Effect of L-triiodothyronine on liver microsomal Δ6 and Δ5 desaturase activity of male rats

The effect of different doses of L-triiodothyronine (T₃) on the activity of 6 and 5 desaturases and lipid fatty acid composition was studied in liver microsomes of male rats. The activity of 6 and 5 desaturases was decreased 24 and 28%, respectively, in animals administered a daily intraperitoneal dose of 1000g T₃/100g body wt. for 5 days, whereas with 500g T₃/100g body wt. only 6 desaturase activity was decreased. On the other hand, no enzyme activity changed at a shorter period of hormone treatment. Changes in microsomal fatty acid composition did not seem to be a direct consequence of desaturation activity, since after 1 and 5 days of T₃ treatment, the concentrations of 18:2 (n-6) and 20:3 (n-6) decreased and only after 1 day that of 20:4 (n-6) increased in spite of unchanged or decreased 6 and 5 desaturase activities. Other factors than desaturation activity must be involved in fatty acid composition of thyroid hormonetreated rats, such as diet, membrane lipid synthesis and degradation, fatty acid turn-over and oxidation. (Mol Cell Biochem121: 149–153, 1993)     

Preformulation Studies of a Prodrug of Δ9-Tetrahydrocannabinol

Preformulation studies were performed on a hemiglutarate ester prodrug of Δ⁹-tetrahydrocannabinol (THC-HG), to facilitate the development of stable formulations by hot-melt methods. The various studies performed included solid-state thermal characterization, pKa, logP, aqueous and pH dependent solubility, pH stability and effect of moisture, temperature and oxygen on solid-state stability. A hot-melt method was utilized to fabricate THC-HG incorporated poly (ethylene oxide) (PEO) matrices and the bioadhesive properties, release profiles and post-processing stability of these matrices were assessed as a function of the polymer molecular weight. The prodrug exhibited a T _g close to 0°C, indicating its amorphous nature. Thermogravimetric analysis revealed a rapid weight loss after 170°C. The prodrug exhibited a seven-fold higher aqueous solubility as compared to the parent drug (THC). Also, the solubility of the compound increased with increasing pH, being maximum at pH 8. The prodrug exhibited a v-shaped pH-rate profile, with the degradation rate minimum between pH 3 and 4. The moisture uptake and drug degradation increased with an increase in relative humidity. Solid-state stability indicated that the prodrug was stable at −18°C but demonstrated higher degradation at 4°C, 25°C and 40°C (51.6%, 74.5% and 90.1%, respectively) at the end of 3-months. THC-HG was found to be sensitive to the presence of oxygen. The release of the active from the polymeric matrices decreased, while bioadhesion increased, with an increase in molecular weight of PEO.     

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.     

Antibacterial activity of Δ9-tetrahydrocannabinol and cannabidiol

The minimum inhibiting concentrations (MIC) of ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD) for staphylococci and streptococci in broth are in the range of 1–5 g/ml. In the same range, both compounds are also bactericidal. In media containing 4% serum or 5% blood the antibacterial activity is strongly reduced (MIC 50g/ml). Gram-negative bacteria are resistant to THC and CBD.     

Diversification of substrate specificities in teleostei Fads2: characterization of Δ4 and Δ6Δ5 desaturases of Chirostoma estor

Currently existing data show that the capability for long-chain PUFA (LC-PUFA) biosynthesis in teleost fish is more diverse than in other vertebrates. Such diversity has been primarily linked to the subfunctionalization that teleostei fatty acyl desaturase (Fads)2 desaturases have undergone during evolution. We previously showed that Chirostoma estor, one of the few representatives of freshwater atherinopsids, had the ability for LC-PUFA biosynthesis from C₁₈ PUFA precursors, in agreement with this species having unusually high contents of DHA. The particular ancestry and pattern of LC-PUFA biosynthesis activity of C. estor make this species an excellent model for study to gain further insight into LC-PUFA biosynthetic abilities among teleosts. The present study aimed to characterize cDNA sequences encoding fatty acyl elongases and desaturases, key genes involved in the LC-PUFA biosynthesis. Results show that C. estor expresses an elongase of very long-chain FA (Elovl)5 elongase and two Fads2 desaturases displaying Δ4 and Δ6/Δ5 specificities, thus allowing us to conclude that these three genes cover all the enzymatic abilities required for LC-PUFA biosynthesis from C₁₈ PUFA. In addition, the specificities of the C. estor Fads2 enabled us to propose potential evolutionary patterns and mechanisms for subfunctionalization of Fads2 among fish lineages.     

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     

The dimerization of Δ1-piperidine-2-carboxylic acid

The l-amino acid oxidase of Mytilus edulis has been used to oxidize l-lysine on a large scale in the presence of catalase. The alpha-oxo acid derived from lysine cyclizes to a Schiff base, which readily dimerizes. The dimer undergoes spontaneous dehydration and decarboxylation to form 1,2,3,4,5,6,7,8-octahydropyrido[3,2-a]-indolizin-10(4bH)-one. This structure was established by a study of its molecular weight and infrared, nuclear-magnetic-resonance and mass spectra.     

The synthesis of Δ2-prostaglandins

The effect of prostaglandin F_2α on ovulation and fertilization was studied in rabbits. The number of ovulation was not affected by subcutaneous injection of PGF_2α but the recovery of ova was significantly decreased when PGF_2α was given either at 12 or 16 h after HCG injection and autopsied 24 h latter. The results suggest that exogenous PGF_2α accelerates ovum transport and expels the eggs prematurely from the female tract and does not impair ovulation or the fertilization processes when given to rabbit at 1 mg/kg B.W.     

Production of 5,8,11-eicosatrienoic acid by a Δ5 and Δ6 desaturation activity-enhanced mutant derived from a Δ12 desaturation activity-defective mutant of Mortierella alpina 1S-4

Enhanced production of 5,8,11-eicosatrienoic acid (Mead acid, 20:3omega9) was attained with a mutant fungus, Mortierella alpina JT-180, derived from delta12 desaturation activity-defective and delta6 desaturation activity-enhanced M. alpina M209-7. Production of 20:3omega9 by JT-180 was 1.4 times greater than that of the parent strain M209-7. This is thought to be due to its enhanced Delta5 desaturation activity, which was 3.3 times higher than that of M209-7. In both strains, 78.5-80.4% of the total lipids comprised triacylglycerol (TG), and 76.6-79.0% of 20:3omega9 was present in TG. Comparing the fatty acid compositions among various lipid species, the highest percentages (24.1-37.6%) of 20:3omega9 in total lipids were found in phosphatidylcholine. For optimization of 20:3omega9 production by JT-180, a glucose concentration of 4% in the culture medium and shifting of the growth temperature from 28 degrees C to 20 degrees C on the 2nd day were shown to be effective. Under optimal conditions, 20:3omega9 production by JT-180 reached 1.92 g/l culture medium in a 10-l jar fermentor (corresponding to 81.5 mg/g dry mycelia and 18.3% of total fatty acids), which is greater than that reported previously from M209-7 (1.65 g/l).     

Recovery of ΔF508-CFTR function by analogs of hyaluronan disaccharide

We recently discovered that hyaluronan was exported from fibroblasts by MRP5 and from epithelial cells by cystic fibrosis (CF) transmembrane conductance regulator (CFTR) that was known as a chloride channel. On this basis we developed membrane permeable analogs of hyaluronan disaccharide as new class of compounds to modify their efflux. We found substances that activated hyaluronan export from human breast cancer cells. The most active compound 2-(2-acetamido-3,5-dihydroxyphenoxy)-5-aminobenzoic acid (Hylout4) was tested for its influence on the activity of epithelial cells. It activated the ion efflux by normal and defective ΔF508-CFTR. It also enhanced the plasma membrane concentration of the ΔF508-CFTR protein and reduced the transepithelial resistance of epithelial cells. In human trials of healthy persons, it caused an opening of CFTR in the nasal epithelium. Thus compound Hylout4 is a corrector that recovered ΔF508-CFTR from intracellular degradation and activated its export function.     

Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Δptp2 Δmsg5 protein phosphatase double disruptant

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Ca^s). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1–S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Δbck1, Δmkk1, Δslt2/Δmpk1, Δmck1, Δssk2 and Δyak1) suppressed the Ca^s-phenotype of the Δptp2 Δmsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Δssk2 and Δyak1 but not Δbck1, Δmkk1, Δslt2 and Δmck1 was able to suppress the delayed G1–S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1–S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Δptp2 Δmsg5 double disruptant.     

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.     

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.     

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.     

Immunocytochemical localization of Δ3, Δ2-enoyl-CoA isomerase and NADPH-dependent-2,4-dienoyl-CoA reductase in rat kidney

Localization of ³, ²-enoyl-CoA isomerase (ECI) and NADPH-dependent-2,4-dienoyl-CoA reductase (DCR) in the rat kidney was investigated by immunocytochemical techniques. The kidneys were perfusion-fixed and embedded in Epon or LR White. For light microscopy, semi-thin sections of Epon-embedded materials were stained by the immunoenzyme technique after the epoxy resin was removed by treatment with sodium ethoxide. For electron microscopy, ultra-thin sections of LR White-embedded materials were stained by the protein A-gold technique. By light microscopy, the S1 segment of the proximal tubule was most heavily stained for ECI and DCR whilst S2 and S3 segments showed intermediate staining. A weak staining reaction was observed in the distal tubule and the medullary collecting tubule. In the cortical collecting tubule, heavily stained cells were present between weakly stained cells. By electron microscopy, gold particles showing the antigenic sites for ECI were confined mainly to the mitochondria, but few particles were observed in the peroxisomes. Gold labeling for DCR was localized both in the mitochondria and the peroxisomes. The labeling intensity of the peroxisomes was much higher than that of the mitochondria. The results suggest that metabolism of unsaturated fatty acids occurs mainly in the mitochondria and the peroxisomes of the proximal tubule in the kidney.     

Role of microsomal steroid hydroxylases in Δ7-steroid biosynthesis

CYP17 (steroid 17α-hydroxylase/17,20-lyase) is a key enzyme in steroid hormone biosynthesis. It catalyzes two independent reactions at the same active center and has a unique ability to differentiate Δ⁴-steroids and Δ⁵-steroids in the 17,20-lyase reaction. The present work presents a complex experimental analysis of the role of CYP17 in the metabolism of 7-dehydrosteroids. The data indicate the existence of a possible alternative pathway of steroid hormone biosynthesis using 7-dehydrosteroids. The major reaction products of CYP17 catalyzed hydroxylation of 7-dehydropregnenolone have been identified. Catalytic activity of CYP17 from different species with 7-dehydropregnenolone has been estimated. It is shown that CYP21 cannot use Δ⁵–Δ⁷ steroids as a substrate.