Comparison of two techniques for quantifying environmental contaminants in human serum

Peak area matching and linear regression were used to quantify eight chlorinated pesticides and polychlorinated biphenyls (as Aroclor 1260) in human serum. There are no statistically significant differences in data obtained by these two quantifying techniques which were indicated by the paired t-test. For chlorinated pesticides, p = 0.053-0.62, and for polychlorinated biphenyls (as Aroclor 1260), p = 0.64. Analyte residues for the chlorinated pesticides ranged from 0.5 ppb for hexachlorobenzene (HCB) to 186 ppb for dichlorodiphenyldichloroethylene (DDE). Analyte residues for the polychlorinated biphenyls (as Aroclor 1260) ranged from 5-114 ppb. The absolute mean percent difference between the two quantifying techniques ranged from 0.06% for DDE to 8.06% for dieldrin (HEOD) among the chlorinated pesticides. The absolute mean percent difference between the two quantifying techniques for the polychlorinated biphenyls (as Aroclor 1260) was 3.4%. Peak area matching and linear regression were found to be comparable for quantifying these environmental residues in serum when the following conditions apply: 1) the concentration of the chlorinated pesticides is greater than or equal to 0.5 ppb (e.g., HCB, hexachlorocyclohexane (HCCH), oxychlordane (OC), heptachlor epoxide (HE), transnonachlor (TN), HEOD, and dichlorodiphenyltrichloroethane (DDT); 2) the concentration of the chlorinated pesticide is greater than or equal to 3 ppb (e.g., DDE); and 3) the total concentration of polychlorinated biphenyls (e.g., as Aroclor 1260) is greater than or equal to 5 ppb.     

Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Diel emigration and colonization responses of blackfly larvae (Diptera: Simuliidae) to ultraviolet radiation

1. Total counts of blackfly larvae densities over 30- and 57-h periods in experimental channels during May of 1996 & 1997 indicate that ultraviolet radiation (UV; 290–400 nm) may be important in stimulating emigration.
2. Under experimentally controlled solar UV exposure, larval densities at dawn in UV-shielded channels were 161% and 168% higher than in the UV-exposed channels. Larval densities in UV-exposed channels then decreased by 68.2% and 81.1% between dawn and early afternoon of the two days; density decreases in UV-shielded channels were slight, and not statistically significant, during the same periods.
3. Larvae within UV-exposed channels occupied shaded microhabitats during hours of intense solar radiation, suggesting that simuliid larvae can detect and respond to UV radiation over very short periods of time.
4. A cyclical pattern of UV-induced emigration during hours of increasing solar flux (06.30–13.30) and net immigration in the hours of decreasing solar flux and at night emerged. Thus stream invertebrates may be very sensitive to environmental changes, resulting in either increased UV flux or decreased shading of streams. Diel cycles in invertebrate densities should be taken into account in research designs and sampling protocols in order to identify and interpret correctly results of both periodic surveys and experiments.     

Immunochemical studies of human erythropoietin using site-specific anti-peptide antibodies. Identification of a functional domain

Anti-peptide antibodies that bind to the amino terminus of human erythropoietin (residues 1-26) do not inhibit the hormone's biological activity, indicating that this region of the protein does not play a role in receptor recognition (Sytkowski, A. J., and Fisher, J. W. (1985) J. Biol Chem. 260, 14727-14731). We have now identified six other regions of the primary sequence that are relatively hydrophilic and, therefore, have a higher probability of being accessible to such antibody probes. Antibodies raised against synthetic peptides homologous to five of these regions, corresponding to residues 40-59, 80-99, 99-118, 111-129, and 131-150 recognize erythropoietin, confirming the prediction based upon relative hydrophilicity. Antibodies to a carboxyl terminal peptide 147-166 failed to bind the hormone, presumably due to steric hindrance imposed by a disulfide bond between Cys161 and one of the other cysteinyl residues. The antibodies were affinity purified on the relevant immobilized peptide and their capacity to inhibit (neutralize) erythropoietin's activity was assessed. Only anti-peptide 99-118 and anti-peptide 111-129 antibodies inhibited erythropoietin. This effect was reversed by excess peptide, demonstrating that the neutralizing action of the antibody was due to its antigen-specific binding. The results strongly suggest that the portion of erythropoietin's amino acid sequence represented by these peptides plays a functional role in the hormone's action, most probably by forming part of the receptor-binding domain.     

Mutational analysis of the HIS4 translational initiator region in Saccharomyces cerevisiae.

We have mutated various features of the 5' noncoding region of the HIS4 mRNA in light of established Saccharomyces cerevisiae and mammalian consensus translational initiator regions. Our analysis indicates that insertion mutations that introduce G + C-rich sequences in the leader, particularly those that result in stable stem-loop structures in the 5' noncoding region of the HIS4 message, severely affect translation initiation. Mutations that alter the length of the HIS4 leader from 115 to 39 nucleotides had no effect on expression, and sequence context changes both 5' and 3' to the HIS4 AUG start codon resulted in no more than a twofold decrease of expression. Changing the normal context at HIS4 5'-AAUAAUGG-3' to the optimal sequence context proposed for mammalian initiator regions 5'-CACCAUGG-3' did not result in stimulation of HIS4 expression. These studies, in conjunction with comparative and genetic studies in S. cerevisiae, support a general mechanism of initiation of protein synthesis as proposed by the ribosomal scanning model.     

Serotonin (5-hydroxytryptamine) turnover in adult female Ascaris suum tissue

1. The 5-hydroxytryptamine (5-HT, serotonin) turnover was examined in the tissues of adult female Ascaris suum. The 5-HT turnover was highest in the intestine at 34.7 ng 5-HT produced/mg protein/hr and 13.8 ng 5-HT produced/mg protein/hr in muscle tissue. 2. The levels of 5-HT metabolites namely tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindole acetic acid and 5-hydroxytryptophol were measured in muscle and intestinal tissue of adult A. suum. 3. Parachlorophenylalanine inhibited 5-HT production in muscle and intestinal tissue providing in situ evidence for the presence of tryptophan hydroxylase in this tissue. 4. Pargyline increased 5-HT production in muscle and intestinal tissue providing in situ evidence for the presence of monoamine oxidase in this tissue.     

Viral genetic determinants of T-cell killing and immunodeficiency disease induction by the feline leukemia virus FeLV-FAIDS.

Within the fatal immunodeficiency disease-inducing strain of feline leukemia virus, FeLV-FAIDS, are viruses which range in pathogenicity from minimally (clone 61E is the prototype) to acutely pathogenic, most of the latter of which are also replication defective (clone 61C is the prototype). Mixtures of 61E and 61C virus and chimeras generated between them, but not 61E alone, killed feline T cells. T-cell killing depended on changes within a 7-amino-acid region near the C terminus of the gp70 env gene or was achieved independently by changes within a 109-amino-acid region encompassing the N terminus of gp70. The carboxy-terminal change was also sufficient for induction of fatal immunodeficiency disease in cats. Other changes within the 61C gp70 gene enhanced T-cell killing, as did changes in the long terminal repeat, the latter of which also enhanced virus replication. T-cell killing correlated with high levels of intracellular unintegrated and proviral DNA, all of which were blocked by treatment of infected cells with sera from 61C-immune cats or with a neutralizing monoclonal antibody. These findings indicate that T-cell killing is a consequence of superinfection and that the mutations in env critical to pathogenicity of the immunosuppressive variant result in a failure to establish superinfection interference in infected cells.     

Differential effects of parathyroid hormone and its analogues on cytosolic calcium ion and cAMP levels in cultured rat osteoblast-like cells

While the stimulatory effect of parathyroid hormone (PTH) on osteoblast-like cell adenylate cyclase is well known, the effect of PTH on cytosolic calcium ion ([Ca2+]i) mobilization is controversial, one group finding no effect but others reporting various increases. We investigated the effects on [Ca2+]i of synthetic rat PTH fragment 1-34 (rPTH(1-34)) and two bovine PTH analogues that inhibit PTH's stimulation of adenylate cyclase (bovine 8,18Nle, 34Tyr-PTH(3-34) and 34Tyr-PTH(7-34]. [Ca2+]i was measured before, during, and after exposure to PTH analogues in perifused, attached osteoblast-like rat osteosarcoma cells (ROS 17/2.8) that had been scrape-loaded with the luminescent photoprotein aequorin. Resting [Ca2+]i was 0.094 +/- 0.056 microM (mean +/- S.D., n = 103) and rose in a time- and dose-specific way after exposure to rPTH(1-34). At 10(-10) M rPTH(1-34), [Ca2+]i rose 100% within 30 s to a plateau; higher concentrations of PTH yielded increasing initial peaks of [Ca2+]i followed by lower plateaus. At 10(-6) M, the initial peak was 5-fold basal, or 0.64 +/- 0.07 microM. Both analogues of PTH were at least partial agonists for [Ca2+]i mobilization and did not reduce peak [Ca2+]i when co-perifused with rPTH(1-34). However, the analogues did reduce significantly rPTH(1-34)-induced cAMP accumulation and did not increase cAMP accumulation by themselves. Thus, rPTH(1-34) strongly mobilizes [Ca2+]i in ROS 17/2.8 cells, at near-physiologic concentrations. Failure of the PTH analogues to block the effect of PTH on [Ca2+]i while inhibiting the effect on cAMP accumulation suggests separate pathways for PTH activation of adenylate cyclase and mobilization of calcium.     

The bacterial flora of the intestine of Ascaris suum and 5-hydroxytryptamine production

Representative facultative anaerobes of the bacterial flora from the intestine of female Ascaris suum were isolated and identified. The number of bacteria in the intestine was approximately 4 X 10(9) per g wet weight of intestine. Seventeen of 19 of the isolated colonies were found to secrete 5-hydroxytryptamine in culture. Holding A. suum in an antibiotic-containing medium did not affect the levels of 5-hydroxytryptamine in the worm, which were 231 +/- 14 ng/g in antibiotic-media as compared to 250 +/- 16 ng/g in control media. This implied that the bacteria may not be contributing to the level of 5-hydroxytryptamine in the tissues of A. suum.