The unusual intersubunit ferroxidase center of Listeria innocua Dps is required for hydrogen peroxide detoxification but not for iron uptake. A study with site-specific mutants

The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.     

Effects of in vitro production on horse embryo morphology,cytoskeletal characteristics,and blastocyst capsule formation

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.     

An elongation factor Tu (EF-Tu) resistant to the EF-Tu inhibitor GE2270 in the producing organism Planobispora rosea

SecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner membrane, contains two ATP-binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high-affinity nucleotide-binding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross-linking with [α-³²P]-ATP. This analysis revealed that the nucleotide was cross-linked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP-binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low-affinity ATP-domain showed a normal distribution of cytosolic, peripheral and integral membrane forms. Interestingly, the SecA51(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high-affinity ATP-binding site. These results delimit more precisely the high-affinity ATP-binding domain of SecA, indicate the importance of the early amino-terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de-insertion from, the inner membrane by the activity of the high-affinity ATP-binding domain.     

γ-Tocopherol biokinetics and transformation in humans

Background: The uptake and biotransformation of γ-tocopherol (γ-T) in humans is largely unknown. Using a stable isotope method we investigated these aspects of γ-T biology in healthy volunteers and their response to γ-T supplementation.

Methods: A single bolus of 100 mg of deuterium labeled γ-T acetate (d²-γ-TAC, 94% isotopic purity) was administered with a standard meal to 21 healthy subjects. Blood and urine (first morning void) were collected at baseline and a range of time points between 6 and 240 h post-supplemetation. The concentrations of d² and d⁰-γ-T in plasma and its major metabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (-γ-CEHC) in plasma and urine were measured by GC-MS. In two subjects, the total urine volume was collected for 72 h post-supplementation. The effects of γ-T supplementation on α-T concentrations in plasma and α-T and γ-T metabolite formation were also assessed by HPLC or GC-MS analysis.

Results: At baseline, mean plasma α-T concentration was approximately 15 times higher than γ-T (28.3 vs. 1.9 µmol/l). In contrast, plasma γ-CEHC concentration (0.191 µmol/l) was 12 fold greater than α-CEHC (0.016 µmol/l) while in urine it was 3.5 fold lower (0.82 and 2.87 µmol, respectively) suggesting that the clearance of α-CEHC from plasma was more than 40 times that of γ-CEHC. After d²-γ-TAC administration, the d² forms of γ-T and γ-CEHC in plasma and urine increased, but with marked inter-individual variability, while the d⁰ species were hardly affected. Mean total concentrations of γ-T and γ-CEHC in plasma and urine peaked, respectively, between 0–9, 6–12 and 9–24 h post-supplementation with increases over baseline levels of 6–14 fold. All these parameters returned to baseline by 72 h. Following challenge, the total urinary excretion of d²-γ-T equivalents was approximately 7 mg. Baseline levels of γ-T correlated positively with the post-supplementation rise of (d⁰ + d²) – γ – T and γ-CEHC levels in plasma, but correlated negatively with urinary levels of (d⁰ + d²)-γ-CEHC. Supplementation with 100 mg γ-TAC had minimal influence on plasma concentrations of α-T and α-T-related metabolite formation and excretion.

Conclusions: Ingestion of 100mg of γ-TAC transiently increases plasma concentrations of γ-T as it undergoes sustained catabolism to CEHC without markedly influencing the pre-existing plasma pool of γ-T nor the concentration and metabolism of α-T. These pathways appear tightly regulated, most probably to keep high steady-state blood ratios α-T to γ-T and γ-CEHC to α-CEHC.     

Effects of Sirolimus treatment on patients with β-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4⁺ and CD8⁺ T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4⁺ and CD8⁺ T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4⁺ and CD8⁺ T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency.     

Wilms’ Tumor Gene 1 (WT1) Silencing Inhibits Proliferation of Malignant Peripheral Nerve Sheath Tumor sNF96.2 Cell Line

Wilms’ tumor gene 1 (WT1) plays complex roles in tumorigenesis, acting as tumor suppressor gene or an oncogene depending on the cellular context. WT1 expression has been variably reported in both benign and malignant peripheral nerve sheath tumors (MPNSTs) by means of immunohistochemistry. The aim of the present study was to characterize its potential pathogenetic role in these relatively uncommon malignant tumors. Firstly, immunohistochemical analyses in MPNST sNF96.2 cell line showed strong WT1 staining in nuclear and perinuclear areas of neoplastic cells. Thus, we investigated the effects of silencing WT1 by RNA interference. Through Western Blot analysis and proliferation assay we found that WT1 knockdown leads to the reduction of cell growth in a time- and dose-dependent manner. siWT1 inhibited proliferation of sNF96.2 cell lines likely by influencing cell cycle progression through a decrease in the protein levels of cyclin D1 and inhibition of Akt phosphorylation compared to the control cells. These results indicate that WT1 knockdown attenuates the biological behavior of MPNST cells by decreasing Akt activity, demonstrating that WT1 is involved in the development and progression of MPNSTs. Thus, WT1 is suggested to serve as a potential therapeutic target for MPNSTs.     

Sex differences in platelet thromboxane and arterial prostacyclin production in control and n-6 fatty acid supplemented rats

Sex differences in eicosanoid production in platelets and vessel walls have been studied in control and n-6 fatty acid supplemented rats. In platelet rich plasma (PRP) of control female rats, arachidonic acid (AA) levels in phospholipids (PL), thromboxane B₂ (TxB₂) formation following collagen stimulation and aggregatory responses to collagen were higher than in PRP of male rats. 6 keto PGF_1α release from PRP-perfused isolated aortas were the same for both sexes, but the antiaggregatory activity of the wall was higher in males than in females, in association with a greater sensitivity of male platelets to prostacyclin.The administration of n-6 fatty acid supplements increased AA level in PL, TxB₂ production and aggregation only in male platelets. Production of 6 keto PGF_1α and the antiaggregatory activity of aortic walls were reduced after dietary treatment in males, but biochemical and functional parameters were not correlated in females.The results indicate complex sex-related differences in fatty acid metabolism and eicosanoid production, and in responses to n-6 dietary fatty acids in platelets and the vascular system in the rat.     

Assessing coral bleaching and recovery with a colour reference card in Watamu Marine Park,Kenya

With this study we estimated the changes in colour, bleaching and mortality of coral colonies from February to December 2007, using the colour reference card method. The study was developed in the Watamu Marine Park lagoon (Kenya), bridging the local summer when seawater temperatures were highest and coral bleaching risk was at its maximum. Seven coral genera were selected, and their colour recorded using a colour reference card (Coral Watch card). Seven different scenarios of bleaching and mortality were observed, varying among the coral genera and between two species in the genus Pocillopora. Twenty percent of the colonies bleached, of which 50% died. Only 15% of the coral that did not bleach died. Branching genera had a higher bleaching incidence than massive and sub-massive genera. Pocillopora showed the highest bleaching susceptibility, followed by Acropora, and the highest level of mortality. Of the two species of Pocillopora considered in this study, P. eydouxi showed higher bleaching and mortality levels, while P. verrucosa bleached less and experienced only partial mortality. Our results evidenced different patterns of coral bleaching and mortality which were easily and clearly detected with the colour card method during both bleaching and a post-bleaching events.     

The genetic defect in multiple endocrine neoplasia type 2A maps next to the centromere of chromosome 10

Multiple endocrine neoplasia type 2A (MEN2A) is a rare cancer syndrome that is inherited in an apparently autosomal dominant fashion. Previous linkage studies had assigned the MEN2A locus to chromosome 10 in the pericentromeric region. We recently have described several new easily scorable RFLPs for the chromosome 10-specific alpha satellite DNA (the D10Z1) locus that is known, on the basis of previous in situ hybridization experiments, to lie at the centromere. We report here tight linkage between MEN2A and D10Z1, as demonstrated by a maximum lod score of 12.02 at the recombination frequency of zero (1-lod-unit support interval 0-4 cM), indicating that the genetic defect in MEN2A lies in the immediate vicinity of the centromere. By means of a set of ordered polymorphic DNA markers from the pericentromeric region, multipoint as well as pairwise linkage analyses place the MEN2A locus at the middle of a small region (approximately 11 cM) bracketing the centromere with FNRB (at 10p11.2) and RBP3 (at 10q11.2) on either side, providing further support for the centromeric location of the MEN2A locus. Marked sex difference in recombination frequencies exists in this pericentromeric region: significantly (P less than .01) more female than male crossovers were observed across all of the adjacent intervals D10S24-FNRB, FNRB-D10Z1, and D10Z1-RBP3. However, a sex difference was not seen in the 7-cM interval from RBP3 to D10S5, suggesting that large variation in the sex difference in recombination can occur over small chromosomal regions.(ABSTRACT TRUNCATED AT 250 WORDS)