Climate Change and the Sea: A Major Disruption in Steady State and the Master Variables.

Since the beginning of the industrial revolution, humans have burned enormous quantities of coal, oil, and natural gas, rivaling nature's elemental cycles of C, N, and S. The result has been a disruption in a steady state of CO and other greenhouse gases in the atmosphere, a warming of the planet, and changes in master variables (temperature, pH, and pε) of the sea affecting critical physical, chemical, and biological reactions. Humans have also produced copious quantities of N and P fertilizers producing widespread coastal hypoxia and low dissolved oxygen conditions, which now threaten even the open ocean.

Phytoremediation, Bioaugmentation, and the Plant Microbiome.

Understanding plant biology and related microbial ecology as a means to phytoremediate soil and groundwater contamination has broadened and advanced the field of environmental engineering and science over the past 30 years. Using plants to transform and degrade xenobiotic organic pollutants delivers new methods for environmental restoration. Manipulations of the plant microbiome through bioaugmentation, endophytes, adding various growth factors, genetic modification, and/or selecting the microbial community via insertion of probiotics or phages for gene transfer are future areas of research to further expand this green, cost-effective, aesthetically pleasing technology─phytoremediation.

Bioaugmenting the poplar rhizosphere to enhance treatment of 1,4-dioxane.

1,4-Dioxane is a highly mobile and persistent groundwater pollutant that often forms large dilute plumes. Because of this, utilizing aggressive pump-and-treat and ex-situ technologies such as advanced oxidation can be prohibitively expensive. In this study, we bioaugmented the poplar rhizosphere with dioxane-degrading bacteria Mycobacterium dioxanotrophicus PH-06 or Pseudonocardia dioxanivorans CB1190 to enhance treatment of 1,4-dioxane in bench-scale experiments.

A potent erythropoietin-mimicking human antibody interacts through a novel binding site.

Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the erythropoietin receptor (EPOR) in erythroid progenitor cells, leading to proliferation and differentiation into mature red blood cells. To allow less frequent dosing, a hyperglycosylated version of EPO has been developed with a longer half-life. In principle, an agonistic antibody targeting EPOR would offer an even longer half-life, support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell aplasia.

Chkl binds and phosphorylates BAD protein.

Chk1 (checkpoint kinase 1) is a serine-threonine kinase that is critical for G2/M arrest in response to DNA damage. Chk1 phosphorylates Cdc25C at serine-216, a major regulatory site, in response to DNA damage. Furthermore, Chk1 also phosphorylates Cdc25A on serine 123 which accelerates its degradation through the ubiquitin-proteasome pathway and arrests cells in late G2-phase after DNA damage.

Divergence of Genbank and human tumor Bcl-2 sequences and implications for binding affinity to key apoptotic proteins.

Heterodimerization of antiapoptotic and pro-apoptotic Bcl-2 family of proteins provides an important mechanism for apoptosis regulation. Knowledge about key amino acids in the binding groove of native Bcl-2 contributing to this interaction will greatly facilitate the design of Bcl-2-specific inhibitors. There are two different Bcl-2 sequences, M13994 and M14745, in Genbank.

X-ray crystallographic structure of ABT-378 (lopinavir) bound to HIV-1 protease.

The crystal structure of ABT-378 (lopinavir), bound to the active site of HIV-1 protease is described. A comparison with crystal structures of ritonavir, A-78791, and BILA-2450 shows some analogous features with previous reported compounds. A cyclic urea unit in the P(2) position of ABT-378 is novel and makes two bidentate hydrogen bonds with Asp 29 of HIV-1 protease.

Blocking Chk1 expression induces apoptosis and abrogates the G2 checkpoint mechanism.

Checkpoint kinase 1 (Chk1) is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates the Cdc2 activating phosphatase Cdc25C. This in turn inactivates Cdc2, which leads to G2/M arrest.

Abrogation of G2 checkpoint specifically sensitize p53 defective cells to cancer chemotherapeutic agents.

Background: Chkl is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates Cdc25C at the late G2 phase. The inactivation of Cdc25C and consequently, the inactivation of Cdc2, are required for the G2 arrest induced by DNA damage.

Molecular characterization of human SUR2-containing K(ATP) channels.

The distribution of human sulfonylurea receptor-2 (SUR2)-containing K(ATP) channels was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR). mRNA for SUR2B was detected in a variety of tissues including brain, skeletal, cardiac and smooth muscle, whereas SUR2A message was restricted to cardiac and skeletal muscle. An additional splice variant of SUR2 that lacked exon 17 was also identified by RT-PCR in tissues expressing both SUR2A and SUR2B or SUR2B alone.

Development of a p38 Kinase Binding Assay for High Throughput Screening.

p38 is a member of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases, which is activated by cellular stressors and has been shown to be a critical enzyme in the synthesis and action of proinflammatory cytokines, tumor necrosis factor-a (TNF-alpha) and interleukin-1beta (IL-1beta). A group of pyridinyl imidazole compounds such as SB202190 have been identified as selective inhibitors of p38 that bind directly to the ATP pocket of the enzyme. These compounds inhibit the p38 kinase activity, block TNF-alpha and IL-1beta secretion both in vivo and in vitro and are found to be effective in animal models of arthritis, bone resorption, and endotoxin shock.

Cloning and expression of the adenosine kinase gene from rat and human tissues.

Adenosine kinase is ubiquitous in eukaryotes and is a key enzyme in the regulation of the intracellular levels of adenosine, an important physiological effector of many cells and tissues. In this paper we report the cloning of cDNAs encoding adenosine kinase from both rat and human tissues. Two distinct forms of adenosine kinase mRNA were identified in human tissues.

Solution structure of the cyclosporin A/cyclophilin complex by NMR.

Cyclosporin A, a cyclic undecapeptide, is a potent immunosuppressant that binds to a peptidyl-prolyl cis-trans isomerase of 165 amino acids, cyclophilin. The cyclosporin A/cyclophilin complex inhibits the calcium- and calmodulin-dependent phosphatase, calcineurin, resulting in a failure to activate genes encoding interleukin-2 and other lymphokines. The three-dimensional structures of uncomplexed cyclophilin, a tetrapeptide/cyclophilin complex, and cyclosporin A when bound to cyclophilin have been reported.

1H, 13C and 15N backbone assignments of cyclophilin when bound to cyclosporin A (CsA) and preliminary structural characterization of the CsA binding site.

The backbone 1H, 13C and 15N chemical shifts of cyclophilin (CyP) when bound to cyclosporin A (CsA) have been assigned from heteronuclear two- and three-dimensional NMR experiments involving selectively 15N- and uniformly 15N- and 15N,13C-labeled cyclophilin. From an analysis of the 1H and 15N chemical shifts of CyP that change upon binding to CsA and from CyP/CsA NOEs, we have determined the regions of cyclophilin involved in binding to CsA.

NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding.

Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin.

Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin.

We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin. The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A.

Isotope-edited NMR of cyclosporin A bound to cyclophilin: evidence for a trans 9,10 amide bond.

The binding of a 13C-labeled cyclosporin A (CsA) analog to cyclophilin (peptidyl prolyl isomerase) was examined by means of isotope-edited nuclear magnetic resonance (NMR) techniques. A trans 9,10 peptide bond was adopted when CsA was bound to cyclophilin, in contrast to the cis 9,10 peptide bond found in the crystalline and solution conformations of CsA. Furthermore, nuclear Overhauser effects (NOEs) were observed between the zeta 3 and epsilon 3 protons of the methylleucine (MeLeu) residue at position 9 of CsA and tryptophan121 (Trp121) and phenylalanine (Phe) protons of cyclophilin, suggesting that the MeLeu9 residue of CsA interacts with cyclophilin.

Affinity purification of HIV-1 and HIV-2 proteases from recombinant E. coli strains using pepstatin-agarose.

A procedure is described which employs pepstatin-agarose for the affinity purification of either HIV-1 or HIV-2 protease from two similar recombinant E. coli constructs that were developed for the expression of these enzymes. HIV-2 protease was routinely expressed at much higher levels than the HIV-1 enzyme and pepstatin-agarose was the only chromatography step required to isolate pure HIV-2 protease from crude bacterial lysates.

Molecular cloning of a gene belonging to the carcinoembryonic antigen gene family and discussion of a domain model.

Carcinoembryonic antigen (CEA) is a glycoprotein important as a tumor marker for colonic cancer. Immunological and biochemical studies have shown it to be closely related to a number of other glycoproteins, which together make up a gene family. We have cloned a member of this gene family by using long oligonucleotide probes (42-54 nucleotides) based on our protein sequence data for CEA and NCA (nonspecific cross-reacting antigen) and on human codon usage.

Sequence of the promoter region of the gene for human X-linked 3-phosphoglycerate kinase.

We have determined the sequence of an 812-bp BamHI-EcoRI restriction fragment containing the 5' region of the human gene for PGK (3-phosphoglycerate kinase or ATP:3-phospho-D-glycerate 1-phosphotransferase; EC 2.7.2.

Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe.

We have obtained a cDNA clone encoding most of human X-linked 3-phosphoglycerate kinase (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.

Protease-nexin: a cellular component that links thrombin and plasminogen activator and mediates their binding to cells.

This report identifies a component of normal human fibroblasts that forms a covalent linkage with thrombin and urokinase (urinary plasmingoen activator) and mediates most of the specific cellular binding of these proteases. This component, here named protease-nexin (PN), is both associated with the cell surface and released into the culture medium. In several ways PN resembles antithrombin III (AT3), a prominent inhibitor of thrombin in serum: PN links thrombin, probably via an ester bond; PN does not link thrombin blocked at its catalytic site serine; PN has a high-affinity heparin-binding site; and heparin greatly accelerates the rate of linkage between soluble PN and thrombin.