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.      

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.     

Aerobic Biodegradation of High Explosives, Phase I - HMX

The Pantex facility near Amarillo, Texas, has soil and groundwater contaminated with differing combinations of high explosives (HEs), including hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4,6-trinitrotoluene (TNT). This project was concerned with direct treatment of HMX in groundwater withdrawn at this plant. Several physical and chemical treatment schemes for the treatment of HMX have been successful. However, the successful biological treatment of HMX has been limited to anaerobic environments. The objective of this work was to identify microbial consortia and amendments capable of aerobically biodegrading HMX in water. Microbial consortia and amendments employed were provided as livestock manure and soil with its indigenous flora from nearby historically contaminated sites. Possible losses of HMX by nonbiological means such as adsorption and photolysis were accounted for by appropriate abiotic experiments. Loss of the parent compound was measured by high-performance liquid chromatography, using a modification of U.S. Environmental Protection Agency (EPA) Method 8330. Results varied from no degradation to a reduction of parent HMX from 6 to 1 mg/L in 5.2 days. Evidence for biodegradation was supported by the appearance of metabolites. Metabolite identification was performed at Oak Ridge National Laboratory. Five metabolites (four intermediate and one final) were identified.     

Regulation of Endothelial-Specific Transgene Expression by the LacI Repressor Protein In Vivo

Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2) with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI^R, and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI^R protein.     

Morphological and Behavioral Impact of AAV2/5-Mediated Overexpression of Human Wildtype Alpha-Synuclein in the Rat Nigrostriatal System

The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson’s disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 10¹³) or varying titers (2.2 x 10¹², 1.0 x 10¹³, 5.9 x 10¹³, or 1.0 x 10¹⁴) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 10¹⁴ titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 10¹³ titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 10¹³ and 1.0 x 10¹⁴ rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 10¹³ rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.     

A procedure for biolistic transformation and regeneration of transgenic cotton from meristematic tissue

We have optimized methods for transformation of cotton meristem tissue using the Bio-Rad PDS/1000/He gene gun, selection of transformed tissue, and regeneration of transformed cotton plants. We have used either single or multiple bombardments of cotton tissue with 1.6-Å particles at rupture pressures of 90 or 110 kg/cm². The distance between the tissue and the source of particles can be varied between 3 and 6 cm. After bombardment, transformed cotton tissue is identified by selection for growth on media supplemented with 50 μg/mL kanamycin. Tissue sections that form leaves, shoots and at least two roots are then transferred to media supplemented with 100 mg indoleacetic acid (IAA) to favor formation of extensive root systems. The plantlets are then transferred to soil, hardened off, and grown in the greenhouse. These plants have been confirmed to be transgenic by western-blot analysis of leaf protein extracts with polyclonal antiserum to the neomycin phosphotransferase II gene product.     

Novel, potent THC/anandamide (hybrid) analogs

The structure–activity relationship (SAR) of the end pentyl chain in anandamide (AEA) has been established to be very similar to that of Δ⁹-tetrahydrocannabinol (Δ⁹-THC). In order to broaden our understanding of the structural similarities between AEA and THC, hybrid structures 1–3 were designed. In these hybrids the aromatic ring of THC–DMH was linked to the AEA moiety through an ether linkage with the oxygen of the phenol of THC. Hybrid 1 (O-2220) was found to have very high binding affinity to CB1 receptors (K_i = 8.5 nM), and it is interesting to note that the orientation of the side chain with respect to the oxygen in the phenol is the same as in THCs. To further explore the SAR in this series the terminal carbon of the side chain was modified by adding different substituents. Several such analogs were synthesized and tested for their CB1 and CB2 binding affinities and in vivo activity (tetrad tests). The details of the synthesis and the biological activity of these compounds are described.     

Connexins 37 and 40 transduce purinergic signals mediating renal autoregulation

Our previous data indicated that various subtypes of connexin (Cx) were expressed in the juxtaglomerular apparatus. Experiments were performed to characterize the effects on renal autoregulation of specific mimetic peptides that inhibit these Cx subtypes in Wistar-Kyoto rats. Intrarenal infusion of (Cx37,43)GAP27 increased autoregulatory index of renal plasma flow (0.06 +/- 0.05 to 0.47 +/- 0.06, n = 6, P < 0.05) and glomerular filtration rate (GFR; 0.01 +/- 0.07 to 0.49 +/- 0.07, P < 0.05). The additional administration of 8-cyclopentyl- 1,3-dipropylxanthine (CPX) produced a further elevation of autoregulatory index of RPF (0.86 +/- 0.07, P < 0.05) and GFR (0.88 +/- 0.09, P < 0.05), compared with (Cx37,43)GAP27 alone. However, the addition of pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid (PPADS) to (Cx37,43)GAP27 did not. Combined treatment with CPX and PPADS markedly worsened autoregulatory index of RPF (0.04 +/- 0.10 to 0.81 +/- 0.06, n = 6 P < 0.01) and GFR (0.05 +/- 0.08 to 0.79 +/- 0.05, P < 0.01). (Cx40)GAP27 induced similar changes to (Cx37,43)GAP27. Renal autoregulation was preserved in the presence of (Cx43)GAP26. Our results indicate that the inhibition of gap junction impaired renal autoregulation. Furthermore, the present data provide evidence that both adenosine and purinergic receptors contribute to glomerular autoregulation. Finally, our findings suggest that gap junctions, at least in part, transduce purinergic signals mediating renal autoregulation.     

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.     

BIOSYNTHESIS OF PHOSPHATIDIC ACID IN RAT BRAIN VIA ACYL DIHYDROXYACETONE PHOSPHATE

Abstract— The enzymes for the biosynthesis of phosphatidic acid from acyl dihydroxyacetone phosphate were shown to be present in rat brain. These enzymes were mainly localized in the microsomal fraction of 12–14 day old rat brains. The brain microsomal acyl CoA: dihydroxyacetone phosphate acyl transferase (EC 2.3.1.42), exhibited a broad pH optimum between pH 5 and 9 with maximum activity at pH 5.4. K _m for DHAP at pH 5.4 was 0.1 m m and V _max was 0.86nmol/min/mg of microsomal protein. The corresponding microsomal enzyme for the glycerophosphate pathway (acyl CoA: sn -glycerol-3-phosphate acyl transferase EC 2.3.1.15) was shown to have a different pH optimum (pH 7.6). On the basis of the differences in pH optima, differential effects of sodium cholate in the enzymes and a common substrate competition study, these acyl transferases were postulated to be two different microsomal enzymes.
Acyl DHAP:NADPH oxidoreductase (EC 1.1.1.101) in brain microsomes was found to be quite specific for NADPH as cofactor, being able to utilize NADH only at very high concentrations. This enzyme exhibited a K _m of 8.6 μ m with NADPH and V _mx of 0.81 nmol/min/mg protein. The presence of these two enzymes and the known presence of l-acyl- sn -glycerol-3-phosphate: acyl CoA acyl transferase in brain (F leming & H ajra , 1977) demonstrated the biosynthesis of phosphatidic acid in brain via acyl dihydroxyacetone phosphate. Phosphatidic acid was shown to form when dihydroxyacetone phosphate, acyl CoA, NADPH and other cofactors were incubated together with brain microsomes. Further properties of the enzymes and the probable importance of the presence of this pathway in brain were discussed.