Biochemistry and cell biology

Negative regulators of angiogenesis play a major role in maintaining vascular homeostasis. Thrombospondin-1 (TSP1) is a natural inhibitor of angiogenesis. This report examines the presence of TSP1 in ocular samples and determines whether its production is altered in diabetes. Western blot analysis detected a 140 kDa antiangiogenic fragment of TSP1(gp140) in vitreous samples prepared from normal human and rat eyes. Intact TSP1 was detected in aqueous humor samples prepared from normal rat and bovine eyes. In contrast, TSP1 was virtually absent in vitreous and aqueous humor samples prepared from diabetic rat eyes. Furthermore, production of TSP1 by microvascular endothelial cells in culture was sensitive to high concentrations of glucose. Retinal blood vessels appeared nonuniform and dilated in diabetic animals when compared to control animals. These results demonstrate that TSP1 and its antiangiogenic fragment are present in aqueous humor and vitreous of normal rat eyes and are dramatically reduced in diabetes. Thus, TSP1 may play a role in ocular vascular homeostasis and its absence may contribute to vascular dysfunctions associated with diabetes.

Cancer is a complex disease arising from multiple genetic lesions. Cellular transformation is accompanied by changes in numerous processes, including DNA repair, cellular proliferation, cell-cycle control and apoptosis, and acquired capabilities such as tissue invasion and metastasis . The complex interplay between genes altered in these different processes can lead ultimately to tumorigenesis. This conference covered some of the latest discoveries that address the molecular causes of cancer, and its broad subject base reflects the diversity of genes involved. As changes in gene expression are a major driving force behind tumorigenesis, this was a prominent topic of discussion, as were cell-cycle control and the maintenance of genome integrity. The influence of the local cellular environment in determining the normal versus oncogenic functions of genes was a key concept to emerge and, finally, new technologies that permit us to study genes and cancer in a more global context were covered. Changes in gene expression profiles are among the key determinants of tumorigenesis. One of the first steps in activating gene expression is altering the accessibility of chromatin to the transcription machinery. This can involve either modification of histone tails and/or changes in the chromatin structure (Figure ). Proteins related to components of the SWI/SNF complex form parts of important chromatin remodelling machines. Studies by T. Owen-Hughes (Dundee, UK) on one such protein, the chomatin remodelling ATPase, ISWI, suggest how it may play a role in transcriptional regulation by mobilizing nucleosomes along the DNA. The key finding presented was that ISWI binds to free DNA next to the nucleosome and promotes translocation of the DNA, which then loops around the nucleosome in a stepwise manner. Obviously, these remodelling machines must be targeted to the correct promoters. Work on the yeast homologue, ISW1, by C. Goding (Oxted, UK) suggests a mechanism by which this could be accomplished. His studies on the PHO8 promoter demonstrate a role for ISW1 in repressing transcription and implicate the basic helix-loop-helix leucine zipper (bHLHZip) transcription factor Cbf-1 in recruiting it to the DNA in vivo. The importance of the histone H4 tail for the repressive activity mediated by ISW1 was also demonstrated by Goding. Histone tails are subject to covalent modification with a number of different functional groups, including phosphate, acetyl, methyl and ubiquitin groups. These modifications can act positively or negatively to regulate gene expression and often act in a combinatorial manner.

Elk-1 belongs to the Ternary Complex Factors (TCFs) subfamily of the ETS domain proteins, and plays a critical role in the expression of immediate-early genes (IEGs) upon mitogen stimulation and activation of the mitogen-activated protein kinase (MAPK) cascade. The association of TCFs with Serum Response Elements (SREs) on IEG promoters has been widely studied and a role for Elk-1 in promoting cell cycle entry has been determined. However, the presence of the ETS domain transcription factor Elk-1 in axons and dendrites of post-mitotic adult brain neurons has implications for an alternative function for Elk-1 in neurons other than controlling proliferation. In this study, possible alternative roles for Elk-1 in neurons were investigated, and it was demonstrated that blocking TCF-mediated transactivation in neuronal cells leads to apoptosis through a caspase-dependent mechanism. Indeed RNAi-mediated depletion of endogenous Elk-1 results in increased caspase activity. Conversely, overexpression of either Elk-1 or Elk-VP16 fusion proteins was shown to rescue PC12 cells from chemically-induced apoptosis, and that higher levels of endogenous Elk-1 correlated with longer survival of DRGs in culture. It was shown that Elk-1 regulated the Mcl-1 gene expression required for survival, and that RNAimediated degradation of endogenous Elk-1 resulted in elimination of the mcl-1 message. We have further identified the survival-ofmotor neuron-1 (SMN1) gene as a

Alternative splicing plays a major role in transcriptome diversity and plasticity, but it is largely unknown how tissue-specific and embryogenesis-specific alternative splicing is regulated. The highly conserved splicing factor Slu7 is involved in 3′ splice site selection and also regulates alternative splicing. We show that Slu7 has a unique spatial pattern of expression among human and mouse embryonic and adult tissues. We identified several functional Ets binding sites and GC-boxes in the human Slu7 (hSlu7) promoter region. The Ets and GC-box binding transcription factors, Elk-1 and Sp1, respectively, exerted opposite effects on hSlu7 transcription: Sp1 protein enhances and Elk-1 protein represses transcription in a dose-dependent manner. Sp1 protein bound to the hSlu7 promoter in vivo, and depletion of Sp1 by RNA interference (RNAi) repressed hSlu7 expression. Elk-1 protein bound to the hSlu7 promoter in vivo, and depletion of Elk-1 by RNAi caused an increase in the endogenous l...

02-evolving photosystem II complexes, which were isolated from the cyanobacterium Anacystis nidulans as described previously [(1986) B&him. Biophys. Acta 849, 203-2101, were further purified on a Sepharose 6B column. These complexes retain the L-amino acid oxidase protein. Moreover, various types of photosystern II inhibitors were tested for their effect on L-amino acid oxidase activity. The results show that chlorpromazine, 2,4,6,2',4',6'-hexanitrodiphenylamine, and o-phenanthroline inhibited photosynthetic 02 evolution as well as L-amino acid oxidase activity, while 3-(3,4-dichlorophenyl)-1,1-dimethylurea and 4-hydroxy-3,%diiodobenzonitrile only inhibited photosynthetic O2 evolution. These results further support a functional role of the L-amino acid oxidase protein in photosynthetic 02 evolution. We suggest that a flavin-derived molecule -possibly a 10, I@-ring-opened derivative of FAD -is the organic prosthetic group of the watersplitting complex in A. nidulans and that this flavin derivative is able to chelate mangenese.

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/ PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance.

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor Nix is responsive to lipotoxicity and accumulates in response to diacylglycerols induced by high-fat (HF) feeding. In addition, previous studies have implicated autophagy and mitophagy in muscle insulin sensitivity. To provide a better understanding of these observations, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes including Beclin-1, ATG3, and -5. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the previously reported roles of these phospholipids and Nix in mitochondrial dynamics, we investigated aberrant mitochondrial fission and turn-over as a mechanism of myocyte in...

Background-Six known steps are required for the circulating thyroid hormone (TH) to exert its action on target tissues. For three of these steps, human mutations and distinct phenotypes have been identified. The clinical, laboratory, genetic and molecular characteristics of these three defects of TH action are the subject of this review. The first defect, recognized 45 years ago, produces resistance to TH and carries the acronym, RTH. In the majority of cases it is caused by TH receptor β gene mutations. It has been found in over 3,000 individuals belonging to approximately 1,000 families. Two relatively novel syndromes presenting reduced sensitivity to TH involve membrane transport and metabolism of TH. One of them, caused by mutations in the TH cell-membrane transporter MCT8, produces severe psychomotor defects. It has been identified in more than 170 males from 90 families. A defect of the intracellular metabolism of TH in 10 individuals from 8 families is caused by mutations in the SECISBP2 gene required for the synthesis of selenoproteins, including TH deiodinases. Major Conclusions-Defects at different steps along the pathway leading to TH action at cellular level can manifest as reduced sensitivity to TH. General Significance-Knowledge of the molecular mechanisms involved in TH action allows the recognition of the phenotypes caused by defects of TH action. Once previously known defects have been ruled out, new molecular defects could be sought, thus opening the avenue for novel insights in thyroid physiology.

In addition to its role as a strong antioxidant, vitamin C regulates the differentiation of several cell lineages. In vertebrate skeletal muscle, the vitamin C transporter SVCT2 is preferentially expressed in slow muscle fibers. To gain insights into the possible involvement of intracellular vitamin C on early myogenesis, we investigated the regulation of SVCT2 expression in cultures of chick fetal myoblasts. SVCT2 expression increases in cultures of both, slow and fast muscle-derived myoblasts, as they fuse to form mainly fast myotubes. Interestingly, we found that SVCT2 could be positively modulated by potassium-induced depolarization of myotubes. These findings suggest that SVCT2-mediated uptake of vitamin C could play diverse roles on skeletal muscle development and physiology.

In addition to its role as a strong antioxidant, vitamin C regulates the differentiation of several cell lineages. In vertebrate skeletal muscle, the vitamin C transporter SVCT2 is preferentially expressed in slow muscle fibers. To gain insights into the possible involvement of intracellular vitamin C on early myogenesis, we investigated the regulation of SVCT2 expression in cultures of chick fetal myoblasts. SVCT2 expression increases in cultures of both, slow and fast muscle-derived myoblasts, as they fuse to form mainly fast myotubes. Interestingly, we found that SVCT2 could be positively modulated by potassium-induced depolarization of myotubes. These findings suggest that SVCT2-mediated uptake of vitamin C could play diverse roles on skeletal muscle development and physiology.

During the reproductive phase of a plant, shoot meristems follow one of two developmental programs to produce either flowers or vegetative shoots. The decision as to which meristems give rise to flowers, and when they do so, determines the general morphology of an inflorescence. Molecular and genetic research in Arabidopsis and other model species has identified several genes that control the identity that a meristem will adopt. These meristem identity genes are activated in response to developmental and environmental cues, and can be assigned to three basic categories: those required either to initiate or maintain the floral program in some meristems and those required to maintain the vegetative program in others.

We have examined the floral morphology and ontogeny of three mutants of Arabidopsis thaliana, Ap2-5, Ap2-6, and Ap2-7, that exhibit homeotic changes of the perianth organs because of single recessive mutations in the AP2 gene. Homeotic conversions observed are: sepals to carpels in all three mutants, petals to stamens in Ap2-5, and petals to carpels in Ap2-6. Our analysis of these mutants suggests that the AP2 gene is required early in floral development to direct primordia of the first and second whorls to develop as perianth rather than as reproductive organs. In addition, our results support one of the two conflicting hypotheses concerning the structures of the calyx and the gynoecium in the Brassicaceae.

SkyLine, a high-throughput homology modeling pipeline tool, detects and models true sequence homologs to a given protein structure. Structures and models are stored in SkyBase with links to computational function annotation, as calculated by MarkUs. The SkyLine/SkyBase/MarkUs technology represents a novel structure-based approach that is more objective and versatile than other protein classification resources. This structure-centric strategy provides a multidimensional organization and coverage of protein space at the levels of family, function, and genome. The concept of "modelability", the ability to model sequences on related structures, provides a reliable criterion for membership in a protein family ("leverage") and underlies the unique success of this approach. The overall procedure is illustrated by its application to START domains, which comprise a Biomedical Theme for the Northeast Structural Genomics Consortium (NESG) as part of the Protein Structure Initiative (PSI). START domains are typically involved in the non-vesicular transport of lipids. While 19 experimentally determined structures are available, the family, whose evolutionary hierarchy is not well determined, is highly sequence diverse, and the ligand-binding potential of many family members is unknown. The SkyLine/SkyBase/MarkUs approach provides significant insights and predicts: 1) many more family members (~4,000) than any other resource; 2) the function for a large number of unannotated proteins; 3) instances of START domains in genomes from which they were thought to be absent; and 4) the existence of two types of novel proteins, those containing dual START domain and those containing N-terminal START domains.

Background: There is a great need for a high throughput computational tool for predicting the function of lipid binding domains on a genomic scale. Results: A newly developed computation protocol allows for genome-wide prediction of membrane binding properties of ANTH domains. Membrane binding properties of proteins can be systematically and reliably predicted by our combinatorial approach. Significance: A novel functionality of lipid binding domains can be computationally predicted.

The role of phosphorylation for regulating the Rac1 GEF PREX2 is not understood. Results: PAK phosphorylation of PREX2 downstream of PIP 3 and G␤␥ reduces PREX2 GEF activity. Second messengers can initiate negative feedback to decrease Rac1 activation through PAK phosphorylation of PREX2. Significance: PAK negative regulation of GEFs could represent a broadly utilized mechanism to attenuate Rac1 activation and its outputs. Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 )-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP-bound Rac1 then activates its downstream effectors, including p21-activated kinases (PAKs). PREX2 and Rac1 are frequently mutated in cancer and have key roles within the insulin-signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2-mediated activation of Rac1 by the second messengers PIP 3 or G␤␥, we found that PREX2 was phosphorylated through a PAK-dependent mechanism. PAK-mediated phosphorylation of PREX2 reduced GEF activity toward Rac1 by inhibiting PREX2 binding to PIP 3 and G␤␥. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Furthermore, the onset of insulin-induced phosphorylation of PREX2 was delayed compared with AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAKs phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated.

We discuss recent approaches for structure-based protein function annotation. We focus on template-based methods where the function of a query protein is deduced from that of a template for which both the structure and function are known. We describe the different ways of identifying a template. These are typically based on sequence analysis but new methods based on purely structural similarity are also being developed that allow function annotation based on structural relationships that cannot be recognized by sequence. The growing number of available structures of known function, improved homology modeling techniques and new developments in the use of structure allow template-based methods to be applied on a proteome-wide scale and in many different biological contexts. This progress significantly expands the range of applicability of structural information in function annotation to a level that previously was only achievable by sequence comparison.

Emerging evidence indicates that membrane lipids regulate protein networking by directly interacting with protein-interaction domains (PIDs). As a pilot study to identify and functionally annodate lipid-binding PIDs on a genomic scale, we performed experimental and computational studies of PDZ domains. Characterization of 70 PDZ domains showed that 40% had submicromolar membrane affinity. Using a computational model built from these data, we predicted the membrane binding properties of 2000 PDZ domains from 20 species. The accuracy of the prediction was experimentally validated for 26 PDZ domains. We also subdivided lipidbinding PDZ domains into three classes based on the interplay between membrane and protein binding sites. For different classes of PDZ domains, lipid binding regulates their protein interactions by different mechanisms. Functional studies of a PDZ domain protein, rhophilin2 suggest that all classes of lipid binding PDZ domains serve as genuine dual-specificity modules regulating protein interactions at the membrane under physiological conditions.

SkyLine, a high-throughput homology modeling pipeline tool, detects and models true sequence homologs to a given protein structure. Structures and models are stored in SkyBase with links to computational function annotation, as calculated by MarkUs. The SkyLine/SkyBase/MarkUs technology represents a novel structure-based approach that is more objective and versatile than other protein classification resources. This structure-centric strategy provides a multidimensional organization and coverage of protein space at the levels of family, function, and genome. The concept of "modelability", the ability to model sequences on related structures, provides a reliable criterion for membership in a protein family ("leverage") and underlies the unique success of this approach. The overall procedure is illustrated by its application to START domains, which comprise a Biomedical Theme for the Northeast Structural Genomics Consortium (NESG) as part of the Protein Structure Initiative (PSI). START domains are typically involved in the non-vesicular transport of lipids. While 19 experimentally determined structures are available, the family, whose evolutionary hierarchy is not well determined, is highly sequence diverse, and the ligand-binding potential of many family members is unknown. The SkyLine/SkyBase/MarkUs approach provides significant insights and predicts: 1) many more family members (~4,000) than any other resource; 2) the function for a large number of unannotated proteins; 3) instances of START domains in genomes from which they were thought to be absent; and 4) the existence of two types of novel proteins, those containing dual START domain and those containing N-terminal START domains.

Background: There is a great need for a high throughput computational tool for predicting the function of lipid binding domains on a genomic scale. Results: A newly developed computation protocol allows for genome-wide prediction of membrane binding properties of ANTH domains. Membrane binding properties of proteins can be systematically and reliably predicted by our combinatorial approach. Significance: A novel functionality of lipid binding domains can be computationally predicted.

The tetramerization domain of p53 is required for efficient tumor suppressor activity. This domain, however, also allows wild-type p53 to heterooligomerize with dominant negative tumor-derived p53 mutants. We explored the feasibility of substituting the native tetramerization domain of wild-type p53 with an engineered leucine zipper that assembles as a four-stranded coiled coil. The engineered zipper drove p53 tetramerization in vitro and p53 function in vivo. Furthermore, it alleviated transdominant inhibition by tumor-derived p53 mutants, implying that dominant negative mutants act by hetero-oligomerizing with wild-type p53. The ability of the engineered zipper to drive tetramerization was critical for p53 function, since p53 dimers, formed by substituting the p53 tetramerization domain with a native leucine zipper, were weak tumor suppressors.

Efficient delivery of heterologous molecules for treatment of cells is a great challenge in modern medicine and pharmacology. Cellpenetrating peptides (CPPs) may improve efficient delivery of a wide range of macromolecular cargos, including plasmid DNA, small interfering RNA, drugs, nanoparticulate pharmaceutical carriers, and anticancer drugs. In this paper, we present the history of CPPs' discovery with special attention drawn to sequences of viral origin. We also describe different CPP families with regard to their physicochemical properties and numerous mechanisms of CPP cell uptake by direct penetration and endocytotic pathways. A detailed description is focused on formation of carrier-cargo complexes, which are needed for practical use of CPPs in medicine and biotechnology. Examples of successful application of CPPs in treatment of human diseases are also presented, including decreased tumor growth and induction of cancer cell death. Finally, we review modern design appro...

Efficient delivery of heterologous molecules for treatment of cells is a great challenge in modern medicine and pharmacology. Cellpenetrating peptides (CPPs) may improve efficient delivery of a wide range of macromolecular cargos, including plasmid DNA, small interfering RNA, drugs, nanoparticulate pharmaceutical carriers, and anticancer drugs. In this paper, we present the history of CPPs' discovery with special attention drawn to sequences of viral origin. We also describe different CPP families with regard to their physicochemical properties and numerous mechanisms of CPP cell uptake by direct penetration and endocytotic pathways. A detailed description is focused on formation of carrier-cargo complexes, which are needed for practical use of CPPs in medicine and biotechnology. Examples of successful application of CPPs in treatment of human diseases are also presented, including decreased tumor growth and induction of cancer cell death. Finally, we review modern design approaches to novel CPPs and prediction of their activity. To sum up, the current review presents a thorough and up-to-date knowledge of CPPs and may be a valuable source of information for researchers in pharmacology designing new therapeutic agents.

Background The narrow-leafed lupin, Lupinus angustifolius L., is a grain legume species with a relatively compact genome. The species has 2n = 40 chromosomes and its genome size is 960 Mbp/1C. During the last decade, L. angustifolius genomic studies have achieved several milestones, such as molecular-marker development, linkage maps, and bacterial artificial chromosome (BAC) libraries. Here, these resources were integratively used to identify and sequence two gene-rich regions (GRRs) of the genome. Results The genome was screened with a probe representing the sequence of a microsatellite fragment length polymorphism (MFLP) marker linked to Phomopsis stem blight resistance. BAC clones selected by hybridization were subjected to restriction fingerprinting and contig assembly, and 232 BAC-ends were sequenced and annotated. BAC fluorescence in situ hybridization (BAC-FISH) identified eight single-locus clones. Based on physical mapping, cytogenetic localization, and BAC-end annotation, ...

The diving and thermoregulatory metabolic rates of two species of diving seabird, common (Uria aalge) and thick-billed murres (U. lomvia), were studied in the laboratory. Post-absorptive resting metabolic rates were similar in both species, averaging 7.8 W.kg-1, and were not different in air or water (15-20 ~ These values were 1.5-2 times higher than values predicted from published allometric equations. Feeding led to increases of 36 and 49%, diving caused increases of 82 and 140%, and preening led to increases of 107 and 196% above measured resting metabolic rates in common and thick-billed murres, respectively. Metabolic rates of both species increased linearly with decreasing water temperature; lower critical temperature was 15 ~ in common murres and 16 ~ in thick-billed murres. Conductance (assuming a constant body temperature) did not change with decreasing temperature, and was calculated at 3.59W.m-2.~ -1 and 4.68W.m-2.~ -1 in common and thick-billed murres, respectively. Murres spend a considerable amount of time in cold water which poses a significant thermal challenge to these relatively small seabirds. If thermal conductance does not change with decreasing water temperature, murres most likely rely upon increasing metabolism to maintain body temperature. The birds probably employ activities such as preening, diving, or food-induced thermogenesis to meet this challenge.

EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His 6 ) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. In vivo QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All proteins were solubilized in the same buffered n-dodecyl-β-D-maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. In vitro analysis of protein multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants (K d ) and maximum specific one-site binding (B max ) values for particular QCCs. In vitro analyses of both proteins demonstrated subtle but significant differences in multimerization and QCC binding. In vivo analysis indicates differences caused by the addition of the tag, we also observed differences in vitro that could be a result of the tag and/or the different purification methods.

A simple and fast procedure that allows the separation of small (1-3 kDa) peptides on glycine-SDS gels is described. Peptides were separated by glycine-SDS/PAGE as a result of in situ complexation of peptide/SDS during elecla'ophoretic migration and visualized by Coomassie blue staining. The data presented here shows the separation of small peptides of different isoelectric points, sizes, and hydrophobicity on polyacrylamide mini gels. Ten different peptides have been tested with this method. The data suggest the dependence of SDS/peptide complex formation and migration due to the number of basic amino acid residues, length of peptide and the hydrophobicity/hydrophilicity ratio.

We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI). In the moeBA228Tmutant strain, E. coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC. Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228Tmutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not. MoeB functions to sulfurylate MoaD, and in the structure of the MoeB–MoaD complex, Ala-228 is located in the interface region between the tw...

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane α-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (≤ 1 μM) in SDS-EmrE is monomeric, but upon increasing EmrE concentration, a variety of multimeric states can be observed by SDS-Tricine polyacrylamide gel electrophoresis (PAGE). Addition of the (QCC), tetraphenyl phosphonium (TPP), to SDS-EmrE samples enhanced EmrE multimer formation using SDS-Tricine PAGE. The relative shapes of EmrE multimers in SDS with or without TPP addition were determined by small angle neutron scattering (SANS) analysis and revealed that EmrE dimers altered in conformation depending on the SDS concentration. SANS analysis also revealed that relative shapes of larger EmrE multimers (≥ 100 nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS-EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.

The small multidrug resistance (SMR) protein family is a bacterial multidrug transporter family. As suggested by their title, SMR proteins are composed of four transmembrane α-helices of approximately 100-140 amino acids in length. Since their designation as a family, many homologues have been identified and characterized both structurally and functionally. In this review the topology, structure, drug resistance, drug binding, and transport mechanisms of the entire SMR protein family are examined. Additionally, updated bioinformatic analysis of predicted and characterized SMR protein family members was also conducted. Based on SMR sequence alignments and phylogenetic analysis of current members, we propose that this small multidrug resistance transporter family should be expanded into three subclasses: (i) the small multidrug pumps (SMP), (ii) suppressor of groEL mutation proteins (SUG), and a third group (iii) paired small multidrug resistance proteins (PSMR). The roles of these three SMR subclasses are examined, and the well-characterized members, such as Escherichia coli EmrE and SugE, are described in terms of their function and structural organization.

Small multidrug resistance (SMR) protein family members confer bacterial resistance to toxic antiseptics and are believed to function as dual topology oligomers. If dual topology is essential for SMR activity, then the topology bias should change as bacterial membrane lipid compositions alter to maintain a "neutral" topology bias. To test this hypothesis, a bioinformatic analysis of bacterial SMR protein sequences was performed to determine a membrane protein topology based on charged amino acid residues within loops, and termini regions according to the positive inside rule. Three bacterial lipid membrane parameters were examined, providing the proportion of polar lipid head group charges at the membrane surface (PLH), the relative hydrophobic fatty acid length (FAL), and the proportion of fatty acid unsaturation (FAU). Our analysis indicates that individual SMR pairs, and to a lesser extent SMR singleton topology biases, are significantly correlated to increasing PLH, FAL and FAU differences validating the hypothesis. Correlations between the topology biases of SMR proteins identified in Gram + compared to Gram-species and each lipid parameter demonstrated a linear inverse relationship.

The twin-arginine translocase (Tat) system is used by many bacteria and plants to move folded proteins across the cytoplasmic or thylakoid membrane. In most bacteria, the TatA protein is believed to form a defined pore in the membrane through homo-oligomerization with other TatA protomers. The predicted secondary structure of TatA includes a transmembrane helix, an amphipathic helix, and an unstructured C-terminal region. Here biophysical and structural investigations were performed on a synthetic peptide representing the amphipathic region of TatA (residues 22 to 44, abbreviated TatAH2). The C-terminal region of TatA (residues 44-89) was previously shown to be accessible from both the cytoplasmic and periplasmic sides of the membrane only when the membrane potential was intact, suggesting dependence of its topology on an energized membrane (Chan et al. 2007 Biochemistry 46: 7396-404). Such observation suggests that the TatAH2 region would have unique lipid interactions that may be related to the function of TatA during translocation and thus warranted further investigations. NMR and CD spectroscopy of TatAH2 show that it adopts a predominantly helical structure in a membrane environment while remaining unstructured in aqueous solution. Differential scanning calorimetry studies also reveal that TatAH2 interacts with DPPG lipids but not with DPPC, suggesting that negatively charged phospholipid head groups contribute to the membrane interactions with TatA.

Small multidrug resistance (SMR) protein family member, SugE, is an integral inner membrane protein that confers host resistance to antiseptic quaternary cation compounds (QCC). SugE studies generally focus on its resistance to limited substrates in comparison to SMR protein EmrE. This study examines the conformational characteristics of SugE protein in two detergents, sodium dodecyl sulphate (SDS) and dodecyl maltoside (DDM), commonly used to study SMR proteins. The influence of cetylpyridinium (CTP) and cetrimide (CET) using SugE aromatic residues (4W, 2Y, 1F) as intrinsic spectroscopic probes was also determined. Organically extracted detergent solubilized Escherichia coli SugE protein was examined by SDS-Tricine PAGE and various spectroscopic techniques. SDS-Tricine PAGE analysis of SugE in either detergent demonstrates the protein predominates as a monomer but also dimerizes in SDS. Far-UV region circular dichroism (CD) analysis determined that the overall α-helix content SugE in SDS and DDM was almost identical and unaltered by QCC. Near-UV region CD, fluorescence, and second-derivative ultraviolet absorption (SDUV) indicated that only DDM-SugE promoted hydrophobic environments for its Trp and Tyr residues that were perturbed by QCC addition. This study identified that only the tertiary structure of SugE protein in DDM is altered by QCC.

To investigate the pro-oxidative activity of nutmeg, safflower, and lemongrass, methanolic extracts of each were incubated with bovine serum albumin (BSA) in the absence or presence of Fe 3+ /EDTA/H 2 O 2 , Fe 2+ /ascorbate, or Cu 2+ /H 2 O 2 at 37°C. The protein cleavage by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), protein and free amino groups contents were determined. As a result, BSA degradation was observed on SDS-PAGE. This corresponded with decrease in protein concentration and free amino group elevation in the reaction mixture. Such degradation was inhibited in the presence of mannitol, the hydroxyl radical scavenger, indicating that the pro-oxidative activity in these spices caused hydroxyl radical formation.

The bioeconomy era will rely on efficient fractionation of renewable resources via integrated biorefineries. The food supply chain waste, despite its inherent variability, could evolve into an important industrial feedstock on account of its availability, versatility, and sustainability, for the production of bio-based products. Waste streams generated from all stages of the life cycle of food products could be refined into different fractions, which will be either purified to high-value molecules or converted via green chemical and/or biotechnological routes for the production of biobased products. A working group of the EUBis COST Action TD1203 is taking steps to gather a critical mass of knowledge and expertise to create innovation and technological breakthroughs.

Objective. To determine by the Next-Generation Sequencing Illumina method to identify the genes related to the pigmentation of the fungus in the DNA sequencing of enzymes and secondary metabolites. As well as in determining the phenotypic and genotypic characteristics in general of the species A. salvadorensis. Methodology. For the identification of cluster genes in enzymes, proteins, and secondary metabolites, MACROGEN For the identification of cluster genes in enzymes, proteins, and secondary metabolites, MACROGEN used the following systems: the EggNOG system summary: Orthology Frequency within COG (Clusters of Orthologous Groups) Categories in sequencing reading., MetaCyc is a comprehensive database that provides in-depth information on metabolic pathways, biochemical reactions, enzymes, and associated compounds. UniRef (UniProt Reference Clusters) organizes groups of protein sequences from the UniProt Knowledgebase including isoforms and selected UniParc entries into clustered sets to enhance sequence analysis and annotation efficiency. EggNOG: Relative Abundance in Hierarchical Categories of COG (Clusters of Orthologous Groups) using CPM, KEGG Orthology (KO) and KEGG summary: Orthology Frequency within Main and Sub-Categories. DNA-seq. Conclusions. 14 enzymes and secondary metabolites were found in the production of black pigments produced by the fungus by oxidative stress.

During inflammation of the gums, resident cells of the periodontium, gingival fibroblasts (GFs), interact with heterogeneous cell populations of the innate and adaptive immune system that play a crucial role in protecting the host from pathogenic infectious agents. We investigated the effects of chronic inflammation, by exposing peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocyte (PBL) cultures, and GF–PBMC cocultures to Toll-like receptor 2 (TLR2) and TLR4 activators for 21 days and assessed whether this influenced leukocyte retention, survival, and proliferation. Chronic stimulation of PBMC–GF cocultures with TLR2 and TLR4 agonists induced a reduction of NK (CD56+CD3−), T (CD3+), and B (CD19+) cells, whereas the number of TLR-expressing monocytes were unaffected. TLR2 agonists doubled the T cell proliferation, likely of a selective population, given the net decrease of T cells. Subsequent chronic exposure experiments without GF, using PBMC and PBL cultures, sho...

Extracellular acidification by osteoclasts is essential to bone resorption. During proton pumping, intracellular pH (pH i ) is thought to be kept at a near-neutral level by chloride/bicarbonate exchange. Here we show that the Na + -independent chloride/bicarbonate anion exchanger 2 (Ae2) is relevant for this process in the osteoclasts from the long bones of Ae2 a,b -/-mice (deficient in the main isoforms Ae2a, Ae2b 1 and Ae2b 2 ). Although the long bones of these mice had normal numbers of multinucleated osteoclasts, these cells lacked a ruffled border and displayed impaired bone resorption activity, resulting in an osteopetrotic phenotype of long bones. Moreover, in vitro osteoclastogenesis assays using long-bone marrow cells from Ae2 a,b -/-mice suggested a role for Ae2 in osteoclast formation, as fusion of pre-osteoclasts for the generation of active multinucleated osteoclasts was found to be slightly delayed. In contrast to the abnormalities observed in the long bones, the skull of Ae2 a,b -/-mice showed no alterations, indicating that calvarial osteoclasts may display normal resorptive activity. Microfluorimetric analysis of osteoclasts from normal mice showed that, in addition to Ae2 activity, calvarial osteoclasts -but not long-bone osteoclasts -possess a sodiumdependent bicarbonate transporting activity. Possibly, this might compensate for the absence of Ae2 in calvarial osteoclasts of Ae2 a,b -/-mice.

Osteoclasts mediate bone resorption, which is critical for bone development, maintenance, and repair. Proper control of osteoclast development and function is important and deregulation of these processes may lead to bone disease, such as osteoporosis. Previous studies have shown that the cytosolic protein tyrosine phosphatase SHP-1 acts as a suppressor of osteoclast differentiation and function, but putative inhibitory receptors that mediate recruitment and activation of SHP-1 in osteoclasts have remained unknown. In the present study, we identify the SHP-1-recruiting inhibitory immunoreceptor signal regulatory protein (SIRP) ␣ as a negative regulator of osteoclast activity. SIRP␣ is expressed by osteoclasts, and osteoclasts from mice lacking the SIRP␣ cytoplasmic tail and signaling capacity display enhanced bone resorption in vitro. Consequently, SIRP␣-mutant mice have a significantly reduced cortical bone mass. Furthermore, osteoclasts from SIRP␣-mutant mice show an enhanced formation of actin rings, known to be instrumental in bone resorption. SIRP␣ mutation did not significantly affect osteoclast formation, implying that the role of SIRP␣ was limited to the regulation of mature osteoclast function. This identifies SIRP␣ as a bona fide inhibitory receptor that regulates the bone-resorption activity and supports a concept in which osteoclast function is balanced by the signaling activities of activating and inhibitory immunoreceptors.-

The risk of nutrient deficiencies increases with age in our modern Western society, and vitamin B 12 deficiency is especially prevalent in the elderly and causes increased homocysteine (Hcy) and methylmalonic acid (MMA) levels. These three factors have been recognized as risk factors for reduced bone mineral density and increased fracture risk, though mechanistic evidence is still lacking. In the present study, we investigated the influence of B 12 , Hcy, and MMA on differentiation and activity of bone cells. B 12 deficiency did not affect the onset of osteoblast differentiation, maturation, matrix mineralization, or adipocyte differentiation from human mesenchymal stem cells (hMSCs). B 12 deficiency caused an increase in the secretion of Hcy and MMA into the culture medium by osteoblasts, but Hcy and MMA appeared to have no effect on hMSC osteoblast differentiation. We further studied the effect of B 12 , Hcy, and MMA on the formation of multinucleated tartrate-resistant acid phosphatase-positive osteoclasts from mouse bone marrow. We observed that B 12 did not show an effect on osteoclastogenesis. However, Hcy as well as MMA were found to induce osteoclastogenesis in a dose-dependent manner. On the basis of these results, we conclude that B 12 deficiency may lead to decreased bone mass by increased osteoclast formation due to increased MMA and Hcy levels.

The multinucleated osteoclast has a unique function: degradation of mineralized tissues. It is generally taken that all osteoclasts are alike, independent of the skeletal site where they exert their activity. Recent data, however, question this view as they show that osteoclasts at different bony sites appear to differ, for example in the machinery responsible for resorption. Support for the notion that there may be heterogeneity in osteoclasts is obtained from studies in which osteoclast activity is inhibited and from observations in osteopetrosis and inflammatory bone conditions. In this review we discuss the available evidence and propose the existence of bone-site-specific osteoclast heterogeneity.

Adhesion of circulating tumor cells to vascular endothelium is mediated by specialized molecules that are functional under shear forces exerted by hematogenous flow. Endothelial E-selectin binding to glycoforms of CD44 mediates shear-resistant cell adhesion in numerous physiological and pathological conditions. However, this pathway is poorly understood in breast cancer and is the focus of the present investigation. All breast cancer cell lines used in this study strongly expressed CD44. In particular, BT-20 cells expressed CD44s and multiple CD44v isoforms, whereas MDA-MB-231 cells predominantly expressed CD44s but weakly expressed CD44v isoforms. CD44 expressed by BT-20, but not MDA-MB-231, cells possessed E-selectin ligand activity as detected by Western blotting and antigen capture assays. Importantly, CD44 expressed by intact BT-20 cells were functional E-selectin ligands, regulating cell rolling and adhesion under physiological flow conditions, as found by shRNA-targeted silen...

Introduction: DNA methylation constitutes one important epigenetic mechanism that regulates gene expression in human cells. With regard to obesity, bariatric surgery-induced weight loss has been associated with promoter methylation changes in several genes. Hyperleptinemia is a characteristic feature of obesity. The underlying regulating mechanisms have not yet been completely elucidated. Methods: We investigated the methylation of the promoters of the leptin gene (LEP) and the leptin receptor gene (LEPR) as well as leptin expression in pre- and postbariatric surgery patients using a comparative cross-sectional design. Results: Our results revealed significantly higher LEP promoter methylation patterns in prebariatric surgery patients compared to postoperatively. DNA methylation of the LEPR promoter was significantly higher in the postoperative group. Moreover, we found significantly higher leptin serum levels in patients before the bariatric surgery than afterwards. Discussion: The...

Background: Diabetes increases the risk of periodontal disease. Integrating periodontal care with the treatment of type 2 diabetes (T2DM) may facilitate the management of both diseases. To evaluate the periodontal status at two years in patients with T2DM and assess glycated hemoglobin (HbA1c) according to the severity and extension of periodontitis. Methods: This is a descriptive cohort study. Full-mouth periodontal evaluation was performed and blood samples were obtained to analyze HbA1c at baseline, 3, 12 and 24 months. Clinical attachment level, probing depth, bleeding on probing and oral hygiene were measured. We analyzed mean HbA1c according to severity and extension of periodontitis. We included 160 patients with T2DM. Basal HbA1c was 7.7 ± 2.2%. Periodontal health increased 27.5 % at 24 months. At baseline, moderate and severe periodontitis was present in 29.9% and 13.4% patients, respectively. At two years, 22.5% and 11.8% had moderate and severe periodontitis, respectively. Bleeding on probing decreased from 15.7% to 6.4% at 24 months. HbA1c was higher in subjects with severe and generalized periodontitis. The periodontal status in patients with newly diagnosis of T2DM who received a comprehensive diabetes care improved at 3 months. This improvement was maintained at 24 months of follow-up.

Introduction The Roux-en-Y gastric bypass (RYGB) is one of the most widely used techniques for bariatric surgery. After RYGB, weight loss up to 50%-70% of excess body weight, improvement of insulin-resistance, changes in food preferences and improvements in cognitive performance have been reported. This protocol describes a longitudinal study of the neural correlates associated with food-processing and cognitive performance in patients with morbid obesity before and after RYGB relative to lean controls. Methods and analysis This study is a pre-post casecontrol experiment. Using functional MRI, the neural responses to food stimuli and a working memory task will be compared between 25 patients with obesity, pre and post RYGB, and a matched, lean control group. Resting state fMRI will be measured to investigate functional brain connectivity. Baseline measurements for both groups will take place 4 weeks prior to RYGB and 12 months after RYGB. The effects of RYGB on peptide tyrosine tyrosine and glucagon-like polypeptide-1 will also be determined. The project has received ethical approval by the local medical ethics committee of the Carl-von-Ossietzky University of Oldenburg, Germany (registration: 2017-073). Results will be published in a peer-reviewed journal as original research and on international conferences. trial registration number DRKS00012495; Pre-results.

Intracellular signaling pathways not only control cell proliferation and survival, but also regulate the provision of cellular energy and building blocks through mitochondrial and non-mitochondrial metabolism. Wild-type and oncogenic RAF kinases have been shown to prevent apoptosis following the removal of interleukin 3 (IL-3) from mouse pro-myeloid 32D cells by reducing mitochondrial reactive oxygen species production. To study primary effects of RAF on mitochondrial energy metabolism, we applied high-resolution respirometry after short-term IL-3 deprivation (8 h), before 32D cells show detectable signs of cell death. Respiration in intact 32D cells was suppressed as an early event following removal of IL-3, but remained more stable in 32D cells expressing the v-RAF oncogene. In permeabilized 32D cells deprived of IL-3, respiratory capacities of the NADH-pathway, the convergent NADH&succinate-pathway, and Complex IV activity were decreased compared to cells grown in the presence of...