Staining of glycoproteins in polyacrylamide and agarose gels with fluorescent lectins.

We describe a simple and sensitive method for staining of the carbohydrate moiety of glycoproteins in polyacrylamide or agarose electrophoretic gels. Gels are incubated in a solution of fluorescein-labeled concanavalin A. Following destaining with a neutral buffer, glycoproteins exhibit fluorescence under long-range ultraviolet light. Thus, the glucose/mannose containing β- and γ-chains of human fibrinogen give fluorescent bands, whereas the carbohydrate-free α-chain does not react. Less than 100 ng of hexose bound to fibrinogen β- or γ-chains could be detected. The procedure is suitable for staining of other carbohydrate residues in glycoproteins, which can be recognised by specific agglutinins, as shown by binding of fluorescein-labeled lectins from Ricinus communis to galactose residues of fibrinogen.     

Variations in oxygen consumption of Demidoff's bushbaby (Galago demidovii, lorisidae, primates) in captivity

Summary In captivity,Galago demidovii shows annual variations in oxygen consumption which are independent of daylength cycles. This rhythm is characterized by two periods during which metabolic and sexual activities are increased: a short period in November/December; and a longer period from March to June inclusive. These two periods alternate with periods of decreased metabolic rate, of which the most pronounced extends from July to September. Another point of interest is that the basal metabolism of captive Galagos is 17.5% above the value calculated from its body weight.The physiological cycle observed in captivity is synchronized with climatic variations in Makokou (Gaboon), from where the animals originated: higher metabolic and sexual activities are correlated with rainy seasons. This study suggests that an endogenous rhythm may exist inGalago demidovii.     

Environmental and social determinants of sexual function in the male lesser mouse lemur (Microcebus murinus).

Environmental factors that regulate the sexual activity of male lesser mouse lemurs have been studied experimentally with more than 60 captive animals over an 8-year period. In this nocturnal Malagasy prosimian, variation in day length is the primary factor controlling seasonal sexual activity. Plasma testosterone concentrations were low (= 9 ng/ml) during short days and reached 60 ng/ml during long days (> 12-hour day). This cyclic pattern persists unchanged when artificial photoperiodic rhythms are applied and is not altered by ageing. The timing of puberty is also regulated by photoperiodic changes. Nevertheless, the sexual activity of the male lesser mouse lemur can be dramatically modified by the social environment. In heterosexual groups, behavioural and physiological components of sexual activity are depressed in all males except the dominant one, whose aggressive interactions are always successful. Intermale sexual inhibition was shown to be mediated by chemical cues present in the urine of dominant or isolated males but not in urine of subordinate individuals. The inhibitory signals possess lipophilic properties and are not contingent on the gonadal activity of the urine donor but are linked to adrenocortical activity. By contrast, chemical signals stimulating the reproductive function of all males are found in the urine of females, the presence of which is required for the establishment of clear dominance among grouped males. Endocrine mechanisms underlying intermale sexual inhibition by chemical cues were analysed. Variations in prolactin strongly suggest that olfaction interacts with the photoperiodic regulation of reproductive function, leading to changes in the sensitivity of the negative feedback effect of testosterone on gonadotrophin secretion. Inhibitory or stimulatory effects of chemical signals are discussed in the context of their functional significance for wild populations.     

Thermal behavior and elastic properties of phospholipid bilayers under the effect of a synthetic flavonoid derivative, LEW-10.

We have investigated the effect on phospholipidic bilayers of LEW-10, a synthetic flavonoid, derivative of diosmin. Two optical techniques, Quasi-elastic Light Scattering (QLS) and Fourier Transform Infrared Spectroscopy (FT-IR) were used. The results show that in the presence of LEW-10, the phase transition of the bilayers is lowered and that the elastic modulus is decreased. The FT-IR results indicate interactions in the aqueous interface regions of the bilayers. We also discuss LEW-10 comparatively with another derivative, LEW-7/S1, whose effect has been previously studied.     

What are the consequences of being a downy oak (quercus pubescens) or a holm oak (Q. ilex) for breeding blue tits (Parus caeruleus)?

By comparison with deciduous oaks, the lower yearly production of new leaves in sclerophyllous oaks is hypothesized to have several consequences on animal communities. In particular, the production of arthropod communities that feed upon the leaves should be lower in sclerophyllous than in deciduous oaks, this causing changes in breeding patterns and the demographic balance in insectivorous birds. Studies in both deciduous and sclerophyllous habitats in southern France have shown that: 1) the spring development of new leaves occurs later and more slowly in sclerophyllous than in deciduous oaks, 2) the biomass of caterpillars is much lower in sclerophyllous oak forests, and 3) there is a large variation in life history traits of the Blue Tit depending in which type of habitat they breed. Laying date occurs later and clutch size is lower in sclerophyllous habitats than in deciduous habitats. The evolution of life history traits is discussed according to whether poor sclerophyllous habitats are isolated (e.g. on Corsica) or are parts of landscapes including both habitat types.     

The role of proline residues in the folding kinetics of the bovine pancreatic trypsin inhibitor derivative RCAM(14–38)

The role of proline residues in the folding of the trypsin inhibitor derivative RCAM(14–38) has been studied by testing for slow-folding species of the unfolded protein, which could result from the introduction of wrong proline isomers after unfolding. The unfolded protein at 25 °C contains chiefly fast-folding (U_F) molecules: they refold with a time constant of 40 milliseconds at pH 6.8 in 1.9 m-guanidinium chloride. At least one minor slow-folding (U_s) species has been found, using fluorescence to monitor refolding. The reaction in which this U_s species is formed after unfolding shows the properties expected for the cis: Irans isomerization of a proline residue. When refolding is monitored by tyrosine absorbance, two minor slow reactions are found. The faster reaction is in the same time range (15 s at 25 °C) as that studied by fluorescence, and the slower reaction is quite slow (200 s at 25 °C). It is not known whether the slower reaction results from a second U_s species. There are four trans proline residues in bovine pancreatic trypsin inhibitor: the proportion of slow-folding molecules (not more than 25% at 25 °C) is smaller than expected if every proline residue can produce a U_s species and if the cis to trans ratio of each residue after unfolding is at least 0.1:0.9.Criteria based on folding kinetics are given for classifying the types of folding reaction shown by unfolded molecules containing a single wrong proline isomer. Levitt (1980) has classified three types of proline residues according to the energy difference (small, intermediate or large) between the native protein and the predicted minimum energy structure containing a wrong proline isomer. He suggests that these three types of proline residues can be recognized by the types of folding reactions they produce. Only type II (intermediate) folding reactions have thus far been characterized by the criteria introduced here. We point out that the type of folding reaction depends also on the folding conditions, and a possible explanation for this effect is given.     

Studies on factor VIII-related protein. I. Ultrastructural and electrophoretic heterogeneity of human factor VIII-related protein.

Human factor VIII-related protein precipitates with specific heterologous anti-bodies directed against purified factor VIII and supports ristocetin-induced aggregation of washed platelets. We purified human factor VIII from cryoprecipitate by subsequent gel filtration on crosslinked large-pore agarose. Factor VIII-related protein appeared as a large aggregate following electrophoresis on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The same material was separated into multiple bands (molecular weight in excess of several millions) following electrophoresis on SDS-1% agarose gels. After complete disulfide reduction of factor VIII-related protein and electrophoresis on SDS-5% polyacrylamide gels a single subunit chain (Mr approximately equal to 200 000) was revealed. Analysis of this protein, in its non-reduced state, by negative contrast electron microscopy showed filaments of markedly variable size. The calculated molecular weight of such filaments ranged from about 0.6.10(6) to 20.10(6). We conclude that size heterogeneity is an essential feature of human factor VIII-related protein.     

Asymmetric distribution of arachidonic acid in the plasma membrane of human platelets. A determination using purified phospholipases and a rapid method for membrane isolation.

1. Non-lytic degradation of human platelet phospholipids have been performed using a combination of bee venom phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) and Staphylococcus aureus sphingomyelinase C (sphingomyelin choline phosphohydrolase). Under these conditions, 25.4% of total phospholipds are degraded and 6.4% of total platelet arachidonic acid is released. 2. A new method for rapid isolation of platelet plasma membrane is described, based on the use of [3H]concanavalin A as a membrane marker and of self-generating gradients of Percoll. Plasma membranes are enriched 5.2 fold in lectin marker and 0.43 in N-acetyl-beta-D-glucosaminidase, the main contaminant. This method allows to estimate that 57% of the total cell phospholipids and 61% of the total arachidonic acid content are located in the plasma membrane. 3. The distribution of phospholipids and arachidonic acid between the two leaflets of the plasma membrane has been deduced by using these values and those obtained from non-lytic treatment of intact platelets by phospholipases. It is concluded that 45% of plasma membrane phospholipids, comprising 93% of sphingomyelin, 45% of phosphatidylcholine, 9% of phosphatidylserine, 16% of phosphatidylinositol and 20% of phosphatidylethanolamine form the outer half of the human platelet plasma membrane. The phospholipids appear to bear only 10% of the total membrane arachidonic acid.     

Dérivés d'acides 3-(β-d-érythrofuranosyl)-pyrazolecarboxyliques

Treatment of 2,5-anhydro-1-bromo-1-deoxy-2,3-O-isopropylidene-1-p-nitrophenylhydrazono-d-ribose with methyl acetylenecarboxylate gave methyl 3-(2,3-O-isopropylidene-β-d-erythrofuranosyl)-1-p-nitrophenylpyrazole- (8) and 5-carboxylate (9). Amidification at C-5 of 8 was easier than at C-4 of 9. Similarly, dimethyl 3-(2,3-O-isopropylidene-β-d-erythrofuranosyl)- 1 - p-nitrophenylpyrazole-4,5-dicarboxylate gave specifically a 5-carbamoyl derivative, the structure of which was established by comparison of the ¹³C-n.m.r.spectrum with those of a series of glycosylpyrazoles. The correlation between the experimental values of the chemical shifts of the carbon atoms of the pyrazole ring and the values calculated by addition of the contributions of the various groups linked to the ring was better (R 0.98) than the correlations obtained by calculation by the CNDO/2 method of the total electron population (R 0.92) or of the π-electron population of each carbon atom (R 0.85).