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A recently Danish study reported that no vapour barrier is needed in ceilings, if the attic is well ventilated and the ceiling towards the dwelling is airtight. Based on that study, new investigations were initiated with focus on the hygrothermal behaviour in ventilated attics with different air change rates. A test house with three sets of four different ceiling constructions – all airtight – was used in this study. The ventilation rate was reduced in two of the sets with approx. 35 % and 50 %, respectively. Air change rates were measured with tracer gas. Furthermore, temperature and relative humidity was measured every hour. Measurements in similar ceilings with mineral wool or cellulose-based insulation material show that hygroscopic properties of the insulation have very limited effect on relative humidity. Furthermore, only at low ventilation rate the effect of a vapour barrier could be measured with minor impact. Based on the short-measured period the calculations of the risk of mould growth showed no risk. The results indicate that even when the ventilation is reduced by 50 %, the ventilated attic still performs well if the ceiling is highly airtight. However, the importance of vapour barriers becomes more important at lower air change rates.

To remove moisture by ventilating constructions is a well-known strategy, used successfully in numerous facades and roofs. However, simulating ventilation is often difficult as convection in this case is in another dimension than what is usually in focus in 1D hygrothermal simulations. There are strategies for simulating ventilation in 1D programs assuming a fixed air change rate (ACH). Unfortunately, ACH in roofs highly depend on wind direction and speed, hence a constant rate is a gross simplification. The article describes a comparative study between simulations with a 1D hygrothermal simulation program (WUFI) and simulations with BSim, a program in which the indoor climate can be simulated in different zones, in this case the ventilated attic. Focus is the effect of ACH in the attic on temperature and relative humidity. Finally, measurements of ACH in attics of ten single-family houses are presented. These measurements show that average ACH measured over more than two weeks differ very much from house to house although the ventilation openings in all houses seemed to be in accordance with current guidelines. Even measurements of the same attic differ considerably from time to time. The paper discusses what implications this should have on simulations.

To date, not many studies have presented evidence of SARS-CoV-2 infecting the female reproductive system. Furthermore, so far, no effect of the administration of anti-COVID 19 vaccines has been reported to affect the quality of oocytes retrieved from women who resorted to assisted reproduction technology (ART). The FF metabolic profiles of women who had been infected by SARS-CoV-2 before IVF treatments or after COVID-19 vaccination were examined by 1 H NMR. Immunochemical characterization of proteins and cytokines involved in the redox and inflammatory pathways was performed. The increased expression of SOD2 and NQO1, the lack of alteration of IL-6 and CXCL10 levels, as well as the increased expression of CD39, suggested that, both sharing similar molecular mechanisms or proceeding along different routes, the redox balance is controlled in the FF of both vaccinated and recovered women compared to controls. The lower amount of metabolites known to have proinflammatory activity, i.e., TMAO and lipids, further supported the biochemical results, suggesting that the FF microenvironment is controlled so as to guarantee oocyte quality and does not compromise the outcome of ART. In terms of the number of blastocysts obtained after ICSI and the pregnancy rate, the results are also comforting.

There is growing evidence that various ATP-binding cassette (ABC) transporters contribute to the growth and development of tumors, but relatively little is known about how the ABC transporter family behaves in hepatocellular carcinoma (HCC), one of the most common cancers worldwide. Cellular model studies have shown that ABCC6, which belongs to the ABC subfamily C (ABCC), plays a role in the cytoskeleton rearrangement and migration of HepG2 hepatocarcinoma cells, thus highlighting its role in cancer biology. Deep knowledge on the molecular mechanisms underlying the observed results could provide therapeutic insights into the tumors in which ABCC6 is modulated. In this study, differential expression levels of mRNA transcripts between ABCC6 -silenced HepG2 and control groups were measured, and subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. Real-Time PCR and Western blot analyses confirmed bioinformatics; functional studies support the molecular mechanisms underlying the observed effects. The results provide valuable information on the dysregulation of fundamental cellular processes, such as the focal adhesion pathway, which allowed us to obtain detailed information on the active role that the down-regulation of ABCC6 could play in the biology of liver tumors, as it is involved not only in cell migration but also in cell adhesion and invasion.

Breast cancer (BC) is the most common type of cancer among women in almost all countries worldwide and is one of the oncological pathologies for which is indicated fertility preservation, a type of procedure used to help keep a person's ability to have children. Follicular fluid (FF) is a major component of oocyte microenvironment, which is involved in oocyte growth, follicular maturation, and in communication between germ and somatic cells; furthermore, it accumulates all metabolites during oocytes growth. To obtain information about changes on fertility due to cancer, we aimed at investigating potential biomarkers to discriminate between FF samples obtained from 16 BC patients and 10 healthy women undergoing in vitro fertilization treatments. An NMR-based metabolomics approach was performed to investigate the FF metabolic profiles; ELISA and western blotting assays were used to investigate protein markers of oxidative and inflammatory stress, which are processes closely related to cancer. Our results seem to suggest that FFs of BC women display some significant metabolic alterations in comparison to healthy controls, and these variations are also related with tumor staging.

The aim of this pilot study is to evaluate if SARS-CoV-2 infection or vaccination against SARS-CoV-2 infection induce observable metabolic effects in follicular fluid of women who are following in vitro fertilization (IVF) treatments. The possible impact of coronavirus disease 2019 (COVID-19) on fertility and IVF outcome is considered. We have selected for this study: six women vaccinated against SARS-CoV-2 infection, five recovered COVID-19 patients, and we used nine healthy women as the control group. At the time of oocytes retrieval from participants in the study, follicular fluids were collected and metabolomic analysis was performed by 1 H NMR spectroscopy in combination with multivariate analysis to interpret the spectral data. The search for antibody positivity in the follicular fluid aspirates was also carried out, together with the western blotting analysis of some inflammatory proteins, interleukin-6, tumor necrosis factor α (TNFα), and the free radical scavenger superoxide dismutase 2. Higher levels of Ala and Pro together with lower levels of lipids and trimethylamine N-oxide (TMAO) were found in follicular fluids (FFs) of vaccinated women while lower levels of many metabolites were detected in FFs of recovered COVID patients. Expression level of TNF-α was significantly lower both in recovered COVID-19 patients and vaccinated women in comparison to healthy controls., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Castiglione Morelli, Iuliano, Schettini, Ferri, Colucci, Viggiani, Matera and Ostuni.)

Pseudoxanthoma elasticum (PXE) is a complex autosomal recessive disease caused by mutations of ABCC6 transporter and characterized by ectopic mineralization of soft connective tissues. Compared to the other ABC transporters, very few studies are available to explain the structural components and working of a full ABCC6 transporter, which may provide some idea about its physiological role in humans. Some studies suggest that mutations of ABCC6 in the liver lead to a decrease in some circulating factor and indicate that PXE is a metabolic disease. It has been reported that ABCC6 mediates the efflux of ATP, which is hydrolyzed in PPi and AMP; in the extracellular milieu, PPi gives potent anti-mineralization effect, whereas AMP is hydrolyzed to Pi and adenosine which affects some cellular properties by modulating the purinergic pathway. Structural and functional studies have demonstrated that silencing or inhibition of ABCC6 with probenecid changed the expression of several genes and proteins such as NT5E and TNAP, as well as Lamin, and CDK1, which are involved in cell motility and cell cycle. Furthermore, a change in cytoskeleton rearrangement and decreased motility of HepG2 cells makes ABCC6 a potential target for anti-cancer therapy. Collectively, these findings suggested that ABCC6 transporter performs functions that modify both the external and internal compartments of the cells.

This study aimed to evaluate donkey seminal plasma metabolites and relate this information to the main characteristics of sperm quality. Sperm kinetics from 10 donkey stallions were analyzed with a computerized system at the time of collection (T0) and after 24 h storage at 4 °C (T24). Seminal plasma was frozen at -80 °C for subsequent proton nuclear magnetic resonance ( 1 H NMR) spectroscopy. On three stallions, semen collection was repeated monthly for three times and sperm analysis also included mitochondrial activity and oxidative status. One stallion was azoospermic and a second semen collection was performed after one month. In the seminal plasma, 17 metabolites were identified; their levels showed numerous significant variations between the azoospermic and the normospermic individuals and grouped in well-defined clusters in a multivariate analysis. Comparing individuals with high and low sperm motility, the only discriminating metabolite was phenylalanine, whose levels were lower in the latter, as in the azoospermic individual. Phenylalanine was also the only metabolite highly correlated with all sperm kinematic parameters at T24. In conclusion, the present study has provided relevant information on the chemical characteristics of donkey semen, identified relationships between seminal metabolites, semen parameters, and sperm kinetics, and offered insights for future technological applications.

Muscari comosum L. bulbs are commonly used as food in South Italy and also in folk medicine. By evaluating in vitro antioxidant activity and biological activities of their aqueous and methanol extracts, we shed light on the potential role, including both the nutraceutical and health benefits, of this plant. Total polyphenol content (TPC) and total flavonoid content (TFC) were evaluated by the Folin-Ciocalteu method and by the aluminum chloride method, respectively. Antioxidant activity was investigated by three in vitro assays and relative antioxidant capacity index (RACI) was calculated to compare results obtained by different tests. The extracts were tested to evaluate their possible involvement in redox homeostasis, using the human hepatoma (HepG2) cell line used as model. The extracts exhibited concentration/solvent dependent radical scavenging activity, as well as dysregulation of some genes involved in redox pathways by promoting Nrf2, SOD-2, GPX1, ABCC6 and ABCG2 expression. NMR metabolomics analysis suggests that HepG2 cells treated with Muscari comosum extracts experience changes in some metabolites involved in various metabolic pathways.

Introduction: The main goal of this retrospective cohort study is the assessment of the effects of administration of recombinant-hCG (r-hCG) versus urinary-hCG (u-hCG) on follicular fluid (FF) composition of women who underwent in vitro fertilization (IVF) treatments., Materials and Methods: We selected 70 patients with infertility attributable to tubal diseases, unexplained infertility, and male factor. Metabolomics analysis of their FFs was performed by 1 H nuclear magnetic resonance ( 1 H NMR) spectroscopy in combination with multivariate analysis to interpret the spectral data. Univariate statistical analysis was applied to investigate the possible correlations between clinical parameters and between clinical parameters and metabolites identified by NMR., Results: According to the type of hCG used, significant differences were detected in FFs of women with male factor and unexplained infertility, both in qualitative and quantitative terms, for some metabolites as cholesterol, citrate, creatine, β-hydroxybutyrate, glycerol, lipids, amino acids (Glu, Gln, His, Val, Lys) and glucose. No significant difference was observed in women with tubal diseases. Besides, the number of MII oocytes in the u-hCG-treated groups correlates positively with glutamate in tubal disease and with glycerol in unexplained infertility. In the r-hCG-treated groups, the number of MII oocytes correlates positively with lipid in tubal disease, positively with citrate and negatively with glucose in male infertility., Conclusions: Metabolite composition of FF changes according to different type of hCG treatment and this can be related to oocyte development and subsequent outcome. According to the data of this study, different types of hCG should be used in relation to the diagnosis of infertility to obtain better results in inducing oocyte maturation in women undergoing IVF.

Mitochondrial carriers catalyse the translocation of numerous metabolites across the inner mitochondrial membrane, playing a key role in different cell functions. For this reason, mitochondrial carrier gene expression needs tight regulation. The human SLC25A13 gene, encoding for the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), catalyses the electrogenic exchange of aspartate for glutamate plus a proton, thus taking part in many metabolic processes including the malate-aspartate shuttle. By the luciferase (LUC) activity of promoter deletion constructs we identified the putative promoter region, comprising the proximal promoter (-442 bp/-19 bp), as well as an enhancer region (-968 bp/-768 bp). Furthermore, with different approaches, such as in silico promoter analysis, gene silencing and chromatin immunoprecipitation, we identified two transcription factors responsible for SLC25A13 transcriptional regulation: FOXA2 and USF1. USF1 acts as a positive transcription factor which binds to the basal promoter thus ensuring SLC25A13 gene expression in a wide range of tissues. The role of FOXA2 is different, working as an activator in hepatic cells. As a tumour suppressor, FOXA2 could be responsible for SLC25A13 high expression levels in liver and its downregulation in hepatocellular carcinoma (HCC).

ABCC6 is a member of the C subfamily of ATP-binding cassette transporters whose mutations are correlated to Pseudoxanthoma elasticum, an autosomal recessive, progressive disorder characterized by ectopic mineralization and fragmentation of elastic fibers. Structural studies of the entire protein have been hindered by its large size, membrane association, and domain complexity. Studies previously performed have contributed to shed light on the structure and function of the nucleotide binding domains and of the N-terminal region. Here we report the expression in E. coli of the polypeptide E 205 -G 279 contained in the cytoplasmic L0 loop. For the first time structural studies in solution were performed. Far-UV CD spectra showed that L0 is structured, assuming predominantly α-helix in TFE solution and turns in phosphate buffer. Fluorescence spectra indicated some flexibility of the regions containing aromatic residues. 1 H NMR spectroscopy identified three helical regions separated by more flexible regions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Several metabolomics studies have correlated follicular fluid (FF) metabolite composition with oocyte competence to fertilization, embryo development and pregnancy but there is a scarcity of research examining the metabolic effects of various gynaecological diseases., Objectives: In this study we aimed to analyze and correlate the metabolic profile of FF from women who were following in vitro fertilization (IVF) treatments with their different infertility pathologies., Methods: We selected 53 women undergoing IVF who were affected by: tubal diseases, unexplained infertility, endometriosis, polycystic ovary syndrome (PCOS). FF of the study participants was collected at the time of oocytes retrieval. Metabolomic analysis of FF was performed by nuclear magnetic resonance (NMR) spectroscopy., Results: FF presents some significant differences in various infertility pathologies. Although it was not possible to discriminate between FF of control participants and women with tubal diseases and unexplained infertility, comparison of FF metabolic profile from control women with patients with endometriosis and PCOS revealed significant differences in some metabolites that can be correlated to the causes of infertility., Conclusion: NMR-based metabolic profiling may be successfully applied to find diagnostic biomarkers for PCOS and endometriosis and it might be also used to predict oocyte developmental potential and subsequent outcome.

Purpose: The purpose of this study was to evaluate the possible application of metabolomics to identify follicular fluid changes in cancer patients undergoing fertility preservation. Although metabolomics have been applied already in cancer studies, this is the first application on follicular fluid of cancer patients., Methods: We selected for the study ten patients with breast cancer and lymphoma who resorted to oocyte cryopreservation to preserve fertility and ten healthy women undergoing in vitro fertilization treatments. Follicular fluid was collected at the time of oocytes retrieval. Metabolomic analysis of follicular fluids was performed by 1 H-nuclear magnetic resonance (NMR) spectroscopy in combination with multivariate analysis to interpret the spectral data. Univariate statistical analysis was applied to find correlations between patients' features and metabolites identified by NMR., Results: Partial least squares discriminant analysis allowed to discriminate samples from cancer patients and healthy controls. Univariate statistical analysis found significant correlations between patients' features and metabolites identified by NMR. This finding allowed to identify biomarkers to differentiate both healthy controls from cancer patients and the two different classes of oncological patients., Conclusion: The follicular fluids of cancer patients display significant metabolic alterations in comparison to healthy subjects. NMR-based metabolomics could be a valid prognostic tool for identifying and selecting the best cryopreserved oocytes and improving the outcome prediction in cancer women undergoing in vitro fertilization.

Inflammatory conditions and oxidative stress have a crucial role in Down syndrome (DS). Emerging studies have also reported an altered lipid profile in the early stages of DS. Our previous works demonstrate that citrate pathway activation is required for oxygen radical production during inflammation. Here, we find up-regulation of the citrate pathway and down-regulation of carnitine/acylcarnitine carrier and carnitine palmitoyl-transferase 1 genes in cells from children with DS. Interestingly, when the citrate pathway is inhibited, we observe a reduction in oxygen radicals as well as in lipid peroxidation levels. Our preliminary findings provide evidence for a citrate pathway dysregulation, which could be related to some phenotypic traits of people with DS., (© 2016 John Wiley & Sons Ltd.)

Up-Regulated Gene 7 (URG7) is a host gene up-regulated in HBV infected hepatocytes that has been suggested to have an anti-apoptotic activity mediated by caspases 3 and 8 and an endoplasmic reticulum localization. Here we report the structural characterization of the encoded protein URG7 by circular dichroism and fluorescence spectroscopy in different solvent media: phosphate buffer and two membrane-mimetic solvents, i.e. 2,2,2-trifluoroethanol (TFE) and SDS micelles. In all solvents URG7 contains substantial amounts of secondary structures. To obtain information about the structural organization and stability of URG7, its thermal denaturation in a membrane environment was studied and intermediate states of thermal unfolding were observed. Furthermore, fluorescence results in SDS micelles could be compatible with different environments for the four tryptophan residues in URG7. Preliminary NMR data indicate that URG7 in TFE solution is quite flexible and not well folded. These data are the first structural information on URG7 and might provide an insight into its structure-function relationships.

The interaction between cisplatin and an 18-residue CCHC zinc finger motif derived from a retroviral nucleocapsid protein (PyrZf18) has been studied using UV-visible, CD and (1)H NMR spectroscopies and ESI-MS spectrometry. Cisplatin irreversibly blocks the cysteine zinc binding groups in the free peptide and is able to slowly eject zinc from the zinc-peptide complex. The observed end product of the reaction with cisplatin is a complex in which only one ammonia molecule is coordinated to platinum. After an initial binding with two cysteine residues and the formation of the (PyrZf18)-platinum-(NH3)2 complex, a release of one ammonia molecule occurs because of trans-labilization, and the third cysteine is coordinated, leading to a mixture of isomers and/or conformers of the (PyrZf18)-platinum-NH3 complex. The results are discussed with respect to the potential antiretroviral activity of platinum(II) compounds and to the possible interaction of cisplatin with the cellular nucleic acid binding proteins., (Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.)

In this study the characterization of white wines produced from the monovarietal 'Greco bianco' grape variety is presented for the first time. A total of 40 commercial wines, from two different southern Italian regions, Calabria and Campania, from the same grape variety and two different vintages, were investigated. The analyses were performed by means of chromatographic methods, conventional analyses, and nuclear magnetic resonance (NMR) spectroscopy. No differentiation was observed according to the year of production but a significant discrimination was achieved using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). In particular, PLS-DA allowed the selection of compounds (total acidity; citric, malic, succinic, and lactic acids; total polyphenol index; glucose and proline/arginine ratio) useful for differentiating the studied wines on the basis of geographical origin.

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) belongs to the ABC transporter family, whose members share many characteristic features including membrane domains and two nucleotide-binding domains (NBD1 and NBD2). These function cooperatively to bind and hydrolyze ATP for the transport of substrates across biological membranes. In this study, MRP6-NBD2 (residues 1252-1503) was expressed in Escherichia coli, purified and structurally and functionally characterized. CD spectra suggested that the protein is folded. Furthermore, NBD2 is shown to be biologically active as it binds ATP and presents ATPase activity although significantly lower compared with isolated NBD1. The mixture of NBD2 and NBD1 exhibited an activity similar to the NBD2 alone, indicating that NBD1 and NBD2 form a heterodimer with the latter limiting ATP hydrolysis. These findings suggest that NBD1 has a higher tendency to form an active homodimer, which is also supported by in silico analysis of energy-minimized dimers of the homology models of the two domains.

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family. Proteins in this family share many characteristic structural features, including two membrane-spanning domains and two nucleotide-binding domains (NBD1 and NBD2), that function cooperatively but not equally bind and hydrolyze ATP. The MRP6 protein is structurally and functionally poorly characterized. Previously, we showed, by NMR spectroscopy, that a fragment of MRP6-NBD1 presents helical structure and fluorescence experiments demonstrated that peptide binds ATP. These data suggested that the study on selected regions could be a valid approach to define the structure of MRP6 . In the present study, to better characterize MRP6-NBD1, we report data of CD spectroscopy, nucleotide binding and ATP hydrolysis on two different polypeptides, one corresponding to the full-length NBD1 domain (residues from Asp-627 to Leu-851) and the other is a shorter polypeptide (residues from Arg-648 to Thr-805) without some key residues. We report that both polypeptides are highly structured in aqueous buffer and in 20% trifluoroethanol showing considerable helical and β-structure content. The ATP hydrolysis activity is exhibited only by the full-length NBD1 domain. Comparison between our findings and the structurally well characterized MRP1-NBD1 supports the role of H-loop for the ATP hydrolysis and of A-loop in stabilizing the ATP binding.

Multidrug-resistance-associated protein 6 (MRP6/ABCC6) is a protein belonging to the ABC transporter family which couple ATP hydrolysis with the transport of molecules across biological membranes. MRP6 topology presents three transmembrane domains and two nucleotide-binding domains (NBDs). The protein is structurally and functionally poorly characterized. Mutations in ABCC6 gene cause Pseudoxanthoma elasticum, a recessive genetic disorder affecting the elastic tissues. Most mutations have been found in NBDs that are critical for ATP binding and hydrolysis. With the aim to better characterize MRP6, we have performed a preliminary study on the fragment E748-A785 of MRP6-NBD1, with the wild type sequence and the R765Q mutation found in PXE affected patients. CD and NMR spectroscopy show the presence of helical structures in both peptides. Fluorescence experiments demonstrate that peptides bind ATP. The NMR structure of the mutated peptide is compared with the corresponding region of the MRP6-NBD1 modeled structure using as a template the X-ray structure of MRP1-NBD1. The finding that both wild type and mutated peptide present the same structure and similar affinity for ATP suggests that the onset of PXE symptoms is a consequence of the different type of interactions involving residue 765 R/Q inside the protein.

Bloom's syndrome (BS) is an autosomal recessive disorder characterized by growth retardation, cancer predisposition, and sterility. BS mutated (Blm), the gene mutated in BS patients, is one of five mammalian RecQ helicases. Although BLM has been shown to promote genome stability by assisting in the repair of DNA structures that arise during homologous recombination in somatic cells, less is known about its role in meiotic recombination primarily because of the embryonic lethality associated with Blm deletion. However, the localization of BLM protein on meiotic chromosomes together with evidence from yeast and other organisms implicates a role for BLM helicase in meiotic recombination events, prompting us to explore the meiotic phenotype of mice bearing a conditional mutant allele of Blm. In this study, we show that BLM deficiency does not affect entry into prophase I but causes severe defects in meiotic progression. This is exemplified by improper pairing and synapsis of homologous chromosomes and altered processing of recombination intermediates, resulting in increased chiasmata. Our data provide the first analysis of BLM function in mammalian meiosis and strongly argue that BLM is involved in proper pairing, synapsis, and segregation of homologous chromosomes; however, it is dispensable for the accumulation of recombination intermediates.

We explored the possibility of differentiating Italian wines produced in different regions by means of nuclear magnetic resonance (NMR) techniques. Ten commercial red Aglianico wines were selected from different areas of the Basilicata region in the south of Italy. Some important components of these wines were identified by the assignments of their (1)H and (13)C resonances using one- and two-dimensional homonuclear and heteronuclear NMR experiments. These data were compared with those obtained from 10 Aglianico wines produced in Campania, another southern Italian region. Differences were found among the wines according to their geographical origin and vintage. A fine discrimination of Aglianico wines from Basilicata and Campania was obtained, suggesting that the selected NMR parameters may be a valuable tool for wine authenticity control.

Events occurring during meiotic prophase I are critical for the successful production of haploid gametes. Many prophase I events are mediated by a meiosis-specific structure called the synaptonemal complex. To date, the limited knowledge we have about the dynamics of these prophase I events in mice comes from fixed, two-dimensional preparations of meiotic cells making it impossible to study the three-dimensional (3D) arrangement of meiotic chromosomes. The current study involves the development of an imaging system to view prophase I events in live mammalian spermatocytes by generating a transgenic mouse, Sycp3-Eyfp ( 21HC ), expressing a fluorescently tagged synaptonemal complex protein, SYCP3. Using this live imaging system, the 3D structural arrangement of chromosomes in the different prophase I substages has been characterized in live spermatocytes, and aspects of the 3D architecture of spermatocytes have been observed that would not be possible with existing techniques. Additionally, chromosome movement in prophase I spermatocytes and meiotic progression from pachynema to diplonema were observed following treatment with the phosphatase inhibitor, okadaic acid (OA), which accelerates the progression of cells through late prophase I. These studies demonstrate that the Sycp3-Eyfp ( 21HC ) live imaging system is a useful tool for the study of mammalian prophase I dynamics.

ABCC6 is a member of the adenosine triphosphate-binding cassette (ABC) gene subfamily C that encodes a protein (MRP6) involved in active transport of intracellular compounds to the extracellular environment. Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), an autosomal recessive disorder of the connective tissue characterized by progressive calcification of elastic structures in the skin, the eyes, and the cardiovascular system. MRP6 is codified by 31 exons and contains 1503 amino acids. In addition to a full-length transcript of ABCC6, we have identified an alternatively spliced variant of ABCC6 from a cDNA of human liver that lacks exons 19 and 24. The novel isoform was named ABCC6 Δ19Δ24. PCR analysis from cDNA of cell cultures of primary human hepatocites and embryonic kidney confirms the presence of the ABCC6Δ19Δ24 isoform. Western blot analysis of the embryonic kidney cells shows a band corresponding to the molecular weight of the truncated protein.

The oxoglutarate carrier (OGC) is a member of the mitochondrial carrier protein superfamily, which includes the ADP/ATP carrier and other functionally characterized members, and exchanges cytosolic malate for 2-oxoglutarate from the mitochondrial matrix. By means of CD and NMR spectroscopy, we previously characterized four synthetic peptides containing transmembrane segments (TMSs) I, II, V and VI of the OGC, respectively, in TFE/water mixtures and SDS micelles. Here, we present data on the remaining transmembrane segments of OGC obtained by performing CD and NMR studies on peptides corresponding to TMS-III and TMS-IV. In TFE/water, alpha-helical structures were found for these peptides in the L121-R146 and T187-S201 regions, respectively. We also evaluated the compatibility between the helical structures of our peptides and a homology model of the OGC based on the available X-ray structure of the ATP/ADP carrier.

Salmon calcitonin (sCT) forms an amphipathic helix in the region 9-19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161-174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu(23),Ala(24)-sCT in which Pro(23) and Arg(24) were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic alpha-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9-19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.

Lentiviral nucleocapsid proteins are a class of multifunctional proteins that play an essential role in RNA packaging and viral infectivity. They contain two CX(2)CX(4)HX(4)C zinc binding motifs connected by a basic linker of variable length. The 3D structure of a 37-aa peptide corresponding to sequence 22-58 from lentiviral EIAV nucleocapsid protein NCp11, complexed with zinc, has been determined by 2D (1)H NMR spectroscopy, simulated annealing, and molecular dynamics. The solution structure consists of two zinc binding domains held together by a five-residue basic linker Arg(38)-Ala-Pro-Lys-Val(42) that allows for spatial proximity between the two finger domains. Observed linker folding is stabilized by H bonded secondary structure elements, resulting in an Omega-shaped central region, asymmetrically centered on the linker. The conformational differences and similarities with other NC zinc binding knuckles have been systematically analyzed. The two CCHC motifs, both characterized by a peculiar Pro-Gly sequence preceding the His residue, although preserving Zn-binding geometry and chirality of other known NC proteins, exhibit local fold differences both between each other and in comparison with other previously characterized retroviral CCHC motifs.

The study of mammalian meiosis is complicated by the timing of meiotic events in females and by the intermingling of meiotic sub-stages with somatic cells in the gonad of both sexes. In addition, studies of mouse mutants for different meiotic regulators have revealed significant differences in the stringency of meiotic events in males versus females. This sexual dimorphism implies that the processes of recombination and homologous chromosome pairing, while being controlled by similar genetic pathways, are subject to different levels of checkpoint control in males and females. This review is focused on the emerging picture of sexual dimorphism exhibited by mammalian germ cells using evidence from the broad range of meiotic mutants now available in the mouse. Many of these mouse mutants display distinct differences in meiotic progression and/or dysfunction in males versus females, and their continued study will allow us to understand the molecular basis for the sex-specific differences observed during prophase I progression.

Dermaseptins, small polycationic peptides synthesized by amphibians, exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, show little hemolytic activity. Dermaseptins S are active only against bacteria and form aggregates at high peptide/lipid ratios, whereas dermaseptins B are active also against fungi and form aggregates at low peptide/lipid ratios. A new dermaseptin, named DS 01, from the skin secretion of Phyllomedusa oreades, showed not only strong antibacterial properties against Gram-positive and Gram-negative bacteria but also antiprotozoan activity in the microM range. An analysis of the sequences of all dermaseptins only shows a common tendency to adopt amphipathic helical conformations but does not hint at significant differences. In order to rationalize the biological differences among dermaseptins, it is necessary to analyze their conformational properties in greater detail. A structural characterization in media that mimic the membrane environment shows that the surface properties of DS 01, as compared to those of dermaseptins S1 and B2, are intermediate, in agreement with its peculiar pharmacological profile. The regular alternation of positive and negative patches on the surface suggests a plausible aggregation mechanism., (Copyright (c) 2005 Wiley Periodicals, Inc.)

The structures of the first and the second transmembrane segment of the bovine mitochondrial oxoglutarate carrier (OGC) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. Peptides 21-46 and 78-108 of its primary sequence were synthesized and structurally characterized in membrane-mimetic environments. CD data showed that at high concentrations of TFE (>50%) and SDS (>2%) both peptides assume alpha-helical structures, whereas in more hydrophilic environments only peptide 78-108 has a helical structure. (1)H-NMR spectra of the two peptides in TFE/water and SDS were fully assigned, and the secondary structures of the peptides were obtained from nuclear Overhauser effects, (3)J(alphaH-NH) coupling constants and alphaH chemical shifts. The three-dimensional solution structures of the peptides in TFE/water were generated by distance geometry calculations. A well-defined alpha-helix was found in the region K24-V39 of peptide 21-46 and in the region A86-F106 of peptide 78-108. We cannot exclude that in intact OGC the extension of these helices is longer. The helix of peptide 21-46 is essentially hydrophobic, whereas that of peptide 78-108 is predominantly hydrophilic.

Calmodulin (CaM)-protein interactions are usually described by studying complexes between synthetic targets of ca 25 amino acids and CaM. To understand the relevance of contacts outside the protein-binding region, we investigated the complex between recombinant human CaM (hCaM) and P7, a 38-residue peptide corresponding to the autoinhibitory domain of human cardiac titin kinase (hTK). To expedite the structure determination of hCaM-P7 we relied upon the high degree of similarity with other CaM-kinase peptide complexes. By using a combined homonuclear NMR spectroscopy and molecular modeling approach, we verified for the bound hCaM similar trends in chemical shifts as well as conservation of NOE patterns, which taken together imply the conservation of CaM secondary structure. P7 was anchored to the protein with 52 experimental intermolecular contacts. The hCaM-P7 structure is very similar to known CaM complexes, but the presence of NOE contacts outside the binding cavity appears to be novel. Comparison with the hTK crystal structure indicates that the P7 charged residues all correspond to accessible side-chains, while the putative anchoring hydrophobic side-chains are partially buried. To test this finding, we also modeled the early steps of the complex formation between Ca(2+)-loaded hCaM and hTK. The calculated trajectories strongly suggest the existence of an "electrostatic funnel", driving the long-range recognition of the two proteins. On the other hand, on a nanosecond time scale, no intermolecular interaction is formed as the P7 hydrophobic residues remain buried inside hTK. These results suggest that charged residues in hTK might be the anchoring points of Ca(2+)/hCaM, favoring the intrasteric regulation of the kinase. Furthermore, our structure, the first of CaM bound to a peptide derived from a kinase whose three-dimensional structure is known, suggests that special care is needed in the choice of template peptides to model protein-protein interactions., (Copyright 2001 Academic Press.)

We previously reported the structural and biological properties of the C-terminal sequence (REGDPSSSQHLPSTPSSPRV) coded by the rarely expressed exon 26A of human elastin. It assumes a stable type II beta-turn structure spanning the REGD sequence and possesses chemotactic and immunological properties. Here the structural characterization of the sequence coded by this exon was completed. Nuclear magnetic resonance and circular dichroism studies on the N-terminal amino acid sequence (GADEGVRRSLSPELREGD) showed the presence of an alpha-helix within VRRSL and a type II beta-turn within SPEL. The smaller peptides GADEGVRRSLSP and LSPELREGD revealed structural features similar to those identified in the parent peptide. No beta-turn was found in the REGD sequence of these peptides and no chemotactic activity was detected, thereby demonstrating that this biological activity is conformation dependent. Structural studies on additional peptides such as LREGD, ELREGD and LSPELREGDPSS showed that the presence of a Glu residue two positions before the Arg residue inhibits the beta-turn formation in the REGD sequence.

Modern chemical control of pests has contributed to a dramatic improvement in public welfare since its introduction 50 years ago. Millions of lives have been saved through the control of disease vectors, and millions more have been improved by the use of chemicals to produce an inexpensive and abundant food supply. Hundreds of pesticidally active ingredients are in commercial use today, and among these are found genotoxic and nongenotoxic carcinogens. In the United States, the Environmental Protection Agency regulates carcinogens using threshold and nonthreshold approaches depending upon the outcome of a weight-of-evidence determination. More than one-half of all pesticides with some evidence of carcinogenic potential are regulated by the nonthreshold approach. The limitations on product use imposed by this approach have restricted the number of products available to growers and to the public. This restriction has had a direct impact on industry with respect to commercial success and financial returns on investment as well as an indirect impact on the industry's ability to fund the discovery and development of new compounds. This paper explores the question of how well regulation by the nonthreshold approach has achieved the goal of protecting public health, whether it does this better than the alternative use of the threshold approach, and whether the incremental protection it affords is a meaningful public benefit that justifies the aforementioned impacts on industry.

Background: Grass pollen allergens are the most important and widespread elicitors of pollen allergy. One of the major plant allergens which millions of people worldwide are sensitized to is Phl p 2, a small protein from timothy grass pollen. Phl p 2 is representative of the large family of cross-reacting plant allergens classified as group 2/3. Recombinant Phl p 2 has been demonstrated by immunological cross-reactivity studies to be immunologically equivalent to the natural protein., Results: We have solved the solution structure of recombinant Phl p 2 by means of nuclear magnetic resonance techniques. The three-dimensional structure of Phl p 2 consists of an all-beta fold with nine antiparallel beta strands that form a beta sandwich. The topology is that of an immunoglobulin-like fold with the addition of a C-terminal strand, as found in the C2 domain superfamily. Lack of functional and sequence similarity with these two families, however, suggests an independent evolution of Phl p 2 and other homologous plant allergens., Conclusions: Because of the high homology with other plant allergens of groups 1 and 2/3, the structure of Phl p 2 can be used to rationalize some of the immunological properties of the whole family. On the basis of the structure, we suggest possible sites of interaction with IgE antibodies. Knowledge of the Phl p 2 structure may assist the rational structure-based design of synthetic vaccines against grass pollen allergy.

We have studied the dynamic properties of human (h) and salmon (s) calcitonin (CT) in solution. For both hormones, distance geometry in torsion-angle space has been used to generate three-dimensional structures consistent with NMR data obtained in sodium dodecyl sulfate micelles. For sCT and hCT we used, respectively, 356 and 275 interproton distances together with hydrogen-bonds as restraints. To better characterize their flexibility and dynamic properties two fully unrestrained 1100-ps molecular dynamics (MD) simulations in methanol were performed on the lowest-energy structures of both hormones. Statistical analyses of average geometric parameters and of their fluctuations performed in the last 1000 ps of the MD run show typical helical values for residues 9-19 of sCT during the whole trajectory. For hCT a shorter helix was observed involving residues 13-21, with a constant helical region in the range 13-19. Angular order parameters S(phi) and S(psi) indicate that hCT exhibits a higher flexibility, distributed along the whole chain, including the helix, while the only flexible amino acid residues in sCT connect three well-defined domains. Finally, our study shows that simulated annealing in torsion-angle space can efficiently be extended to NMR-based three-dimensional structure calculations of helical polypeptides. Furthermore, provided that a sufficient number of NMR restraints describes the system, the method allows the detection of equilibria in solution. This identification occurs through the generation of 'spurious' high-energy structures, which, for right-handed alpha-helices, are likely to be represented by left-handed alpha-helices.

The KH motif has recently been identified in single or multiple copies in a number of RNA associated proteins. Here we review the current knowledge accumulated about the sequence, structure, and functions of the KH. The multidomain architecture of most of the KH-containing proteins inspired an approach based on the production of peptides spanning the sequence of an isolated KH motif. Correct identification of the minimal length necessary for producing a folded peptide has had a number of important consequences for interpreting functional data. The presence of the KH motifs in fmr1, the protein responsible for the fragile X syndrome, and their possible role in the fmr1 functions are also discussed., (Copyright 1999 John Wiley & Sons, Inc.)

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHNalpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic alpha-helix from Leu9 to Phel6, followed by a type-I beta-turn between residues Phel6 and Phel9. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure-activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed.

The structural and biological properties of the amino acid sequence coded by the rarely expressed exon 26A of human elastin were investigated. The C-terminal portion of this sequence, corresponding to residues 600-619 of human tropoelastin, REGDPSSSQHLPSTPSSPRV and three shorter derived peptides, LREGDPSS, SSSQHLPS, and LPSTPSSP, were synthesized and studied. Spectroscopic analyses by CD and NMR have identified a type II beta-turn within the sequence REGD of the octapeptide LREGDPSS. This structural motif was found also in the tetrapeptide REGD in both trifluoroethanol and water. The CD spectrum of the tetrapeptide REGD in trifluoroethanol was consistent with a pure type II beta-turn. A high chemotactic activity for monocytes was exhibited by the structured peptides REGD (CI 0.90 at 10(-)7 M) and LREGDPSS (CI 0.80 at 10(-)11 M), at variance with the unfolded peptides LPSTPSSP and SSSQHLPS, suggesting that this activity is strictly correlated with folded structures. Because the exon 26A of human elastin is expressed in the neointima of hypertensive pulmonary arteries, and macrophages are present in this pathologic tissue [Liptay et al. (1993) J. Clin. Invest. 91, 588-594], the chemotactic activity for human monocytes reported in this paper is consistent with an active role played by the exon 26A in inducing the migration of the monocyte/macrophage cells to the neointima.

Background: The invariant chain (li) is a transmembrane protein that associates with the major histocompatibility complex class II (MHC II) molecules in the endoplasmic reticulum. The cytosolic tail of li contains two leucine-based sorting motifs and is involved in sorting the MHC II molecules to the endosomal pathway where the peptide antigen is bound. This region of li also contributes to phenotypical changes in cells, such as the formation of large endocytic structures., Results: We report here the three-dimensional structure of a 27 amino acid peptide corresponding to the cytosolic tail of li. The structure was determined by nuclear magnetic resonance (NMR) spectroscopy using a computational strategy. At high concentration, this structure reveals a new triple-stranded alpha-helical bundle in which the helices, two parallel and one antiparallel, are almost coplanar. Trimerization is mediated by electrostatic interactions intercalated by three hydrophobic layers., Conclusions: The new trimer fold, the first to be identified by NMR data alone, can be used to improve understanding of protein-protein interactions and to model multiple-helical transmembrane proteins and receptors. We suggest that interactions of the li cytosolic tails may form part of a mechanism that could cause the endosomal retention and enlarged endosomes induced by li.

In an attempt to explore the relationships between conformation of chemotactic peptides related to elastin and their biological activity we have studied five peptides: VGVAPG, VGVPG, VGAPG, GVAPG and GGVPG in solvents of different polarities which may mimic the environmental conditions at the receptor site. CD and NMR studies showed that GVAPG has no preference for structured conformations, while the other peptides may assume folded conformations in organic solvents. All these peptides but GGVPG showed chemotactic activity for monocytes. The chemotactic activity of VGVPG, VGAPG and VGVAPG was inhibited by lactose, while chemotaxis of peptide GVAPG was insensitive to lactose, suggesting the existence of different chemotactic receptors.

The KH module is a sequence motif found in a number of proteins that are known to be in close association with RNA. Experimental evidence suggests a direct involvement of KH in RNA binding. The human FMR1 protein, which has two KH domains, is associated with fragile X syndrome, the most common inherited cause of mental retardation. Here we present the three-dimensional solution structure of the KH module. The domain consists of a stable beta alpha alpha beta beta alpha fold. On the basis of our results, we suggest a potential surface for RNA binding centered on the loop between the first two helices. Substitution of a well-conserved hydrophobic residue located on the second helix destroys the KH fold; a mutation of this position in FMR1 leads to an aggravated fragile X phenotype.

Nebulin, a large protein (600 to 800 kDa) located in the thin filament of striated vertebrate muscle, is assumed to bind and stabilise F-actin. Complete sequence determination of human nebulin has only recently been accomplished showing a uniform modular structure along the whole length of the molecule. Up to 97% of the sequence is assembled from repeats of a sequence motif 35 amino acid residues long. This architecture suggests that a structural and functional understanding of such a large molecule may be possible by characterising single repeats and reconstructing from them the behaviour of the whole molecule. In the present study, we extend and generalise to the whole molecule previous work carried out on single repeats from a limited region of nebulin. Knowledge of the complete sequence allowed extensive analysis of the single repeats revealing a progressive N to C-terminal divergence that is mirrored by an increase of the alpha-helix propensity. A number of synthetic peptides spanning the sequences of selected repeats were obtained and their conformational and binding properties studied in detail. All the peptides showed a tendency to fold as transient helices in aqueous solution with helix content as observed by CD and NMR studies in excellent agreement with predictions. A higher helical tendency of repeats near the C terminus was observed. Analysis of the influence of charged media as well as trifluoroethanol on the folding of single repeats strongly suggested that the mechanism by which the nebulin alpha-helix is stabilised is mostly electrostatic. Peptides with higher helical content also showed a higher binding affinity to F-actin. Considerably varying effects were observed for the peptides on F-actin viscosity and polymerisation. We discuss the divergence in sequence and helical tendency and its correlation to the functional data with regard to their significance for the assembly of the thin filament during myogenesis.

The cytosolic tail of the major histocompatibility complex-associated invariant chain protein contains two Leu-based motifs that both mediate efficient sorting to the endocytic pathway. Nuclear magnetic resonance data on a peptide of 27 residues corresponding to the cytosolic tail of human invariant chain indicate that in water at pH 7.4 the membrane distal motif Leu7-Ile8 lies within a nascent helix, while the membrane proximal motif Met16-Leu17 is part of a turn. The presence of a small amount of methanol stabilizes an alpha helix from Gln4 to Leu17 with a kink on Pro15. Point mutations of the cytosolic tail of the protein suggest that amino-terminal residues located in spatial proximity to the Leu motifs contribute to efficient internalization and targeting to endosomes in transfected COS cells. Residues on the spatially opposite side of the Leu motifs were, on the other hand, mutated with no measurable effect on targeting. Structural and biological data thus suggest that the signals are not continuous but consist of "signal patches" formed by the three-dimensional structure of the cytosolic tail of invariant chain.

The giant muscle proteins of the titin family, which are specific for the striated muscles of vertebrates and invertebrates, contain as a common feature a catalytic protein kinase domain of so far unclear function and regulation. In myosin light chain kinase, a family evolutionarily related to titin, kinase regulation is achieved by calmodulin binding to a region of the kinase C-terminus which bears similarity to the substrate. A calmodulin-binding sequence has also been identified in the C-terminus of the Aplysia twitchin kinase. In analogy, we identified a putative calmodulin-binding site in the titin kinase C-terminal sequence. The expressed catalytic domain itself and a series of synthetic peptides from this region were tested for their ability to bind calmodulin. Biochemical data indicate that titin kinase as well as peptides from its C-terminus bind to calmodulin in an equimolar complex in the presence of calcium. The interaction of truncated peptides with calmodulin is, however, weaker than that of myosin light chain kinase. Nuclear magnetic resonance studies showed that these peptides have a tendency to adopt alpha-helical conformations in solution. Helicity increases upon binding of calmodulin in a calcium-dependent fashion, as judged by circular dichroism spectra. We, therefore, propose that this calmodulin-binding region of titin could play a regulatory role for the enzyme, the substrate of which still remains to be identified.

Recent results with the Drosophila melanogaster period gene suggest that the apparently conserved repetitive motif (Thr-Gly)n encoded by this gene may play an important role in the temperature compensation of the circadian clock. We have therefore initiated both a theoretical and experimental conformational analysis of (Thr-Gly)n peptides. By using a build-up method, it is clear that the hexapeptide (Thr-Gly)3 represents a 'conformational monomer' and generates a stable type II or type III beta-turn. Circular dichroism and nuclear magnetic resonance spectra of synthetic (Thr-Gly)3 and poly(Thr-Gly) peptides revealed that these peptides exhibit flexible conformations, especially in more polar environments and at higher temperatures. We speculate that this flexibility may illuminate our understanding of both the molecular mechanism of temperature compensation and the systematic geographical distribution within Europe of the Thr-Gly length polymorphism in D. melanogaster.

The KH module has recently been identified in a number of RNA associated proteins including vigilin and FMR1, a protein implicated in the fragile X syndrome. In this work, NMR spectroscopy was used to determine the secondary structure in solution of a KH domain (repeat 5 from vigilin). Almost complete assignments were obtained for the 1H and 15N resonances using uniform 15N-labeling of the protein combined with homo-nuclear 2D 1HNMR and 3D 15N correlated 1H NMR. On the basis of NOE patterns, secondary chemical shifts and amide solvent exposure, the secondary structure consists of an antiparallel three stranded beta sheet connected by two helical regions. This domain may also be stabilized by an appended C-terminal helix which is common to many but not all members of the KH family.