cis-Prenyltransferase AtCPT6 produces a family of very short-chain polyisoprenoids in planta

cis-Prenyltransferases (CPTs) comprise numerous enzymes synthesizing isoprenoid hydrocarbon skeleton with isoprenoid units in the cis (Z) configuration. The chain-length specificity of a particular plant CPT is in most cases unknown despite the composition of the accumulated isoprenoids in the tissue of interest being well established. In this report AtCPT6, one of the nine Arabidopsis thaliana CPTs, is shown to catalyze the synthesis of a family of very short-chain polyisoprenoid alcohols of six, seven, and eight isoprenoid units, those of seven units dominating. The product specificity of AtCPT6 was established in vivo following its expression in the heterologous system of the yeast Saccharomyces cerevisiae and was confirmed by the absence of specific products in AtCPT6 T-DNA insertion mutants and their overaccumulation in AtCPT6-overexpressing plants. These observations are additionally validated in silico using an AtCPT6 model obtained by homology modeling. AtCPT6 only partially complements the function of the yeast homologue of CPT-Rer2 since it restores the growth but not protein glycosylation in rer2Δ yeast. This is the first in planta characterization of specific products of a plant CPT producing polyisoprenoids. Their distribution suggests that a joint activity of several CPTs is required to produce the complex mixture of polyisoprenoid alcohols found in Arabidopsis roots.     

Association analysis of vitamin D receptor gene polymorphisms with bone mineral density in young women with Graves' disease

Graves' (GD) hyperthyroidism induces accelerated bone turnover that leads to decreased bone mineral density (BMD). The role of the VDR gene in predisposition to primary osteoporosis has been recognized. Recent studies show associations between the VDR gene polymorphisms and susceptibility to autoimmune diseases. Here we analyzed if VDR gene polymorphisms: BsmI, ApaI, TaqI, and FokI may predispose women with Graves' hyperthyroidism to BMD reduction or to disease development. The subjects were 75 premenopausal female Polish patients with GD and 163 healthy women. The genotyping was performed by the use of the restriction fragment length polymorphism analysis (RFLP). We studied the association of the VDR polymorphisms and their haplotypes with patients' BMD and also SNPs and haplotypes association with Graves' disease. We found a strong linkage disequilibrium for the BsmI, ApaI, and TaqI polymorphims that formed three most frequent haplotypes in Graves' women: baT (47.9%), BAt (34.9%), and bAT (16.4%). We did not show statistically significant association of analyzed VDR polymorphisms or haplotypes with decreased bone mineral density in Graves' patients. However, the presence of F allele had a weak tendency to be associated with Graves' disease (with OR=1.93; 95% CI: 0.97-3.84; p=0.058). In conclusion: VDR gene polymorphisms do not predict the risk of decreased BMD in Polish women with Graves'. It may be speculated that the F allele carriers of the VDR-FokI polymorphism are predisposed to Graves' disease development.     

Differential effects of selenium compounds on glucose synthesis in rabbit kidney-cortex tubules and hepatocytes. In vitro and in vivo studies

Although selenium is taken with diet mainly as selenoamino acids, its hypoglycaemic action on hepatic gluconeogenesis has been studied with the use of inorganic selenium derivatives. The aim of the present investigation was to compare relative efficacies of inorganic and organic selenium compounds in reducing glucose synthesis in hepatocytes and renal tubules, significantly contributing to the glucose homeostasis. In contrast to hepatocytes, both selenite and methylselenocysteine inhibited renal gluconeogenesis by about 40-45% in control rabbits. Selenate did not affect this process, whereas selenomethionine inhibited gluconeogenesis by about 20% in both hepatocytes and renal tubules. In contrast to methylselenocysteine, selenite decreased intracellular ATP content, glutathione reduced/glutathione oxidized (GSH/GSSG) ratio and pyruvate carboxylase, PEPCK and FBPase activities, while methylselenocysteine diminished PEPCK activity due to elevation of intracellular 2-oxoglutarate and GSSG, inhibitors of this enzyme. Experiments in vivo indicate that in 3 of 9 alloxan-diabetic rabbits treated for 14 days with methylselenocysteine (0.182mg/kg body weight) blood glucose level was normalized, whereas in all diabetic rabbits plasma creatinine and urea levels decreased from 2.52+/-0.18 and 87.4+/-9.7 down to 1.63+/-0.11 and 39.0+/-2.8, respectively. In view of these data selenium supplementation might be beneficial for protection against diabetes-induced nephrotoxicity despite selenium accumulation in kidneys and liver.     

Leishmania mexicana: participation of NF-kappaB in the differential production of IL-12 in dendritic cells and monocytes induced by lipophosphoglycan (LPG)

Dendritic cells (DC) and macrophages (Mφ) are well known as important effectors of the innate immune system and their ability to produce IL-12 indicates that they possess the potential of directing acquired immunity toward a Th1-biased response. Interestingly, the intracellular parasite Leishmania has been shown to selectively suppress Mφ IL-12 production and are DC the principal source of this cytokine. The molecular details of this phenomenon remain enigmatic. In the present study we examined the effect of Leishmania mexicana lipophosphoglycan (LPG) on the production of IL-12, TNF-α, and IL-10 and nuclear translocation of NF-κB. The results show that LPG induced more IL-12 in human DC than in monocytes. This difference was due in part to nuclear translocation of NF-κB, since LPG induced more translocation in DC than in monocytes. These results suggest that Leishmania LPG impairs nuclear translocation of NF-κB in monocytes with the subsequent decrease in IL-12 production.     

Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli

An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective s subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon. The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene. These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant. We have used this finding as a basis for selection of new plasmid umuC mutants. The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail. In the umuC122:: Tn 5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC ⁺ plasmid. Interestingly, while the frequency of UV-induced GC → AT transitions is greatly reduced, the frequency of AT → TA transversions is not affected. Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC ⁺; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A. We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame. We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.     

Can membrane-bound carotenoid pigment zeaxanthin carry out a transmembrane proton transfer?

Polar carotenoid pigment zeaxanthin (β,β-carotene-3,3′-diol) incorporated into planar lipid membranes formed with diphytanoyl phosphatidylcholine increases the specific electric resistance of the membrane from ca. 4 to 13 × 10⁷ Ω cm² (at 5 mol% zeaxanthin with respect to lipid). Such an observation is consistent with the well known effect of polar carotenoids in decreasing fluidity and structural stabilization of lipid bilayers. Zeaxanthin incorporated into the lipid membrane at 1 mol% has very small effect on the overall membrane resistance but facilitates equilibration of the transmembrane proton gradient, as demonstrated with the application of the H⁺-sensitive antimony electrodes. Relatively low changes in the electrical potential suggest that the equilibration process may be associated with a symport/antiport activity or with a transmembrane transfer of the molecules of acid. UV-Vis linear dichroism analysis of multibilayer formed with the same lipid-carotenoid system shows that the transition dipole moment of the pigment molecules forms a mean angle of 21° with respect to the axis normal to the plane of the membrane. This means that zeaxanthin spans the membrane and tends to have its two hydroxyl groups anchored in the opposite polar zones of the membrane. Detailed FTIR analysis of β-carotene and zeaxanthin indicates that the polyene chain of carotenoids is able to form weak hydrogen bonds with water molecules. Possible molecular mechanisms responsible for proton transport by polyenes are discussed, including direct involvement of the polyene chain in proton transfer and indirect effect of the pigment on physical properties of the membrane.     

Phosphatidylinositol 4-kinasebeta is critical for functional association of rab11 with the Golgi complex

Phosphatidylinositol 4-kinasebeta (PI4Kbeta) plays an essential role in maintaining the structural integrity of the Golgi complex. In a search for PI4Kbeta-interacting proteins, we found that PI4Kbeta specifically interacts with the GTP-bound form of the small GTPase rab11. The PI4Kbeta-rab11 interaction is of functional significance because inhibition of rab11 binding to PI4Kbeta abolished the localization of rab11 to the Golgi complex and significantly inhibited transport of vesicular stomatitis virus G protein from the Golgi complex to the plasma membrane. We propose that a novel function of PI4Kbeta is to act as a docking protein for rab11 in the Golgi complex, which is important for biosynthetic membrane transport from the Golgi complex to the plasma membrane.     

Is coarse woody debris in lakes a refuge or a trap for benthic invertebrates exposed to fish predation?

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Assignment of the absolute configuration of P-chiral 5' mRNA cap analogues containing phosphorothioate moiety

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