Effects of L-Glutamate Transport Inhibition by a Conformationally Restricted Glutamate Analogue (2S,1'S,2'R)-2-(Carboxycyclopropyl)Glycine (L-CCG III) on Metabolism in Brain Tissue In Vitro Analysed by NMR Spectroscopy

(2S,1'S,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG III) was a substrate of Na⁺-dependent glutamate transporters (GluT) in Xenopus laevis oocytes (IC₅₀ 13 and 2 M for, respectively, EAAT 1 and EAAT 2) and caused an apparent inhibition of [³H]L-glutamate uptake in mini-slices of guinea pig cerebral cortex (IC₅₀ 12 M). In slices (350 M) of guinea pig cerebral cortex, 5 M L-CCG III increased both the flux of label through pyruvate carboxylase and the fractional enrichment of glutamate, GABA, glutamine and lactate, but had no effect on total metabolite pool sizes. At 50 M L-CCG III decreased incorporation of ¹³C from [3-¹³C]-pyruvate into glutamate C4, glutamine C4, lactate C3 and alanine C3. The total metabolite pool sizes were also decreased with no change in the fractional enrichment. Furthermore, L-CCG III was accumulated in the tissue, probably via GluT. At lower concentration, L-CCG III would compete with L-glutamate for GluT and the changes probably reflect a compensation for the missing L-glutamate. At 50 M, intracellular L-CCG III could reach > 10 mM and metabolism might be affected directly.     

Effect of keratin on the lipid composition of Scopulariopsis brevicaulis (Bainier)

Lipid content and composition of Scopulariopsis brevicaulis cultured with or without its natural substrate keratin was investigated. Lipid content was found to be lower in the presence of keratin (maximum 12% against 19% in the absence of keratin), with differences in levels of linoleic acid (C₁₈₌₂), palmitic (C₆₀) and to a lesser extent palmitoleic (C₁₆₌₁) and -linolenic (C₁₈₌₃) acids. The degree of unsaturation was correspondingly lower in the organisms cultured with keratin as substrate.     

Temporal and Spatial Allozyme Variation in the South American Cactophilic Drosophila antonietae (Diptera; Drosophilidae)

Drosophila antonietae is an endemic South American cactophilic species that uses Cereus hildmaniannus rotting cladodes as breeding sites. We assessed temporal and spatial intrapopulational allozyme variation of two natural populations. Our results suggest that environmental variation (rain precipitation) is probably influencing allozyme temporal variation. Moreover, it seems that D. antonietae does not have intrapopulation structure and has N _ev (variance effective size) 83 and N _ec (number of adult flies that colonize each rotting cladode) = 21. The deficiency of heterozygotes found must be due to null alleles, a temporal Wahlund effect, or selection against heterozygotes. Assortative mating and inbreeding are discarded. This is the first report on allozyme variation in D. antonietae. It gives some insight on intrapopulational genetics through space and time for this species. This is important to understand its general genetic variability and will be essential to future works on the natural history and evolution of this species.     

A comparison between the amino acid composition of an egg parasitoid wasp and some of its hosts

Amino acid compositions of the eggs of five lepidopteran hosts for Trichogramma minutum were compared with each other and with a non-host species, Rhodnius prolixus, in which T. minutum oviposits but does not develop. Host eggs are quite homogeneous, particularly when compared according to groupings of potentially interconvertible amino acids. Combined mole percent values for glycine, serine and alanine were higher in hosts (27.5–29.2 mole%) than in R. prolixus eggs (21.5 mole%), in bovine serum albumin (14.9%), which has been used as a protein source in artificial diets for T. minutum, or in many of the mixtures used in published diets for this species. Since these three amino acids make up 26.3 mole% of the adult amino acid content of T. minutum, their deficiency in diets could require metabolic compensation detrimental to development.Adult T. minutum arising from eggs of Manduca sexta, Choristoneura fumiferana, and Sitotroga cerealella are similar in amino acid composition to each other and, in general, to their hosts. Variability appears greater in hosts than in adult wasp composition, suggesting some interconversion of host amino acids to accommodate inflexible nutritional requirements of T. minutum.In the three host species tested, free amino acids constituted 15.8–19.3% by weight of the amino acid in egg contents. In M. sexta eggs, glycine, serine and alanine together make up 28.4% by weight of the total free amino acid, a much higher proportion than in many published diets. The four free amino acids (isoleucine, leucine, phenylalanine and histidine) reported to be oviposition stimulants in experiments on encapsulated diets are present in sufficient concentrations to induce oviposition in the host species tested and in R. prolixus. S. cerealella egg contents having approximately 1.8 g amino acid, yield one or rarely two adult T. minutum (1g amino acid/insect). In contrast, M. sexta eggs with 94 g amino acid each yield an average of 10–12 adults (8.2g amino acid/insect). This suggests that small hosts are allocated few eggs which can only develop into small adults because of nutrient supply (parasitoid size in metabolically restricted), whereas much larger hosts are allocated proportionately fewer eggs than the former resulting in larger, and presumably more viable and fecund, adults (parasitoid size is established behaviourally).     

Protein metabolism and strength performance after bovine colostrum supplementation

Summary. This study was designed to determine the responses of muscle protein, serum amino acids, and strength performance to bovine colostrum supplementation in physically active men. The rest (R) group (n=6) and the exercise (E) group (n=6) carried out twice a 2-week experiment randomly assigned in a double-blind fashion with either placebo (PLA; consuming daily 20g maltodextrin) or bovine colostrum (COL; consuming daily 20g colostrum supplement) treatment with one month between. On the test day after the treatment period the measurements were carried out in fasting conditions and E carried out a strength training session (STS). The methods involved the infusion of ring-²H₅-phenylalanine, femoral arterial and venous blood sampling, and biopsies from the vastus lateralis muscle. Serum concentration of essential amino acids during recovery was greater (p<0.05) in the COL groups compared with the PLA groups. Both muscle protein synthesis and breakdown increased (p<0.05) with COL. There were no differences in phenylalanine net balance or strength performance between the PLA and COL groups. It was concluded that a 2-week supplementation with bovine colostrum in physically active men increases serum concentration of essential amino acids but has no effect either on strength performance or protein net balance in fasting conditions during recovery after STS.     

Conditional lysis ofEscherichia coli by the fusion of extracellular (STA) to periplasmic (LTB) enterotoxins: Apparent phenotypic suppression of lactose permease

The expression of a methanol-soluble, heat-stable enterotoxin (ST_A) fused to the B subunit of the heat-labile enterotoxin (LT_B) at 35°C or higher temperatures caused strains ofEscherichia coli deficient in lactose permease to behave on indicator media as Lac⁺; however, at 33°C or lower temperatures the original Lac^– phenotype of the host strains was maintained. The apparent phenotypic suppression oflacY was shown to be due to lysis of a fraction of the bacteria and the consequent release of active-galactosidase to the culture supernatant. After incubation at 37°C for 1 h, the cultures were committed to lyse. Plasmid and chromosomal mutants that do not show this phenotype were isolated by selecting Lac^– colonies at the unpermissive temperature. The mutations on the plasmids were localized in both the heat-stable and the heat-labile enterotoxin genes. Chromosomal mutants that show normal levels of-galactosidase and fused toxins have also been isolated.     

The rate of microbial degradation of oil in a beach gravel column

An automatic, continuous flow respirometer was used to follow the bio-oxidation of a crude oil in a column of fairly coarse beach material. A number of water percolation rates were employed, with and without inorganic nutrient supplementation (nitrate and phosphate). Initially, nutrient supplementation was required to allow significant oxidation rates, but a capacity for biodegradation in the absence of continued supplementation developed slowly. The increase in oxidation rate with nutrient supplementation was in the proportion 0.23 mg oxygen permol nitrate. This proportionality was similar to previous results in this laboratory (using different systems), as was the effect of temperature. The mean of two Q₁₀ values in this work was 2.7.     

Neuropathological Responses to Chronic NMDA in Rats Are Worsened by Dietary n-3 PUFA Deprivation but Are Not Ameliorated by Fish Oil Supplementation

Background

Dietary long-chain n-3 polyunsaturated fatty acid (PUFA) supplementation may be beneficial for chronic brain illnesses, but the issue is not agreed on. We examined effects of dietary n-3 PUFA deprivation or supplementation, compared with an n-3 PUFA adequate diet (containing alpha-linolenic acid [18:3 n-3] but not docosahexaenoic acid [DHA, 22:6n-3]), on brain markers of lipid metabolism and excitotoxicity, in rats treated chronically with NMDA or saline.

Methods

Male rats after weaning were maintained on one of three diets for 15 weeks. After 12 weeks, each diet group was injected i.p. daily with saline (1 ml/kg) or a subconvulsive dose of NMDA (25 mg/kg) for 3 additional weeks. Then, brain fatty acid concentrations and various markers of excitotoxicity and fatty acid metabolism were measured.

Results

Compared to the diet-adequate group, brain DHA concentration was reduced, while n-6 docosapentaenoic acid (DPA, 22:5n-6) concentration was increased in the n-3 deficient group; arachidonic acid (AA, 20:4n-6) concentration was unchanged. These concentrations were unaffected by fish oil supplementation. Chronic NMDA increased brain cPLA₂ activity in each of the three groups, but n-3 PUFA deprivation or fish oil did not change cPLA₂ activity or protein compared with the adequate group. sPLA₂ expression was unchanged in the three conditions, whereas iPLA₂ expression was reduced by deprivation but not changed by supplementation. BDNF protein was reduced by NMDA in N-3 PUFA deficient rats, but protein levels of IL-1β, NGF, and GFAP did not differ between groups.

Conclusions

N-3 PUFA deprivation significantly worsened several pathological NMDA-induced changes produced in diet adequate rats, whereas n-3 PUFA supplementation did not affect NMDA induced changes. Supplementation may not be critical for this measured neuropathology once the diet has an adequate n-3 PUFA content.     

Silicon amelioration of aluminium toxicity in teosinte (Zea mays L. ssp. mexicana)

The influence of different Al concentrations, (0, 60 and 120 M Al) on growth and internal concentrations of Al, Si and selected organic acids was analysed in plants of teosinte (Zea mays L. ssp. mexicana), a wild form of maize from acid soils from Mexico. The plants were grown in nutrient solutions (pH 4.0) with or without 4 M silicon. Analysis with the GEOCHEM speciation program did not reveal differences between free activities of Al³⁺ in solutions with and without 4 M Si, but solutions with Si yielded lower concentrations of monomeric Al species, [Al]_mono, when analysed by a modified aluminon method. Plants grown on solutions with similar [Al]_mono, but differing in silicon, showed highly significant differences in growth and tissue concentrations of Al and organic acids. Silicon prevented growth inhibition at [Al]_mono concentrations as high as 35 M, while plants grown without Si suffered severe growth reductions with 33 M [Al]_mono. In solutions with similar [Al]_mono concentrations plants with Si had lower tissue Al concentrations and higher concentrations of malic acid than plants without Si. In view of both the significant influence of Si on the response of plants to Al toxicity and the fact that some soluble Si is always present in soil solutions, the addition of low Si concentrations to nutrient solutions used for Al-tolerance screening is recommended.     

Cytotaxonomy of the triatominae (Reduviidae: Hemiptera)

The chromosome number and meiotic cycle of 20 species of Triatominae have been investigated. In the male, there are five types of chromosome complement: 20+XY, 20+X₁X₂Y, 20+X₁X₂X₃Y, 18+XY and 22+XY.The cytological data suggest that the type number for the subfamily is 22 (20+XY). In the hybrids: Triatoma barberi () and T. protracta (), anomalous behavior of certain chromosomes has been observed. Phylogenetic relationships based on chromosome evidence in the subfamily have been discussed. It is suggested that fragmentation is the major factor for chromosome evolution in the group.     

New cases of somatic conversion (paramutation) in tomato (Lycopersicon esculentum Mill.)

Summary The genetic behaviour of three chlorophyll variegated F₁ tomato plants, derived from irradiated gametophytes, was analyzed over several generations of selfing and backcrossing. The results suggest that irradiation has put genes, different in all three mutants, into a labile state, n*, remaining so after fertilization. This state had the power of converting the associated wild allele N into a deficient form.Somatic conversion was soon followed, in Plant C₁₁ always and nearly always in Plant C₁₂, by stabilization of both alleles in a conversion-inactive recessive state, genetically similar, and stable except in special conditions.In the other type, found seldom in C₁₂, always in C₆, the n* state was permanent and transmissible. Conversion occurred with a certain frequency determined by developmental and genotypic influences, and the converted allele also acquired conversion power, so that gametes from an N n* plant were of three kinds: N, n* and n*. This process corresponds to paramutation (Brink 1958).Results were compared and contrasted with other published data.Contribution nr. 748 of the Biology Division of Euratom.     

Elevated atmospheric partial pressure of carbon dioxide and dry matter production of konjak (Amorphophallus konjac K. Koch)

Konjak (Amorphophallus konjac K. Koch) was grown under normal (350 bar) or enriched (700 bar) CO₂ partial pressure in glasshouses kept at 33/26 °C. Doubling the CO₂ partial pressure resulted in twice the yield of corm because the net CO₂ assimilation rate doubled and, due to the simple source-sink relationship, the increased production was partitioned to the corm. The response to CO₂ of assimilation by konjak is discussed in relation to its original habitat in the tropics.     

Antide B,an antagonist of LHRH with cis-3-(4-pyrazinylcarbonylaminocyclohexyl)alanine in position 5

Summary Several LHRH antagonists with trans-3-(4-pyrazinylcarbonylaminocyclohexyl)alanine (trans-PzACAla) in the position 5 were synthesized and their antiovulatory activity was compared with the activity of the analogs containing cis-PzACAla in this position. In all cases cis-isomer produced more potent analogs. Introduction of cis-PzACAla in the position 5 of Antide gave Antide B which completely inhibits ovulation at a dose of 0.5µg/rat. Antide B releases negligible histamine (ED₅₀ = 104µg/mL), and has excellent solubility in water. Also, an improved synthesis of cis-PzACAla is reported, involving the hydrogenation of 4-aminophenylalanine on a rhodium catalyst to give the desired cis-isomer with a 53% yield.Abbreviations Cpa 3-(4-chlorophenyl)alanine - ILys N-isopropyllysine - Nal 3-(2-naphthyl)alanine - NicLys N-nicotinoyllysine - Pal 3-(3-pyridyl)alanine - PicLys N-picolinoyllysine - PzACAla 3-(4-pyrazinylcarbonylaminocyclohexyl)alanine - Qal 3-(3-quinolyl)alanine     

Alterations in the activity of phospholipases A2 in postmortem white matter from patients with multiple sclerosis

Activities toward arachidonyl-labelled phospholipase A₂ substrates were assayed in fractions of white matter and cerebral cortex from control subjects and in fractions of demyelinated plaque, normal-appearing white matter and cerebral cortex from subjects who died with multiple sclerosis. Membranous activity at pH 8.6 in the presence of Ca²⁺, characteristic of 14 kDa secretory phospholipase A₂, in either multiple sclerosis white matter or cortex did not differ from controls, whereas membranous activity at pH 4.5 in the absence of added Ca²⁺, characteristic of lysosomal enzymes was increased over controls in both plaque and normal-appearing white matter but not cerebral cortex. Activity in the cytosol fraction, at pH 8.6 in the presence of Ca²⁺ and glycerol characteristic of the cytosolic 85 kDa enzyme was decreased by greater than 50% in both white matter and cortex samples from multiple sclerosis subjects. Immuno-precipitation and-blotting confirmed that the deficient activity was largely attributable to the 85 kDa enzyme although the enzyme protein was not similarly reduced.Special issue dedicated to Dr. Leon S. Wolfe.     

Comparative studies of hepatotoxicity and fumonisin B1 and B2 content of water and chloroform/methanol extracts of Fusarium moniliforme strain MRC 826 culture material

Fusarium moniliforme has been associated with several diseases including equine leukoencephalomalacia, human esophageal cancer and hepatotoxicity/hepatocarcinogenicity in laboratory animals. The potential health risks to animals and humans posed by F. moniliforme contaminated grains cannot be assessed until the toxins are identified and toxicologically evaluated. As part of a systematic approach to identifying the hepatotoxins produced by F. moniliforme, diets containing aqueous and chloroform/methanol (11) extracts of F. moniliforme strain MRC 826 culture material (CM) and/or the extracted CM residues were fed to male Sprague-Dawley rats for four weeks. Serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activities were increased after two and four weeks and microscopic liver lesions were found in those animals fed aqueous CM extract and the CM residue after chloroform/ methanol extraction. Fumonisins B₁ and B₂ were extracted from the CM by water, but not chloroform/ methanol, and were present in the toxic diets at concentrations of 93–139 and 82–147 ppm, respectively. Nontoxic diets contained 22 ppm fumonisin B₁ and 65 ppm fumonisin B₂.Abbreviations CM culture material - ELEM equine leukoencephalomalacia Mention of a trademark, proprietory name or vendor does not imply its approval by the US Department of Agriculture to the exclusion of others that may also be suitable.     

Seed oil fatty acid patterns of theAconitum-Delphinium-Helleborus complex (Ranunculaceae)

Many members ofRanunculaceae contain unusual fatty acids in their seed oils. This leads to rather typical genus-specific fatty acid patterns or fingerprints in these seed oils. The members of theDelphinioideae and/orHelleboroideae, however, do not contain highly unusual fatty acids. Nevertheless, their seed oil fatty acid fingerprints are also fairly typical and genus-specific, and the patterns found are rather consistent throughout several species of one genus. It was found that species ofAconitum do not contain fatty acids with 20 carbon atoms.Delphinium, Consolida, Helleborus, Nigella and others do contain C₂₀ fatty acids. In allHelleborus species, for example, there was a consistent C₂₀ fatty acid pattern of 20:020:120:2>20:3. Species ofNigella andGaridella contain high levels,Helleborus low levels, of 20:2n-6 in their seed oils.Delphinium andAconitum both contain low levels of 18:3n-3, whereasHelleborus spp. consistently show high levels of this fatty acid. The genus-specific fatty acid patterns found are discussed, and a correlation with the subfamily and tribe affiliation of the genera investigated here is attempted.     

Frequent Seizures Are Associated with a Network of Gray Matter Atrophy in Temporal Lobe Epilepsy with or without Hippocampal Sclerosis

Objective

Patients with temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) have diffuse subtle gray matter (GM) atrophy detectable by MRI quantification analyses. However, it is not clear whether the etiology and seizure frequency are associated with this atrophy. We aimed to evaluate the occurrence of GM atrophy and the influence of seizure frequency in patients with TLE and either normal MRI (TLE-NL) or MRI signs of HS (TLE-HS).

Methods

We evaluated a group of 172 consecutive patients with unilateral TLE-HS or TLE-NL as defined by hippocampal volumetry and signal quantification (122 TLE-HS and 50 TLE-NL) plus a group of 82 healthy individuals. Voxel-based morphometry was performed with VBM8/SPM8 in 3T MRIs. Patients with up to three complex partial seizures and no generalized tonic-clonic seizures in the previous year were considered to have infrequent seizures. Those who did not fulfill these criteria were considered to have frequent seizures.

Results

Patients with TLE-HS had more pronounced GM atrophy, including the ipsilateral mesial temporal structures, temporal lobe, bilateral thalami and pre/post-central gyri. Patients with TLE-NL had more subtle GM atrophy, including the ipsilateral orbitofrontal cortex, bilateral thalami and pre/post-central gyri. Both TLE-HS and TLE-NL showed increased GM volume in the contralateral pons. TLE-HS patients with frequent seizures had more pronounced GM atrophy in extra-temporal regions than TLE-HS with infrequent seizures. Patients with TLE-NL and infrequent seizures had no detectable GM atrophy. In both TLE-HS and TLE-NL, the duration of epilepsy correlated with GM atrophy in extra-hippocampal regions.

Conclusion

Although a diffuse network GM atrophy occurs in both TLE-HS and TLE-NL, this is strikingly more evident in TLE-HS and in patients with frequent seizures. These findings suggest that neocortical atrophy in TLE is related to the ongoing seizures and epilepsy duration, while thalamic atrophy is more probably related to the original epileptogenic process.     

Feeding ecology of the sand smelt <Emphasis Type="Italic">Atherina boyeri</Emphasis> (Risso 1810) (Osteichthyes,Atherinidae) in the western Mediterranean: evidence for spatial variability based on stable carbon and nitrogen isotopes

Synopsis The feeding ecology of the sand smelt Atherina boyeri Risso 1810 (Osteichthyes, Atherinidae) was investigated in a Mediterranean coastal basin (Stagnone di Marsala, Italy) by means of carbon and nitrogen stable isotope ratios. Sampling was carried out seasonally in 1999 in two locations characterised by different depth and vegetal coverage. Throughout the year the sand smelt showed enriched ¹³C and ¹⁵N values in both sampling locations (¹³C about –12 and ¹⁵N about 12). This result suggests the overall importance of the benthic pathway in the food web leading to A. boyeri. Our isotopic picture is only in partial agreement with stomach content data from the literature. Stomach contents highlighted that, while A. boyeri is benthivore in the location with high vegetal coverage, it is able to shift its diet toward zooplankton in the location with higher depth and lower complexity. In our opinion, this discrepancy can be a consequence of the different temporal scale which stomach contents and stable isotopes are referred to. Contrarily to stomach contents, stable isotope analysis provides time-integrated information on the real diet averaged over some months or more. We suggest that long-term and short-term diets of the sand smelt differ. Zooplankton, due to its low biomass in the study site seems to be exploited scarcely by A. boyeri and its trophic role is detectable only considering the short term diet with stomach contents, while the time-integrated diet of A. boyeri seems to be based on benthic prey.     

Inter-simple-sequence repeat (ISSR)-PCR for the identification of saprophytic strains of Leptospira

The inter-simple-sequence repeat (ISSR) primers that anneal to a simple repeat of various length and at non-repetitive motifs at 3 and 5 end were attempted for PCR amplification of Leptospira genome. Of the six ISSR primers tested, namely, (AG)₈T, (AG)₈C, (AG)₈G, (CA)₈A, (TG)₈C and (TG)₈G, only primer (AG)₈T produced amplification of 1000 bp in the two non-pathogenic Leptospira species tested, viz; Leptospira biflexa serovar patoc and L. meyeri serovar ranarum, with no amplification in any of the 16 standard pathogenic serovars tested. The remaining five ISSR primers did not exhibit any amplification of the Leptospira genome in either pathogenic or non-pathogenic species. From among 35 Leptospira isolates recovered from hospitalized patients with pyrexia of unknown origin and/or febrile jaundice (12 in number) and from different environmental water sources (23 in number), (AG)₈T ISSR-PCR correctly identified all the 22 isolates from water sources that were confirmed to be non-pathogenic by conventional tests. The results therefore, confirmed the ability of a primer, based on simple-sequence repeat motif, to produce a fragment that is useful as a group genetic marker in Leptospira species. The single nucleotide anchor, T, at the 3 end of the primer appeared to play an important role in differentiation of pathogenic and non-pathogenic species of Leptospira. Multiplex PCR, using ISSR primer, (AG)₈T and the reported 16S rRNA gene primers, specific for pathogenic Leptospira species, or the 23S rRNA Leptospira genus specific primers, provided clear identification of serovars and isolates into pathogenic or non-pathogenic groups.     

Activation of phospholipases A2 and D of a human neuroblastoma cell line (LA-N-2) by N-dodecyl-L-lysine amide (compound 24), a putative G protein activator: characteristics of inhibition by (-)-nicotine

Compound 24, an alkyl-substituted amino acid amide, previously found to activate pertussis toxin-sensitive G proteins in cell membranes and membrane protein fractions, was used as a tool to determine the mechanism/location of nicotine inhibition of amyloid peptide-stimulated phospholipase A₂ and D activities in a human neuroblastoma cell line, LA-N-2, in vitro. In contrast to our previous findings with amyloid peptide, these phospholipase activations by compound 24 were not inhibited by (–)-nicotine, cholera toxin or tetanus toxin pretreatment. The contrasting activation of these phospholipases by amyloid peptide and compound 24 are discussed.