Alexandre Lobo-da-Cunha

Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH: ubiquinone reductase of Neurospora mitochondria.

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH:ubiquinone reductase (Complex I) fromNeurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain ofN. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functionalnuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH:ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.

Rhodopirellula lusitana sp. nov. and Rhodopirellula rubra sp. nov., isolated from the surface of macroalgae☆☆☆

Twenty two strains of Rhodopirellula were isolated from the epiphytic community of several marine macroalgae and separated into two groups, designated as group B and group C. In this study, we characterized these groups as two novel species belonging to the genus Rhodopirellula. These strains were represented by pleomorphic cells that were arranged in rosettes and formed pink- or red-pigmented colonies. The organisms were chemoorganotrophic and required vitamin B12 for growth. Their optimal temperature for growth was around 25°C. Major fatty acids were C18:1 ω9c, C16:0 and C16:1 ω7c/C16:1 ω6c. Phosphatidylcholine and phosphatidylglycerol were the major polar lipids. Unidentified phospholipids were also present. The 16S rDNA sequence analysis confirmed the affiliation of these organisms to the order Planctomycetales, genus Rhodopirellula, with R. baltica as the closest phylogenetic relative. The analysis of a partial sequence of the gene encoding the β-subunit of RNA polymerase (rpoB) confirmed the phylogenetic separation of the isolates into two different species of the genus Rhodopirellula. The 16S rRNA sequences from strains of group B revealed their widespread occurrence across the world, whereas strains of group C were not observed before. On the basis of physiological, biochemical, chemotaxonomic and genetic characteristics we propose that our isolates represent two new species of Rhodopirellula, Rhodopirellula rubra sp. nov. (type strain is LF2(T)=DSM 25,459=CECT 8075) and Rhodopirellula lusitana sp. nov. (type strain is UC17(T)=DSM 25,457=LMG 27,777).

Bacillus purgationiresistans sp. nov., isolated from a drinking-water treatment plant

A Gram-positive, aerobic, non-motile, endospore-forming rod, designated DS22(T), was isolated from a drinking-water treatment plant. Cells were catalase- and oxidase-positive. Growth occurred at 15-37 °C, at pH 7-10 and with <8% (w/v) NaCl (optimum growth: 30 °C, pH 7-8 and 1-3% NaCl). The major respiratory quinone was menaquinone 7, the G+C content of the genomic DNA was 36.5 mol% and the cell wall contained meso-diaminopimelic acid. On the basis of 16S rRNA gene sequence analysis, strain DS22(T) was a member of the genus Bacillus. Its closest phylogenetic neighbours were Bacillus horneckiae NRRL B-59162(T) (98.5% 16S rRNA gene sequence similarity), Bacillus oceanisediminis H2(T) (97.9%), Bacillus infantis SMC 4352-1(T) (97.4%), Bacillus firmus IAM 12464(T) (96.8%) and Bacillus muralis LMG 20238(T) (96.8%). DNA-DNA hybridization, and biochemical and physiological characterization allowed the differentiation of strain DS22(T) from its closest phylogenetic neighbours. The data supports the proposal of a novel species, Bacillus purgationiresistans sp. nov.; the type strain is DS22(T) (=DSM 23494(T)=NRRL B-59432(T)=LMG 25783(T)).

Seasonal and gender variation of peroxisome proliferator activated receptors expression in brown trout liver.

PPAR isotypes have been previously identified in the teleost brown trout (Salmo trutta f. fario) and their organ distribution pattern established. Being that the liver is a vital metabolic organ presenting expression of all isotypes and also knowing that estrogens/estrogen receptors seem to interact with PPARs, we hypothesized that the latter may very well change seasonally. So, we studied the expression of these receptors in the liver, along the annual reproductive cycle and in both genders. According to real-time RT-PCR, PPARalpha mRNA expression in females was significantly higher in May and lower in September than in other seasons. No significant variation was observed along the year in males. A significant difference between genders occurred in May, when PPARalpha expression was higher for females. PPARbeta expression showed little variation along the reproductive cycle in females, but in males it was significantly higher in December than in the other seasons. No significant differences existed between genders. PPARgamma was more expressed in February than in September and December, for females. As to males, it was more expressed in February than in all other seasons. No significant differences were observed between genders. The study proved our hypothesis that PPARs gene expression varies along the year. Moreover, PPARalpha expression in females followed the same annual variation pattern as peroxisome volumes and enzyme activities, and an inverse pattern relatively to the salmonid type annual plasma estradiol levels. The data agrees with the idea that PPARalpha is under estradiol modulation and that cross-talk between this receptor and the estrogen receptor possibly exists.

Insights into the ultrastructural morphology of novel Planctomycetes

Knowledge of the interesting phylum of Planctomycetes has increased in the last decades both due to cultural and molecular methods. Although a restricted number of species have been described to date, this group presents a much larger diversity that has been mainly revealed by molecular ecology studies. Isolation experiments allowed us to get a number of new Planctomycetes taxa that extend the already described ones. In this work we present the ultrastructural morphological characterization of these new taxa as well as we give new details of Aquisphaera giovannonii ultrastructure. Furthermore, our interpretation on Planctomycetes cell envelope is provided.

The peroxisomes of the hepatopancreas in two species of chitons

Abstract. This paper presents the first description of peroxisomes in polyplacophorans. As in other molluscs, the hepatopancreas of chitons is composed of basophilic and digestive cells. In the basophilic cells, the endoplasmic reticulum is abundant and several Golgi stacks can be observed. These cells also possess secretion granules and vacuoles with spherites. The digestive cells are mainly characterized by the presence of many food vacuoles. Several peroxisomes were observed in the basophilic cells of Acanthochiton crinita, most of them almost spherical. The matrix is filled with tubular structures and a crystalline nucleoid is also present in these organelles. In the digestive cells of A. crinita, peroxisomes are also almost spherical and possess two kinds of nucleoids. One of them presents a diamond shape and a bundle of tubular structures forms a second kind of nucleoid, which shows an elongated form. In Lepidochitona cinerea, the peroxisomes of basophilic cells are spherical or oval. Within the matrix, a cluster of dense rods and a prismatic nucleoid were observed. In the digestive cells of this species, almost spherical or oval peroxisomes are common, but they are smaller than the peroxisomes of the preceding cells. Nucleoids were not detected, but a few dense rods could be observed in the matrix. In both cell types of the two species, catalase activity was detected in the peroxisomal matrix. In addition, the elongated nucleoid of A. crinita digestive-cell peroxisomes and the nucleoid of L. cinerea basophilic-cell peroxisomes also present catalase activity.

The hepatocytes of the brown trout (Salmo trutta fario): a stereological study of some cytoplasmic components with the breeding cycle.

Sex differences exist in fish hepatocytes, but studies for characterizing their cytology throughout the breeding cycle are still scarce; suggesting changes, but most lacking quantitative data. To address this limitation, to complement baseline data generated from the brown trout model, and to prove that sex‐specific seasonal changes exist, we made an unbiased stereological evaluation of the hepatocytic cytoplasm. Unprecedentedly for fish liver, the stereological design was exempt from model (biased) assumptions. Five (3 years old) animals per sex were studied in endogenous vitellogenesis, exogenous vitellogenesis, and spawning season end. Liver pieces for analysis were systematically sampled. Stereology was done in transmission electron microscopy (TEM) micrographs. Primary data generated relative volume estimates of the major cytoplasmic components. Such values were used for deriving absolute volumes (per cell and per liver). Lipid droplets did not show changes. As to other targets, trends at cell and liver levels were not always equal. If the hepatocyte was the reference space, the contents in mitochondria, dense bodies, glycogen, and cytosol changed seasonally, in both sexes. If taking the liver as the reference, changes attained the Golgi apparatus and rough endoplasmic reticulum (RER), besides dense bodies, glycogen (in females), and cytosol. The components volumes (namely per liver) were often positively (negatively for glycogen) correlated with the ovary weight, disclosing new associations and implications in fish. While also offering gold‐standard data for backing morphofunctional correlations and pathology, we revealed a new process by which females increase the amount of RER and Golgi throughout vitellogenesis, breaking from the idea on how this event happens in fish. Microsc. Res. Tech. 73:766–778, 2010.

The 17β-hydroxysteroid dehydrogenase 4: Gender-specific and seasonal gene expression in the liver of brown trout (Salmo trutta f. fario)

Previously, it was documented that liver peroxisomes display seasonal size changes in the adult Salmo trutta fario, especially in females (and negatively correlated with ovary maturation). It was then hypothesized that decreases in peroxisome size could be paralleled by changes in peroxisomal beta-oxidation and estradiol catabolism actions. The 17beta-hydroxysteroid dehydrogenase 4 has been portrayed as playing an important role in both processes. To elucidate its function in the described peroxisomal pattern, we isolated the cDNA and predicted the protein sequence of the enzyme in that species. The seasonal gene expression pattern in both genders was addressed through quantitative PCR. Fish sampling was in post-spawning period, early and advanced gonad maturation, and pre-spawning. Males did not vary seasonally. As to females, a seasonal pattern was evidenced according to our previous hypothesis. We suggest that the decreased levels observed during vitellogenesis are related to lipid needs for ovary maturation, and, additionally, with the need of modulating estradiol titers.

Measurement of peroxisomal enzyme activities in the liver of brown trout (Salmo trutta), using spectrophotometric methods

BackgroundThis study was aimed primarily at testing in the liver of brown trout (Salmo trutta) spectrophotometric methods previously used to measure the activities of catalase and hydrogen peroxide producing oxidases in mammals. To evaluate the influence of temperature on the activities of those peroxisomal enzymes was the second objective. A third goal of this work was the study of enzyme distribution in crude cell fractions of brown trout liver.ResultsThe assays revealed a linear increase in the activity of all peroxisomal enzymes as the temperature rose from 10° to 37°C. However, while the activities of hydrogen peroxide producing oxidases were strongly influenced by temperature, catalase activity was only slightly affected. A crude fraction enriched with peroxisomes was obtained by differential centrifugation of liver homogenates, and the contamination by other organelles was evaluated by the activities of marker enzymes for mitochondria (succinate dehydrogenase), lysosomes (aryl sulphatase) and microsomes (NADPH cytochrome c reductase). For peroxisomal enzymes, the activities per mg of protein (specific activity) in liver homogenates were strongly correlated with the activities per g of liver and with the total activities per liver. These correlations were not obtained with crude peroxisomal fractions.ConclusionsThe spectrophotometric protocols originally used to quantify the activity of mammalian peroxisomal enzymes can be successfully applied to the study of those enzymes in brown trout. Because the activity of all studied peroxisomal enzymes rose in a linear mode with temperature, their activities can be correctly measured between 10° and 37°C. Probably due to contamination by other organelles and losses of soluble matrix enzymes during homogenisation, enzyme activities in crude peroxisomal fractions do not correlate with the activities in liver homogenates. Thus, total homogenates will be used in future seasonal and toxicological studies of brown trout peroxisomes.

Light and electron microscopy study of the salivary glands of the carnivorous opisthobranch Philinopsis depicta (Mollusca, Gastropoda)

Cephalaspideans are a group of opisthobranch gastropods that comprises carnivorous and herbivorous species, allowing an investigation of the relationship between these diets and the morphofunctional features of the salivary glands. In this study, the salivary glands of the carnivorous cephalaspidean Philinopsis depicta were observed by light and electron microscopy. The secretory epithelium of these ribbon-shaped glands is formed by ciliated cells, granular cells and cells with apical vacuole. In ciliated cells the nucleus and most cytoplasmic organelles are located in the wider apical region and a very thin stalk reaches the base of the epithelium. These cells possess significant amounts of glycogen. Granular cells are packed with electron-dense secretory granules and also contain several cisternae of rough endoplasmic reticulum and Golgi stacks. The other type of secretory cell is mainly characterized by the presence of a large apical vacuole containing secretion. These cells possess high amounts of rough endoplasmic reticulum cisternae and several Golgi stacks. Vesicles with peripheral electron-dense material are also abundant, and seem to fuse to form the apical vacuole. The available data point out to a significant difference between the salivary glands of carnivorous and herbivorous cephalaspidean opisthobranchs, with an intensification of protein secretion in carnivorous species.