Fra1 activity in the frog, Rana esculenta, testis: a new potential role in sperm transport

Using an anti-Fos family member antibody, we have previously described in Rana esculenta testis the presence of a nuclear, 43 kDa protein that we hypothesized to be Fra1. With the assistance of an antibody against Fra1 that does not cross-react with other Fos family members, here we report data on Fra1 expression, localization, and putative activity in Rana esculenta testis during its annual reproductive cycle. Western blot analysis confirms that the nuclear, 43 kDa protein is Fra1. Immunocytochemistry validates the Western blot results and shows cytoplasmic and nuclear immunostaining of Fra1 in peritubular myoid cells, efferent ducts, and blood vessels. We report for the first time in a vertebrate, experimental evidence showing that the expression of Fra1 is related to peritubular myoid cells during sperm transport from the tubular compartment to efferent ducts.     

Glucosylceramidase mass and subcellular localization are modulated by cholesterol in Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values. GCase is activated and stabilized by saposin C (Sap C) and anionic phospholipids. Here we show by immunofluorescence microscopy that in normal fibroblasts, GCase, Sap C, and lysobisphosphatidic acid (LBPA), the most abundant anionic phospholipid in the endolysosomal system, reside in the same intracellular vesicular structures. In contrast, the colocalization of GCase, Sap C, and LBPA is markedly impaired in NPC fibroblasts but can be re-established by cholesterol depletion. These data show for the first time that the level of cholesterol modulates the interaction of GCase with its protein and lipid activators, namely Sap C and LBPA, regulating the GCase activity and stability.     

L-type Ca2+ channels in Ca2+ channelopathies

Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.     

Hyperphosphorylation and aggregation of tau in experimental autoimmune encephalomyelitis

Axonal damage is a major morphological correlate and cause of permanent neurological deficits in patients with multiple sclerosis (MS), a multifocal, inflammatory and demyelinating disease of the central nervous system. Hyperphosphorylation and pathological aggregation of microtubule-associated protein tau is a common feature of many neurodegenerative diseases with axonal degeneration including Alzheimer's disease. We have therefore analyzed tau phosphorylation, solubility and distribution in the brainstem of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Tau was hyperphosphorylated at several sites also phosphorylated in Alzheimer's disease and became partially detergent-insoluble in EAE brains. Morphological examination demonstrated accumulation of amorphous deposits of abnormally phosphorylated tau in the cell body and axons of neurons within demyelinating plaques. Hyperphosphorylation of tau was accompanied by up-regulation of p25, an activator of cyclin-dependent kinase 5. Phosphorylation of tau, activation of cdk5, and axonal pathology were significantly reduced when diseased rats were treated with prednisolone, a standard therapy of acute relapses in MS. Hyperphosphorylation of tau was not observed in a genetic or nutritional model of axonal degeneration or demyelination, suggesting that inflammation as detected in the brains of rats with EAE is the specific trigger of tau pathology. In summary, our data provide evidence that axonal damage in EAE and possibly MS is linked to tau pathology.     

Flow characterization of a wavy-walled bioreactor for cartilage tissue engineering

Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0-0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties.     

Prevalence of human papillomavirus and Epstein-Barr virus DNA in penile cancer cases from Brazil

Penile cancer is a potentially mutilating disease. Although its occurrence is relatively rare worldwide, penile cancer rates can be high in developing countries. A few studies have been conducted on the involvement of human papillomavirus (HPV) in penile carcinoma, which have found HPV present in 30-70% of penile malignant lesions, with a higher prevalence of HPV 16 and 18. It has been assumed that cofactors, such as Epstein-Barr virus (EBV) infections, may play a role in the progression of penile neoplasia. The aim of this study was to determine HPV and EBV prevalence in 135 penile malignant lesions from Brazilian men through the use of MY09/11 polymerase chain reaction (PCR), type-specific PCR and restriction fragment length polymorphism analysis. HPV prevalence among the men tested was 60.7%. Of the men who tested positive, 27 presented with HPV 16 (29.7%), five with HPV 18 (5.5%), 21 with HPV 45 (23.1%) and nine with HPV 6 (9.9%). Seven mixed infections were detected (9.2%), while 11 cases remained untyped (13.4%). Regarding EBV positivity, 46.7% of the samples contained EBV DNA with EBV-1 as the most prevalent type (74.6%). More than 23% of the men were co-infected with both HPV and EBV, while 35% presented exclusively with HPV DNA and 20% presented only with EBV DNA. Penile carcinoma aetiology has not been fully elucidated and the role of HPV and EBV infections individually or synergistically is still controversial. Hence, more studies are needed to determine their possible role in carcinogenesis.     

Genome sequences of the ethanol-tolerant Lactobacillus vini strains LMG 23202T and JP7.8.9

We report on the genome sequences of Lactobacillus vini type strain LMG 23202(T) (DSM 20605) (isolated from fermenting grape musts in Spain) and the industrial strain L. vini JP7.8.9 (isolated from a bioethanol plant in northeast Brazil). All contigs were assembled using gsAssembler, and genes were predicted and annotated using Rapid Annotation using Subsystem Technology (RAST). The identified genome sequence of LMG 23202(T) had 2.201.333 bp, 37.6% G+C, and 1,833 genes, whereas the identified genome sequence of JP7.8.9 had 2.301.037 bp, 37.8% G+C, and 1,739 genes. The gene repertoire of the species L. vini offers promising opportunities for biotechnological applications.     

Impacts of Brain Serotonin Deficiency following Tph2 Inactivation on Development and Raphe Neuron Serotonergic Specification

Brain serotonin (5-HT) is implicated in a wide range of functions from basic physiological mechanisms to complex behaviors, including neuropsychiatric conditions, as well as in developmental processes. Increasing evidence links 5-HT signaling alterations during development to emotional dysregulation and psychopathology in adult age. To further analyze the importance of brain 5-HT in somatic and brain development and function, and more specifically differentiation and specification of the serotonergic system itself, we generated a mouse model with brain-specific 5-HT deficiency resulting from a genetically driven constitutive inactivation of neuronal tryptophan hydroxylase-2 (Tph2). Tph2 inactivation (Tph2-/-) resulted in brain 5-HT deficiency leading to growth retardation and persistent leanness, whereas a sex- and age-dependent increase in body weight was observed in Tph2+/- mice. The conserved expression pattern of the 5-HT neuron-specific markers (except Tph2 and 5-HT) demonstrates that brain 5-HT synthesis is not a prerequisite for the proliferation, differentiation and survival of raphe neurons subjected to the developmental program of serotonergic specification. Furthermore, although these neurons are unable to synthesize 5-HT from the precursor tryptophan, they still display electrophysiological properties characteristic of 5-HT neurons. Moreover, 5-HT deficiency induces an up-regulation of 5-HT(1A) and 5-HT(1B) receptors across brain regions as well as a reduction of norepinephrine concentrations accompanied by a reduced number of noradrenergic neurons. Together, our results characterize developmental, neurochemical, neurobiological and electrophysiological consequences of brain-specific 5-HT deficiency, reveal a dual dose-dependent role of 5-HT in body weight regulation and show that differentiation of serotonergic neuron phenotype is independent from endogenous 5-HT synthesis.     

Accumulation of bis(monoacylglycero)phosphate and gangliosides in mouse models of neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses comprise a group of inherited severe neurodegenerative lysosomal disorders characterized by lysosomal dysfunction and massive accumulation of fluorescent lipopigments and aggregated proteins. To examine the role of lipids in neurodegenerative processes of these diseases, we analysed phospho- and glycolipids in the brains of ctsd−/− and nclf mice, disease models of cathepsin D and CLN6 deficiency, respectively. Both ctsd−/− and nclf mice exhibited increased levels of GM2 and GM3 gangliosides. Immunohistochemically GM2 and GM3 staining was found preferentially in neurons and glial cells, respectively, of ctsd−/− mice. Of particular note, a 20-fold elevation of the unusual lysophospholipid bis(monoacylglycero)phosphate was specifically detected in the brain of ctsd−/− mice accompanied with sporadic accumulation of unesterified cholesterol in distinct cells. The impaired processing of the sphingolipid activator protein precursor, an in vitro cathepsin D substrate, in the brain of ctsd−/− mice may provide the mechanistic link to the storage of lipids. These studies show for the first time that cathepsin D regulates the lysosomal phospho- and glycosphingolipid metabolism suggesting that defects in the composition, trafficking and/or recycling of membrane components along the late endocytic pathway may be critical for the pathogenesis of early onset neuronal ceroid lipofuscinoses.