Generation of excited species catalyzed by horseradish peroxidase or hemin in the presence of reduced glutathione and H2O2

Horseradish peroxidase (HRP) (EC 1.11.1.7) catalyzes the oxidation of reduced glutathione. This reaction is accompanied by light emission, which is attributed to the generation of singlet oxygen. The chemiluminescence is directly related to thiyl radical formation, as deduced from the correlation between the time course of HRP-compound II formation and light emission in the presence of different amounts of H2O2. Superoxide dismutase has an inhibitory effect on the chemiluminescence without affecting the HRP-compound II formation. This indicates the direct involvement of superoxide radicals in the production of photoemissive species. Replacement of HRP by hemin is also accompanied by chemiluminescence.     

Biliverdin IX delta and neobiliverdin IX delta, isolated from the ovaries of the marine snail, Turbo cornutus

The ovaries of the marine snail Turbo cornutus contain a number of pigments. So far, the presence of carotenoids and a chromoprotein with a bile pigment, called turboverdin (= 3(2)-hydroxy-mesobiliverdin IX alpha), as its prosthetic group are known. The present work describes the isolation and structure elucidation of two further bile pigments, biliverdin IX delta and neobiliverdin IX delta. This is the first report of naturally occurring bile pigments with IX delta structure.     

High efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts

An efficient system has been established that allows well controlledDNA microinjection into tobacco (Nicotiana tabacum) mesophyllprotoplasts with partially regenerated cell walls and subsequentanalysis of transient as well as stable expression of injectedreporter genes in particular targeted cells or derived clones.The system represents an effective tool to study parametersimportant for the successful transformation of plant cells bymicroinjection and other techniques. Protoplasts were immobilizedin a very thin layer of medium solidified with agarose or alginate.DNA microinjection was routinely monitored by coinjecting FITC-dextranand aimed at the cytoplasm of target cells. The injection procedurewas optimized for efficient delivery of injection solution intothis compartment. Cells were found to be at the optimal stagefor microinjection about 24 h after immobilization in solidmedium. Embedded cells could be kept at this stage for up to4 d by incubating them at 4 C in the dark. Within 1 h successfuldelivery of injection, solution was routinely possible into20–40 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913,a plasmid containing the neo (neomycin phosphotransferase II)gene, stably transformed, paromomycin-resistant clones couldbe recovered through selection. Transgenic tobacco lines havebeen established from such clones. Injection solutions containingpSHI913 at a concentration of either 50 µg ml^–1or 1 mg ml^–1 have been tested. With 1 mg ml^–1 plasmidDNA the percentage of resistant clones per successfully injectedcell was determined to be about 3.5 times higher. Incubationof embedded protoplasts at 4C before microinjection was foundto reduce the percentage of resistant clones obtained per injectedcell Protoplasts were immobilized above a grid pattern and the locationof injected cells was recorded by Polaroid photography. Thefate of particular targeted cells could be observed. Isolationand individual culture of clones derived from injected cellswas possible. Following cytoplasmic coinjection of FITC-dextranand 1 mg ml^–1 plasmid DNA on average about 20% of thetargeted cells developed into microcalli and roughly 50% ofthese calli were stably transformed. Transient expression ofthe firefly luciferase gene (Luc) was nondestructively analysed24 h after injection of pAMLuc. Approximately 50% of the injectedcells that were alive at this time point expressed the Luc genetransiently. Apparently, stable integration of the injectedgenes occurred in essentially all transiently expressing cellsthat developed into clones. Key words: DNA microinjection, firefly luciferase, FITCdextran, Nicotiana tabacum, protoplast transformation     

Protection against reactive oxygen species by NAD(P)H: quinone reductase induced by the dietary antioxidant butylated hydroxyanisole (BHA). Decreased hepatic low-level chemiluminescence during quinone redox cycling

Menadione elicits low-level chemiluminescence (lambda greater than 620 nm) associated with redox cycling of the quinone in mouse hepatic postmitochondrial fractions. This photoemission is suppressed when the animals are fed a diet containing the anticarcinogenic antioxidant, 2[3]-(tert-butyl)-4-hydroxyanisole (BHA), which leads to a 13-fold increase in NAD(P)H: quinone reductase (EC 1.6.99.2). Inhibition of the enzyme by dicoumarol completely abolishes the protective effect of BHA treatment and leads to higher chemiluminescence, reaching similar photoemission for BHA-treated and control animals. These findings indicate that the two-electron reduction promoted by quinone reductase prevents redox cycling and that BHA protects against reactive oxygen species by elevating the activity of this enzyme.     

Straw utilization for biofuel production: A consequential assessment of greenhouse gas emissions from bioethanol and biomethane provision with a focus on the time dependency of emissions

The shift from straw incorporation to biofuel production entails emissions from production, changes in soil organic carbon (SOC) and through the provision of (co‐)products and entailed displacement effects. This paper analyses changes in greenhouse gas (GHG) emissions arising from the shift from straw incorporation to biomethane and bioethanol production. The biomethane concept comprises comminution, anaerobic digestion and amine washing. It additionally provides an organic fertilizer. Bioethanol production comprises energetic use of lignin, steam explosion, enzymatic hydrolysis and co‐fermentation. Additionally, feed is provided. A detailed consequential GHG balance with in‐depth focus on the time dependency of emissions is conducted: (a) the change in the atmospheric load of emissions arising from the change in the temporal occurrence of emissions comparing two steady states (before the shift and once a new steady state has established); and (b) the annual change in overall emissions over time starting from the shift are assessed. The shift from straw incorporation to biomethane production results in net changes in GHG emissions of (a) ?979 (?436 to ?1,654) and (b) ?955 (?220 to ?1,623) kg CO₂‐eq. per t_{dry matter} straw converted to biomethane (minimum and maximum). The shift to bioethanol production results in net changes of (a) ?409 (?107 to ?610) and (b) ?361 (57 to ?603) kg CO₂‐eq. per t_{dry matter} straw converted to bioethanol. If the atmospheric load of emissions arising from different timing of emissions is neglected in case (a), the change in GHG emissions differs by up to 54%. Case (b) reveals carbon payback times of 0 (0–49) and 19 (1–100) years in case of biomethane and bioethanol production, respectively. These results demonstrate that the detailed inclusion of temporal aspects into GHG balances is required to get a comprehensive understanding of changes in GHG emissions induced by the introduction of advanced biofuels from agricultural residues.     

Engagement with HIV Prevention Treatment and Care among Female Sex Workers in Zimbabwe: a Respondent Driven Sampling Survey

Objective(S)

To determine the HIV prevalence and extent of engagement with HIV prevention and care among a representative sample of Zimbabwean sex workers working in Victoria Falls, Hwange and Mutare.

Design

Respondent driven sampling (RDS) surveys conducted at each site.

Methods

Sex workers were recruited using respondent driven sampling with each respondent limited to recruiting 2 peers. Participants completed an interviewer-administered questionnaire and provided a finger prick blood sample for HIV antibody testing. Statistical analysis took account of sampling method.

Results

870 women were recruited from the three sites. HIV prevalence was between 50 and 70%. Around half of those confirmed HIV positive were aware of their HIV status and of those 50-70% reported being enrolled in HIV care programmes. Overall only 25-35% of those with laboratory-confirmed HIV were accessing antiretroviral therapy. Among those reporting they were HIV negative, 21-28% reported having an HIV test in the last 6 months. Of those tested HIV negative, most (65-82%) were unaware of their status. Around two-thirds of sex workers reported consistent condom use with their clients. As in other settings, sex workers reported high rates of gender based violence and police harassment.

Conclusions

This survey suggests that prevalence of HIV is high among sex workers in Zimbabwe and that their engagement with prevention, treatment and care is sub-optimal. Intensifying prevention and care interventions for sex workers has the potential to markedly reduce HIV and social risks for sex workers, their clients and the general population in Zimbabwe and elsewhere in the region.     

Alterations of Red Cell Membrane Properties in Nneuroacanthocytosis

Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington’s disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an “acanthocytic state” of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration.     

Pancreatic carcinoma, pancreatitis, and healthy controls: metabolite models in a three-class diagnostic dilemma

Metabolomics as one of the most rapidly growing technologies in the “-omics” field denotes the comprehensive analysis of low molecular-weight compounds and their pathways. Cancer-specific alterations of the metabolome can be detected by high-throughput mass-spectrometric metabolite profiling and serve as a considerable source of new markers for the early differentiation of malignant diseases as well as their distinction from benign states. However, a comprehensive framework for the statistical evaluation of marker panels in a multi-class setting has not yet been established. We collected serum samples of 40 pancreatic carcinoma patients, 40 controls, and 23 pancreatitis patients according to standard protocols and generated amino acid profiles by routine mass-spectrometry. In an intrinsic three-class bioinformatic approach we compared these profiles, evaluated their selectivity and computed multi-marker panels combined with the conventional tumor marker CA 19-9. Additionally, we tested for non-inferiority and superiority to determine the diagnostic surplus value of our multi-metabolite marker panels. Compared to CA 19-9 alone, the combined amino acid-based metabolite panel had a superior selectivity for the discrimination of healthy controls, pancreatitis, and pancreatic carcinoma patients $ [ {\text{volume under ROC surface}}\;\left( {\text{VUS}} \right) = 0. 8 9 1 { }\left( { 9 5\,\% {\text{ CI }}0. 7 9 4- 0. 9 6 8} \right)]. $ We combined highly standardized samples, a three-class study design, a high-throughput mass-spectrometric technique, and a comprehensive bioinformatic framework to identify metabolite panels selective for all three groups in a single approach. Our results suggest that metabolomic profiling necessitates appropriate evaluation strategies and—despite all its current limitations—can deliver marker panels with high selectivity even in multi-class settings.