whereas EGF-induced activation of these early response genes was not inhibited in FOS-expressing PC-12 cells. Ca-ionophore- and db cAMP-induced egr-1 activation in PC-12 fos cells was completely inhibited. Both parent and PC-12 fos cells expressed similar high basal levels of jun-D, whose expression was the least regulatable by all of these agents. Transfection of fos promoter-chloramphenicol acetyltransferase (promoter-CAT) plasmid into these stable FOS-expressing PC-12 cells revealed that these effects are exerted at the fos promoter level.
CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast.
Authors: Neiman A M , Chang F , Komachi K , Herskowitz I
Published: August 2013

Mutations in either the CDC36 or CDC39 gene cause yeast cells to arrest in G1 of the cell cycle at the same point as treatment with mating pheromone. We demonstrate here that strains harboring temperature-sensitive mutations in CDC36 or CDC39 activate expression of the pheromone-inducible gene FUS1 when shifted to nonpermissive temperature. We show further that cell-cycle arrest and induction of FUS1 are dependent on known components of the mating factor response pathway, the STE genes. Thus, the G1-arrest phenotype of cdc36 and cdc39 mutants results from activation of the mating factor response pathway. The CDC36 and CDC39 gene products behave formally as negative elements in the response pathway: they are required to block response in the absence of pheromone. Epistasis analysis of mutants defective in CDC36 or CDC39 and different STE genes demonstrates that activation requires the response pathway G protein and suggests that CDC36 and CDC39 products may control synthesis or function of the G alpha subunit.
Membrane traffic between secretory compartments is differentially affected during mitosis.
Authors: Kreiner T , Moore H P
Published: August 2013

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.
H-ras(val12) induces cytoplasmic but not nuclear events of the cell cycle in small Xenopus oocytes.
Authors: Johnson A D , Cork R J , Williams M A , Robinson K R , Smith L D
Published: August 2013

Microinjection of H-ras(val12) protein into fully grown Xenopus oocytes has been shown to induce meiotic maturation. In the present study, mRNA encoding the mutant ras protein was injected into both fully grown (stage 6) and growing (stage 4) oocytes. The mRNA induced nuclear breakdown in stage 6 oocytes, as expected. However, the mRNA induced neither nuclear breakdown nor maturation promoting factor when injected into stage 4 oocytes. Instead, the response in stage 4 oocytes included an activation pulse of calcium, cortical granule breakdown, elevation of the vitelline envelope, and abortive cleavage furrows, all of which are characteristics of the activation response in mature eggs. In addition, the injected mRNA led to increased rates of endogenous protein synthesis and the migration of subcortical organelles into the oocyte interior. These observations are discussed relative to the suggestion that oncogenic ras protein leads to an increase in both diacylglycerol and inositol trisphosphate, which then regulate the various cytoplasmic events described.
Mapping of the functional determinants of the integrin beta 1 cytoplasmic domain by site-directed mutagenesis.
Authors: Marcantonio E E , Guan J L , Trevithick J E , Hynes R O
Published: August 2013

We describe here the expression of deletion mutants of the cytoplasmic domain of the avian integrin beta 1 subunit. These mutants, which contain termination codons at positions 767, 776, 791, and 800, were transfected into mouse 3T3 cells to determine which sequences were essential for localization of integrins into focal contact sites. In all cases, high-level expression of the truncated avian integrins was obtained. Heterodimers were formed between the exogenous truncated avian beta 1 subunits and endogenous mouse alpha subunits, and these heterodimers were efficiently exported to the cell surface. The longest truncated beta 1 subunit tested, which is only four amino acids shorter than the wild type, does localize to focal contacts. In contrast, beta 1 subunits with moderately long truncations of the cytoplasmic domain failed to localize to focal contacts, including one which contains the consensus sequence for tyrosine phosphorylation. Surprisingly, a mutant subunit in which the bulk of the cytoplasmic domain was missing (but the segment nearest the membrane including the dibasic residues (RR) remained) did localize weakly to focal contacts. These results implicate the peptide segment nearest to the transmembrane region in focal contact localization. In addition, mutant subunits that included this segment together with a larger portion of the cytoplasmic domain did not localize as well as the shorter form, suggesting that these cytoplasmic domain segments are defective, presumably because of abnormal folding.
Fibronectin receptor overexpression and loss of transformed phenotype in a stable variant of the K562 cell line.
Authors: Symington B E
Published: August 2013

A variant of the K562 erythroleukemia cell line, FA-K562, was selected by cycles of adhesion to solid-phase plasma fibronectin (FN). FA-K562 expresses fourfold more cell-surface alpha 5 beta 1 fibronectin receptor (FNR) than parental K562. In addition to expected differences in adhesion to FN, other differences between FA-K562 and K562 implicate this FNR in the regulation of cell growth and morphology. FA-K562 proliferates slowly in liquid culture, its cloning efficiency in soft agar is only approximately 10% compared with approximately 85% for parental K562, and it is nontumorigenic in nude mice. The reduced soft agar growth potential of FA-K562 involves FNR function, because either glycine-arginine-glycine-aspartate-serine (GRGDS) or monoclonal anti-alpha 5 antibody in the agar medium increased cloning efficiency of FA-K562 about fivefold. Morphologically, FN-adherent FA-K562 become fibroblastoid in appearance, assemble filamentous actin, and differ from K562 in vimentin staining intensity and pattern. Soluble GRGDS peptide inhibits both FA-K562 adhesion to FN and the associated cytoskeletal changes. These findings link the alpha 5 beta 1 FNR to both the transformed phenotype and morphology of FA-K562.
Steroid-induced epithelial-fibroblastic conversion associated with syndecan suppression in S115 mouse mammary tumor cells.
Authors: Leppä S , Härkönen P , Jalkanen M
Published: August 2013

Cell-matrix interactions play an important role in the maintenance of cell shape, supposed to be mediated by the anchorage of cellular cytoskeleton to extracellular matrix via matrix receptors. In this work the expression of one of the known matrix receptors, syndecan, was studied during the hormone-induced change in the phenotype of Shionogi 115 (S115) mouse mammary tumor cells. In the presence of testosterone, when S115 cells express fibroblastic phenotype, they increased their growth rate and became gradually anchorage independent. These cells, however, revealed strong RGDS-dependent binding to fibronectin (FN) but not binding to the heparin-binding domain of FN. Instead, S115 cells growth without testosterone showed epithelial morphology and binding to the heparin-binding domain of FN, suggesting an alteration of syndecan expression in hormone-treated S115 cells. As quantitated by radioimmunoassay and by Western blot, the amounts of both matrix-binding ectodomain of syndecan and syndecan mRNA (2.6 kb) declined in hormone-treated S115 cells. The addition of antiandrogen cyproterone acetate to culture medium opposed the effect of testosterone on syndecan mRNA. We thus propose that the inactivation of syndecan gene and the consequent suppression of syndecan expression is related to the altered adhesion properties, the disappearance of epithelial phenotype, and, on the other hand, to the appearance of transformed-like phenotype in hormone-treated S115 cells.
Epidermal growth factor-stimulated calcium ion transients in individual A431 cells: initiation kinetics and ligand concentration dependence.
Authors: Cheyette T E , Gross D J
Published: August 2013

The A431 epidermoid carcinoma cell line responds to epidermal growth factor (EGF) stimulation with a number of rapid changes, including alterations in free cytosolic calcium ion concentration ([Ca2+]i). At the single cell level, these changes in [Ca2+]i are known to proceed after a clear lag phase subsequent to EGF stimulus (Gonzalez et al., 1988). The present study explores the dependence on EGF concentration of this early [Ca2+]i signal. High levels of EGF (9.0-4.3 nM) produce a [Ca2+]i spike followed by an elevation of [Ca2+]i above basal levels. The time of initiation of the spike varies from 5 to 9 s at the high dose and from 8 to 32 s at the low dose in cells that respond. A lower level of EGF (1.5 nM) produces [Ca2+]i oscillations with no prolonged elevation over basal [Ca2+]i. The initiation of response at this [EGF] ranges from 20 to 410 s. Intermediate stimulus levels generate [Ca2+]i responses that are kinetic admixtures of these limiting responses. A simple model based on the enzymatically amplified signal cascade from ligand binding through Ca2+ release or influx is examined. The model predicts a prolonged lag phase followed by a rapid increase in the [CA2+]i signal that compares favorably with the data reported here.
The promoter of DNA puff gene II/9-1 of Sciara coprophila is inducible by ecdysone in late prepupal salivary glands of Drosophila melanogaster.
Authors: Bienz-Tadmor B , Smith H S , Gerbi S A
Published: August 2013

DNA puffs occur in Sciarid salivary gland chromosomes; they are sites of DNA amplification and intense transcription and they appear to encode secreted structural proteins needed for pupation. In this report we have used P-element transformation of Drosophila to study regulation of a Sciara DNA puff gene. We found that a 718-bp promoter fragment of DNA puff gene II/9-1 from Sciara coprophila directs expression of the bacterial reporter gene CAT in late prepupal salivary glands of transgenic Drosophila melanogaster. The identical tissue and analogous stage specificity indicate that some aspects of the ecdysone response are evolutionarily conserved between Drosophila and Sciara. When transgenic salivary glands are cultured in vitro, CAT activity is rapidly induced by ecdysone, suggesting direct control of gene expression by the ecdysone receptor. Putative stage-specific factors limit expression of the chimeric Sciara-CAT gene in transgenic Drosophila to late prepupae but not to third instar larvae when ecdysone titers are also high.
Endothelin-1 activates phospholipase D and thymidine incorporation in fibroblasts overexpressing protein kinase C beta 1.
Authors: Pai J K , Dobek E A , Bishop W R
Published: August 2013

Endothelins (ETs) are a family of extremely potent vasoconstrictor peptides. In addition, ET-1 acts as a potent mitogen and activates phospholipase C in smooth muscle cells and fibroblasts. We examined the effects of ET-1 on phosphatidylcholine (PC) metabolism and thymidine incorporation in control Rat-6 fibroblasts and in cells that overexpress protein kinase C beta 1 (PKC). PC pools were labeled with [3H]myristic acid, and formation of phosphatidylethanol (PEt), an unambiguous marker of phospholipase D (PLD) activation, was monitored. ET-1 stimulated much greater PEt formation in the PKC overexpressing cells. ET-1 action was dose-dependent with a half-maximal effect at 1.0 x 10(-9) M. With increasing ethanol concentrations, [3H]PEt formation increased at the expense of [3H]phosphatidic acid (PA). Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA accumulation and decreased [3H]diacylglycerol (DAG) formation. These data are consistent with the formation of [3H]DAG from PC by the sequential action of PLD and PA phosphohydrolase. Phorbol esters are known to stimulate thymidine incorporation and PLD activity to a greater extent in PKC overexpressing cells than in control cells. ET-1 also stimulates thymidine incorporation to a greater extent in the PKC overexpressing cells. The effect of ET-1 on thymidine incorporation into DNA in the overexpressing cells was also dose-dependent with a half-maximal effect at 0.3 x 10(-9) M. Enhanced PLD activity induced by ET-1 in the overexpressing cells may contribute to the mitogenic response, especially in light of a possible role of the PLD product, PA, in regulation of cell growth.
Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme.
Authors: Hellmich M R , Strumwasser F
Published: August 2013

An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.
ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.
Authors: Lee H C , Aarhus R
Published: August 2013

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.
Transduction of the bradykinin response in human fibroblasts: prolonged elevation of diacylglycerol level and its correlation with protein kinase C activation.
Authors: Etscheid B G , Albert K A , Villereal M L , Palfrey H C
Published: August 2013

Stimulation of quiescent human fibroblasts with the peptide mitogen bradykinin (BK) led to a biphasic elevation in cellular 1,2-diacylglycerol (DAG), as estimated by either measurement of total DAG mass or [3H]arachidonate incorporation. A rapid initial transient that peaked 15 s after BK addition was followed by a decline to near basal levels then a second rise to a plateau phase during which DAG levels remained elevated for less than or equal to 45 min. The source of the initial DAG transient appeared to be primarily polyphosphoinositides as these phospholipids were rapidly hydrolyzed after BK addition. This transient correlates well temporally with previous observations of the kinetics of inositol trisphosphate accumulation and intracellular free [Ca2+] observed in the same cells. Cultures preincubated with [3H]myristic acid incorporated label predominantly into the phosphatidylcholine (PC) pool. Subsequent addition of BK under these conditions caused only a relatively slow accumulation of [3H]DAG to a plateau level, without an initial transient. Together with the observation that PC was found to decrease upon BK stimulation, these observations suggest that the late phase of DAG accumulation may involve breakdown of other phospholipids including PC. To investigate the consequences of DAG elevation we examined the phosphorylation of an acidic 80 kDa protein, whose phosphorylation is solely dependent on the activation of protein kinase C (PK-C). The 80 kDa fibroblast protein could be immunoprecipitated by an antibody to bovine brain "myristoylated and alanine-rich C-kinase substrate" (MARCKS) and phosphopeptide maps of brain and fibroblast MARCKS were similar. Stimulation of [32P]-prelabeled fibroblasts with serum, BK, vasopressin, or 12-O-tetradecanoyl phorbol acetate, but not epidermal growth factor or calcium ionophores, resulted in the rapid phosphorylation of MARCKS. With BK or serum this phosphorylation showed an initial transient peak at less than 1 min then rose again to a plateau level that was sustained for less than or equal to 45 min. Removal of BK resulted in a rapid decline in MARCKS phosphorylation. These studies show that the biphasic DAG signal in BK-stimulated human fibroblasts correlates well with the state of activation of PK-C. However, the persistent activation of PK-C does not appear to require continued high levels of Ca2+.
Differential dependence of early and late increases in 1,2-diacylglycerol on the presence of catalytically active alpha-thrombin: evidence for regulation at the level of 1,2-diacylglycerol generation.
Authors: Rangan L A , Wright T M , Raben D M
Published: August 2013

alpha-Thrombin stimulates a biphasic increase in cellular 1,2-diacylglycerol mass in quiescent IIC9 fibroblasts. This report describes the use of hirudin, a high-affinity inhibitor of alpha-thrombin that renders it catalytically inactive, to investigate the dependence of elevated 1,2-diacylglycerol levels on the presence of catalytically active alpha-thrombin. When cultures were incubated in the presence of alpha-thrombin, 1,2-diacylglycerol levels remained elevated for greater than or equal to 4 h. Inactivation of alpha-thrombin after 15 s did not alter the kinetics of 1,2-diacylglycerol formation occurring over the next 1 h. However, sustained (1-4 h) increases in this lipid were eliminated. Inactivation of alpha-thrombin after 1 h of stimulation resulted in 1) an immediate and reversible decline in 1,2-diacylglycerol levels, 2) elimination of the sustained phase of 1,2-diacylglycerol production, 3) inhibition of the alpha-thrombin-stimulated generation of choline metabolites, and 4) a blunted mitogenic response to alpha-thrombin. These data indicate that early (0-1 h) and late (1-4 h) increases in 1,2-diacylglycerol are differentially dependent on the presence of catalytically active alpha-thrombin. Furthermore, sustained increases in 1,2-diacylglycerol in response to alpha-thrombin are regulated at least in part at the level of generation (via phosphatidylcholine hydrolysis). Our results also support a role for sustained 1,2-diacylglycerol levels in the mitogenic response.
Evidence that interleukin-1 and phorbol esters activate NF-kappa B by different pathways: role of protein kinase C.
Authors: Bomsztyk K , Rooney J W , Iwasaki T , Rachie N A , Dower S K , Sibley C H
Published: August 2013

Nuclear factor kappa B (NF-kappa B) is a ubiquitous transcription factor that affects expression of many genes, including immunoglobulin kappa (kappa), the interleukin-2 receptor alpha chain, and two genes in HIV-1. NF-kappa B can be activated by a number of stimuli, including pharmacological stimulation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and treatment in vitro with either protein kinase C or protein kinase A. This has lead to the proposal that these kinases are key enzymes in the physiological activation of NF-kappa B as well. We have used a murine B cell line, 70Z/3, and T cell line, EL-4 6.1 C10, to study the activation of NF-kappa B by two physiological activators, interleukin-1 alpha (IL-1) and lipopolysaccharide (LPS). There are four reasons to propose that these agents activate pathways that do not include protein kinase C as a major component in these cell lines. First, the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) strongly inhibited PMA-induced activation of NF-kappa B in 70Z/3 cells but had no effect on NF-kappa B activated by IL-1 or LPS. Second, depletion of protein kinase C by prolonged growth of 70Z/3 in PMA abrogated the capacity of the cells to activate NF-kappa B in response to further PMA treatment. However, these same cells activated NF-kappa B normally after either IL-1 or LPS treatment. Third, IL-1 effectively activated NF-kappa B in EL-4 6.1 C10 cells, but PMA did not. Fourth, interferon-gamma is a potent activator of protein kinase C in 70Z/3 cells, but is completely inactive in the mobilization of NF-kappa B. These results suggest that the physiological inducers IL-1 and LPS activate NF-kappa B by pathways independent of protein kinase C in both 70Z/3 and EL-4 6.1 C10 cells.
Genetic fine-structural analysis of the Saccharomyces cerevisiae alpha-pheromone receptor.
Authors: Konopka J B , Jenness D D
Published: August 2013

The alpha-pheromone receptor encoded by the STE2 gene contains seven potential transmembrane domains. Its ability to transduce the pheromone signal is thought to require the action of a G protein. As an initial step toward defining the structural features of the receptor required for its activity, we examined the phenotypic consequences of linker insertion mutations (12 bp) at 10 different sites in the STE2 gene. Three mutant classes, which correspond to three different regions of the receptor protein, were observed. 1) The two mutants affecting the C-terminal region (C-terminal mutants) were essentially wild type for mating efficiency, pheromone binding, and pheromone sensitivity. 2) The three mutants in the N-terminus mated with reduced efficiency, showed reduced pheromone binding capacity, and were partially defective in pheromone induction of agglutinin production and cell division arrest. Increased gene dosage of these N-terminal alleles suppressed their mutant phenotypes, whereas the sst2-1 mutation, which blocks adaptation to pheromone, did not result in suppression. Thus, the N-terminal mutants were apparently limited by receptor production, but not by the adaptation function SST2. 3) The five mutants in the central region containing the seven transmembrane segments (central mutants) were completely defective for mating and did not respond to pheromone, but could be distinguished by their ability to bind pheromone. Inserts in or near transmembrane domains 2 and 4 blocked pheromone binding, whereas inserts into transmembrane domains 1, 5, and 6 retained partial pheromone binding activity even though they failed to transduce a signal. The central mutants were not suppressed by increased gene dosage, and one mutant (ste2-/101) was partially suppressed by sst2-1. Furthermore, the central core mutants were also distinguished from one another in that three of the five mutants were able to partially complement the temperature sensitivity of ste2-3.
Localization of transforming growth factor-beta 1 in mitochondria of murine heart and liver.
Authors: Heine U I , Burmester J K , Flanders K C , Danielpour D , Munoz E F , Roberts A B , Sporn M B
Published: August 2013

Using both electron microscopic immunohistochemistry and cell fractionation techniques, we show that transforming growth factor-beta 1 (TGF-beta 1) is found in mitochondria of rat and mouse cardiac myocytes and rat hepatocytes. Four different polyclonal antibodies, raised against various epitopes encompassing the mature portion of the TGF-beta 1 molecule as well as the pro-region of its precursor, were used for the electron microscopy studies. The localization of TGF-beta 1 in mitochondria was confirmed by detection of the native peptide in mitochondria isolated from rat heart and liver; the majority of native TGF-beta 1 found in liver homogenates was recovered in highly pure mitochondrial fractions. The functional role of TGF-beta in the mitochondrion is unknown at present.
Influenza virus hemagglutinin trimers and monomers maintain distinct biochemical modifications and intracellular distribution in brefeldin A-treated cells.
Authors: Russ G , Bennink J R , Bächi T , Yewdell J W
Published: August 2013

Brefeldin A (BFA) induces the retrograde transport of proteins from the Golgi complex (GC) to the endoplasmic reticulum (ER). It is uncertain, however, whether the drug completely merges the ER with post-ER compartments, or whether some of their elements remain physically and functionally distinct. We investigated this question by the use of monoclonal antibodies specific for monomers and trimers of the influenza virus hemagglutinin (HA). In untreated influenza virus-infected cells, monomers and trimers almost exclusively partition into the ER and GC, respectively. In BFA-treated cells, both monomers and trimers are detected in the ER by immunofluorescence. Cell fractionation experiments indicate, however, that whereas HA monomers synthesized in the presence of BFA reside predominantly in vesicles with a characteristic density of the ER, HA trimers are primarily located in lighter vesicles characteristic of post-ER compartments. Biochemical experiments confirm that in BFA-treated cells, trimers are more extensively modified than monomers by GC-associated enzymes. Additional immunofluorescence experiments reveal that in BFA-treated cells, HA monomers can exist in an ER subcompartment less accessible to trimers and, conversely, that trimers are present in a vesicular compartment less accessible to monomers. These findings favor the existence of a post-ER compartment for which communication with the ER is maintained in the presence of BFA and suggest that trimers cycle between this compartment and the ER, but have access to only a portion of the ER.
Carboxy-terminal truncations of epidermal growth factor (EGF) receptor affect diverse EGF-induced cellular responses.
Authors: Li W , Hack N , Margolis B , Ullrich A , Skorecki K , Schlessinger J
Published: August 2013

The binding of epidermal growth factor (EGF) to its receptor induces tyrosine phosphorylation of phospholipase C gamma (PLC gamma), which appears to be necessary for its activation leading to phosphatidyl inositol (PI) hydrolysis. Moreover, EGF-receptor (EGF-R) activation and autophosphorylation results in binding of PLC gamma to the tyrosine phosphorylated carboxy-terminus of the receptor. To gain further insights into the mechanisms and interactions regulating these processes, we have analyzed transfected NIH-3T3 cells expressing two EGF-R carboxy-terminal deletion mutants (CD63 and CD126) with reduced capacity to stimulate PI hydrolysis, Ca2+ rises, and DNA synthesis. In fact, the CD126 mutant lacking 126 carboxy-terminal amino acids, including four tyrosine autophosphorylation sites, was unable to stimulate PI hydrolysis or Ca2+ rise in response to EGF. Surprisingly, EGF binding to the cell lines expressing CD63 or CD126 mutants was followed by similar stimulation of tyrosine phosphorylation of PLC gamma. Our results suggest that although necessary, tyrosine phosphorylation of PLC gamma may not be sufficient for stimulation and PI hydrolysis. It is clear, however, that the carboxy-terminal region of EGF-R is involved in regulation of interactions with cellular targets and therefore plays a crucial role in postreceptor signaling pathways.
Down-regulation of cellular platelet-derived growth factor receptors induced by an activated neu receptor tyrosine kinase.
Authors: Lehtola L , Nistér M , Hölttä E , Westermark B , Alitalo K
Published: August 2013

The functional integration of growth factor signaling occurs at several levels in target cells. One of the most proximal mechanisms is receptor transmodulation, by which one activated receptor can regulate the expression of other receptors in the same cells. Well-established transregulatory loops involve platelet-derived growth factor (PDGF) down-regulation of epidermal growth factor (EGF) receptors and beta-type transforming growth factors modulation of PDGF receptors. We have studied the relationship between neu tyrosine kinase activation and the expression of the PDGF receptors in transfected NIH/3T3 cells. Expression of the neu oncogene, but not of the neu proto-oncogene, was associated with a decrease of PDGF alpha- and beta-receptors on the cell surface, as measured by [125-I]PDGF-AA and -BB binding. These results were corroborated by metabolic labeling and immunoprecipitation of the PDGF beta-receptors. PDGF alpha- and beta-receptor mRNAs were strongly decreased in the neu oncogene-transformed cells in comparison with control cells expressing the neu proto-oncogene. Down-regulation of the PDGF receptors and their mRNAs was also observed after EGF treatment of cells expressing a chimeric EGF receptor/neu receptor, where the neu tyrosine kinase is activated by EGF binding. These results show that the neu tyrosine kinase can down-modulate PDGF receptor expression, and the effect is mediated via decreased PDGF receptor mRNA levels.
Regulation of Cl/HCO3 exchange in gastric parietal cells.
Authors: Thomas H A , Machen T E
Published: August 2013

Microspectrofluorimetry of the fluorescent indicators 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein and 6-methoxy-N-(3-sulfopropyl)-quinolinium was used to measure intracellular pH (pHi), intracellular Cl (Cli), and transmembrane fluxes of HCO3 and Cl in single parietal cells (PC) in isolated rabbit gastric glands incubated in HCO3/CO2-buffered solutions. Steady-state pHi was 7.2 in both resting (50 microM cimetidine) and stimulated (100 microM histamine) PCs. Transmembrane anion (HCO3 or Cl) flux rates during Cl removal from or readdition to the perfusate were the same in resting and stimulated PCs. These rates increased at alkaline pHi, though this pHi dependence was small in the physiological range. Maximum velocity (Vmax) for Cl influx or HCO3 efflux was 80-110 mM/min at pHi 7.6-7.8, and the Km for extracellular concentrations of Cl (Clo) was 25 mM; in the physiological range (pHi 7.1-7.3), Vmax for anion fluxes was approximately 50 mM/min. Steady-state Cli in the unstimulated PC was 62 +/- 5 mM, but on histamine stimulation, Cli decreased rapidly to 25 mM and then increased back to a steady-state level of 44 mM. HCO3 fluxes due to Cl removal or readdition were completely blocked by 0.5 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS), but Cl fluxes were only inhibited by 80%. H2DIDS did not inhibit the decrease in Cli that occurred with histamine treatment. Diphenylamine carboxylate (0.5 mM) inhibited Cl flux by only 50% and caused no additional inhibition of Cl flux when used in conjunction with H2DIDS. Transmembrane anion fluxes during solution Cl removal or readdition occurred 80% through the anion exchanger at the basal membrane and 20% through other pathway(s), presumably the Cl channel in the apical membrane. We conclude that the increase in transport activity via the Cl/HCO3 exchanger that occurs during histamine-induced increases in HCl secretion is due mostly to the decrease in Cli. In the resting cell with Cli = 62 mM, Clo = 120 mM, pHi = 7.2, and extracellular pH = 7.4, the anion exchanger is poised near its thermodynamic equilibrium. During histamine stimulation Cli drops from 62 mM to 44 mM, the thermodynamic equilibrium of the anion exchanger at the basolateral membrane is disturbed, and the anion exchanger then exchanges cellular HCO3 for extracellular Cl. Cli serves a crucial regulatory role in stimulus-secretion coupling in the PC.
Yeast proteins associated with microtubules in vitro and in vivo.
Authors: Barnes G , Louie K A , Botstein D
Published: August 2013

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.