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ABSTRACTCells sense and appropriately respond to the physical conditions and availability of nutrients in their environment. This sensing of the environment and consequent cellular responses are orchestrated by a multitude of signaling pathways and typically involve changes in transcription and metabolism. Recent discoveries suggest that the signaling and transcription machineries are regulated by signals which are derived from metabolism and reflect the metabolic state of the cell. Acetyl coenzyme A (CoA) is a key metabolite that links metabolism with signaling, chromatin structure, and transcription. Acetyl-CoA is produced by glycolysis as well as other catabolic pathways and used as a substrate for the citric acid cycle and as a precursor in synthesis of fatty acids and steroids and in other anabolic pathways. This central position in metabolism endows acetyl-CoA with an important regulatory role. Acetyl-CoA serves as a substrate for lysine acetyltransferases (KATs), which catalyze the transfer of acetyl groups to the epsilon-amino groups of lysines in histones and many other proteins. Fluctuations in the concentration of acetyl-CoA, reflecting the metabolic state of the cell, are translated into dynamic protein acetylations that regulate a variety of cell functions, including transcription, replication, DNA repair, cell cycle progression, and aging. This review highlights the synthesis and homeostasis of acetyl-CoA and the regulation of transcriptional and signaling machineries in yeast by acetylation.

Concrete pavement thickness data were collected from sections of two concrete pavements with a nondestructive testing (NDT) device and by measuring concrete core thickness. The NDT device allowed thickness measurements every 15 ft compared with 1,000 ft for each core. Comparison of the core and NDT data showed that core thickness data do not capture the extreme peaks and valleys of pavement thickness variation. Further analysis of the core and NDT thickness data with the Minnesota Department of Transportation's concrete pavement thickness specifications highlighted that contractor pay deductions based on thickness deficiencies were not realized. Autocorrelation analysis of the data showed that concrete pavement thickness variation is not random but is correlated to thicknesses of lag between 30 and 270 ft. This correlation lag is a starting point for answering the question, how often must a thickness measurement be taken to accurately characterize the thickness variation of a concrete pavement?

The SHRP 2 R21 project on composite pavement investigated the durability of various mixtures of portland cement concrete (PCC) used in the construction of a two-layer composite PCC pavement. Project consultants in Europe, where composite PCC over PCC pavement was more common than in the United States, advised the R21 research team to consider using the CIF (capillary suction, internal damage, and freeze-thaw) standard of the International Union of Laboratories and Experts in Construction Materials, Systems, and Structures (RILEM), Paris, rather than the familiar ASTM standards. As a result, the R21 project adopted the RILEM CIF standard to evaluate the freeze-thaw durability and salt scaling resistance of concretes. The research also explored a modified RILEM CIF test (using pure water instead of a sodium chloride solution in scaling tests) alongside the standard RILEM CIF tests. The paper describes this experience to expose other institutions and agencies in the United States to the RILEM standards for the freeze-thaw durability and salt scaling resistance testing of concretes.

Pervious concrete pavement must perform as both a stormwater management tool and a pavement. While much research has focused on porosity and permeability of pervious concrete, few guidelines exist for structural analysis and design of such pavement. In this paper, an existing pervious concrete pavement was analyzed for stiffness and fatigue by means of Westergaard's theory of a medium-thick plate on a Winkler foundation. The stiffness evaluation compared the responses of pervious and conventional concrete pavements to falling weight deflectometer stresses and to models created in ISLAB2005. Although pervious concrete had a lower modulus of elasticity than conventional concrete pavement, Westergaard's model adequately described the behavior of pervious pavements. Additionally, a fatigue analysis of pervious concrete was completed through use of the StreetPave fatigue model.

State departments of transportation began using recycled concrete aggregate (RCA) as aggregate in portland cement concrete pavement in the United States in the late 1970s. Although RCA is rarely used in current U.S. rigid pavement slabs, the impetus for its continued use remains the same: a lack of landfill space, a shortage of nearby quality natural aggregates, or both. However, as American pavement engineers and researchers place a greater emphasis on sustainable, reusable roadways, the status quo for RCA in American roadways should be reconsidered along with these new priorities. This study proposes to revisit the use of recycled concrete as aggregate in rigid pavement slabs by using overlooked research to address the concerns that prevented the wide-scale adoption of recycled concrete as an aggregate in pavement slabs by state departments of transportation. Experiences encountered in countries (mostly restricted to Europe) where the use of RCA in rigid pavement is more common are also described. New opportunities for the use of RCA as a structural component in pavement concretes are detailed.

ABSTRACTPhosphatidylinositol phosphates are involved in signal transduction, cytoskeletal organization, and membrane trafficking. Inositol polyphosphates, produced from phosphatidylinositol phosphates by the phospholipase C-dependent pathway, regulate chromatin remodeling. We used genome-wide expression analysis to further investigate the roles of Plc1p (phosphoinositide-specific phospholipase C in Saccharomyces cerevisiae) and inositol polyphosphates in transcriptional regulation. Plc1p contributes to the regulation of approximately 2% of yeast genes in cells grown in rich medium. Most of these genes are induced by nutrient limitation and other environmental stresses and are derepressed in plc1? cells. Surprisingly, genes regulated by Plc1p do not correlate with gene sets regulated by Swi/Snf or RSC chromatin remodeling complexes but show correlation with genes controlled by Msn2p. Our results suggest that the increased expression of stress-responsive genes in plc1? cells is mediated by decreased cyclic AMP synthesis and protein kinase A (PKA)-mediated phosphorylation of Msn2p and increased binding of Msn2p to stress-responsive promoters. Accordingly, plc1? cells display other phenotypes characteristic of cells with decreased PKA activity. Our results are consistent with a model in which Plc1p acts together with the membrane receptor Gpr1p and associated Gaprotein Gpa2p in a pathway separate from Ras1p/Ras2p and converging on PKA.

Phosphatidylinositol phosphates are involved in signal transduction, cytoskeletal organization, and membrane trafficking. Inositol polyphosphates, produced from phosphatidylinositol phosphates by the phospholipase C-dependent pathway, regulate chromatin remodeling. We used genome-wide expression analysis to further investigate the roles of Plc1p (phosphoinositide-specific phospholipase C in Saccharomyces cerevisiae) and inositol polyphosphates in transcriptional regulation. Plc1p contributes to the regulation of approximately 2% of yeast genes in cells grown in rich medium. Most of these genes are induced by nutrient limitation and other environmental stresses and are derepressed in plc1 cells. Surprisingly, genes regulated by Plc1p do not correlate with gene sets regulated by Swi/Snf or RSC chromatin remodeling complexes but show correlation with genes controlled by Msn2p. Our results suggest that the increased expression of stress-responsive genes in plc1 cells is mediated by decreased cyclic AMP synthesis and protein kinase A (PKA)-mediated phosphorylation of Msn2p and increased binding of Msn2p to stress-responsive promoters. Accordingly, plc1 cells display other phenotypes characteristic of cells with decreased PKA activity. Our results are consistent with a model in which Plc1p acts together with the membrane receptor Gpr1p and associated Gprotein Gpa2p in a pathway separate from Ras1p/Ras2p and converging on PKA.

The article offers information about the family of Frederick Lanz and Magdalene Ruchte Lanz. Frederick and Magdalene went to the U.S. with their nine children in the U.S. in 1850. He was, from a religious standpoint, opposed to all war. He married into a family in Switzerland. A charts is presented that shows details about the family of Frederick.

In Saccharomyces cerevisiae, many osmotically inducible genes are regulated by the Sko1p-Ssn6p-Tup1p complex. On osmotic shock, the MAP kinase Hog1p associates with this complex, phosphorylates Sko1p, and converts it into an activator that subsequently recruits Swi/Snf and SAGA complexes. We have found that phospholipase C (Plc1p encoded by PLC1) is required for derepression of Sko1p-Ssn6p-Tup1p–controlled osmoinducible genes upon osmotic shock. Although plc1Δ mutation affects the assembly of the preinitiation complex after osmotic shock, it does not affect the recruitment of Hog1p and Swi/Snf complex at these promoters. However, Plc1p facilitates osmotic shock–induced recruitment of the SAGA complex. Like plc1Δ cells, SAGA mutants are osmosensitive and display compromised expression of osmotically inducible genes. The reduced binding of SAGA to Sko1p-Ssn6p-Tup1p–repressed promoters in plc1Δ cells does not correlate with reduced histone acetylation. However, SAGA functions at these promoters to facilitate recruitment of the TATA-binding protein. The results thus provide evidence that Plc1p and inositol polyphosphates affect derepression of Sko1p-Ssn6p-Tup1p–controlled genes by a mechanism that involves recruitment of the SAGA complex and TATA-binding protein.

This paper describes the design, manufacture and properties of membrane structures with capacitive detection of deflection. The purpose of the study is to find a sensing organ to be used in an integrated tactile sensor for medical applications. The designed structures are implemented in a commercial CMOS process with a small number of post-micromachining steps. The process used offers two possibilities to achieve capacitors with a moving membrane, namely sacrificial etching of aluminium or silicon dioxide. Both methods have their strengths, but also limitations. These issues, which are strongly affecting the design flow, are discussed together with measurement results of the fabricated structures. The membranes from sacrificial oxide etching showed reliable dielectric gap formation. However, the large variation in their stress properties together with limited available pitch rendered them inferior to membranes from sacrificial aluminium etching. Moreover, the aluminium release etch can be achieved with a simpler etch-opening formation procedure than the oxide release etch.

Forestry is considered the sustainable utilization of forests. Rapid changes in society have determined the future development of research and education including forestry and forestry science. Due to the wide range of human demands on forests, universities and other institutions need to recognize the role of forestry in society and organize specific courses for educating professionals. Some international processes and changes such as globalization and integration impose new requirements related to the role, organization and function of education at the European level. As the result of these global initiatives, there have been many reforms of the education system in Central and Eastern European countries (CEECs). This paper presented provides some general information on forestry and its respective research and education of CEECs in transition over the last decade. Using an example the author's country, the Czech Republic, this paper examines the basic problems that foresters have faced since the creation of the market economy in the beginning of the 1990s. Important problems as well as the challenges to find solutions are mentioned. This concerns those countries joining the EU this year, as well as those undertaking the same path. A seminar on the challenges of forest science 5 years ago, which underlined some of the most important problems, was dedicated to forestry in developing countries. However, there is one question remaining, “What has changed since that time?”.

Providing education and development to patient care personnel with minimal disruption to patient care activities is a daily challenge to staff educators. This article describes how a small nursing development department planned and coordinated the orientation to a new facility and the education and training on new equipment to over 1,000 nurses over a period of several months. The strategies used at this large, urban, academic medical center can be applied to other healthcare facilities.

To investigate the effects of neurotrophin‐3 (NT‐3) on postnatal proprioceptive neurons and their targets, transgenic mice were generated that use the myosin light chain 1 (mlc) promoter to overexpress NT‐3 in skeletal muscle. Ribonuclease protection assays revealed that NT‐3 overexpression in hindlimb skeletal muscle began at embryonic day 14 (E14) and continued throughout adulthood. Overexpression of NT‐3 during late embryogenesis resulted in increased numbers of large sensory and small fusimotor axons. Within a week of birth, mlc/NT‐3mice retract their limbs to the torso when lifted by the tail. Footprint analysis revealed that mlc/NT‐3mice had significant abnormalities in their gait compared with wild‐types. Beam walking and rotorod analysis confirmed the poor limb control by mlc/NT‐3mice. These locomotive deficits progressively worsened with age and were likely related to the formation of morphologically abnormal muscle spindles. The most common spindle anomaly was the presence of excessive intrafusal bag fibers within individual muscle spindles. To assess the role of NT‐3 in recovery from nerve injury, sciatic nerve crushes were performed in young adult mice. Two days after injury, mlc/NT‐3mice displayed significantly improved sciatic functional indexes and a significant increase in muscle spindles that remained associated with axons. The latter finding suggests that excess NT‐3 in muscle may retard the degeneration of proprioceptive axons after nerve crush. Long‐term survival after nerve injury in mlc/NT‐3mice did not induce further changes in spindle number or morphology. These findings demonstrate that, in addition to promoting embryonic proprioceptive neuron survival, postnatal overexpression of NT‐3 in muscle leads to abnormal spindle formation and deficits in locomotive control. However, our results also show that NT‐3 may be therapeutic for proprioceptive axons immediately after nerve injury by delaying axon degeneration. J. Comp. Neurol. 432:244–258, 2001. © 2001 Wiley‐Liss, Inc.

ABSTRACTThe budding yeast PLC1gene encodes a homolog of the d isoform of mammalian phosphoinositide-specific phospholipase C. Here, we present evidence that Plc1p associates with the kinetochore complex CBF3. This association is mediated through interactions with two established kinetochore proteins, Ndc10p and Cep3p. We show by chromatin immunoprecipitation experiments that Plc1p resides at centromeric loci in vivo. Deletion of PLC1, as well asplc1mutations which abrogate the interaction of Plc1p with the CBF3 complex, results in a higher frequency of minichromosome loss, nocodazole sensitivity, and mitotic delay. Overexpression of Ndc10p suppresses the nocodazole sensitivity of plc1mutants, implying that the association of Plc1p with CBF3 is important for optimal kinetochore function. Chromatin extracts fromplc1? cells exhibit reduced microtubule binding to minichromosomes. These results suggest that Plc1p associates with kinetochores and regulates some aspect of kinetochore function and demonstrate an intranuclear function of phospholipase C in eukaryotic cells.

The topography of formation and migration of phosphatidic acid (PA) in the transverse plane of rat liver mitochondrial outer membrane (MOM) were investigated. Isolated mitochondria and microsomes, incubated with sn-glycerol 3-phosphate and an immobilized substrate palmitoyl-CoA-agarose, synthesized both lyso-PA and PA. The mitochondrial and microsomal acylation of glycerophosphate with palmitoyl-CoA-agarose was 80-100% of the values obtained in the presence of free palmitoyl-CoA. In another series of experiments, both free polymyxin B and polymyxin B-agarose stimulated mitochondrial glycerophosphate acyltransferase activity approximately 2-fold. When PA loaded mitochondria were treated with liver fatty acid binding protein, a fifth of the phospholipid left the mitochondria. The amount of exportable PA reduced with the increase in the time of incubation. In another approach, PA-loaded mitochondria were treated with phospholipase A(2). The amount of phospholipase A(2)-sensitive PA reduced when the incubation time was increased. Taken together, the results suggest that lysophosphatidic acid (LPA) and PA are synthesized on the outer surface of the MOM and that PA moves to the inner membrane presumably for cardiolipin formation.

Solutions of [35S]bromosul-phthalein ([35S]BSP) in heparinized canine plasma, in the proportions established in vivoafter injecting BSP intravenously to test liver function, were ultracentrifuged at 226,000 g for 24 hr at 5°. Protein-free supernatant was replaced by Krebs-Ringer buffer (pH 7.40), the protein sediment resuspended, and the mixture recentrifuged. That process was repeated several times, and the radioactivity of each resulting supernatant was measured. Since [35S]BSP could not be adequately purified, supernatant radioactivities reflected both [35S]BSP and radioim-purity. Therefore, a model was derived that (i) interpreted the rapid decrease in supernatant radioactivities of initial centrifugations and the gradual fall thereafter; and (ii) allowed us to determine picomoles of non-protein-bound [35S]BSP. Results indicated that only 0.053% (SD 0.0013%) of BSP in our system was not protein-bound.

The Brno municipal waste incinerator (MWI), a Project TOCOEN model source of PAHs, PCBs, PCDDs/Fs was observed and the soil contamination in its surroundings was determined. The total observed concentrations of PAHs, PCBs PCDDs/Fs were found in the ranges of 369.2 to 5,077.9 ng•g-1, 2.0 to 111 ng•g-1, and 0.018 to 0.140 pg•g-1 TEQ, respectively.

As a part of cooperation between Chemical Time Bombs Program and Project TOCOEN, the contamination of sediments in Moravian and Slovakian part of Danube river catchment area by PAHs, PCBs, PCDDs/Fs was determined. The total contents of PAHs ranged between 25.5 and 4,690.3 ng•g-1, PCBs between 4.9 and 232 ng•g-1 and PCDDs/Fs were detected at one site in concentration 0.0005 pg•g-1 TEQ.

We constructed mutants of the prototypical, nuclear-accumulating protein nucleoplasmin and used them in bothin vivoandin vitronuclear transport assays to search for transport-influencing domains distinct from this protein's recognized nuclear localization sequence. We identified the polyglutamic acid tract on the amino flank of the nuclear localization sequence as being involved in two stages of nuclear transport. This poly-glu tract is required for the facilitated translocation of nucleoplasmin through the nuclear pore complex, and it also enhances the subsequent binding of nucleoplasmin within the nucleus.

This article highlights recent studies into the roles of the G proteins in two processes required for axon growth: growth cone motility and vesicular transport. Heterotrimeric G proteins are involved in growth cone motility, but their precise roles remain controversial. The small GTP-binding proteins are clearly established regulators of the actin cytoskeleton in fibroblasts, and their functions are just beginning to be explored in the growth cone. Members of the rab subfamily of small GTP-binding proteins have been shown to regulate vesicular transport in every cell type examined thus far, including neurons.

A soluble fragment of YCK2, a casein kinase-1 isoform from Saccharomyces cerevisiae, has been purified and characterized in vitro. The procedure enriches enzyme activity to a final specific activity of 4.7 µmol min−1 mg−1 (when assayed with casein as substrate). Structural analysis reveals that the preparation arises from N-terminal modification and C-terminal proteolysis of the initially synthesized 546-residue protein, consisting of residues 2-495 ± 1. Kinetic analysis demonstrates that YCK2 is similar to casein kinase-1 isolated from other organisms in its inability to use GTP as nucleotide substrate, in its sensitivity to heparin and ribofuranosylbenzimidazole inhibitors, and in its peptide substrate selectivity. The enzyme is unusual, however, in that it is insensitive to the potent mammalian casein kinase-1 inhibitor N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide.Copyright 1993, 1999 Academic Press

Ultraviolet emission lines in supernova remnants (SNRs) provide important clues to the shock velocities, densities, grain destruction, and thermal structure of the cooling regions in these objects. However, several of the brightest ultraviolet emission lines seen in SNRs, including C II l1335, C IV l1550, and O VI l1034, are resonance lines whose brightnesses will be reduced by scattering if the column density along the line of sight is sufficiently large. Scattering is particularly important in the bright filaments of SNRs because these shock waves are seen nearly edge-on. To assess the importance of resonant scattering in the UV, we have taken advantage of the enormous diffuse source sensitivity of the Hopkins Ultraviolet Telescope to observe the UV emission from a face-on shock in the Vela supernova remnant. The position chosen is seen in projection near the center of the remnant and corresponds to a bright soft X-ray region. A 165 km s-1 shock with a 30% carbon depletion matches most of the line intensities, but the weakness of the N V l1240 line may suggest a departure from the simple shock models. We compare the spectrum of the face-on shock to that of a nearby bright filament and find some differences in shock velocity as well as in resonance-line scattering. We also derive the ram pressure of the shock from Fabry-Perot observations, and find that it greatly exceeds the thermal pressure derived from the [S II] doublet. This result strongly suggests pressure support by magnetic fields or cosmic rays.

TheSaccharomyces cerevisiae PLC1gene encodes a homolog of the δ isoform of mammalian phosphoinositide-specific phospholipase C. Cells deleted forPLC1gene (plc1Δ) are viable but display several phenotypes, including temperature and osmotic sensitivity and defects in utilization of carbon sources other than glucose. We have used the two hybrid screen to identify Plc1p-interacting proteins. One of the identified proteins was Tor2p, a putative phosphatidylinositol (PtdIns) kinase involved in regulation of protein synthesis, cell cycle progression and organization of the actin cytoskeleton. This interaction was confirmed biochemically by coprecipitation of Plc1p and Tor2p. The results suggest that Tor2p, as a PtdIns kinase, produces phosphorylated PtdIns, which is then hydrolyzed by the associated Plc1p. The proximity of Tor2p to Plc1p may therefore result in a regulated spatial and temporal coupling of synthesis and hydrolysis of phosphorylated forms of PtdIns.

Glycerophosphate acyltransferase (GAT) catalyzes the conversion of sn-glycerol 3-phosphate to lysophosphatidic acid (LPA), the first and committed step of triacylglycerol and phospholipid synthesis. In spite of the important regulatory roles GAT may play in this biosynthetic pathway, little information is available on the structure, biochemical properties, and regulation of GAT from eukaryotic cells. We solubilized GAT from rat liver mitochondrial membranes and purified it to an apparent homogeneity by hydroxylapatite chromatography, preparative isoelectric focusing, and gel filtration. The enzyme is composed of a single polypeptide of 85 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography of the native protein. The GAT activity was completely lost during the purification procedure and required addition of exogenous phospholipids for its reconstitution. Since a high phospholipid to detergent ratio was needed for full reactivation, it is concluded that GAT requires “lipid boundary” for reconstitution. The ability of different phospholipids to reactivate GAT decreased in the following order: phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), asolectin, phosphatidylinositol (PI), phosphatidylserine (PS), and cardiolipin. 1,2-Dioleoyl derivatives of PG and PE were more effective in reconstituting the GAT activity than corresponding dipalmitoyl derivatives. The GAT activation was further increased by using a combination of PG and PE or PG and PC. Regardless of the phospholipid used for reconstitution, palmitoyl-CoA was the best acyl donor and LPA was the only reaction product.

Phosphatidylinositol ()P 5-kinase (PtdIns(4)P 5-kinase) catalyzes the last step in the synthesis of phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2). PtdIns(4,5)P2 is a precursor of diacylglycerol and inositol 1,4,5-trisphosphate and is also involved in regulation of actin cytoskeleton remodeling and membrane traffic. To satisfy such varied demands in several aspects of cell physiology, synthesis of PtdIns(4,5)P2 must be stringently regulated. In this paper we describe extraction, purification, and characterization of PtdIns(4)P 5-kinase from the plasma membranes of Schizosaccharomyces pombe. We also provide evidence that PtdIns(4)P 5-kinase is phosphorylated and inactivated by Cki1, the S. pombe homolog of casein kinase I. Phosphorylation by Cki1 in vitro decreases the activity of PtdIns(4)P 5-kinase. In addition, and most importantly, overexpression of Cki1 in S. pombe results in a reduced synthesis of PtdIns(4,5)P2 and in a lower activity of PtdIns(4)P 5-kinase associated with the plasma membrane. These results suggest that PtdIns(4)P 5-kinase is a target of Cki1 in S. pombe and that Cki1 is involved in regulation of PtdIns(4, 5)P2 synthesis by phosphorylating and inactivating PtdIns(4)P 5-kinase.

Phosphatidylinositol (4)P 5-kinase (PtdIns(4)P 5-kinase) catalyzes the last step in the synthesis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). PtdIns(4,5)P2is a precursor of diacylglycerol and inositol 1,4,5-trisphosphate and is also involved in regulation of actin cytoskeleton remodeling and membrane traffic. To satisfy such varied demands in several aspects of cell physiology, synthesis of PtdIns(4,5)P2must be stringently regulated. In this paper we describe extraction, purification, and characterization of PtdIns(4)P 5-kinase from the plasma membranes of Schizosaccharomyces pombe.We also provide evidence that PtdIns(4)P 5-kinase is phosphorylated and inactivated by Cki1, the S. pombehomolog of casein kinase I. Phosphorylation by Cki1 in vitrodecreases the activity of PtdIns(4)P 5-kinase. In addition, and most importantly, overexpression of Cki1 in S. pomberesults in a reduced synthesis of PtdIns(4,5)P2and in a lower activity of PtdIns(4)P 5-kinase associated with the plasma membrane. These results suggest that PtdIns(4)P 5-kinase is a target of Cki1 in S. pombeand that Cki1 is involved in regulation of PtdIns(4, 5)P2synthesis by phosphorylating and inactivating PtdIns(4)P 5-kinase.

An overview of sources of PAHs in the Czech Republic and that of the values of emission factors and annual emissions are given. Residential heating, coal fired stations, and production of PAHs, carbon black, and coke were identified as the major sources of PAHs in Czech Republic. The total annual emission of 16 PAHs suggested by US EPA was assessed at 700 to 735 tons per annum.

The GCS1 gene of the budding yeast Saccharomyces cerevisiae mediate the resumption of cell proliferation from the starved, stationary-phase state. Here we identify yeast genes that, in increased dosages, overcome the growth defect of gcs1 delta mutant cells. Among these are YCK1 (CK12) and YCK2 (CKI1), encoding membrane-associated casein kinase I, and YCK3, encoding a novel casein kinase I isoform. Some Yck3p gene product was found associated with the plasma membrane, like Yck1p and Yck2p, but most confractionated with the nucleus, like another yeast casein kinase I isoform, Hrr25p. Genetic studies showed that YCK3 and HRR25 constitute an essential gene family and that Yck3p can weakly substitute for Yck1p-Yck2p. For gcs1 delta suppression, both a protein kinase domain and a C-terminal prenylation motif were shown to be necessary. An impairment in endocytosis was found for gcs1 delta mutant cells, which was alleviated by an increased YCK2 gene dosage. The ability of an increased casein kinase I gene dosage to suppress the effects caused by the absence of Gcs1p suggests that Gcs1p and Yck1p-Yck2p affect parallel pathways.

The six high-mountain soil samples (more than 1,100 m above sea level) from National Park Krkonose (North Bohemia, Czech-Polish border) were analyzed for contents of PCBs, non-ortho and mono-ortho PCBs and PCDDs/Fs. The content of PCBs ranged between 31 and 137 ng.g-1 dry soil. PCB 77 was predominant coplanar PCBs. The contents of PCDDs/Fs ranged between 17 and 29 pg.g-1 1-TEQ of PCDDs/Fs and were between 20.1 and 41.2 pg.g-1 T-TEQ of PCDDs/Fs and coplanar PCBs. The contents of PCDFs were significantly higher than those of PCDDs. The predominant congeners were OCDF and OCDD. The contribution of non-ortho and mono-ortho PCBs to the total 1-TEQ ranged between 9.3 and 49.3%.

The possible negative effluents of model source of PAHs on the environment were observed. The model source of PAHs was Urx Chemical Factory, Valasske Mezifici, Czechoslovakia, who produced aromatic compounds from benzene to phenanthrene and some of their derivatives and carbon black.

The human platelet receptors for normal, nonimmune IgG and its F(ab')2 and Fc fragments were studied by the use of a cleavable, bifunctional, photoactivable, 125I-labeled cross-linking agent. Derivatization of the ligands with N-[4-(p-azido-m-[125I]iodophenylazo) benzoyl]-3-aminopropyl-N'-oxysuccinimide ester (Denny-Jaffe reagent) reduced their binding to platelets by greater than or equal to 20%. Cleavage of the azo linkage of the Denny-Jaffe reagent, which splits the molecule so that its 125I-labeled portion becomes associated with the receptor half of the cross-linked ligand-receptor complex, was utilized to directly identify receptors for the various immunoglobulin ligands. Specificity of the binding reaction could be demonstrated by suppressing the iodination of the receptors with excess nonderivatized ligand. Two principal IgG-related receptors could be identified by high-resolution NaDodSO4/PAGE and subsequent analysis of the electrophoretically transferred peptides to nitrocellulose filters for localization of radioactivity and immunological characterization. Intact monomeric IgG and F(ab')2 fragments derived from it appeared to have the glycoprotein IIIa as the major receptor, whereas Fc fragments bound predominantly to a peptide of Mr approximately 200,000 (Mr, approximately 50,000 under rigorous reducing conditions).

The glutathione S-transferases (GSTs) represent a significant group of detoxification enzymes that play an important role in drug resistance in all eukaryotic species. In this paper we report an identification and characterization of the twoSaccharomyces cerevisiaegenes, GTT1and GTT2(glutathionetransferase 1 and 2), coding for functional GST enzymes. Despite only limited similarity with GSTs from other organisms (∼50%), recombinant Gtt1p and Gtt2p exhibit GST activity with 1-chloro-2,4-dinitrobenzene as a substrate. Both Gtt1p and Gtt2p are able to form homodimers, as determined by two hybrid assay. Subcellular fractionation demonstrated that Gtt1p associates with the endoplasmic reticulum. Expression of GTT1is induced after diauxic shift and remains high throughout the stationary phase. Strains deleted for GTT1and/or GTT2are viable but exhibit increased sensitivity to heat shock in stationary phase and limited ability to grow at 39 °C.

Topsoil samples from Project TOCOEN model sites and some other locations in Czechoslovakia were collected and analyzed for PCBs, PCDDs and PCDFs. The various types of contaminated areas and sites are compared as first describing the situation of contamination of Czechoslovak soils by chlorinated aromatic pollutants.

In the yeast Saccharomyces cerevisiae, mutations in either of two unlinked genes, RAM1 or RAM2, abolish the farnesyltransferase activity responsible for prenylation of Ras proteins and the a-factor mating pheromone. Here we report that the function of RAM1 and RAM2 genes is required for the membrane localization of Ras proteins and a-factor. The RAM2 gene was sequenced and can encode a 38-kDa protein. We examined the functional interaction of RAM2 and RAM1 by expressing the genes in Escherichia coli. Extracts derived from an E. coli strain that coexpressed RAM1 and RAM2 efficiently farnesylated a-factor peptide and Ras protein substrates. In contrast, extracts derived from E. coli strains that expressed either RAM gene alone were devoid of activity; however, when the latter extracts were mixed, protein farnesyltransferase activity was reconstituted. These results indicate that the yeast farnesyl-protein transferase is comprised of Ram1 and Ram2 polypeptides. Although Ram1 is a component of the enzyme, disruption of the RAM1 gene in yeast was not lethal, indicating that the Ram1-Ram2 farnesyltransferase is not essential for viability. In contrast, disruption of RAM2 was lethal, suggesting that Ram2 has an essential function in addition to its role with Ram1 in protein farnesylation.

Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.

Two cDNAs encoding casein kinase-1 have been isolated from a yeast cDNA library and termed CKI1 and CKI2. Each clone encodes a protein of approximately 62,000 Da containing a highly conserved protein kinase domain surrounded by variable amino- and carboxy-terminal domains. The proteins also contain two conserved carboxy-terminal cysteine residues that comprise a consensus sequence for prenylation. Consistent with this posttranslational modification, cell fractionation experiments demonstrate that intact CKI1 is found exclusively in yeast cell membranes. Gene disruption experiments reveal that, although neither of the two CKI genes is essential by itself, at least one CKI gene is required for yeast cell viability. Spores deficient in both CKI1 and CKI2 fail to grow and, therefore, either fail to germinate or arrest as small cells before bud emergence. These results suggest that casein kinase-1, which is distributed widely in nature, plays a pivotal role in eukaryotic cell regulation.

To gain a greater understanding of the factors that drive spatial organization in multicellular aggregates of cancer cells, we investigate the segregation patterns of 6 breast cell lines (MDA-MB-231, MDA-MB-468, MDA-MB-436, MDA-MB-157, ZR-75-1, and MCF-10A) of varying degree of mesenchymal character during formation of mixed aggregates. We consider cell sorting in the context of available adhesion proteins and cellular contractility, biophysical properties that are typically considered in models of cell sorting. We characterize the mechanisms of spheroid formation as being primarily cadherin- or integrin-driven. The primary compaction mediator for a given cell type plays an important role in compaction speed, which in turn is the major factor dictating preference for interior or exterior position within mixed aggregates. In particular, cadherin-deficient, invasion-competent cells tend to position towards the outside of aggregates, facilitating access to extracellular matrix. We show that reducing actomyosin contractility has a differential effect on spheroid formation depending on the compaction mechanism. Inhibition of contractility has a significant stabilizing effect on cell-cell adhesions in integrin-driven aggregation and a mildly destabilizing effect in cadherin-based aggregation. This differential response is exploited as a spheroid formation method and as a method through which to statically control aggregate organization and dynamically rearrange cells in pre-formed aggregates. Sequestration of invasive cells in the interior of spheroids provides a physical barrier that reduces invasion in three-dimensional culture, revealing a potential strategy for containment of invasive cell types.Movie S1Movie S1MDA‐MB‐231 (green) and MCF‐10A (magenta) co‐culture spheroid formed in a low adhesion plate. Images are taken every 10 min for 20 hr.Movie S2Movie S2(left) Untreated MDA‐MB‐231 cells allowed to aggregate and imaged every 30 minutes for 30 hours. (right) MDA‐MB‐231 cells treated with 5 μM para‐nitroblebbistatin and imaged under the same conditions. Para‐nitroblebbistatin was used in place of blebbistatin in live‐cell imaging due to poor photostability of and background fluorescence contributed by conventional blebbistatin.Movie S3Movie S3MDA‐MB‐231 (magenta) and MDA‐MCF‐157 (green) co‐culture spheroid formed with BME for 24 hours, treated with cell recovery solution for 1 hour, and subsequently treated with 5 μM para‐nitroblebbistatin. Images are taken every 6 hrs for 120 hrs. Para‐nitroblebbistatin was used in place of blebbistatin in live‐cell imaging due to poor photostability of and background fluorescence contributed by conventional blebbistatin.

Tumor-educated platelets (TEPs) are potential biomarkers for cancer diagnostics. We employ TEP-derived RNA panels, determined by swarm intelligence, to detect and monitor glioblastoma. We assessed specificity by comparing the spliced RNA profile of TEPs from glioblastoma patients with multiple sclerosis and brain metastasis patients (validation series, n = 157; accuracy, 80%; AUC, 0.81 [95% CI, 0.74–0.89; p < 0.001]). Second, analysis of patients with glioblastoma versus asymptomatic healthy controls in an independent validation series (n = 347) provided a detection accuracy of 95% and AUC of 0.97 (95% CI, 0.95–0.99; p < 0.001). Finally, we developed the digitalSWARM algorithm to improve monitoring of glioblastoma progression and demonstrate that the TEP tumor scores of individual glioblastoma patients represent tumor behavior and could be used to distinguish false positive progression from true progression (validation series, n = 20; accuracy, 85%; AUC, 0.86 [95% CI, 0.70–1.00; p < 0.012]). In conclusion, TEPs have potential as a minimally invasive biosource for blood-based diagnostics and monitoring of glioblastoma patients.

The article discusses the architectural design of the New World Center, a concert hall located in South Beach, Miami Bleach, Florida that was designed by firm Gehry Partners LLP.

The article reviews the architectural design of a tower that is being designed by Morris Adjmi Architects and constructed by Sciame Construction for Thor Equities and Taconic Investment Partners in Manhattan, New York City.

Provides tips for principals on how to create a positive school culture in the U.S. Understanding of the developmental processes that occur for students during the high school years; Philosophy of open-ended hope and possibility.

A personal narrative is presented which explores the author's working experiences including one as a design engineer at industrial and agricultural equipment manufacturer Vermeer Corp. where he realized the importance to never stop learning and to live life like there is no tomorrow.