Rpe65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.     

Differential regulation of central vasopressin receptors in transgenic rats with low brain angiotensinogen

The consequences of permanent alteration to the brain renin-angiotensin system (RAS) on central vasopressinergic system was studied in transgenic rats with low brain angiotensinogen [TGR(ASrAOGEN)]. Levels of vasopressin (AVP) and V1a receptor mRNAs were measured by ribonuclease protection assay (RPA) and AVP by radioimmunoassay (RIA). AVP (100 pmol/50 nl) was microinjected into the nucleus tractus solitarii (NTS) of urethane-anesthetized TGR(ASrAOGEN) and Sprague-Dawley (SD) rats and the mean arterial pressure (MAP) and heart rate (HR) baroreflex induced by phenylephrine were evaluated. AVP but not its mRNA levels were significantly lower in the hypothalamus and hypophysis of TGR(ASrAOGEN) rats. Brainstem V1a mRNA levels were significantly higher in TGR(ASrAOGEN) in comparison to SD rats (5.2+/-0.4% vs. 3.3+/-0.2% of beta-actin mRNA, P<0.05). In contrast, the hypothalamic V1a mRNA levels in TGR(ASrAOGEN) were not different from those found in SD rats. AVP microinjections induced a greater decrease in MAP in TGR(ASrAOGEN) in comparison with SD rats (-19.9+/-5.2 vs. -7.5+/-0.7 mm Hg, P<0.01). The significantly higher baroreflex sensitivity observed in TGR compared to that of SD rats was normalized after AVP microinjection. The increased brainstem V1a mRNA levels and sensitivity to AVP in TGR(ASrAOGEN) rats indicates a functional upregulation of AVP receptors in the NTS. The fact that the hypothalamic V1a mRNA levels are not altered indicates that these receptors are differentially regulated in different brain regions. This study demonstrates that a permanent deficit in brain angiotensinogen synthesis can alter the functionality of central vasopressinergic system.     

C-kit immunopositive interstitial cells (Cajal-type) in human myometrium

Previous reports describing Cajal-like interstitial cells in human uterus are contradictory in terms of c-kit immunoreactivity: either negative (but vimentin-positive) in pregnant myometrium, or positive, presumably in the endometrium. The aim of this study was to verify the existence of human myometrial Cajal-like interstitial cells (m-CLIC). Six different, complementary approaches were used: 1) methylene-blue supravital staining of tissue samples (cryosections), 2) methylene blue and Janus green B vital staining (m-CLIC and mitochondrial markers, respectively), and 3) extracellular single-unit electrophysiological recordings in cell cultures, 4) non-conventional light microscopy on glutaraldehyde/osmium fixed, Epon-embedded semi-thin sections (less than 1 microm) stained with toluidine blue (TSM), 5) transmission electron microscopy (TEM), and 6) immunofluorescence (IF). We found m-CLIC in myometrial cryosections and in cell cultures. In vitro, m-CLIC represented approximately 7% of the total cell number. m-CLIC had 2-3 characteristic processes which were very long (approximately 60 microm), very thin (< or =0.5 microm) and moniliform. The dilated portions of processes usually accommodated mitochondria. In vitro, m-CLIC exhibited spontaneous electrical activity (62.4+/-7.22 mV membrane potentials, short duration: 1.197+/-0.04 ms). Moreover, m-CLIC fulfilled the usual TEM criteria, the so-called 'gold' or 'platinum' standards (e.g. the presence of discontinuous basal lamina, caveolae, endoplasmic reticulum, and close contacts between each other, with myocytes, nerve fibers and/or capillaries etc.). IF showed that m-CLIC express CD117/c-kit, sometimes associated with CD34, with vimentin along their processes. In conclusion, we describe myometrial Cajal-like interstitial cells that have affinity for methylene blue and Janus green B vital dyes, fulfill (all) TEM criteria, express CD117/c-kit and have spontaneous electric activity.     

Is sperm FISH analysis still useful for Robertsonian translocations? Meiotic analysis for 23 patients and review of the literature

Background

Robertsonian translocations (RobT) are common structural chromosome rearrangements where carriers display a majority of chromosomally balanced spermatozoa from alternate segregation mode. According to some monotony observed in the rates of balanced segregation, is sperm FISH analysis obsolete for RobT carriers?

Methods

Retrospective cohort research study on 23 patients analyzed in our center from 2003 to 2017 and compared to the data of 187 patients in literature from 1983 to 2017.Robertsonian translocation carriers were divided in six groups according to the chromosomes involved in the translocation: 9 patients from our center and 107 from literature carrying 45,XY,der(13;14) karyotype, 3 and 35 patients respectively with 45,XY,der(14;21), 5 and 11 patients respectively with 45,XY,der(13;15), 4 and 7 patients respectively with 45,XY,der(14;15), 1 and 4 patients respectively with 45,XY,der(13;22),and 1 and 10 patients respectively with 45,XY,der(14;22).

Results

Alternate segregation mode is predominant in our group of Robertsonian translocation carriers with 73.45% ±8.05 of balanced spermatozoa (min 50.92%; max 89.99%). These results are compliant with the data from literature for all translocations types (p?>?0.05) and are consistent among the different types of Robertsonian translocations (p?>?0.05) except for der(13;15) that exhibit lower balanced spermatozoa rates (p?<?0.05 versus der(13;14), der(14;21), (13;21) and der(15;22)). Normozoospermic patients also display a significantly (p?<?0.01) higher rate of balanced sperm cells than patients with abnormal seminograms whatever the defect implied.

Conclusions

According to the discrepancies observed between der(13;15) and all the other Rob T carriers, the differences observed among patients presenting normal and abnormal sperm parameters and the input in genetical counselling, sperm FISH does not seem obsolete for these patients. Moreover, it seems important to collect more data for rare RobT.

     

A Sequential Scenario for the Origin of Biological Chirality

A sequential model is proposed regarding the origin of biological chirality. Three major stages are presumed: a symmetry breaking (prebiotic chiral disruption in enantiomeric mixtures of monomers), a chiral amplification (prebiotic increase of the chiral character of the monomers affected first by the symmetry breaking), and a chiral expansion (proto biological increase of the chiral character and spread of the chirality to molecules which were less affected by prebiotic chiralizations). As a symmetry-breaking mechanism, the model proposed by Deutsch (1991) is used, which involves a dissymmetric exposure of amino acids (AA) to ultraviolet circularly polarized light (UV-CPL) on evaporative seashores. It is presumed that the chiral amplification, up to a protobiologic significance, was influenced by a periodic overlapping of two abiotic events, a synchronization between tidal-based hydrous–anhydrous cycles, and littoral asymmetric photolysis cycles. This long-term astronomic asymmetry acted around 3.8–4.2 billion years ago and was unique to the Earth in our solar system. It is also presumed that the abiotic symmetry breaking is heterogenous, that only a few l-AAs were used in the beginning, and that the chirality expanded later to all 20 AAs based on a coevolutionary strategy of the genetic code and on a physiological relationship between AAs. In this scenario the d-chirality of pentoses in polynucleotides was attributed to both d-pentose/l-AA relationships and to a structural evolution. Received: 10 May 1996 / Accepted: 13 August 1996     

Low-Temperature Growth of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of ≈35°C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature (≈22°C) MR-1 grows with a doubling time of about 40 min, but when moved from 22°C to 3°C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of ≈67 h. In comparison to cells grown at 22°C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22°C.     

An improved method for discrimination of cell populations in tissue sections using microscopy-based multicolor tissue cytometry.

BACKGROUND: In tissue context, researchers and pathologists lack a generally applicable standard for quantitative determination of cytological parameters. Increasing knowledge of disease-specific markers calls for an appropriate in situ tissue cytometry. METHODS: Microscopy-based multicolor tissue cytometry (MMTC) permits multicolor analysis of single cells within tissue context. RESULTS: Tissue specimens stained for CD45/CD3/CD4/CD8 were analyzed. Specificity as well as reproducibility of MMTC is demonstrated and a novel MMTC-based function to improve visual discrimination of subpopulations is introduced. CONCLUSIONS: Our data demonstrate that MMTC constitutes an important step toward automated and quantitative fluorometry of solid tissues and cell monolayers.     

Red cell H-deficient,salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin

Based on the genetic model proposing thatH andSe are two structural genes, we predicted that the red cell H-deficient, salivary ABH secretor phenotype should be found on Reunion island, where a large series of H-deficient non-secretor families have been previously described. Two such Reunion individuals are now reported. POU [A_h, Le(a–b+), secretor of A, H, Le^a and Le^b in saliva] and SOU [O_h, Le(a–b+), secretor of H, Le^a and Le^b in saliva]. Both are devoid of H -2-fucosyltransferase activity in serum. In addition, the preparation of total non-acid glycosphingolipids from plasma and red cells of POU revealed the type 1ALe^b heptaglycosylceramide and small amounts of the monofucosylated type 1 A hexaglycosylceramide. Both glycolipids possess an H structure probably synthesised by the product of theSe gene. No other blood group A glycolipids, with types 2, 3 or 4 chains, normally present in the presence of the product of theH gene, were found on red cells or plasma of POU.TheH,Se andLe genetic control of the expression of ABH and related antigens in different tissue structures of the skin is described in 54 H-normal individuals of known ABO, secretor and Lewis phenotypes; in one red cell H-deficient salivary secretor (SOU); and in one H-deficient non-secretor (FRA). Sweat glands express ABH under the control of theSe gene. Sweat ducts express ABH under the control of bothH andSe genes and Lewis antigens under the control ofLe and bothH andSe genes. Epidermis, vascular endothelium and red cells express ABH under the control of theH gene. The products ofH andSe genes are usually expressed in different cells. However, the results illustrate that in some structures, like the epithelial cells of sweat ducts, both the products ofH andSe genes can contribute to the synthesis of the same Le^b structure.     

An efficient method for medium throughput screening of cuticular wax composition in different plant species

Introduction

Most aerial plant organs are covered by a cuticle, which largely consists of cutin and wax. Cuticular waxes are mixtures of dozens of compounds, mostly very-long-chain aliphatics that are easily extracted by solvents. Over the last four decades, diverse cuticular wax analysis protocols have been developed, most of which are complex and time-consuming, and need to be adapted for each plant species or organ. Plant genomics and breeding programs often require mid-throughput metabolic phenotyping approaches to screen large numbers of individuals and obtain relevant biological information.

Objectives

To generate a fast, simple and user-friendly methodology able to capture most wax complexity independently of the plant, cultivar and organ.

Methods

Here we present a simple GC–MS method for screening relatively small wax amounts, sampled by short extraction with a versatile, uniform solvent. The method will be tested and validated in leaves and fruits from three different crop species: tomato (Solanum lycopersicum), apple (Malus domestica) and hybrid aspen (Populus tremula × tremuloides).

Results

Consistent results were obtained in tomato cultivar M82 across three consecutive years (2010–2012), two organs (leaf and fruit), and also in two different tomato (M82 and MicroTom) and apple (Golden Delicious and Granny Smith) cultivars. Our results on tomato wax composition match those reported previously, while our apple and hybrid aspen analyses provide the first comprehensive cuticular wax profile of these species.

Conclusion

This protocol allows standardized identification and quantification of most cuticular wax components in a range of species.