Transmission of porcine circovirus 2 (PCV2) by semen and viral distribution in different piglet tissues

Porcine circovirus infections are caused by the porcine circovirus 2 (PCV2). Among six different clinical manifestations involving respiratory, enteric, nervous and reproductive signs, the postweaning multisystemic wasting syndrome (PMWS) is the most important and studied disease. However, reproductive failures associated with PCV2 have been increasingly reported. Some studies have shown the possible contamination of sows by semen of PCV2 positive boars. In order to investigate the transmission of PCV2 by contaminated semen and its ability to infect the sow and piglets, 20 PCV2 negative sows were inseminated, 10 with negative boar semen and 10 with previously nested-PCR tested positive boar semen. The sows were weekly monitored and blood samples were collected. Based on the results, 4 out 20 sows were selected (1 sow was PCR negative and inseminated with a negative semen, 2 sows were PCR negative and inseminated with a positive semen and 1 sow was PCR negative and inseminated with a positive semen, but became PCR positive around the 30 days of pregnancy). After weaning, 12 male piglets, 3 of each sow, were selected and maintained under isolation. In order to investigate which organs harbored the virus, the young pigs were necropsied around 9 months of age. Samples of serum collected monthly were tested by immunocitochemistry (ICC), and all 12 pigs serum converted. Samples of lymphoid, systemic and reproductive organs were analyzed by nested-PCR and immunohistochemistry (IHC). Evaluation of the samples by nested-PCR, revealed that several tissues were positive in 10 of 12 pigs, mainly the lymph nodes, bone marrow and spleen. Various samples were positive by IHC in 8 of 12 piglets, being the lymph nodes, tonsils and bulbourethral glands the most frequently positive. Thus, the results of testing different samples, in the 3 tests (ICC, nested-PCR and IHC) were complementary. These results show that PCV2 transmission through semen to the sows and piglets may occur and may also represent a potential risk for the herd.

Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues

Eighteen young steers were inoculated with Toxoplasma gondii and randomly distributed into three groups of six animals each: GI, 2.5x10(5) "P" strain oocysts, GII, 5.0x10(6) "RH" strain tachyzoites, and GIII (Control). Clinical, serological and parasitemia exams were realized. Parasite investigation by bioassay and PCR was realized on semen and fragments of skeletal musculature, lymph nodes, brain, retina, spleen, liver, lung, testicle, epididymis and seminal vesicle. Blood and semen samples were collected on days -2, -1, 1, 3, 5, 7, 14 and weekly thereafter, up to postinfection day (PID) 84. The inoculated steers (GI and GII) presented hyperthermia from PID 3 to 16. Antibodies against T. gondii were detected through the indirect fluorescence antibody test (IFAT) on PID 5 (1:16) in both inoculated groups (oocysts and tachyzoites), reaching peaks of 1:4096 on PID 7. Parasitemia outbursts occurred in all infected bovines, principally from PID 7 to 28, independent of the strain and inoculate used. Bioassays revealed the presence of parasites in semen samples of animals infected with oocysts (GI) and tachyzoites (GII) on several experimental days between PID 7 and 84. Tissue parasitism by T. gondii was diagnosed by bioassay and the PCR technique in several organ and tissue fragments. These findings suggest the possibility of sexual transmission of T. gondii in the bovine species.

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Microwave-assisted solvent extraction and analysis of shikimic acid from plant tissues

A better method for determination of shikimate in plant tissues is needed to monitor exposure of plants to the herbicide glyphosate [N-(phosphonomethyl)glycine] and to screen the plant kingdom for high levels of this valuable phytochemical precursor to the pharmaceutical oseltamivir. A simple, rapid, and efficient method using microwave-assisted extraction (MWAE) with water as the extraction solvent was developed for the determination of shikimic acid in plant tissues. High performance liquid chromatography was used for the separation of shikimic acid, and chromatographic data were acquired using photodiode array detection. This MWAE technique was successful in recovering shikimic acid from a series of fortified plant tissues at more than 90% efficiency with an interference-free chromatogram. This allowed the use of lower amounts of reagents and organic solvents, reducing the use of toxic and/or hazardous chemicals, as compared to currently used methodologies. The method was used to determine the level of endogenous shikimic acid in several species of Brachiaria and sugarcane (Saccharum officinarum) and on B. decumbens and soybean (Glycine max) after treatment with glyphosate. The method was sensitive, rapid and reliable in all cases.

Allelopathic effect of black mustard tissues and root exudates on some crops and weeds

Laboratory and greenhouse experiments were conducted to evaluate the phytotoxic effect of black mustard extracts and root exudates on two crops: Trifolium alexandrinum and Triticum aestivum, and two weeds: Phalaris paradoxa and Sisymbrium irio. The seeds were treated with aqueous and ethanolic extracts and chloroform for eight days, or subjected to root exudates of just harvested mustard in a greenhouse for five weeks. High-performance liquid chromatography (HPLC) was used to quantify phytotoxins from plant tissues. Seed germination of P. paradoxa was reduced with the lowest concentration of the different extracts. However, the aqueous extract at 4% completely curtailed the germination of all the target species. In general, plant extracts had a concentration-dependent reduction of seedling growth of the target species. However, the ethanolic extract, at the lowest concentration, has stimulated the shoot length of both T. alexandrinum and T. aestivum, and the root length of the former. Mustard root exudates inhibited emergence and growth of the target species throughout the experiment. Ferulic and syringic acids were the dominant allelochemicals found when HPLC was used.

Storage substances in the androgametogenesis and mature pollen grain of Ilex paraguariensis St. Hil. (Aquifoliaceae)

Storage substances such as starch grains, proteins and lipids were studied during the male gametogenesis and in the mature pollen grain of Ilex paraguariensis St. Hil. (Aquifoliaceae). There are two cycles of amylogenesis and amylolyse. The first cycle lasts until the vacuolated stage when the starch is hydrolyzed and amorphous proteins are stored inside the single vacuole. The next cycle begins after mitosis with the formation of the vegetative and generative cells. At this point, the young vegetative cell stores many starch grains that are bigger than in the first cycle. During the maturation of the male gametophyte, the starch is hydrolyzed and it is absent in the mature pollen grain. Small lipid droplets surround the young generative cell after the mitosis of the androspore and are dispersed in the vegetative cytoplasm during its maturity. The relationship between the pollen storage substances and the ontogeny of the layers in the sporoderm, formation of the generative cell, and the male germ unit were discussed.

Leaf anatomy of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)

Immature and mature leaves of juvenile and adult plants of Araucaria angustifolia (Araucariaceae) were observed with the objective of updating the morphoanatomical data of the leaves of this species, which were restricted to basic descriptions in previous studies. The observations, made in optical allowed to establish anatomical differences among mature leaves of juvenile and adult plants in relation to the number of palisade parenchimal layers, the number of compartmented cells and the transfusion tissue development. Epidermis, the albuminous cells, the phloem, and the transfusion tissue descriptions are in disagreement with the data obtained data by different authors. The epidermal tissue and the hypodermis differ entirely when the plant is still juvenile, being inferred that these tissues would soon perform the protection function against mechanical damages and water loss, the vital characteristics during the first development months of young offspring.

Diurnal variations of non-structural carbohydrates in vegetative tissues of Melinis minutiflora, Echinolaena inflexa and Lolium multiflorum (Poaceae)

Echinolaena inflexa (Poir.) Chase is a native C3 grass species with high biomass production and abundance in the cerrado comparable to Melinis minutiflora Beauv., a C4 forage grass of African origin, widespread in the cerrado, displacing some native herbaceous species. These species accumulate mainly starch, differently of Lolium multiflorum Lam. ssp. italicum Beck cv. Lema (ryegrass), a C3 temperate forage grass that accumulates fructans as the major storage carbohydrate in the vegetative tissues. In the present study, we analyzed diurnal variations in the content and composition of non-structural carbohydrates (NSC) in the aerial vegetative parts of the three species. Results showed similarity in water-soluble carbohydrates (WSC) and starch contents between Melinis minutiflora and Echinolaena inflexa, with the exotic grass accumulating higher amounts of NSC. However, the levels of sucrose, glucose and fructose were 10 fold higher in Lolium multiflorum, that accumulates fructan as the main storage carbohydrate. Although the contents of WSC and starch were similar in the tropical species E. inflexa and M. minutiflora, their diurnal variations were different and could be related to starch metabolism, especially in M. minutiflora.

Allelopathic interference of Sapindus saponaria root and mature leaf aqueous extracts on diaspore germination and seedling growth of Lactuca sativa and Allium cepa

Sapindus saponaria (soapberry) is a species that presents a great diversity of chemical compounds, such as saponins; however, few studies have examined the allelopathic effect of this species. Therefore, this study provides an evaluation of the allelopathic potential of aqueous extracts of the roots and mature leaves of S. saponaria on the germination of diaspores and seedlings growth of lettuce (Lactuca sativa) and onion (Allium cepa). The aqueous extract was prepared in the proportion of 100 g of dry plant material in 1,000 mL of distilled water (a concentration of 10% w v-1), and diluted with distilled water to 7.5, 5.0 and 2.5% concentrations. The mature leaf extracts caused delay and decrease in the germination process of the lettuce and onion diaspores, with inhibitory effect concentration-dependent, while the root extracts showed no allelopathic effects on the germination process. Both extracts caused abnormalities and inhibited the growth of shoot and root seedlings.

Transient expression of a reporter gene introduced by bioballistic bombardment into Racosperma mangium (Leguminosae family) tissues

We report on an assay of direct transfer of DNA into calli and seeds of Racosperma (ex-Acacia) mangium, using a bioballistic method. We observed transient expression of the GUS gene in the treated tissues

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK) by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

Gender differences in the activities of aspirin-esterases in rat tissues

The activities of aspirin (acetylsalicylic acid)-esterases were measured in several tissues (liver, kidney, adrenal glands, brain and serum) from adult male and female Wistar rats. In males, both aspirin-esterase I (assayed at pH 5.5) and II (assayed at pH 7.4) activities were higher in liver homogenates when compared to females (aspirin-esterase I: males 48.9 ± 4.8 (N = 8) and females 29.3 ± 4.2 (N = 8) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 41.4 ± 4.1 (N = 8) and females 26.1 ± 4.5 (N = 8) nmol of salicylic acid formed min-1 mg protein-1, P<0.001). In serum, enzyme activity was higher in females than in males (aspirin-esterase I: males 0.85 ± 0.06 (N = 6) and females 1.18 ± 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 1.03 ± 0.13 (N = 6) and females 1.34 ± 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, P<0.001). In the other tissues assayed, no statistically significant difference between males and females was found. There were no statistically significant differences when the enzymes were assayed in different phases of the estrous cycle in liver and serum. These results show that the differences in aspirin-esterase activity observed between males and females are not due to the estrous cycle. The gender difference obtained in our study may indicate an involvement of gonadal hormones in the control of the hydrolysis of aspirin. This possibility is currently under investigation.

Effect of Walker 256 tumor growth on intestinal absorption of leucine, methionine and glucose in newly weaned and mature rats

In tumor-bearing rats, most of the serum amino acids are used for synthesis and oxidation processes by the neoplastic tissue. In the present study, the effect of Walker 256 carcinoma growth on the intestinal absorption of leucine, methionine and glucose was investigated in newly weaned and mature rats. Food intake and carcass weight were decreased in newly weaned (NT) and mature (MT) rats bearing Walker 256 tumor in comparison with control animals (NC and MC). The tumor/carcass weight ratio was higher in NT than in MT rats, whereas nitrogen balance was significantly decreased in both as compared to control animals. Glucose absorption was significantly reduced in MT rats (MT = 47.3 ± 4.9 vs MC = 99.8 ± 5.3 nmol min-1 cm-1, Kruskal-Wallis test, P<0.05) but this fact did not hamper the evolution of cancer. There was a significant increase in methionine absorption in both groups (NT = 4.2 ± 0.3 and MT = 2.0 ± 0.1 vs NC = 3.7 ± 0.1 and MC = 1.2 ± 0.2 nmol min-1 cm-1, Kruskal-Wallis test, P<0.05), whereas leucine absorption was increased only in young tumor-bearing rats (NT = 8.6 ± 0.2 vs NC = 7.7 ± 0.4 nmol min-1 cm-1, Kruskal-Wallis test, P<0.05), suggesting that these metabolites are being used for synthesis and oxidation processes by the neoplastic cells, which might ensure their rapid proliferation especially in NT rats.

Pp2a: At The Crossroads Between Growth and Defence In Plants

Living organisms manage their resources in well evolutionary-preserved manner to grow and reproduce. Plants are no exceptions, beginning from their seed stage they have to perceive environmental conditions to avoid germination at wrong time or rough soil. Under favourable conditions, plants invest photosynthetic end products in cell and organ growth to provide best possible conditions for generation of offspring. Under natural conditions, however, plants are exposed to a multitude of environmental stress factors, including high light and insufficient light, drought and flooding, various bacteria and viruses, herbivores, and other plants that compete for nutrients and light. To survive under environmental challenges, plants have evolved signaling mechanisms that recognise environmental changes and perform fine-tuned actions that maintain cellular homeostasis. Controlled phosphorylation and dephosphorylation of proteins plays an important role in maintaining balanced flow of information within cells. In this study, I examined the role of protein phosphatase 2A (PP2A) on plant growth and acclimation under optimal and stressful conditions. To this aim, I studied gene expression profiles, proteomes and protein interactions, and their impacts on plant health and survival, taking advantage of the model plant Arabidopsis thaliana and the mutant approach. Special emphasis was made on two highly similar PP2A-B regulatory subunits, B’γ and B’ζ. Promoters of B’γ and B’ζ were found to be similarly active in the developing tissues of the plant. In mature leaves, however, the promoter of B’γ was active in patches in leaf periphery, while the activity of B’ζ promoter was evident in leaf edges. The partially overlapping expression patterns, together with computational models of B’γ and B’ζ within trimeric PP2A holoenzymes suggested that B’γ and B’ζ may competitively bind into similar PP2A trimmers and thus influence each other’s actions. Arabidopsis thaliana pp2a-b’γ and pp2a-b’γζ double mutants showed dwarfish phenotypes, indicating that B’γ and B’ζ are needed for appropriate growth regulation under favorable conditions. However, while pp2a-b’γ displayed constitutive immune responses and appearance of premature yellowings on leaves, the pp2a-b’γζ double mutant supressed these yellowings. More detailed analysis of defense responses revealed that B’γ and B’ζ mediate counteracting effects on salicylic acid dependent defense signalling. Associated with this, B’γ and B’ζ were both found to interact in vivo with CALCIUM DEPENDENT PROTEIN KINASE 1 (CPK1), a crucial element of salicylic acid signalling pathway against pathogens in plants. In addition, B’γ was shown to modulate cellular reactive oxygen species (ROS) metabolism by controlling the abundance of ALTERNATIVE OXIDASE 1A and 1D in mitochondria. PP2A B’γ and B’ζ subunits turned out to play crucial roles in the optimization of plant choices during their development. Taken together, PP2A allows fluent responses to environmental changes, maintenance of plant homeostasis, and grant survivability with minimised cost of redirection of resources from growth to defence.

Incorporation of dietary trans monounsaturated fatty acids into tissues of Walker 256 tumor-bearing rats

The correlation between dietary trans fatty acids and neoplasia was examined in the present study. Walker 256 tumor-bearing and control rats were fed a trans monounsaturated fatty acid (MUFA)-rich diet for 8 weeks and the incorporation of trans fatty acids by tumor tissue was examined. Also, the effect of tumor growth on trans fatty acid composition of plasma and liver, and the content of thiobarbituric acid-reactive substances (TBARS) was determined. Walker 256 tumor cells presented both trans and cis MUFAs given in the diet. The equivalent diet proportions were 0.66 for trans and 1.14 for cis. Taking into consideration the proportion of trans MUFAs in plasma (11.47%), the tumor incorporated these fatty acids in a more efficient manner (18.27%) than the liver (9.34%). Therefore, the dietary trans fatty acids present in the diet are actively incorporated by the tumor. Tumor growth itself caused marked changes in the proportion of polyunsaturated fatty acids in the plasma and liver but provoked only slight modifications in both trans and cis MUFAs. Tumor growth also reduced the unsaturation index in both plasma and liver, from 97.79 to 86.83 and from 77.51 to 69.64, respectively. This effect was partially related to an increase in the occurrence of the lipid oxidation/peroxidation process of TBARS content which was increased in both plasma (from 0.428 to 0.505) and liver (from 9.425 to 127.792) due to tumor growth.