Practical considerations regarding the use of streptolysin-O as a permeabilising agent for cells in the investigation of exocytosis

Streptolysin-O is widely used in cell biological investigations in order to make large (>12 nm) pores in the plasma membrane and so to render the cytosol directly accessible to experimental manipulation. We have compared the effect of streptolysin-O commercially formulated (Murex Diagnostics) as a diagnostic reagent in pathology with two pure reagents (a conventional purified protein, and a recombinant protein generated in E.coli) on exocytotic secretion from mast cells. For mast cells permeabilised by streptolysin obtained from the commercial source, exocytosis (of -D-N-acetylglucosaminidase) is dependent on provision of both Ca²⁺ and a guanine nucleotide. In contrast, for cells permeabilised by either of the two pure proteins, a substantial extent of Ca²⁺-independent exocytosis can be elicited. When the Murex material is subject to dialysis or ultrafiltration, some secretion can be induced in the absence of Ca²⁺, indicating a modulatory function of the low mol wt additives of formulation, mainly phosphate and cysteine. However, Ca²⁺-independent exocytosis is still manifest when the pure proteins are reconstituted with ultrafiltrates from the Murex material. These observations indicate that reagents used to permeabilise cells should be characterised thoroughly and used with great care. Confirmation that the cytolytic activity of the Murex material derives from a cholesterol directed factor was demonstrated by inhibition of exocytosis when red blood cell derived (and hence cholesterol containing) sonicated liposomes were provided.     

Le débitage Levallois du site d’Amane Oukider (Sud-Est du Maroc) : analyse technologique d’un assemblage standardisé

The Amane Oukider site (Tinghir, Anti-Atlas) was discovered by an Italo-Moroccan field crew during an archeological mapping project in South-East Morocco. It is an open-air site located near a outcrop of raw materials composed of dolerite. The only debitage method in evidence is the Levallois (lineal and recurrent centripetal) method; the plein débitage products were systematically exported from the site. The Levallois preferential flakes are characterized by a very high level of standardization and they were probably used as blanks for cleaver production.     

Neutrophil elastase (NE)-deficient mice demonstrate a nonredundant role for NE in neutrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo

Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE(-/-)) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE(-/-) mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1beta and TNF-alpha and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE(-/-) mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1beta, KC, and macrophage inflammatory protein-1alpha in NE(-/-) mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.     

Down co-regulation of light absorption, photochemistry, and carboxylation in Fe-deficient plants growing in different environments

The regulation of photosynthesis through changes in light absorption, photochemistry, and carboxylation efficiency has been studied in plants grown in different environments. Iron deficiency was induced in sugar beet (Beta vulgaris L.) by growing plants hydroponically in controlled growth chambers in the absence of Fe in the nutrient solution. Pear (Pyrus communis L.) and peach (Prunus persica L. Batsch) trees were grown in field conditions on calcareous soils, in orchards with Fe deficiency-chlorosis. Gas exchange parameters were measured in situ with actual ambient conditions. Iron deficiency decreased photosynthetic and transpiration rates, instantaneous transpiration efficiencies and stomatal conductances, and increased sub-stomatal CO₂ concentrations in the three species investigated. Photosynthesis versus CO₂ sub-stomatal concentration response curves and chlorophyll fluorescence quenching analysis revealed a non-stomatal limitation of photosynthetic rates under Fe deficiency in the three species investigated. Light absorption, photosystem II, and Rubisco carboxylation efficiencies were down-regulated in response to Fe deficiency in a coordinated manner, optimizing the use of the remaining photosynthetic pigments, electron transport carriers, and Rubisco.     

Doxycycline reduces plasma VEGF-C/sVEGFR-3 and improves pathology in lymphatic filariasis

Lymphatic filariasis is a disease of considerable socioeconomic burden in the tropics. Presently used antifilarial drugs are able to strongly reduce transmission and will thus ultimately lower the burden of morbidity associated with the infection, however, a chemotherapeutic principle that directly induces a halt or improvement in the progression of the morbidity in already infected individuals would constitute a major lead. In search of such a more-effective drug to complement the existing ones, in an area endemic for bancroftian filariasis in Ghana, 33 microfilaremic and 18 lymphedema patients took part in a double-blind, placebo-controlled trial of a 6-wk regimen of 200 mg/day doxycycline. Four months after doxycycline treatment, all patients received 150-200 microg/kg ivermectin and 400 mg albendazole. Patients were monitored for Wolbachia and microfilaria loads, antigenemia, filarial dance sign (FDS), dilation of supratesticular lymphatic vessels, and plasma levels of lymphangiogenic factors (vascular endothelial growth factor-C [VEGF-C] and soluble vascular endothelial growth factor receptor-3 [(s)VEGFR-3]). Lymphedema patients were additionally monitored for stage (grade) of lymphedema and the circumferences of affected legs. Wolbachia load, microfilaremia, antigenemia, and frequency of FDS were significantly reduced in microfilaremic patients up to 24 mo in the doxycycline group compared to the placebo group. The mean dilation of supratesticular lymphatic vessels in doxycycline-treated patients was reduced significantly at 24 mo, whereas there was no improvement in the placebo group. Preceding clinical improvement, at 12 mo, the mean plasma levels of VEGF-C and sVEGFR-3 decreased significantly in the doxycycline-treated patients to a level close to that of endemic normal values, whereas there was no significant reduction in the placebo patients. The extent of disease in lymphedema patients significantly improved following doxycycline, with the mean stage of lymphedema in the doxycycline-treated patients being significantly lower compared to placebo patients 12 mo after treatment. The reduction in the stages manifested as better skin texture, a reduction of deep folds, and fewer deep skin folds. In conclusion, a 6-wk regimen of antifilarial treatment with doxycycline against W. bancrofti showed a strong macrofilaricidal activity and reduction in plasma levels of VEGF-C/sVEGFR-3, the latter being associated with amelioration of supratesticular dilated lymphatic vessels and with an improvement of pathology in lymphatic filariasis patients.     

The HOXB4 homeoprotein promotes the ex vivo enrichment of functional human embryonic stem cell-derived NK cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34(+)CD45RA(+) precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells.     

Mice Doubly-Deficient in Lysosomal Hexosaminidase A and Neuraminidase 4 Show Epileptic Crises and Rapid Neuronal Loss

Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the α-subunit of lysosomal β-hexosaminidase A, which converts G_M2 to G_M3 ganglioside. Hexa^−/− mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise G_M2 ganglioside via a lysosomal sialidase into glycolipid G_A2, which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is not effective in humans, infantile Tay-Sachs disease is fatal in the first years of life. Previously, we identified a novel ganglioside metabolizing sialidase, Neu4, abundantly expressed in mouse brain neurons. Now we demonstrate that mice with targeted disruption of both Neu4 and Hexa genes (Neu4 ^−/−;Hexa ^−/−) show epileptic seizures with 40% penetrance correlating with polyspike discharges on the cortical electrodes of the electroencephalogram. Single knockout Hexa ^−/− or Neu4 ^−/− siblings do not show such symptoms. Further, double-knockout but not single-knockout mice have multiple degenerating neurons in the cortex and hippocampus and multiple layers of cortical neurons accumulating G_M2 ganglioside. Together, our data suggest that the Neu4 block exacerbates the disease in Hexa^−/− mice, indicating that Neu4 is a modifier gene in the mouse model of Tay-Sachs disease, reducing the disease severity through the metabolic bypass. However, while disease severity in the double mutant is increased, it is not profound suggesting that Neu4 is not the only sialidase contributing to the metabolic bypass in Hexa ^−/− mice.     

Compound heterozygosity at the sphingomyelin phosphodiesterase-1 (SMPD1) gene is associated with low HDL cholesterol

Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders caused by deficient activity of the enzyme acid sphingomyelinase (aSMase) and the resulting accumulation of sphingomyelin in tissues. In the present study, we investigated two family members who had been diagnosed with Type B NPD and who had a severe decrease in plasma high density lipoprotein cholesterol (HDL-C). The proband (a 48-year-old male) had an HDL-C of 0.30 mmol/l (12 mg/dl) and his sister had values of 0.45 mmol/l (17 mg/dl) with severe premature coronary artery disease (CAD). Hypertriglyceridemia was found in both cases. aSMase activity measured in skin fibroblasts appeared markedly depressed. The SMPD1 gene, coding for aSMase, was sequenced in affected subjects and all family members. Compound heterozygosity (DeltaR608 and R441X) was identified in both affected patients. Carriers of the DeltaR608 mutation tended to have moderately to severe decreased HDL-C levels, whereas carriers of the R441X mutation, although present only in young subjects (<20 years of age) had normal HDL-C levels. To investigate the cause of the low HDL-C level in these patients, we studied apoA-I-mediated cellular cholesterol efflux in fibroblasts. Unlike patients with Tangier disease, cholesterol efflux was found to be normal under the experimental conditions used in the present study. On the other hand, we observed a significant increase in the free cholesterol:esterified cholesterol ratio in HDL fraction from these patients and a decrease in endogenous lecithin-cholesterol acyltransferase (LCAT) activity, as determined by the fractional esterification rate. Taken together, these results suggest that (1) compound heterozygosity at the SMPD1 gene causes a severe decrease in aSMase activity and in HDL-C and increases the risk of CAD, (2) this lipoprotein abnormality is not attributable to defective cellular cholesterol efflux, (3) abnormal HDL composition might cause a decrease in LCAT activity and a lack of HDL maturation.