Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Effects of environmentally hazardous chemicals on the emergence and early growth of selected Australian plants

The effect of soil-incorporated copper, tri-allate, and anthracene on the emergence and early growth of three Australian native species (Banksia ericifolia, Casuarina distyla andEucalyptus eximia) and three crop species (Avena sativa, Cucumis sativus andGlycine max), was assessed using OECD Test Guideline 208. The crop species are sensitive species used in overseas phytotoxicity testing, and their responses were compared with those of the native species. Seeds were grown in pots in a glasshouse in a sandy loam soil at the chemical concentrations of 0, 10, 100, 1000 and 2000 mg kg^–1. LC50 and EC50 values were determined for each species. The most sensitive species was the monocotyledonA. sativa, while among the five dicotyledonsC. distyla was most sensitive. All three chemicals delayed emergence and affected seedling growth. The results indicate that the conditions of the OECD Test Guideline can be met under Australian conditions, but that the Guideline requires modification for use with Australian native species.     

Growth and respiration in two mangrove species at a range of salinities

Growth and dark respiration rates were measured in leaves and roots of seedlings of Avicennia marina (Forsk.) Vierh, (grey mangrove), and Aegiceras corniculatum (L.) Blanco (river mangrove). Plants were grown in a soil mixture at ambient temperatures and watered with 0.25 and 100% sea-water. Oxygen uptake was measured in excised root and leaf samples. In both species growth was maximal in 25% sea-water, and root respiration was lowest in 100% sea-water. Differences were found between the two species in the responses of leaf respiration to salinity. In A. corniculatum leaf respiration was raised in both 25 and 100% sea-water, while in A. marina only leaves in 100% sea-water showed higher rates of respiration. These results are consistent with the view that A. marina is the more salt-tolerant of the two species. In A. corniculatum the respiration rates of the hypocotyl were also measured, and were much higher in 100% sea-water than in the other two treatments. The results suggest that at high salinities there is a high metabolic cost in the shoots of both species, and that at such salinities rates of root respiration may be limited by the supply of substrate from the shoots.     

Evaluation of a vermicompost and leachates on <i>Solidago x hybrida</i> and organic carbon mineralization under aerobic incubations

In the floriculture region of Tenancingo in the State of Mexico, the application of stabilized organic matter, such as vermicompost and leachates, contributes to improve the quality of the soil and plant nutrition. However, it is important to know the chemical composition of a vermicompost and the mineralization process. This is because the amount and speed of nutrient release which will be available to the crop will depend on that knowledge. The objective of the study was to evaluate the effect of the application of a vermicompost and leachates on various quantitative variables of Solidago x hybrid, and the mineralization of organic carbon under aerobic incubations. Vermicompost (74 and 36 g/kg soil), leachates (5 and 10 L/kg soil), 0.33 g/kg soil of chemical fertilizer Ca (NO₃)₂, and not treated soil (control) were applied under greenhouse conditions to evaluate their effects on plant growth variables. Mixtures of 100 g of soil with vermicompost and leachates were made in the laboratory which were incubated during 9 weeks to obtain the potentially mineralizable organic carbon (Corg PM) and the rate of mineralization (k) after adjusting an exponential model. In the greenhouse experiment there were no statistical differences after applying vermicompost and leachates on the quantitative variables (number of stems per plant, diameter of the panicle, fresh weight, plant length and stem diameter) with respect to the control (p>0.05). The effect among the applied doses was evident only for variables such as fresh weight, panicle length and stem diameter with respect to the control. In incubated soils, k values ranged between 0.209-0.325 C mg/kg soil/week. Only with the application of leachates in high doses two pools of organic matter were shown: one soluble labile (102.9 mg/kg soil) and the other hydrolysable (819 mg/kg soil). The soluble, labile fraction favored nutrient availability immediately after its application to the soil. However, a single pool of hydrolysable organic C (987-1074 mg/kg soil) was found when vermicompost was applied. It was associated with a release of organic matter during crop development, and to a possible stimulation of microbial activity. High values of electrical conductivity in vermicompost and leachates (8.2-11.7 mS/m) suggest a moderate application of both products.     

Viruses: agents of coral disease?

The potential role of viruses in coral disease has only recently begun to receive attention. Here we describe our attempts to determine whether viruses are present in thermally stressed corals Pavona danai, Acropora formosa and Stylophora pistillata and zoanthids Zoanthus sp., and their zooxanthellae. Heat-shocked P. danai, A. formosa and Zoanthus sp. all produced numerous virus-like particles (VLPs) that were evident in the animal tissue, zooxanthellae and the surrounding seawater; VLPs were also seen around heat-shocked freshly isolated zooxanthellae (FIZ) from P. danai and S. pistillata. The most commonly seen VLPs were tail-less, hexagonal and about 40 to 50 nm in diameter, though a diverse range of other VLP morphotypes (e.g. rounded, rod-shaped, droplet-shaped, filamentous) were also present around corals. When VLPs around heat-shocked FIZ from S. pistillata were added to non-stressed FIZ from this coral, they resulted in cell lysis, suggesting that an infectious agent was present; however, analysis with transmission electron microscopy provided no clear evidence of viral infection. The release of diverse VLPs was again apparent when flow cytometry was used to enumerate release by heat-stressed A. formosa nubbins. Our data support the infection of reef corals by viruses, though we cannot yet determine the precise origin (i.e. coral, zooxanthellae and/or surface microbes) of the VLPs seen. Furthermore, genome sequence data are required to establish the presence of viruses unequivocally.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

High sero-prevalence of caseous lymphadenitis identified in slaughterhouse samples as a consequence of deficiencies in sheep farm management in the state of Minas Gerais, Brazil

Background

Multiple congenital ocular anomalies (MCOA) syndrome is a hereditary congenital eye defect that was first described in Silver colored Rocky Mountain horses. The mutation causing this disease is located within a defined chromosomal interval, which also contains the gene and mutation that is associated with the Silver coat color (PMEL17, exon 11). Horses that are homozygous for the disease-causing allele have multiple defects (MCOA-phenotype), whilst the heterozygous horses predominantly have cysts of the iris, ciliary body or retina (Cyst-phenotype). It has been argued that these ocular defects are caused by a recent mutation that is restricted to horses that are related to the Rocky Mountain Horse breed. For that reason we have examined another horse breed, the Icelandic horse, which is historically quite divergent from Rocky Mountain horses.

Results

We examined 24 Icelandic horses and established that the MCOA syndrome is present in this breed. Four of these horses were categorised as having the MCOA-phenotype and were genotyped as being homozygous for the PMEL17 mutation. The most common clinical signs included megaloglobus, iris stromal hypoplasia, abnormal pectinate ligaments, iridociliary cysts occasionally extending into the peripheral retina and cataracts. The cysts and pectinate ligament abnormalities were observed in the temporal quadrant of the eyes. Fourteen horses were heterozygous for the PMEL17 mutation and were characterized as having the Cyst-phenotype with cysts and occasionally curvilinear streaks in the peripheral retina. Three additional horses were genotyped as PMEL17 heterozygotes, but in these horses we were unable to detect cysts or other forms of anomalies. One eye of a severely vision-impaired 18 month-old stallion, homozygous for the PMEL17 mutation was examined by light microscopy. Redundant duplication of non-pigmented ciliary body epithelium, sometimes forming cysts bulging into the posterior chamber and localized areas of atrophy in the peripheral retina were seen.

Conclusions

The MCOA syndrome is segregating with the PMEL17 mutation in the Icelandic Horse population. This needs to be taken into consideration in breeding decisions and highlights the fact that MCOA syndrome is present in a breed that are more ancient and not closely related to the Rocky Mountain Horse breed.     

The antidepressant-like effect of Ocimum basilicum in an animal model of depression

We investigated the efficacy of Ocimum basilicum (OB) essential oils for treating depression related behavioral, biochemical and histopathological changes caused by exposure to chronic unpredictable mild stress (CUMS) in mice and to explore the mechanism underlying the pathology. Male albino mice were divided into four groups: controls; CUMS; CUMS plus fluoxetine, the antidepressant administered for pharmacological validation of OB; and CUMS plus OB. Behavioral tests included the forced swim test (FST), elevated plus-maze (EPM) and the open ?eld test (OFT); these tests were performed at the end of the experiment. We assessed serum corticosterone level, protein, gene and immunoexpression of brain-derived neurotropic factor (BDNF) and glucocorticoid receptors (GRs) as well as immunoexpression of glial fibrillary acidic protein (GFAP), Ki67, caspase-3 in the hippocampus. CUMS caused depression in the mice as evidenced by prolonged immobility in the FST, prolonged time spent in the open arms during the EPM test and reduction of open field activity in the OFT. OB ameliorated the CUMS induced depressive status. OB significantly reduced the corticosterone level and up-regulated protein and gene expressions of BDNF and GR. OB reduced CUMS induced hippocampal neuron atrophy and apoptosis, and increased the number of the astrocytes and new nerve cells. OB significantly increased GFAP-positive cells as well as BDNF and GR immunoexpression in the hippocampus.