The Domon Family of Plant Plasma Membrane B-Type Cytochromes: Heterologous Expression, Biochemical Characterization and Physiological Roles in Vivo

The DOMON domain is a domain widespread in nature, predicted to fold in a β-sandwich structure. In plants, AIR12 is constituted by a single DOMON domain located in the apoplastic space and is GPI-modified for anchoring to the plasma membrane. Arabidopsis thaliana AIR12 has been heterologously expressed as a recombinant protein (recAtAIR12) in Pichia pastoris. Spectrophotometrical analysis of the purified protein showed that recAtAir12 is a cytochrome b. RecAtAIR12 is highly glycosylated, it is reduced by ascorbate, superoxide and naftoquinones, oxidised by monodehydroascorbate and oxygen and insensitive to hydrogen peroxide. The addition of recAtAIR12 to permeabilized plasma membranes containing NADH, FeEDTA and menadione, caused a statistically significant increase in hydroxyl radicals as detected by electron paramagnetic resonance. In these conditions, recAtAIR12 has thus a pro-oxidant role. Interestingly, AIR12 is related to the cytochrome domain of cellobiose dehydrogenase which is involved in lignin degradation, possibly via reactive oxygen species (ROS) production. In Arabidopsis the Air12 promoter is specifically activated at sites where cell separations occur and ROS, including •OH, are involved in cell wall modifications. air12 knock-out plants infected with Botrytis cinerea are more resistant than wild-type and air12 complemented plants. Also during B. cinerea infection, cell wall modifications and ROS are involved. Our results thus suggest that AIR12 could be involved in cell wall modifying reactions by interacting with ROS and ascorbate. CyDOMs are plasma membrane redox proteins of plants that are predicted to contain an apoplastic DOMON fused with a transmembrane cytochrome b561 domain. CyDOMs have never been purified nor characterised. The trans-membrane portion of a soybean CyDOM was expressed in E. coli but purification could not be achieved. The DOMON domain was expressed in P. pastoris and shown to be itself a cytochrome b that could be reduced by ascorbate.

Functional characterization of modified vaccinia virus Ankara-encoded anti-apoptotic proteins

Candidate vaccines based on the highly attenuated orthopoxvirus strain MVA are tested against various infectious and cancer diseases and, more profound, vaccines based on wildtype and recombinant viruses have been found safe and immunogenic in clinical trials. Compared to conventional vaccine strains, MVA lacks many functional genes for potentially important regulators of virus-host interactions. However, some gene functions responsible for counteraction of cellular antiviral pathways are still conserved in the genome of MVA and the inhibition of apoptosis seems to be one important mechanism, the virus is still able to interact with.rnrnVaccinia viruses encode several proteins which prevent the induction of virus-induced apoptosis. The vaccinia virus anti-apoptotic protein F1 was shown to counteract the activation of the mitochondrial pathway of apoptosis in a highly effective manner. Another vaccinia virus protein, N1, like F1 shows structural and functional similarity to members of the cellular anti-apoptotic bcl-2 family and was also shown to inhibit apoptosis. The vaccinia virus early protein E3 inhibits programmed cell death by binding to and sequestration of dsRNA molecules, normally inducing cellular antiviral pathways also driving the induction of apoptosis. All three anti-apoptotic genes were functionally analyzed during this work.rn

Varying spin state composition by the choice of capping ligand in a family of molecular chains: detailed analysis of magnetic properties of chromium(III) horseshoes

We report a detailed physical analysis on a family of isolated, antiferro-magnetically (AF) coupled, chromium(III) finite chains, of general formula (Cr(RCO(2))(2)F)(n) where the chain length n = 6 or 7. Additionally, the chains are capped with a selection of possible terminating ligands, including hfac (= 1,1,1,5,5,5-hexafluoropentane-2,4-dionate(1-)), acac (= pentane-2,4-dionate(1-)) or (F)(3). Measurements by inelastic neutron scattering (INS), magnetometery and electron paramagnetic resonance (EPR) spectroscopy have been used to study how the electronic properties are affected by n and capping ligand type. These comparisons allowed the subtle electronic effects the choice of capping ligand makes for odd member spin 3/2 ground state and even membered spin 0 ground state chains to be investigated. For this investigation full characterisation of physical properties have been performed with spin Hamiltonian parameterisation, including the determination of Heisenberg exchange coupling constants and single ion axial and rhombic anisotropy. We reveal how the quantum spin energy levels of odd or even membered chains can be modified by the type of capping ligand terminating the chain. Choice of capping ligands enables Cr-Cr exchange coupling to be adjusted by 0, 4 or 24%, relative to Cr-Cr exchange coupling within the body of the chain, by the substitution of hfac, acac or (F)(3) capping ligands to the ends of the chain, respectively. The manipulation of quantum spin levels via ligands which play no role in super-exchange, is of general interest to the practise of spin Hamilton modelling, where such second order effects are generally not considered of relevance to magnetic properties.

Increased parasite resistance and recurrent airway obstruction in horses of a high-prevalence family

BACKGROUND: Equine recurrent airway obstruction (RAO) shares many characteristics with human asthma. In humans, an inverse relationship between susceptibility to asthma and resistance to parasites is suspected. HYPOTHESIS/OBJECTIVES: Members of a high-incidence RAO half-sibling family (F) shed fewer strongylid eggs compared with RAO-unaffected pasture mates (PM) and that RAO-affected horses shed fewer eggs than RAO-unaffected half-siblings. ANIMALS: Seventy-three F and 73 unrelated, age matched PM. METHODS: Cases and controls kept under the same management and deworming regime were examined. Each individual was classified as RAO affected or RAO unaffected and fecal samples were collected before and 1-3 weeks and 3 months after deworming. Samples were analyzed by combined sedimentation-flotation and modified McMaster methods and classified into 3 categories of 0 eggs per gram of feces (EpG), 1-100 EpG, and > 100 EpG, respectively. RESULTS: PM compared with RAO-affected F had a 16.7 (95% confidence interval [CI]: 2.0-136.3) times higher risk for shedding > 100 EpG compared with 0 EpG and a 5.3 (95% CI: 1.0-27.4) times higher risk for shedding > 100 EpG compared with 0 EpG. There was no significant effect when RAO-unaffected F were compared with their PM. RAO-unaffected compared with RAO-affected offspring had a 5.8 (95% CI: 0.0-1.0) times higher risk for shedding 1-100 EpG. Age, sex, breed, and sharing pastures with other species had no significant confounding effects. CONCLUSION AND CLINICAL IMPORTANCE: RAO is associated with resistance against strongylid parasites in a high-prevalence family.

Proton-coupled oligopeptide transporter family SLC15: physiological, pharmacological and pathological implications

Mammalian members of the proton-coupled oligopeptide transporter family (SLC15) are integral membrane proteins that mediate the cellular uptake of di/tripeptides and peptide-like drugs. The driving force for uphill electrogenic symport is the chemical gradient and membrane potential which favors proton uptake into the cell along with the peptide/mimetic substrate. The peptide transporters are responsible for the absorption and conservation of dietary protein digestion products in the intestine and kidney, respectively, and in maintaining homeostasis of neuropeptides in the brain. They are also responsible for the absorption and disposition of a number of pharmacologically important compounds including some aminocephalosporins, angiotensin-converting enzyme inhibitors, antiviral prodrugs, and others. In this review, we provide updated information on the structure-function of PepT1 (SLC15A1), PepT2 (SLC15A2), PhT1 (SLC15A4) and PhT2 (SLC15A3), and their expression and localization in key tissues. Moreover, mammalian peptide transporters are discussed in regard to pharmacogenomic and regulatory implications on host pharmacology and disease, and as potential targets for drug delivery. Significant emphasis is placed on the evolving role of these peptide transporters as elucidated by studies using genetically modified animals. Whenever possible, the relevance of drug-drug interactions and regulatory mechanisms are evaluated using in vivo studies.

The Family Partners Credit Card: A Token Economy System Adapted for Intensive Family Preservation Services to Enable Families to Manage Difficult Behavior of Adolescents

Increasingly, families referred for Intensive Family Preservation Services have not experienced a crisis of maltreatment, focused on the parent; rather these families have children with chronic behavioral difficulties for which their parents lack the skills to cope. These are the same families whose children were formerly placed in residential programs. This paper presents The Family Partners Credit Card System, incorporating behavioral techniques developed to treat children in out-of-home placements into a family preservation model. Two case examples illustrate how the system has been modified to train biological or adoptive parents in parenting skills, enable them to teach their children pro-family behaviors, and reinforce new behaviors through a credit card that monitors an ongoing balance of credits and fines.

Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction

Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.

Translation and cultural adaptation of the Stigma-Devaluation scale for use among family caregivers of mentally ill relatives in Jordan

Background/significance. Mental illness stigma is a matter of great concern to family caregivers. Few research studies have been conducted in the Arab World on family caregivers' perception of stigma associated with caring for a mentally ill relative. Review of the literature on measurement of the concept of stigma related to caring for a mentally ill relative yielded no instrument appropriate for use in a Jordanian sample. Reliable and valid instruments to measure stigma perception among family caregivers are needed for research and practice, particularly in Arabic speaking populations. ^ Purpose. The purposes of this study were: (1) translate the Stigma-Devaluation scale (SDS) into Arabic, modifying it to accurately reflect the cultural parameters specific to Jordan, and (2) test the reliability, the content and construct validity of the Arabic version of the SDS for use among a sample of family members of mentally ill relatives in Jordan. ^ Design. Methodologic, cross-sectional. ^ Methods. The SDS was translated into Arabic language, modified and culturally adapted to the Jordanian culture by a translation model which incorporates a cultural adaptation process. The Arabic SDS was evaluated in a sample of 164 family caregivers in the outpatient mental health clinic in Irbid-Jordan. Cronbach's alpha estimation of internal consistency was used to assess the reliability of the SDS. Construct validity was determined by confirmatory factor analysis (CFA). Measurements of content validity and reading level of the Arabic SDS were included. ^ Findings. Content Validity Index was determined to be 1.0. Reading level of the Arabic SDS was considered at a 6th grade or lower Cronbach's alpha coefficient of the modified Arabic SDS total scale was .87. Initial results of CFA did not fully support the proposed factor structures of the SDS or its subscales. After modifications, the indices indicated that the modified model of each subscale had satisfactory fit. ^ Conclusion. This study provided psychometric evidence that the modified Arabic SDS translated and culturally adapted instrument, is valid and conceptually consistent with the content of the original English SDS in measuring stigma perception among families of mentally ill relatives in Jordan. ^

Effectiveness of Community Case Management in Family Risk Reduction

This study evaluated a modified home-based model of family preservation services, the long-term community case management model, as operationalized by a private child welfare agency that serves as the last resort for hard-to-serve families with children at severe risk of out-of-home placement. The evaluation used a One-Group Pretest-Posttest design with a modified time-series design to determine if the intervention would produce a change over time in the composite score of each family's Child Well-Being Scales (CWBS). A comparison of the mean CWBS scores of the 208 families and subsets of these families at the pretest and various posttests showed a statistically significant decrease in the CWBS scores, indicating decreased risk factors. The longer the duration of services, the greater the statistically significant risk reduction. The results support the conclusion that the families who participate in empowerment-oriented community case management, with the option to extend service duration to resolve or ameliorate chronic family problems, have experienced effective strengthening in family functioning.

Complete structure of the human alpha-albumin gene, a new member of the serum albumin multigene family.

The nucleotide sequence of the human alpha-albumin gene, including 887 bp of the 5'-flanking region and 1311 bp of the 3-flanking region (24,454 in total), was determined from three overlapping lambda phage clones. The sequence spans 22,256 bp from the cap site to the polyadenylylation site, revealing a gene structure of 15 exons separated by 14 introns. The methionine initiation codon ATG is within exon 1; the termination codon TGA is within exon 14. Exon 15 is entirely untranslated and contains the polyadenylylation signal AATAAA. The deduced polypeptide chain is composed of a 21-amino-acid leader peptide, followed by 578 amino acids of the mature protein. There are seven repetitive DNA elements (Alu and Kpn) in the introns and 3-flanking region. The sizes of the 15 alpha-albumin exons match closely those of the albumin, alpha-fetoprotein, and vitamin D-binding protein genes. The exons are symmetrically placed within the three domains of the individual proteins, and they share a characteristic codon splitting pattern that is conserved among members of the gene family. The results provide strong evidence that alpha-albumin belongs to, and most likely completes with, the serum albumin gene family. Based on structural similarity, alpha-albumin appears to be most closely related to alpha-fetoprotein. The complete structure of this family of four tandemly linked genes provides a well-characterized approximately 200 kb locus in the 4q subcentromeric region of the human genome.

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues.

Yeast synaptobrevin homologs are modified posttranslationally by the addition of palmitate.

Yeast possess two homologs of the synaptobrevin family of vesicle-associated membrane proteins that function in membrane recognition and vesicle fusion. Yeast proteins Snc1 and Snc2 localize to secretory vesicles and are required for constitutive exocytosis. They also form a physical complex with a plasma membrane protein, Sec9, which is necessary for vesicle docking and fusion to occur in vivo. Formation of this molecular complex, as a prerequisite for vesicle fusion, appears to have been conserved evolutionarily. Here we demonstrate that Snc proteins undergo a single posttranslational modification with the addition of a palmitate moiety to Cys-95 in Snc1. Modification of Cys-95 (which is located proximal to the transmembrane domain) is rapid, occurs in the endoplasmic reticulum, and is long-lasting. Mutation of Cys-95 to Ser-95 blocks palmitoylation and appears to affect Snc protein stability. This provides evidence that synaptobrevin-like proteins are modified posttranslationally, and we predict that fatty acylation may be common to those found in higher eukaryotes.

Biochemical characterization and DNA binding properties of the MocR family member GabR, a PLP-dependent transcription factor

GabR è un fattore di trascrizione chimerico appartenente alla famiglia dei MocR/GabR, costituito da un dominio N-terminale elica-giro-elica di legame al DNA e un dominio effettore e/o di oligomerizzazione al C-terminale. I due domini sono connessi da un linker flessibile di 29 aminoacidi. Il dominio C-terminale è strutturalmente omologo agli enzimi aminotransferasici fold-type I, i quali, utilizzando il piridossal-5’-fosfato (PLP) come cofattore, sono direttamente coinvolti nel metabolismo degli aminoacidi. L’interazione contemporanea di PLP e acido γ-aminobutirrico (GABA) a GabR fa sì che questa promuova la trascrizione di due geni, gabT e gabD, implicati nel metabolismo del GABA. GabR cristallizza come un omodimero con una configurazione testa-coda. Il legame con la regione promotrice gabTD avviene attraverso il riconoscimento specifico di due sequenze dirette e ripetute (ATACCA), separate da uno spacer di 34 bp. In questo studio sono state indagate le proprietà biochimiche, strutturali e di legame al DNA della proteina GabR di Bacillus subtilis. L’analisi spettroscopica dimostra che GabR interagisce con il PLP formando l’aldimina interna, mentre in presenza di GABA si ottiene l’aldimina esterna. L’interazione fra il promotore gabTD e le forme holo e apo di GabR è stata monitorata mediante Microscopia a Forza atomica (AFM). In queste due condizioni di legame è stata stimata una Kd di circa 40 ηM. La presenza di GABA invece, determinava un incremento di circa due volte della Kd, variazioni strutturali nei complessi GabR-DNA e una riduzione del compattamento del DNA alla proteina, indipendentemente dalla sequenza del promotore in esame. Al fine di valutare il ruolo delle caratteristiche topologiche del promotore, sono state inserite cinque e dieci bp all’interno della regione spacer che separa le due sequenze ripetute dirette riconosciute da GabR. I significativi cambiamenti topologici riscontrati nel frammento aggiunto di cinque bp si riflettono anche sulla forte riduzione dell’affinità di legame verso la proteina. Al contrario, l’inserzione di 10 bp provoca solamente l’allontanamento delle sequenze ripetute dirette. L’assenza quindi di cambiamenti significativi nella topologia di questo promotore fa sì che l’affinità di legame per GabR rimanga pressoché inalterata rispetto al promotore non mutato. L’analisi del potenziale elettrostatico superficiale di GabR mostra la presenza di una fascia carica positivamente che si estende lungo un’intera faccia della proteina. Per verificare l’importanza di questa caratteristica di GabR nel meccanismo di interazione al DNA, sono stati preparati ed indagati i mutanti R129Q e K362-366Q, in cui la carica positiva superficiale risultava indebolita. L’affinità di legame dei mutanti di GabR per il DNA era inferiore rispetto alla proteina non mutata, in particolar modo nel mutante K362-366Q. Le evidenze acquisite suggeriscono che la curvatura intrinseca del promotore ed il corretto orientamento delle sequenze sulla doppia elica, più della distanza che le separa, siano critici per sostenere l’interazione con GabR. Oltre a questo, la superficie positiva di GabR è richiesta per accomodare la curvatura del DNA sul corpo della proteina. Alla luce di questo, l’interazione GabR-gabTD è un esempio di come il riconoscimento specifico di sequenze, la topologia del DNA e le caratteristiche strutturali della proteina siano contemporaneamente necessarie per sostenere un’interazione proteina-DNA stabile.

Haloferax mediterranei GlnK proteins are post-translationally modified by uridylylation

In this work we report for the first time a post-translational modification of PII homologues from the Archaea Domain. Haloferax mediterranei is the first haloarchaea whose PII proteins have been studied, it possesses two of them (GlnK1 and GlnK2), both encoded adjacent to a gene for the ammonia transporter Amt. An approach based on 2DE, anti-GlnK immunoblot and peptide mass fingerprint (MALDI-TOF-MS) of the reactive spots showed that GlnK proteins in H. mediterranei are post-translationally uridylylated. A third spot with lower pI suggests the existence of a non-descript post-translational modification in this protein family.

Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR

G-protein coupled receptors (GPCRs) typically have a functionally important C-terminus which, in the largest subfamily (family A), includes a membrane-parallel eighth helix. Mutations of this region are associated with several diseases. There are few C-terminal studies on the family B GPCRs and no data supporting the existence of a similar eighth helix in this second major subfamily, which has little or no sequence homology to family A GPCRs. Here we show that the C-terminus of a family B GPCR (CLR) has a disparate region from N400 to C436 required for CGRP-mediated internalization, and a proximal region of twelve residues (from G388 to W399), in a similar position to the family A eighth helix, required for receptor localization at the cell surface. A combination of circular and linear dichroism, fluorescence and modified waterLOGSY NMR spectroscopy (SALMON) demonstrated that a peptide mimetic of this domain readily forms a membrane-parallel helix anchored to the liposome by an interfacial tryptophan residue. The study reveals two key functions held within the C-terminus of a family B GPCR and presents support for an eighth helical region with striking topological similarity to the nonhomologous family A receptor. This helix structure appears to be found in most other family B GPCRs.