Acute and chronic toxicity of Nigella sativa fixed oil

We investigated the toxicity of the fixed oil of Nigella sativa L seeds in mice and rats through determination of LD50 values and examination of possible biochemical, hematological and histopathological changes. The acute toxicity of Nigella sativa fixed oil was investigated in mice. LD50 values, obtained by single doses, orally and intraperitoneally administered in mice, were 28.8 ml/kg body wt. p.o. [26.2-31.6] and 2.06 ml/kg body wt. i.p. [1.86-2.26], respectively. Chronic toxicity was studied in rats treated daily with an oral dose of 2 ml/kg body wt. for 12 weeks. Changes in key hepatic enzymes levels, including aspartate-aminotransferase, alanine-aminotranferase, and gamma-glutamyltransferase and histopathological modifications (heart, liver, kidneys and pancreas) were not observed in rats treated with Nigella sativa after 12 weeks of treatment. The serum cholesterol, triglyceride and glucose levels and the count of leukocytes and platelets decreased significantly, compared to control values, while hematocrit and hemoglobin levels increased significantly. A slowing of body weight gain was also observed in Nigella sativa treated rats, as compared to control animals. The low toxicity of Nigella sativa fixed oil, evidenced by high LD50 values, key hepatic enzyme stability and organ integrity, suggests a wide margin of safety for therapeutic doses of Nigella sativa fixed oil, but the changes in hemoglobin metabolism and the fall in leukocyte and platelet count must be taken into consideration.     

Memo-RhoA-mDia1 signaling controls microtubules, the actin network, and adhesion site formation in migrating cells

Actin assembly at the cell front drives membrane protrusion and initiates the cell migration cycle. Microtubules (MTs) extend within forward protrusions to sustain cell polarity and promote adhesion site turnover. Memo is an effector of the ErbB2 receptor tyrosine kinase involved in breast carcinoma cell migration. However, its mechanism of action remained unknown. We report in this study that Memo controls ErbB2-regulated MT dynamics by altering the transition frequency between MT growth and shortening phases. Moreover, although Memo-depleted cells can assemble the Rac1-dependent actin meshwork and form lamellipodia, they show defective localization of lamellipodial markers such as α-actinin-1 and a reduced number of short-lived adhesion sites underlying the advancing edge of migrating cells. Finally, we demonstrate that Memo is required for the localization of the RhoA guanosine triphosphatase and its effector mDia1 to the plasma membrane and that Memo–RhoA–mDia1 signaling coordinates the organization of the lamellipodial actin network, adhesion site formation, and MT outgrowth within the cell leading edge to sustain cell motility.     

Découverte de charophytes et ostracodes de l’Yprésien inférieur dans les Monts des Ksour (Algérie) : biostratigraphie et paléoécologie

A stratigraphic, palaeontological and sedimentological study was carried out in the red beds cropping out on the left side of Oued Tafarahit, south-east of the Ksour Mountains (Algeria). The studied succession consists of a non-marine fining-upward detrital formation, including microconglomerate, sandstone and clay beds, which were previously attributed to the Cenozoic sensu lato, since no palaeontological evidence was available. Sedimentology suggests deposition in a fluvial environment. The clayey levels yielded the assemblage formed by the charophytes Peckichara atlasensis, Maedleriella cristellata, Nitellopsis (Tectochara) thaleri, Grovesichara sp., and Lamprothamnium papulosum and the ostracods Neocyprideis meguerchiensis, Herpetocypris? sp. and Cyprinotus? sp. This assemblage allows constraining the age of the detrital series from Oued Tafarahit to the Ypresian. Ostracods are typical of freshwater to euryhaline environments. The freshwater taxa (Herpetocypris? sp. and Cyprinotus? sp.) would indicate phases of desalinisation during periods of flood. However, the brackish water species (Neocyprideis meguerchiensis) is characteristic of saline phases related to low water table periods. Moreover the charophyte and ostracod assemblage confirms a close palaeobiogeographic relationship between North Africa and Southern Europe during the Lower Eocene.     

Genotyping of Cre-lox mice and detection of tissue-specific recombination by multiplex PCR.

Conditional gene targeting, based on Cre-lox or other systems, requires frequent genotyping of transgenic mouse populations and monitoring of tissue-specific Cre recombinatory efficiency. This is currently achieved by Southern analysis from tail- and tissue-derived DNA. Multiplex PCR amplification of the floxed (flanked by loxP sites) genomic region, combined with the PCR detection of the Cre transgene, simplifies this task. Here, we show that complete genotyping of a floxed locus is possible with three appropriately placed primers and that this triplex PCR can be performed simultaneously with a universal PCR assay for the detection of Cre transgenes. Using this approach, we also determined the ratios of recombined versus non-recombined floxed genomic segments in genomic DNA samples. This allowed us to estimate the efficiency of in vivo conditional inactivation from biopsy material and tissue samples that were too small for Southern analysis. As many new conditional knockouts are spatiotemporally restricted, such assays will become increasingly useful. The proposed PCR strategy is flexible and may be adapted to the structural specificities of any target gene.     

Red cell enzyme polymorphisms in Moroccans and southern Spaniards: new data for the genetic history of the western Mediterranean.

Population samples from Morocco (El Jadida, south Atlantic coast) and La Alpujarra (Granada mountains, Spain), located on both shores of the western Mediterranean, were typed for 8 erythrocyte genetic markers: ACP1, ESD, PGD, AK1, GLO1, PGM1, SODA, and DIA. Genetic heterogeneity within western Mediterranean groups was investigated on the basis of allele frequencies of these 8 polymorphisms plus ABO and Rh (CDE). Only slight peculiarities for the ACP1, GLO1, and AK1 systems were observed in the 2 samples compared with other Mediterranean data. The new data are consistent with a main north to south genetic differentiation in the Mediterranean region. However, with regard to other European groups, the La Alpujarra population shows a particular affinity with North Africans that may be compatible with both an ancient common substratum and/or a special historical influence during the Muslim domination of the Iberian Peninsula.     

Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene

Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially Cre-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.     

High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR)

Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies. The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, we have previously introduced a technology--linear amplification-mediated (LAM) PCR--for detecting and sequencing unknown DNA flanking sequences down to the single cell level (Supplementary Note online). LAM-PCR analyses have enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies, and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. The reliability and robustness of this method results from the initial preamplification of the vector-genome junctions preceding nontarget DNA removal via magnetic selection. Subsequent steps are carried out on a semisolid streptavidin phase, including synthesis of double complementary strands, restriction digest, ligation of a linker cassette onto the genomic end of the fragment and exponential PCR(s) with vector- and linker cassette-specific primers. LAM-PCR can be adjusted to all unknown DNA sequences adjacent to a known DNA sequence. Here we describe the use of LAM-PCR analyses to identify 5' long terminal repeat (LTR) retroviral vector adjacent genomic sequences.     

Identification of Roles for Peptide: N-Glycanase and Endo-β-N-Acetylglucosaminidase (Engase1p) during Protein N-Glycosylation in Human HepG2 Cells

Background

During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo β-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells.

Methods/Principal Findings

During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Δ deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man₈GlcNAc₂, corresponding to ∼70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc₁Man₉GlcNAc₂ and Man₉GlcNAc₂ that corresponds to ∼30% of total fOS.

Conclusions/Significance

As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.