Effects of Dietary Form of Selenium on Its Distribution in Eggs

The objective of this experiment was to investigate the selenium distribution in eggs from hens fed diets supplemented with Se from sodium selenite (SS) or selenium-enriched yeast (SY). One-day-old female chickens of Hy-Line Brown breed were randomly divided into four groups according to dietary treatments and, for the subsequent 9?months, were fed diets which differed only in the form or amount of Se supplemented. During the whole experiment, group 1 (control) was fed basal diet (BD) with only background Se level of 0.13?mg/kg dry matter (DM). Diets for groups 2 and 3 consisted of BD supplemented with an Se dose of 0.4?mg/kg DM either in the form of SS or SY, respectively. Group 4 was fed BD supplemented with 0.9?mg Se/kg DM from SY. After 9?months of dietary treatments, the Se levels in egg yolk and albumen from hens fed unsupplemented diet were almost identical whereas eggs from hens given diet supplemented with SS showed significantly higher Se deposition in yolk than in albumen (P?相似文献   

High mitochondrial mutation rates estimated from deep-rooting Costa Rican pedigrees

Estimates of mutation rates for the noncoding hypervariable Region I (HVR-I) of mitochondrial DNA vary widely, depending on whether they are inferred from phylogenies (assuming that molecular evolution is clock-like) or directly from pedigrees. All pedigree-based studies so far were conducted on populations of European origin. In this article, we analyzed 19 deep-rooting pedigrees in a population of mixed origin in Costa Rica. We calculated two estimates of the HVR-I mutation rate, one considering all apparent mutations, and one disregarding changes at sites known to be mutational hot spots and eliminating genealogy branches which might be suspected to include errors, or unrecognized adoptions along the female lines. At the end of this procedure, we still observed a mutation rate equal to 1.24 × 10(-6) , per site per year, i.e., at least threefold as high as estimates derived from phylogenies. Our results confirm that mutation rates observed in pedigrees are much higher than estimated assuming a neutral model of long-term HVRI evolution. We argue that until the cause of these discrepancies will be fully understood, both lower estimates (i.e., those derived from phylogenetic comparisons) and higher, direct estimates such as those obtained in this study, should be considered when modeling evolutionary and demographic processes.     

A Small Multidrug Resistance-like Transporter Involved in the Arabinosylation of Arabinogalactan and Lipoarabinomannan in Mycobacteria

The biosynthesis of the major cell envelope glycoconjugates of Mycobacterium tuberculosis is topologically split across the plasma membrane, yet nothing is known of the transporters required for the translocation of lipid-linked sugar donors and oligosaccharide intermediates from the cytoplasmic to the periplasmic side of the membrane in mycobacteria. One of the mechanisms used by prokaryotes to translocate lipid-linked phosphate sugars across the plasma membrane relies on translocases that share resemblance with small multidrug resistance transporters. The presence of an small multidrug resistance-like gene, Rv3789, located immediately upstream from dprE1/dprE2 responsible for the formation of decaprenyl-monophosphoryl-β-d-arabinose (DPA) in the genome of M. tuberculosis led us to investigate its potential involvement in the formation of the major arabinosylated glycopolymers, lipoarabinomannan (LAM) and arabinogalactan (AG). Disruption of the ortholog of Rv3789 in Mycobacterium smegmatis resulted in a reduction of the arabinose content of both AG and LAM that accompanied the accumulation of DPA in the mutant cells. Interestingly, AG and LAM synthesis was restored in the mutant not only upon expression of Rv3789 but also upon that of the undecaprenyl phosphate aminoarabinose flippase arnE/F genes from Escherichia coli. A bacterial two-hybrid system further indicated that Rv3789 interacts in vivo with the galactosyltransferase that initiates the elongation of the galactan domain of AG. Biochemical and genetic evidence is thus consistent with Rv3789 belonging to an AG biosynthetic complex, where its role is to reorient DPA to the periplasm, allowing this arabinose donor to then be used in the buildup of the arabinan domains of AG and LAM.     

Biological and structural characterization of the Mycobacterium smegmatis nitroreductase NfnB,and its role in benzothiazinone resistance

Tuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl‐β‐d ‐ribose 2′‐epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro‐group to an amino‐group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB‐BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.     

Immunomodulation targeting abnormal protein conformation reduces pathology in a mouse model of Alzheimer's disease

Many neurodegenerative diseases are characterized by the conformational change of normal self-proteins into amyloidogenic, pathological conformers, which share structural properties such as high β-sheet content and resistance to degradation. The most common is Alzheimer''s disease (AD) where the normal soluble amyloid β (sAβ) peptide is converted into highly toxic oligomeric Aβ and fibrillar Aβ that deposits as neuritic plaques and congophilic angiopathy. Currently, there is no highly effective treatment for AD, but immunotherapy is emerging as a potential disease modifying intervention. A major problem with most active and passive immunization approaches for AD is that both the normal sAβ and pathogenic forms are equally targeted with the potential of autoimmune inflammation. In order to avoid this pitfall, we have developed a novel immunomodulatory method that specifically targets the pathological conformations, by immunizing with polymerized British amyloidosis (pABri) related peptide which has no sequence homology to Aβ or other human proteins. We show that the pABri peptide through conformational mimicry induces a humoral immune response not only to the toxic Aβ in APP/PS1 AD transgenic mice but also to paired helical filaments as shown on AD human tissue samples. Treated APP/PS1 mice had a cognitive benefit compared to controls (p<0.0001), associated with a reduction in the amyloid burden (p = 0.0001) and Aβ40/42 levels, as well as reduced Aβ oligomer levels. This type of immunomodulation has the potential to be a universal β-sheet disrupter, which could be useful for the prevention or treatment of a wide range of neurodegenerative diseases.     

Identification of a Polyprenylphosphomannosyl Synthase Involved in the Synthesis of Mycobacterial Mannosides

We report on the identification of a glycosyltransferase (GT) from Mycobacterium tuberculosis H37Rv, Rv3779, of the membranous GT-C superfamily responsible for the direct synthesis of polyprenyl-phospho-mannopyranose and thus indirectly for lipoarabinomannan, lipomannan, and the higher-order phosphatidyl-myo-inositol mannosides.The mycobacterial cell envelope consists of a multilayered structure of covalently linked peptidoglycan, arabinogalactan, and mycolic acids (the mAGP complex) and, among other important constituents, various noncovalently bound glycosylated lipids, notably the phosphatidyl-myo-inositol mannosides (PIMs) and their more glycosylated end products lipomannan (LM) and lipoarabinomannan (LAM) (6, 8). These glycolipids and lipoglycans exhibit a broad range of immunomodulatory activities implicated in the pathogenesis of tuberculosis and leprosy (for recent reviews, see references 5, 8, and 10).Many steps in the biosynthesis of these phosphoinositides have been defined (for recent reviews, see references 3 and 12). Mannosyltransferases (ManTs) responsible for the polymerization aspects of the synthesis of the higher-order extracytoplasmic PIMs (PIM₄ to PIM₆), LM and LAM, are of the glycosyltransferase C (GT-C) multi-transmembrane domain superfamily, whose members are dependent on polyprenyl-phospho-mannopyranose (polyprenyl-P-Manp), specifically C₃₅-P-Man or C₅₀-P-Man, as the Manp donor, which is in contrast with what occurs in the early steps of the synthesis of the simpler PIMs, which directly utilize GDP-Man (3, 4, 12). However, the biosynthetic origins of C₃₅/C₅₀-P-Man have not been fully explored. In this report, we identify Rv3779, an unassigned GT-C, as a ManT directly responsible for the origins of polyprenyl-P-Man and indirectly for the synthesis of the more polar PIMs, LM and LAM.A survey of the Mycobacterium tuberculosis H37Rv genome for genes with predicted (poly)saccharide-associated functions led to the identification of a cluster of 31 “cell wall biosynthetic genes” (2, 3), including not only arabinogalactan synthetic genes (embCA and embCB, glf, glfT1, and glfT2) but the genes for the putative ABC transporter proteins and mycolyl transferases and Rv3779, among five open reading frames, apparently encoding polyprenyl-P sugar-dependent GT-Cs (3). In particular, the integral membrane protein Rv3779 (666 amino acids) was identified as a putative GT-C, due to a conserved hallmark DXD motif and 12 to 14 predicted membrane-spanning domains (3). However, unlike with other GT-C enzymes, which typically carry the signature DXD motif on extracytoplasmic loops, the DLD motif of Rv3779 (at amino acid position 82) is predicted to map to the second loop and to be on the cytosolic side of the plasma membrane. This observation suggests that unlike other GT-C superfamily enzymes, Rv3779 utilizes a cytosolic sugar donor, presumably a nucleotide sugar. Rv3779 has orthologs in all slow-growing mycobacteria whose genome sequences are available but is not found in Mycobacterium smegmatis or in any fast-growing mycobacteria, with the notable exception of Mycobacterium abscessus. Orthologs of this gene are also not found in any other members of the suborder Corynebacterineae.Since Rv3779 is not naturally present in M. smegmatis, M. smegmatis strain mc²155 was transformed with a multicopy plasmid (pVV16-Rv3779), allowing the expression of a recombinant C-terminal His₆-tagged Rv3779 protein under the control of the phsp60 promoter (13). A cell-free ManT assay using membranes or whole lysate, similar to the one described by Korduláková et al. (13), was then conducted to determine if Rv3779 is involved in some aspect of PIM/LM/LAM biosynthesis. Thin-layer chromatography (TLC) autoradiography demonstrated an approximately threefold increase in a time-dependent manner in the incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into C₃₅-P-Man and C₅₀-P-Man by mc²155/pVV16-Rv3779 extracts compared with that of the control (Fig. ​(Fig.1A).1A). The two accumulated Man-containing glycolipids were mildly alkali stable and mildly acid labile (data not shown), with TLC mobility properties confirming their identities as precursor glycolipids of the mycobacterial polyisoprenyl-P class (4). An important and substrate concentration-dependent increase in mannosyl transfer following Rv3779 overexpression was also observed when C₅₀-P, the only form of polyprenylphosphate apparently produced by M. tuberculosis (7), was added as an acceptor substrate to the reaction mixture (Fig. 1B and C). This increase in activity over the control was about 27-fold during the first 30 min of the reaction when 0.05 mM C₅₀-P was used in the assay (Fig. ​(Fig.1C).1C). The background polyprenyl-P-Manp syntase activity detected in the control strain most likely resulted from MSMEG_3859/MSMEG_3860 (Ppm1/Ppm2), an M. smegmatis ortholog of the Ppm1 synthase from M. tuberculosis (68% identity, as determined with a 781-amino-acid overlap) (1, 9, 11). Unfortunately but not unexpectedly in light of the reported difficulty of expressing polytopic membrane GTs in Escherichia coli (14), attempts to produce Rv3779 in an active form in E. coli using different expression systems, host strains, growth conditions, and induction protocols were unsuccessful.Open in a separate windowFIG. 1.Effect of Rv3779 overexpression on the incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into mannolipids by cell extracts from M. smegmatis. (A) TLC analysis of an in vitro cell-free assay using GDP-[¹⁴C]Man and crude cell lysates from mc²155/pVV16 and mc²155/pVV16-Rv3779. Lysates were incubated with GDP-[¹⁴C]Man at 37°C for the indicated periods of time. The synthesized mannolipids were extracted with CHCl₃-CH₃OH (2:1, vol/vol), and a 10% aliquot of each sample was analyzed by TLC followed by autoradiography. The TLC plate was developed in CHCl₃-CH₃OH-H₂O-NH₄OH (65:25:4:0.5). Lanes: C, mc²155/pVV16; O, mc²155/pVV16-Rv3779. (B and C) Incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into exogenous decaprenyl-phosphate (C₅₀)-P using membrane extracts from mc²155/pVV16 and mc²155/pVV16-Rv3779. (B) TLC analysis of the first 15 min of the in vitro cell-free assays using 0.5 mM of (C₅₀)-P. (C) Quantification of the Man incorporated by the control (open symbols) and overexpressor (filled symbols) into C₅₀-P-Man over time. The concentrations of (C₅₀)-P used in the assays were 0 (circles), 0.005 (rectangles), and 0.05 (diamonds) mM. The inset shows a closeup of results of the assays using 0 and 0.005 mM (C₅₀)-P.Incubation of whole-cell lysate with GDP-[¹⁴C]Man for more-extended periods (4 to 24 h) and extraction of products with more-polar solvents (e.g., hot phenol) provides a ready estimate of the degree of synthesis of at least the LM metabolic end product. A comparison of the products synthesized by mc²155/pVV16 and mc²155/pVV16-Rv3779 (Fig. ​(Fig.2)2) showed little synthesis of [¹⁴C]LM at 4 h but the production of some low-molecular-weight [¹⁴C]LM at 24 h by the control lysate. In contrast, there was a distinct production of [¹⁴C]LM populations by the lysate from mc²155/pVV16-Rv3779 at both times, indicative of a secondary stimulatory effect of Rv3779 overexpression on LM, and presumably LAM, synthesis.Open in a separate windowFIG. 2.In vitro LM biosynthesis in M. smegmatis overexpressing Rv3779. Crude cell extracts (4 mg protein) of mc²155/pVV16 and mc²155/pVV16-Rv3779 were incubated with 1.0 μCi of GDP-[¹⁴C]Man (specific activity, 305 mCi/mmol) for 4 and 24 h. The reaction was stopped by adding CHCl₃-CH₃OH (2:1, vol/vol), and the LM/LAM contained in the cell pellet was extracted with hot phenol. LM/LAM separated on 10 to 20% Tricine gel and subsequently blotted to a nitrocellulose membrane was revealed by autoradiography. The leftmost column shows molecular mass markers (in kilodaltons).To confirm these effects of Rv3779, an M. tuberculosis H37Rv Rv3779 knockout mutant (H37RvΔRv3779) was generated by homologous recombination using standard procedures (15) (see Fig. S1 in the supplemental material). H37RvΔRv3779 grew at a much lower rate than WT H37Rv (see Fig. S2 in the supplemental material). Moreover, when examined by scanning electron microscopy, the mutant cells were found to be significantly shorter than their wild-type (WT) parent (Fig. ​(Fig.3).3). Normal growth rate and cell length were restored in the mutant upon complementation with a WT copy of Rv3779 (Fig. ​(Fig.3;3; see Fig. S2 in the supplemental material). TLC profiles of lipid extracts and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses showed a profound decrease in the amounts of acyl-PIM₆ (AcPIM₆) and Ac₂PIM₆ in H37RvΔRv3779 compared to those of the WT strain (Fig. ​(Fig.4A;4A; see Fig. S3 in the supplemental material). Importantly, the synthesis of the simpler PIMs, those arising directly from GDP-Man, was not altered in the mutant (Fig. ​(Fig.4A;4A; see Fig. S3 in the supplemental material). The examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with the monoclonal antibody CS-35 and concanavalin A of the phenol-extracted LM/LAM fraction from WT H37Rv and H37RvΔRv3779 also revealed considerably smaller amounts of both LM and LAM in the mutant (Fig. ​(Fig.4B).4B). Complementation of H37RvΔRv3779 with a WT copy of Rv3779 restored both a normal polar PIM and a normal LM/LAM profile to the mutant.Open in a separate windowFIG. 3.Scanning electron micrographs of M. tuberculosis H37Rv (WT), the mutant H37RvΔRv3779, and the complemented mutant H37RvΔRv3779/pVV16-Rv3779 cultured in 7H9-oleic acid-albumin-dextrose-catalase-Tween 80 broth at 37°C. The length of the H37RvΔRv3779 mutant cells was on average 1.2 ± 0.1 μm, compared to 2.3 ± 0.1 μm for the WT strain and 2.1 ± 0.3 μm for the complemented mutant.Open in a separate windowFIG. 4.Analysis of polar PIMs LM and LAM from WT M. tuberculosis H₃₇Rv, the H37RvΔRv3779 mutant, and H37RvΔRv3779/pVV16-Rv3779. (A) Equal amounts of total cellular lipids from WT H₃₇Rv (lane 1), the Rv3779 mutant H37RvΔRv3779 (lane 2), the mutant carrying an empty plasmid, H37RvΔRv3779/pVV16 (lane 3), and the complemented mutant H37RvΔRv3779/pVV16-Rv3779 (lane 4) were analyzed by TLC developed in CHCl₃-CH₃OH-H₂O-NH₄OH (65:25:4:0.5). (B) LM and LAM extracted from equal-weight cells of WT H₃₇Rv (lane 1), the Rv3779 mutant H37RvΔRv3779 (lane 2), and the complemented mutant H37RvΔRv3779/pVV16-Rv3779 (lane 3) were separated on a 10 to 20% Tricine gel and revealed by periodic acid-Schiff staining. The Western blot analyses were performed on the same samples using concanavalin A (ConA) reacting with the t-Manp residues of LM/LAM, as well as the CS-35 monoclonal antibody known to react with the arabinan segment of LAM.From this evidence, we conclude that Rv3779 is involved in the synthesis of the polar PIMs LM and LAM through its primary role as a polyprenyl-P-Man synthase. The disruption of this gene, which results in important alterations in PIM, LM, and LAM profiles, also has profound effects on cell growth and shape. Rv3779 is the second polyprenyl-P-Man synthase involved in PIM/LM/LAM biosynthesis identified in M. tuberculosis (11).