Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.     

Apoptosis, cell proliferation and serotonin immunoreactivity in gut of Liza aurata from natural heavy metal polluted environments: preliminary observations

In the present paper, the effect of natural environment non-lethal heavy metal concentration on cell renewal of Liza aurata intestinal epithelium, was studied by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling) method and anti-PCNA (proliferating cell nuclear antigen) immunohistochemistry, in order to detect, respectively, apoptosis and cell proliferation. In addition, the presence and distribution of the cell renewal regulator, serotonin, was immunohistochemically investigated. In order to reduce variability, only immature specimens were considered. The results indicated that in the control specimens from non-polluted areas, the PCNA immunoreactive nuclei of the proximal intestinal epithelium were only located at the bottom of the intestinal folds, together with a few TUNEL-positive nuclei, and goblet mucous differentiated cells. In the specimens from polluted areas, the number of PCNA immunoreactive cells was greatly enhanced, and they extended along the mid portion of the intestinal folds; the number of TUNEL-positive nuclei was enhanced as well, but they were almost exclusively detected in the third apical portion of the intestinal folds. Serotonin immunoreactive nerve elements were more frequently detected in the intestinal wall of L. aurata specimens from polluted areas, and besides that, some serotonin immunoreactive endocrine cells were also present. Variations in distribution and frequency of TUNEL-positive nuclei, PCNA immunoreactive nuclei, and serotonin immunoreactivity put in evidence an alteration of cell renewal with an enhancement of cell proliferation, probably leading to morphological intestinal fold changes.     

A Specific Real-Time Quantitative PCR Detection System for Event MON810 in Maize YieldGard® Based on the 3′-Transgene Integration Sequence

The increasing presence of transgenic plant derivatives in a wide range of animal and human consumables has provoked in western Europe a strong demand for appropriate detection methods to evaluate the existence of transgenic elements. Among the different techniques currently used, the real-time quantitative PCR is a powerful technology well adapted to the mandatory labeling requirements in the European Union (EU). The use of transgene flanking genomic sequences has recently been suggested as a means to avoid ambiguous results both in qualitative and quantitative PCR-based technologies. In this study we report the identification of genomic sequences adjacent to the 3-integration site of event MON810 in transgenic maize. This genetically modified crop contains transgene sequences leading to ectopic expression of a synthetic CryIA(b) endotoxin which confers resistance to lepidopteran insects especially against the European corn borer. The characterization of the genome–transgene junction sequences by means of TAIL-PCR has facilitated the design of a specific, sensitive and accurate quantification method based on TaqMan chemistry. Cloning of event MON810 3-junction region has also allowed to compare the suitability of plasmid target sequences versus genomic DNA obtained from certified reference materials (CRMs), to prepare standard calibration curves for quantification.     

Prediction of gestational age by ultrasonic fetometry in llamas (Lama glama) and alpacas (Lama pacos)

Fetal biparietal diameter (BPD) and thorax height (TH) were measured by ultrasound during intrauterine growth in pregnant llamas (Lama glama) and alpacas (Lama pacos). The goal was to establish representative curves that allows estimation of gestational age (GA) from real-time ultrasonic measurements of these fetal structures at any stage of gestation. Llamas and alpacas were mated under controlled conditions. Ultrasound exams were conducted to determine pregnancy status 1 month later. Measurements of fetal BPD and TH were conducted from the second month of pregnancy until term. Observation and assessment of fetal TH was difficult during the last 3 months of pregnancy, specially in llamas. Regression curves were calculated from the data as a function of GA, with the best fit represented by the following equations: llama GA=(BPD-0.002399)43.02293,r=0.98,P<0.001; llama GA=(TH-0.07137)46.94485, r=0.95,P<0.001; alpaca GA=(BPD-0.11376)47.23287, r=0.98,P<0.001; alpaca GA=(TH-0.36436)52.87663, r=0.96,P<0.001, where GA was measured in days and BPD and TH in centimeters. Results indicate that ultrasonic measurement of these fetal biometric variables constitute a valuable tool to estimate GA at any stage of pregnancy in these domestic South American camelids.     

土壤低剂量芘污染对蚯蚓若干生化指标的影响

通过人工污染土壤的方法,设计芘的暴露浓度为0、60、120、240、480、960μg.kg-1.暴露实验进行1、3、7和14d后,分别检测蚯蚓内脏中细胞色素P450含量、谷胱甘肽转移酶(GST)、超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量.结果表明,在供试浓度范围内,蚯蚓内脏中各生化指标对污染物暴露指示的敏感性存在差异:其中P450含量、GST和SOD活性最为敏感;POD和CAT活性次之;而MDA含量未对低剂量的芘暴露起到明显的指示作用.研究同时发现,低剂量污染物暴露的时间效应要强于剂量效应的影响.因而,在进行生态毒性诊断时,采用多指标和多时段的检测对增强指示的灵敏性和有效性尤为重要.     

Short-term hypoxia/reoxygenation activates the angiogenic pathway in rat caudate putamen

In response to hypoxia, tissues have to implement numerous mechanisms to enhance oxygen delivery, including the activation of angiogenesis. This work investigates the angiogenic response of the hypoxic caudate putamen after several recovery times. Adult Wistar rats were submitted to acute hypoxia and analysed after 0 h, 24 h and 5 days of reoxygenation. Expression of hypoxia-inducible factor-1 alfa (HIF-1α) and angiogenesis-related genes including vascular endothelial growth factor (VEGF), adrenomedullin (ADM) and transforming growth factor-beta 1 (TGF-β1) was determined by both RT-PCR and ELISA. For vessel labelling, lectin location and expression were analysed using histochemical and image processing techniques (fractal dimension). Expression of Hif-1α, Vegf, Adm and Tgf-β1 mRNA rose immediately after hypoxia and this increase persisted in some cases after 5 days post-hypoxia. While VEGF and TGF-β1 protein levels increased parallel to mRNA expression, ADM remained unaltered. The quantification of the striatal vessel network showed a significant augmentation at 24 h of reoxygenation. These results reveal that not only short-term hypoxia, but also the subsequent reoxygenation period, up-regulate the angiogenic pathway in the rat caudate putamen as a neuroprotective mechanism to hypoxia that seeks to maintain a proper blood supply to the hypoxic tissue, thereby minimizing the adverse effects of oxygen deprivation.     

The deleterious effects of bed rest on human skeletal muscle fibers are exacerbated by hypercortisolemia and ameliorated by dietary supplementation

Prolonged inactivity associated with bed rest in a clinical setting or spaceflight is frequently associated with hypercortisolemia and inadequate caloric intake. Here, we determined the effect of 28 days of bed rest (BR); bed rest plus hypercortisolemia (BRHC); and bed rest plus essential amino acid (AA) and carbohydrate (CHO) supplement (BRAA) on the size and function of single slow- and fast-twitch muscle fibers. Supplementing meals, the BRAA group consumed 16.5 g essential amino acids and 30 g sucrose at 1100, 1600, and 2100 h, and the BRHC subjects received 5 daily doses of 10–15 mg of oral hydrocortisone sodium succinate throughout bed rest. Bed rest induced atrophy and loss of force (mN) and power (µN·FL·s^–1) in single fibers was exacerbated by hypercortisolemia where soleus peak force declined by 23% in the type I fiber from a prevalue of 0.78 ± 0.02 to 0.60 ± 0.02 mN post bed rest (compared to a 7% decline with bed rest alone) and 27% in the type II fiber (1.10 ± 0.08 vs. 0.81 ± 0.05 mN). In the BRHC group, peak power dropped by 19, 15, and 11% in the soleus type I, and vastus lateralis (VL) type I and II fibers, respectively. The AA/CHO supplement protected against the bed rest-induced loss of peak force in the type I soleus and peak power in the VL type II fibers. These results provide evidence that an AA/CHO supplement might serve as a successful countermeasure to help preserve muscle function during periods of relative inactivity. isotonic contractile properties; peak force and power; calcium sensitivity; essential amino acids     

Melanin in <Emphasis Type="Italic">Fonsecaea pedrosoi</Emphasis>: a trap for oxidative radicals

Background  

The pathogenic fungus Fonsecaea pedrosoi constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus.     

The effect of time on physiological changes in eel Anguilla anguilla, induced by lindane.

1. Eel were exposed to a sublethal concentration of lindane (0.335 ppm) for 6, 12, 24, 48, 72 and 96 hr. 2. Concentrations of glycogen, glucose, lactate, pyruvate and lipids were determined in gill tissue after lindane exposure. 3. Gill glycogen decreased and glucose levels increased at 6 hr of treatment, lactate and pyruvate concentration increased between 6 and 48 hr. Total lipid values decreased between 6 and 24 hr; thereafter, the levels increased up to 72 hr of exposure. 4. Clear changes were found in all parameters tested in gill tissues. The observed effects of lindane on metabolism in fish are discussed in relation to acute stress syndrome.