Autophagy facilitates secretion and protects against degeneration of the Harderian gland

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.     

High concordance between marker profiles of 22 human leukemia-lymphoma cell lines tested with the same monoclonal antibodies before and during the second international workshop on human differentiation antigens

Summary Our laboratory participated in the Second International Workshop and Conference on Human Leucocyte Differentiation Antigens. In this international study the reactivity profiles of monoclonal antibodies were analyzed on normal and malignant hematopoietic cells. The Workshop was divided into three categories: the T-cell, B-cell and myelomonocytic cell studies. We blindly tested 159 coded monoclonal antibodies of the panel for the T-cell study on 22 permanently established leukemia cell lines. The monoclonal antibodies were provided by the Workshop Committee and their reactivity with the target cells was visualized by standardized indirect immunofluorescence. After decoding it was recognized that 11 monoclonal antibodies had been examined on these cell lines prior to the Workshop. The reactivity of these 11 monoclonal antibodies was analyzed and compared with the earlier results. From a total of 217 paired tests done blindly in the Workshop study and prior to the Workshop, 191 tests (88%) did not show significantly different data. The possible reasons for discrepancies include nonspecific Fc-receptor-binding on some cell lines and a relatively nonspecific reactivity of some monoclonal antibodies.This analysis demonstrates the stability of the antigen expression on human leukemia-lymphoma cell lines grown at consistently optimal conditions, for the tests, using the same monoclonal antibodies as in the Workshop, had been performed 0.5–5 years prior to the Workshop study. On the other hand, nonspecific Fc-binding, wide specificity of monoclonal antibodies and a shift in antigen expression of the cells (due to poor growth conditions, involuntary induction of differentiation and other factors) must be taken into consideration upon immunological analysis.This study was supported in part by Veterans Administration RAG GrantH. G. D. is recipient of a Research Training Fellowship awarded by the Deutsche Forschungsgemeinschaft (Dr 157/1-1)     

The EMBL nucleotide sequence database

The European Molecular Biology Laboratory Nucleotide Sequence Database receives sequence and sequence annotation data from genome projects, sequencing centers, individual scientists, and patent offices. Data may be most efficiently submitted to the database using the Internet based submission tool WEBIN or via previously established genome project accounts. Biologist curators will review the data and provide accession numbers within two working days. Non-confidential data are exchanged daily in an international collaboration between EMBL, DDBJ (the DNA Databank of Japan) and GenBank (USA) and may be accessed and retrieved via the Internet with the Sequence Retrieval System (SRS). Sequence database searching algorithms (e.g., Blitz, Fasta, Blast) are available for comparison of query to database sequences.     

Phase Transitions of Frozen Camu-camu (Myrciaria dubia (H.B.K.) McVaugh) Pulp: Effect of Cryostabilizer Addition

Camu-camu is a tropical fruit with very high vitamin C content and commercialized as frozen pulp. Enthalpies of freezing, temperatures of the onset of ice melting, and glass transition temperatures of the maximally freeze-concentrated phase () of camu-camu pulp and of samples containing maltodextrin (DE20) and sucrose were measured by differential scanning calorimetry. Maltodextrin exhibited the largest freeze stabilization potential, increasing from −58.2 °C (natural pulp) to −39.6 °C when 30% (w/w) maltodextrin DE 20 was added. Sucrose showed negligible effect on but enhanced considerably the freezing point depression and less amount of ice was formed.     

Long-term resistance to tomato spotted wilt virus in transgenic tobacco cultivars expressing the viral nucleoprotein gene: greenhouse and field tests

We examined the resistance phenotype of a large number of transgenic tobacco plants originating from 12 commercial (Nicotiana tabacum) cultivars expressing the sense form of the nucleoprotein (N) gene of L3, a Bulgarian isolate of tomato spotted wilt virus (TSWV). The analysis revealed that transgenic plants are completely protected against the homologous L3 isolate of TSWV irrespective of whether or not they contain detectable levels of translational product. The effectiveness of protection against the virus was investigated upon mechanical inoculation under greenhouse conditions and in field trials. Non-segregating resistant lines were selected and the inheritance of the resistance to TSWV was analysed in successive generations (R₃–R₆). Extensive tests under controlled conditions and two-year field trials proved that the resistance to TSWV is stable in different environments and is a stably inherited trait.     

Effect of phytoestrogens on gene expression of carbonic anhydrase II in rat uterus and liver

The aim of this study was to characterize carbonic anhydrase II (CA2), as novel estrogen responsive gene, towards its usefulness to elucidate the molecular mechanisms of phytoestrogen action. Effects of estradiol-17beta (E2), and the phytoestrogens genistein (Gen), daidzein (Dai), as well as 8-prenylnaringenin (8PN) on CA2 mRNA expression were investigated in vivo in the uterus and liver of Wistar rats, and in vitro in Fe33 hepatoma cells. Relative amounts of mRNA levels of CA2 were measured by real-time RT-PCR. In vivo CA2 expression in uterus and liver is down-regulated by estrogen in time dependent manner with the most pronounced effect detectable 72 h after treatment. Treatment with Gen results in a slight down-regulation of CA2 expression in the uterus. In liver a response to Gen is detectable only after 7 h, where the expression of the gene is down-regulated to 60%. Treatment with Dai and 8PN for 72 h results in a slight down-regulation of CA2 in both tissues. In contrast in Fe 33 cells CA2 gene expression was up-regulated in response to the treatment with E2 for 7 h. In summary, we could demonstrate that the modulation of CA2 gene expression following treatment with E2 and Gen in rat uterus is comparable to the uterotrophic response of these substances, but with an inverted pattern. Remarkably, of all phytoestrogens 8PN exhibited the strongest uterotrophic response but only induced a very faint decrease of CA2 expression. In addition, we provide the first pieces of evidence that 8PN, like Gen and Dai, cannot be considered as a pure agonist. In conclusion, CA2 shows estrogen sensitivity not only in both tissues studied, but also in many others. Further, it exhibits a differential sensitivity thereby being capable to discriminate between different molecular qualities of phytoestrogens, like demonstrated for Gen and 8PN.     

Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle.

Hormone-sensitive lipase (HSL) is believed to play an important role in the mobilization of fatty acids from triglycerides (TG), diglycerides, and cholesteryl esters in various tissues. Because HSL-mediated lipolysis of TG in adipose tissue (AT) directly feeds non-esterified fatty acids (NEFA) into the vascular system, the enzyme is expected to affect many metabolic processes including the metabolism of plasma lipids and lipoproteins. In the present study we examined these metabolic changes in induced mutant mouse lines that lack HSL expression (HSL-ko mice). During fasting, when HSL is normally strongly induced in AT, HSL-ko animals exhibited markedly decreased plasma concentrations of NEFA (-40%) and TG (-63%), whereas total cholesterol and HDL cholesterol levels were increased (+34%). Except for the increased HDL cholesterol concentrations, these differences were not observed in fed animals, in which HSL activity is generally low. Decreased plasma TG levels in fasted HSL-ko mice were mainly caused by decreased hepatic very low density lipid lipoprotein (VLDL) synthesis as a result of decreased NEFA transport from the periphery to the liver. Reduced NEFA transport was also indicated by a depletion of hepatic TG stores (-90%) and strongly decreased ketone body concentrations in plasma (-80%). Decreased plasma NEFA and TG levels in fasted HSL-ko mice were associated with increased fractional catabolic rates of VLDL-TG and an induction of the tissue-specific lipoprotein lipase (LPL) activity in cardiac muscle, skeletal muscle, and white AT. In brown AT, LPL activity was decreased. Both increased VLDL fractional catabolic rates and increased LPL activity in muscle were unable to provide the heart with sufficient NEFA, which led to decreased tissue TG levels in cardiac muscle. Our results demonstrate that HSL deficiency markedly affects the metabolism of TG-rich lipoproteins by the coordinate down-regulation of VLDL synthesis and up-regulation of LPL in muscle and white adipose tissue. These changes result in an "anti-atherogenic" lipoprotein profile.     

Verification of performance with the automated direct optical TIRF immunosensor (River Analyser) in single and multi-analyte assays with real water samples

In order to verify the reproducibility, precision, and robustness of the optical immunosensor River Analyser (RIANA), we investigated two common statistical methods to evaluate the limit of detection (LOD) and the limit of quantification (LOQ). Therefore, we performed a simultaneous multi-analyte calibration with atrazine, bisphenol A, and estrone in Milli-Q water. Using an automated biosensor, it was possible for the first time to achieve a LOD below 0.020 microg L(-1) using a common statistically based method without sample pre-treatment and pre-concentration for each of the analytes in a simultaneous multi-analyte calibration. This biosensor setup shows values comparable to those obtained by more classical analytical methods. Based on this calibration, we measured spiked and un-spiked real water samples with complex matrices (samples from different water bodies, from ground water sources, and tap water samples). The comparison between our River Analyser and common analytical methods (like GC-MS and HPLC-DAD) shows overall comparable values for all three analytes. Furthermore, a calibration of isoproturon (IPU) (in single analyte mode) resulted in a LOD of 0.016 microg L(-1), and a LOQ of 0.091 microg L(-1). In compliance with guidelines of the Association of Analytical Communities International (AOAC), six out of nine recovery rates (recovery rate: measured concentration divided by real concentration in percent) for three surface water samples with different matrices (spiked and un-spiked) could be obtained between 70 and 120% (recovery rates between 70 and 120%, as demanded by the guidelines of the AOAC International). The reproducibility was checked by measuring replica of each sample within independent repetitions. Robustness could be demonstrated by long-term stability tests of the biosensor surface. These studies show that the biosensor used offers the necessary reproducibility, precision, and robustness required for an analytical method.     

Genotypic and Phenotypic Characterization of Staphylococcus aureus Isolates from Wild Boars

Eight Staphylococcus aureus isolates collected from 117 wild boars were characterized and compared to livestock isolates. They belonged to sequence types ST133, ST425, and the new type ST1643. The spa types were t1181, t6782, and the new types t6384, t6385, and t6386. Antimicrobial susceptibility testing and microarray-based genotyping confirmed the absence of important virulence/resistance genes.