The effects of Δ9-tetrahydrocannabinol Δ9-THC) on the regional concentrations of spermidine in the rat brain

The effects of intravenous administration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC, 2 mg/kg, i.v.) on the regional brain spermidine concentrations of the rat were examined. Thirty minutes after vehicle treatment, the spermidine concentrations were: for the medulla oblongata/pons, 68.2 ± 7.7 μg/g; the hypothalamus, 67.7 ± 2.6 μg/g; the midbrain, 59.1 ± 4.4 μg/g; the cerebellum, 47.3 ± 5.9 μg/g and for the cortex, 13.8 ± 0.8 μg/g. Thirty minutes after Δ⁹-THC, these concentrations were reduced in the midbrain (47.0 ± 8.0% of control, P < 0.0001) and cortex (69.4 ± 7.4% of control, P < 0.009). The spermidine concentrations were not significantly altered in the medulla oblongata/pons (86.5 ± 13.3%, P > 0.36), hypothalamus (107.2 ± 11.8, P > 0.36) or cerebellum (89.0 ± 14.4%, P < 0.48). These results suggest that spermidine within the midbrain and cortex may be involved in the expression of some of the actions of Δ⁹-THC.     

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

Two relatively rare fatty acids, γ-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, α-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Δ⁶ desaturase and an Arabidopsis Δ¹⁵ desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Δ⁶ desaturase event with the Δ¹⁵ desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean β-conglycinin promoter. Soybean events that carried only the Δ¹⁵ desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.     

Identification of conserved domains in the Δ12 desaturases of cyanobacteria

Cyanobacterial genes for enzymes that desaturate fatty acids at the 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by crosshybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.Abbreviations X:Y(Z) fatty acid containing X carbon atoms with Y double bonds in the cis configuration at position Z counted from the carboxyl terminus     

Dominance of Δ5-sterols in eight species of the cactaceae

The sterols from eight species in seven genera of the Cactaceae are 24-alkyl-Δ⁵-sterols. In all eight species, Echinopsis tubiflora, Pereskia aculeata, Hylocereus undatus, Notocactus scopa, Epiphyllum sp., Schlumbergera bridgesii, Opuntia comonduensis and O. humifusa, the dominant sterol is sitosterol (24α-ethylcholest-5-en-3β-ol) at 66–87% of the total sterol composition with the 24ξ-methylcholest-5-en-3β-ol present at 8–33%. Stigmasterol (24α-ethylcholesta-5,22E-dien-3β-ol) is present at 2–8% of the total sterol in P. aculeata, H. undatus, N. scopa and Epiphyllum sp. whereas cholesterol (cholest-5-en-3β-ol) is present in six species at levels of <0.1–5.0%. Avenasterol (24-ethylcholesta-7,24(28)Z-dien-3/gb-ol) and sitostanol (24α-ethyl-5α-cholestan-3β-ol) are each present in two species.     

Absence of the Δccr5 mutation in indigenous populations of the Brazilian Amazon

Carriers of the deltaccr5 allele, which contains a deletion of 32 bases in relation to the normal allele of the beta-chemokine receptor gene (CCR5), have increased resistance to HIV-1 infection. The higher frequency of this mutation in Europeans than in Blacks and Asians, has generated interest in determining its distribution in other populations. The population of this study involved 300 Amerindians from four Brazilian Amazon tribes (Tikuna, Baniwa, Kashinawa, and Kanamari). All of the individuals were homozygous for the normal allele, which corroborates the hypothesis that the deltaccr5 allele has a European origin, and that its occurrence in urban populations in South America is the result of immigration.     

Substitutions in the BamA β-barrel domain overcome the conditional lethal phenotype of a ΔbamB ΔbamE strain of Escherichia coli

BamA interacts with the BamBCDE lipoproteins, and together they constitute the essential β-barrel assembly machine (BAM) of Escherichia coli. The simultaneous absence of BamB and BamE confers a conditional lethal phenotype and a severe β-barrel outer membrane protein (OMP) biogenesis defect. Without BamB and BamE, wild-type BamA levels are significantly reduced, and the folding of the BamA β-barrel, as assessed by the heat-modifiability assay, is drastically compromised. Single-amino-acid substitutions in the β-barrel domain of BamA improve both bacterial growth and OMP biogenesis in a bamB bamE mutant and restore BamA levels close to the BamB(+) BamE(+) level. The substitutions alter BamA β-barrel folding, and folding in the mutants becomes independent of BamB and BamE. Remarkably, BamA β-barrel alterations also improve OMP biogenesis in cells lacking the major periplasmic chaperone, SurA, which, together with BamB, is thought to facilitate the transfer of partially folded OMPs to the soluble POTRA (polypeptide-transport-associated) domain of BamA. Unlike the bamB bamE mutant background, the absence of BamB or SurA does not affect BamA β-barrel folding. Thus, substitutions in the outer membrane-embedded BamA β-barrel domain overcome OMP biogenesis defects that occur at the POTRA domain of BamA in the periplasm. Based on the structure of FhaC, the altered BamA residues are predicted to lie on a highly conserved loop that folds inside the β-barrel and in regions pointing outside the β-barrel, suggesting that they influence BamA function by both direct and indirect mechanisms.     

A practical Δ-dehydrogenation of Δ-3-keto-steroids with DDQ in the presence of TBDMSCl at room temperature

A mild and efficient Δ¹-dehydrogenation of Δ⁴-3-keto-steroids with DDQ in the presence of tertbutyldimethylchlorosilane at room temperature was developed.     

Identification of the substrate recognition region in the Δ6-fatty acid and Δ8-sphingolipid desaturase by fusion mutagenesis

Δ⁸-sphingolipid desaturase and Δ⁶-fatty acid desaturase share high protein sequence identity. Thus, it has been hypothesized that Δ⁶-fatty acid desaturase is derived from Δ⁸-sphingolipid desaturase; however, there is no direct proof. The substrate recognition regions of Δ⁶-fatty acid desaturase and Δ⁸-sphingolipid desaturase, which aid in understanding the evolution of these two enzymes, have not been reported. A blackcurrant Δ⁶-fatty acid desaturase and a Δ⁸-sphingolipid desaturase gene, RnD6C and RnD8A, respectively, share more than 80 % identity in their coding protein sequences. In this study, a set of fusion genes of RnD6C and RnD8A were constructed and expressed in yeast. The Δ⁶- and Δ⁸-desaturase activities of the fusion proteins were characterized. Our results indicated that (1) the exchange of the C-terminal 172 amino acid residues can lead to a significant decrease in both desaturase activities; (2) amino acid residues 114–174, 206–257, and 258–276 played important roles in Δ⁶-substrate recognition, and the last two regions were crucial for Δ⁸-substrate recognition; and (3) amino acid residues 114–276 of Δ⁶-fatty acid desaturase contained the substrate recognition site(s) responsible for discrimination between ceramide (a substrate of Δ⁸-sphingolipid desaturase) and acyl-PC (a substrate of Δ⁶-fatty acid desaturase). Substituting the amino acid residues 114-276 of RnD8A with those of RnD6C resulted in a gain of Δ⁶-desaturase activity in the fusion protein but a loss in Δ⁸-sphingolipid desaturase activity. In conclusion, several regions important for the substrate recognition of Δ8-sphingolipid desaturase and Δ⁶-fatty acid desaturase were identified, which provide clues in understanding the relationship between the structure and function in desaturases.     

Validation of the determination of ΔF508 mutations of the cystic fibrosis gene in over 11000 mouthwashes

Mouthwashes can be used as a DNA resource for mutation detection and, because collection and DNA isolation is simple and cheap, they could in particular, be used for large numbers of samples. To determine the failure rate (the proportion of mouth samples in which no PCR product was obtained) and the specificity of buccal epithelial cell mutation detection in large numbers of samples, we collected mouthwashes and blood samples from 11413 blood donors and tested the mouthwashes for the F508 mutation, which has an estimated frequency of 75% among cystic fibrosis chromosomes in The Netherlands. Blood samples were tested for the F508 mutations only if the mutation was identified in the mouthwash or in the case of a failure to obtain PCR products. The sensitivity of the test was determined in mouthwashes of 75 F508 carriers known from earlier family studies. These samples were offered blindly between the mouthwashes of the blood donors. Both specificity and sensitivity of the mouthwash procedure were 100%. The overall failure rate was 5.6%. This large figure was caused mainly by insufficient rinsing of the mouth in one particular blood bank. Exclusion of the results of this blood bank reduced the failure rate to 1.8%. Our results also confirm that for a large number of samples the mouthwash procedure is suitable for mutation detection and, with proper instructions, can be used in community screening.     

Contribution of ΔpH and ΔE to Photosynthesis of Chlamydomonas Reinhardtii

The contribution of two components (pH and E) of the proton motive force to photosynthesis of C. reinhardtii was studied. Valinomycin, a photophosphorylation uncoupler, decreased significantly the fast phase (related mainly to the membrane electric potential) of millisecond delayed light emission (ms-DLE) of C. reinhardtii. Nigericin, another photophosphorylation uncoupler, decreased the slow phase (related mainly to the proton gradient) and partly also the fast phase of ms-DLE. Both valinomycin and nigericin decreased the net ATP content and photosynthetic rate of C. reinhardtii, but the inhibition by nigericin was stronger than that by valinomycin. Hence both components of the proton motive force contribute to photosynthesis and although the contribution of pH is larger than that of E, the latter is not negligible in photosynthesis of C. reinhardtii.     

Frequency of the cystic fibrosis ΔF508 mutation in a large sample of the French population

Summary We have determined the frequency of the cystic fibrosis (CF) ΔF508 mutation in a large sample of CF patients originating from different areas of France, including the greater Paris, Brittany, Alsace, Lorraine and Rh?ne-Alpes regions. A total of 422 CF chromosomes were studied, and the defect was found to account for 75% of the mutant alleles. In the course of the survey, a rare nucleotide sequence polymorphism leading to an isoleucine to valine substitution at position 506 of the CF transmembrane conductance regulator protein has been characterized in an unaffected individual. Our data enable the evaluation of the probabilities that a chromosome negative for the ΔF508 mutation carriers another CF defect.     

Effects of the ΔF508 mutation on the structure,function, and folding of the first nucleotide-binding domain of CFTR

The fatal autosomal recessive disease cystic fibrosis (CF) is caused by mutations in the gene which encodes the cystic fibrosis transmembrane conductance regulator (CFTR). Many of these disease-causing mutations, including the deletion of F508 (F508) which accounts for approximately 70% of the disease alleles, occur in one of the two consensus nucleotide binding sequences. Peptide studies have directly demonstrated that the N-terminal nucleotide binding sequences bind adenine nucleotides. Structurally, circular dichroism spectropolarimetry indicates that this region of CFTR assumes a -stranded structure in solution. The F508 mutation causes a diminution in the amount of -stranded structure and a concomitant increase in the amount of random coil structure present, indicating that either the mutant peptide has a different native structure or that the conformational equilibrium is shifted toward a more disordered form. Furthermore, the mutant peptide is more sensitive to denaturation, indicating that F508 is a stability, or protein-folding mutant. Here we review these results and discuss their implications for interpreting the behavior of F508in situ and for the rational design of new CF drugs.     

ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability.     

Reversal of the ΔdegP phenotypes by a novel rpoE allele of Escherichia coli

RseA sequesters RpoE (σ(E)) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σ(E) to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σ(E) regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σ(E) levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σ(E)-dependent RybB::LacZ construct showed only a weak activation of the σ(E) pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σ(E) and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrA(L222Q), it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σ(E)-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σ(E)-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σ(E) may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σ(E) levels.     

Occurrence of Δ5-sterols in plants producing predominantly Δ7-sterols: Studies on the sterol compositions of six cucurbitaceae seeds

The sterol compositions of six Cucurbitaceae seeds, Cucurbita pepo, C. lagenaria, Citrullus lanatus, Cucumis sativus, C. melo and Luffa aegyptiaca, were examined by TLC, GLC, HPLC and m some cases by mass spectrometry and ¹HNMR spectroscopy. Thirteen components were identified. They were codisterol, 25(27)-dehydroporiferasterol, clerosterol, isofucosterol, stigmasterol, campesterol, 22-dihydrobrassicasterol, sitosterol, 25(27)-dehydrofungisterol, 25(27)-dehydrochondrillasterol, 24β-ethyl-25(27)-dehydrolathosterol, avenasterol, spinasterol, 24ξ-methyllathosterol and 22-dihydrospinasterol. 24-Methylenecholesterol may also have been present. The Δ⁵-sterols were observed in all species. The pattern of sterols suggests the existence of four biosynthetic pathways operating from a 24(25)-dehydroprecursor. This work represents only the second time either codisterol or 25(27)-dehydrofungisterol has been isolated from a higher plant.     

Isolation of the ERG2 Gene,Encoding Δ8 → Δ7 Sterol Isomerase,from the Maize Smut Pathogen Ustilago maydis

Bailey, A. M., Burden, R. S., James, C. S., Keon, J. P. R., Croxen, R., Bard, M., and Hargreaves, J. A. 1993. Isolation of the ERG2 gene, encoding Δ⁸ → Δ⁷ sterol isomerase, from the maise smut pathogen Ustilago maydis. Experimental Mycology 18, 87-92. The ERG2 gene encoding Δ⁸ → Δ⁷ sterol isomerase has been isolated from the fungal plant pathogen Ustilago maydis. This was accomplished by screening an U. maydis genomic library with a fragment of the Saccharomyces cerevisiae ERG2 gene. The identity of the U. maydis ERG2 gene was confirmed by complementation of an U. maydis Erg2 mutant and by comparing the deduced amino acid sequence encoded by the U. maydis ERG2 gene with that of the S. cerevisiae ERG2 gene product.