Transgenic mouse model of tauopathies with glial pathology and nervous system degeneration

Frontotemporal dementias (FTDs), including corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), are neurodegenerative tauopathies characterized by widespread CNS neuronal and glial tau pathologies, but there are no tau transgenic (Tg) mice that model neurodegeneration with glia tau lesions. Thus, we generated Tg mice overexpressing human tau in neurons and glia. No neuronal tau aggregates were detected, but old mice developed Thioflavin S- and Gallyas-positive glial tau pathology resembling CBD astrocytic plaques. Tau-immunoreactive and Gallyas-positive oligodendroglial coiled bodies (similar to CBD and PSP), glial degeneration, and motor deficits were associated with age-dependent accumulations of insoluble hyperphosphorylated human tau and tau immunopositive filaments in degenerating glial cells. Thus, tau-positive glial lesions similar to human FTDs occur in these Tg mice, and these pathologies are linked to glial and axonal degeneration.     

The relationship between oxidative/nitrative stress and pathological inclusions in Alzheimer's and Parkinson's diseases

Alzheimer's (AD) and Parkinson's diseases (PD) are late-onset neurodegenerative diseases that have tremendous impact on the lives of affected individuals, their families, and society as a whole. Remarkable efforts are being made to elucidate the dominant factors that result in the pathogenesis of these disorders. Extensive postmortem studies suggest that oxidative/nitrative stresses are prominent features of these diseases, and several animal models support this notion. Furthermore, it is likely that protein modifications resulting from oxidative/nitrative damage contribute to the formation of intracytoplasmic inclusions characteristic of each disease. The frequent presentation of both AD and PD in individuals and the co-occurrence of inclusions characteristic of AD and PD in several other neurodegenerative diseases suggests the involvement of a common underlying aberrant process. It can be surmised that oxidative/nitrative stress, which is cooperatively influenced by environmental factors, genetic predisposition, and senescence, may be a link between these disorders.     

Phylogenetic position of Mediterranean Astereae and character evolution of daisies (Bellis,Asteraceae) inferred from nrDNA ITS sequences

Phylogenetic analyses of nrITS sequences of Asteraceae revealed that the Bellis group is a natural assemblage comprising all the species of Bellis and Bellium, but not Rhynchospermum. In contrast, we propose to include the genera Bellis, Bellium, and Bellidastrum in the subtribe Bellidinae in the interest of circumscribing natural groups. Our results also suggest an early diversification in the western Mediterranean Basin of two monophyletic lineages, Bellis and Bellium. Three major groups can be distinguished within BELLIS: (1) the B. perennis group, containing five annual and perennial species with three ploidy levels (diploid, octoploid, and decaploid), which are distributed throughout the Mediterranean Basin despite lack of pappus; (2) the Bellis sylvestris group, with five annual and perennial species primarily from the western Mediterranean, in which there are five ploidy levels (diploid, tetraploid, hexaploid, octoploid, and decaploid); and (3) a basal grade consisting of three diploid, perennial species which displays remarkable diversification of morphologies. Striking characteristics, such as an annual life form, polyploidy, and loss of pappus, seem to have occurred in parallel several times and in different geographical areas during the early diversification of Bellis species in the western Mediterranean. Character evolution reconstructions allow us to describe a putative ancestor of the genus Bellis (proto-Bellis).     

Integrated effects of multiple modulators on human liver glycogen phosphorylase a

Hepatic glucose production is increased in people with type 2 diabetes. Glucose released from storage in liver glycogen by phosphorylase accounts for approximately 50% of the glucose produced after an overnight fast. Therefore, understanding how glycogenolysis in the liver is regulated is of great importance. Toward this goal, we have determined the kinetic characteristics of recombinant human liver glycogen phosphorylase a (HLGPa) (active form) and compared them with those of the purified rat enzyme (RLGPa). The Michaelis-Menten constant (K(m)) of HLGPa for P(i), 5 mM, was about fivefold greater than the K(m) of RLGPa. Two P(i) (substrate) concentrations were used (1 and 5 mM) to cover the physiological range for P(i). Other effectors were added at estimated intracellular concentrations. When added individually, AMP stimulated, whereas ADP, ATP and glucose inhibited, activity. These results were similar to those of the RLGPa. However, glucose inhibition was about twofold more potent with the human enzyme. UDP-glucose, glucose 6-phosphate, and fructose 1-phosphate were only minor inhibitors of both enzymes. We reported previously that when all known effectors were present in combination at physiological concentrations, the net effect was no change in RLGPa activity. However, the same combination reduced HLGPa activity, and the inhibition was glucose dependent. We conclude that a combination of the known effectors of phosphorylase a activity, when present at estimated intracellular concentrations, is inhibitory. Of these effectors, only glucose changes greatly in vivo. Thus it may be the major regulator of HLGPa activity.     

Characterization and mutagenesis of Gal/GlcNAc-6-O-sulfotransferases

The installation of sulfate groups on the carbohydrate residues of glycoproteins, glycolipids, and glycosaminoglycans is a critical posttranslational modification that occurs in all higher eukaryotes. The Gal/GalNAc/GlcNAc-6-O-sulfotransferases (GSTs) are a recently discovered family of carbohydrate sulfotransferases that share significant sequence homology at the amino acid level and mediate a number of different biological processes such as leukocyte adhesion at sites of chronic inflammation. Structural and mechanistic studies of this family of sulfotransferases have been hindered by the lack of a productive recombinant protein expression system. We developed a baculovirus expression system for five of the seven cloned GSTs and determined their kinetic parameters using both thin-layer chromatography and a recently developed polymer dot-blot assay. We used these tools to perform the first site-directed mutagenesis study of a member of this sulfotransferase family, GST2. Using sequence alignments with other carbohydrate and cytosolic sulfotransferases, we selected residues within the putative binding regions for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the carbohydrate substrate for mutagenesis. Kinetic analysis of the mutants identified residues that are essential for catalytic activity. These results should facilitate mechanistic studies and the development of small molecule inhibitors of this enzyme family to ameliorate chronic inflammatory diseases.     

Androstenedione interferes in luteal regression by inhibiting apoptosis and stimulating progesterone production

Androgens, in concert with lactogenic hormones, contribute to the maintenance of function of the corpus luteum (CL) in pregnant rats. Whereas some of the androgenic actions in the CL are clearly mediated by intracrine conversion to estrogen, pure androgenic effects are also implicated in the regulation of this transient endocrine gland. In this report, we have established, to our knowledge for the first time, the expression of androgen receptor (AR) mRNA and protein throughout gestation in the rat CL. We have found that the AR remains expressed in the CL of gestation on Day 4 postpartum and becomes expressed in the newly formed CL after postpartum ovulation. An AR immunoreactive protein was identified in the CL of pregnancy as well as in prostate and epididymis, which were used as positive controls. The luteal AR protein had mainly nuclear localization, yet some diffuse cytoplasmic staining was also observed. Moreover, we have established that androstenedione, the main circulating androgen in pregnant rats, significantly reduces the decline in luteal weight observed during postpartum structural regression. This effect was correlated with a decrease in the number of cells undergoing apoptosis and with enhanced levels of circulating progesterone. In addition, in vivo administration of androstenedione delayed the occurrence of DNA fragmentation in postpartum CL incubated in serum-free conditions. Finally, we have shown that the interference with apoptosis in vitro elicited by androstenedione is accompanied by an increased capacity of the CL to secrete progesterone. In summary, the results of this study have established that the rat CL expresses AR throughout pregnancy and after parturition, and they have defined a potential role for androstenedione in opposing postpartum luteal regression through inhibition of apoptosis and stimulation of progesterone production.     

Mutations in erg4 affect the sensitivity of Saccharomyces cerevisiae to medium-chain fatty acids

We have found that the medium-chain fatty acids (MCFAs) undecanoic acid (11:0), 10-undecenoic acid (11:1 Delta 10), and lauric acid (12:0) can affect the growth of Saccharomyces cerevisiae in a dose-dependent manner. The principal effect was a longer lag phase in MCFA-containing medium, although higher concentrations of 11:1 Delta 10 inhibited growth. Their relative order of inhibitory action was 11:1 Delta 10>11:0>12:0. Cellular content with MCFA supplementation was dependent on the concentration and the particular species of fatty acid, with 12:0 showing the highest relative accumulation and 11:1 Delta 10 the lowest at all concentrations. We have isolated and characterized a mutant that is hypersensitive to MCFA supplementation and is unable to grow at the normally permissive condition of 1 mM 11:1 Delta 10. However, it does not appear to accumulate higher relative levels of the fed MCFA compared to wild-type cells. Complementation of the mutant revealed that the ERG4 gene, encoding the enzyme that catalyzes the last step in ergosterol biosynthesis, had been mutated. The fatty acid composition of the erg4 Delta mutant differs only slightly from wild-type cells, mainly involving an increase in the relative amount of 12:0. These results indicate that yeast require ergosterol for optimal growth on certain MCFAs. We discuss the role ergosterol may have in cells responding to exogenous MCFAs and in supporting optimal cell growth.     

The kinetics of translocation of Smac/DIABLO from the mitochondria to the cytosol in HeLa cells

Smac (second mitochondrial activator of caspases) is released from the mitochondria during apoptosis to relieve inhibition of caspases by the inhibitor of apoptosis proteins (IAPs). The release of Smac antagonizes several IAPs and assists the initiator caspase-9 and effector caspases (caspase-3, caspase-6, and caspase-7) in becoming active, ultimately leading to death of the cell. Translocation of Smac along with cytochrome c and other mitochondrial pro-apoptotic proteins represent important regulatory checkpoints for mitochondria-mediated apoptosis. Whether Smac and cytochrome c translocate by the same mechanism is not known. Here, we show that the time required for Smac efflux from the mitochondria of cells subjected to staurosporine-induced apoptosis is approximately four times longer than the time required for cytochrome c efflux. These results suggest that Smac and cytochrome c may exit the mitochondria by different pathways.     

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L-null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H-null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.     

Primary immune deficiencies unravel the molecular basis of immune response

Primary immune deficiencies (PID) represent inborn errors of immunity. Over the years, detailed analysis of the clinical and laboratory features associated with these unique and rare disorders have shed light on the complex array of signals and processes that govern development and activation of the immune system. While the first examples of PID pertained to severe defects in lymphoid development, more recently a variety of gene defects have been identified in humans that do not compromize the ability to generate lymphocytes, but rather result in profound immune dysregulation. In many cases, identification of the molecular and cellular bases of PID has preceeded development of animal models by gene targeting. Finally, since the very first cases reported in humans, PID have also represented a unique tool to investigate the efficacy of novel therapeutic approaches (from molecular therapy to hematopoietic stem cell transplantation to somatic cells gene therapy), that have been applied or may apply to a variety of more common human diseases.