First molecular characterization of canine parvovirus strains in Sardinia, Italy.

Canine parvovirus type 2 (CPV-2) is responsible of acute hemorrhagic gastroenteritis in young dogs. CPV-2 emerged in 1978 in the USA, but new antigenic types, CPV-2a, 2b and 2c, have completely replaced the original type. In this study, we analyzed 81 animals collected in Sardinia, Italy. The VP2 sequence analysis of 27 positive samples showed that all antigenic CPV-2 types are circulating. CPV-2b seems to be the most widespread variant, followed by CPV-2a. Furthermore, 12 CPV-2b strains displayed further amino acid substitutions and formed a separate cluster in a phylogenetic tree, indicating regional genetic variation.

Proteomic changes in the ileum of sheep infected with Mycobacterium avium subspecies paratuberculosis.

Johne's disease (JD) is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). To identify the processes activated in the sheep intestine during natural MAP infection, and to provide a panel of differential host and pathogen proteins with diagnostic and prognostic potential, a differential shotgun proteomics workflow, including mass spectrometry, label-free quantisation and pathway analysis, was applied to ileal tissues of ewes with and without JD. Out of 2889 total proteins identified, 384 were differentially expressed and 341 were expressed at a higher level in JD. On the basis of Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis, these proteins were involved in numerous relevant biological networks and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including inhibition of phagosome acidification (such as V-ATPase), bacterial invasion, leucocyte recruitment and activation, and antimicrobial activity (such as haptoglobin, lactoferrin, cathelicidins, calgranulins and interleukins). A total of 28 MAP proteins were identified, including bacterioferritin, ß-lactamase and heparin-binding haemagglutinin (HBHA), a mycobacterial adhesin crucial for dissemination of infection.

Mycobacteriosis caused by Mycobacterium marinum in reared mullets: first evidence from Sardinia (Italy).

Mycobacterium marinum is a slow-growing non-tuberculous mycobacterium, and it is considered the most common aetiologic agent of mycobacteriosis in wild and cultured fish. The diagnosis is principally made by histology when positive Ziehl-Neelsen stain granulomas are detected. The aim of this study was to investigate the occurrence of mycobacteriosis in extensively cultured Mugilidae of two lagoons (Cabras and San Teodoro) from Sardinia by the use of histology, microbiology, PCR and DNA sequencing. Nine of 106 mullets examined were affected by mycobacteriosis, and the spleen was the most affected organ. The histology detected higher rate (100%) of infection in spleen than the culture and PCR (75% and 62.5%, respectively). The sequencing of hsp65 gene identified M. marinum as the primary cause of mycobacteriosis in the mullets examined. Mullets affected by mycobacteriosis were mainly fished in the San Teodoro lagoon characterized by critical environmental conditions. Histology remains the most common method in detecting fish affected by mycobacteriosis, and PCR-based methods are essential for species identification. Our finding are worthy of attention because mycobacteriosis caused by M. marinum in reared mullets was evidenced for the first time in Sardinia, suggesting that this disease may be underestimated also in other cultured fish species.

Australian freshwater sponges with a new species of Pectispongilla (Porifera: Demospongiae: Spongillida).

This paper focus on the biodiversity assessment of Australian inland water. Checklists of Australian Spongillida are also provided with biogeographic notes together with the geographic range of all species of freshwater sponges in Australia. New discoveries on freshwater sponges are reported from ephemeral freshwater habitats in Kakadu National Park (Australia Northern Territory). Morphological analyses show that the sponges belong to Radiospongilla and Pectispongilla in the family Spongillidae. Radiospongilla cfr. philippinensis shows a single layer of radial gemmuloscleres and the absence of tangential gemmuloscleres in the gemmular theca. Pectispongilla gagudjuensis n. sp. diverges from the diagnostic traits of the four species currently assigned to the genus i.e. skeletal megascleres are dominant acanthostrongyles and less frequent acanthoxeas shorter than in the other species, microscleres are absent, and gemmules are larger than in the other species of the genus.

LGI1 microdeletion in autosomal dominant lateral temporal epilepsy.

OBJECTIVES: To characterize clinically and genetically a family with autosomal dominant lateral temporal epilepsy (ADLTE) negative to LGI1 exon sequencing test. METHODS: All participants were personally interviewed and underwent neurologic examination. Most affected subjects underwent EEG and neuroradiologic examinations (CT/MRI). Available family members were genotyped with the HumanOmni1-Quad v1.0 single nucleotide polymorphism (SNP) array beadchip and copy number variations (CNVs) were analyzed in each subject. LGI1 gene dosage was performed by real-time quantitative PCR (qPCR). RESULTS: The family had 8 affected members (2 deceased) over 3 generations. All of them showed GTC seizures, with focal onset in 6 and unknown onset in 2. Four patients had focal seizures with auditory features. EEG showed only minor sharp abnormalities in 3 patients and MRI was unremarkable in all the patients examined. Three family members presented major depression and anxiety symptoms. Routine LGI1 exon sequencing revealed no point mutation. High-density SNP array CNV analysis identified a genomic microdeletion about 81 kb in size encompassing the first 4 exons of LGI1 in all available affected members and in 2 nonaffected carriers, which was confirmed by qPCR analysis. CONCLUSIONS: This is the first microdeletion affecting LGI1 identified in ADLTE. Families with ADLTE in which no point mutations are revealed by direct exon sequencing should be screened for possible genomic deletion mutations by CNV analysis or other appropriate methods. Overall, CNV analysis of multiplex families may be useful for identifying microdeletions in novel disease genes.

Increased degradation in rat liver induced by antilipolytic agents: a model for studying autophagy and protein degradation in liver?

A dramatic increase in the plasma glucagon/insulin ratio can be induced by treating fasted rats with antilipolytic drugs (e.g., with 3,5-dimethylpyrazole, 12 mg/kg body wt). These hormone changes are the physiologically appropriate response to a rapid decrease in free fatty acids and glucose plasma levels. Under this experimental condition, many vacuolated lysosomes can be observed at the electron microscopic level as early as 30 min and autophagic vacuoles are detectable in the liver cells 1 hr after the administration of the drug. By 1 hr and 45 min, vacuoles often contain recognizable peroxisomes. At the biochemical level, liver proteolysis in vitro is increased significantly. Very interestingly, changes in peroxisomal (but not mitochondrial or reticulum or cytosolic) enzyme activities are detected that are preventable by the administration of glutamine (i.e., of an inhibitor of proteolysis in vivo) but not by an isocaloric amount of glycine or alanine. It is concluded that the administration of antilipolytic agents to fasted animals may provide a convenient (i.e., an inexpensive, highly reproducible and timable) physiologic model to study hormone-induced autophagy in liver cells.

Protective effect of 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase, against streptozotocin-induced diabetes.

The addition of 3-aminobenzamide (a potent inhibitor of poly(ADP-ribose)synthetase) into the incubation medium, prevents streptozotocin-induced inhibition of glucose-stimulated insulin release from isolated islets [control 142 +/- 14 microU X islet-1 X h-1; streptozotocin (0.5 mg/ml) 31 +/- 8; 3-aminobenzamide (1.0 mg/ml) 96 +/- 11; streptozotocin plus 3-aminobenzamide 122 +/- 19]. In vivo, intraperitoneal 3-aminobenzamide 300 mg/kg body weight prevents the appearance of overt diabetes in streptozotocin-treated rats. These protective effects of 3-aminobenzamide are dose-dependent and are similar to those exerted by nicotinamide. Taking into account that poly ADP-ribosylation is involved in the repair of damaged DNA, the protection exerted by 3-aminobenzamide against the diabetogenic effect of streptozotocin strongly supports the view that this acute effect may be a major consequence of the activation of DNA repair mechanisms in islet cells.

Effects of antilipolytic agents on rat liver peroxisomes and peroxisomal oxidative activities.

The mechanisms involved in the inhibitory effects of antilipolytic agents on rat liver peroxisomal fatty acid oxidative activity have been explored. Treatment of fasting rats with antilipolytic drugs (either 3,5-dimethylpyrazole (12 mg/kg body weight) or Acipimox (25 mg/kg body weight] resulted in a decrease in free fatty acid and glucose plasma levels within 5-10 and in a significant increase in the plasma glucagon to insulin ratio within 15. Changes in the fatty acid oxidative activity appeared with a 2.5-3 h delay and were then very rapid (a 30-40% decrease in the activity occurred in additional 2 h). Many peroxisomal enzyme activities (including non-beta-oxidative activities such as uricase and D-amino acid oxidase) exhibited similar changes with the same delay. Simultaneously with the enzyme changes, at the electron microscope level many autophagic vacuoles were detected in the liver cells, often containing peroxisomal structures. Glutamine, an inhibitor of proteolysis in vivo, prevented the decrease in enzyme activities. It was concluded that the decrease in peroxisomal enzyme activities may be the consequence of enhanced peroxisome degradation due to the stimulation of autophagic processes in liver cells.

Effect of cold adaptation on liver peroxisomes and peroxisomal oxidative activities of rat. A morphometric/stereologic and biochemical study.

The variations in liver peroxisomes and in peroxisomal enzymes were studied in the rat during adaptation to cold (5 degrees C). An increase in the number and in the volume and surface fractions of peroxisomes was detected by day 7. Qualitative ultrastructural changes of the peroxisome compartment were observed. Several peroxisomal enzyme activities were found to exhibit a significant increase with different temporal patterns. These results are discussed with regard to the possible contribution of liver peroxisomes to non-shivering thermogenesis.

Transmural gradient of glycogen metabolism in the normal rat left ventricle.

The changes of glycogen metabolism with the location of tissue within the ventricle wall have been explored in the rat myocardium. The hearts were cut in 100 microns thick serial sections and all sections were analyzed for their content in glycogen, glucose-6-phosphate, UDPG and glycogen enzymes and for glucose incorporation into glycogen and for the 2-deoxyglucose uptake after the intravenous injection of the 14C-labelled sugars. The rate of glycogen turnover was significantly higher in the subendocardial myocardium (P less than 0.01) and the levels of glucose-6-phosphate and the total (i.e. a + b) activity of glycogen phosphorylase were significantly higher in the subepicardial tissue (P less than 0.01 in both instances). No significant transmural gradient of UDPG was found and transmural changes of total (i.e. I + D) synthase activity were barely significant. These changes in glycogen metabolism may be related to regional differences in the cardiac work load and to a differentiation of the subendocardial and subepicardial heart fibers.

[Cold adaptation and changes in peroxisome enzyme in various organs of the rat]. / Adattamento al freddo e comportamento delle attivita enzimatiche perossisomiali in alcuni organi del ratto.

In the rat brown fat peroxisomes - thermogenetic organules - an peroxisomal enzyme activities undergo remarkable changes during the adaptation to cold of the animals (see 3). In this paper was show that changes of peroxisomal enzyme activities occur also in liver and kidney during cold-adaptation. Catalase, L-hydroxyacid oxidase, uricase and D-aminoacid oxidase (DAO) were assayed as in (6). During cold-adaptation, the activity of the former three enzymes (Table 2) increases with the weight of the organs (Table 1) whereas that of DAO exhibits a much larger increase (Table 3). Results are discussed with regard to the contribution of the liver to non-shivering thermogenesis.

Peroxisomal enzyme activities in regenerating liver of rats.

The activities of five peroxisomal enzymes (fatty acyl-CoA oxidase, FAO; uricase, U; L-hydroxyacid oxidase, LHAO; D-aminoacid oxidase, DAO; and catalase, C and the triglyceride content were determined in the regenerating liver of rats after partial hepatectomy. The FAO activity was significantly increased on day 3. Thus, its rise followed the increase of the triglyceride content and preceded the disappearance of steatosis. For U and LHAO a similar progressive increase in activities was obtained on days 3, 5, and 7 of regeneration. DAO exhibited a highly significant increase on day 1. C increased on day 1 and then again after day 3. It is concluded that peroxisomal functions are not restored at parallel rates during liver regeneration.

The accumulation of 2-deoxyglucose-6-phosphate activates glycogen synthase (and inactivates glycogen phosphorylase) in rat skeletal muscle.

In the muscle loaded with 2-dGlc "in vitro" (this sugar is accumulated as hexosephosphate) glycogen synthase I levels are changed by a mechanism which is additive to those of hormones such as insulinor epinephrine. The levels of glycogen phosphorylase are decreased only at the highest 2-dGlc-6-P concentration. The role of this effect of sugar phosphate - which has been attributed to the activation of muscle phosphatase (6) - is discussed with regard to glycogen metabolism during muscle function.

Developmental changes of glycogen enzymes in fast and slow muscles of the rat.

Glycogen synthase (GS), Branching enzyme (BE), Glycogen Phosphorylase (GPho) and the Debranching enzyme (DE) activities were studied in the white extensor digitorum longus (ELD) and in the red soleus (S) muscles of rats during the developmental period from birth to the young adulthood. During the first days of postnatal life all enzyme activities similarly increase in both ELD and in S. BE and DE seem to rise faster and reach the adult levels earlier than GS and GPho. Significant differences can be seen between ELD and S by age 14--21 days: glycogenolytic enzymes rise higher in the ELD, GS activity is similar in both muscles whereas BE activity is higher in S. All these develop changes are completed in 25 days. Thereafter, only GPho activity in the ELD continues to slowly increase.

[Effects of catecholamine treatment on UDPG levels in white and red skeletal muscle]. / Effetti del trattamento con catecolamine sui livelli di UDPG nel muscolo scheletrico bianco e rosso del ratto.

Adrenaline causes of 5 fold increase of glucose-6-phosphate and of glucose-1-phosphate both in the white extensor digitorum longus and in the red soleus (where the levels of the two sugar phosphates are significantly lower). On the other hand, UDPG levels--which are similar in the two muscles--are significantly decreased after adrenaline. It has been concluded that the levels of glucose-1-phosphate and of UDPG in muscle are not bound to change together.

Effects of adrenaline administration on the activity of glycogen metabolizing enzymes in different muscle types.

Adrenaline administration which leads to glycogen changes different in fast and slow twitch muscles (Gorski, 1978) causes very similar acute changes in the activities of glycogen synthase I and of glycogen phosphorylase 'a' in these two types of rat muscle.