A Lysine-to-Arginine Change Found in Natural Alleles of the Human T-Cell Lymphotropic/Leukemia Virus Type 1 p12I Protein Greatly Influences Its Stability

The HTLV-1 singly spliced open reading frame I protein, p12(I), is highly unstable and appears to be necessary for persistent infection in rabbits. Here we demonstrate that p12(I) forms dimers through two putative leucine zipper domains and that its stability is augmented by specific proteasome inhibitors. p12(I) is ubiquitylated, and mutations of its unique carboxy-terminus lysine residue to an arginine greatly enhance its stability. Interestingly, analysis of 53 independent HTLV-1 strains revealed that the natural p12(I) alleles found in ex vivo samples of tropical spastic paraparesis-HTLV-1-associated myelopathy patients contain a Lys at position 88 in some cases, whereas arginine is consistently found at position 88 in HTLV-1 strains from all adult T-cell leukemia-lymphoma (ATLL) cases and healthy carriers studied. This apparent segregation of different alleles in tropical spastic paraparesis-HTLV-associated myelopathy and ATLL or healthy carriers may be relevant in vivo, since p12(I) binds the interleukin-2 receptor beta and gammac chains, raising the possibility that the two natural alleles might affect differently the regulation of these molecules.     

Differential effects of various growth factors and cytokines on the syntheses of DNA,type I collagen,laminin, fibronectin,osteonectin/secreted protein,acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor-β (TGF-β) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3- to 10-fold. Western and Northern blots showed that TGF-β enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet-derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [³H]thymidine incorporation into DNA. TGF-β, EGF, and TNF-α had less effect on DNA synthesis, whereas IL-1β inhibited DNA synthesis. These findings demonstrated that TGF-β, bFGF, EGF, PDGF, TNF-α, and IL-1β have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells. J. Cell. Physiol. 174:194–205, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis,SAR, and X-ray structure of tricyclic compounds as potent FBPase inhibitors

With the aim of discovering a novel class of fructose-1,6-bisphosphatase (FBPase) inhibitors, a series of compounds based on tricyclic scaffolds was synthesized. Extensive SAR studies led to the finding of 8l with an IC₅₀ value of 0.013 μM against human FBPase. An X-ray crystallographic study revealed that 8l bound at AMP binding sites of human liver FBPase with hydrogen bonding interactions similar to AMP.     

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.     

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.