Sandhoff disease in Argentina: high frequency of a splice site mutation in the HEXB gene and correlation between enzyme and DNA-based tests for heterozygote detection

The level of -hexosaminidase activity in plasma and leukocytes and the frequency of three known HEXB mutations were studied in an Argentinean deme with high incidence of infantile Sandhoff disease. Two mutations were previously identified in one of two Sandhoff patients from the region, a splice mutation, IVS-2+1 GA, and a 4-bp deletion, CTTT_782–785. These mutations, and a 16kb deletion from the 5' end of the HEXB gene common in non-Argentineans, were screened in 9 Sandhoff patients (all unrelated), 24 obligate heterozygotes, 33 additional individuals belonging to families with affected members, and 64 randomly ascertained individuals from the high risk region. Of 31 independent alleles examined, including those of the two patients previously reported, 30 had the IVS-2 splice mutation and only the originally reported patient had the CTTT deletion. The 16-kb deletion was not observed. Further, among the 57 unaffected members of families with a previous history of Sandhoff disease, and absolute correlation was found between carrier diagnosis by enzyme assay of leukocytes and the DNA-based tests for mutation. One of the 64 controls was classified as a carrier by enzyme assay but did not have one of the three mutations screened. We conclude that a single mutation predominates in this Argentinean population and that the DNA-based test can be an effective supplement or alternative to enzyme-based testing.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p<0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p<0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Plasma membrane Ca2+ channels in roots of higher plants and their role in aluminium toxicity

Single Ca²⁺ channel records were obtained from plasma membrane-enriched fractions of wheat roots incorporated into artificial planar lipid bilayers. The channel had a unitary conductance of 15 pS for a 10 to 95 mM CaCl₂ gradient (cytoplasm: outside of the cell). The voltage dependence displayed by the channel agreed with that expected for Ca²⁺ channels in the plasma membrane. The channel gating was strongly modified by addition of 20 M extracellular verapamil (a Ca²⁺ channel antagonist). Extracellular AlCl₃ (70 M, pH 4.9) almost completely blocked the channel.     

Genotypic variation in biological nitrogen fixation by common bean

Field experiments were performed in Austria, Brazil, Chile, Colombia, Guatemala, Mexico and Peru as part of an FAO/IAEA Co-ordinated Research Programme to investigate the nitrogen fixing potential of cultivars and breeding lines of common bean (Phaseolus vulgaris L.). Each experiment included approximately 20 bean genotypes which were compared using the ¹⁵N isotope dilution method. Great differences in nitrogen fixation were observed between and within experiments, with average values of 35% N derived from atmosphere (% Ndfa) and highest values of 70% Ndfa being observed. These values which were larger than had been reported previously for common bean, were observed only when environmental factors were favorable. Therefore, common bean lines are available, which can support high biological nitrogen fixation. These can be used either directly as cultivars for production or in breeding programmes to enhance nitrogen fixation in other cultivars.     

Production and initial characterisation of the xylan-degrading system of Phanerochaete chrysosporium

We report the optimum conditions for the degradation of oat spelt arabinoxylan and a preliminary characterisation of the inducible xylan-degrading system of the lignin-degrading white-rot fungus Phanerochaete chrysosporium. Xylanase activity was optimal at pH 5.0 and 50°C; see attached sheet the maximum reaction velocity (V_max) of the system was 3.86 units (U) mg^–1 protein with arabinoxylan as substrate and the substrate concentration giving half V_max (S_0.5) was 0.52 mg ml^–1. At concentrations of arabinoxylan greater than 15 mg ml^–1 excess substrate inhibition was observed. Xylose at 0.9 mm inhibited activity to the extent of 50%. Xylanase activity increased as a function of the dilution of the enzyme preparation prior to assay. It was resolved into four peaks by using a DEAE-Biogel column; the material in these peaks differed with respect to xylan solubilisation and the formation of reducing sugars. Electrofocusing gels allowed visualisation of several bands of activity corresponding to each peak. The arabinoxylan degradation system of P. chrysosporium is therefore composed of multiple components. Correspondence to: P. Broda     

In vitro survival of murine morulae after quick freezing in the presence of chemically defined macromolecules and different cryoprotectants

We studied the ability of frozen-thawed mouse morulae to develop in vitro when the cryoprotectant proteins were substituted with one of the following nonorganic macromolecules: polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and ficoll. We also determined how these agents interacted with 3 different cryoprotectants: glycerol (GLY), propylene glycol (PG), and ethylene glycol (EG). The influence of both of the above factors was measured on the basis of post-thaw morphological appearance, the percentage of development to the expanded blastocyst stage and the total cell count. Morulae (n=950) were collected from superovulated mice. Those classified as good or excellent were distributed among the 12 different freezing solutions, obtained by combining the 3 cryoprotectants with the 4 macromolecules (the 3 mentioned above, plus a control of 5% fetal calf serum) in phosphate buffered saline (PBS). Embryos frozen in PVA, PVP and ficoll tended to be a little difficult to recover from the straws. Development to the expanded blastocyst stage was significantly lower (P<0.05) in propylene glycol (43.6%) than in ethylene glycol (79.5%) or in glycerol (76.1%). Polyvinyl alcohol provided a higher survival rate when combined with glycerol (90.3) or ethylene glycol (95.0), but when it was combined with propylene glycol, only 56.5% of embryos survived after thawing. A positive interaction was observed between glycerol and PVA and between ethylene glycol and PVA or ficoll. The results indicate that fetal serum could be successfully substituted for any of the 3 chemically defined macromolecules. However, our findings also suggest that the use of PG as a cryoprotectant should be avoided when mouse morulae are frozen using the quick freezing method.     

Life-history studies of heterophyid trematodes in the Neotropical Region: Ascocotyle (Phagicola) diminuta (Stunkard & Haviland, 1924) and A. (P.) angrense Travassos, 1916

The life-cycle of Ascocotyle (Phagicola) diminuta (Stunkard & Haviland, 1924) was reproduced experimentally, starting from cercariae from naturally infected Littoridina castellanosae and L. parchappei (Hydrobiidae) collected from artificial ponds in the Zoological Garden in Buenos Aires and from Los Ranchos stream, Buenos Aires Province, respectively. Metacercariae were found encysted in the gills of experimentally exposed Cnesterodon decemmaculatus (Poecilidae) and of other naturally infected freshwater fishes. Adults were obtained experimentally in chicks and mice and from a naturally infected egret, Egretta thula. A. (P.) angrense Travassos, 1916 was found parasitising the egret Ixobrychus involucris; it is considered a valid species and the morphological differences between it and A. (P.) diminuta were established. The “Phagicola-form” of the cercaria in the present life-cycle is also known in the genus Pygidiopsis.     

Protein phosphatases in higher plants: multiplicity of type 2A phosphatases in Arabidopsis thaliana

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen. A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical proteins (97% identity). Both proteins are 306 amino acids in length and are very similar (79–80% identity) to the mammalian isotypes of the catalytic subunit of protein phosphatase 2A. Therefore, they have been designated PP2A-1 and PP2A-2. A third cDNA clone, EP7, was isolated and sequenced. The polypeptide encoded (308 amino acids, lacking the initial Met codon) is 80% identical with human phosphatases 2A and was named PP2A-3. The PP2A-3 protein is extremely similar (95% identity) to the predicted protein from a cDNA clone previously found in Brassica napus. Southern blot analysis of genomic DNA using AP-1 and AP-2 probes, as well as probes derived from clones EP7, EP8a and EP14a strongly indicates that at least 6 genes closely related to type 2A phosphatases are present in the genome of A. thaliana. Northern blot analysis using the same set of probes demonstrates that, at the seedling stage, the mRNA levels for PP2A-1, PP2A-3 and the gene containing the AP-1 sequence are much higher than those of PP2A-2 and AP-2. These results demonstrate that a multiplicity of type 2A phosphatases might be differentially expressed in higher plants.     

A pulsing device for packed-bed bioreactors: II. Application to alcoholic fermentation

When the immobilized cells are employed in packed-bed bioreactors several problems appear. To overcome these drawbacks, a new bioreactor based on the use of pulsed systems was developed [1]. In this work, we study the glucose fermentation by immobilized Saccharomyces cerevisiae in a packed-bed bioreactor. A comparative study was then carried out for continuous fermentation in two packed-bed bioreactors, one of them with pulsed flow. The determination of the axial dispersion coefficients indicates that by introducing the pulsation, the hydraulic behaviour is closer to the plug flow model. In both cases, the residence time tested varied from 0.8 to 2.6 h. A higher ethanol concentration and productivity (increases up to 16%) were achieved with the pulsated reactors. The volumes occupied by the CO₂ were 5.22% and 9.45% for fermentation with/without pulsation respectively. An activity test of the particles from the different sections revealed that the concentration and viability of bioparticles from the two bioreactors are similar. From the results we conclude that the improvements of the process are attributable to a mechanical effect rather than to physiological changes of microorganisms.List of Symbols D m²/s dispersion coefficient - K _is l/g inhibition substrate constant - K _ip l/g inhibition ethanol constant - K _s g/l Apparent affinity constant - P g/l ethanol concentration - q _p g/(gh) specific ethanol productivity - Q _p g/(lh) overall ethanol productivity - q _s g/(gh) specific glucose consumption rate - Q _s g/(lh) glucose consumption rate - S g/l residual glucose concentration - S(in0) g/l initial glucose concentration - V _max g/(lh) maximum rate - Y _p/s g/g yield in product     

Decreased Electroconvulsive Shock-Induced Diacylglycerols and Free Fatty Acid Accumulation in the Rat Brain by Ginkgo biloba Extract (EGb 761): Selective Effect in Hippocampus as Compared with Cerebral Cortex

Abstract: The effect of Ginkgo biloba extract (EGb 761) treatment (100 mg/kg/day, per os, for 14 days) on electroconvulsive shock (ECS)-induced accumulation of free fatty acids (FFA) and diacylglycerols (DAG) was analyzed in rat cerebral cortex and hippocampus. EGb 761 reduced the FFA pool size by 33% and increased the DAG pool by 36% in the hippocampus. These endogenous lipids were unaffected in cerebral cortex. During the tonic seizure (10 s after ECS) the fast accumulation of FFA, mainly 20:4, was similar in sham- and EGb 761 -treated rats, in both the cerebral cortex and hippocampus. However, further accumulation of free 18:0 and 20:4, observed in the hippocampus of sham-treated rats during clonic seizures (30 s to 2 min after ECS), did not occur in EGb 761-treated animals. The rise in DAG content triggered in the cortex and hippocampus by ECS was delayed by EGb 761 treatment from 10 s to 1 min, when values similar to those in sham animals were attained. Moreover, in the hippocampus the size of the total DAG pool was decreased by 19% during the tonic seizure. At later times, DAG content showed a faster decrease in EGb 761-treated rats. By 2 min levels of all DAG acyl groups decreased to values significantly lower than in sham animals in both cortex and hippocampus. This study shows that EGb 761 treatment affects, with high selectivity, lipid metabolism and lipid-derived second messenger release and removal in the hippocampus, while affecting to a lesser extent the cerebral cortex.