Formation and structure of reaction products of cis-PtCl2(NH3)2 with d(ApG) and/or d(GpA) in di-, tri- and penta-nucleotides. Preference for GpA chelation over ApG chelation

The reaction products of cis-PtCl2(NH)3)2 with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high-performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300-MHz 1H-NMR (pH dependence of the non-exchangeable base-protons, T1 relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments. The smallest of the investigated nucleotides, d(ApG) and d(GpA), both formed a variety of different platinum chelates. In the reaction with d(ApG) 15% cis-Pt(NH3)2-[d(ApG)N1(1),N7(2)] and 78% cis-Pt(NH3)2[d(ApG)N7(1),N7(2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1:1 product. From the main product two rotamers were found to occur: at room temperature, 81% anti,anti and 19% anti,syn product is present. With d(GpA) about equal amounts of N1,N7 and N7,N7 products were found; for both products the anti,anti and anti,syn conformations were found, respectively. Upon reaction of cis-PtCl2(NH3)2 with d(pApG) and d(pGpA) only the N7,N7 products were found; at room temperature and pH greater than 1.5 these products were present in anti,anti conformation. However, for the d(pApG)-platinum chelate at -20 degrees C a small amount (less than 5%) of a second product could be observed in NMR. For the d(pGpA)-platinum chelate a second N7,N7-coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5'-phosphate group is protonated). With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra-molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no N1-coordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed.     

Unilateral Lesion of Dopamine Neurons Induces Grooming Asymmetry in the Mouse

Grooming behaviour is the most common innate behaviour in animals. In rodents, it consists of sequences of movements organized in four phases, executed symmetrically on both sides of the animal and creating a syntactic chain of behavioural events. The grooming syntax can be altered by stress and novelty, as well as by several mutations and brain lesions. Grooming behaviour is known to be affected by alterations of the dopamine system, including dopamine receptor modulation, dopamine alteration in genetically modified animals, and after brain lesion. While a lot is known about the initiation and syntactic modifications of this refined sequence of movements, effects of unilateral lesion of dopamine neurons are unclear particularly regarding the symmetry of syntactic chains. In the present work we studied grooming in mice unilaterally lesioned in the medial forebrain bundle by 6-hydroxydopamine. We found a reduction in completion of grooming bouts, associated with reduction in number of transitions between grooming phases. The data also revealed the development of asymmetry in grooming behaviour, with reduced tendency to groom the contralateral side to the lesion. Symmetry was recovered following treatment with L-DOPA. Thus, the present work shows that unilateral lesion of dopamine neurons reduces self-grooming behaviour by affecting duration and numbers of events. It produces premature discontinuation of grooming chains but the sequence syntax remains correct. This deficient grooming could be considered as an intrinsic symptom of Parkinson’s disease in animal models and could present some similarities with abnormalities of motor movement sequencing seen in patients. Our study also suggests grooming analysis as an additional method to screen parkinsonism in animal models.     

L63, the Drosophila PFTAIRE,interacts with two novel proteins unrelated to cyclins

L63 encodes a CDK-like protein homologous to the mammalian PFTAIRE. We showed previously that L63 provides a CDK-related function critical to development (Dev. Biol. 221 (2000) 23). We present here the first biochemical characterization of L63 kinase. In addition, we describe two novel Drosophila proteins, PIF-1 and PIF-2 (for PFTAIRE Interacting Factor-1 and -2), identified in a two-hybrid screen for their ability to interact with the amino-terminal region of L63. The full-length PIF-1 cDNA shows an unusual dicistronic organization. PIF-1A and PIF-1B (the L63 interactor) predicted proteins are expressed in vivo, and show a distinct expression profile during development. Interaction between L63 and PIF-1B was confirmed in vitro and in vivo. The role of this interaction remains to be demonstrated, but our data suggest that PIF-1B might serve as a regulator of L63.     

Variable inter and intraspecies alkaline phosphatase activity within single cells of revived dinoflagellates

Adaptation of cell populations to environmental changes is mediated by phenotypic variability at the single-cell level. Enzyme activity is a key factor in cell phenotype and the expression of the alkaline phosphatase activity (APA) is a fundamental phytoplankton strategy for maintaining growth under phosphate-limited conditions. Our aim was to compare the APA among cells and species revived from sediments of the Bay of Brest (Brittany, France), corresponding to a pre-eutrophication period (1940’s) and a beginning of a post-eutrophication period (1990’s) during which phosphate concentrations have undergone substantial variations. Both toxic marine dinoflagellate Alexandrium minutum and the non-toxic dinoflagellate Scrippsiella acuminata were revived from ancient sediments. Using microfluidics, we measured the kinetics of APA at the single-cell level. Our results indicate that all S. acuminata strains had significantly higher APA than A. minutum strains. For both species, the APA in the 1990’s decade was significantly lower than in the 1940’s. For the first time, our results reveal both inter and intraspecific variabilities of dinoflagellate APA and suggest that, at a half-century timescale, two different species of dinoflagellate may have undergone similar adaptative evolution to face environmental changes and acquire ecological advantages.Subject terms: Climate-change ecology, Microbial ecology     

EP-cadherin in muscles and epithelia of Xenopus laevis embryos.

EP-cadherin is a novel Xenopus Ca+2-dependent adhesion molecule, which shares comparable homology with mouse E- and P-cadherins (Ginsberg, De Simone and Geiger; 1991, Development 111, 315-325). We report here the patterns of expression of this molecule in Xenopus laevis embryos at different developmental stages ranging from cleavage to postmetamorphic. EP-cadherin is already expressed in the oocyte and egg and can then be detected in close association with the membrane of all blastomeres up to late blastula stages. Starting at late gastrula stages, the level of EP-cadherin expression increases sharply in non-neural ectodermal cells, in the somites and in the notochord; it persists in endodermal cells and decreases rapidly in all migratory cells. During neurulation the level of EP-cadherin expression declines gradually in the nervous system and is undetectable here throughout later development except in the optic nerve and in the neural part of the olfactory organ. This pattern continues during later development so that in the tailbud stage and up to metamorphosis the most prominent staining is detected in the epidermis and skeletal muscle. After metamorphosis, the molecule gradually disappears from the muscle tissue and the major site of expression remains the skin. EP-cadherin is invariably present in close association with the cell membrane. In the muscle it is associated with the sarcolemma at regions of myoblast-myoblast or myotube-myotube contact. In epidermal cells, EP-cadherin is usually coexpressed with E-cadherin. Yet, while E-cadherin staining is always restricted to the basolateral aspects of the cells, EP-cadherin is often distributed throughout the plasmalemma including the apical surface.     

Melatonin secretion occurs at a constant rate in both young and older men and women

The magnitude and duration of melatonin (MLT) secretion were measured over a period of 25 h with pharmacokinetic studies employing administration of D(7) MLT at midday and at midnight in two separate studies and two groups of subjects, 12 young and 11 older men and women. Plasma levels of endogenous MLT and D(7) MLT were quantified separately by use of a specific and sensitive method (gas chromatography-mass spectrometry) previously developed in our laboratory, enabling us to measure endogenous and exogenous MLT levels down to 0.5 pg/ml in plasma. In the two groups of subjects, MLT secretion occurred only at night: onset time of secretion was from 1915 to 2205 (Greenwich mean time), and offset was from 0305 to 0545. No MLT peak was observed in individual nocturnal MLT profiles that were similar to curves obtained for a rate-constant infusion. Modelization demonstrated the superimposition of observed data and simulated curves. MLT concentrations decreasing from the offset of secretion might correspond to the elimination of MLT present in the body at the end of nocturnal secretion. By use of the MLT clearance given by pharmacokinetics, the amount of secreted MLT was found to be 35.7 and 21.6 microg for men and women, respectively, and the rate of secretion was 4.6 and 2.8 microg/h, respectively. No significant gender difference was observed for these two parameters when normalized to body weight. No significant gender difference was observed for onset times of secretion or duration of secretion (7.6-8.6 h) within the two groups, or between young and older subjects.     

Biochemical properties and cellular localization of Plasmodium falciparum protein disulfide isomerase

We have previously reported the isolation of a 52,000 M(r) protein (Pf52) displaying consensus sequences for thiol:disulfide oxidoreductases. Pf52 therefore represents the plasmodial protein disulfide isomerase (PDI). It has been renamed PfPDI and correlates to MAL8P1.17 in the annotated genome of P. falciparum (3D7 strain). Antibodies were raised against recombinant (His)(6)-tagged forms of PfPDI devoid of its signal peptide sequence, demonstrating a major co-localization of PfPDI with endoplasmic reticulum-resident proteins, PfBIP and PfERC, but not with the Golgi marker PfERD2. Recombinant PfPDI displayed typical biochemical functions of PDIs: oxidase/isomerase and reductase activities, as well as a chaperone-like behavior on the denaturated protein rhodanese. These activities were comparable to those measured for the purified native bovine PDI and the human recombinant PDI. The antiplasmodial compound DS61 does inhibit the recombinant PfPDI oxidase/isomerase activity but not that of the human recombinant PDI, suggesting structural differences between both enzymes. However, a discrepancy between the inhibitory activity of DS61 on the recombinant PfPDI (IC(50) of 430 microM) and its in vitro antiplasmodial activity (IC(50) of 0.1 microM) was observed, suggesting that PfPDI is not the only target of DS61. Taking into account its biochemical properties and its intracellular localization, the involvement of PfPDI in the parasite protein folding is discussed, as well as its potential for the development of alternative antimalarial chemotherapy strategies.