Full length ryanodine receptor subtype 3 encodes spontaneous calcium oscillations in native duodenal smooth muscle cells

Two isoforms of the ryanodine receptor subtype 3 (RYR3) have been described in smooth muscle. The RYR3 short isoform (RYR3S) negatively regulates the calcium-induced calcium release mechanism encoded by the RYR2, whereas the role of the full length isoform of RYR3 (RYR3L) was still unclear. Here, we describe RYR-dependent spontaneous Ca(2+) oscillations measured in 10% of native duodenum myocytes. We investigated the role of RYR3 isoforms in these spontaneous Ca(2+) signals. Inhibition of RYR3S expression by antisense oligonucleotides revealed that both RYR2 and RYR3L were able to propagate spontaneous Ca(2+) waves that were distinguishable by frequency analysis. When RYR3L expression was inhibited, the spontaneous Ca(2+) oscillations were never observed, indicating that RYR3S inhibited the function of RYR2. RYR2 expression inhibition led to Ca(2+) oscillations identical to those observed in control cells suggesting that RYR3S did not functionally interact with RYR3L. The presence and frequency of RYR3L-dependent Ca(2+) oscillations were dependent on sarcoplasmic reticulum Ca(2+) content as revealed by long-term changes of the extracellular Ca(2+) concentration. Our study shows that, in native duodenal myocytes, the spontaneous Ca(2+) waves are encoded by the RYR3L alone, which activity is regulated by sarcoplasmic reticulum Ca(2+) loading.     

The Pleistocene Ma U’Oi cave, northern Vietnam: palaeontology, sedimentology and palaeoenvironments

In November 2001, a Vietnamese-French team undertook the excavation of the Ma U’Oi cave in northern Vietnam. This limestone karst cave is located in the province of Hoà Binh, 70 km ESE from Hanoi and is typical of the northern Vietnam landscape. The site yielded an in situ mammalian fauna of a relatively modern composition. We also found a mixed fauna with a lower molar attributed to an archaic Homo(Demeter et al., in press). We estimate the age of Ma U’Oi fauna between 169 kyr, the age of Thum Wiman Nakin (Esposito et al., 1998) estimated by U/Th method and 80-60 kyr, the biochronological age of Lang Trang (Long et al., 1996), or even Holocene. The Ma U’Oi site is important because of the scarcity of Vietnamese sites of those particular levels. For that reason, it fills a gap in the biostratigraphy of Vietnam and permits new correlations with other sites of the mainland, especially those well documented from Thailand.     

Bacterial symbionts in insects or the story of communities affecting communities

Bacterial symbionts are widespread in insects and other animals. Most of them are predominantly vertically transmitted, along with their hosts' genes, and thus extend the heritable genetic variation present in one species. These passengers have a variety of repercussions on the host's phenotypes: besides the cost imposed on the host for maintaining the symbiont population, they can provide fitness advantages to the host or manipulate the host's reproduction. We argue that insect symbioses are ideal model systems for community genetics. First, bacterial symbionts directly or indirectly affect the interactions with other species within a community. Examples include their involvement in modifying the use of host plants by phytophagous insects, in providing resistance to natural enemies, but also in reducing the global genetic diversity or gene flow between populations within some species. Second, one emerging picture in insect symbioses is that many species are simultaneously infected with more than one symbiont, which permits studying the factors that shape bacterial communities; for example, horizontal transmission, interactions between host genotype, symbiont genotype and the environment and interactions among symbionts. One conclusion is that insects' symbiotic complements are dynamic communities that affect and are affected by the communities in which they are embedded.     

Three-dimensional 1H NMR structure of the nucleocapsid protein NCp10 of Moloney murine leukemia virus

Summary The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX₂CysX₄HisX₄Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX₂CysX₄HisX₄Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz ¹H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747±0.209 Å for backbone atoms and 1.709±0.187 Å when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr²⁸, Trp³⁵, Lys³⁷, Lys⁴¹ and Lys⁴² residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.     

Cyclodecapeptides to mimic the radical site of tyrosyl-containing proteins.

Tyrosyl radicals are involved in many biologically important processes. The development of model compounds to mimic radical enzyme active sites, such as galactose oxidase (GO), has widely contributed to an enhanced understanding of their spectral properties, structural attributes and even reactivity. An emerging approach towards the synthesis of such active site mimetics is the use of peptidic ligands. The potential of cyclodecapeptides to bear phenoxyl radicals has been evaluated through three compounds. LH(4) (2+) is a cyclodecapetide containing two histidine residues (mimicking His(496) and His(581) of GO) and two tyrosine residues (mimicking Tyr(495) and the Tyr(272)* radical of GO). L(tBu)H(4) (2+) and L(OMe)H(4) (2+) incorporate 2,4,6-protected phenols in place of each tyrosine in LH(4) (2+). The deprotonation constants of each peptide determined by potentiometric titrations showed that there are some interactions between the acido-basic residues. Cyclic voltammetric studies revealed that only the peptides incorporating 2,4,6-protected phenolates exhibit reversible redox couples and are thus precursors of radicals stable enough to persist in solution. These studies also showed L(OMe2-) to possess the lower oxidation potential, indicating that this peptide, in its radical form, is the most stabilized. The electrochemically generated radical species have been characterized by EPR spectroscopy.     

Control of IgG glycosylation by in situ and real-time estimation of specific growth rate of CHO cells cultured in bioreactor

The cell-specific growth rate (µ) is a critical process parameter for antibody production processes performed by animal cell cultures, as it describes the cell growth and reflects the cell physiological state. When there are changes in these parameters, which are indicated by variations of µ, the synthesis and the quality of antibodies are often affected. Therefore, it is essential to monitor and control the variations of µto assure the antibody production and achieve high product quality. In this study, a novel approach for on-line estimation of µ was developed based on the process analytical technology initiative by using an in situ dielectric spectroscopy. Critical moments, such as significant µ decreases, were successfully detected by this method, in association with changes in cell physiology as well as with an accumulation of nonglycosylated antibodies. Thus, this method was used to perform medium renewals at the appropriate time points, maintaining the values of µ close to its maximum. Using this method, we demonstrated that the physiological state of cells remained stable, the quantity and the glycosylation quality of antibodies were assured at the same time, leading to better process performances compared with the reference feed-harvest cell cultures carried out by using off-line nutrient measurements.     

Detection of heterozygous carriers of the ataxia-telangiectasia (ATM) gene by G2 phase chromosomal radiosensitivity of peripheral blood lymphocytes

In ataxia-telangiectasia (A-T) patients, mutations in a single gene, ATM, result in an autosomal recessive syndrome that embraces a variety of clinical features and manifests extreme radiosensitivity and a strong predisposition to malignancy. Heterozygotes for the ATM gene have no clinical expression of A-T but may be cancer prone with a moderate increase in in vitro radiosensitivity. We performed a blind chromosomal analysis on G₂-phase lymphocytes from 7 unrelated A-T patients, 13 obligate A-T heterozygotes (parents of the patients), and 14 normal controls following X-irradiation with 1 Gy in order to evaluate this cytogenetic method as a tool for detection of ATM carriers. Both A-T homozygotes and heterozygotes showed significantly increased levels of radiation-induced chromatid damage relative to that of normal controls. These results show that the G₂-phase chromosomal radiosensitivity assay can be used for the detection of A-T heterozygotes. In combination with molecular genetic analyses, this test may be of value in studies of familial and sporadic cancers aimed at determination of the potential involvement of ATM mutations in tumor risk or development. Received: 5 May 1997 / Accepted: 26 August 1997     

Development of a cyclosporin A derivative with excellent anti-hepatitis C virus potency

Synthetic modification of cyclosporin A at P3-P4 positions led to the discovery of NIM258, a next generation cyclophilin inhibitor with excellent anti-hepatitis C virus potency, with decreased transporter inhibition, and pharmacokinetics suitable for coadministration with other drugs. Herein is disclosed the evolution of the synthetic strategy to from the original medicinal chemistry route, designed for late diversification, to a convergent and robust development synthesis. The chiral centers in the P4 fragment were constructed by an asymmetric chelated Claisen rearrangement in the presence of quinidine as the chiral ligand. Identification of advanced crystalline intermediates enabled practical supply of key intermediates. Finally, macrocyclization was carried out at 10% weight concentration by a general and unconventional “slow release” concept.