Engineering Tocopherol Selectivity in alpha-TTP: A Combined In Vitro/In Silico Study

"We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of a-tocopherol transfer" "protein (a-TTP) towards a-tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for a-tocopherol to a-TTP 8.26+2.13 kcal mol{1 lower than that of c-tocopherol. Our calculations show that c-tocopherol binds to a-TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of a-TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to c-tocopherol, with striking structural similarities to the wild-type- a-tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of a-TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover," "the identification of a c-tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for a-tocopherol selection in omnivorous animals."

Synthetic peptides corresponding to a repetitive sequence of malarial histidine rich protein bind haem and inhibit haemozoin formation in vitro

Synthetic peptides containing a repetitive hexapeptide sequence (Ala-His-His-Ala-Ala-Asp) of malarial histidine-rich protein II were evaluated for binding with haem in vitro. The pattern of haem binding suggested that each repeat unit of this sequence provides one binding site for haem. Chloroquine inhibited the haem-peptide complex formation with preferential formation of a haem chloroquine complex. In vitro studies on haem polymerisation showed that none of the peptides could initiate haemozoin formation. However, they could inhibit haemozoin formation promoted by a malarial parasite extract, possibly by competitively binding free haem. These results indicate this hexapeptide sequence represents the haem binding site of the malarial histidine-rich protein and possibly the site of nucleation for haem polymerisation.

High expression of octamer-binding transcription factor 4A, prominin-1 and aldehyde dehydrogenase strongly indicates involvement in the initiation of lung adenocarcinoma resulting in shorter disease-free intervals

The increasing relevance of the cancer stem cell (CSC) hypothesis and the impact of CSC-associated markers in the carcinogenesis of solid tumours may provide potential prognostic implications in lung cancer. We propose that a collective genetic analysis of established CSC-related markers will generate data to better define the role of putative CSCs in lung adenocarcinoma (LAC).

Insulin-like growth factor-I treatment in primary growth hormone insensitivity: effect of recombinant human IGF-I (rhIGF-I) and rhIGF-I/rhIGF-binding protein-3 complex

Growth hormone insensitivity syndrome (GHIS) is a rare cause of growth retardation characterized by high serum GH levels, and low serum insulin-like growth factor I (IGF-I) levels associated with a genetic defect of the GH receptor (GHR) as well post-GHR signaling pathway. Based on clinical, as well as biochemical characteristics, GHIS can be genetically classified as classical/Laron's syndrome and nonclassical/atypical GHIS. Recombinant human IGF-I (rhIGF-I) treatment is effective in promoting growth in subjects who have GHIS. Further, pharmacological studies of a IGF-I compound containing a 1:1 molar complex of rhIGF-I and rhIGF-binding protein-3 (BP-3) demonstrated that the complex was effective in increasing levels of circulating total and free IGF-I and that the administration in patients with GHIS should be safe, well-tolerated and more effective than rhIGF-I on its own.

Sensitive and selective detection of free FXIII activation peptide: a potential marker of acute thrombotic events

Coagulation factor XIII (FXIII) stabilizes fibrin fibers and is therefore a major player in the maintenance of hemostasis. FXIII is activated by thrombin resulting in cleavage and release of the FXIII activation peptide (AP-FXIII). The objective of this study was to characterize the released AP-FXIII and determine specific features that may be used for its specific detection. We analyzed the structure of bound AP-FXIII within the FXIII A-subunit and interactions of AP-FXIII by hydrogen bonds with both FXIII A-subunit monomers. We optimized our previously developed AP-FXIII ELISA by using 2 monoclonal antibodies. We determined high binding affinities between the antibodies and free AP-FXIII and demonstrated specific binding by epitope mapping analyses with surface plasmon resonance and enzyme-linked immunosorbent assay. Because the structure of free AP-FXIII had been characterized so far by molecular modeling only, we performed structural analysis by nuclear magnetic resonance. Recombinant AP-FXIII was largely flexible both in plasma and water, differing significantly from the rigid structure in the bound state. We suggest that the recognized epitope is either occluded in the noncleaved form or possesses a structure that does not allow binding to the antibodies. On the basis of our findings, we propose AP-FXIII as a possible new marker for acute thrombotic events.

Deficient CEBPA DNA binding function in normal karyotype AML patients is associated with favorable prognosis

CCAAT/enhancer binding protein-α (CEBPA) mutations in acute myeloid leukemia (AML) patients with a normal karyotype (NK) confer favorable prognosis, whereas NK-AML patients per se are of intermediate risk. This suggests that blocked CEBPA function characterizes NK-AML with favorable outcome. We determined the prognostic significance of CEBPA DNA binding function by enzyme-linked immunosorbent assay in 105 NK-AML patients. Suppressed CEBPA DNA binding was defined by 21 good-risk AML patients with inv(16) or t(8;21) (both abnormalities targeting CEBPA) and 8 NK-AML patients with dominant-negative CEBPA mutations. NK-AML patients with suppressed CEBPA function showed a better overall survival (P = .0231) and disease-free survival (P = .0069) than patients with conserved CEBPA function. Suppressed CEBPA DNA binding was an independent marker for better overall survival and disease-free survival in a multivariable analysis that included FLT3-ITD, NPM1 and CEBPA mutation status, white blood cell count, age and lactate dehydrogenase. These data indicate that suppressed CEBPA function is associated with favorable prognosis in NK-AML patients.

Lactococcus lactis HemW (HemN) is a haem-binding protein with a putative role in haem trafficking

Lactococcus lactis cannot synthesize haem, but when supplied with haem, expresses a cytochrome bd oxidase. Apart from the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in haem metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is known for hemW. HemW-like proteins occur in bacteria, plants and animals, and are usually annotated as CPDHs (coproporphyrinogen III dehydrogenases). However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly produced, purified HemW contained an Fe-S (iron-sulfur) cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound haem b in vitro, with a stoichiometry of one haem per monomer and a KD of 8 μM. In vivo, HemW occurred as a haem-free cytosolic form, as well as a haem-containing membrane-associated form. Addition of L. lactis membranes to haem-containing HemW triggered the release of haem from HemW in vitro. On the basis of these findings, we propose a role of HemW in haem trafficking. HemW-like proteins form a distinct phylogenetic clade that has not previously been recognized.

Corticosteroid-binding globulin: a possible early predictor of infection in acute necrotizing pancreatitis

OBJECTIVE: Infected pancreatic necrosis is the main cause of death in patients with acute pancreatitis, and therefore its early prediction is of utmost importance. Endogenous cortisol metabolism plays a basic role both in the course of acute pancreatitis and in the process of infection. The purpose of this study was to analyze corticosteroid-binding globulin (CBG), total cortisol, calculated free cortisol and adrenocorticotropic hormone as potential early predictors in order to differentiate between infected pancreatic necrosis and sterile pancreatic necrosis in patients with acute pancreatitis. MATERIAL AND METHODS: Serum levels of CBG, total cortisol, calculated free cortisol, and plasma levels of adrenocorticotropic hormone were determined in 109 consecutive patients with acute pancreatitis. C-reactive protein was measured as the control parameter. Thirty-five patients developed necrotizing pancreatitis and 10 developed infection of the necrosis. Blood was monitored for 6 days after the onset of pain; 30 healthy individuals served as controls. RESULTS: Of all parameters only CBG showed a significant difference (p = 0.0318) in its peak levels measured in the first 48 h in patients with sterile (26.5 microg/ml, range 21.3-34.7) and infected (16.0 microg/ml, range 15.2-25.0) necrosis at a cut-off level of 16.8 microg/ml. That difference was further preserved for the first 6 days after onset of pain. CONCLUSIONS: In our group of patients, a decreased CBG level below 16.8 g/ml within the initial 48 h of acute pancreatitis was an early predictor of later infected pancreatic necrosis, with a positive predictive value of 100% and a negative predictive value of 87.5%.

Prognosis in pediatric hematologic malignancies is associated with serum concentration of mannose-binding lectin-associated serine protease-2 (MASP-2)

BACKGROUND: Mannose-binding lectin (MBL) and MBL-associated serine protease-2 (MASP-2) are key components of the lectin pathway of complement activation. Their serum concentrations show a wide interindividual variability. This study investigated whether the concentration of MBL and MASP-2 is associated with prognosis in pediatric patients with cancer. METHODS: In this retrospective multicenter study, MBL and MASP-2 were measured by commercially available ELISA in frozen remnants of serum taken at diagnosis. Associations of overall survival (OS) and event-free survival (EFS) with MBL and MASP-2 were assessed by multivariate Cox regression accounting for prognostically relevant clinical variables. RESULTS: In the 372 patients studied, median serum concentration of MBL was 2,808 microg/L (range, 2-10,060) and 391 microg/L (46-2,771) for MASP-2. The estimated 4-year EFS was 0.60 (OS, 0.78). In the entire, heterogeneous sample, MBL and MASP-2 were not significantly associated with OS or EFS. In patients with hematologic malignancies, however, higher MASP-2 was associated with better EFS in a significant and clinically relevant way (hazard ratio per tenfold increase (HR), 0.22; 95% CI, 0.09-0.54; P = 0.001). This was due to patients with lymphoma (HR, 0.11; 95% CI, 0.03-0.47; P = 0.003), but less for those with acute leukemia (HR, 0.35; 95% CI, 0.11-1.15; P = 0.083). CONCLUSION: In this study, higher MASP-2 was associated with better EFS in pediatric patients with hematologic malignancies, especially lymphoma. Whether MASP-2 is an independent prognostic factor affecting risk stratification and anticancer therapy needs to be assessed in prospective, disease-specific studies.

Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls. The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts. This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts. Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting. In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation.

Effect of zinc binding residues in growth hormone (GH) and altered intracellular zinc content on regulated GH secretion.

Endocrine cells store hormones in concentrated forms (aggregates) in dense-core secretory granules that are released upon appropriate stimulation. Zn(2+) binding to GH through amino acid residues His18, His21, and Glu174 are essential for GH dimerization and might mediate its aggregation and storage in secretory granules. To investigate whether GH-1 gene mutations at these positions interfere with this process, GH secretion and intracellular production were analyzed in GC cells (rat pituitary cell line) transiently expressing wt-GH and/or GH Zn mutant (GH-H18A-H21A-E174A) in forskolin-stimulated vs nonstimulated conditions. Reduced secretion of the mutant variant (alone or coexpressed with wt-GH) compared with wt-GH after forskolin stimulation was observed, whereas an increased intracellular accumulation of GH Zn mutant vs wt-GH correlates with its altered extracellular secretion. Depleting Zn(2+) from culture medium using N,N,N',N'-tetrakis(2-pyridylemethyl)ethylenediamine, a high-affinity Zn(2+) chelator, led to a significant reduction of the stimulated wt-GH secretion. Furthermore, externally added Zn(2+) to culture medium increased intracellular free Zn(2+) levels and recovered wt-GH secretion, suggesting its direct dependence on free Zn(2+) levels after forskolin stimulation. Confocal microscopy analysis of the intracellular secretory pathway of wt-GH and GH Zn mutant indicated that both variants pass through the regulated secretory pathway in a similar manner. Taken together, our data support the hypothesis that loss of affinity of GH to Zn(2+) as well as altering intracellular free Zn(2+) content may interfere with normal GH dimerization (aggregation) and storage of the mutant variant (alone or with wt-GH), which could possibly explain impaired GH secretion.

Adsorption interaction of carrier-free thallium species with gold and quartz surfaces

The adsorption interactions of thallium and its compounds with gold and quartz surfaces were investigated. Carrier-free amounts of thallium were produced in nuclear fusion reactions of alpha particles with thick gold targets. The method chosen for the studies was gas thermochromatography and varying the redox potential of the carrier gases. It was observed that thallium is extremely sensitive to trace amounts of oxygen and water, and can even be oxidized by the hydroxyl groups located on the quartz surface. The experiments on a quartz surface with O2, He, H2 gas in addition with water revealed the formation and deposition of only one thallium species – TlOH. The adsorption enthalpy was determined to be Δ HSiO2ads(TlOH) = −134 ± 5 kJ mol−1. A series of experiments using gold as stationary surface and different carrier gases resulted in the detection of two thallium species – metallic Tl (H2 as carrier gas) and TlOH (O2, O2+H2O and H2+H2O as pure carrier gas or carrier gas mixture) with Δ HAuads(Tl) = −270 ± 10 kJ mol− and Δ HAuads(TlOH) = −146 ± 3 kJ mol−1. These data demonstrate a weak interaction of TlOH with both quartz and gold surfaces. The data represent important information for the design of future experiments with the heavier homologue of Tl in group 13 of the periodic table – element 113 (E113).

A low affinity penicillin-binding protein 2x is required for heteroresistance in Streptococcus pneumoniae

Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the minimal inhibitory concentration (MIC). This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin binding proteins PBP2b and PBP2x and secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from heteroresistant strain Spain(23F)2349 in non-heteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression is not observed and therefore expression does not correlate with an increased proportion of resistant subpopulations. Additional ciaR disruption mutants which have been described to mediate PBP-independent β-lactam resistance revealed no heteroresistant phenotype by PAP.We also showed, that the highly resistant subpopulations (HOM*) of transformants containing low affinity pbp2x undergo an increase in resistance upon selection on penicillin plates which partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins pstS, phoU, pstB and pstC in these highly resistant subpopulations.In conclusion, the presence of low affinity pbp2x enables certain pneumococcal colonies to survive in the presence of beta lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaption to antibiotic stress.