Distribution of endoplasmic reticulum and calciosome markers in membrane fractions isolated from different regions of the canine brain

Four regions of the canine brain (frontal lobe, parieto-occipital lobe, brainstem, and cerebellum) were each fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). Markers of endoplasmic reticulum (glucose-6-phosphate phosphatase and rotenone-insensitive NADPH cytochrome c reductase) and markers of the 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store ([3H]IP3 binding and IP3-induced Ca2+ release) were measured. No correlation was found between the two classes of markers, which suggests that the IP3 receptor does not belong to the endoplasmic reticulum in canine brain. Cerebellum P2 and P3 fractions displayed levels of [3H]IP3 binding 10- to 30-fold higher, and rates of IP3-induced Ca2+ release greater than 15-fold faster than the homologous cerebrum and brainstem fractions. Actively accumulated Ca2+ was only partially released by IP3, both before and after saponin disruption of the plasma membrane compartment. The proportion of the IP3-sensitive Ca2+ store relative to that of the total (IP3-sensitive and IP3-insensitive) Ca2+ store was variable; i.e., it was larger in cerebellum P2 (approximately 90%) than in cerebrum fractions (less than 30%). Cerebellum fractions constitute the best source from which an IP3-sensitive Ca2+ storing organelle can be purified.     

Protein oxidative damage and redox imbalance induced by ionising radiation in CHO cells

Reactive oxygen species (ROS) are important mediators of the cytotoxicity induced by the direct reaction of ionising radiation (IR) with all critical cellular components, such as proteins, lipids, and nucleic acids. The derived oxidative damage may propagate in exposed tissues in a dose- and spatiotemporal dependent manner to other cell compartments, affecting intracellular signalling, and cell fate. To understand how cell damage is induced, we studied the oxidative events occurring immediately after cell irradiation by analysing the fate of IR-derived ROS, the intracellular oxidative damage, and the modification of redox environment accumulating in Chinese hamster ovary (CHO) within 1?h after cell irradiation (dose range 0–10?Gy). By using the immuno-spin trapping technique (IST), spectrophotometric methods, and electron paramagnetic resonance (EPR) spectroscopy, we showed that IR-derived ROS (i) induced an IST-detectable, antioxidant-inhibitable one-electron oxidation of specific intracellular proteins; (ii) altered the glutathione (GSH) content (which was found to increase below 2?Gy, and decrease at higher doses, leading to a redox imbalance); (iii) decreased glutathione peroxidase and glutaredoxin activity; (iv) modified neither glutathione reductase nor thioredoxin reductase activity; (v) were detected by spin trapping technique, but adduct intensity decreased due to cell competition for ROS; and (vi) induced no EPR-detectable radicals assignable to oxidised cellular components. In conclusion, our results showed that IR generated an early high oxidising potential (protein radical intermediates, redox imbalance, modified redox enzyme activity) in irradiated cells potentially able to propagate the damage and induce oxidative modification of secondary targets.     

Leaf age affects the efficacy of insecticides to control Asian citrus psyllid,Diaphorina citri (Hemiptera: Liviidae)

Huanglongbing (HLB), the most destructive citrus disease worldwide, is associated with the bacteria transmitted by the psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae). One of the main tactics used to manage this disease is the chemical control of D. citri. In this study, the efficacy of four insecticides commonly used by the citrus growers against the D. citri adults was assessed in two vegetative growth stages of flush: young shoots (~ 10 cm in length) and mature leaves. The bioassays were carried out under the greenhouse and field conditions. The insecticides bifenthrin, dimethoate, imidacloprid and thiamethoxam were tested at the recommended rates of 15, 450, 40 and 25 mg/L of water, respectively. The insecticides were sprayed onto the leaves of each stage until runoff. Psyllid mortality was assessed at different periods after insecticide application (0, 1 and 2 weeks). In both greenhouse and field trees, the period of control was shorter in the young shoots than in the mature leaves. In addition, the efficacy of insecticides rapidly decreased in the young shoots over time, mainly for imidacloprid and bifenthrin (less than 1 week). Thus, these results suggest that the number of spray applications to control D. citri should be more frequent during the flushing period.     

Correlation between synthesis and methylation of DNA in HeLa cells

For the whole cell cycle the methylation of DNA was studied in synchronized $\text{HeLa}$ cells and in nuclei isolated from them. In the intact cells the methylation of DNA cytosine runs parallel to DNA synthesis. The pattern of DNA cytosine methylation by the isolated nuclei is almost identical to that obtained with the whole cells. Since the isolated nuclei do not synthesize DNA, it is shown that DNA methylation continues for at least 30 min after DNA synthesis is over. No DNA minor thymine is found in the isolated nuclei.     

Ultrastructure of the developing thymus of the leopard frog (Rana pipiens)

Summary A study of the ultrastructure of the developing thymus of the leopard frog (Rana pipiens) revealed that the thymus had undergone all of the major changes which would persist through larval life and metamorphosis by the time that the animals had reached larval stage IV of Taylor and Kollros (1946). These changes included development of an outer, lymphoid cortical region and an inner, essentially nonlymphoid medulla; mitotic activity among lymphoid cell precursors and the formation of the first small lymphocytes; development of complex cysts containing PAS-positive material and the appearance of other signs of secretory activity among epithelial cells of the medulla; and differentiation of large myoid cells containing bundles of striated muscle fibrils. The changes are particularly noteworthy because they first appear during a period in which the animals are known to be developing the capacity to respond immunologically to allografts.Supported by a grant from the National Institutes of Health number GM-11782 to E.P.V.     

Cytokine expression and ultrastructural alterations in fresh-frozen,freeze-dried and γ-irradiated human amniotic membranes

The aim of this work was to compare the effects on human amniotic membrane of freeze-drying and γ-irradiation at doses of 10, 20 and 30 kGy, with freezing. For this purpose, nine cytokines (interleukin 10, platelet-derived growth factor-AA, platelet-derived growth factor-BB, basic fibroblast growth factor, epidermal growth factor, transforming growth factor beta 1, and tissue inhibitors of metalloproteinase-1, -2, and -4) were titrated in 5 different preparations for each of 3 amniotic membranes included in the study. In addition, the extracellular matrix structure of each sample was assessed by transmission electron microscopy. While freeze-drying did not seem to affect the biological structure or cytokine content of the different amniotic membrane samples, γ-irradiation led to a significant decrease in the tissue inhibitors of metalloproteinase-4, basic fibroblast growth factor and epidermal growth factor, and induced structural damage to the epithelium, basement membrane and lamina densa. The higher the irradiation dose the more severe the damage to the amniotic membrane structure. In conclusion, the Authors recommend processing amniotic membrane under sterile conditions to guarantee safety at every step rather than final sterilization with γ-irradiation, thereby avoiding alteration to the biological characteristics of the amniotic membrane.