Using tuberous sclerosis complex to understand the impact of MTORC1 signaling on mitochondrial dynamics and mitophagy in neurons

Constitutive activation of the MTOR pathway is a key feature of defects in the tuberous sclerosis complex and other genetic neurodevelopmental diseases, collectively referred to as MTORopathies. MTORC1 hyperactivity promotes anabolic cell functions such as protein synthesis, yet at the same time catabolic processes such as macroautophagy/autophagy are suppressed. Mitochondria are major substrates of autophagy; however, their role in MTORopathies remains largely undefined. Here, we review our recent study showing that several aspects of mitochondrial function, dynamics and turnover are critically impaired in neuronal models of TSC. We discuss the relevance of these findings to neurological manifestations associated with TSC and speculate on autophagy as a novel treatment target for MTORopathies.     

Identification of antimony resistance markers in Leishmania tropica field isolates through a cDNA-AFLP approach

Pentavalent antimonial compounds have been the first line therapy for leishmaniasis; unfortunately the rate of treatment failure of anthroponotic cutaneous leishmaniasis (ACL) is increasing due to emerging of drug resistance. Elucidation of the molecular mechanisms operating in antimony resistance is critical for development of new strategies for treatment. Here, we used a cDNA-AFLP approach to identify gene(s) which are differentially expressed in resistant and sensitive Leishmania tropica field isolates. We identified five genes, aquaglyceroporin (AQP1) acts in drug uptake, ATP-binding cassette (ABC) transporter (MRPA) involved in sequestration of drug, phosphoglycerate kinase (PGK) implicated in glycolysis metabolism, mitogen activated protein kinase (MAPK) and protein tyrosine phosphatase (PTP) responsible for phosphorylation pathway. The results were confirmed using real time RT-PCR which revealed an upregulation of MRPA, PTP and PGK genes and downregulation of AQP1 and MAPK genes in resistant isolate. To our knowledge, this is the first report of identification of PTP and PGK genes potentially implicated in resistance to antimonials. Our findings support the idea that distinct biomolecules might be involved in antimony resistance in L. tropica field isolates.     

Inhibition by heparin of protein kinase C activation and hydroxyl radical generation in puromycin aminonucleoside treated isolated rat hepatocytes

Heparin has been reported to have many actions similar to calcium-dependent protein kinase (PKC) inhibitors. We have found that puromycin aminonucleoside (PAN) increases hydroxyl radical generation and this was prevented by H-7, a PKC inhibitor in isolated rat hepatocytes. In this study, we investigate the effect of heparin on the increased hydroxyl radical generation as well as PKC activation by PAN in isolated rat hepatocytes. To estimate the amount of hydroxyl radical generation, we measured methylguanidine (MG) and creatol which are the products from the reaction of creatinine and hydroxyl radical. Synthetic rate of MG plus creatol in isolated rat hepatocytes incubated in Krebs-Henseleit bicarbonate buffer containing creatinine and tested reagents were recorded. This rate with or without PAN was 231 ± 11 or 112 ± 5.6 nmol/g wet cells/4 h (mean ± S.E., n = 5), respectively. Heparin concentrations of 3.3, 6.6 and 10 U/ml inhibited MG plus creatol synthesis in the presence of PAN by 30, 38 and 39%, and without PAN by 8.4, 27 and 34%, respectively. Statistical significance was observed except for 3.3 U/ml without PAN. The ratio of PKC in membrane/cytoplasmic fraction, an indicator of PKC activation, increased 2.8- and 3-fold that of the 0 time after 60 and 120 min incubation with PAN while heparin at 10 U/ml almost completely suppressed this increase in the ratio of PKC. The PKC ratio of the membrane/cytoplasmic fraction obtained from hepatocytes with heparin alone or without PAN and heparin was almost unchanged during the tested period. Variation of PKC levels in membrane fraction is similar to that of PKC ratio of the membrane/cytoplasmic fraction. Increased creatol synthesis by PAN and its inhibition by heparin were observed in the same samples as those used for the PKC study.These results indicate that heparin inhibits the increase in hydroxyl radical generation induced by PAN through inhibition of PKC activation in isolated rat hepatocytes.     

TRAPPopathies: An emerging set of disorders linked to variations in the genes encoding transport protein particle (TRAPP)‐associated proteins

The movement of proteins between cellular compartments requires the orchestrated actions of many factors including Rab family GTPases, Soluble NSF Attachment protein REceptors (SNAREs) and so‐called tethering factors. One such tethering factor is called TRAnsport Protein Particle (TRAPP), and in humans, TRAPP proteins are distributed into two related complexes called TRAPP II and III. Although thought to act as a single unit within the complex, in the past few years it has become evident that some TRAPP proteins function independently of the complex. Consistent with this, variations in the genes encoding these proteins result in a spectrum of human diseases with diverse, but partially overlapping, phenotypes. This contrasts with other tethering factors such as COG, where variations in the genes that encode its subunits all result in an identical phenotype. In this review, we present an up‐to‐date summary of all the known disease‐related variations of genes encoding TRAPP‐associated proteins and the disorders linked to these variations which we now call TRAPPopathies.      

Controlled microchannelling in dense collagen scaffolds by soluble phosphate glass fibers

A problem with tissue engineering scaffolds is maintaining seeded cell viability and function due to limitations of oxygen and nutrient transfer. An approach to maintain suitable oxygen concentrations throughout the scaffold would be to controllably incorporate microchannelling within these scaffolds. This study investigated the incorporation of unidirectionally aligned soluble phosphate based glass fibers (PGF) into dense collagen scaffolds. PGF are degradable, and their degradation can be controlled through their chemistry and dimensions. Plastic compression was used to produce composite scaffolds at three different weight percentage while maintaining greater than 80% resident cell viability. PGF-collagen scaffold composition was quantified through thermogravimetric analysis as well as being morphologically and mechanically characterized. PGF degradation was measured through ion chromatography, and channel formation was verified with ultrasound imaging and SEM. The free movement of coated microbubble agents confirmed the channels to be continuous in nature and of 30-40 microm diameter. These microchannels in dense native collagen matrices could play an important role in hypoxia/perfusion limitations and also in the transportation of nutrients and potentially forming blood vessels through dense implants.     

Pathogenicity of three entomopathogenic nematodes against the onion thrips,Thrips tabaci Lind. (Thys.; Thripidae)

Pathogenicity of a native isolate of Steinernema feltiae (H1) and two exotic strains, Heterorhabditis bacteriophora and Steinernema carpocapsae was assessed under laboratory conditions using different concentrations i.e. 4000, 6000, 8000 and 10,000 infective juveniles/ml against second instar larvae, prepupa and pupa of Thrips tabaci Lindeman. The mortality data were recorded 24 and 48?h post-inoculation. The highest mortality rate was recorded for prepupa (62%) than second instar (12.5%) by H. bacteriophora and S. carpocapsae, respectively, 24?h after treatment. No significant differences were found in mortality between prepupa and pupa with increasing the nematodes concentrations (from 4000 to 10,000 nematode/ml) but increasing nematode concentrations increased the mortality of second instar. At the end of the experiment (48?h.), S. feltiae H1 caused the highest mortality on second instar larvae (74%), whereas all other species caused 80–83% mortalities on pupa. This study suggests that native isolate of S. feltiae (H1) had high potential to infect soil-dwelling stages of T. tabaci.     

Effect of improved diabetes control on the expression of lipoprotein lipase in human adipose tissue.

Patients with diabetes commonly manifest hypertriglyceridemia along with decreased adipose tissue lipoprotein lipase (LPL) activity, and improved diabetes control tends to reverse these abnormalities. To better understand the mechanism of regulation of LPL in diabetes, 11 diabetic patients (3 type I, 8 type II) were brought under improved glycemic control, and adipose tissue LPL gene expression was assessed by performing paired fat biopsies. Six of the 11 patients attained improved control with insulin, with a decrease in glycohemoglobin (glyc Hgb) from 13.8 +/- 0.9 to 10.4 +/- 0.6%; 5 patients attained improved control with glyburide (glyc Hgb fell from 14.2 +/- 2.4 to 8.8 +/- 0.6%), and together they demonstrated a lowering of serum triglycerides and total cholesterol. No changes were observed in HDL cholesterol. Improved diabetes control resulted in a significant increase in LPL activity in both the heparin-releasable (HR) and extractable (EXT) fractions of adipose tissue, as well as in LPL immunoreactive mass. The change in LPL activity with improved control was variable, and showed a positive correlation with the HDL levels prior to treatment (r = 0.74, P less than 0.02). When adipose tissue was pulse-labeled with [35S]methionine, there was an increase in isotope incorporation into LPL after treatment, indicating an increase in LPL synthetic rate. However, improved diabetes control resulted in no significant change in LPL mRNA levels. Thus, improved glycemic control resulted in an increase in LPL activity which correlated with each patient's basal high density lipoprotein. This increase in LPL activity was accompanied by an increase in LPL immunoreactive mass, and an increase in LPL synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chaperone Potential of Pulicaria undulata Extract in Preventing Aggregation of Stressed Proteins

This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease.