Loss of Purkinje cells in the PKCγ H101Y transgenic mouse

Spinocerebellar ataxia type 14 (SCA14) is an autosomal, dominant neurodegenerative disorder caused by mutations in PKCγ. The objective of this study was to determine effects of PKCγ H101Y SCA14 mutation on Purkinje cells in the transgenic mouse. Results demonstrated that wild type PKCγ-like Purkinje cell localization of HA-tagged PKCγ H101Y mutant proteins, altered morphology and loss of Purkinje cells were observed in the PKCγ H101Y SCA14 transgenic mouse at four weeks of age. Failure of stereotypical clasping responses in the hind limbs of transgenic mice was also observed. Further, PKCγ H101Y SCA14 mutation caused lack of total cellular PKCγ enzyme activity, loss of connexin 57 phosphorylation on serines, and activation of caspase-12 in the PKCγ H101Y SCA14 transgenic mouse. Results clearly demonstrate a need for PKCγ control of gap junctions for maintenance of Purkinje cells. This is the first transgenic mouse to our knowledge which models a human SCA14 mutation.     

Rapid determination of tacrine and other drug metabolites in microsomal incubate by newly developed targeting algorithm on UHPLC/TOFMS

A rapid simultaneous determination method for in vitro Cytochrome P450 (CYP) activity assay of 1,2,3,4-tetrahydroacridin-9-amine (tacrine) metabolites using ultra high performance liquid chromatography (UHPLC) coupled with computer-assisted in-source collision induced dissociation (CID) monitoring was investigated. In general, enzyme inhibition assays require quantitative analysis of incubates with drugs using various concentrations of substrates/inhibitors. The assay of CYP isozyme inhibition is an important informational step in the drug discovery process and, with the many substrates listed by the FDA, high-throughput qualitative and quantitative analyses are desired. Based on sub-2-micron packing material with reversed phase chromatography combined with a single liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS), a less than 1 min analysis time is presented for two additional drugs. We successfully determined seven of the eight potential isomer metabolites for the drug tacrine in 2.5 min using a 2 mm internal diameter × 100 mm length column and applying in-source CID with our newly developed chromatographic peak deconvolution technique. Although two of the peaks were heavily fused at a peak width of less than 300 ms, we could clearly identify these peaks by monitoring the chromatographic intensity difference of their fragment peaks on the mass spectrum.     

DPP-4 inhibition with linagliptin ameliorates the progression of premature aging in klotho−/− mice

Background

The potential of anti-aging effect of DPP-4 inhibitors is unknown. This study was performed to determine whether linagliptin, a DPP-4 inhibitor, could protect against premature aging in klotho?/? mice.

Methods

Klotho?/? mice exhibit multiple phenotypes resembling human premature aging, including extremely shortened life span, cognitive impairment, hippocampal neurodegeneration, hair loss, muscle atrophy, hypoglycemia, etc. To investigate the effect of linagliptin on these aging-related phenotypes, male klotho?/? mice were divided into two groups: (1) control group fed the standard diet, and (2) linagliptin group fed the standard diet containing linagliptin. Treatment with linagliptin was performed for 4 weeks. The effect of linagliptin on the above mentioned aging-related phenotypes was examined.

Results

Body weight of klotho?/? mice was greater in linagliptin group than in control group (11.1 ± 0.3 vs 9.9 ± 0.3 g; P < 0.01), which was associated with greater gastrocnemius muscle weight (P < 0.01) and greater kidney weight (P < 0.05) in linagliptin group. Thus, linagliptin significantly prevented body weight loss in klotho?/? mice. Survival rate of klotho?/? mice was greater in linagliptin group (93%) compared to control group (67%), although the difference did not reach statistical significance (P = 0.08). None of linagliptin-treated klotho?/? mice had alopecia during the treatment (P < 0.05 vs control klotho?/? mice). Latency of klotho?/? mice in passive avoidance test was larger in linagliptin group than in control group (P < 0.05), indicating the amelioration of cognitive impairment by linagliptin. Cerebral blood flow of klotho?/? mice was larger in linagliptin group than in control group (P < 0.01), being associated with greater cerebral phospho-eNOS levels (P < 0.05) in linagliptin group. Neuronal cell number in hippocampal CA1 region was greater in linagliptin group than in control group (P < 0.05). Linagliptin group had greater cerebral phospho-Akt (P < 0.05) and phospho-CREB (P < 0.05) than control group. Thus, linagliptin ameliorated brain aging in klotho?/? mice. The degree of hypoglycemia in klotho?/? mice was less in linagliptin group than in control group, as estimated by the findings of OGTT.

Conclusions

Out work provided the evidence that DPP-4 inhibition with linagliptin slowed the progression of premature aging in klotho?/? mice, and provided a novel insight into the potential role of DPP-4 in the mechanism of premature aging.

     

Conformational dynamics of wild-type Lys-48-linked diubiquitin in solution

Proteasomal degradation is mediated through modification of target proteins by Lys-48-linked polyubiquitin (polyUb) chain, which interacts with several binding partners in this pathway through hydrophobic surfaces on individual Ub units. However, the previously reported crystal structures of Lys-48-linked diUb exhibit a closed conformation with sequestered hydrophobic surfaces. NMR studies on mutated Lys-48-linked diUb indicated a pH-dependent conformational equilibrium between closed and open states with the predominance of the former under neutral conditions (90% at pH 6.8). To address the question of how Ub-binding proteins can efficiently access the sequestered hydrophobic surfaces of Ub chains, we revisited the conformational dynamics of Lys-48-linked diUb in solution using wild-type diUb and cyclic forms of diUb in which the Ub units are connected through two Lys-48-mediated isopeptide bonds. Our newly determined crystal structure of wild-type diUb showed an open conformation, whereas NMR analyses of cyclic Lys-48-linked diUb in solution revealed that its structure resembled the closed conformation observed in previous crystal structures. Comparison of a chemical shift of wild-type diUb with that of monomeric Ub and cyclic diUb, which mimic the open and closed states, respectively, with regard to the exposure of hydrophobic surfaces to the solvent indicates that wild-type Lys-48-linked diUb in solution predominantly exhibits the open conformation (75% at pH 7.0), which becomes more populated upon lowering pH. The intrinsic properties of Lys-48-linked Ub chains to adopt the open conformation may be advantageous for interacting with Ub-binding proteins.     

The functional traits of host fish can act as good predictors for parasite composition in a neotropical floodplain

Parasite diversity can be influenced by the interaction of environmental factors and host traits, but understanding which traits can be decisive for the establishment of the parasite may provide subsidies for a better understanding of the host-parasite relationship. In this study, we investigated whether functional traits, diet, and host phylogeny can predict the similarity of the endoparasite composition of a fish assemblage in a Brazilian floodplain. Of the three evaluated components, the host's diet was the factor that showed the greatest influence on the composition and similarity of endoparasites, demonstrating the highest value of the explanation. The functional traits and phylogeny, despite presenting significant values (unique effect and global effect), showed low explainability in the composition of the endoparasites. When analyzing the joint effects, all components showed significant influence. Hosts that live in the same environment that are phylogenetically related and have a similar ecology have a certain degree of homogeneity in their parasite assemblages and, because they are endoparasites (which are acquired trophically along the chain), diet is the main driver of parasite richness and similarity. Overall, host traits can be one of the main determinants of parasite composition, so studies that address the functional traits of the host provide a representation of local diversity and define the possible patterns of these parasite communities.     

Cardiovascular actions of l-cysteine and l-cysteine sulfinic acid in the nucleus tractus solitarius of the rat

The sulfur-containing excitatory amino acid (EAA) l-cysteine sulfinic acid (CSA), a neurotransmitter candidate, is endogenously synthesized from l-cysteine (Cys). Exogenous Cys administration into the brain produces cardiovascular effects; these effects likely occur via synaptic stimulation of central nervous system (CNS) neurons that regulate peripheral cardiovascular function. However, the cardiovascular responses produced by CNS Cys administration could result from CSA biosynthesized in synapse. The present study examined the role of CSA in Cys-induced cardiovascular responses within the nucleus tractus solitarius (NTS) of anesthetized rats. The NTS receives input from various visceral afferents that gate autonomic reflexes, including cardiovascular reflexes. Within the NTS, both Cys and CSA microinjections produced decrease responses in arterial blood pressure and heart rate that were similar to those produced by l-glutamate. Co-injection of the ionotropic EAA receptor antagonist kynurenic acid abolished Cys-, but not CSA-, induced cardiovascular responses. This finding suggests that only Cys-induced cardiovascular responses are mediated by kynurenate-sensitive receptors. This study provides the first demonstration that Cys- and CSA-induced cardiovascular responses occur via different mechanisms in the NTS of rats. Further, this study also indicates that Cys-induced cardiovascular responses do not occur via CSA. Thus, within the NTS, endogenous Cys and/or CSA might be involved in cardiovascular regulation.     

Long-Term Oral Administration of Hop Flower Extracts Mitigates Alzheimer Phenotypes in Mice

Coincident with the expanding population of aged people, the incidence of Alzheimer disease (AD) is rapidly increasing in most advanced countries. At present, no effective prophylactics are available. Among several pathological mechanisms proposed for AD, the “amyloid hypothesis” has been most widely accepted, in which accumulation or deposition of Aβ is considered to be the initial event. Thus, prevention of Aβ production would be an ideal strategy for the treatment or prevention of AD. Aβ is produced via the proteolytic cleavage of its precursor protein, APP (amyloid precursor protein), by two different enzymes, β and γ-secretases. Indeed, inhibitors against either or both enzymes have been developed and tested for clinical efficacy. Based on the “amyloid hypothesis”, we developed a luciferase-based screening method to monitor γ-secretase activity, screened more than 1,600 plant extracts, most of which have long been used in Chinese medicine, and observed that Hop extracts significantly inhibit Aβ production in cultured cells. A major component of the inhibitory activity was purified, and its chemical identity was determined by NMR to be Garcinielliptone HC. In vivo, oral administration of Hop extracts to AD model mice decreased Aβ depositions in the cerebral cortex of the parietal lobe, hippocampus, and artery walls (amyloid angiopathy) in the brains. In a Morris water maze test, AD model mice that had daily consumed Hop extracts in their drinking water showed significant mitigation of memory impairment at ages of 9 and 12 months. Moreover, in the open field test oral administration of Hop extracts also prevented an emotional disturbance that appeared in the AD mice at 18 months. Despite lifelong consumption of Hop extracts, no deleterious side effects were observed at any age. These results support the “amyloid hypothesis”, and indicate that Hop extract is a promising candidate for an effective prophylactic for AD.     

Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.