Historical contamination of Yukon Lake sediments by PCBs and organochlorine pesticides: influence of local sources and watershed characteristics.

PCBs and other persistent organochlorine (OC) pesticides were analyzed in sediment cores collected from six lakes in Yukon Territory and one in northern British Columbia, Canada, with the objective of establishing sources and historical trends of these contaminants. DDT was found to be the most prominent OC in the sediment profiles of most of the lakes. Maximum sigmaDDT levels (3.47-2680 ng g(-1) dw) were observed in sediment slices dated to the 1950s from lakes near populated areas. In contrast, in more remote lakes (Hanson, Kusawa and Lindeman), the maximum sigmaDDT concentrations were observed in the sediments dated to the 1970s. Highest sigmaPCB and sigmaDDT concentrations were measured in sediments from Watson Lake, near a suspected PCB waste disposal site and in a region where DDT was heavily applied in the 1950s and 1960s. Elevated sigmaPCB concentrations [16.1-93.6 ng g(-1) dry weight (dw)] were also observed in sediments from lakes situated near populated areas, relative to Kusawa and Lindeman (11.1 and 12.7 ng g(-1) dw, respectively). Recent sigmaPCB fluxes ranged from 621 ng m(-2) y(-1) in Kusawa Lake to 16400 ng m(-2) y(-1) in Little Atlin Lake. The extremely high sedimentation rate (2050 g m(-2) y(-1)) in glacial fed Lindeman Lake gave rise to elevated fluxes of sigmaPCB (2410 ng m(-2) y(-1)) and other OCs, despite much lower concentrations in the sediment. Levels of hexachlorocyclohexanes (sigmaHCH), chlordane-related compounds (sigmaCHL), and chlorobenzenes (sigmaCBz) were in the low ng g(-1) (dw) range in all lake sediments, similar to concentrations previously reported for Arctic lakes in Canada, indicating that their major source was long range atmospheric transport. Contamination of the lakes with PCBs and DDT near populated areas of the Yukon Territory appears to be a result of regional activities rather than long range transport and deposition. The results also point to glacial runoff as a significant source of OCs to small, high elevation lakes (Lindeman), but not to larger lakes within the Yukon River drainage basin that are also affected by glacial sources (Kusawa, Laberge).

Consideration of conformational transitions and racemization during process development of recombinant glucagon-like peptide-1.

Physicochemical characterization of dry, excipient-free recombinant glucagon-like peptide-1 (rGLP-1) indicates the conformation and purity of the bulk peptide is dependent on the purification scheme and the in-process storage and handling. The recombinant peptide preparations were highly pure and consistent with the expected primary structure and bioactivity. However, variations in solubility were observed for preparations processed by different methods. The differences in solubility were shown to be due to conformational differences induced during purification. A processing scheme was identified to produce rGLP-1 in its native, soluble form, which exhibits FT-IR spectra, consistent with glucagon-like peptide-1 synthesized by solid-state peptide synthesis. rGLP-1 was also found to undergo base-catalyzed amino acid racemization. Racemization can impact the yield and impurity profile of bulk rGLP-1, since the peptide is exposed to alkali during its purification. A combination of enzymatic digestion using leucine aminopeptidase (which cleaves N-terminal L-amino acids >> D-amino acids) and matrix-assisted laser desorption ionization mass spectrometry was used to identify racemization as a degradation pathway. The racemization rate increased with increasing temperature and base concentration, but decreased with increasing peptide concentration. The racemized peptides were shown to be less bioactive than rGLP-1.

Aqueous stability of recombinant human thrombopoietin as a function of processing schemes.

Preformulation studies conducted with recombinant human thrombopoietin (rhTPO), a 332 amino acid glycoprotein which stimulates platelet production, show distinctions in degradation profiles as a function of processing schemes. The stability-limiting degradation pathways change as a function of purification stage and method and are dependent upon the presence of contaminating protease. The stability-limiting degradation pathway of affinity-purified and in-process rhTPO preparations is primarily attributed to proteolysis initiated by a protease present as a fermentation contaminant. The proteolysis increases with increasing pH as a function of temperature. The degradation profiles for these preparations show that bioactivity initially increases and then decreases with increasing pH as a function of temperature. This is consistent with proteolysis to active forms which ultimately undergo degradation to less active forms. Similar studies conducted with rhTPO preparations purified by a combination of more conventional chromatographic steps show different stability-limiting degradation pathways and a different pH-stability profile when compared to affinity purified or in-process preparations. In this case, degradation is accompanied by decreases in activity under all conditions, consistent with the conversion to less active forms. These results illustrate the importance of preformulation and stability characterization of protein pharmaceuticals in support of both process and formulation development. Issues related to storage and handling of inprocess preparations differ from those with formulated product since the stability-limiting degradation pathways change as a function of purification stage.

Antibodies against the N-terminus of IL-8 receptor A inhibit neutrophil chemotaxis.

Neutrophil migration and activation are the cornerstones of the acute inflammatory response. Interleukin-8 triggers several functions of neutrophils in host defense: chemotaxis, degranulation and enzyme release, and superoxide production. Interleukin-8 is most potent as a chemoattractant, so chemotaxis is likely the most important of these functions. The effects of interleukin-8 on neutrophils are mediated through two receptors, IL-8RA and IL-8RB. To investigate the role of these receptors in neutrophil chemotaxis, we produced inhibitory antibodies to IL-8RA. These antibodies inhibit neutrophil chemotaxis toward IL-8 in vitro. These findings show that IL-8RA mediates a chemotactic signal in neutrophils and suggest that an anti-receptor strategy may be a useful approach to limit neutrophil migration in inflammation.

A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.

Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.

The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization.

Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [125I]glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3',5'-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns.

Expression cloning and signaling properties of the rat glucagon receptor.

Glucagon and the glucagon receptor are a primary source of control over blood glucose concentrations and are especially important to studies of diabetes in which the loss of control over blood glucose concentrations clinically defines the disease. A complementary DNA clone for the glucagon receptor was isolated by an expression cloning strategy, and the receptor protein was expressed in several kidney cell lines. The cloned receptor bound glucagon and caused an increase in the intracellular concentration of adenosine 3', 5'-monophosphate (cAMP). The cloned glucagon receptor also transduced a signal that led to an increased concentration of intracellular calcium. The glucagon receptor is similar to the calcitonin and parathyroid hormone receptors. It can transduce signals leading to the accumulation of two different second messengers, cAMP and calcium.

Clinical evaluation of an EIA for the sensitive and specific detection of serum antibody to Trypanosoma cruzi (Chagas' disease).

A commercial EIA for the detection of antibody to Trypanosoma cruzi was clinically evaluated. The primary use of this test is in the diagnosis and screening of donated blood in Latin America. When compared with sera positive by xenodiagnosis, the assay had a clinical sensitivity of 100%. When tested against matched hemagglutination (HA) and immunofluorescence (IFA) results (i.e., when both tests gave negative results) the EIA had a specificity of 99.03% (305/308). The cross-reactivity of this test was determined using sera from malaria and leishmaniasis patients (obtained from Africa, ensuring that the sera did not contain Chagasic antibodies) and from schistosomiasis, toxoplasmosis, tuberculosis, syphilis, and systemic lupus erythematosus samples. The EIA was 100% specific whereas IFA or commercially available HA kits from Latin America cross-reacted with several of the samples. In this investigation, the EIA appeared to be at least as sensitive and more specific than IFA or HA in the serodiagnosis of Chagas' disease.

Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulin-dependent enzymes.

Nanomolar concentrations of synthetic peptides corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase were found to inhibit calmodulin activation of seven well-characterized calmodulin-dependent enzymes: brain 61 kDa cyclic nucleotide phosphodiesterase, brain adenylate cyclase, Bordetella pertussis adenylate cyclase, red blood cell membrane Ca++-pump ATPase, brain calmodulin-dependent protein phosphatase (calcineurin), skeletal muscle phosphorylase b kinase, and brain multifunctional Ca++ (calmodulin)-dependent protein kinase. Inhibition could be entirely overcome by the addition of excess calmodulin. Thus, the myosin light chain kinase peptides used in this study may be useful antagonists for studying calmodulin-dependent enzymes and processes.

Immunological distinction between calmodulin-sensitive and calmodulin-insensitive adenylate cyclases.

Previous studies using calmodulin-Sepharose affinity chromatography have suggested that bovine brain may contain a mixture of calmodulin-sensitive and -insensitive adenylate cyclase activities (Wescott, K. R., La Porte, D. C., and Storm, D. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 82, 3086-3090). In this study, mice were immunized with a purified preparation of the calmodulin-sensitive adenylate cyclase from bovine brain, and a polyclonal antiserum was obtained which was specific to the calmodulin-sensitive form of the enzyme. The antiserum was not inhibitory and precipitated enzyme activity from a homogeneous preparation of the calmodulin-sensitive adenylate cyclase catalytic subunit. Furthermore, the antiserum did not interact with calmodulin-insensitive adenylate cyclase which was resolved from the calmodulin-sensitive form of the enzyme by calmodulin-Sepharose affinity chromatography. Since the only polypeptide specifically precipitated by the antiserum had an Mr of 135,000, which was identical to the Mr of the catalytic subunit of the enzyme, it is concluded that the antiserum interacted directly and specifically with the catalytic subunit of the calmodulin-sensitive isozyme of adenylate cyclase. Detergent-solubilized membranes from several rat tissues were examined for the presence of calmodulin-sensitive adenylate cyclase using anti-calmodulin-sensitive adenylate cyclase antiserum. Approximately 40-60% of the total adenylate cyclase activity of rat brain and kidney were immunoprecipitated by the antiserum, whereas liver and testes contained no detectable calmodulin-sensitive adenylate cyclase. Approximately 15% of the total adenylate cyclase activity in rat heart and lung was the calmodulin-sensitive form. These data indicate that the calmodulin-sensitive and insensitive adenylate cyclases from bovine brain are immunologically distinct and support the proposal that there may be two or more distinct adenylate cyclase isozymes in brain.

Purification of the calmodulin-sensitive adenylate cyclase from bovine cerebral cortex.

A calmodulin-sensitive adenylate cyclase was purified 3000-fold from bovine cerebral cortex using DEAE-Sephacel, calmodulin-Sepharose, and two heptanediamine-Sepharose column steps. The purified enzyme activity was stimulated by calmodulin, forskolin, 5'-guanylyl imidodiphosphate, and NaF. The molecular weight of the protein component was estimated as 328 000 with a smaller form of Mr 153 000 obtained in the presence of Mn2+. The most highly purified preparations contained major polypeptides of 150 000, 47 000, and 35 000 daltons on sodium dodecyl sulfate (SDS) gels. Photoaffinity labeling of the preparation with azido[125I]iodocalmodulin gave one product of 170 000 daltons on SDS gels. It is proposed that the catalytic subunit of the calmodulin-sensitive enzyme is 150 000 +/- 10 000 daltons and that the enzyme exists as a complex of one catalytic subunit and the stimulatory guanyl nucleotide regulatory complex. These data are consistent with the previous report that the catalytic subunit of this enzyme has a molecular weight of 150 000 +/- 10 000 [Andreasen, T.J., Heideman, W., Rosenberg, G.B., & Storm, D.R. (1983) Biochemistry 22,2757].

Photoaffinity labeling of brain adenylate cyclase preparations with azido[125 I]iodocalmodulin.

A partially purified calmodulin-sensitive adenylate cyclase from bovine cerebral cortex was photoaffinity labeled with azido[125 I]iodocalmodulin. Sodium dodecyl sulfate gel electrophoresis followed by autoradiography revealed several cross-linked polypeptides ranging in molecular weight from 37000 to 200000. The calmodulin-sensitive enzyme was submitted to a number of purification steps to determine if any of the calmodulin binding polypeptides copurified with adenylate cyclase activity. Fractionation procedures used included Bio-Gel A5M and Ultragel AcA 34 gel chromatography, isoelectric focusing, and native gradient gel electrophoresis. One cross-linked peptide having a molecular weight of 170000 correlated with adenylate cyclase activity through all purification steps. Native gradient gel electrophoresis in the presence of 0.03% deoxycholate gave one peak of adenylate cyclase activity with a Stokes radius of 40 A, consistent with a molecular weight of 140000-150000. It is proposed that the molecular weight of the adenylate cyclase catalytic subunit is 150,000 and that each catalytic subunit interacts with one calmodulin.

Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa.

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.

Reconstitution of adenylate cyclase in Neurospora from two components of the enzyme.

The ATP-Mg2+-dependent, guanine nucleotide-stimulated adenylate cyclase of Neurospora crassa was shown to be composed of distinct components which, when mixed, can reconstitute a holoenzyme. After brief heat treatment, the adenylate cyclase activity in crude homogenates of Neurospora was found to have reduced activity and greatly reduced sensitivity to stimulation by guanyl-5'-yl-imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity could be restored by addition of a homogenate from a crisp-1 (cr-1) mutant of Neurospora which was shown to have little or no adenylate cyclase activity. These results show that the adenylate cyclase of Neurospora resembles the enzyme of animal systems in that it is composed of distinct regulatory and catalytic components. The data presented also show that the cr-1 mutant is defective in the adenylate cyclase catalytic component but not in the regulatory component. Therefore, cr-1 is unlike most other such eucaryotic mutants which appear to be defective in the regulatory component.