Peripheral distribution of a neuropeptide recognized by an antiserum raised against the mammalian CRF41, in the mollusc Helix pomatia

Immunocytochemical methods using an antiserum raised against ovine corticoliberin revealed perikarya and processes in the central and peripheral nervous system of the Pulmonate Gastropod Helix pomatia. The coexistence of immunoreactive nerve fibres and primary sensory neurons in the intestinal wall and in the tentacles appeared particularly significant. The distribution of this peptide suggests that it could act as a sensory neurotransmitter at the central and peripheral levels.     

Contribution to the knowledge of <Emphasis Type="Italic">Peziza</Emphasis> with multiguttulate ascospores,including <Emphasis Type="Italic">P. retrocurvatoides</Emphasis> sp. nov.

A few Peziza species have multiguttulate ascospores. Our study of several collections made during the year 2015 allows us to improve our knowledge of these species morphologically and phylogenetically, especially P. retrocurvata. A new species, morphologically close to P. retrocurvata, is also described and illustrated under the name P. retrocurvatoides. Finally, our examination of the type collection of P. brunneoatra proved this to be a distinct species.     

Mutations in NDUFB11, Encoding a Complex I Component of the Mitochondrial Respiratory Chain,Cause Microphthalmia with Linear Skin Defects Syndrome

Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject’s mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.     

CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification

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Oxygen debt in submaximal and supramaximal exercise in children at high and low altitude

The effect of high altitude (HA) on O2 debt and blood lactate concentration [( L]) was examined in 10- to 13-yr-old children who exhibited the same level of physical fitness. Fifty-one children acclimatized to HA (3,700 m) were compared with 40 children living at low altitude (LA, 330 m) during submaximal (20-95% maximal aerobic power, MAP), maximal and supramaximal (115% MAP) bicycle exercise. Results showed that 1) maximal O2 uptake (VO2max) and maximal heart rate were significantly (P less than 0.001) lower at HA than at LA by 15% and 11 beats X min-1, respectively; 2) for a given absolute work load, O2 debt was higher at HA than at LA, and the slopes of the linear relationships between O2 debt and O2 uptake were significantly higher at HA; 3) when related to percent of VO2max, O2 debts in HA and LA were similar; for 115% MAP maximal O2 debt and [L] were not significantly different (maximal O2 debt, 45.7 +/- 2.7 and 45.9 +/- 3.8 ml X kg-1; [L], 6.0 +/- 0.3 and 6.7 +/- 0.5 mM); and 4) linear relationships between maximal O2 debt and [L] were the same at HA and LA. This suggests that HA did not modify the anaerobic capacity in children.     

Transmembrane movement of diether phospholipids in human erythrocytes and human fibroblasts

We have synthesized spin-labeled (SL) and fluorescently labeled diacyl, 1-alkyl-2-acyl-, and di-alkyl glycerophospholipids. The sn-2 chain was a short chain with either a nitroxide group or a 7-nitro-2, 1,3-benzoxadiazol-4-yl (NBD). After incorporation in the exoplasmic leaflet of human erythrocytes, we found that SL-phosphatidylcholine (PC) redistributed very slowly across the plasma membrane, less than 20% reaching the cytoplasmic leaflet in 3 h at 37 degrees C. In contrast, SL-phosphatidylserine (PS) accumulated on the cytoplasmic leaflet with the same plateau corresponding to 90% of the probes inside. The characteristic times for inward redistribution were different for the three PS analogues: at 37 degrees C, the t(1/2) for the diacyl, alkyl-acyl, and dialkyl compounds were 2.3, 3.5, and 41 min, respectively. ATP depletion or incubation with N-ethylmaleimide inhibited the rapid translocation of the PS derivatives. The diether PS bearing an NBD group translocated very slowly in human erythrocytes and no acceleration by ATP could be measured. On the other hand, in human fibroblasts, the diether NBD-PS and SL-PS were both transported from the exoplasmic to the cytoplasmic monolayer of the plasma membrane as it is the case for the transport of the respective diester PS analogues. These results prove that the ether bonds do not prevent completely PS binding and translocation by the aminophospholipid translocase despite a probable hindrance due to the ether linkage on the sn-2 chain. Because of the high stability of the ether linkage, SL and NBD diether analogues should be useful to investigate lipid traffic in cultured cells.     

Quantitative immunocytochemical studies on the gonadotrophs isolated from the pituitary of the male rat

Summary Quantitative studies on the population of the gonadotropic cells in the pituitary of adult male rats were performed after trypsic dissociation of the pituitary glands and immunoenzymatic staining with anti--LH or anti--FSH antisera. Number, area and extinction of labelled cells were measured by use of an image analyser and a cytophotometer. The gonadotrophs represent approximately 14% of the pituitary cells. The mean area of gonadotrophs is significantly larger after staining with anti--LH serum than after staining with anti--FSH serum. Planimetric measurement of the gonadotrophs reveals their variability in size ranging between 30 and 160 m². Moreover, the size distribution depends on the staining serum used: more numerous small-sized cells (<75 m²) are stained with anti--FSH serum than with anti--LH serum, which conversely stains more numerous large-sized cells. Cytophotometric measurements indicate that immunostaining varies greatly among cells of the same size class and that the staining intensity appears to increase according to the cell size. These results emphasize the morphofunctional heterogeneity of the gonadotropic cell population.     

A simple method for coupling in situ hybridization and immunocytochemistry: application to the study of peptidergic neurons

We have devised a simple procedure for immunostaining of sections that have previously undergone autoradiographic visualization of mRNAs by in situ hybridization. Classical hybridocytochemistry techniques were performed first on cryostat sections of formaldehyde-fixed tissue. Standard methods were used for slide coating by emulsion dipping and for revelation, fixation, and coverslipping steps. The key to this method is the emulsion removal, or permeabilization, by a short trypsin incubation (0.2% for 20-30 sec) which facilitates the good access of antibodies used in a subsequent immunocytochemical technique to section epitopes. Usual immunofluorescence and immunoperoxidase procedures were successfully performed after this treatment. The immunoreactivity of several neuropeptides was well preserved after this procedure. In addition to its usefulness in our studies, this general method should be applicable to many other situations in which autoradiographic and immunocytochemical detections must be coupled.