Pancreatic hepatocytes in hamsters (Mesocricetus auratus) infected with Trypanosoma cruzi

Hepatocytic metaplasia may be induced in hamsters by carcinogens, and associated with aging, diabetes or chronic pancreatitis. By means of histopathologic and immunohistochemic studies, we observed pancreatic hepatocytes in hamsters infected and reinfected with Trypanosoma cruzi. The change was seen in 18 (19%) out of 94 infected animals, and was not found among 53 controls, Normal islet cells were immunoreactive for neuron-specific enolase and not reactive for NCL-HAS. Metaplastic cells were immunoreactive for NCL-HAS and not reactive for islet hormones and enolase. No relationship was observed between number of inoculations and metaplasia; however, the intensity of the inflammatory process and sequels seems to favor the development of metaplastic cells. Hamsters infected with T. cruzi may be useful to study hepatocytic metaplasia, and contribute to clarify aspects of Chagas' disease and pancreatic changes. Our data indicate that aging, in addition to inflammation and atrophy, plays a role in this change.     

Conservation of the mosaic structure of the four internal transcribed spacers and localisation of the rrn operons on the Streptococcus pneumoniae genome

The detection of heterogeneity of the 16S-23S ribosomal intergenic transcribed spacer (ITS) region has become rather common over the past years for identification and typing purposes of bacteria. The ITS not only varies in sequence and length, but also in number of alleles per genome and in their position on the chromosome together with the ribosomal clusters. The ITS characterisation has allowed discrimination of several species within a genus and variation in ITS sequences between the multiple rrn operons present within a genome may be as high or greater than between strains of the same species or subspecies. It is important to understand the variability of ITS sequences in a given genome to gain insights into bacterial physiology and taxonomy. The present study describes the possibility to type Streptococcus pneumoniae by PCR-ribotyping of the spacer region, the determination of the molecular structure of the ITS, and the determination of the number and localisation of rrn operons in this microorganism. Our results show that the genome of S. pneumoniae contains four ribosomal operons, showing the same genomic organisation among strains, each containing a single ITS allele of 270 bp. The ITS sequence presents a mosaic organisation of blocks highly conserved intra- and inter-species within the genus Streptococcus, giving no possibility for variations to arise.     

Distinctive pitted enamel hypoplasia and short stature

We report a case of postnatal onset short stature and a distinctive pitted enamel hypoplasia in a 19-year-old woman. Growth hormone deficiency and other endocrine deficiencies were excluded. Additional observations of similar cases might outline a newly recognized syndrome.     

Expression of the alpha(2)delta subunit interferes with prepulse facilitation in cardiac L-type calcium channels

We investigated the role of the accessory alpha(2)delta subunit on the voltage-dependent facilitation of cardiac L-type Ca(2+) channels (alpha(1C)). alpha(1C) Channels were coexpressed in Xenopus oocytes with beta(3) and alpha(2)delta calcium channel subunits. In alpha(1C) + beta(3), the amplitude of the ionic current (measured during pulses to 10 mV) was in average approximately 1.9-fold larger after the application of a 200-ms prepulse to +80 mV. This phenomenon, commonly referred to as voltage-dependent facilitation, was not observed when alpha(2)delta was coexpressed with alpha(1C) + beta(3). In alpha(1C) + beta(3), the prepulse produced a left shift ( approximately 40 mV) of the activation curve. Instead, the activation curve for alpha(1C) + beta(3) + alpha(2)delta was minimally affected by the prepulse and had a voltage dependence very similar to the G-V curve of the alpha(1C) + beta(3) channel facilitated by the prepulse. Coexpression of alpha(2)delta with alpha(1C) + beta(3) seems to mimic the prepulse effect by shifting the activation curve toward more negative potentials, leaving little room for facilitation. The facilitation of alpha(1C) + beta(3) was associated with an increase of the charge movement. In the presence of alpha(2)delta, the charge remained unaffected after the prepulse. Coexpression of alpha(2)delta seems to set all the channels in a conformational state from where the open state can be easily reached, even without prepulse.     

Reduced mitochondrial Ca2+ transients stimulate autophagy in human fibroblasts carrying the 13514A>G mutation of the ND5 subunit of NADH dehydrogenase

Mitochondrial disorders are a group of pathologies characterized by impairment of mitochondrial function mainly due to defects of the respiratory chain and consequent organellar energetics. This affects organs and tissues that require an efficient energy supply, such as brain and skeletal muscle. They are caused by mutations in both nuclear- and mitochondrial DNA (mtDNA)-encoded genes and their clinical manifestations show a great heterogeneity in terms of age of onset and severity, suggesting that patient-specific features are key determinants of the pathogenic process. In order to correlate the genetic defect to the clinical phenotype, we used a cell culture model consisting of fibroblasts derived from patients with different mutations in the mtDNA-encoded ND5 complex I subunit and with different severities of the illness. Interestingly, we found that cells from patients with the 13514A>G mutation, who manifested a relatively late onset and slower progression of the disease, display an increased autophagic flux when compared with fibroblasts from other patients or healthy donors. We characterized their mitochondrial phenotype by investigating organelle turnover, morphology, membrane potential and Ca²⁺ homeostasis, demonstrating that mitochondrial quality control through mitophagy is upregulated in 13514A>G cells. This is due to a specific downregulation of mitochondrial Ca²⁺ uptake that causes the stimulation of the autophagic machinery through the AMPK signaling axis. Genetic and pharmacological manipulation of mitochondrial Ca²⁺ homeostasis can revert this phenotype, but concurrently decreases cell viability. This indicates that the higher mitochondrial turnover in complex I deficient cells with this specific mutation is a pro-survival compensatory mechanism that could contribute to the mild clinical phenotype of this patient.Mitochondrial disorders include a wide range of pathological conditions characterized by defects in organelle homoeostasis and energy metabolism, in particular in the electron transport chain (ETC) complexes. They are mostly caused by mutations in nuclear- or mtDNA-encoded genes of the respiratory chain complexes leading to a variety of clinical manifestations, ranging from lesions in specific tissues, such as in Leber''s hereditary optic neuropathy, to complex multisystem syndromes, such as myoclonic epilepsy with ragged-red fibers, Leigh syndrome or the mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS).^{1, 2} Despite the detailed knowledge of the molecular defects in these diseases, their pathogenesis remains poorly understood. The heterogeneity of signs and symptoms depends on the diversity of the genetic background and on patient-specific compensatory mechanisms. Several studies investigated the consequences of nuclear DNA mutations on intracellular organelle physiology and Ca²⁺ homeostasis.^{3, 4} Here we analyzed a cohort of patients with mutations in the mtDNA-encoded ND5 subunit of NADH dehydrogenase in order to correlate the clinical phenotype with relevant intracellular parameters involved in mitochondrial physiology, such as the rate of autophagy and mitophagy fluxes, mitochondrial Ca²⁺ dynamics, mitochondrial membrane potential and their functional relationship.Mitochondrial Ca²⁺ is a key regulator of organelle physiology, and impairment of cation homeostasis is a general feature of many pathological conditions, including mitochondrial diseases.⁵ In addition, Ca²⁺ uptake in this organelle has recently been demonstrated to be a fundamental regulator of autophagy.^{6, 7} Autophagy is involved in physiological organelle turnover and in the removal of damaged or non-functional mitochondria by autophagy (called ‘mitophagy'')^{8, 9, 10, 11} and is critical for organelle quality control. Given the pivotal role of mitochondrial Ca²⁺ in the adaptation of adenosine triphosphate (ATP) production to cellular energy demand, the recent identification of the channel responsible for Ca²⁺ entry into the organelle, the mitochondrial Ca²⁺ uniporter (MCU), is instrumental for the understanding of the regulation of mitochondrial Ca²⁺ transport in both physiological and pathological conditions. MCU was identified in 2011,^{12, 13} and in the following years, molecular insight on its complex regulatory mechanism was obtained. The pore region is composed of MCU, its isoform MCUb¹⁴ and essential MCU regulator (EMRE).¹⁵ The channel is gated by the Ca²⁺-sensitive proteins mitochondrial Ca²⁺ uptake 1 (MICU1) and MICU2^{16, 17, 18, 19} and further regulated by the SLC25A23 protein.²⁰ As to its cellular function, mitochondrial Ca²⁺ has been shown to stimulate ATP production by positive regulation of three key dehydrogenases of the tricarboxylic acid cycle²¹ and of the ETC.²² In parallel, unregulated and sustained organelle Ca²⁺ overload can also lead to the opening of the mitochondrial permeability transition pore,^{23, 24} with consequent dissipation of mitochondrial membrane potential (ΔΨ_mt), release of caspase cofactors and activation of the apoptotic cascade.⁵ Despite the significant molecular understanding of all these cellular processes, their role in the pathogenesis of mitochondrial diseases is still poorly understood. Here we investigated the interplay of these pathways and the possibility of their contribution to determine the severity of the pathology in a cellular model consisting of fibroblasts from patients carrying mutations in the mitochondrial ND5 gene.     

CD44v10: an antimetastatic membrane glycoprotein for pancreatic cancer

AIMS: The aims of this study were 1) to investigate the mRNA pattern of CD44 variants in three primary (MIA PaCa 2, PANC-1, PSN-1) and two metastatic (CAPAN-1, SUIT-2) pancreatic cancer (PC) cell lines; 2) to ascertain whether the genetic transfer of CD44s and CD44v10 modifies the adhesion of PC cells to the extracellular matrix (ECM) in vitro and their metastatic behavior in vivo. METHODS: CD44 mRNA analysis was done by means of RT-PCR. Adhesion to ECM the was assessed using coated microtiter plates. For the study of CD44v10 insertion in the CAPAN-1 line, liposome-mediated DNA transfer was used. SCID mice were employed for in vivo experiments. RESULTS: CD44v10 mRNA was not expressed by the CAPAN-1 nor by four of the six SUIT-2-derived clones. The stable expression of CD44v10 by modified CAPAN-1 significantly enhanced fibronectin adhesion. Mice without either liver or pancreatic metastases were more frequently found among the animals injected with modified (CD44v10 expressing) than with non-modified CAPAN-1. CONCLUSIONS: 1) It is possible to differentiate between metastatic and non-metastatic PC cells on the basis of CD44v10 expression; 2) CD44v10 seems to be involved in mediating fibronectin adhesion in vitro and in counteracting metastases in vivo.     

Membrane composition of jetted lipid vesicles: a Raman spectroscopy study

Microfluidic jetting is a promising method to produce giant unilamellar phospholipid vesicles for mimicking living cells in biomedical studies. We have investigated the chemical composition of membranes of vesicles prepared using this approach by means of Raman scattering spectroscopy. The membranes of all jetted vesicles are found to contain residuals of the organic solvent decane used in the preparation of the initial planar membrane. The decane inclusions are randomly distributed over the vesicle surface area and vary in thickness from a few to several tens of nanometers. Our findings point out that the membrane properties of jetted vesicles may differ considerably from those of vesicles prepared by other methods and from those of living cells. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular insights into cell toxicity of a novel familial amyloidogenic variant of β2‐microglobulin

The first genetic variant of β₂‐microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild‐type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1‐enriched domains only, thus establishing a link between aggregate‐membrane contact and cell damage.     

Biodistribution of 137Cs in a mouse model of chronic contamination by ingestion and effects on the hematopoietic system

The aim of this work was to define the possible occurrence of hematological changes during the course of a chronic ingestion of ¹³⁷Cs. A mouse model was used, with ingestion through drinking water with a cesium concentration of 20 kBq l⁻¹. Ingestion started in parent animals before mating, and ¹³⁷Cs intake and its effect on the hematopoietic system was studied in offspring at various ages between birth and 20 weeks. ¹³⁷Cs content was measured in various organs, indicating that ¹³⁷Cs was distributed throughout the organism including lympho-hematopoietic organs, i.e., femurs, spleen and thymus. However, we did not observe any effect on the hematopoietic system, whatever the parameter used. In fact, blood cell counts, mononuclear cell counts and progenitor frequency in bone marrow and spleen, and Flt3-ligand, Erythropoietin, G-CSF and SDF-1 concentration in plasma remained unchanged when compared to control animals. Moreover, phenotypic analysis did not show any change in the proportions of bone marrow cell populations. These results indicate that, although ¹³⁷Cs was found in all organs implicated in the hematopoietic system, this did not induce any changes in bone marrow function.