Natural competence in strains of Actinobacillus pleuropneumoniae

The fatty acid (FA) profiles of myxobacteria contain FA species with double bonds at the Δ⁵ and Δ¹¹ positions, the latter being rather unusual among bacteria. Despite this knowledge, the mechanism for introduction of these double bonds has never been described before in myxobacteria. Searches for candidate genes in the genome of the model organism Myxococcus xanthus revealed 16 genes, which have been annotated as FA desaturases. However, due to redundant substrate specificity, functional analyses of these enzymes by construction of inactivation mutants did not lead to the identification of their function or substrate specificity. Therefore, we elucidated the regioselectivity of the desaturation reactions by heterologous expression of eight desaturases from M. xanthus in Pseudomonas putida and thus could prove five of them to be indeed active as desaturases, with three (MXAN_1742, MXAN_3495 and MXAN_5461) and two (MXAN_0317 and MXAN_6306) acting as Δ⁵ and Δ¹¹ desaturases, respectively. This is the first report about the heterologous expression and regioselectivity of FA desaturases in myxobacteria.     

NIDA522131, a new radioligand for imaging extrathalamic nicotinic acetylcholine receptors: in vitro and in vivo evaluation

A novel radioligand, 6-chloro-3-((2-( S )-azetidinyl)methoxy)-5-(2-fluoropyridin-4-yl)pyridine (NIDA522131), for imaging extrathalamic nicotinic acetylcholine receptors (nAChRs) was characterized in vitro and in vivo using positron emission tomography. The K_d and T_1/2 of dissociation of NIDA522131 binding measured at 37°C in vitro were 4.9 ± 0.4 pmol/L and 81 ± 5 min, respectively. The patterns of radioactivity distribution in monkey brain in vivo was similar to that of 2-[¹⁸F]fluoro-3-(2( S )-azetidinylmethoxy)pyridine (2FA), a radioligand that has been successfully used in humans, and matched the α₄β₂* nAChRs distribution. Comparison between [¹⁸F]NIDA522131 and 2FA demonstrated better in vivo binding properties of the new radioligand and substantially greater radioactivity accumulation in brain. Consistent with [¹⁸F]NIDA522131 elevated affinity for nAChRs and its increased lipophilicity, both, the total and non-displaceable distribution volumes were substantially higher than those of 2FA. Estimated binding potential values in different brain regions, characterizing the specificity of receptor binding, were 3–4 fold higher for [¹⁸F]NIDA522131 than those of 2FA. Pharmacological evaluation in mice demonstrated a toxicity that was comparable to 2FA and is in agreement with a 2300 fold higher affinity at α₄β₂* versus α₃β₄* nAChRs. These results suggest that [¹⁸F]NIDA522131 is a promising positron emission tomography radioligand for studying extrathalamic nAChR in humans.     

The Effect of Temperature on the Fatty Acid Composition of Tetrahymena pyriformis WH-14

SYNOPSIS. A reduction in the growth temperature of Tetrahymena pyriformis strain WH-14 from 35 C to 15 C resulted in distinct alterations in the fatty acid composition of the glycerophospholipids. The proportion of normal saturated acids declined from 26 to 19%; palmitoleic acid increased by 6%, and the composition of the polyunsaturated fatty acids increased in 18:2 Δ^6,11(n) and decreased in 18:2 Δ^9,12(n) and 18:3 Δ^6,9,12(n). The unsaturation index (the average number of double bonds/100 molecules) did not change with a shift in temperature.
Two biosynthetic pathways exist in Tetrahymena for the formation of unsaturated fatty acids. The observed changes in fatty acid composition that accompany a lowering of the environmental temperature can be accounted for by a reduction in the accumulation of products of the fatty acid pathway leading to the formation of γ-linolenic acid [16:0(n) → 18:0(n) → 18:1 Δ⁹(n) → 18:2 Δ^9,12(n) → 18:3 Δ^6,9,12(n)] and an increase in the components of the pathway leading to the formation of 18:2 Δ^6,11(n) [16:0(n) → 16:1 Δ⁹(n) → 18:1 Δ¹¹(n) → 18:2 Δ^6,11(n)]. The data suggest that the regulatory mechanism in Tetrahymena differs from that found in some bacteria where a simple substitution of unsaturated fatty acids for saturated fatty acids occurs at low culture temperatures.     

Evidence for the synthesis of the multi-positional isomers of monounsaturated fatty acid in Methylococcus capsusatus by the anaerobic pathway

Abstract The biosynthesis of the positional isomers of the monounsaturated fatty acids of Methylococcus capsulatus (Bath) has been investigated by studying the incorporation of [2-¹⁴C]malonyl CoA into long-chain fatty acids in vitro. The major unsaturated products were Δ ⁹ 16:1 and Δ ¹¹ 18:1; however, Δ ⁸, Δ ¹⁰ and Δ ¹¹ 16:1, as well as, Δ ¹⁰, Δ ¹² and Δ ¹³ 18:1 were also synthesized. The exclusion of O₂ from the reaction vessel did not affect the synthesis of unsaturated fatty acids or the double bonds positions. Cerulenin inhibited the synthesis of unsaturated fatty acid more than saturated fatty acid. The use of both [1-¹⁴C] octanoate and [1-¹⁴C] decanoate as substrate resulted in the synthesis of long-chain fatty acids, however, unsaturates were only synthesized from octanoate. These results imply that the unique positional isomers of M. capsulatus are not synthesized by an aerobic mechanism.     

Phosphatidylcholine molecular species involved in Γ-linolenic acid biosynthesis in microsomes from borage seeds

Boraginaceae seeds are particularly rich in Γ -linolenic acid (6,9,12-octadecatrienoic acid, Γ -18:3). In microsomes, the analysis of phosphatidylcholine (PC) molecular species by HPLC led to identification of 15 different molecular species; among them 4 contained Γ -18:3, mostly at position 2 of sn -glycerol. Time courses of acylation and desaturation in PC molecular species were examined when [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA was provided as substrates to isolated microsomes. With [¹⁴C]oleoyl-CoA or [¹⁴C]linoleoyl-CoA and in the absence of NADH, 3 main labelled PC molecular species were found: 18:2/[¹⁴C]18:1, 16:0/[¹⁴C]18:1 and 18:1/[¹⁴C]18:1. When NADH was present in the incubation medium, the fatty acids were progressively desaturated by the Δ12- and Δ6-desaturases successively (with [¹⁴C]oleoyl-CoA as precursor) or by the Δ6-desaturase alone (with [¹⁴C]linoleoyl-CoA as precursor). In both types of experiments, 7 final desaturation products in microsomes were evidenced; among them, 3 contained radioactive Γ -18:3, i.e . 18:2/[¹⁴C] Γ -18:3, 18:1/[¹⁴C] Γ -18:3 and 16:0/[¹⁴C] Γ -18:3. While the Δ12-desaturase had no specificity for position on the glycerol backbone, labelled Γ -linolenic acid was recovered exclusively in the sn -2 position.     

Pressure-stress effects on the ultrastructure of cells of the dimorphic yeast Candida tropicalis

Abstract Starved cells of cadmium-sensitive Staphylococcus aureus 17810S accumulated ¹⁰⁹Cd via the Mn²⁺ porter energized by the membrane potential (ΔΨ) generated by l-lactate oxidation. However, Cd²⁺ accumulation did not result in inhibition of respiration and consequent generation of electrochemical proton gradient (Δμ_H+) via the respiratory chain. Thus, Δμ_H+-consuming processes, such as ATP synthesis and [¹⁴C]glutamate transport proceeded normally, despite the presence of Cd²⁺ in the cytoplasm. The mechanism of the intrinsic cadmium-insensitivity of the l-lactate oxidizing system is discussed.     

Vaccination with SIVmac239Δnef activates CD4+ T cells in the absence of CD4+ T-cell loss

Background  Pathogenic HIV and SIV infections characteristically deplete central memory CD4⁺ T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4⁺ T-cell play roles in protection against wild-type virus challenge.
Methods  Rhesus macaques were vaccinated with SIVmac239Δnef and quantitative and phenotypic polychromatic flow cytometry analyses were performed on mononuclear cells from blood, lymph nodes and rectal biopsies.
Results  Animals vaccinated with SIVmac239Δnef demonstrated no loss of CD4⁺ T cells in any tissue, and in fact CCR5⁺ and CD28⁺CD95⁺ central memory CD4⁺ T cells were significantly increased. In contrast, CD4⁺ T-cell numbers and CCR5 expression significantly declined in unvaccinated controls challenged with SIVmac239. Also, intracellular Ki67 increased acutely as much as 3-fold over baseline in all tissues after SIVmac239Δnef vaccination then declined following primary infection.
Conclusion  We demonstrated in this study that SIVmac239Δnef vaccination did not deplete CD4⁺ T cells but transiently activated and expanded the memory cell population. However, increases in numbers and activation of memory CD4⁺ T cells did not appear to influence protective immunity.     

Novel role of Wag31 in protection of mycobacteria under oxidative stress

Wag31 of Mycobacterium tuberculosis belongs to the DivIVA family of proteins known to regulate cell morphology in Gram-positive bacteria. Here we demonstrate an unrecognized, novel role of Wag31 in oxidatively stressed mycobacteria. We report the cleavage of penicillin-binding protein 3 (PBP3) by the intramembrane metalloprotease Rv2869c (MSMEG_2579) in oxidatively stressed cells. Amino acids ¹⁰²A and ¹⁰³A of PBP3 are required for Rv2869c-mediated cleavage. Wag31_MTB, by virtue of its interaction with PBP3 through amino acid residues ⁴⁶NSD⁴⁸, protects it from oxidative stress-induced cleavage. PBP3 undergoes cleavage in Mycobacterium smegmatis (strain PM2) harbouring wag31 (Δ⁴⁶NSD⁴⁸) instead of the wild type, with concomitant reduction in ability to withstand oxidative stress. Overexpression of Wag31(Δ⁴⁶NSD⁴⁸) attenuates the survival of M. tuberculosis in macrophages with concomitant cleavage of PBP3, and renders the organism more susceptible towards hydrogen peroxide as well as drugs which generate reactive oxygen species, namely isoniazid and ofloxacin. We propose that targeting Wag31 could enhance the activity of mycobactericidal drugs which are known to generate reactive oxygen species.     

Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of σ32in vivo

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of σ³², an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades σ³² and has a central role in the control of the σ³² level. The ftsH null mutant was isolated, and establishment of the Δ ftsH mutant allowed us to investigate control mechanisms of the stability and the activity of σ³² separately in vivo . Loss of the FtsH function caused marked stabilization and consequent accumulation of σ³² (≈20-fold of the wild type), leading to the impaired downregulation of the level of σ³². Surprisingly, however, Δ ftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of σ³² when it is accumulated. Overproduction of DnaK/J reduces the activity of σ³² in Δ ftsH cells without any detectable changes in the level of σ³², indicating that the DnaK chaperone system is responsible for the activity control of σ³² in vivo . In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of σ³².     

A role for p53 in the regulation of lysosomal permeability by delta 9-tetrahydrocannabinol in rat cortical neurones: implications for neurodegeneration

The psychoactive ingredient of marijuana, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), can evoke apoptosis in cultured cortical neurones. Whilst the intracellular mechanisms responsible for this apoptotic pathway remain to be fully elucidated, we have recently identified a role for the CB₁ type of cannabinoid (CB) receptor and the tumour suppressor protein, p53. In the current study, we demonstrate the Δ⁹-THC promotes a significant increase in lysosomal permeability in a dose- and time-dependent manner. The increase in lysosomal permeability was blocked by the CB₁ receptor antagonist, AM251. Δ⁹-THC increased the localization of phospho-p53Ser15 at the lysosome and stimulated the release of the lysosomal cathepsin enzyme, cathepsin-D, into the cytosol. The p53 inhibitor, pifithrin-α and small interfering RNA-mediated knockdown of p53 prevented the Δ⁹-THC-mediated increase in lysosomal permeability. Furthermore, the Δ⁹-THC -mediated induction of apoptosis was abrogated by a cell-permeable cathepsin-D inhibitor (10 μM). Thus, the study demonstrates that Δ⁹-THC impacts on the lysosomal system, via p53, to evoke lysosomal instability as an early event in the apoptotic cascade. This provides evidence for a novel link between the CB₁ receptor and the lysosomal branch of the apoptotic pathway which is crucial in regulating neuronal viability following exposure to Δ⁹-THC.     

Neuropeptide Y inhibits [Ca2+]i changes in rat retinal neurons through NPY Y1, Y4, and Y5 receptors

Neuropeptide Y (NPY) and NPY receptors are widely distributed in the CNS, including the retina, but the role of NPY in the retina is largely unknown. The aim of this study was to investigate whether NPY modulates intracellular calcium concentration ([Ca²⁺]_i) changes in retinal neurons and identify the NPY receptors involved. As NPY decreased the [Ca²⁺]_i amplitudes evoked by 30 mM KCl in only 50% of neurons analyzed, we divided them in two populations: NPY-non-responsive neurons (Δ2/Δ1 ≥ 0.80) and NPY-responsive neurons (Δ2/Δ1 < 0.80), being the Δ2/Δ1 the ratio between the amplitude of [Ca²⁺]_i increase evoked by the second (Δ2) and the first (Δ1) stimuli of KCl. The NPY Y₁/Y₅, Y₄, and Y₅ receptor agonists (100 nM), but not the Y₂ receptor agonist (300 nM), inhibited the [Ca²⁺]_i increase induced by KCl. In addition, the inhibitory effect of NPY on evoked-[Ca²⁺]_i changes was reduced in the presence of the Y₁ or the Y₅ receptor antagonists. In conclusion, NPY inhibits KCl-evoked [Ca²⁺]_i increase in retinal neurons through the activation of NPY Y₁, Y₄, and Y₅ receptors. This effect may be viewed as a potential neuroprotective mechanism of NPY against retinal neurodegeneration.     

Parentage testing in alpacas (Vicugna pacos) using semi-automated fluorescent multiplex PCRs with 10 microsatellite markers

The aim of this study was to assess and apply a microsatellite multiplex system for parentage determination in alpacas. An approach for parentage testing based on 10 microsatellites was evaluated in a population of 329 unrelated alpacas from different geographical zones in Perú. All microsatellite markers, which amplified in two multiplex reactions, were highly polymorphic with a mean of 14.5 alleles per locus (six to 28 alleles per locus) and an average expected heterozygosity ( H _E) of 0.8185 (range of 0.698–0.946). The total parentage exclusion probability was 0.999456 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.999991 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. In a case test of parentage assignment, the microsatellite panel assigned 38 (from 45 cases) offspring parentage to 10 sires with LOD scores ranging from 2.19 × 10⁺¹³ to 1.34 × 10⁺¹⁵ and Δ values ranging from 2.80 × 10⁺¹² to 1.34 × 10⁺¹⁵ with an estimated pedigree error rate of 15.5%. The performance of this multiplex panel of markers suggests that it will be useful in parentage testing of alpacas.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.     

Effects of supplemental UV-B radiation on growth and leaf photosynthetic reactions of soybean (Glycine max)

Experiments were conducted under greenhouse conditions to investigate the effects of enhanced UV-B radiation (280 to 320 nm) on height, fresh and dry weights, leaf chlorophyll and carotenoids, CO₂ uptake rates, and Hill activity in soybean ( Glycine max L. cv. Bragg). Plants were exposed for 6 h continuously from midmorning to midafternoon each day to UV-B radiation which was provided by Westinghouse FS-40 sun lamps filtered with 0.127-mm cellulose acetate film (UV-B enhanced) or 0.127-mm Mylar S film (UV-B Mylar control). Three different UV-B enhanced radiation levels were tested: 1.09 (treatment T₁), 1.36 (treatment T₂), and 1.83 (treatment T₃) UV-B sun equivalent units (UV-B_sec) where 1 UV-B_sec= 15.98 mW·m⁻² of solar UV-B obtained by applying EXP -[(α-265)/21]², a weighting function that simulates the DNA absorption spectrum, to the UV-B lamp systems. These UV-B levels correspond to a calculated decrease in stratospheric ozone content of 6%, 21%, and 36% for treatment T₁, T₂, and T₃, respectively.
Daily exposure of soybean plants to UV-B radiation significantly decreased height, fresh and dry weights, leaf chlorophyll and carotenoid contents, and CO₂ uptake rates. Leaf pigment extracted in 80% acetone from UV-B-treated soybean plants showed considerable increase in absorption in the wavelength region of 330 to 400 nm with increased UV-B radiation levels. Chloroplast preparations from leaves of T₂ and T₃ plants showed significant reductions in Hill reaction measurements.     

Endogenous cannabinoid anandamide inhibits nicotinic acetylcholine receptor function in mouse thalamic synaptosomes

The effects of the endogenous cannabinoid anandamide [arachidonylethanolamide (AEA)] on the function of nicotinic acetylcholine receptor (nAChR) were investigated using the ⁸⁶Rb⁺ efflux assay in thalamic synaptosomes. AEA reversibly inhibited ⁸⁶Rb⁺ efflux induced by 300 μM ACh with an IC₅₀ value of 0.9 ± 2 μM. Pre-treatment with the cannabinoid (CB₁) receptor antagonist SR141716A (1 μM), the CB₂ receptor antagonist SR144528 (1 μM), or pertussis toxin (0.2 mg/mL) did not alter the inhibitory effects of AEA, suggesting that known CB receptors are not involved in AEA inhibition of nAChRs. AEA inhibition of ⁸⁶Rb⁺ efflux was not reversed by increasing acetylcholine (ACh) concentrations. In radioligand binding studies, the specific binding of [³H]-nicotine was not altered in the presence of AEA, indicating that AEA inhibits the function of nAChR in a non-competitive manner. Neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor, indomethacin, (5 μM) affected AEA inhibition of nAChRs, suggesting that the effect of AEA is not mediated by its metabolic products. Importantly, the extent of AEA inhibition of ⁸⁶Rb⁺ efflux was significantly attenuated by the absence of 1% fatty acid free bovine serum albumin pre-treatment, supporting previous findings that fatty acid-like compounds modulate the activity of nAChRs. Collectively, the results indicate that AEA inhibits the function of nAChRs in thalamic synaptosomes via a CB-independent mechanism and that the background activity of these receptors is affected by fatty acids and AEA.     

Cloning, molecular characterization and heterologous expression of AMY1, an α-amylase gene from Cryptococcus flavus

A Cryptococcus flavus gene ( AMY1 ) encoding an extracellular α-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) ¹⁴⁴DVVVNH¹⁴⁹, (II) ²³⁵GLRIDSLQQ²⁴³, (III) ²⁶³GEVFN²⁶⁷, (IV) ³²⁷FLENQD³³², placed the enzyme in the GH13 α-amylase family. Furthermore, sequence comparison suggests that the C. flavus α-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae . The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL⁻¹) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme ( c . 67 kDa), part of which was due to N-glycosylation.     

Selective production of cis-9,trans-11 isomer of conjugated linoleic acid from trans-vaccenic acid methyl ester by Delacroixia coronata

Aims:  Bio-process development for isomer selective and efficient production of cis -9, trans -11-octadecadienoic acid (CLA) from trans -vaccenic acid ( t -VA, trans -11-octadecenoic acid) through microbial fatty acid Δ9-desaturation reaction.
Methods and Results:  A total of 550 strains of fungi and yeasts were screened for CLA production from t -VA through Δ9 desaturation. Delacroixia coronata IFO 8586 was selected as a potent producer of CLA from t -VA. Efficient CLA production was observed during cultivation in medium supplemented with the methyl ester of t -VA ( t -VAME). Under the optimal conditions with 33·3 mg ml⁻¹ of t -VAME as substrate, 10·5 mg ml⁻¹ CLA was produced by D. coronata IFO 8586 after 7 days of cultivation in the medium containing dextrin (5·0%), tryptone (2·0%) and thiourea (0·83 μmol ml⁻¹). The strain produced the cis -9, trans -11 isomer of CLA selectively (98% of total CLA), with a small amount of the trans -9, trans -11 isomer (2% of total CLA), mainly in the form of triacylglycerols (69% of total CLA).
Conclusions:  A practical bio-process for selective production of cis -9, trans -11 isomer of CLA using filamentous fungus D. coronata IFO 8586 was successfully established.
Significance and Impact of the Study:  Isomer selective bio-process for the practical production of cis -9, trans -11-CLA was first established. The process is benefitable for expanding the application of CLA for medicinal and nutraceutical purposes.