Specific immunosuppression of IgE response to hapten DNP by DNP linked to monoclonal IgG1 in rats

Summary The induction of the anti-DNP IgE in rat was suppressed by pretreatment of rats with the tolerogen synthesized by coupling DNP to rat IgG, i.e.; DNP_7–10-IgG. It was found that DNP₁₀-IgG₁ was an effective tolerogen, whereas other DNP conjugates, i.e. DNP₉-IgM, DNP₉-IgA, DNP₁₀-IgE, DNP₁₀-IgG_2c and DNP₁₀-IgG_2a were ineffective.This work was partly supported by a research grant from the Medical Council of Canada to W.Y.L.     

Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes

Tolerance against oxidative stress generated by high light intensities or the catalase inhibitor aminotriazole (AT) was induced in intact tobacco plants by spraying them with hydrogen peroxide (H₂O₂). Stress tolerance was concomitant with an enhanced antioxidant status as reflected by higher activity and/or protein levels of catalase, ascorbate peroxidase, guaiacol peroxidases, and glutathione peroxidase, as well as an increased glutathione pool. The induced stress tolerance was dependent on the dose of H₂O₂ applied. Moderate doses of H₂O₂ enhanced the antioxidant status and induced stress tolerance, while higher concentrations caused oxidative stress and symptoms resembling a hypersensitive response. In stress-tolerant plants, induction of catalase was 1.5-fold, that of ascorbate peroxidase and glutathione peroxidase was 2-fold, and that of guaiacol peroxidases was approximately 3-fold. Stress resistance was monitored by measuring levels of malondialdehyde, an indicator of lipid peroxidation. The levels of malondialdehyde in all H₂O₂-treated plants exposed to subsequent high light or AT stress were similar to those of unstressed plants, whereas lipid peroxidation in H₂O₂-untreated plants stressed with either high light or AT was 1.5- or 2-fold higher, respectively. Although all stress factors caused increases in the levels of reduced glutathione, its levels were much higher in all H₂O₂-pretreated plants. Moreover, significant accumulation of oxidized glutathione was observed only in plants that were not pretreated with H₂O₂. Extending the AT stress period from 1 to 7 days resulted in death of tobacco plants that were not pretreated with H₂O₂, while all H₂O₂-pretreated plants remained little affected by the prolonged treatment. Thus, activation of the plant antioxidant system by H₂O₂ plays an important role in the induced tolerance against oxidative stress. Received 11 December 2001; received after revision 25 January 2002; accepted 4 February 2002     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B₃ to bovine serum albumin. Aflatoxin B₃ was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B₁ (AFB₁) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B₁, B₂, G₁, and G₂ when tritiated AFB₁ was used as the marker ligand. The concentrations causing 50% inhibition of binding of³H-AFB₁ to the antibodies by unlabeled aflatoxins B₁, B₂, G₁, G₂ and B₃ were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B₁ and G₁, two of the most important toxic metabolites produced byAspergillus flavus andA. parasiticus.     

Radioimmunological determination of prostaglandin D2 synthesis in human thrombocytes

Summary A specific radioimmunoassay for prostaglandin D₂ was developed. Using the radioimmunoassay, prostaglandin D₂ synthesis by human thrombocytes was measured. While the cyclooxygenase inhibitor indomethacin inhibits formation of prostaglandin D₂, increased formation of prostaglandin D₂ was observed in the presence of the thromboxane synthetase inhibitor imidazole.This work was supported by the Deutsche Forschungsgemeinschaft.     

Effects of pCai and pHi on cell-to-cell coupling

Summary Internal longitudinal resistance (r_i), a determinant of cardiac conduction, is affected by changes in intracellular calcium and protons. However, the role and mechanism by which H⁺ and Ca²⁺ may modulate r_i is uncertain. Cable analysis was performed in cardiac Purkinje fibers to measure r_i during various interventions. In some experiments, intracellular pH (pH_i) was recorded simultaneously to study the pH_i-r_i relation. Both intracellular Ca²⁺ and H⁺ independently modified r_i. However, internal resistance of cardiac fibers was insensitive to pH_i changes compared to other tissues. A latent period preceded the pH_i-related changes in r_i and the amount of change depended upon methodology. The results suggest that direct action of protons on r_i may be subordinate to other regulatory processes. Ionic regulation of internal longitudinal resistance may occur by more than one mechanism: i) direct cationic binding to sites on junctional membrane proteins; and ii) H⁺- or Ca²⁺-dependent phosphorylation of junctional proteins.     

Production and characterization of antibody against aflatoxin M1

Summary Antibody against aflatoxin M₁ was obtained after immunization of rabbits with bovine serum albumin-afla M₁ oxime conjugate. The antibody has greatest binding efficiency for afla M₁, and was less efficient for afla B₁. Cross-reaction of antibody with aflatoxin Q₁, aflatoxicol, and aflatoxin B_2a was weak. Aflatoxin B₂, G₁, and G₂ and afla B₁-guanine adduct showed almost no cross-reaction with the antibody. The sensitivity of the binding assay for aflatoxin M₁ detection is in the range of 1–10 ng per assay. Detailed methods for the preparation of the conjugate, production of immune serum, and methods for antibody determination are described.Supported by the College of Agricultural and Life Sciences, North Central Regional project NC-129, the University of Wisconsin-Madison, and by Public Health Service research grant number CA 15064 from the National Cancer Institute, NIH.The authors wish to thank Dr R.C. Garner for providing aflatoxin-B-guanine adduct, and Dr Dennis H. Hseih for providing aflatoxicol and aflatoxin Q₁.     

Role of the ubiquitin–proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y₁₃ receptor (P2Y₁₃-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y₁₃-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y₁₃-R. When transiently expressed, P2Y₁₃-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y₁₂ receptor (P2Y₁₂-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y₁₃-R in the ER, suggesting a close link between ubiquitination of P2Y₁₃-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y₁₃-R and P2Y₁₂-R strongly restored plasma membrane expression of P2Y₁₃-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y₁₃-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y₁₃-R in hepatocytes, and consequently stimulated P2Y₁₃-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y₁₃-R-deficient mice, thus reinforcing the role of P2Y₁₃-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin–proteasome system might be a novel powerful HDL therapy to enhance P2Y₁₃-R expression and consequently promote the overall RCT.     

Über die Wirkung von Sr-Ionen auf den Ca2+-Austausch am Meerschweinchenvorhof

Summary When CaCl₂ in Tyrode solution was replaced by SrCl₂,⁴⁵Ca efflux from guinea-pig auricles did not change significantly. In Na-free solution, however,⁴⁵Ca influx was reduced when, in addition to CaCl₂, SrCl₂ was present in the solution, suggesting an equivalent replacement of Ca by Sr at membrane sites.     

Is ATP synthesized by a vacuolar-ATPase in the extremely halophilic bacteria?

The proton-dependent synthesis of ATP was demonstrated in representative members of the generaHalobacterium, Haloarcula, andHaloferax. In all cases, synthesis was not inhibited by nitrate or N-ethylmaleimide, inhibitors of the vacuolar-like ATPase found in Archaea, but was affected by azide, an inhibitor of F₀F₁-ATP syntheses. These observations extend the earlier observations withHalobacterium saccharovorum and suggest that ATP synthesis in these organisms is brought about by an F₀F₁-APT synthase.     

Plasma triiodothyronine,thyroxine and thyrotrophin levels in germfree rats

Summary Plasma T₃, T₄ and TSH levels in developing germfree rats were high, low and normal as compared with those in conventional counterparts. The high T₃/T₄ ratio in germfree rat plasma was lowered by cholestyramine feeding.This work was supported in part by a grant from the Imanaga Foundation, Nagoya, Japan.     

Effect of iron and hemoproteins on hydrogen peroxide-supported styrene oxidation to styrene oxide

Summary Fe²⁺, Fe³⁺ and their complexes with EDTA and hemin, methemalbumin and methemoglobin were active catalyzers of H₂O₂ supported styrene oxidation to styrene oxide. Methemoglobin was the most active compound; its peroxidative activity was comparable to that of cytochrome P-450 in liver microsomes of phenobarbital-treated rats. Cumene hydroperoxide supported styrene oxidation with methemoglobin and microsomal hemoproteins and was found to be more efficient than H₂O₂.This work was supported by C.N.R. (National Research Council) contract No. 79.03197.04.     

Superoxide dismutase activity during the plasmodial life cycle ofPhysarum polycephalum

Summary Superoxide dismutase activity was slow throughout the cell cycle of surface cultures ofPhysarum polycephalum. This activity increased markedly when the organism was induced to spherulate. Glutathione (GSH) and hydrogen peroxide (H₂O₂) concentrations changed very little during the cell cycle. During spherulation GSH decreased; H₂O₂ and the cyanide-resistant respiration of plasmodial homogenates increased.     

Sigmoid shape of the oxygen equilibrium curve and the P50 of human hemoglobin

The fractional saturation of hemoglobin with oxygen was plotted against P/P₅₀ and the slope of the abscissa at 1 was calculated for 38 OEC data sets of human Hb A. There was a linear correlation between the slope and the Hill coefficient (n_max), and the slope was about one-forth that of n_max. This implies that the slope of the abscissa at 1 of Y vs P/P₅₀ plot can provide information about the magnitude of cooperativity in hemoglobin oxygen binding.     

Interchange trisomics in pearl millet

Summary In the progeny of interchange heterozygotes ofPennisetum typhoides, 2 interchange trisomic plants were obtained. These were selfed over 2 generations and the cytology of the parents, s₁ and s₂ plants was studied. The types of multiple associations in the s₁ and s₂ generations differed from those of the parent. A significant decrease in mean chiasma frequency was noted in s₁ and s₂.Acknowledgment. The author is grateful to Prof. em. J. V. Pantulu, Department of Botany, Andhra University, Waltair, for his suggestions and encouragement.     

Cell-to-cell coupling studied in isolated ventricular cell pairs

Summary Cell pairs isolated from adult rat and guinea pig ventricles were used to study the electrical properties of the nexal membrane. Each cell of a pair was connected to a voltage-clamp system so as to enable whole-cell, tight-seal recording. The current-voltage relationship of the nexal membrane was found to be linear, revealing a resistance r_n of 2–4 M. r_n was insensitive to the sarcolemmal membrane potential (range:–90 to +30 mV), and exerted no time-dependent gating behavior (range: 0.1 to 10 s). Lowering pH_i yielded a small increase in r_n. Vigorous elevations in [Ca²⁺]_i gave rise to an increase in r_n which was associated with a cell shortening. Uncoupling caused by aliphatic alcohols or halothane did not produce cell shortening. Cell pairs were also used to study action potential transfer.     

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H₂ O₂ ) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incu-bated with 3 mM or higher H₂ O₂ concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expres-complex sion of cyclin D1 and D2 upon H₂ O₂ treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H₂ O₂ was not due to induction of protein synthesis. These results indicate that H₂ O₂ reversibly inhibits the ubiquitin-proteasome dependent degra-dation of cyclin D1 and D2, probably by transiently in-hibiting ubiquitination and/or the proteasome. Received 12 March 2001; received after revision 5 April 2001; accepted 9 April 2001     

Efficacy of tumor cell vaccine after incorporating monophosphoryl lipid A (MPL) in tumor cell membranes containing tumor-associated ganglioside

Murine B16 melanoma expresses the ganglioside. GM₃. GM₃ shed from tumor cells is immunosuppressive and promotes tumor growth¹. Reduction or elimination of the shed GM₃ could be therapeutic, and the anti-GM₃ antibodies may reduce and clear the shed ganglioside. To test this hypothesis, mice were challenged with tumor cells, with or without inducing anti-GM₃ antibody response. Since gangliosides are poor immunogens and T-cell independent antigens, an adjuvant (monophosphoryl lipid A (MPL), a non-toxic lipid A ofSalmonella), directed against B-cells, was employed. MPL was incorporated onto liposomes and into the surface membrane of B16 mouse melanoma cells; both are rich in GM₃. C57BL/6J mice immunized with MPL-liposomes or MPL-B16 cells responded with elevated levels of anti-GM₃ IgM. Non-immunized mice or mice immunized with B16 cells alone or ganglioside GM₃ alone (without MPL) elicited poor anti-GM₃ IgM response, confirming the GM₃'s immunologic crypticity and MPL's immunopotentiating effect. MPL's immunopotentiating effect was improved by coupling it to melanoma cell membranes C57BL/6J mice were immunized with irradiated B16 alone or MPL alone or MPL-conjugated irradiated B16. After three weekly immunizations, each mouse received a challenge dose of viable syngeneic B16. Neither MPL alone nor B16 alone had a significant effect on tumor growth or host survival; however, administration of MPL-conjugated B16 cells significantly prevented tumor growth and prolonged survival. Our results indicate that MPL-incorporated B16 cells augment the anti-GM₃ IgM response, which may reverse GM₃-induced immunosuppression by eliminating tumor-derived GM₃, and restore immunocompetence.     

Effects of photoperiod,temperature and testosterone-treatment on plasma T3 and T4 levels in the Djungarian hamster,Phodopus sungorus

Summary The effects of photoperiod, temperature and testosterone treatment on plasma T₃ and T₄ levels were investigated in the Djungarian hamster. Plasma T₃ level was affected by temperature (25°C<7°C) but not by photoperiod. Plasma T₄ level was affected by photoperiod (short day < long day) at 25°C. Administration of testosterone increased plasma T₄ level under short photoperiod at 25°C. Thus, higher plasma T₄ level under long photoperiod at 25°C might be induced by testosterone.     

Composition of epicuticular wax of rice,Oryza sativa

Summary The epicuticular wax of rice, varietyRibe, comprised n-alkanes, esters, aldehydes and free alcohols. The nalkanes contained 4 major chain lengths, C₂₇, C₂₉, C₃₁ and C₃₃. Triacontanal and dotriacontanal were the major aldehydes. Octacosanol comprised 89% of the free alcohols. The esters were mainly esters of C₁₆ to C₂₄ acids with C₂₂ to C₃₀ alcohols.This research was supported by the Consiglio Nazionale delle Ricerche, Rome.