13C Nuclear magnetic relaxation study of segmental dynamics of hyaluronan in aqueous solutions

(13)C spin-lattice relaxation times (T(1)) and nuclear Overhauser enhancements (NOE) were measured as a function of temperature and magnetic field strength for the hetero-polysaccharide hyaluronan in water solutions. The relaxation data of the endocyclic ring carbons were successfully interpreted in terms of chain segmental motions by using the bimodal time-correlation function of Dejean de la Batie, Laupretre and Monnerie. On the basis of the calculated correlation times for segmental motion and amplitudes of librational motions of the C-H vectors at the various carbon sites of the HA repeating unit, we concluded that intramolecular hydrogen bonding of the secondary structure of HA plays a major role in the conformational flexibility of this carbohydrate molecule. The internal rotation of the free hydroxymethyl groups about the exocyclic C-5-C-6 bonds superimposed on segmental motion has been described as a diffusion process of restricted amplitude. The rate and amplitude of the internal rotation indicate that the hydroxymethyl groups are not involved in intramolecular hydrogen bonding. Finally, the motional parameters describing the local dynamics of the HA chain were correlated with the secondary structure of HA in aqueous solutions.     

Reversible site-selective labeling of membrane proteins in live cells

Chemical and biological labeling is fundamental for the elucidation of the function of proteins within biochemical cellular networks. In particular, fluorescent probes allow detection of molecular interactions, mobility and conformational changes of proteins in live cells with high temporal and spatial resolution. We present a generic method to label proteins in vivo selectively, rapidly (seconds) and reversibly, with small molecular probes that can have a wide variety of properties. These probes comprise a chromophore and a metal-ion-chelating nitrilotriacetate (NTA) moiety, which binds reversibly and specifically to engineered oligohistidine sequences in proteins of interest. We demonstrate the feasibility of the approach by binding NTA-chromophore conjugates to a representative ligand-gated ion channel and G protein-coupled receptor, each containing a polyhistidine sequence. We investigated the ionotropic 5HT(3) serotonin receptor by fluorescence measurements to characterize in vivo the probe-receptor interactions, yielding information on structure and plasma membrane distribution of the receptor.     

Genito-sexual dysfunction in patients with a medullary lesion

Multiple dramatic consequences follow medullary lesions. Not only are voluntary motor control and sensitivity of the body segment below the lesion lost, but it also becomes impossible to control erection and ejaculation as well as urinary and faecal continency. The first investigations into genito-sexual function in paraplegics have brought about the idea, commonly admitted in the medical world, that this kind of patient is impotent and sterile. Fortunately this idea is disappearing gradually and many data have demonstrated that appropriate treatment is required and some therapies efficient. This is particularly important in the case of the population concerned, namely young men in 70% of the cases, since the usual age bracket at trauma is between 25 and 35 years old. At this time of life, sexual activity is often at its peak, so that the fertility potential becomes erased.     

Determination of linezolid in plasma and bronchoalveolar lavage by high-performance liquid chromatography with ultraviolet detection using a fully automated extraction method

The aim of this study was to develop a specific and sensitive high-performance liquid chromatographic assay for the determination of linezolid in human plasma, and bronchoalveolar lavage. The sample extraction was based on a fully automated solid-phase extraction with an OASIS HLB cartridge. The method used ultraviolet detection set at a wavelength of 254 nm and a separation with a Zorbax Eclipse XDB C8 column. The assay has been found linear over the concentration range 0.02-30 microg/ml and 0.04-30 microg/ml for linezolid, respectively, in plasma and bronchoalveolar lavage. It provided good validation data for accuracy and precision (CV <4.64% and 5.08%, accuracy in the range 96.93-102.67% and 97.33-105.67%, respectively, for intra- and inter-day). The assay will be applied to determine the penetration of linezolid in human bronchoalveolar lavage during pharmacokinetic steady-state.     

Fine analysis of the Pneumocystis carinii f. sp. carinii genome by two-dimensional pulsed-field gel electrophoresis

Pneumocystis carinii is a general designation for a group of unusual unicellular fungal parasites responsible of pneumopathy in animal hosts. Divided into several subgroups termed the 'special forms', P. carinii is prone to an extensive karyotype variation. In previous studies, the nuclear genome of these organisms has been considered to be haploid and a set of 16 chromosomes has been assigned to P. carinii f. sp. carinii, a special form known to infect rats. We report the analysis of the genome of an isolate representative of the karyotype 1 of this special form, using two-dimensional pulsed-field gel electrophoresis procedures. The 'karyotype and restriction display' (KARD) fingerprints indicated the presence of 17 different chromosomes. The haploid genome size was estimated to be 8.4 Mbp. Some homologous chromosomes were distinguished on the basis of a single restriction fragment length polymorphism, which raises the possibility of a diploid nucleus. A restriction map of the chromosome 15, characterized by two homologues with a size difference of 7 kb, was constructed. Hybridization data indicated that insertion/deletion events may have occurred within subtelomeric regions which carry genes encoding the major surface glycoprotein (MSG) of Pneumocystis.     

Expression of interleukin-1 receptors and their role in interleukin-1 actions in murine microglial cells

Interleukin (IL)-1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL-1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL-1 receptors [IL-1 type-I receptor (IL-1RI), IL-1 type-II receptor (IL-1RII) and IL-1 receptor accessory protein (IL-1RAcP)] on microglia. In the present study we investigated whether microglia express IL-1 receptors and whether they present target or modulatory properties for IL-1 actions. RT-PCR analysis demonstrated lower expression of IL-1RI and higher expression of IL-1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL-1RI, IL-1RII and IL-1RAcP mRNAs, induced the release of IL-1beta, IL-6 and prostaglandin-E2 (PGE2), and activated nuclear factor kappaB (NF-kappaB) and the mitogen-activated protein kinases (MAPKs) p38, and extracellular signal-regulated protein kinase (ERK1/2), but not c-Jun N-terminal kinase (JNK) in microglial cultures. In comparison, IL-1beta induced the release of PGE2, IL-6 and activated NF-kappaB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL-1beta also failed to affect LPS-primed microglial cells. Interestingly, a neutralizing antibody to IL-1RII significantly increased the concentration of IL-1beta in the medium of LPS-treated microglia and exacerbated the IL-1beta-induced IL-6 release in mixed glia, providing the first evidence that microglial IL-1RII regulates IL-1beta actions by binding excess levels of this cytokine during brain inflammation.     

The crown and stem of the V3 loop play distinct roles in human immunodeficiency virus type 1 envelope glycoprotein interactions with the CCR5 coreceptor

Human immunodeficiency virus type 1 envelope glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor in order to mediate viral entry. A CD4-induced surface on gp120, primarily composed of residues in the V3 loop and the C4 domain, interacts with CCR5. In the present study, we generated envelope glycoproteins comprising chimeric V3 loops and/or V3 loops with deletions and studied their binding to CCR5 amino-terminal domain (Nt)-based sulfopeptides and cell surface CCR5, as well as their ability to mediate viral entry. We thus delineated two functionally distinct domains of the V3 loop, the V3 stem and the V3 crown. The V3 stem alone mediates soluble gp120 binding to the CCR5 Nt. In contrast, both the V3 stem and crown are required for soluble gp120 binding to cell surface CCR5. Within the context of a virion, however, the V3 crown alone determines coreceptor usage. Our data support a two-site gp120-CCR5 binding model wherein the V3 crown and stem interact with distinct regions of CCR5 in order to mediate viral entry.