Effects of habitat on mercury concentrations in fish: a case study of Nile perch (Lates niloticus) in Lake Nabugabo, Uganda.

This study focused on variation in fish mercury (Hg) concentrations in 185 Nile perch (Lates niloticus) samples collected across four different habitat types in Lake Nabugabo, Uganda, a tropical lake located proximate to Lake Victoria. We quantified the stomach contents of Nile perch using the % index of relative importance, as well as, nitrogen and carbon isotopic concentrations to assess the role of diet and trophic level on Hg concentrations. In each habitat, we also evaluated a suite of chemical and physical characteristics that are commonly associated with variation in Hg bioavailability in temperate systems. Using linear mixed models and ANOVA, we demonstrate that habitat of capture is an important predictor of Hg concentrations in Nile perch from Lake Nabugabo and that the relationship between habitat and Hg is size and diet dependent. Nile perch diet as well as dissolved oxygen concentration and pH were also correlated with observed differences in fish Hg. Overall, Hg concentrations in Nile perch were all well below the WHO/FAO recommended guideline of 500 ng/g (mean 13.6 ± 0.4 ng/g wet weight; range 4.9 and 29.3 ng/g wet weight). This work contributes to a growing awareness of intra-lake divergence in Nile perch, as well as, divergence in Hg concentrations between varying aquatic habitat types, particularly wetlands.

Casein kinase II is required for cell cycle progression during G1 and G2/M in Saccharomyces cerevisiae.

The catalytic subunit of Saccharomyces cerevisiae casein kinase II (Sc CKII) is encoded by the CKA1 and CKA2 genes, which together are essential for viability. Five independent temperature-sensitive alleles of the CKA2 gene were isolated and used to analyze the function of CKII during the cell cycle. Following a shift to the nonpermissive temperature, cka2ts strains arrested within a single cell cycle and exhibited a dual arrest phenotype consisting of 50% unbudded and 50% large-budded cells. The unbudded half of the arrested population contained a single nucleus and a single focus of microtubule staining, consistent with arrest in G1. Most of the large-budded fraction contained segregated chromatin and an extended spindle, indicative of arrest in anaphase, though a fraction contained an undivided nucleus with a short thick intranuclear spindle, indicative of arrest in G2 and/or metaphase. Flow cytometry of pheromone-synchronized cells confirmed that CKII is required in G1, at a point which must lie at or beyond Start but prior to DNA synthesis. Similar analysis of hydroxyurea-synchronized cells indicated that CKII is not required for completion of previously initiated DNA replication but confirmed that the enzyme is again required for cell cycle progression in G2 and/or mitosis. These results establish a role for CKII in regulation and/or execution of the eukaryotic cell cycle.

Purification and characterization of casein kinase II (CKII) from delta cka1 delta cka2 Saccharomyces cerevisiae rescued by Drosophila CKII subunits. The free catalytic subunit of casein kinase II is not toxic in vivo.

Casein kinase II (CKII) is composed of a catalytic (alpha) and a regulatory (beta) subunit which unite to form an alpha 2 beta 2 holoenzyme. Saccharomyces cerevisiae CKII consists of two distinct catalytic (Sc alpha and Sc alpha') and regulatory (Sc beta and Sc beta') subunits. Simultaneous disruption of the CKA1 and CKA2 genes (encoding the alpha and alpha' subunits, respectively) is lethal. Such double disruptions can be rescued by GAL1, 10-induced expression of the Drosophila alpha and beta subunits (Dm alpha+beta) together or by GAL10-induced expression of the Drosophila alpha subunit (Dm alpha) alone (Padmanabha, R., Chen-Wu, J. L.-P., Hanna, D. E., and Glover, C. V. C. (1990) Mol. Cell. Biol. 10, 4089-4099). Here we report quantitation, purification, and characterization of casein kinase II activity from such rescued strains. Casein kinase II activity from a strain rescued by Dm alpha alone purifies as a free, catalytically active alpha subunit monomer, whereas that from a strain rescued by Dm alpha/beta purifies as a mixture of tetrameric holoenzyme and monomeric alpha subunit. Interestingly, neither Sc beta nor Sc beta' is present at detectable levels in the enzyme obtained from either strain, raising the possibility that rescue by Dm alpha alone may be mediated via the free, monomeric catalytic subunit. Overexpression of total casein kinase II activity from 6- to 18-fold is not toxic and indeed has no overt phenotypic consequences. Production of large amounts of free catalytic subunit also appears to be without effect, even though free catalytic subunit is normally undetectable in S. cerevisiae.

Expression and purification of the alpha and beta subunits of Drosophila casein kinase II using a baculovirus vector.

Two recombinant baculoviruses that express the alpha and beta subunits of Drosophila melanogaster casein kinase II, respectively, have been constructed. The expressed proteins are similar to the authentic Drosophila subunits in size and are recognized by antisera raised against the Drosophila holoenzyme. Extracts derived from cells infected with the alpha subunit-expressing virus display elevated casein kinase II activity in vitro. This activity is markedly enhanced in extracts of cells infected with both viruses, or when alpha and beta subunit-containing extracts are mixed in vitro following lysis. Recombinant holoenzyme and the alpha subunit were purified to near homogeneity using phosphocellulose column chromatography. The specific activity of the purified recombinant holoenzyme was very similar to that of the native enzyme, and was fivefold higher than that of the purified free alpha subunit. The Stokes radius of the recombinant holoenzyme was estimated to be 50 A, a value similar to that reported for the native enzyme, whereas the alpha subunit demonstrated a Stokes radius of 26.5 A. Studies using sucrose density gradient centrifugation showed that, under conditions of high ionic strength, the quaternary structure of the purified holoenzyme was tetrameric (like the native enzyme), whereas the structure of the alpha subunit was monomeric. At lower ionic strength the recombinant holoenzyme had a significantly higher sedimentation coefficient, characteristic of the formation of filaments found for the native enzyme. Interestingly, the purified catalytic subunit also displayed a higher S value under conditions of low ionic strength, revealing the formation of alpha subunit aggregates.

Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinase II is essential for viability in Saccharomyces cerevisiae.

Casein kinase II of Saccharomyces cerevisiae contains two distinct catalytic subunits, alpha and alpha', which are encoded by the CKA1 and CKA2 genes, respectively. Null mutations in the CKA1 gene do not confer a detectable phenotype (J. L.-P. Chen-Wu, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 8:4981-4990, 1988), presumably because of the presence of the CKA2 gene. We report here the cloning, sequencing, and disruption of the CKA2 gene. The alpha' subunit encoded by the CKA2 gene is 60% identical to the CKA1-encoded alpha subunit and 55% identical to the Drosophila alpha subunit (A. Saxena, R. Padmanabha, and C. V. C. Glover, Mol. Cell. Biol. 7:3409-3417, 1987). Deletions of the CKA2 gene were constructed by gene replacement techniques. Haploid cells in which the CKA2 gene alone is disrupted show no detectable phenotype, but haploid cells carrying disruptions in both the CKA1 and CKA2 genes are inviable. Cells in which casein kinase II activity is depleted increase substantially in size prior to growth arrest, and a significant fraction of the arrested cells exhibit a pseudomycelial morphology. Disruption of the activity also results in flocculation. Yeast strains lacking both endogenous catalytic subunit genes can be rescued by expression of the alpha and beta subunits of Drosophila casein kinase II or by expression of the Drosophila alpha subunit alone, suggesting that casein kinase II function has been conserved through evolution.

A new conjugating agent for radioiodination of proteins: low in vivo deiodination of a radiolabeled antibody in a tumor model.

A new radioiodinating agent, N-(p-[125I]iodophenyl)maleimide, has been synthesized for its potential utility in the radioiodination of monoclonal antibodies and other proteins. The efficiency of incorporation of 125I in the B72.3 antibody by iodine monochloride iodination or by maleimide conjugation was 19% and 43%, respectively. The thyroid uptake following intraperitoneal administration of the two 125I-labeled antibody preparations was evaluated in nude mice implanted with LS174T colon carcinoma xenografts. The iodine monochloride preparation showed substantially greater uptake in the thyroid with values of 2.1% ID at 6 h after injection and reaching a maximum of 4.3% ID after 6 days. In contrast, the maleimide preparation showed a uniformly low uptake in thyroid of 0.1%-0.2% ID. The results of these preliminary studies demonstrate that the N-(p-[125I]iodophenyl)maleimide-labeled monoclonal antibodies showed markedly less (less than 10-fold) uptake of radioiodine in the thyroid, indicating significantly less in vivo deiodination than iodine monochloride-labeled monoclonal antibodies, while retaining some tumor localization in vivo. Studies are in progress to optimize N-(p-[125I]iodophenyl)maleimide radioiodination conditions and to improve the tumor localization.

Comparative binding and preclinical localization and therapy studies with radiolabeled human chimeric and murine 17-1A monoclonal antibodies.

Murine MoAb 17-1A is an IgG2a antibody reactive with a gastrointestinal cancer-associated cell surface antigen. Human-mouse chimeric 17-1A MoAbs were constructed in which the murine variable region of 17-1A was joined with human IgG1, IgG2, IgG3, and IgG4 constant regions. Human-mouse IgG1, IgG2, and IgG4 chimeric antibodies were compared with the parental murine antibody and its F(ab')2 fragments for their ability to bind to colon carcinoma cells in vitro, for their blood clearance in normal nude mice, and for their localization and tumor growth inhibition of colon carcinoma xenografts in nude mice. Indirect immunofluorescence experiments with fluorescein-conjugated goat anti-mouse or goat anti-human antibody verified that the substitution of human constant regions in the chimeric MoAbs did not significantly alter the ability of the murine variable region to bind to colon adenocarcinoma cell lines (LS174T, SW948, and C0112). The immunoreactivities of 125I-labeled murine and chimeric 17-1A MoAbs measured in a live cell-binding assay with LS174T, SW948, and C0112 cells revealed that chimeric IgG1, IgG4, and 17-1A F(ab')2 were comparable to murine 17-1A while chimeric IgG2 showed lower binding. The blood half-lives of 125I-labeled murine 17-1A, its F(ab')2 fragments, and chimeric IgG1, IgG2, and IgG4 in normal nude mice determined by serial eye bleeding were 7.5, 0.5, 5.2, 6.9, and 1.9 days, respectively. In biodistribution studies at 4 days after injection of 125I-labeled MoAbs in nude mice bearing LS174T tumors, chimeric IgG1 had the highest tumor concentration of 20.5% injected dose/g with a tumor/blood ratio of 3.2. 131I-labeled murine 17-1A administered in a single injection of 300 microCi or 3 injections of about 300 microCi each to nude mice bearing established LS174T tumors inhibited tumor growth, whereas a comparable amount of unlabeled murine 17-1A did not inhibit tumor growth. 131I-labeled chimeric IgG1 MoAb showed a similar level of tumor growth inhibition. The results of the present study indicate that 17-1A chimeric IgG1 antibody may be the best choice for clinical radioimmunodetection and radioimmunotherapy studies.

Cytotoxic effects of anti-CD5 radioimmunotoxins on human tumors in vitro and in a nude mouse model.

An immunoconjugate, consisting of both toxin and radionuclide on the same antibody molecule, was synthesized by cross-linking the phytotoxin ricin to the T101 monoclonal antibody recognizing the CD5 cluster expressed on normal and malignant T-cells. The hybrid molecule was then labeled with iodine-125 by an iodine monochloride procedure. This radioimmunotoxin (RIT), which selectively bound to the CD5-positive CEM human leukemia cell line, was selectively inhibitory to antigen-positive cells in protein synthesis inhibition assays. RIT was only 3.0-7.8-fold less toxic and was 1.1-1.6-fold slower than unlabeled immunotoxin in inhibiting protein synthesis. Because of the radionuclide moiety, the RIT also provided information related to biodistribution and pharmacokinetics. Four days following intratumoral injection, more than 125-fold greater activity was found in CEM tumors implanted in nude mice as compared to normal tissues. The mean blood half-life for RIT was 25.7 h and for radiolabeled antibody, 91.3 h. Intratumoral injections of RIT selectively induced regression of established CEM tumors. To our knowledge, these studies are the first to demonstrate that a single immunoconjugate can combine the advantages of both a catalytic toxin and radionuclide for cancer therapy.

Localization and imaging with radioiodine-labeled monoclonal antibodies in a xenogeneic tumor model for human B-cell lymphoma.

Two MoAbs directed towards human B-cell malignancies have been studied in a preclinical animal model to evaluate their potential for in vivo imaging and therapy of B-cell lymphomas. Anti-B1 reacts with virtually all immunoglobulin-bearing malignancies and non-T acute lymphoblastic leukemia. Anti-J5 reacts with the common acute lymphoblastic leukemia antigen found on non-T acute lymphoblastic leukemia and follicular lymphomas. Anti-T1 which recognizes the CD5 antigen on most T-cell leukemias and lymphomas was used as a control antibody. These monoclonal antibodies were radiolabeled with 125I or 131I by the ICl method. Namalwa (B-cell) and MOLT-4 (T-cell) tumors were grown s.c. in irradiated nude mice. The highest tissue concentration of 125I-labeled anti-J5 in Namalwa-bearing mice was in blood and tumor. The tumor/blood ratio ranged from 0.7-1.2, with the highest ratio 4 days after injection. Pharmacokinetic analysis indicated that the t1/2 beta of anti-J5 from blood and other tissues ranged from 40-50 h, while the t1/2 beta for tumor averaged 65 h. The area under the curve of tumor was 2- to 5-fold higher than the area under the curve of liver, kidney, skin, and muscle. The peak tissue levels of 125I-labeled anti-B1 in Namalwa-bearing mice were again in blood and tumor and 6 days following injection more than 5-fold greater activity was found in tumor compared to normal tissues other than blood. The tumor/blood ratio was 1.2 and 0.7 at 4 and 6 days after injection. 125I-labeled anti-B1 showed minimal uptake in antigen-negative MOLT-4 tumors and 125I-labeled anti-T1 showed little uptake in Namalwa tumors. Scintigraphic images were obtained following the injection of 131I-labeled anti-J5 and anti-B1 in nude mice bearing Namalwa tumors. These results indicate that radiolabeled anti-J5 and anti-B1 show promise as diagnostic and possibly therapeutic agents for human B-cell lymphoma, although there may be a limitation to clinical utility due to cross-reactivity with some normal cells.

Human leukemia cell binding and killing by anti-CD5 radioimmunotoxins.

We have synthesized a reagent for antibody directed cell targeting composed of the monoclonal antibody (MoAb) T101 linked to the potent toxin ricin. The immunotoxin (IT) was subsequently radiolabeled by a cyclic anhydride procedure with 90Yttrium (90Y) to construct a radioimmunotoxin (RIT) that may have potential for cancer therapy. We evaluated the reagent for selectivity in binding and protein synthesis inhibition (PSI) assays. The RIT selectively bound antigen positive leukemia T-cell lines, with minimal binding to antigen negative control lines. The IT inhibited 87% or greater protein synthesis activity at 1 microgram/ml and exhibited an IC50 (the dose inhibiting 50% activity) of 0.18 +/- 0.08 microgram/ml in the presence of lactose. RIT and nonlabeled IT showed comparable degrees of PSI at 1 microgram/ml and 10 micrograms/ml, suggesting that labeling had little overall effect on the activity of the immunoconjugate. However, indirect evidence showed that the galactose binding site of ricin was inhibited 10-fold by its exposure to 90Y. Control RIT were minimally inhibitory. IT labeled with 131Iodine (131I) by an iodine monochloride technique also retained its capability to selectively inhibit protein synthesis. When RIT were tested for potency in a clonogenic assay against human leukemia T-cell lines, they inhibited 3.61 logs of tumor cell growth at 10 micrograms/ml. This did not represent an improvement over the log elimination with radiolabeled antibody alone, which showed 4.19 log elimination of tumor cells. Our observation that the 90Y-labeled RIT and labeled antibody can selectively eliminate about four logs of tumor cells in an in vitro clonogenic assay is unique. The ability of RIT to kill several logs of tumor cells in vitro renders RIT interesting anti-tumor reagents.

Hyperventilation syndrome.

Hyperventilation is a common, though often unrecognized, disorder of adolescents. While relatively benign, the lack of recognition may lead to extensive, expensive, and unnecessary medical work-ups. The single most important factor in making the diagnosis of HVS lies in the awareness of the disorder's existence. HVS may be diagnosed through a positive response to the provocation test. Patients are asked to hyperventilate and questioned as to whether they experience the symptoms of which they have complained. Successful treatment involves reassurance, education, and giving the patient a strategy for controlling the hyperventilation. If treatment is not successful in a short period, patients should be referred to a qualified mental health professional. While the relationship between hyperventilation and anxiety disorders is unclear, some correlation between them does appear to exist.

Antigenic modulation by anti-CD5 immunotoxins.

We evaluated the modulation of T101 immunotoxins (IT) and free T101 antibody from the surface of normal and leukemic cells to determine whether the presence of toxin on antibody affected antigenic modulation. Reagents were made by conjugating T101, which binds to the T cell antigen CD5, to either intact ricin or purified ricin A chain. We found that T101-A chain modulated CD5 more efficiently than T101-ricin, which modulated CD5 more efficiently than T101 alone. Kinetic studies showed that maximal modulation of IT was reached within 3 hr. When toxicity of the reagents was tested in protein synthesis inhibition assays, T101-ricin in the presence of lactose inhibited 99% of the protein synthesis of CEM cells. T101-A chain was less toxic, inhibiting protein synthesis only 23 to 43%. The addition of the potentiating agent monensin nearly doubled the toxicity of T101-A chain, but did not affect T101-A chain modulation. To determine the fate of bound IT, T101 and T101-ricin were labeled with 125I. Cells were incubated under modulating conditions in the presence of radiolabeled reagents. T101 and T101-ricin were internalized into CEM cells. In contrast, T101, but not T101-ricin, appeared to be shed from peripheral blood mononuclear cells. Our findings show clearly that: 1) the presence of toxin on antibody does not inhibit--and may actually enhance--modulation; 2) T101-IT are internalized, not shed from the cell surface; 3) the lack of toxicity of T101-A chain is not attributed to inability to modulate; 4) there is no correlation between enhancement of T101-A chain toxicity by monensin and antigenic modulation by A chain reagents; and 5) modulation, which is undesirable in monoclonal antibody therapy, may be advantageous in the therapeutic use of IT.

Radiolabeling of monoclonal antibody against carcinoembryonic antigen with 88Y and biodistribution studies.

The biodistribution of the 202 monoclonal antibody against CEA labeled with 88Y by the bicyclic DTPA anhydride method was studied in normal Balb/c mice. The in vitro binding to 1 X 10(7) CO112, LS174T and WiDR colon cancer cells was 21.0, 27.3 and 18.8%, respectively. The binding to an equal number of KM-3 leukemia cells and normal human lymphocytes was 8.9 and 3.2%, respectively. Liver, spleen, kidney and blood were the tissues that showed the highest uptake of radiolabeled antibody in vivo.