Studying the impact of geometrical and cellular cues on myogenesis with a skeletal muscle-on-chip.

In the skeletal muscle tissue, cells are organized following an anisotropic architecture, which is both required during myogenesis when muscle precursor cells fuse to generate myotubes and for its contractile function. To build an in vitro skeletal muscle tissue, it is therefore essential to develop methods to organize cells in an anisotropic fashion, which can be particularly challenging, especially in 3D. In this study, we present a versatile muscle-on-chip system with adjustable collagen hollow tubes that can be seeded with muscle precursor cells. The collagen acts both as a tube-shaped hollow mold and as an extracellular matrix scaffold that can house other cell types for co-culture. We found that the diameter of the channel affects the organization of the muscle cells and that proper myogenesis was obtained at a diameter of 75 µm. In these conditions, muscle precursor cells fused into long myotubes aligned along these collagen channels, resulting in a fascicle-like structure. These myotubes exhibited actin striations and upregulation of multiple myogenic genes, reflecting their maturation. Moreover, we showed that our chip allowed muscle tissue culture and maturation over a month, with the possibility of fibroblast co-culture embedding in the collagen matrix.

Association of bud and anther morphology with developmental stages of the male gametophyte of melon (Cucumis melo L.).

Correlations between the morphological features of f lower buds and the developmental stages of the male gametophyte are of great practical interest as a reliable marker that accelerates and simplif ies the selection of appropriate plant material for isolated microspore culture. Microspore culture enables one to quickly obtain many pure lines of different vegetable crops, but it has not yet been widely applied in the melon (Cucumis melo L.). To successfully apply this technique in a new culture, one has to optimize many of its elements: f irst, f ind the biological markers for selecting the f lower buds containing the microspores of certain development stages. The paper presents the results of research estimating the correlations between the length and diameter of the f lower buds, the length of the visual part of the corolla, the length of the anthers and the development stages of the male gametophyte in the F1 hybrid of the Kim Hong Ngoc melon. The strongest correlation (CC = 0.885) was found for the f lower bed diameter and a strong correlation (CC = 0.880), for the bud length. The corolla's visual part was a less reliable morphological feature, and the anther's length should not be used as a parameter to predict the developmental stages of the melon's male gametophyte. It was also found that one anther could contain the microspores and pollen grains of different developmental stages. In the f lower buds less than 4 mm in length and 1.51 ± 0.02 mm in diameter prevailed tetrads, and in the buds 4.0-4.9 mm in length and 2.30 ± 0.02 mm in diameter, early microspores. The microspores of a middle stage of development prevailed in the f lower buds 5.0-5.9 mm in length and 2.32 ± 0.00 mm in diameter; mid and late vacuolated microspores, in the buds 6.0-8.9 mm in length and 2.96 ± 0.37 mm in diameter; and two-celled pollen, in the buds more than 9 mm in length and more than 3.97 ± 0.34 mm in diameter.

[Acute right heart failure]. / L'insuffisance cardiaque droite aiguë.

Even though, it has long been considered as a passive channel allowing communication between the systemic and pulmonary circulations, it is now clearly established that the right ventricle plays an essential role in cardio-pulmonary couple physiology. Its failure results in a clinical presentation that reflects the systemic congestion and reduced cardiac output. It is the consequence of two pathological situations frequently encountered in intensive care including pulmonary vascular resistance increase and right ventricle contraction alteration. Mechanical ventilation, certain drugs and volume overload can also participate. The management of the acute right heart failure is based on the combination of supportive treatment and causal treatment, specific to the etiology. Supportive therapy aims to optimize filling pressures, reduce afterload and support cardiac contractibility. With the growing number of therapeutic options used according to co-morbidities, decision-making by a multidisciplinary heart team seems essential.

Même s'il a été longtemps considéré comme un conduit passif permettant la communication entre les circulations systémique et pulmonaire, il est maintenant clairement établi que le ventricule droit joue un rôle essentiel dans la physiologie du couple cardio-pulmonaire. Sa défaillance est à l'origine d'un tableau clinique secondaire à la congestion systémique et à la réduction du débit cardiaque. Elle est la conséquence de deux situations pathologiques fréquemment rencontrées incluant l'augmentation des résistances vasculaires pulmonaires et l'altération de la contraction du ventricule droit. La ventilation mécanique, certains médicaments et la surcharge volémique peuvent également y participer. La prise en charge de l'insuffisance cardiaque aiguë repose sur l'association d'un traitement supportif et d'un traitement causal, spécifique à l'étiologie. Le traitement supportif vise à optimaliser les pressions de remplissage, réduire la postcharge et soutenir la contractilité myocardique. Avec le nombre croissant d'options thérapeutiques utilisées en fonction des comorbidités, la prise de décision par une équipe cardiologique multidisciplinaire est indispensable.

[Cardiovascular effects of alcohol consumption]. / Effets cardiovasculaires indésirables de l'alcool.

Alcohol consumption is a serious problem in terms of public health because it remains a major cause of illness and premature death. The effects of alcohol on cardiovascular system are particularly complex. Current data report both favourable and adverse effects depending on the level of consumption, the type of alcohol consumed and the pathology considered. The goal of this review is to describe the spectrum of cardiovascular events associated with alcohol consumption.

La consommation d'alcool représente une problématique sérieuse en termes de santé publique puisqu'elle demeure une cause majeure de maladie et de décès prématuré. Les effets de l'alcool sur le système cardiovasculaire sont particulièrement complexes. Les données actuelles rapportent à la fois des effets favorables et des effets néfastes en fonction du niveau de consommation, du type d'alcool consommé et de la pathologie considérée. L'objectif de cet article est de décrire le spectre des manifestations cardiovasculaires les plus fréquentes liées à la prise d'alcool.

[Maternal and neonatal outcomes of unplanned deliveries]. / Conséquences maternelles et néonatales des accouchements inopinés extrahospitaliers.

OBJECTIVES: Know the impact of the unplanned deliveries in a town of medium size, the characteristics of these women and maternal and neonatal risks. MATERIALS AND METHODS: This was a retrospective study conducted between January 2002 and December 2009. Unexpected delivery was defined as any delivery taking place outside of a non-elective way maternity. Each unexpected delivery was matched at nearest delivery of equivalent term, at the CHU maternity, with an onset of spontaneous labour. RESULTS: Ninety-four women gave birth unexpectedly for a total of 48,721 births (incidence of 0.19%). There was a significant difference between cases and controls for parity (1.8 versus 0.9), the lack of follow-up of pregnancy (21.3% versus 1.1%), tobacco (57.4% versus 25.5%), the socio-economic level, the type of feeding (artificial: 61.7% versus 30.6%), the home-hospital distance and obstetric follow-up. We found a significant increase in perinatal mortality (6.4% versus 1%) and stay in Neonatal ICU (19.1% versus 9.2%). The main neonatal morbidity was hypothermia. CONCLUSION: It is difficult to target a population at risk because the type of these women is non-specific. Prevention of unplanned deliveries and their morbidities through information of patients on the grounds of urgent consultation and support of the newborn to limit hypothermia.

[Extravasation of radiopharmaceuticals: preventive measures and management recommended by SoFRa (Société Française de Radiopharmacie)]. / Extravasation des médicaments radiopharmaceutiques : mesures préventives et prise en charge recommandées par la SoFRa (Société française de radiopharmacie).

Radiopharmaceuticals extravasation is rare but may have serious clinical issues. Because no specific recommendations are being proposed to date, the goals of our working group created within the French Society of Radiopharmacy are to determine preventive measures and to establish a pragmatic management of extravasation of these drugs. Our preventive measures are to recognize the symptoms (erythema, venous discoloration, swelling), to know the risk factors (which are related to radiopharmaceutical, patient, site of injection, injection technique) and severity (from erythema to skin necrosis, depending on the radionuclide) and how to avoid them (training and awareness of staff, choice of injection site, route of drug administration test, use of a catheter for administration of therapeutic radiopharmaceuticals). Management should be immediate. It can be facilitated by a specific emergency kit. General measures recommended are the immediate cessation of injection, aspiration of fluid extravasation, delimitation of the extravasated area with an indelible pen, informing the doctor. Specific measures taking into account the radiotoxicity of the radionuclide and the type of radiopharmaceutical were also established. The patient should be informed by the doctor about the risks and how to take care of. Traceability of the incident must be ensured. A multidisciplinary reflexion is essential to manage the extravasation as early and effectively as possible.

Indications and pregnancy outcomes when multidisciplinary centers for prenatal diagnosis refuse request for termination of pregnancy: a retrospective French study.

OBJECTIVES: To report indications and risk factors for effective termination of pregnancy (TOP) when multidisciplinary prenatal diagnosis centers refuse request for TOP. METHODS: This is a retrospective study from a French national registry between 2005 and 2009. Indication groups for TOP refusal were created. Risk factors for pregnancy outcomes and effective TOP were determined by multinomial logistic regression model. RESULTS: The overall number of TOP refusals was 573. Indications were single malformations (37%), chromosomal abnormalities (16.6%), hygroma (5.2%), potential fetopathies (11%), maternal indications (17.4%), and other indications (12.7%). Pregnancy outcomes were live infants (35.1%), effective TOP (36.3%), intra-uterine fetal death (5.4%), and lost to follow-up (23.2%). Logistic regression model showed an increased likelihood of effective TOP in groups with single malformations [odds ratio (OR) 3.4, 95% confidence interval (CI) 1.6-7.4], chromosomal abnormalities (OR 3, 95% CI 1.2-7.2), and hygromas (OR 19.6, 95% CI 2.3-16.8) compared with other groups. An increased likelihood of effective TOP was also reported when the request was made at first (OR 29.5, 95% CI 10-16.7) and second (OR 6.3, 95% CI 2.3-16.7) trimesters compared with the third trimester. CONCLUSIONS: Effective TOP is particularly frequent when requested during the first trimester and in cases of structural anomalies of the fetus.

Pancreatic inflammation and increased islet macrophages in insulin-resistant juvenile primates.

Chronic high caloric intake has contributed to the increased prevalence of pediatric obesity and related morbidities. Most overweight or obese children, however, do not present with frank metabolic disease but rather insulin resistance or subclinical precursors. The innate immune system plays a role in the pathophysiology of type 2 diabetes but how it contributes to early metabolic dysfunction in children on chronic high-fat diet (HFD) is unclear. We hypothesize that such inflammation is present in the pancreas of children and is associated with early insulin resistance. We used nonhuman primate (NHP) juveniles exposed to chronic HFD as a model of early pediatric metabolic disease to demonstrate increased pancreatic inflammatory markers before the onset of significant obesity or glucose dysregulation. Pancreata from 13-month-old Japanese macaques exposed to a HFD from in utero to necropsy were analyzed for expression of cytokines and islet-associated macrophages. Parameters from an intravenous glucose tolerance test were correlated with cytokine expression. Before significant glucose dysregulation, the HFD cohort had a twofold increase in interleukin 6 (IL6), associated with decreased first-phase insulin response and a sexually dimorphic (male) increase in IL1ß correlating with increased fasting glucose levels. The number of islet-associated macrophages was also increased. Pancreata from juvenile NHP exposed to HFD have increased inflammatory markers and evidence of innate immune infiltration before the onset of significant obesity or glucose dysregulation. Given the parallel development of metabolic disease between humans and NHPs, these findings have strong relevance to the early metabolic disease driven by a chronic HFD in children.

The effect of urease and nitrification inhibitors on ammonia and nitrous oxide emissions from simulated urine patches in pastoral system: a two-year study.

This field study evaluated the effects of applying a combination of urease (UI) and nitrification inhibitors (NI) on ammonia (NH3) and nitrous oxide (N2O) emission from urine patches, using zeolite, single superphosphate (SSP) and urea fertilizer as a carrier. The trial was conducted on a Typic Haplustepts silt loam soil, near Lincoln, Canterbury, New Zealand during 2009-11. The treatments in 2009 included: a control (no urine or inhibitor), urine alone at 600 kg N ha(-1), and urine with either double inhibitor (DI) a mixture (1:7 ratio w/w) of UI (N-(n-butyl) thiophosphoric triamide (nBTPT - trade name Agrotain®) and NI, dicyandiamide (DCD) or DCD alone at 10 kg ha(-1) using zeolite and SSP as carriers. In 2010 trials, both zeolite and urea were used as carriers for DI and DCD. DI-zeolite and DI-urea were equally effective and reduced the average NH3 losses from applied urine over two years by 34.6% in autumn and 40% in spring respectively. The nBTPT in DI-SSP was decomposed by the free acid produced during its dissolution and therefore increased NH3 emission as does DCD alone. DCD consistently increased NH3 emissions by 39% and 15.6% in autumn and spring respectively. Spring application resulted in NH3-N losses of 16.9% as a percentage of the total N applied compared to 8.4% in autumn. Over the two years, the DI reduced N2O emissions by 53% in autumn and 46% in spring over urine alone treatment; the equivalent reductions for DCD were 42% and 39% for autumn and spring, respectively. These results suggest that applying DI in autumn and spring using zeolite or urea carrier five days prior to grazing has the most potential to reduce NH3 and N2O losses from specific urination event than using DCD alone; and therefore warrants further research to improve its longevity.

Outcomes and follow-up of patients treated for multidrug-resistant tuberculosis in Orel, Russia, 2002-2005.

SETTING: Multidrug-resistant tuberculosis (MDR-TB) treatment facility, Orel Oblast, Russian Federation. OBJECTIVES: To determine factors associated with poor outcome and to document status of patients after recording of TB outcomes. DESIGN: Retrospective review of prospective single cohort. RESULTS: Among 192 patients, factors significantly associated with poor outcome in multivariate analysis include three or more treatment interruptions during the intensive phase of therapy and alcohol or drug addiction (adjusted OR [aOR] 2.1, 95%CI 1.0-4.3 and aOR 1.9, 95%CI 1.0-3.7). Previous treatment was associated with poor outcome, but only among smear-positive patients (aOR 3.1, 95%CI 1.3-7.3). Ten patients (5%) developed extensively drug-resistant TB (XDR-TB) during treatment; of 115 patients with at least 6 months of follow-up data after outcomes were recorded, 13 (11%) developed XDR-TB. CONCLUSION: Interventions focused on supporting patient adherence during the intensive phase of treatment; the management of drug and alcohol addiction should be developed and studied. A substantial proportion of patients developed XDR-TB during and after treatment. Longer term follow-up data of patients treated for MDR-TB are needed to better inform programmatic policy.

Fallout radionuclide-based techniques for assessing the impact of soil conservation measures on erosion control and soil quality: an overview of the main lessons learnt under an FAO/IAEA Coordinated Research Project.

This paper summarizes key findings and identifies the main lessons learnt from a 5-year (2002-2008) coordinated research project (CRP) on "Assessing the effectiveness of soil conservation measures for sustainable watershed management and crop production using fallout radionuclides" (D1.50.08), organized and funded by the International Atomic Energy Agency through the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. The project brought together nineteen participants, from Australia, Austria, Brazil, Canada, Chile, China, Japan, Morocco, Pakistan, Poland, Romania, Russian Federation, Turkey, United Kingdom, United States of America and Vietnam, involved in the use of nuclear techniques and, more particularly, fallout radionuclides (FRN) to assess the relative impacts of different soil conservation measures on soil erosion and land productivity. The overall objective of the CRP was to develop improved land use and management strategies for sustainable watershed management through effective soil erosion control practices, by the use of ¹³7Cs (half-life of 30.2 years), ²¹°Pb(ex) (half-life of 22.3 years) and 7Be (half-life of 53.4 days) for measuring soil erosion over several spatial and temporal scales. The environmental conditions under which the different research teams applied the tools based on the use of fallout radionuclides varied considerably--a variety of climates, soils, topographies and land uses. Nevertheless, the achievements of the CRP, as reflected in this overview paper, demonstrate that fallout radionuclide-based techniques are powerful tools to assess soil erosion/deposition at several spatial and temporal scales in a wide range of environments, and offer potential to monitor soil quality. The success of the CRP has stimulated an interest in many IAEA Member States in the use of these methodologies to identify factors and practices that can enhance sustainable agriculture and minimize land degradation.

Water requirements for livestock production: a global perspective.

Water is a vital but poorly studied component of livestock production. It is estimated that livestock industries consume 8% of the global water supply, with most of that water being used for intensive, feed-based production. This study takes a broad perspective of livestock production as a component of the human food chain, and considers the efficiency of its water use. Global models are in the early stages of development and do not distinguish between developing and developed countries, or the production systems within them. However, preliminary indications are that, when protein production is adjusted for biological value in the human diet, no plant protein is significantly more efficient at using water than protein produced from eggs, and only soybean is more water efficient than milk and goat and chicken meat. In some regions, especially developing countries, animals are not used solely for food production but also provide draught power, fibre and fertiliser for crops. In addition, animals make use of crop by-products that would otherwise go to waste. The livestock sector is the fastest-growing agricultural sector, which has led to increasing industrialisation and, in some cases, reduced environmental constraints. In emerging economies, increasing involvement in livestock is related to improving rural wealth and increasing consumption of animal protein. Water usage for livestock production should be considered an integral part of agricultural water resource management, taking into account the type of production system (e.g. grain-fed or mixed crop-livestock) and scale (intensive or extensive), the species and breeds of livestock, and the social and cultural aspects of livestock farming in various countries.

Factors associated with positive tuberculin skin test results among HIV-infected persons in Orel Oblast, Russia.

BACKGROUND: The treatment of persons living with human immunodeficiency virus/acquired immune-deficiency syndrome (PLWHAs) for latent tuberculosis infection (LTBI) reduces tuberculosis (TB) morbidity. Despite a high TB burden and an expanding human immunodeficiency virus epidemic, Russia had limited data on the utility of the tuberculin skin test (TST) for LTBI diagnosis in PLWHAs. OBJECTIVE: To determine the prevalence and predictors of positive TSTs in PLWHAs in Orel Oblast. METHODS: A total of 150 consenting PLWHAs being followed up at the AIDS Center were administered a TST and a questionnaire for risk factors for LTBI. A positive TST result was defined as >or=5 mm induration. RESULTS: Of the 150 subjects, 67% were male and 74% were aged <30 years. Of the PLWHAs tested, 26% had a positive TST result, while among PLWHAs with CD4(+) >500 cells/ml, 36% were TST-positive. TST positivity varied inversely with CD4(+) cell count. Among PLWHAs with a history of injection drug use, the primary risk factor for HIV, 29 (31.9%) were positive. CONCLUSIONS: A high proportion of tested PLWHAs had a positive TST and could benefit from preventive therapy (PT) to reduce the risk of TB. A TB control programme in Russia should therefore include TST screening among PLWHAs and PT, besides active TB case finding and treatment.

HPV16 E6 confers p53-dependent and p53-independent phenotypes in the epidermis of mice deficient for E6AP.

High-risk human papillomaviruses are the causative agents of cervical and other anogenital cancers. In these cancers, two viral oncogenes, E6 and E7, are expressed. E6 is best known for its ability to inactivate the tumor suppressor p53, which is thought to arise through ubiquitin-mediated degradation of p53 and involve a ternary complex between E6, p53 and the E3 ligase, E6AP. In mice transgenic for wild-type HPV16 E6, its expression leads to epithelial hyperplasia and an abrogation of normal cellular responses to DNA damage. Whereas only the latter phenotype is dependent upon E6's inactivation of p53, both are reduced in transgenic mice expressing an E6 mutant severely reduced in its binding to E6AP and other cellular proteins that bind E6 through a shared alpha-helix motif. Here, we investigated whether E6AP is required for the induction of the above phenotypes through the use of both E6AP-mutant and E6AP-null mice. E6, in the absence of E6AP retains an ability to induce epithelial hyperplasia, abrogate DNA damage responses and inhibit the induction of p53 protein following exposure to ionizing radiation. We conclude that E6 is able to induce both p53-dependent and p53-independent phenotypes through E6AP-independent pathways in the mouse.

Constructed wetland attenuation of nitrogen exported in subsurface drainage from irrigated and rain-fed dairy pastures.

Nitrogen removal performance is reported for constructed wetlands treating subsurface drainage from irrigated and rain-fed dairy pastures in North Island, New Zealand. Flow-proportional sampling of inflow and outflow concentrations were combined with continuous flow records to calculate mass balances for the wetlands. Drainage flows from the irrigated catchment were 2.5-4 fold higher and N exports up to 5 fold higher per unit area than for the rain-fed catchment. Hydraulic and associated N loadings to the wetlands were highly pulsed, associated with rainfall, soil water status, and irrigation events. Transient pulses of organic nitrogen were an important form of N loss from the rain-fed landscape in the first year, and were very effectively removed in the wetland (> 90%). Median nitrate concentrations of approximately 10 g m(-3) in the drainage inflows were reduced by 15-67% during passage through the wetlands and annual nitrate-N loads by 16-61% (38-31 7 g N m(-2)y(-1)). Generation in the wetlands of net ammoniacal-N and organic-N (irrigated site) partially negated reduction in nitrate-N loads. The results show that constructed wetlands comprising 1-2% of catchment area can provide moderate reductions in TN export via pastoral drainage, but performance is markedly influenced by variations in seasonal loading and establishment/maturation factors.

Nitrate removal in riparian wetlands: interactions between surface flow and soils.

Riparian wetlands containing springs are thought to be ineffective at removing nitrate because contact times between the upwelled ground water and the underlying microbially active soils are short. Tracer experiments using lithium bromide (LiBr) and nitrate (NO3-N) injected at the surface were used to quantify residence times and NO3-N removal in a riparian swale characteristic of New Zealand hill-country pasture. An experimental enclosure was used with collecting trays at the downstream end to measure flow and concentration, shallow wells to measure subsurface concentrations, and an array of logging conductivity probes to monitor tracer continuously. The majority of added tracer reached the outlet more slowly than could be explained by surface flow, but more quickly than could be explained by Darcy seepage flow. There was evidence from the wells of tracer diffusing vertically to a depth of at least 5 cm into the surface soil layer, which was permanently saturated and highly porous. During dry weather 24 +/- 9% of added NO3-N was removed over a distance of 1.5 m largely by denitrification. The net uptake length coefficient for this wetland (K = 0.08 +/- 0.03 m(-1)) is slightly higher than the range (K = 0.01-0.07 m(-1)) measured in a small stream channel infested with macrophytes. Nitrate removal is expected to decrease with increasing flow. Seepage flow is estimated to have removed only 7 +/- 4% of the added NO3-N and we hypothesize that vertical diffusion substantially increases NO3-N removal in this type of wetland. Riparian wetlands with springs and surface flows should not be dismissed as having low NO3-N removal potential without checking whether there is significant vertical mixing.

Evaluation of the nutritional and inflammatory status in cancer patients for the risk assessment of severe haematological toxicity following chemotherapy.

BACKGROUND: The toxicity outcome of cancer patients receiving chemotherapy is difficult to predict. In this study the influence of malnutrition and inflammation on acute haematological toxicity was investigated. PATIENTS AND METHODS: Between January 1999 and January 2000, 48 consecutive cancer patients experienced severe haematological toxicity (SHT), either neutropenic fever or severe thrombocytopenia, following various chemotherapy regimens. Their baseline characteristics were compared with those of 59 control patients. Previous chemotherapy regimens, type of chemotherapy, performance status (PS), calculated creatinine clearance, bilirubin, C-reactive protein (1), alpha-1 acid glycoprotein (2), albumin (3), pre-albumin (4) and the nutritional and inflammatory status (NIS) ratio [NIS = (1 x 2)/(3 x 4)] were studied. Statistical analysis was carried out using either a t-test or a chi-square test. A receiver operating characteristic (ROC) curve determined the cut-off value for NIS. RESULTS: Patients experiencing SHT had a higher PS (P <0.001), inflammatory serum protein levels (P <0.001) and NIS ratio (P <0.0001), but lower haemoglobin (P <0.05) and serum-albumin levels (P <0.0001). Using a cut-off of 0 or 1 for PS and 1 for NIS, sensitivity was 98%, 43% and 89%; specificity was 38%, 90% and 66%, respectively. In 37 patients treated with topotecan as single agent, the determinants for SHT were PS (P <0.0001) and NIS (P <0.0001). CONCLUSIONS: Altered nutritional and inflammatory status correlates with increased risk of severe haematological toxicity following anticancer chemotherapy.

Immunocytochemical characterization of viruses and antigenic macromolecules in viral vaccines.

Gold immunolabeling combined with negative staining (GINS) provides a valuable immunocytochemical approach that allows a direct ultrastructural definition of all viral vaccine constituents that share common antigenic features with pathogenic viral particles. These results have implications for the development of viral vaccines since it has been demonstrated that incomplete viral particles such as natural empty capsides and Rotavirus-like particles lacking the infective genome are potential candidates for the production of neutralizing antibodies. Furthermore comparative results of the application of GINS to either inactivated vaccines or unfixed samples provide direct evidence that even after inactivation specific antigenic sites are still available for gold immunolabeling.

Analysis of lycopene geometrical isomers in biological microsamples by liquid chromatography with coulometric array detection.

Methods of analysis for determining low quantities of lycopene cis-trans isomers in biological tissues are needed. Development of two liquid chromatography (LC) methods based on the polymeric C30 stationary phase equipped with coulometric electrochemical array detection (ED) is described. Separation of 13 lycopene isomers including prolycopene, (a novel tetra-cis-lycopene found in Tangerine tomatoes) was accomplished with both isocratic and gradient methods using different proportions of methanol, methyl tert.-butyl ether, water and 1 M ammonium acetate buffer. Carotenoids were detected at potential settings between 200 and 620 mV. Differences in generated current-voltage curves aided in tentative identification of trans carotenoid species and select cis isomers of lycopene. These methods were successfully applied in the analysis of small quantities of plasma, buccal mucosal cells, prostate and cervical tissues. Limits of detection for trans-lycopene by ED were found to be 50 fmol representing a 10- to 100-fold increase over conventional UV-Vis absorbance methods.

Relationship of delta-aminolevulinic acid-induced protoporphyrin IX levels to mitochondrial content in neoplastic cells in vitro.

Protoporphyrin IX, induced by the exogenous addition of delta-aminolevulinic acid, reaches different levels in different tumor cells. Because many of the steps in heme biosynthesis, of which protoporphyrin IX is penultimate, are located in the mitochondria, we surmised that the mitochondrial content of cells may relate to the amount of protoporphyrin IX synthesized in response to excess delta-aminolevulinic acid. We observed that accumulation of MitoTracker, a fluorescent mitochondrial probe, delta-aminolevulinic acid-induced protoporphyrin IX levels, and porphobilinogen deaminase activity all presented with the same cell-line-dependent rank order among the four different neoplastic cells. This rank order, however, differed for cytochrome c oxidase activity, the final enzyme in mitochondrial electron transport, and for accumulation of radioactive label from [(14)C]delta-aminolevulinic acid. The data demonstrate that enzymes involved in heme biosynthesis, in general, display a rank order associated with mitochondrial content. These data imply that such parameters may have value as prognosticators of cells to produce delta-aminolevulinic acid-induced protoporphyrin IX, a photosensitizer for photodynamic therapy of cancer.