Corneal alterations during combined therapy with cyclodextrin/allopregnanolone and miglustat in a knock-out mouse model of NPC1 disease

Background

Niemann Pick disease type C1 is a neurodegenerative disease caused by mutations in the NPC1 gene, which result in accumulation of unesterified cholesterol and glycosphingolipids in the endosomal-lysosomal system as well as limiting membranes. We have previously shown the corneal involvement in NPC1 pathology in form of intracellular inclusions in epithelial cells and keratocytes. The purpose of the present study was to clarify if these inclusions regress during combined substrate reduction- and by-product therapy (SRT and BPT).

Methodology/Principal Findings

Starting at postnatal day 7 (P7) and thereafter, NPC1 knock-out mice (NPC1^−/−) and wild type controls (NPC1^+/+) were injected with cyclodextrin/allopregnanolone weekly. Additionally, a daily miglustat injection started at P10 until P23. Starting at P23 the mice were fed powdered chow with daily addition of miglustat. The sham group was injected with 0.9% NaCl at P7, thereafter daily starting at P10 until P23, and fed powdered chow starting at P23. For corneal examination, in vivo confocal laser-scanning microscopy (CLSM) was performed one day before experiment was terminated. Excised corneas were harvested for lipid analysis (HPLC/MS) and electron microscopy.In vivo CLSM demonstrated a regression of hyperreflective inclusions in all treated NPC1^−/−mice. The findings varied between individual mice, demonstrating a regression, ranging from complete absence to pronounced depositions. The reflectivity of inclusions, however, was significantly lower when compared to untreated and sham-injected NPC1^−/− mice. These confocal findings were confirmed by lipid analysis and electron microscopy. Another important CLSM finding revealed a distinct increase of mature dendritic cell number in corneas of all treated mice (NPC1^−/− and NPC1^+/+), including sham-treated ones.

Conclusions/Significance

The combined substrate reduction- and by-product therapy revealed beneficial effects on the cornea. In vivo CLSM is a non-invasive tool to monitor disease progression and treatment effects in NPC1 disorder.     

Role of FAT/CD36 in novel PKC isoform activation in heart of spontaneously hypertensive rats

Disruption to the sensitive balance of long-chain fatty acids and glucose in the heart could cause cardiovascular diseases. Searching for a possible role of novel protein kinase C (nPKC) in heart with disrupted energy balance, we compared the insulin-resistant spontaneously hypertensive rats (SHR), which carry a nonfunctional variant of the fatty acid transporter FAT/CD36, with the less insulin-resistant congenic strain SHR-4 that is genetically identical except for a segment on chromosome 4 including a wild-type gene for a functional FAT/CD36. We analyzed expression of the nPKC-δ and -ε isoforms plus triacylglycerols (TAG) content in the myocardium of both FAT/CD36 strains and after a high sucrose diet (HSD). Two weeks before killing, males of both strains were randomly divided into two groups and fed either a standard laboratory chow or an HSD. PKC was determined by Western blotting in particulate and cytosolic fractions from left ventricles. The SHR-4 rats exhibited lower serum levels of insulin and free fatty acids than did SHR rats and higher amounts of PKC-ε in the heart particulate fraction. HSD caused accumulation of heart TAG in SHR but not in SHR-4. HSD increased PKC-δ and decreased PKC-ε expression in particulate fraction from left ventricles of SHR-4 while having no effects in SHR. These results demonstrate that reduced insulin resistance in SHR-4 rats with wild-type FAT/CD36 is associated with the insulin signaling pathway involving nPKCs.     

Porphyrin Binding to Gun4 Protein,Facilitated by a Flexible Loop,Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.     

Bioprospecting of Turbinaria Macroalgae as a Potential Source of Health Protective Compounds

The present study aims to focus on the bioprospecting of marine macroalgae of Turbinaria species, plenteous biomass of the world ocean. Three types of solvents, i.e., H₂O, MeOH/H₂O (80:20, v/v) and hexane/i‐PrOH (50:50, v/v), were used for extraction. Both the biological activity and the pattern of present chemicals were characterized. For the cell proliferation assay, the human embryonic kidney 293 cells, cervix/breast/pancreatic adenocarcinoma, and osteosarcoma cells were used. For the antioxidant activity determination, both intracellular assay with human embryonic kidney and cervix adenocarcinoma cells, as well as the biochemical DPPH test, were employed. To complete the information about macroalgae composition, organic compounds were characterized by the liquid chromatography coupled with high resolution tandem mass spectrometry. Attention was concentrated mainly on the lipidomic profile characterization. In spite the fact that any significant antiproliferative effect was not observed for cancer cells, both the Turbinaria species were shown to be good protectors against the oxidative stress of the non‐cancer cells. Most of the antioxidants were determined in the hexane/i‐PrOH extract. As regards the lipids identified, most of them belonged to the triacylglycerols followed by sphingomyelins, diacylglycerols, and polar (lyso)phospholipids. Additionally to fatty acids with 14, 16 and 18 carbons, also those with odd carbon numbers were frequently present.     

Production of Hyaluronic Acid by Mutant Strains of Group C Streptococcus

This study addresses the influence of upstream region sequence on the strength of has operon promoter in highly encapsulated S. equi subsp. zooepidemicus (SEZ). For this purpose, seven different strains were constructed. Each strain carries a point mutation in one of the following positions upstream of the has promoter: ?43, ?44, ?49, and ?50 bp. To facilitate measuring of the recombinant promoter relative strength, ß-glucuronidase gene was used as a reporter gene. Three mutations located in positions ?49 and ?50: AT, GT, and AG, positively impacted has promoter strength when compared to the wild type sequence GG. Conversely, two other mutations: TG and TT, exhibited a slight inhibitory effect. Further, three different strains carrying chromosomal mutations in the has promoter region were constructed. In two cases, the has operon is under the control of a stronger promoter and in the third strain the has operon is controlled by a weaker promoter. The laboratory fermenter scale cultivations confirmed the increase of hyaluronan yields for SEZPhasAG and SEZPhas2G, resulting 116 and 105 %, respectively. As expected, the yield of the hyaluronic acid of SEZPhas2B strain fell to 41 %.     

Evolution of karyotype,sex chromosomes,and meiosis in mygalomorph spiders (Araneae: Mygalomorphae)

Spider diversity is partitioned into three primary clades, namely Mesothelae, Mygalomorphae, and Araneomorphae. Mygalomorph cytogenetics is largely unknown. Our study revealed a remarkable karyotype diversity of mygalomorphs. Unlike araneomorphs, they show no general trend towards a decrease of 2n, as the chromosome number was reduced in some lineages and increased in others. A biarmed karyotype is a symplesiomorphy of mygalomorphs and araneomorphs. Male meiosis of some mygalomorphs is achiasmatic, or includes the diffuse stage. The sex chromosome system X₁X₂0, which is supposedly ancestral in spiders, is uncommon in mygalomorphs. Many mygalomorphs exhibit more than two (and up to 13) X chromosomes in males. The evolution of X chromosomes proceeded via the duplication of chromosomes, fissions, X–X, and X‐autosome fusions. Spiders also exhibit a homomorphic sex chromosome pair. In the germline of mygalomorph males these chromosomes are often deactivated; their deactivation and pairing is initiated already at spermatogonia. Remarkably, pairing of sex chromosomes in mygalomorph females is also initiated at gonial cells. Some mygalomorphs have two sex chromosome pairs. The second pair presumably arose in early‐diverging mygalomorphs, probably via genome duplication. The unique behaviour of spider sex chromosomes in the germline may promote meiotic pairing of homologous sex chromosomes and structural differentiation of their duplicates, as well as the establishment of polyploid genomes. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 377–408.     

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Accurate species-level identifications underpin many aspects of basic and applied biology;however,identifications can be hampered by a lack of discriminating morphological characters,taxonomic expertise or time.Molecular approaches,such as DNA"barcoding"of the cytochrome c oxidase(COI)gene,are argued to overcome these issues.However,nuclear encoding of mitochondrial genes(numts)and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications.One insect group for which these molecular and morphological problems are significant are the dacine fruit flies(Diptera:Tephritidae:Dacini).We addressed these issues associated with COI barcoding in the dacines by first assessing several"universal"COI primers against public mitochondrial genome and numt sequences for dacine taxa.We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region.Our new primers were tested alongside universal primers on a selection of dacine species,including both fresh preserved and decades-old dry specimens.Additionally,Bactrocera tiyoni mitochondrial and nuclear genomes were compared to identify putative numts.Four numt clades were identified,three of which were amplified using existing universal primers.In contrast,our new primers preferentially amplified the"true"mitochondrial COI barcode in all dacine species tested.The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable.Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.