The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction.

Quinone reductase [NAD(P)H:(quinone acceptor) oxidoreductase, EC 1.6.99.2], also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. These two-electron reductions participate in the reductive bioactivation of cancer chemotherapeutic agents such as mitomycin C in tumor cells. Thus, surprisingly, the same enzymatic reaction that protects normal cells activates cytotoxic drugs used in cancer chemotherapy. The 2.1-A crystal structure of rat liver quinone reductase reveals that the folding of a portion of each monomer is similar to that of flavodoxin, a bacterial FMN-containing protein. Two additional portions of the polypeptide chains are involved in dimerization and in formation of the two identical catalytic sites to which both monomers contribute. The crystallographic structures of two FAD-containing enzyme complexes (one containing NADP+, the other containing duroquinone) suggest that direct hydride transfers from NAD(P)H to FAD and from FADH2 to the quinone [which occupies the site vacated by NAD(P)H] provide a simple rationale for the obligatory two-electron reductions involving a ping-pong mechanism.

Disulfide oxidoreductase activity of Shigella flexneri is required for release of Ipa proteins and invasion of epithelial cells.

Secretion of IpaB, IpaC, and IpaD proteins of Shigella flexneri, essential for the invasion of epithelial cells, requires a number of proteins encoded by the spa and mxi loci on the large plasmid. Introduction of dsbA::Tn5 into S.flexneri from Escherichia coli K-12 reduced invasiveness, which resulted from a decrease in the capacity to release IpaB, IpaC, and IpaD proteins into the external medium. Examination of the surface-presented Ipa proteins of the dsbA mutant, however, revealed Ipa proteins at levels similar to those on wild-type cells. Since the defective phenotype was similar to that of the spa32 mutant of S. flexneri and the Spa32 sequence possessed two Cys residues, the effect of dsbA mutation of the folding structure of Spa32 under reducing conditions and on the surface expression of Spa32 was investigated. The results indicated that Spa32 was a disulfide-containing protein whose correctly folded structure was required for its presentation on the outer membrane. Indeed, replacing either one of the two Cys residues in Spa32 with Ser by site-directed mutagenesis reduced its capacity to release Ipa proteins into the external medium and led to the accumulation of Spa32 protein in the periplasm. These results indicated that the DsbA protein performs an essential function during the invasion of mammalian cells, by facilitating transport of the Spa32 protein across the outer membrane.

HMG box transcription factor gene Hbp1 is expressed in germ cells of the developing mouse testis

HMG box containing protein 1 (HBP1) is a high mobility group domain transcriptional repressor that regulates proliferation in differentiated tissues. We have found mouse Hbp1 to be expressed strongly in the embryonic mouse testis from approximately 12.5 days post coitum, compared with low levels of expression in the embryonic ovary. Expression of Hbp1 is maintained in the developing testis beyond the onset of spermatogenesis after birth. Whole-mount in situ hybridisation analysis showed that expression of Hbp1 in the XY gonad is localized within the developing testis cords, the precursors of the seminiferous tubules. Expression of Hbp1 is not apparent in testis cords of gonads from homozygous We mutant embryos, which lack germ cells. In situ hybridisation analysis on cryosectioned embryonic testis indicated that Hbp1 expression resembles that of the germ cell marker Oct4. We conclude that Hbp1 is up-regulated specifically in germ cells of the developing XY gonad. The expression of Hbp1 in XY germ cells appears to correlate with the onset of mitotic arrest in these cells. (C) 2004 Wiley-Liss, Inc.

Expression of the tudor-related gene Tdrd5 during development of the male germline in mice

Homologues of Drosophila germ cell determinant genes such as vasa, nanos and tudor have recently been implicated in development of the male germline in mice. In the present study, the mouse gene encoding Tudor domain containing protein 5 (TDRD5) was isolated from a 12.5-13.5 days post coitum (dpc) male-enriched subtracted cDNA library. Whole-mount in situ hybridization analysis of Tdrd5 expression in the mouse embryonic gonad indicated that this gene is upregulated in the developing testis from 12.5 dpc, with expression levels remaining higher in testis than ovary throughout embryogenesis. Expression of Tdrd5 was absent in testes isolated from W-e/W-e embryos, which lack germ cells. In situ hybridization (ISH) on cryosectioned 13.5 dpc testes suggests that expression of Tdrd5, like that of Oct4, is restricted to germ cells. Northern hybridization analysis of expression in adult tissues indicated that Tdrd5 is expressed in the testis only, implying that expression of this gene is restricted to the male germline throughout development to adulthood. (C) 2004 Elsevier B.V. All rights reserved.

Particulate carriers as immunological adjuvants

In recent years, much interest has focused on the significance of inducing not only systemic immunity but also good local immunity at susceptible mucosal surfaces. A new field of mucosal immunity has been established as information accumulates on gut-associated lymphoid tissue, bronchus-associated lymphoid tissue and nasal-associated lymphoid tissue (GALT, BALT and NALT, respectively) and on their role in both local and systemic immune responses. This project, following the line of investigation started by other workers, was designed to study the use of microspheres to deliver antigens by the mucosal routes (oral and nasal). Antigen-containing microspheres were prepared with PLA and PLGA, by either entrapment within the particles or adsorption onto the surface. The model protein antigens used in this work were mainly tetanus toxoid (TT), bovine serum albumin (BSA) and γ-globulins.In vitro investigations included the study of physicochemical properties of the particulate carriers as well as the assessment of stability of the antigen molecules throughout the formulation procedures. Good loading efficiencies were obtained with both formulation techniques, which did not affect the immunogenicity of the antigens studied. The influence of the surfactant employed on the microspheres' surface properties was demonstrated as well as its implications on the adsorption of proteins. Preparations containing protein adsorbed were shown to be slightly more hydrophobic than empty PLA microspheres, which can enhance the uptake of particles by the antigen presenting cells that prefer to associate with hydrophobic surfaces. Systemic and mucosal immune responses induced upon nasal, oral and intramuscular administration have been assessed and, when appropriate, compared with the most widely used vaccine adjuvant, aluminium hydroxide. The results indicate that association of TT with PLA microspheres through microencapsulation or adsorption procedures led to an enhancement of specific mucosal IgA and IgG and systemic IgG responses to the mucosal delivered antigens. Particularly, nasal administration of TT produced significantly higher serum levels of specific IgG in test animals, as compared to control groups, suggesting that this is a potential route for vaccination. This implies the uptake and transfer of particles through the nasal mucosa, which was further demonstrated by the presence in the blood stream of latex particles as early as 10 min after nasal administration.

A comparison of spatial patterns of TDP-43 cellular inclusions in familial and sporadic frontotemporal lobar degeneration with TDP-43 proteinopathy

Abnormal protein aggregates of transactive response (TAR) DNA-binding protein (TDP-43) in the form of neuronal cytoplasmic inclusions (NCI), oligodendroglial inclusions (GI), neuronal internuclear inclusions (NII), and dystrophic neurites (DN) are the pathological hallmark of frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP). To investigate the role of phosphorylated TDP-43 (pTDP-43) in neurodegeneration in FTLD-TDP, the spatial patterns of the pTDP-43-immunoreactive NCI, GI, NII, and DN were studied in frontal and temporal cortex in three groups of cases: (1) familial FTLD-TDP caused by progranulin (GRN) mutation, (2) a miscellaneous group of familial cases containing cases caused by valosin-containing protein (VCP) mutation, ubiquitin associated protein 1 (UBAP1) mutation, and cases not associated with currently known genes, and (3) sporadic FTLD-TDP. In a significant number of brain regions, the pTDP-43-immunoreactive inclusions developed in clusters and the clusters were distributed regularly parallel to the tissue boundary. The spatial patterns of the inclusions were similar to those revealed by a phosphorylation-independent anti-TDP-43 antibody. The spatial patterns and cluster sizes of the pTDP-43-immunoreactive inclusions were similar in GRN mutation cases, remaining familial cases, and in sporadic FTLD-TDP. Hence, pathological changes initiated by different genetic factors in familial cases and by unknown causes in sporadic FTLD-TDP appear to follow a parallel course resulting in very similar patterns of degeneration of frontal and temporal lobes.

Acute phase proteins and biomarkers for health in chickens

Acute phase proteins (APPs) are proteins synthesised predominantly in the liver, whose plasma concentrations increase (positive APP) or decrease (negative APP) as a result of infection, inflammation, trauma and tissue injury. They also change as a result of the introduction of immunogens such as bacterial lipopolysaccharide (LPS), turpentine and vaccination. While publications on APPs in chickens are numerous, the limited availability of anti-sera and commercial ELISAs has resulted in a lot of information on only a few APPs. Disease is a threat to the poultry industry, as pathogens have the potential to evolve, spread and cause rapid onset of disease that is detrimental to the welfare of birds. Low level, sub-acute disease with non-specific, often undiagnosed causes can greatly affect bird health and growth and impact greatly on productivity and profitability. Developing and validating methods to measure and characterise APPs in chickens will allow these proteins to be used diagnostically for monitoring flock health. Using immune parameters such as APPs that correlate with disease resistance or improvements in production and welfare will allow the use of APPs as selection parameters for breeding to be evaluated. For APPs to be useful parameters on which to evaluate chicken health, information on normal APP concentrations is required. Ceruloplasmin (Cp) and PIT54 concentrations were found to be much lower in healthy birds form commercial production farms than the reported normal values obtained from the literature. These APPs were found to be significantly higher in culled birds from a commercial farm and Cp, PIT54 and ovotransferrin (Ovt) were significantly higher in birds classified as having obvious gait defects. Using quantitative shotgun proteomics to identify the differentially abundant proteins between three pools: highly acute phase (HAP), acute phase (AP) and non-acute phase (NAP), generated data from which a selection of proteins, based on the fold difference between the three pools was made. These proteins were targeted on a individual samples alongside proteins known to be APPs in chickens or other species: serum amyloid A (SAA), C-reactive protein (CRP), Ovt, apolipoprotein A-I (apo-AI), transthyretin (Ttn), haemopexin (Hpx) and PIT54. Together with immunoassay data for SAA, Ovt, alpha-1-acid glycoprotein (AGP) and Cp the results of this research reveal that SAA is the only major APP in chickens. Ovotransferrin and AGP behave as moderate APPs while PIT54 and Cp are minor APPs. Haemopexin was not significantly different between the three acute phase groups. Apolipoprotein AI and Ttn were significantly lower in the HAP and AP groups and as such can be classed as negative APPs. In an effort to identify CRP, multiple anti-sera cross reacting with CRP from other species were used and a phosphorylcholine column known to affinity purify CRP were used. Enriched fractions containing low molecular weight proteins, elutions from the affinity column together with HAP, AP and NAP pooled samples were applied to a Q-Exactive Hybrid Quadrupole–Orbitrap mass spectrometer (Thermo Scientific) for Shotgun analysis and CRP was not identified. It would appear that CRP is not present as a plasma protein constitutively or during an APR in chickens and as such is not an APP in this species. Of the proteins targeted as possible novel biomarkers of the APR in chickens mannan binding lectin associated serine protease-2, α-2-HS-glycoprotein (fetuin) and major facilitator superfamily domain-containing protein 10 were reduced in abundance in the HAP group, behaving as negative biomarkers. Myeloid protein and putative ISG(12)2 were positively associated with the acute phase being significantly higher in the HAP and AP groups. The protein cathepsin D was significantly higher in both HAP and AP compared to the NAP indicating that of all the proteins targeted, this appears to have the most potential as a biomarker of the acute phase, as it was significantly increased in the AP as well as the HAP group. To evaluate APPs and investigate biomarkers of intestinal health, a study using re-used poultry litter was undertaken. The introduction of litter at 12 days of age did not significantly increase any APPs measured using immunoassays and quantitative proteomics at 3, 6 and 10 days post introduction. While no APP was found to be significantly different between the challenged and control groups at anytime point, the APPs AGP, SAA and Hpx did increase over time in all birds. The protein apolipoprotein AIV (apo-AIV) was targeted as a possible APP and because of its reported role in controlling satiety. An ELISA was developed, successfully validated and used to measure apo-AIV in this study. While no significant differences in apo-AIV plasma concentrations between challenged and control groups were identified apo-AIV plasma concentrations did change significantly between certain time points in challenged and control groups. Apoliporotein AIV does not appear to behave as an APP in chickens, as it was not significantly different between acute phase groups. The actin associated proteins villin and gelsolin were investigated as possible biomarkers of intestinal health. Villin was found not to be present in the plasma of chickens and as such not a biomarker target. Gelsolin was found not to be differentially expressed during the acute phase or as a result of intestinal challenge. Finally a proteomic approach was undertaken to investigate gastrocnemius tendon (GT) rupture in broiler chickens with a view of elucidating to and identify proteins associated with risk of rupture. A number of proteins were found to be differentially expressed between tendon pools and further work would enable further detailing of these findings. In conclusion this work has made a number of novel findings and addressed a number of data poor areas. The area of chicken APPs research has stagnated over the last 15 years with publications becoming repetitive and reliant on a small number of immunoassays. This work has sought to characterise the classic APPs in chickens, and use a quantitative proteomic approach to measure and categorise them. This method was also used to take a fresh approach to biomarker identification for both the APR and intestinal health. The development and validation of assays for Ovt and apo-AIV and the shotgun data mean that these proteins can be further characterised in chickens with a view of applying their measurement to diagnostics and selective breeding programs.

Transcriptome, carbohydrate, and phytohormone analysis of Petunia hybrida reveals a complex disturbance of plant functional integrity under mild chilling stress

Cultivation of chilling-tolerant ornamental crops at lower temperature could reduce the energy demands of heated greenhouses. To provide a better understanding of how sub-optimal temperatures (12 degrees C vs. 16 degrees C) affect growth of the sensitive Petunia hybrida cultivar 'SweetSunshine Williams', the transcriptome, carbohydrate metabolism, and phytohormone homeostasis were monitored in aerial plant parts over 4 weeks by use of a microarray, enzymatic assays and GC-MS/MS. The data revealed three consecutive phases of chilling response. The first days were marked by a strong accumulation of sugars, particularly in source leaves, preferential up-regulation of genes in the same tissue and down-regulation of several genes in the shoot apex, especially those involved in the abiotic stress response. The midterm phase featured a partial normalization of carbohydrate levels and gene expression. After 3 weeks of chilling exposure, a new stabilized balance was established. Reduced hexose levels in the shoot apex, reduced ratios of sugar levels between the apex and source leaves and a higher apical sucrose/hexose ratio, associated with decreased activity and expression of cell wall invertase, indicate that prolonged chilling induced sugar accumulation in source leaves at the expense of reduced sugar transport to and reduced sucrose utilization in the shoot. This was associated with reduced levels of indole-3-acetic acid and abscisic acid in the apex and high numbers of differentially, particularly up-regulated genes, especially in the source leaves, including those regulating histones, ethylene action, transcription factors, and a jasmonate-ZIM-domain protein. Transcripts of one Jumonji C domain containing protein and one expansin accumulated in source leaves throughout the chilling period. The results reveal a dynamic and complex disturbance of plant function in response to mild chilling, opening new perspectives for the comparative analysis of differently tolerant cultivars.

Estudo da proteína humana centrossomal TBCCD1 em condições de stress oxidativo causado por H2O2 em estado estacionário

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.

Synthesis, biological studies of linear and branched arabinofuranoside-containing glycolipids and their interaction with surfactant protein A

Oligoarabinofuranoside-containing glycolipids relevant to mycobacterial cell wall components were synthesized in order to understand the functional roles of such glycolipids. A series of linear tetra-, hexa-, octa-and a branched heptasaccharide oligoarabinofuranosides, with 1 -> 2 and 1 -> 5 a-linkages between the furanoside residues, were synthesized by chemical methods from readily available monomer building blocks. Upon the synthesis of glycolipids, constituted with a double alkyl chain-substituted sn-glycerol core and oligosaccharide fragments, biological studies were performed to identify the effect of synthetic glycolipids on the biofilm formation and sliding motilities of Mycobacterium smegmatis. Synthetic glycolipids and arabinofuranosides displayed an inhibitory effect on the growth profile, but mostly on the biofilm formation and maturation. Similarly, synthetic compounds also influenced the sliding motility of the bacteria. Further, biophysical studies were undertaken, so as to identify the interactions of the glycolipids with a pulmonary surfactant protein, namely surfactant protein A (SP-A), with the aid of the surface plasmon resonance technique. Specificities of each glycolipid interacting with SP-A were thus evaluated. From this study, glycolipids were found to exhibit higher apparent association constants than the corresponding oligosaccharide portion alone, without the double alkyl group-substituted glycerol core.

The N-terminal region containing the zinc finger domain of tobacco streak virus coat protein is essential for the formation of virus-like particles

Tobacco streak virus (TSV), a member of the genus Ilarvirus (family Bromoviridae), has a tripartite genome and forms quasi-isometric virions. All three viral capsids, encapsidating RNA 1, RNA 2 or RNA 3 and subgenomic RNA 4, are constituted of a single species of coat protein (CP). Formation of virus-like particles (VLPs) could be observed when the TSV CP gene was cloned and the recombinant CP (rCP) was expressed in E. coli. TSV VLPs were found to be stabilized by Zn2+ ions and could be disassembled in the presence of 500 mM CaCl2. Mutational analysis corroborated previous studies that showed that an N-terminal arginine-rich motif was crucial for RNA binding; however, the results presented here demonstrate that the presence of RNA is not a prerequisite for assembly of TSV VLPs. Instead, the N-terminal region containing the zinc finger domain preceding the arginine-rich motif is essential for assembly of these VLPs.

Negative Cooperativity and High Affinity in Chitooligosaccharide Binding by a Mycobacterium smegmatis Protein Containing LysM and Lectin Domains

LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides has been underexplored. Binding of a Mycobacterium smegmatis protein containing a lectin (MSL) and one LysM domain to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration that demonstrate the presence of two binding sites of nonidentical affinities per dimeric MSL-LysM molecule. The affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in the MSL-LysM molecule, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the case presented here, two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of the MSL-LysM molecule for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.

The effects of supplemental feeds containing different protein: Energy ratios on the growth and survival of Tilapia nilotica (Oreochromis niloticus) in brackishwater ponds

A research was conducted in thirty approximately 100 sq.m earthern ponds of the Brackishwater Aquaculture Centre (BAC), College of Fisheries, University of the Philippines, Leganes Iloilo from November 7, 1982 to March 7, 1983 to evaluate the effects of nine supplemental feeds containing different protein: energy ratios on the growth and survival of Tilapia nilotica in brackishwater ponds. Nine supplemental feeds formulated were with protein levels of 20%, 25%, and 30% each at three energy levels of 3,000 kcals; 3,500 kcals; and 4,000 kcals. There was a control treatment with no feeding so that mean weight gain growth rate, feed conversion rate, and survival were determined. Fish fingerlings were acclimated from 0-29 ppt. salinity before the experiment and 20% of fish in each treatment were sampled after every 30 days. Growth rates were significantly different and increased with increasing energy level at the 30% protein feeds but decreased at high energy levels in the 20% and 25% protein feeds. Feed conversion was significantly different due to interaction between protein and energy levels in the feeds, and was better at the 30:3,500 kcals feeds having a feed conversion of 1.55 g. Survival was not significantly different