Acute effect of aflatoxin B1 on different inbred mouse strains II

The acute effects of Aflatoxin B₁(AFB₁) were evaluated on C57B1/6, CBA/J, B10A and Balb/c mice challenged with a single intraperitoneal dose of the mycotoxin (60 mg/Kg animal weight). 90 mice per strain were divided into three groups of 30 animals each: the intoxicated group and control groups I and II. Intoxicated mice were injected intraperitonealy with AFB₁ dissolved in corn oil, while control I mice received corn oil only (0.01 ml/g) by the same route. Lots of 10 animals from the intoxicated and control groups were sacrificed 24, 72 and 168 hours after challenge. Control mice II remained untreated and were used as standards of normality for biochemical (hepatic and renal function) and hematological evaluation. AFB₁ was detected in the liver of C57B1/6 and CBA/J mice 24 hours (1.46 and 0.75 ng/g, respectively), 72 hours (2.30 and 0.08 ng/g, respectively), and 168 hours (2.18 and 0.25 ng/g, respectively) after challenge. The mycotoxin was also observed in the liver of B10A mice (6.20 ng/g) 72 hours post-injection. The most evident histological lesions were observed 168 hours after treatment in C57B1/6 and B10A mice. Serum levels of alkaline phosphatase in intoxicated C57B1/6 and B10A mice were significantly higher than those of control I and II animals. The histopathologic lesions and biochemical changes were very discrete in Balb/c and CBA/J mice. It is included that strains C57B1/6 and B10A are more susceptible than strains CBA/J and Balb/c to the acute effects of AFB₁. Such difference probably reflects each strains's ability to biotransform and eliminate AFB₁ and its metabolites.     

Reversibility of chilling injury to corn seedlings

Seedlings of corn (Zea mays) were tested for recovery from chilling injury incurred at 0.3 ± 0.3 C. At 0.3 C visual leaf injury appeared in 36 hours, whereas stem and root injuries appeared later. Appearance of leaf injury was preceded by a rise in O₂ uptake and a lessened effect of 2,4-dinitrophenol on O₂ uptake by leaf segments and was accompanied by increased ion leakage from the leaves. These effects were reversible, in that transfer of seedlings to 21 C after 36 hours at 0.3 C produced a return of O₂ uptake, 2,4-dinitrophenol stimulation, and ion leakage to the levels of unchilled leaves, as well as a disappearance of leaf symptoms, within 72 hours. For most seedlings, transfer to 21 C after 48 to 60 hours at 0.3 C reversed the chilling effects on O₂ uptake, 2,4-dinitrophenol stimulation, and injury symptoms but not on ion leakage within 108 hours. However, some seedlings collapsed during 48 to 60 hours of chilling, and these never recovered. Transfer to 21 C after 72 hours at 0.3 C did not produce recovery from any symptom of chilling injury examined, and these seedlings soon died. No growth occurred at 0.3 C, but growth began soon after transfer to 21 C. Seedlings chilled 24 or 36 hours grew at reduced rates during the first 72 hours at 21 C, but within 96 hours at 21 C were growing at the same rate as nonchilled seedlings. These results demonstrate considerable capacity of growing plants to recover from short chilling treatments even though significant physiological changes occurred at low temperatures.     

Effect of temperature on production of aflatoxin on rice by Aspergillus flavus

Summary The effect of temperature on formation of aflatoxin on solid substrate (rice) byAspergillus flavus NRRL 2999 has been studied in some detail. The optimum temperature for production of both aflatoxin B₁ and G₁ under the conditions employed is 28° C. Comparable yields of B₁ were obtained at 32° C, but considerably less G₁ was produced at this temperature. Both B₁ and G₁ were found in lesser amounts at temperatures above 32° C, and the aflatoxin content of rice incubated at 37° C was low (300–700 ppb) even though growth was good.Reducing the temperature from 28° to 15° C resulted in progressively less aflatoxin, but 100 ppb of B₁ was detected in cultures incubated 3 weeks at 11° C. No aflatoxin was produced at 8° C.The ratio of the four aflatoxins is affected by temperature. At the lower temperatures, essentially equal amounts of aflatoxin B₁ and G₁ were produced, whereas at 28° C, approximately four times as much B₁ was detected as G₁. At the higher temperatures, relatively less G was formed, until at 37° C, less than 10 ppb was detected.This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

The intensity of respiration and heat emission from seedlings of Festuca pratensis (Hud.) and Hordeum vulgare L. during pathogenesis caused by Bipolaris sorokiniana (Sacc.) Shoem

The presented work was conducted on seedlings of spring barley and meadow fescue which differ in the degree of sensitivity to leaf spot pathogen Bipolaris sorokiniana (Sacc.) Shoem. The seedling reaction to inoculation with mycelium and conidia was examined in glasshouse conditions on the basis of respiration intensity and heat production. The leaf respiration was measured using Clark-type electrode, while heat emission was evaluated by means of isotermic microcalorimeter. The measurements were performed after 1, 3, 6, 10, 24, 48, 72, 168 and 240 hours since the inoculation moment. Leaves of meadow fescue were characterized by the most intense respiration at the 6^th hour, while barley leaves at the 24^th and 72^nd hour after inoculation. In the case of meadow fescue the greatest heat production was noted in the period between 24 and 168 hours after inoculation. Simultaneously, at the 48^th hour the smallest rate of respiration was observed. Barley leaves emitted the greatest amount of heat only in the first 3 hours of the pathogenesis. In these hours the smallest respiration rate was noted. The observed, opposing reaction of respiration intensity and heat emission in the infected seedlings of both species may illustrate a disorder in metabolic processes in plants during pathogenesis. The plants studied differed in the time of their reaction to pathogen attack: barley responded earlier in heat production, while fescue extended respiration rate in the first hours after inoculation. This is clearly observable, when coefficients of metabolic inefficiency (heat rates per mole O₂) are compared. In the case of barley the highest rates were noticed just after inoculation, whereas in fescue at the 48^th hour. In both species attack of pathogen caused high metabolic efficiency.     

Selective vitamin B12 malabsorption in two siblings

Two siblings with megaloblastic anemia responsive to parenteral vitamin B₁₂ were studied to elucidate the cause of the B₁₂ deficiency. Gastric juice from both contained acid and functional intrinsic factor. Serum contained transcobalamin II and lacked antibodies to intrinsic factor. Schilling tests showed vitamin B₁₂ malabsorption uncorrected by hog intrinsic factor or pancreatic extract. Other parameters of small intestinal function were normal. Proteinuria was initially present in both but cleared in one following treatment with B₁₂. These patients with “familial selective vitamin B₁₂ malabsorption” are the first reported from Canada. Only 37 cases have been reported in the world literature to date.     

Appearance of an Alternate Pathway Cyanide-resistant during Germination of Seeds of Cicer arietinum

The combined action of the inhibitors antimycin A and cyanide with benzohydroxamic acid indicates the presence of a cyanide-resistant pathway of respiration in chick pea (Cicer arietinum L.) seeds. The appearance of this pathway takes place during germination. During the first 12 hours of germination, the respiration is predominantly cyanide-sensitive, showing after this time a shift to an “alternate” respiration which is sensitive to benzohydroxamic acid, reaching the maximal cyanide resistance between 72 and 96 hours of germination. The appearance of the alternate pathway is initiated by high O₂ concentrations and depends on cytoplasmic protein synthesis, since its appearance is inhibited by cycloheximide but not by chloramphenicol. Actinomycin D has no effect on the appearance of the alternate pathway. Our results indicate, in agreement with other authors, that the branching point is located between the flavoproteins and cytochromes b, probably at the level of ubiquinone, but the possibility of more than one branching point of the electron flow is also considered.     

The heat shock protein hsp70 binds in vivo to subregions 2-48BC and 3-58D of the polytene chromosomes ofDrosophila hydei

Multiple interactions of members of the hsp70 family with cellular components have already been described. We present, however, the first evidence that upon heat shock treatment hsp70 molecules interact with specific chromosomal subdivisions of the polytene chromosomes ofDrosophila hydei. After a heat shock treatment of 20 min the protein binds to subdivision 3-58D₁ and to the heat shock inducible subdivisions 2-48B_3–6 and 2-48C_1–2. Hsp70 molecules were also observed in subdivision 3-58D₁ during recovery at 25°C but not in subdivisions 2-48B_3–6 and 2-48C_1–2. Our data suggest that this interaction is stress specific. DNase and RNase experiments suggest, moreover, that the hsp70 molecules bind to RNA from ribonucleoproteins (RNPs) in subdivisions 2-48B_3–6 and 2-48C_1–2 and to DNA in subdivision 3-58D₁. The DNA sequences in subdivision 3-58D₁ seem to have the potential to adopt the Z-DNA conformation.     

Nucleic Acid Metabolism in Germinating Onion: I. Changes in Root Tip Nucleic Acid during Germination

Nucleic acid synthesis in the G₁ cell population of the 1-millimeter apex of the Allium cepa embryo was studied during the initial 73 hours of germination. Quantitative data indicate that the total amount of RNA per cell began to increase after 18 hours of germination while the initial DNA per cell increase did not occur until some 20 hours later. Polyacrylamide gel electrophoresis patterns of ³H-uridine-labeled total nucleic acid samples indicated that synthesis of all detectable RNA fractions present in the pre-emergent 1-millimeter apex (i.e., cytoplasmic and “chloroplast-like” RNA) began at approximately the same time (18 hours). Synthesis of the various cytoplasmic RNA fractions continued throughout the germination period. Data indicating synthesis of the “chloroplast-like” RNA were obtained only for the initial 36 hours of germination. Specific radioactivity of ³H-uridine-labeled total nucleic acid increased during the first 41.5 hours of germination but then decreased while the accumulation of RNA per cell continued to increase throughout the 73-hour period. In addition, a method is described which reduced the bacterial contamination of Allium seed to a level not detectable by incorporation of radioactive precursors into bacterial ribosomal RNA.     

Mobilization of respiratory metabolism in potato tubers by carbon dioxide

Applying high concentrations of CO₂ to whole potato tubers stimulated a rapid and pronounced respiratory gas exchange, which persisted for a prolonged time. The upsurge in respiration was proportional to the applied CO₂ concentrations and was further augmented by high O₂ levels. Tests using whole potatoes, or potato tissue slices from tubers previously treated with CO₂, indicated that the rapid CO₂-induced respiration is sensitive to cyanide during the first 24 hours of CO₂ application. The respiratory rise cannot be attributed to the emergence of a cyanide-resistant alternative electron transport pathway, although prolonged applications of CO₂, up to 72 hours, led to a gradual development of the pathway. CO₂-stimulated respiration was accompanied by a pronounced decline in the content of starch and glucose 6-phosphate, suggesting an active utilization of respiratory substrates. The ATP content in the CO₂-treated potatoes increased markedly, resembling similar increases in tissues undergoing respiratory upsurge.     

Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells

Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10⁶ cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF₅₀ and DMF₃₇) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective “off the shelf” therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia.     

Validation and Comparison of the Therapeutic Efficacy of Boron Neutron Capture Therapy Mediated By Boron-Rich Liposomes in Multiple Murine Tumor Models

Boron neutron capture therapy (BNCT) was performed at the University of Missouri Research Reactor in mice bearing CT26 colon carcinoma flank tumors and the results were compared with previously performed studies with mice bearing EMT6 breast cancer flank tumors. Mice were implanted with CT26 tumors subcutaneously in the caudal flank and were given two separate tail vein injections of unilamellar liposomes composed of cholesterol, 1,2-distearoyl-sn-glycer-3-phosphocholine, and K[nido-7-CH₃(CH₂)₁₅–7,8-C₂B₉H₁₁] in the lipid bilayer and encapsulated Na₃[1-(2`-B₁₀H₉)-2-NH₃B₁₀H₈] within the liposomal core. Mice were irradiated 30 hours after the second injection in a thermal neutron beam for various lengths of time. The tumor size was monitored daily for 72 days. Despite relatively lower tumor boron concentrations, as compared to EMT6 tumors, a 45 minute neutron irradiation BNCT resulted in complete resolution of the tumors in 50% of treated mice, 50% of which never recurred. Median time to tumor volume tripling was 38 days in BNCT treated mice, 17 days in neutron-irradiated mice given no boron compounds, and 4 days in untreated controls. Tumor response in mice with CT26 colon carcinoma was markedly more pronounced than in previous reports of mice with EMT6 tumors, a difference which increased with dose. The slope of the dose response curve of CT26 colon carcinoma tumors is 1.05 times tumor growth delay per Gy compared to 0.09 times tumor growth delay per Gy for EMT6 tumors, indicating that inherent radiosensitivity of tumors plays a role in boron neutron capture therapy and should be considered in the development of clinical applications of BNCT in animals and man.     

Varying patterns of protein synthesis in bread wheat during heat shock

High temperatures during seedling growth are considered as one of the factors that can modify surviving properties in wheat (Triticum aestivum L.) plant. This work attempts to evaluate the heat shock responses of seedling of winter wheat (Bezostaya-1) using growth parameters (seedling length, embryonal root length and embryonal root number), membrane stability index (MSI) and two dimensional (2D) gel electrophoresis analysis of heat shock proteins (HSPs) during heat shock. Seedlings grown until first leaf opening at controlled conditions (23 degrees C, 200 micromol m(-2) s(-1), 16h day/8h night, 50-60% humidity) were exposed to 37 degrees C or 45 degrees C high temperatures for 2, 4 and 8 hours. While 37 degrees C did not cause any significant change, 45 degrees C heat treatments caused significant decrease in terms of seedling and root length, and leaf MSI for all exposure times. However, all the plants from 45 degrees C heat treatments continued to grow during recovery period. 2D protein analysis indicated that 37 degrees C, 8 hours exposure caused stronger and more diverse heat shock response than the other treatments, followed by 37 degrees C, 4 hours, 45 degrees C, 8 hours, 45 degrees C, 4 hours, 45 degrees C, 2 hours treatments. 5 protein spots, ranging from 6-7.8 pl (isoelectric point) and 27-31.7 kDA molecular weight, were expressed at 37 degrees C, 2 hours and continued at 37 and 45 degrees C for all exposure times. This suggests that these early proteins and other newly synthesized proteins may have protective effects at 37 and 45 degrees C and provide plants for healthy growth during the recovery period.     

水淹和恢复对凤丹光合特性和根系活力的影响

选用3年生油用牡丹凤丹(Paeonia ostii)实生苗为试验材料,以常规供水管理的凤丹苗为对照,测定凤丹光合特性以及根系活力和形态随着水淹和水淹恢复处理时间的变化,探究牡丹的耐湿机制。结果表明:(1)水淹后,凤丹叶片净光合速率、蒸腾速率、气孔导度及根系活力均较对照组显著下降,且下降幅度随着水淹时间的延长而增大;在持续水淹过程中,叶片胞间CO_2浓度则呈先下降后上升的变化趋势;水淹168h时,根系活力在所测定指标中下降幅度最大,仅为对照组的2.34%,此时根系变黑且腐烂,大部分根系脱落。(2)凤丹叶片净光合效率的下降在水淹120h过程中与气孔限制性调节密切相关,而到水淹168h时非气孔限制性成为光合作用的限制因子。(3)水淹72h后进行恢复处理120h后,叶片净光合速率、蒸腾速率、胞间CO_2浓度和根系活力均显著上升;恢复处理168h后,净光合速率和胞间CO_2浓度恢复到正常的生长水平,同时绝大部分根尖呈现白色且发育良好,根系生长向正常的生命状态恢复。研究表明,油用牡丹凤丹对水淹胁迫的应答迅速且灵敏,虽不耐水淹,但恢复能力强,这为其栽培管理以及在相对湿润地区推广种植提供了理论依据。     

T-lymphocyte mediated cytolysis: Temperature dependence of killer cell dependent and independent phases and lack of recovery from the lethal hit at low temperatures

Using alloantiserum and complement to inactivate cytolytic T-lymphocytes after they had administered the “lethal hit” to target cells, the rate of killer-cell independent lysis (KCIL) as measured by radiochromium release was followed at various temperatures. Under usual conditions, KCIL was half-completed on the average after 1.7 hr at 37 °C. The average Q₁₀ of KCIL is about 1.6 during the first few hours after cooling, but near 0 °, lysis slows down at later times. Thus, the extent of KCIL after 6–8 hr at 0 ° is frequently less than one-tenth of that at 37 °C. The Q₁₀ of the whole killing process is 2.5 near 37 °C but exceeds 6 near 22 °C.Evidence has been presented elsewhere suggesting that recovery from complement mediated damage may occur under appropriate conditions. Since KCIL can largely be arrested at low temperatures, we tested for possible recovery from or repair of the T-cell administered “lethal hit” during incubations at low temperature following (i) inactivation of killer cells by antiserum and complement or (ii) detachment of killer cells with EDTA and prevention of subsequent killer-target cell contact with dextran. No evidence for recovery from the “lethal hit” was found during incubations from 0.3 to 5 hr at 20 °, 15 °, or 0 °C. The temperature dependence of KCIL raises the possibility that metabolic events are of importance during KCIL. However, the previous finding that lysis following damage mediated by antiserum and complement is equally temperature sensitive leaves no basis for postulating such metabolic events. Hence, although unequivocal direct evidence has been difficult to obtain, colloid osmotic lysis is at present the simplest and most plausible explanation of killer-cell independent lysis.     

Role of Third Serum Vitamin B12 Binding Protein in Vitamin B12 Transport

A third vitamin B₁₂ binding protein present in normal serum has been shown to participate in transport of labelled vitamin B₁₂ absorbed from the gut. All three vitamin B₁₂ binding proteins in serum were labelled at the same time after oral administration of vitamin B₁₂, implying that “free” vitamin B₁₂ reached the portal blood from the gut mucosa.     

Intracellular Endothelin Type B Receptor-driven Ca2+ Signal Elicits Nitric Oxide Production in Endothelial Cells

Endothelin-1 exerts its actions via activation of ET_A and ET_B G_q/11 protein-coupled receptors, located in the plasmalemma, cytoplasm, and nucleus. Although the autocrine/paracrine nature of endothelin-1 signaling has been extensively studied, its intracrine role has been largely attributed to interaction with receptors located on nuclear membranes and the nucleoplasm. Because ET_B receptors have been shown to be targeted to endolysosomes, we used intracellular microinjection and concurrent imaging methods to test their involvement in Ca²⁺ signaling and subsequential NO production. We provide evidence that microinjected endothelin-1 produces a dose-dependent elevation in cytosolic calcium concentration in ET_B-transfected cells and endothelial cells; this response is sensitive to ET_B but not ET_A receptor blockade. In endothelial cells, the endothelin-1-induced Ca²⁺ response is abolished upon endolysosomal but not Golgi disruption. Moreover, the effect is prevented by inhibition of microautophagy and is sensitive to inhibitors of the phospholipase C and inositol 1,4,5-trisphosphate receptor. Furthermore, intracellular endothelin-1 increases nitric oxide via an ET_B-dependent mechanism. Our results indicate for the first time that intracellular endothelin-1 activates endolysosomal ET_B receptors and increase cytosolic Ca²⁺ and nitric oxide production. Endothelin-1 acts in an intracrine fashion on endolysosomal ET_B to induce nitric oxide formation, thus modulating endothelial function.     

Trypanosoma cruzi Response to Sterol Biosynthesis Inhibitors: Morphophysiological Alterations Leading to Cell Death

The protozoan parasite Trypanosoma cruzi displays similarities to fungi in terms of its sterol lipid biosynthesis, as ergosterol and other 24-alkylated sterols are its principal endogenous sterols. The sterol pathway is thus a potential drug target for the treatment of Chagas disease. We describe here a comparative study of the growth inhibition, ultrastructural and physiological changes leading to the death of T. cruzi cells following treatment with the sterol biosynthesis inhibitors (SBIs) ketoconazole and lovastatin. We first calculated the drug concentration inhibiting epimastigote growth by 50% (EC₅₀/72 h) or killing all cells within 24 hours (EC₁₀₀/24 h). Incubation with inhibitors at the EC₅₀/72 h resulted in interesting morphological changes: intense proliferation of the inner mitochondrial membrane, which was corroborated by flow cytometry and confocal microscopy of the parasites stained with rhodamine 123, and strong swelling of the reservosomes, which was confirmed by acridine orange staining. These changes to the mitochondria and reservosomes may reflect the involvement of these organelles in ergosterol biosynthesis or the progressive autophagic process culminating in cell lysis after 6 to 7 days of treatment with SBIs at the EC₅₀/72 h. By contrast, treatment with SBIs at the EC₁₀₀/24 h resulted in rapid cell death with a necrotic phenotype: time-dependent cytosolic calcium overload, mitochondrial depolarization and reservosome membrane permeabilization (RMP), culminating in cell lysis after a few hours of drug exposure. We provide the first demonstration that RMP constitutes the “point of no return” in the cell death cascade, and propose a model for the necrotic cell death of T. cruzi. Thus, SBIs trigger cell death by different mechanisms, depending on the dose used, in T. cruzi. These findings shed new light on ergosterol biosynthesis and the mechanisms of programmed cell death in this ancient protozoan parasite.     

Adaptation to CO(2) Level and Changes in the Phosphorylation of Thylakoid Proteins during the Cell Cycle of Chlamydomonas reinhardtii

The photosynthetic performance of synchronously grown Chlamydomonas reinhardtii alternated rhythmically during the cell cycle. The activity of the “CO₂ concentrating mechanism” including the ability to accumulate CO₂ internally and the activity of carbonic anhydrase peaked after 6 to 9 hours of light and reached minimum after 6 to 9 hours of dark. Consequently, the apparent photosynthetic affinity to extracellular CO₂ alternated rhythmically. At the end of the dark period the cells behaved as if they were adapted to high CO₂ even though they were continuously aerated with air. Results from experiments in which the light or dark periods were extended bear on the interaction between the internal (cell cycle or biological clock) and the external (light) signal. The observed rhythmical alterations in photosynthetic V_max may result from changes in PSII activity. The latter may be partly explained by the capacity for phosphorylation of thylakoid proteins, which reached maximum after 9 hours of light and decreased toward the dark period.