RNA Interference Mitigates Motor and Neuropathological Deficits in a Cerebellar Mouse Model of Machado-Joseph Disease

Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease.     

Circadian rhythms in surface molecules of rat blood lymphocytes

The present article examines whether the expression of certain surface molecules that trigger immune responses shows a circadian rhythm. We also analyzed the rhythms in the number and percentage of lymphocyte subpopulations, in the leukocyte differential counts, and in the total red and white blood cell counts. Blood samples obtained from rats at 2-h intervals for 24 h were stained with several mouse monoclonal antibodies directed against lymphocyte surface molecules and processed by flow cytometry. The number of B, total T, Tgammadelta, Th, and Ts/c cells followed a 24-h rhythm with a peak in the first half of the resting period. The expression of CD45, CD5, CD3, and CD4 followed a circadian rhythm. Their acrophases suggested temporal association between CD45 and CD5 at the end of the active phase and between CD4 and CD3 at the beginning of this phase. This temporal organization could have an important role for immune cell function.     

Intrahippocampal infusion of the bombesin/gastrin-releasing peptide antagonist RC-3095 impairs inhibitory avoidance retention

Bombesin (BN)-like peptides regulate cell proliferation and cancer growth as well as neuroendocrine and neural functions. We evaluated the effects of the BN/gastrin-releasing peptide (GRP) antagonist RC-3095 on memory formation. Male Wistar rats were given a bilateral infusion of saline or RC-3095 (0.2, 1.0 or 5.0 microg) into the dorsal hippocampus either immediately or 2 h after training in an inhibitory avoidance (IA) task. Retention test trials were carried out 1.5 h (short-term memory) and 24 h (long-term memory) after training. RC-3095 impaired both short- and long-term retention only when given immediately after training. The results suggest that the hippocampal BN/GRP receptor system modulates IA memory formation.     

The dynamics of exoskeletal-epidermal structure during molt in juvenile lobster by electron microscopy and electron spectroscopic imaging

The exoskeletal-epidermal complex of juvenile lobsters at various stages throughout the molt cycle was examined by conventional electron microscopy, freeze-etch replicas, and electron spectroscopic imaging. This latter technique which enables the direct localization of atomic elements superimposed over morphological fine structure has been applied to this tissue complex to determine the spatial distributions and interrelationships of calcium, phosphorus, and sulphur. Chitin microfibril assembly is visualized in thin sections as occurring at the surface of apical membrane plaques which in freeze-etch replicas invariably possess a rich distribution of intramembrane particles on both P and E faces. In early stages of mineralization the exo- and endocuticular zones of the exoskeleton possess a dense Ca-containing lamellar repeat. These bands are unrelated to the helicoidal arrangement of chitin microfibrils. At later stages of development mineral deposits occur within the exocuticle and advance through to the endocuticle. These deposits align with chitin microfibrils and exhibit a helicoidal pattern. Morphological and chemical alterations associated with mineralization and demineralization of the exoskeleton are discussed.     

Bronchial-associated lymphoid tissue (BALT) response to airway challenge with cigarette smoke, bovine antigen and anti-pulmonary serum.

The bronchial-associated lymphoid tissue (BALT) is a lymphoepithelial organ, related to the immune defence of the lung and to alveolar clearance, which changes size in certain states of disease. Changes in the size of BALT were quantified and compared, and Spearman's test was used to test the relation with the bronchial epithelium. A total of 180 rats were used, divided into 6 groups of 30 as follows: 1) untreated controls; 2) exposed to cigarette smoke for two months; 3) treated with anti-pulmonary serum three doses daily over five days; 4) exposed to cigarette smoke and treated with anti-pulmonary serum; 5) sensitized with bovine albumin and exposed to an environment containing this antigen for two months; 6) exposed to cigarette smoke and bovine albumin. The lungs were processed for histological study, and were stained with the PAS-Alcian blue method. The main left bronchi BALT was studied, and the following were quantified: Lymphatic area (LA), as a percentage of the lung surface occupied by BALT; the flat epithelium (FEp), as the length of bronchial epithelium anatomically related to LA, whose cells tend to adopt a flat shape; the Contact epithelium (Cep), as the length of bronchial epithelium which is in direct contact with the LA. A percentage count of bronchial cells was made in the following classifications: globet cells; globet cells stained with the PAS-Alcian blue method; flat cells; lymphoepithelium cells; columnar cells; and bronchial epithelium cells excluding the above two cell types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variations at regulatory regions of the milk protein genes are associated with milk traits and coagulation properties in the Sarda sheep

Regulatory variation at the ovine casein genes could have important effects on the composition and coagulation properties of milk. Herewith, we have partially resequenced the promoters and the 3′‐UTR of the four casein genes in 25 Sarda sheep. Alignment of these sequences allowed us to identify a total of 29 SNPs. This level of polymorphism (one SNP every 250 bp) is remarkably high if compared with SNP densities estimated in human genic regions (approximately one SNP per bp). The 29 SNPs identified in our resequencing experiment, plus three previously reported SNPs mapping to the lactalbumin, alpha (LALBA) and β‐lactoglobulin (BLG, also known as progestagen‐associated endometrial protein, PAEP) genes, were genotyped with a multiplex TaqMan Open Array Real‐Time PCR assay in 760 Sarda sheep with records for milk composition and coagulation properties. Association analysis revealed the existence of significant associations of CSN1S2 and CSN3 genotypes with milk protein and casein contents. Moreover, genotypes at CSN1S1 were significantly associated with rennet coagulation time, curd firming time and curd firmness, whereas CSN2 was associated with curd firming time. These results suggest that SNPs mapping to the promoters and 3′‐UTRs of ovine casein genes may exert regulatory effects on gene expression and that they could be used for improving sheep milk quality and technological traits at the population level through marker assisted selection.