Krabbe disease: involvement of connexin43 in the apoptotic effects of sphingolipid psychosine on mouse oligodendrocyte precursors.

Krabbe disease is a genetic demyelinating syndrome characterized by deficiency of the enzyme ß-galactosylceramidase, lysosomal psychosine accumulation, and loss of myelin-forming cells. In this study, some apoptotic markers such as apoptotic index (AI), DNA fragmentation, caspase-3, PTEN, Bad, and PI3K were determined in oligodendrocyte precursors from wild type or twitcher mice untreated or treated with psychosine. Twitcher is a natural mouse model of Krabbe disease containing a premature stop codon (W339X) in the ß-galactosylceramidase gene. Moreover, a possible involvement of connexin (Cx)43 in cell death of oligodendrocyte precursors induced by psychosine was investigated with the final aim to provide a contribution to the knowledge of the molecular mechanisms and pathophysiological events that occur in Krabbe disease. Connexins are a multigene family of structurally related trans-membrane proteins able to modulate essential cellular processes such as proliferation, differentiation and migration. Among these, Cx43 is the predominant isoform in many cell types, including neural progenitor cells. Our results showed an increase of AI, DNA fragmentation, caspase-3, PTEN, Bad, and Cx43 associated to a decrease of PI3K, pAKT and pBad. Taken together, these findings suggest an involvement of Cx43 in the psychosine-mediated apoptosis of primary oligodendrocyte progenitors from wild type or twitcher mice, used for the first time as cell models in comparison. It could open unexplored perspective also for other demyelinating diseases.

Distribution of ADP-ribosylation factor-related protein 1 in mouse brain.

ADP-ribosylation factor (Arf)-related protein 1 (ARFRP1) is a membrane-associated GTPase, which inhibits the Arf/Sec7-dependent activation of phospholipase D and belongs to the Arf-like (Arl) GTPases. Although ARFRP1 is involved in post-Golgi membrane trafficking and its lack leads to embryonic lethality, little is known about its possible function in the central nervous system. To obtain more knowledge about ARFRP1, we have characterized its mRNA distribution in adult mouse brain by in situ hybridization and real-time PCR. We observed a widespread distribution of ARFRP1-mRNA, with the highest levels in cerebral cortex, thalamic nuclei, colliculus, substantia nigra and granule cell layer of cerebellum. Moderate levels were observed in some amygdaloid nuclei, CA2 area and dentate gyrus of hippocampus, endopiriform nuclei, globus pallidus, striatum, molecular layer of cerebellum, and locus coeruleus, whereas no expression was detected in hypothalamic nuclei, CA1 and CA3 areas of hippocampus, zona incerta. A significant decrease of ARFRP1-mRNA was observed in cerebral cortex following sleep deprivation, whereas no change was observed in cerebellar cortex, locus courelus, brainstem, hippocampus and pontine nuclei. This study provides the first detailed analysis of the regional distribution of ARFRP1 in the mouse brain and a quantitative view of its changes following sleep deprivation.

Expression of pannexin1 in the CNS of adult mouse: cellular localization and effect of 4-aminopyridine-induced seizures.

The expression pattern of pannexin1, a gene coding for a protein that forms gap junction channels, was studied as both mRNA and protein in the CNS of adult mouse. Pannexin1 was widely expressed in the CNS by neuronal cell types but not glial cells, except for Bergmann glial cells of the cerebellar cortex. Cells positive to Ca-binding proteins, principally parvalbumin, but also calbindin and calretinin, as well as glutamate decarboxylase 67 kDa isoform, were pannexin1-positive. Pannexin1 labeling was found in cells which are known to exhibit spontaneous and synchronous discharge, such as neurons of the inferior olivary complex and the reticular thalamic nucleus, and also in neurons whose electrical activity is not coupled with neighboring cells, such as motoneurons of the spinal cord. The analysis of cellular localization showed puncta that surrounded cell bodies (e.g. the pyramidal cells of hippocampus) or restricted areas inside the cell bodies (e.g. the spinal motoneurons). In Bergmann glial cells the staining was present as fine grains that covered a large part of the cellular surface. Pannexin1 stained cells that previous studies have reported as expressing connexin36, another protein forming gap junction channels. Thus, it was possible that these two proteins could be integrated in the same functions. Since connexin36 expression levels change after seizures, we examined the expression of both pannexin1 and connexin36 in cerebral cortex, hippocampus, cerebellum and brain stem at different time intervals (2, 4 and 8 h) after i.p. injection of 4-aminopyridine, which resulted in systemic seizures. The only modification of the expression levels observed in this study concerned the progressive decrement of the connexin36 in the hippocampus, while pannexin1 expression was unchanged. This finding suggested that pannexin1 and connexin36 are involved in different functional roles or that they are expressed in different cell types and that only those expressing the Cx36 are induced to apoptosis by epileptic seizures.

Analytical approaches to the diagnosis and treatment of aging and aging-related disease: redox status and proteomics.

Basal levels of oxidants are indispensible for redox signaling to produce adaptive cellular responses such as vitagenes linked to cell survival; however, at higher levels, they are detrimental to cells, contributing to aging and to the pathogenesis of numerous age-related diseases. Aging is a complex systemic process and the major gap in aging research reminds the insufficient knowledge about pathways shifting from normal "healthy" aging to disease-associated pathological aging. The major complication of normal "healthy" aging is in fact the increasing risk of age-related diseases such as cardiovascular diseases, diabetes mellitus, and neurodegenerative pathologies that can adversely affect the quality of life in general, with enhanced incidences of comorbidities and mortality. In this context, global "omics" approaches may help to dissect and fully study the cellular and molecular mechanisms of aging and age-associated processes. The proteome, being more close to the phenotype than the transcriptome and more stable than the metabolome, represents the most promising "omics" field in aging research. In the present study, we exploit recent advances in the redox biology of aging and discuss the potential of proteomics approaches as innovative tools for monitoring at the proteome level the extent of protein oxidative insult and related modifications with the identification of targeted proteins.

Genomic organization and chromosomal localization of the mouse Connexin36 (mCx36) gene.

Connexin36 (Cx36) is a new connexin that was recently cloned in mouse, rat and human. It is highly expressed in neurons of the CNS. To gain insight into the transcriptional regulation of this gene, we have cloned the genomic region containing the entire mCx36 gene and sequenced about 7.6kb around the coding region. The computer analysis of this sequence was helpful in defining putative regulative sequences. Using both 5'-RACE and RNAse protection assay, we have mapped the transcription starting site commonly used in both adult olfactory bulb and brain, in position -479 from the ATG. By 3'-RACE, we defined the polyadenylation site used that is located 1436nt downstream the stop codon. The expected transcript is 2875nt long and is consistent with the 2.9kb transcript found in the same tissues by Northern blot. Finally, we have mapped mCx36 on chromosome 2 in the position F3 in a region that is synthenic to human chromosome 15q14, where the human Cx36 gene has been recently mapped.

Multiple zonal projections of the basilar pontine nuclei to the cerebellar cortex of the rat.

This study revealed a sagittal zonal pattern of projections to the cerebellar cortex after hydraulic or iontophoretic injections of anterograde tracers (tritiated leucine, wheat germ agglutinin-horseradish peroxidase, or biotinylated dextrane amine) in the basilar pontine nuclei of Wistar rats. The zonal pattern of projection was observed only after injections of small size, whereas large injections labeled diffusely wide areas of the cerebellar cortex, masking the zonal projection because the fusion of contiguous stripes. Diverging projections to discrete sets of sagittal stripes in the two sides of the cerebellar cortex arose from single injections. The stripes of fiber terminals were sharply delimited on both sides by areas, interstripes, either virtually void of labeling or with a much lower density of labeling. Thus, the areas of the cerebellar cortex were parceled in sets of sagittal compartments, stripes and interstripes, by the pontine projections. Up to five compartments (three stripes and two interstripes) were observed in the paraflocculus, in the copula pyramidis, and in vermal lobule IX. Up to nine compartments (five stripes and four interstripes) were found in the crus I, the lobulus simplex, the paramedian lobule, and vermal lobules VI-VIII. Up to seven compartments (four stripes and three interstripes) were found in the crus II. Single injections into the basilar pontine nuclei usually labeled symmetric areas of the cerebellar cortex, which, in some cases, showed similar number of stripes. When this was not the case, the stripes were usually more numerous in the contralateral than in the ipsilateral side. All areas of the cerebellar cortex were projected upon, with zonation patterns from different regions of the basilar pontine nuclei. The projections of the basilar pontine nuclei to the cerebellar cortex were arranged according to a fixed pattern specific for each cortical area, independently of the number of stripes labeled within. The mean width of the stripes visualized in the single cortical areas of different rats was similar, despite the different size of the injections. The length of the stripes ranged widely in the various areas of different rats. The data collected in this study are consistent with the idea that all the mossy afferents to the cerebellar cortex are arranged with a zonal pattern.

Diverging projections of the C2 and D2 olivocorticonuclear cerebellar pathways of the rat.

A divergent mediolateral projection to the cerebellar nuclei of the C2 and the D2 olivocorticonuclear cerebellar pathways was found after segregate injections of a tracer (either WGA-HRP or FR or BDA) in the rostral (D2 area) or caudal side (C2 area) of the rat paraflocculus. The C2 olivary area of the cerebellar cortex sends most of its nuclear projection to the nucleus interpositus posterior (classically perceived as the nuclear target of the C2 olivocorticocerebellar pathway) and a smaller contingent of fibres to the parvocellular region of the nucleus lateralis (classically perceived as the nuclear target of the D2 olivocorticocerebellar pathway). The D2 olivary area of the cerebellar cortex sends most of its nuclear projection to the parvocellular region of the nucleus lateralis (classically perceived as the nuclear target of the D2 olivocorticocerebellar pathway) and a smaller contingent of fibres to the magnocellular region of the nucleus lateralis (classically perceived as the nuclear target of the D1 olivocorticocerebellar pathway). The lateral interaction of the D2 and the C2 olivocerebellar pathways could represent the anatomical substrate for the functional integration of different olivocerebellar compartments.

Cx36 is dynamically expressed during early development of mouse brain and nervous system.

Connexins are structural proteins that are part of the gap junctional channels which couple cells in different tissues. Connexin36 (Cx36) is a new member of the connexin gene family, found to be expressed essentially if not exclusively in neuronal cells in adult CNS of mouse, rat and man. Here we have studied Cx36 expression during murine embryonic development. Cx36 shows a highly dynamic pattern of expression. It is first (E9.5) evident in the forebrain and later its expression expand caudally in the midbrain. At E12.5 its expression correlates with major morphogenetic boundaries in the developing mouse brain, specifically with the dorsoventral telencephalic boundary and the Zona Limitans Intrathalamica. Starting at midgestation (E12.5), it is also expressed in both sympathetic and spinal ganglia, and in two longitudinal stripes along the spinal cord.

Expression of connexin36 mRNA in adult rodent brain.

A new member of the connexin gene family, named Connexin36 (Cx36) has, recently, been identified in rodents and shown to be preferentially, if not exclusively, expressed in neurones of the adult CNS. In this study we present a detailed in situ hybridization analysis of the expression pattern of mouse Connexin 36 (mCx36) mRNA in the adult mouse brain, with particular regards to the correlation of mCx36 expression to specific neuronal cell classes and systems. We found that mCx36 was strongly and widely expressed in the brain, including areas where the presence of gap junctions was never detected before. Quantitative analysis of the hybridization signal indicated varying levels of expression in different areas. In particular mCx36 was highly expressed in the neurones at different levels of the motor pathway, the olfactory pathway, the hippocampus, and areas related to the generation of respiratory rhythm. On the contrary, mCx36 was more heterogeneously expressed in nuclei of the sensory pathways. These findings show that mCx36 is the first connexin specifically expressed in neuronal cells in the adult rodent brain. The profiles of expression clearly indicate that mCx36 might play specific roles within different neuronal systems.

The pontocerebellar projection: longitudinal zonal distribution of fibers from discrete regions of the pontine nuclei to vermal and parafloccular cortices in the rat.

A longitudinal parasagittal organization (alternating labeled and unlabeled stripes) of mossy fiber terminals in the paraflocculus and in the vermal lobule VII of the cerebellum was found after small injections (less than 50 nl) of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into discrete regions of the basilar pontine nuclei (BPN) of rats. Up to three stripes were found within the paraflocculus of both sides, following injections (of about 500 microns in diameter) in either the medial or lateral region of the caudal half of the BPN. Up to five stripes were found in the vermal lobule VII after similar size injections into the rostro-ventral region of the BPN. These results emphasize the possibility that the parasagittal zonal arrangement could be a common pattern of organization shared by climbing and mossy fiber afferents.

Plasma and tonsillar tissue pharmacokinetics of teicoplanin following intramuscular administration to children.

The population pharmacokinetics of teicoplanin in plasma and tonsillar tissue in children was determined following intramuscular administration. Thirty seven patients in all received either a single 5 mg/kg dose; 2 doses of 5 mg/kg, 12 h apart; 3 doses of 5 mg/kg, 12 h apart; or, a single 10 mg/kg dose. Limited data, comprising a maximum of 2 blood samples and 1 tonsillar sample were taken from each patient, with the maximum time being 48 h after the first dose of teicoplanin (in the 3 x 5 mg/kg dosing schedule). All plasma data were analyzed simultaneously by a maximum likelihood method employing a modified EM algorithm. A first-order absorption, one-compartment disposition model was fitted to the data. Mean parameter values (with lower and upper 95% confidence intervals) were: clearance/bioavailability, 0.024 L h(-1) kg(-1) (0.020-0.027); volume of distribution/bioavailability, 0.61 L kg(-1) (0.54-0.70); absorption rate constant, 0.43 h(-1) (0.31-0.61). A first-order transfer model for distribution of teicoplanin between plasma and tonsillar tissue was fitted to the tonsil data. The mean parameter values (95% confidence intervals) were: transfer rate constant between plasma and tonsils 0.49 h(-1) (0.35-0.67); transfer rate constant between tonsils and plasma 0.73 h(-1) (0.52-1.03). These rate constants correspond to a distribution half-life of 0.95 h and an equilibrium distribution concentration ratio between tonsillar tissue and plasma of 0.67. After normalising clearance and volume of distribution for body weight, there was no further influence of body weight on the pharmacokinetic parameters. Also, there was no effect of dose, and as two formulations were used, one for the 5 mg/kg dose and the other for the 10 mg/kg dose, no effect of formulation on the pharmacokinetics of teicoplanin after im (intramuscular) administration was found.

The double contrast barium enema in the identification of proximal colonic adenomas and carcinomas beyond the limits of fiberoptic sigmoidoscopy.

Eight hundred and twenty patients were examined by fiberoptic sigmoidoscopy (SIG) and double contrast barium enema (DCBE) to detect colonic cancers or adenomas. Cancer or adenoma in the bowel tract proximal to the upper limit of SIG insertion was detected in 4 patients and in 12 on DCBE. The DCBE detection rate of proximal colonic lesions varied according to the hemoccult (HO) outcome. This was 1.16% for cancer and 2.03% for adenoma in HO+ patients and null for cancer and 1.23% for adenoma in HO- patients. The detection rate of proximal adenomas was higher in patients who presented adenomas on endoscopy in the distal bowel (SIG+), 2.46% as compared to 0.48% in SIG- patients, independent of the HO reports. Routine DCBE is practically useless in HO-SIG- patients and questionable in HO-SIG+ patients since improvement of the detection rate is null for cancer and moderate for adenoma. It is recommended for HO+ patients because it increases the colonic cancer detection rate (10.5% in this study).

[Malignant tumors of the stomach, colon and rectum. Statistico-epidemiological considerations]. / I tumori maligni dello stomaco e del colon retto. Considerazioni statistico-epidemiologiche.

Mortality figures for tumours of the stomach and colon-rectum in Italy over a 20 year period and divided by age classes, show a reduction in gastric cancer (-33%) and an increase in colo-rectal cancer (+63%). The incidence of mortality by region (greater in the North, less in the South) and town is analysed. Possible relations between varying customs and the onset of tumours in different sites of the gastro-enteric apparatus are recalled.

[Obstructive jaundice caused by solitary nonparasitic cyst of the liver]. / Ittero ostruttivo da cisti solitaria non parassitaria del fegato.

The Authors describe a rare case of obstructive jaundice secondary to solitary nonparasitic cyst of the liver. The anatomopathology, embriological and clinical aspects are analyzed with emphasis on the importance of the instrumental investigation in differential diagnosis of malignancy. Finally they describe the indications to various surgical and non surgical treatment.

Immunocytochemical and RT-PCR analysis of connexin36 in cultures of mammalian glial cells.

The expression of connexin36 (Cx36) was studied in primary cultures of rat brain glial cells: mature astrocytes, ameboid and ramified microglia and immature oligodendrocytes (at middle period of myelinogenesis). The data from these cells were compared with those obtained from cultures of neocortical and hypothalamic neurons. mRNA encoding Cx36 was investigated by RT-PCR, the Cx36 protein by immunocytochemistry using a polyclonal antibody against Cx36 in cells characterized by antibodies specific for the single cell types. The Cx36 was found in oligodendrocytes, both ameboid and ramified microglial cells and in neurons. Astrocytes showed no detectable expression of the Cx36. The expression of Cx36 in oligodendrocytes and microglial cells suggests an involvement of the direct cell-cell communication channels formed by Cx36 in myelin formation and in brain development, damage and repair processes.

Intraparenchymal leiomyoma of the breast: report of a case with emphasis on needle core biopsy-based diagnosis.

OBJECTIVE: We report the clinicopathologic features of a rare case of leiomyoma of the breast parenchyma in a 36-year-old female, diagnosed preoperatively at core biopsy. A complete review of the literature on the topic is provided and differential diagnostic problems are discussed. METHODS: Standard histological examination and immunohistochemical analyses using a large panel of antibodies were performed in both the core biopsy and surgical specimen. RESULTS: Ultrasonography revealed a well-circumscribed tumour mass without calcifications. Histological examination of the core biopsy showed proliferation of bland-looking eosinophilic spindle cells arranged in a fascicular growth pattern. Mitoses, pleomorphism and necrosis were absent. Immunohistochemistry, revealing diffuse staining for a-smooth muscle actin, desmin and h-caldesmon, confirmed the leiomiomatous nature of neoplastic cells. Histological and immunohistochemical analyses of the surgical specimen confirmed the definitive diagnosis of leiomyoma. CONCLUSIONS: The present case emphasizes that diagnosis of leiomyoma of the breast parenchyma can be confidentially rendered on needle core biopsy. We believe that correct diagnosis is primarily dependent on the awareness that this tumour can arise in this unusual site on rare occasions.

Expression of connexin57 in mouse development and in harmaline-tremor model.

Connexin57 (Cx57) was previously reported in retinal cells but not in brain nerve cells. This occurrence was tested in this study, by searching for the expression of Cx57 RNA and protein transcripts during the postnatal development of the mouse CNS. Both the Cx57 RNA (investigated by reverse transcriptase-polymerase chain reaction (RT-PCR)) and the protein (Western-Blot and immunohistochemistry using a polyclonal antibody generated in chicken) transcripts were firstly expressed in the late postnatal development (P12). The expression of Cx57 in adult life (studied at P28, by in situ hybridization and immunohistochemical analysis) concerned few regions of the brain stem (inferior olive, lateral reticular nucleus and motor trigeminal nucleus), the cerebellum (Purkinje cells and cerebellar nuclei) and the spinal cord (alpha-motoneurons). Double immunohistochemical studies using the Cx57 antibody and antibodies, which specifically labelled neuronal nuclei (NeuN) and astrocyte cells glial fibrillary acidic protein (GFAP), showed the expression of Cx57 segregated in neuronal cells. The study also confirmed the expression of Cx57 in the horizontal cells of the retinal outer plexiform layer, reported in previous investigations. Given the expression of Cx57 in the cerebellum and pre-cerebellar nuclei, such as olivary and lateral reticular nuclei, a possible role of Cx57 was hypothesized in the electrical coupling of the cerebellum. This hypothesis was tested by searching for the expression of the Cx57 transcripts in the mouse cerebellum of the harmaline-tremor model. The up-regulation of the Cx57 transcripts reported in this model suggested a possible involvement of Cx57 in the electrotonic coupling of the cerebellar system.

Non-random alkylation of DNA sequences induced in vivo by chemical mutagens.

Previous studies of the interaction of alkylating agents on the eukaryotic genome support the idea that induction of DNA adducts is at specific genomic sites. Here we show molecular and cytological evidence that alkylation is rather specific. Mammalian cell cultures were exposed to different doses of mutagens and the DNA was analyzed by density gradient ultracentrifugation, hydroxylapatite fractionation, and by restriction enzyme analysis. Studies with the labelled mutagens N-ethyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine show that there is a non-random distribution of the adducts. The adducts are found more frequently in A-T, G-C rich satellite DNA and highly repetitive sequences. Analysis with restriction enzymes shows that both methyl and ethyl groups influence the restriction patterns of the enzymes HpaII and MspI that recognize specific endogenous DNA methylation. These data suggest, as a subsequent mechanism, a modification in the pattern of the normal endogenous methylation of 5-methylcytosine.