Microcystic cyanobacteria causes mitochondrial membrane potential alteration and reactive oxygen species formation in primary cultured rat hepatocytes

Cyanobacteria contamination of water has become a growing public health problem worldwide. Microcystis aeruginosa is one of the most common toxic cyanobacteria. It is capable of producing microcystins, a group of cyclic heptapeptide compounds with potent hepatotoxicity and tumor promotion activity. The present study investigated the effect of microcystic cyanobacteria on primary cultured rat hepatocytes by examining mitochondrial membrane potential (MMP) changes and intracellular reactive oxygen species (ROS) formation in cells treated with lyophilized freshwater microcystic cyanobacteria extract (MCE). Rhodamine 123 (Rh-123) was used as a fluorescent probe for changes in mitochondrial fluorescence intensity. The mitochondrial Rh-123 fluorescence intensity in MCE-treated hepatocytes, examined using a laser confocal microscope, responded in a dose- and time-dependent manner. The results thus indicate that the alteration of MMP might be an important event in the hepatotoxicity caused by cyanobacteria. Moreover, the parallel increase of ROS formation detected using another fluorescent probe, 2',7'-dichlorofluorescin diacetate also suggests the involvement of oxidative stress in the hepatotoxicity caused by cyanobacteria. The fact that MMP changes precede other cytotoxic parameters such as nuclear staining by propidium iodide and cell morphological changes suggests that mitochondrial damage is closely associated with MCE-induced cell injury in cultured rat hepatocytes.     

Nutrient removal from piggery effluent using vertical flow constructed wetlands in southern Brazil

Santa Catarina State, southern Brazil, has the greatest swine breeding activities of Latin America. Generally, the piggery wastewater is treated in pond systems that are able to remove organic material according to local environmental legislation. However, these systems do not remove nitrogen and phosphorus efficiently. This work deals with a post-treatment system, using vertical flow constructed wetlands. The experiment was conducted in a swine production farm which has 45,000 animals. Although the pond system was able to partially remove the content of nutrients, their concentration in the effluent was high for environmental disposal. A four-bed vertical flow constructed wetland pilot plant, using Typha spp., was built. The pilot plant operated for 280 days for beds 2-4 (sand 2). However, the experiments with beds 1-3 (sand 1) were stopped after 111 days of operation, when a reduction in the wastewater drainage was observed. The beds with sand 2 showed a 33% COD removal, and about 49% of nitrification was observed from 111 days until the end of the operation. PO(4)-P removal was 45% with a loading rate of around 1.36 g m(-2) d(-1).     

Cyst formation by Toxoplasma gondii in vivo and in brain-cell culture: a comparative morphology and immunocytochemistry study

Formation of Toxoplasma gondii cysts was examined in cultured murine brain cells and was compared with the development of cysts in mouse-brain tissue. Cultures of mixed glial cells from neonatal mouse brain were infected with bradyzoites of the avirulent T. gondii strain DX. The development and maturation of Toxoplasma cysts was monitored for up to 63 days after inoculation. Transmission electron microscopy indicated that in-vitro-derived cysts were morphologically similar to tissue cysts and were located intracellularly, even for up to 63 days postinfection. For immunohistological and immunocytochemical examination of both in-vivo- and in-vitro-infected material, monoclonal antibody (mAb) CC2 was used. MAb CC2 was shown to detect specifically the underlying granular material of the cyst wall without binding to the limiting membrane of the parasitophorous vacuole. This reactivity of mAb CC2 allows the distinction of bradyzoite-containing cysts from parasitophorous vacuoles harboring tachyzoites both in vitro and in vivo.     

The influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol, 25-hydroxycholecalciferol, and 1,25 dihydroxycholecalciferol in young broiler chickens

Three experiments were conducted to determine the influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol (vitamin D3), 25-hydroxycholecalciferol [25-(OH)D3], and 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in young broiler chicks. Two levels of vitamin A (1,500 and 45,000 IU/kg or 450 and 13,500 microg) were fed in all experiments. In Experiment 1, chicks were fed six levels of vitamin D3 (0, 5, 10, 20, 40, and 80 microg/kg). High dietary vitamin A decreased bone ash (P < 0.001), and increased the incidence of rickets (P < or = 0.02). Linear and quadratic responses to vitamin D3 levels were significant (P < 0.01) for body weight, bone ash, incidence and severity of rickets, and plasma calcium. In Experiment 2, six levels of 25-(OH)D3 (0, 5, 10, 20, 40, and 80 microg/kg) were added to the basal diet. Adding 25-(OH)D3 increased (P < 0.001) body weight, bone ash, and plasma calcium, and decreased rickets and plasma vitamin A. Adding 25-(OH)D3 overcame the reduction in bone ash produced by high dietary vitamin A showing a significant (P < 0.02) interaction. In Experiment 3, six levels of 1,25-(OH)2D3 (0, 2, 4, 8, 16, and 32 microg/kg) were added to the basal diet. High dietary vitamin A increased (P < 0.01) the incidence and severity of rickets. Adding 1,25-(OH)2D3 increased (P < 0.01) body weight, bone ash, plasma calcium, and reduced rickets and plasma and liver vitamin A. Adding 1,25-(OH)2D3 overcame the reduction in bone ash, and the increase in rickets produced by high vitamin A was significant (P < or = 0.05). These results indicate that high dietary vitamin A (45,000 IU/kg) interferes with the utilization of vitamin D3, 25-(OH)D3 and 1,25-(OH)2D3, increasing the requirement for each of them. Moreover, 45,000 IU/kg of dietary vitamin A ameliorated the potential toxic effects of feeding high levels of vitamin D3, 25-(OH)D3 and 1,25-(OH)2D3 to young broiler chickens. Further work is necessary to find the minimum levels of these vitamins needed to cause these effects.     

Carbamoyl phosphate synthetase: a crooked path from substrates to products

The formation of carbamoyl phosphate is catalyzed by a single enzyme using glutamine, bicarbonate and two molecules of ATP via a reaction mechanism that requires a minimum of four consecutive reactions and three unstable intermediates. The recently determined X-ray crystal structure of carbamoyl phosphate synthetase has revealed the location of three separate active sites connected by two molecular tunnels that run through the interior of the protein. It has been demonstrated that the amidotransferase domain within the small subunit of the enzyme from Escherichia coli hydrolyzes glutamine to ammonia via a thioester intermediate with Cys269. The ammonia migrates through the interior of the protein, where it reacts with carboxy phosphate to produce the carbamate intermediate. The carboxy phosphate intermediate is formed by the phosphorylation of bicarbonate by ATP at a site contained within the amino-terminal half of the large subunit. The carbamate intermediate is transported through the interior of the protein to a second site within the carboxy-terminal half of the large subunit, where it is phosphorylated by another ATP to yield the final product, carbamoyl phosphate. The entire journey from substrate to product covers a distance of nearly 100 A.     

Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH)/PTHrP receptor

Parathyroid hormone-related peptide (PTHrP) has been identified as the factor responsible for the humoral hypercalcemia of malignancy (HHM). Since the cloning of the cDNA, it has become clear that PTHrP is a prohormone that is posttranslationally cleaved to yield a complex family of peptides. Through its homology to parathyroid hormone (PTH) in the amino-terminus region of the protein, it is able to bind to and activate a common PTH/PTHrP receptor. PTHrP has been shown to be a normal product of many adult and fetal tissues, where it appears to act in an autocrine/paracrine fashion to regulate organogenesis. PTHrP and the PTH/PTHrP receptor seem to be co-expressed in many tissues, but their role in the various systems is uncertain. The use of transgenic and knock-out animal models has contributed to a better understanding of the physiological role of this peptide and its receptor. In this review, the structure of their genes, their expression pattern, and some of their major physiological functions are discussed. Attention is focused on their interaction in the regulation of cartilage and bone development.     

Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11

In a large collaborative screening project, 370 men with idiopathic azoospermia or severe oligozoospermia were analysed for deletions of 76 DNA loci in Yq11. In 12 individuals, we observed de novo microdeletions involving several DNA loci, while an additional patient had an inherited deletion. They were mapped to three different subregions in Yq11. One subregion coincides to the AZF region defined recently in distal Yq11. The second and third subregion were mapped proximal to it, in proximal and middle Yq11, respectively. The different deletions observed were not overlapping but the extension of the deleted Y DNA in each subregion was similar in each patient analysed. In testis tissue sections, disruption of spermatogenesis was shown to be at the same phase when the microdeletion occurred in the same Yq11 subregion but at a different phase when the microdeletion occurred in a different Yq11 subregion. Therefore, we propose the presence of not one but three spermatogenesis loci in Yq11 and that each locus is active during a different phase of male germ cell development. As the most severe phenotype after deletion of each locus is azoospermia, we designated them as: AZFa, AZFb and AZFc. Their probable phase of function in human spermatogenesis and candidate genes involved will be discussed.     

Bacteriophage T4 host range is expanded by duplications of a small domain of the tail fiber adhesin

The adsorption specificity of T4 is determined by the tip of the gene 37 tail fibers which bind to receptors on the bacterial surface. T4 infects only Escherichia coli and closely related Shigella species, but rare host range mutants can be isolated that infect Yersinia pseudotuberculosis I, an evolutionally distant bacterium. Some of these mutations result in amino acid residue substitutions in the C-terminal portion of gene 37, but others involve unequal exchanges between a series of sequence motifs (His boxes) in the same region. The duplication or mutational alteration of this segment apparently suffices for phage adsorption to a Yersinia receptor. It is suggested that recombination between the His box sequences can generate diversity in phage host range by shuffling receptor recognition domains.