Intrinsic properties inhibit axonal outgrowth from neonatal rat spinal cord explant

Lumbar spinal cord explants, harvested from neonatal rat pups aged between postnatal day 0 (P0) and P6, were cultured for a period of 48 hrs in the chemically defined medium R(12) on a poly-ethylene-imine (PEI) and on poly-D-lysin (PDL) coated surface. The outgrowth outside the explant was quantified. Lumbar explants from the same rat and embedded in a collagen matrix, and cortical explants from a P0 rat were used as controls. Statistical analysis demonstrated a clear relation between age-at-explantation and the number of neurites in the corona surrounding the explant. The number of outgrowing neurites decreased sharply with age-at-explantation. The average number of neurites per explant obeyed to the expression log (n) = -0.736x + 3.294 on PEI, and log (n) = -0.721x + 2.295 on PDL; x epsilon in [P0 - P6] (n, the number of neurites per explant; x, the age-at-explantation expressed in postnatal days). A similar observed age-related decrease of outgrowth has been described when culturing the lumbar explant inside a collagen matrix. The phenomenon appears to be an intrinsic property of the explant. We review growth inhibitory properties in different models and propose that the phenomenon occurs here at the interface explant-world.     

In vitro desaturation or elongation of monotrans isomers of linoleic acid by rat liver microsomes

Several nutritional studies have shown the in vivo conversion of the 9c,12t-18:2 and 9t,12c-18:2 into long chain polyunsaturated fatty acids (PUFA) containing 20 carbons (geometrical isomers of eicosadienoic and eicosatetraenoic acids). In the present work, some in vitro studies were carried out in order to have precise information on the conversion of these two isomers.In a first set of experiments, studies were focused on the in vitro 6 desaturation, the first regulatory step of the biosynthesis of n-6 long chain PUFA, from 9c,12c-18:2. Rat liver microsomes were prepared and incubated under desaturation conditions with [1-¹⁴C]-9c,12c-18:2 in presence of unlabelled 9c,12t-, 9t,12c- or 9t,12t-18:2. The data show that each trans isomer induced a decrease of the 6 desaturation of the [1-¹⁴C]-9c,12c-18:2, but the 9c,12t-18:2 was the most potent inhibitor (up to 63%). Rat liver microsomes were also incubated with [1-¹⁴C]-9c,12c-18:2, [1-¹⁴C]-9c,12t-18:2 or [1-¹⁴C]-9t,12c-18:2 under desaturation conditions. The results indicated that 18:2 9c,12t is a much better substrate for desaturase than 9t,12c-18:2. Moreover, the conversion levels of [1-¹⁴C]-9c,12t-18:2 was similar to what was observed for its all cis homologue, at low substrate concentration only. In a second set of experiments, in vitro elongation studies of each mono-trans 18:2 isomers and 9c,12c-18:2 were carried out. For that purpose, rat liver microsomes were incubated with [1-¹⁴C]-9c,12c-18:2, [1-¹⁴C]-9c,12t-18:2 or [1-¹⁴C]-9t,12c-18:2 under elongation conditions. The data show that [1-¹⁴C]-9t,12c-18:2 is better elongated than 9c,12c-18:2 while the amount of product formed from [1-¹⁴C]-9c,12t-18:2 was lower than was produced from the 9c,12c-18:2.Thus, the desaturation enzymes presented a higher affinity for the 9c,12t-18:2 whereas the elongation enzyme presented a higher affinity for the 9t,12c-18:2.     

Effects of annual rainfall and habitat types on the body mass of impala (Aepyceros melampus) in the Zambezi Valley, Zimbabwe

Body mass is often considered as a good indicator of body condition of individuals in ungulates, hence of their fitness, and thus, may be used as an index to monitor the status of populations subject to harvesting schemes. Here, we report the influence of annual rainfall (a proxy for primary production) and habitat on the body mass of impala in a population cropped for meat in a communal area of Zimbabwe. We analyzed the data from 2 contrasted years for rainfall, in two different habitats. In the good year (i.e. high annual rainfall) impala were heavier than in the poor year, and adult females seemed to be less affected than males by variation in primary production. We show that adult males were suffering from a seasonal decrease in body mass, supposedly linked to the rut, particularly in good habitat. Overall, the habitat effect appeared to be dominated by the rainfall effect, and this may be due to the very high animal densities in the good habitat, i.e. fast resource depletion. Our results also suggest that males (juvenile and adult) are more susceptible to changes in food resource abundance and quality than females, which supports previous studies on sexually dimorphic and polygynous species.     

Automated method for the determination of a new matrix metalloproteinase inhibitor in ovine plasma and serum by coupling of restricted access material for on-line sample clean-up to liquid chromatography

A fully automated liquid chromatographic method was developed for the determination of Ro 28-2653, a new synthetic inhibitor of matrix metalloproteinases (MMPs), in ovine serum and plasma. The method was based on the coupling of a pre-column packed with restricted access material, namely LiChrospher RP-8 ADS (alkyl diol silica), for sample clean-up to an analytical column containing octyl silica stationary phase. One hundred microl of biological sample, to which 2-propanol was automatically added, were injected onto the ADS pre-column, which was then washed with a washing liquid consisting of a mixture of 25 mM phosphate buffer (pH 7.0) and acetonitrile (90:10; v/v) for 10 min. By rotation of the switching valve, the analyte was then eluted in the back-flush mode with the LC mobile phase composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 7.0) (57:43; v/v). The UV detection was performed at 395 nm. The main parameters likely to influence the sample preparation technique were investigated. The method was then validated over a concentration range from 17.5 to 1950 ng/ml, the first concentration level corresponding to the lower limit of quantitation. At this concentration level, the mean bias and the R.S.D. value for intermediate precision were -2.4% and 4.2%, respectively.     

Identification of a novel family of G protein-coupled receptor associated sorting proteins

During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein-coupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology.     

Factors influencing the population structure of Aedes aegypti from the main cities in Cambodia

A population genetic analysis was conducted on 47 Aedes aegypti collections from Cambodia. Genetic differentiation at seven polymorphic isoenzyme loci was analysed by starch gel electrophoresis. Low (F(ST)=0.024) but significant (P<10(-6)) differentiation was found when all samples were considered. Whatever the grouping of samples tested, differentiation remained significant but low. The role of human activities (ie insecticide treatments or water storage practices) and environmental factors (ie rainfall) in shaping mosquito differentiation are discussed.     

Effects of three novel metalloproteinases from the venom of the West African saw-scaled viper, Echis ocellatus on blood coagulation and platelets

Two metalloproteinases, a 24-kDa P-I EoVMP1 and a 56-kDa P-III EoVMP2, have recently been isolated from the venom of the West African saw-scaled viper Echis ocellatus. We now reveal a new 65-kDa haemorrhagic group P-III metalloproteinase which we have designated EoVMP3. The aim of this study was to determine whether these three snake venom metalloproteinases (SVMPs) affect platelets and blood coagulation. EoVMP1 had no effect on the aggregation of washed human platelets, whereas EoVMP2 inhibited collagen-induced platelet aggregation. In contrast, EoVMP3 did not inhibit the aggregation of platelets by collagen but instead activated platelets in the absence of any additional co-factors. All three SVMPs were capable of activating prothrombin to varying degrees and can therefore be described as procoagulants. EoVMP1, EoVMP2 and EoVMP3 share sequence identity with other members of the reprolysin family, but differ greatly in their effects on some of the components that control haemostasis.     

Spatial organization of dual-species bacterial aggregates on leaf surfaces

The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves, Pseudomonas syringae, Pantoea agglomerans, and Pseudomonas fluorescens. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of P. fluorescens A506 and P. agglomerans 299R and mixtures of P. syringae B728a and P. agglomerans 299R than in mixtures of two isogenic strains of P. agglomerans 299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a gfp-marked strain of P. agglomerans 299R and a cfp-marked strain of P. agglomerans 299R, or of P. fluorescens A506 and P. agglomerans 299R, were similar. However, the proportion of dead cells in mixed aggregates of P. syringae B728a and P. agglomerans 299R was significantly higher (13.2% +/- 8.2%) than that in monospecific aggregates of these two strains (1.6% +/- 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the P. agglomerans 299R cells located at the interface were dead, while only about 24% of the P. syringae B728a cells were dead. The relevance of our results to understanding bacterial interactions on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed.     

Fate of epoxiconazole and kresoxim-methyl in wheat according to time of application

During the 2000-2001 season a field trial was conducted with the aim of quantifying the distribution and persistence of epoxiconazole and kresoxlm-methyl in the different leaf layers of winter wheat plants. In the case of applications before flag leaf emergence, the redistribution of the two active ingredients in the newly formed leaves following the applications was also measured. Allegro (125 g/L epoxiconazole and 125 g/L kresoxim-methyl, SC) was applied at the manufacturer's recommended rate (1 L/ha) either in a single treatment at stages GS32, GS39 and GS59 or in 2, 3 or 4 split applications. Following spraying, leaf samples were collected over time, from each leaf layer, and the two active ingredients were quantified by gas chromatography with electron capture detection (GC-ECD). Fungicide distribution varies according to time of application. A descending gradient through the leaves was observed in the case of application at GS59. When sprayed at stage GS39, on the other hand, the second leaf intercepted more fungicide than the flag leaf. Kresoxim-methyl was found to degrade faster than epoxiconazole. With split treatments, the last spraying appears to be very significant in terms of final fungicide quantities. Redistribution appears possible, especially in the case of epoxiconazole, though in very small quantities.