Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes.

Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane alpha helices and small ecto- and endodomains. A His6-tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N-dodecyl-beta-d-maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins.     

Contractile differentiation of foetal cattle muscles: intermuscular variability

off actile differentiation was studied in six foetal muscles exhibiting different contractile characteristics in adult cattle: the Masseter, Diaphragma, Biceps femoris, Longissimus thoracis, Semitendinosus and Cutaneus trunci. These muscles were excised from foetuses aged 60-260 days. Fibre types were identified by immunohistochemistry using three monoclonal antibodies raised against types 1, 2a, 2b (or 2x) and foetal myosin heavy chains. The different myosin isoforms were also separated by electrophoresis, identified by immunoblotting and quantified by ELISA. At least two generations of cells were observed in all the muscles studied. The primary, early differentiated one, gave rise to type II fibres in Cutaneus trunci and type I fibres in all remaining muscles. The secondary generation of cells differentiated later than the first generation of cells. Its pattern of differentiation was more complex in particular from 150 to 210 days. It formed slow fibres in slow adult muscles, fast fibres in fast adult muscles and both types in mixed muscles. Precocity of differentiation was muscle-type dependent and related to muscle function at birth.     

Non-Mendelian Female Sterility in DROSOPHILA MELANOGASTER: Principal Characteristics of Chromosomes from Inducer and Reactive Origin after Chromosomal Contamination

Strains of Drosophila melanogaster can be divided into two main classes, inducer and reactive, in relation to non-Mendelian female sterility. The genetic element responsible for the inducer condition (I factor) is chromosomal and may be linked to any inducer-strain chromosome. Each chromosome carrying the I factor (i(+) chromosome) can produce females showing more-or-less reduced fertility when it is introduced by paternal gametes into a reactive oocyte. As long as i(+) chromosomes are transmitted through heterozygous males with reactive originating chromosomes (r chromosomes), I factor strictly follows Mendelian segregation. In contrast, in heterozygous i(+)/r females, a varying proportion of r chromosomes may acquire I factor independently of classical genetic recombination, by a process called chromosomal contamination. This paper reports investigation of the characteristics of the three kinds of chromosomes produced by females in which contamination occurs. It appears that the contaminated reactive chromosomes have irreversibly acquired I factor and behave like i(+) chromosomes, while the i(+) chromosomes used as contaminating elements and the reactive originating chromosomes that have not been contaminated have not undergone any change.     

Detection of anti-Müllerian activity in boar rete testis fluid

Boar rete testis fluid was tested for its capacity to induce Müllerian regression in 14.5-day-old rat Müllerian ducts. Weak activity was present in crude RTF, but after gel filtration 5-fold concentration, greater activity was detected in 1 our of 7 pools of the eluted fractions. The biologically active fraction (mol. wt 160 000-310 000) coincided with the elution of authentic labelled anti-Müllerian hormone, obtained from bovine fetal testes. These results indicate that a small amount of anti-Müllerian hormone is still synthesized in post-natal life.     

Cytochemical duality of neurosecretory material in the hypothalamo-posthypophysial system of the rat as related to hormonal content

Summary Cytochemical methods using silver proteinate, silver methenamine and potassium ferrocyanide + OsO₄ for ultrastructural detection of glycoproteins allow, in the posthypophysis and the magnocellular nuclei of the rat, differentiation of two types of fibres and neurons: one type containing negative granules with a homogeneous content of low electron density, the second type containing granules which demonstrate a ring-shaped deposit either of silver or of potassium ferrocyanide-osmium complex, likely to be related to a glycoproteic component. The difference between these two types is increased by prestaining en bloc with uranyl acetate before the silver proteinate reaction. A similar investigation was carried out on the vasopressin deficient Brattleboro rat; the neurosecretory material, present in some endings and neurones only, is of the nonreactive type, so that it appears justified to correlate the reactivity of granules with vasopressin, and consequently to distinguish neurones and fibres containing vasopressin from those in which oxytocin is quantitatively the main hormonal peptide. This conclusion is strongly supported by the fact that percentages of reactive and negative endings, as determined on this basis in the posthypophysis of normal rats from two different strains, are in good agreement with biochemical data reported in the literature.     

A sensitive LC–MS/MS method for quantification of a nucleoside analog in plasma: Application to in vivo rat pharmacokinetic studies

A LC–MS/MS method was developed and validated for determination of nucleoside analog (NA) in rat plasma. The method run time was 6 min and the limit of quantification (LOQ) was estimated at 100 pg/mL. The extraction procedure consisted on plasma samples protein precipitation with an acetonitrile solution which contained the stable isotope labeled internal standard (IS). Chromatography was performed on a newly developed C₁₆ column (150 mm × 4.6 mm, 5 μm) in order to avoid the use ion pair salts. The samples were eluted at 0.8 mL/min with a gradient of mobile phase made of water and acetonitrile both acidified with 0.5% acetic acid and 0.025% trifluoroacetic acid (TFA). A tandem mass spectrometer was used as a detector for quantitative analysis. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at four concentration levels in rat plasma, over a concentration ranging between 0.1 and 1000 ng/mL. The data indicate that our LC–MS/MS assay is an effective method for the pharmacokinetics study of NA in rat plasma.     

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive

Background information. The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)‐tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. Results. The movement of PAGFP (photoactivatable GFP)‐tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP‐tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)‐sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three‐dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB‐sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. Conclusions. We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.