Parity decreases methamphetamine-induced striatal dopaminergic perturbation

The role of parity upon methamphetamine-induced neurotoxicity of the striatal dopaminergic system was assessed. Female CD-1 mice either remained nulliparous or underwent one or three complete pregnancies and were designated as the 0, 1 or 3 pregnancy groups. The mice were then treated with a neurotoxic regimen of methamphetamine (MA - 40 mg/kg) or its saline vehicle (control) and striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels were measured at 7-days post-MA. Basal levels of striatal DA, DOPAC and the DOPAC/DA ratio were similar among the saline (control) 0, 1 and 3 pregnancy groups. In response to MA, striatal DA and DOPAC were significantly decreased in the 0 and 1 pregnancy as compared with the control group. Mice with 3 pregnancies showed DA and DOPAC levels that did not differ from controls and were significantly greater than the 0 pregnancy group. The DOPAC/DA ratios of the 0 pregnancy group were significantly greater than all other groups (control, 1 and 3 pregnancy) which failed to differ among each other. These results demonstrate that parity decreases MA-induced striatal dopaminergic neurotoxicity, and the degree of this neuroprotection is related to the number of pregnancies experienced.     

PKCδ inhibition enhances tyrosine hydroxylase phosphorylation in mice after methamphetamine treatment

The present study was designed to evaluate the specific role of protein kinase C (PKC) δ in methamphetamine (MA)-induced dopaminergic toxicity. A multiple-dose administration regimen of MA significantly increases PKCδ expression, while rottlerin, a PKCδ inhibitor, significantly attenuates MA-induced hyperthermia and behavioral deficits. These behavioral effects were not significantly observed in PKCδ antisense oligonucleotide (ASO)-treated- or PKCδ knockout (−/−)-mice. There were no MA-induced significant decreases of dopamine (DA) content or tyrosine hydroxylase (TH) expression in the striatum in rottlerin-treated-, ASO-treated- or PKCδ (−/−)-mice. The administration of MA also results in a significant decrease of TH phosphorylation at ser 40, but not ser 31, while the inhibition of PKCδ consistently and significantly attenuates MA-induced reduction in the phosphorylation of TH at ser 40. Therefore, these results suggest that the MA-induced enhancement of PKCδ expression is a critical factor in the impairment of TH phosphorylation at ser 40 and that pharmacological or genetic inhibition of PKCδ may be protective against MA-induced dopaminergic neurotoxicity in vivo.     

Identification of Critical Motifs within HIV-1 Integrase Required for Importin α3 Interaction and Viral cDNA Nuclear Import

The viral cDNA nuclear import is an important requirement for human immunodeficiency virus type 1 (HIV-1) replication in dividing and nondividing cells. Our recent study identified a specific interaction of importin α3 (Impα3) with HIV-1 integrase (IN) and its involvement in viral cDNA nuclear import. In this study, we have performed a more detailed investigation on the molecular mechanism of how HIV-1 IN interacts with Impα3. Our results revealed a reduced interaction between the two IN mutants INKK215,9AA (IN215,9) and INRK263,4AA (IN263,4) with Impα3, while an IN double mutant, IN215,9/263,4, was severely impaired for its Impα3-binding ability, even though it was still found interacting with other cofactors, IN interactor I and Transportin3. Immunostaining and fractionation analysis have shown that YFP-IN215,9/263,4 failed to localize in the nucleus of transfected cells. Also, we found that both major and minor nuclear localization signal binding grooves of Impα3 are involved in interaction with IN. All of these results suggest a cargo protein-import receptor type of interaction. Finally, the effect of IN215,9/263,4 mutations on HIV-1 replication was evaluated, and real-time quantitative PCR analysis showed that, while mutant virus (v215,9/263,4) had a slightly lowered total viral DNA, the 2-long-terminal-repeat DNA, a marker for nuclear import, was greatly reduced during v215,9/263,4 infection in both dividing and nondividing cells. Also, by cell fractionation assay, we found that a significant proportion of viral cDNA was still retained in cytoplasmic fraction of v215,9/263,4-infected cells. Overall, our study provides strong evidence that ²¹¹KELQKQITK and ²⁶²RRKAK regions of IN C-terminal domain are required for Impα3 interaction and HIV-1 cDNA nuclear import.     

Structure and Stoichiometry of Template-Directed Recombinant HIV-1 Gag Particles

Size polydispersity of immature human immunodeficiency virus type 1 (HIV-1) particles represents a challenge for traditional methods of biological ultrastructural analysis. An in vitro model for immature HIV-1 particles constructed from recombinant Gag proteins lacking residues 16-99 and the p6 domain assembled around spherical nanoparticles functionalized with DNA. This template-directed assembly approach led to a significant reduction in size polydispersity and revealed previously unknown structural features of immature-like HIV-1 particles. Electron microscopy and image reconstruction of these particles suggest that the Gag shell formed from different protein regions that are connected by a “scar”—an extended defect connecting the edges of two continuous, regularly packed protein layers. Thus, instead of a holey protein array, the experimental model presented here appears to consist of a continuous array of ∼ 5000 proteins enveloping the core, in which regular regions are separated by extended areas of disorder.     

Up-regulation of the vitamin C transporter SVCT2 upon differentiation and depolarization of myotubes

In addition to its role as a strong antioxidant, vitamin C regulates the differentiation of several cell lineages. In vertebrate skeletal muscle, the vitamin C transporter SVCT2 is preferentially expressed in slow muscle fibers. To gain insights into the possible involvement of intracellular vitamin C on early myogenesis, we investigated the regulation of SVCT2 expression in cultures of chick fetal myoblasts. SVCT2 expression increases in cultures of both, slow and fast muscle-derived myoblasts, as they fuse to form mainly fast myotubes. Interestingly, we found that SVCT2 could be positively modulated by potassium-induced depolarization of myotubes. These findings suggest that SVCT2-mediated uptake of vitamin C could play diverse roles on skeletal muscle development and physiology.     

Nitric oxide increases the invasion of pancreatic cancer cells via activation of the PI3K–AKT and RhoA pathways after carbon ion irradiation

Previous studies have shown that serine proteases and Rho-associated kinase contribute to carbon ion radiation-enhanced invasion of the human pancreatic cancer cell line PANC-1. The results presented here show that nitric oxide synthase (NOS) also plays a critical role in this process. Irradiation of PANC-1 cells promoted invasion and production of nitric oxide (NO), which activated the PI3K–AKT signaling pathway, while independently activating RhoA. Inhibition of PI3K, Rho-associated kinase, and serine protease alone or in conjunction with NOS suppressed the radiation-enhanced invasion of PANC-1 cells, suggesting that they could serve as possible targets for the management of tumor metastasis.     

Analysis of 3 common polymorphisms in the KCNK18 gene in an Australian Migraine Case-control cohort

Migraine is a common neurological disorder characterised by temporary disabling attacks of severe head pain and associated disturbances. There is significant evidence to suggest a genetic aetiology to the disease however few causal mutations have been conclusively linked to the migraine subtypes Migraine with (MA) or without Aura (MO). The Potassium Channel, Subfamily K, member 18 (KCNK18) gene, coding the potassium channel TRESK, is the first gene in which a rare mutation resulting in a non-functional truncated protein has been identified and causally linked to MA in a multigenerational family. In this study, three common polymorphisms in the KCNK18 gene were analysed for genetic variation in an Australian case-control migraine population consisting of 340 migraine cases and 345 controls. No association was observed for the polymorphisms examined with the migraine phenotype or with any haplotypes across the gene. Therefore even though the KCNK18 gene is the only gene to be causally linked to MA our studies indicate that common genetic variation in the gene is not a contributor to MA.     

Calorimetric analysis of the plasma proteome: Identification of type 1 diabetes patients with early renal function decline

Background

Microalbuminuria (MA) has been questioned as a predictor of progressive renal dysfunction in patients with type 1 diabetes (T1D). Consequently, new clinical end points are needed that identify or predict patients that are at risk for early renal function decline (ERFD). The potential clinical utility of differential scanning calorimetry (DSC) analysis of blood plasma and other biofluids has recently been reported. This method provides an alternate physical basis with which to study disease-associated changes in the bulk plasma proteome.

Methods

DSC analysis of blood plasma was applied to identify unique signatures of ERFD in subjects enrolled in the 1st Joslin Study of the Natural History of Microalbuminuria in Type 1 Diabetes, a prospective cohort study of T1D patients. Recent data suggests that differences in the plasma peptidome of these patients correlate with longitudinal measures of renal function. Differences in DSC profile (thermogram) features were evaluated between T1D MA individuals exhibiting ERFD (n = 15) and matched control subjects (n = 14).

Results

The average control group thermogram resembled a previously defined healthy thermogram. Differences were evident between ERFD and control individuals. Heat capacity values of the main two transitions were found to be significant discriminators of patient status.

Conclusions

Results from this pilot study suggest the potential utility of DSC proteome analysis to prognostic indicators of renal disease in T1D.

General significance

DSC shows sensitivity to changes in the bulk plasma proteome that correlate with clinical status in T1D providing additional support for the utility of DSC profiling in clinical diagnostics.     

Hyaluronate coat formation and cell spreading in rat fibrosarcoma cells

Hyaluronate-containing pericellular coats have been demonstrated around rat fibrosarcoma cells by exclusion of particles (fixed red blood cells). The cell coats normally form during spreading of the rat fibrosarcoma cells subsequent to subculturing. Monensin, a drug which disrupts the Golgi and which also inhibits hyaluronate synthesis in these cells, inhibits the regeneration of these coats after hyaluronidase or trypsin treatment but does not inhibit cell spreading. Cycloheximide, a drug which inhibits protein but not hyaluronate synthesis does not prevent coat regeneration but partially inhibits cell spreading. Thus by exploiting the opposing effects of cycloheximide and monensin on coat regeneration and cell spreading, we have been able to dissociate these two phenomena.