A mouse model of intracerebral hemorrhage using autologous blood infusion

The development of controllable and reproducible animal models of intracerebral hemorrhage (ICH) is essential for the systematic study of the pathophysiology and treatment of hemorrhagic stroke. In recent years, we have used a modified version of a murine ICH model to inject blood into mouse basal ganglia. According to our protocol, autologous blood is stereotactically infused in two stages into the right striatum to mimic the natural events of hemorrhagic stroke. Following ICH induction, animals demonstrate reproducible hematomas, brain edema formation and marked neurological deficits. Our technique has proven to be a reliable and reproducible means of creating ICH in mice in a number of acute and chronic studies. We believe that our model will serve as an ideal paradigm for investigating the complex pathophysiology of hemorrhagic stroke. The protocol for establishing this model takes about 2 h.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.     

Rapid recycling of triose phosphates in oak stem tissue

We report the carbon-13 and oxygen-18 isotope ratios in cellulose from the early and late wood of pedunculate oak (Quercus robur L.). The δ¹³ C value of the early wood correlates best with that of the late wood of the previous year. The δ¹⁸O value of the early wood correlates best with that of the late wood of the same year. We suggest that a biochemical explanation of these data is that there is a rapid cycle between hexose monophosphates and triose phosphates in oak stem tissue during cellulose synthesis. Evidence in support of this explanation is provided by the intramolecular distribution of ¹⁴C in labelled fructose extracted from cores of wood that had been supplied with [1?¹⁴C]- and [6-¹⁴C]glucose.     

The effects of low temperature acclimation and photoinhibitory treatments on Photosystem 2 studied by thermoluminescence and fluorescence decay kinetics

The effects of low temperature acclimation and photoinhibitory treatment on Photosystem 2 (PS 2) have been studied by thermoluminescence and chlorophyll fluorescence decay kinetics after a single turnover saturating flash. A comparison of unhardened and hardened leaves showed that, in the hardened case, a decrease in overall and B-band thermoluminescence emissions occurred, indicating the presence of fewer active PS 2 reaction centers. A modification in the form of the B-band emission was also observed and is attributed to a decrease in the apparent activation energy of recombination in the hardened leaves. The acclimated leaves also produced slower Q_A ^– reoxidation kinetics as judged from the chlorophyll fluorescence decay kinetics. This change was mainly seen in an increased lifetime of the slow reoxidation component with only a small increase in its amplitude. Similar changes in both thermoluminescence and fluorescence decay kinetics were observed when unhardened leaves were given a high light photoinhibitory treatment at 4°C, whereas the hardened leaves were affected to a much lesser extent by a similar treatment. These results suggest that the acclimated plants undergo photoinhibition at 4°C even at low light intensities and that a subsequent high light treatment produces only a small additive photoinhibitory effect. Furthermore, it can be seen that photoinhibition eventually gives rise to PS 2 reaction centers which are no longer functional and which do not produce thermoluminescence or variable chlorophyll fluorescence.Abbreviations D1 The 32 kDa protein of Photosystem 2 reaction center - F_m maximum chlorophyll fluorescence yield - F₀ minimal chlorophyll fluorescence yield obtained when all PS 2 centers are open - F_i intermediate fluorescence level corresponding to PS 2 centers which are loosely or not connected to plastoquinone (non-B centers) - F_v maximum variable chlorophyll fluorescence yield (F_v=F_m–F₀) - PS 2 Photosystem 2 - Q_A and Q_B respectively, primary and secondary quinonic acceptors of PS 2 - S1, S2 and S3 respectively, the one, two and three positively charged states of the oxygen evolving system - Z secondary donor of PS 2     

Regulation of Cdc25A half-life in interphase by cyclin-dependent kinase 2 activity

Cdc25A regulates cell cycle progression, has oncogenic and anti-apoptotic activity, and is over-expressed in many human tumors. Phosphorylation by Chk1 and Cds1/Chk2 down-regulates Cdc25A levels in response to genotoxic stresses. Nevertheless, it remains unclear whether Chk1 and Cds1/Chk2 are uniquely responsible for regulating Cdc25A stability during interphase or if other kinase activities contribute. Here we report that treatment of HeLa cells with the cyclin-dependent kinase inhibitor roscovitine caused a concentration- and time-dependent increase in Cdc25A protein levels. Transfection with dominant-negative Cdk mutants demonstrated that only a Cdk2 mutant increased Cdc25A protein levels; Cdk1 and Cdk3 mutants had no effect. The increased Cdc25A protein levels were the result of an increase in the half-life of the protein; no increase in Cdc25A mRNA levels was observed. These results demonstrate Cdk2 kinase activity contributes to the labile nature of Cdc25A during interphase and redefine the nature of the Cdc25A-Cdk2 autoamplification feedback loop.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Identification of an alpha3-fucosyltransferase and a novel alpha2- fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-- >3[Gal(NAc)beta1-->4]GlcNAc sequence

Fucose is a major constituent of the protein- and lipid-linked glycans of the various life-cycle stages of schistosomes. These fucosylated glycans are highly antigenic and seem to play a role in the pathology of schistosomiasis. In this article we describe the identification and characterization of two fucosyltransferases (FucTs) in cercariae of the avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1-- >4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for FucT activity using a variety of acceptor substrates. Type 1 chain (Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those based on a type 2 chain (Galbeta1-->4GlcNAc), whether alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether present as oligosaccharide or contained in a glycopeptide or glycoprotein, all served as acceptor substrates. In this respect the schistosomal alpha3- FucT resembles human FucT V and VI rather than other known FucTs. N- ethylmaleimide, an inhibitor of several human FucTs, had no effect on the activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly inhibitory. Large scale incubations were carried out with Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the products of the incubations were isolated using a sequence of chromatographic techniques. By methylation analysis and 2D-TOCSY and ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1-- >4[Fucalpha1-->2Fucalpha1-->3]GlcNAc, GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1-- >3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2- FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric) Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural element that have been described on schistosomal glycoconjugates.      

Chlorophyll thermofluorescence and thermoluminescence as complementary tools for the study of temperature stress in plants

The photosynthetic apparatus, especially the electron transport chain imbedded in the thylakoid membrane, is one of the main targets of cold and heat stress in plants. Prompt and delayed fluorescence emission originating from photosystem II have been used, most often separately, to monitor the changes induced in the photosynthetic membranes during progressive warming or cooling of a leaf sample. Thermofluorescence of F ₀ and F _M informs on the effects of heat on the chlorophyll antennae and the photochemical centers, thermoluminescence on the stabilization and movements of charges and Delayed Light Emission on the permeability of the thylakoid membranes to protons and ions. Considered together and operated simultaneously, these techniques constitute a powerful tool to characterize the effect of thermal stress on intact photosynthetic systems and to understand the mechanisms of constitutive or induced tolerance to temperature stresses.     

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia

Background

Artemisinin and its derivatives have been used for falciparum malaria treatment in China since late 1970s. Monotherapy and uncontrolled use of artemisinin drugs were common practices for a long period of time. In vitro tests showed that the susceptibility of Plasmodium falciparum to artemisinins was declining in China. A concern was raised about the resistance to artemisinins of falciparum malaria in the country. It has been reported that in vitro artemisinin resistance was associated with the S769N mutation in the PfATPase6 gene. The main purpose of this study was to investigate whether that mutation has occurred in field isolates from China.

Methods

Plasmodium falciparum field isolates were collected in 2006–2007 from Hainan and Yunnan provinces, China. A nested PCR-sequencing assay was developed to analyse the genotype of the PfATPase6 S769N polymorphism in the P. falciparum field isolates.

Results

The genotyping results of six samples could not be obtained due to failure of PCR amplification, but no S769N mutation was detected in any of the 95 samples successfully analysed.

Conclusion

The results indicate that the S769N mutation in the PfATPase6 gene is not present in China, suggesting that artemisinin resistance has not yet developed, but the situation needs to be watched very attentively.     

Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox

Background  

Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults.