Proteome and cytoskeleton responses in osteosarcoma cells with reduced OXPHOS activity

We have recently shown disorganization of the vimentin network in cultured cells deficient in oxidative phosphorylation (OXPHOS). We describe here the cellular responses to OXPHOS deficiency in osteosarcoma cells upon complex I (CI) and complex IV (CIV) inhibition, and upon the lack of mitochondrial DNA (rho0 cells). We examined the cytoskeletal organization and the distribution of mitochondria and analysed total proteome by 2-DE and vimentin expression by ELISA. Upon CIV inhibition and in rho0 cells, the vimentin network had collapsed around the nucleus and formed thick bundles. The mitochondria formed a perinuclear crescent upon CIV inhibition, whereas they accumulated around the nucleus in the rho0 cells, where the amount of vimentin was increased. Analysis of the total proteome revealed that a lack of mitochondrial DNA or inhibition of CI or CIV led to changes in the expression of cytoskeletal and cytoskeleton-associated proteins and proteins involved in apoptosis, OXPHOS, glycolysis, the tricarboxylic acid cycle, and oxidative stress responses. Our findings suggest that a deficiency in the energy converting system and oxidative stress can lead to cytoskeletal changes.     

Inhibition of TGF-beta2 with AP 12009 in recurrent malignant gliomas: from preclinical to phase I/II studies

Transforming growth factor-beta2 (TGF-beta2) is known to suppress the immune response to cancer cells and plays a pivotal role in tumor progression by regulating key mechanisms including proliferation, metastasis, and angiogenesis. For targeted protein suppression the TGF-beta2-specific antisense oligodeoxynucleotide AP 12009 was developed. In vitro experiments have been performed to prove specificity and efficacy of the TGF-beta2 inhibitor AP 12009 employing patient-derived malignant glioma cells as well as peripheral blood mononuclear cells (PBMCs) from patients. Clinically, the antisense compound AP 12009 was assessed in three Phase I/II-studies for the treatment of patients with recurrent or refractory malignant (high-grade) glioma WHO grade III or IV. Although the study was not primarily designed as an efficacy evaluation, prolonged survival compared to literature data and response data were observed, which are very rarely seen in this tumor indication. Two patients experienced long-lasting complete tumor remissions. These results implicate targeted TGF-beta2-suppression using AP 12009 as a promising novel approach for malignant gliomas and other highly aggressive, TGF-beta-2-overexpressing tumors.     

Comparison of the subcellular distribution of G-proteins in hepatocytes in situ and in primary cultures

The subcellular localization of the heterotrimeric G-proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G-proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via G_sα were without effect, which was consistent with the localization of G_sα in the cytoplasmic and nuclear compartments. The “relocalization” of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell-cell and cell-extracellular matrix interactions. © 1996 Wiley-Liss, Inc.     

A systematic review and meta-analysis of the aetiological agents of non-malarial febrile illnesses in Africa

BackgroundThe awareness of non-malarial febrile illnesses (NMFIs) has been on the rise over the last decades. Therefore, we undertook a systematic literature review and meta-analysis of causative agents of non-malarial fevers on the African continent.MethodologyWe searched for literature in African Journals Online, EMBASE, PubMed, Scopus, and Web of Science databases to identify aetiologic agents that had been reported and to determine summary estimates of the proportional morbidity rates (PMr) associated with these pathogens among fever patients.FindingsA total of 133 studies comprising 391,835 patients from 25 of the 54 African countries were eligible. A wide array of aetiologic agents were described with considerable regional differences among the leading agents. Overall, bacterial pathogens tested from blood samples accounted for the largest proportion. The summary estimates from the meta-analysis were low for most of the agents. This may have resulted from a true low prevalence of the agents, the failure to test for many agents or the low sensitivity of the diagnostic methods applied. Our meta-regression analysis of study and population variables showed that diagnostic methods determined the PMr estimates of typhoidal Salmonella and Dengue virus. An increase in the PMr of Klebsiella spp. infections was observed over time. Furthermore, the status of patients as either inpatient or outpatient predicted the PMr of Haemophilus spp. infections.ConclusionThe small number of epidemiological studies and the variety of NMFI agents on the African continent emphasizes the need for harmonized studies with larger sample sizes. In particular, diagnostic procedures for NMFIs should be standardized to facilitate comparability of study results and to improve future meta-analyses. Reliable NMFI burden estimates will inform regional public health strategies.     

Deletion of hypoxia-inducible factor prolyl 4-hydroxylase 2 in FoxD1-lineage mesenchymal cells leads to congenital truncal alopecia

Hypoxia-inducible factors (HIFs) induce numerous genes regulating oxygen homeostasis. As oxygen sensors of the cells, the HIF prolyl 4-hydroxylases (HIF-P4Hs) regulate the stability of HIFs in an oxygen-dependent manner. During hair follicle (HF) morphogenesis and cycling, the location of dermal papilla (DP) alternates between the dermis and hypodermis and results in varying oxygen levels for the DP cells. These cells are known to express hypoxia-inducible genes, but the role of the hypoxia response pathway in HF development and homeostasis has not been studied. Using conditional gene targeting and analysis of hair morphogenesis, we show here that lack of Hif-p4h-2 in Forkhead box D1 (FoxD1)-lineage mesodermal cells interferes with the normal HF development in mice. FoxD1-lineage cells were found to be mainly mesenchymal cells located in the dermis of truncal skin, including those cells composing the DP of HFs. We found that upon Hif-p4h-2 inactivation, HF development was disturbed during the first catagen leading to formation of epithelial-lined HF cysts filled by unorganized keratins, which eventually manifested as truncal alopecia. Furthermore, the depletion of Hif-p4h-2 led to HIF stabilization and dysregulation of multiple genes involved in keratin formation, HF differentiation, and HIF, transforming growth factor β (TGF-β), and Notch signaling. We hypothesize that the failure of HF cycling is likely to be mechanistically caused by disruption of the interplay of the HIF, TGF-β, and Notch pathways. In summary, we show here for the first time that HIF-P4H-2 function in FoxD1-lineage cells is essential for the normal development and homeostasis of HFs.     

The Circadian Clock Gene Circuit Controls Protein and Phosphoprotein Rhythms in Arabidopsis thaliana

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The distribution of microplankton in the McMurdo Dry Valley Lakes,Antarctica: response to ecosystem legacy or present-day climatic controls?

Plankton abundance and biomass were investigated in five lakes of the McMurdo Dry Valleys, Antarctica: Lakes Bonney, Fryxell, Joyce, Hoare and Miers. Despite plankton communities being dominated by organisms <100 m in length, there were striking differences between the lakes, including large variations in plankton vertical distribution and differences in total plankton biomass. Bacterial biomass was highest in the anoxic monimolimnia of the meromictic lakes, reaching 191 g C l^–1 in Lake Fryxell. Photosynthetic nanoflagellates dominated phytoplankton in the five lakes studied. Highest chlorophyll a concentrations were recorded at the chemocline of Lake Fryxell (21 g chl a l^–1). Heterotrophic nanoflagellate concentrations were low, ranging from 2 cells ml^–1 in Hoare to 237 cells ml^–1 in Bonney. By Antarctic standards, ciliates were relatively successful in terms of biomass and diversity in Lakes Fryxell and Hoare. In contrast, Lake Miers possessed extremely low ciliate abundance (<0.04 cells ml^–1). On both sampling occasions, copepod nauplii were observed in Lake Joyce. This is the first recording of crustacean zooplankton within the McMurdo Dry Valley Lakes. Because the foodwebs of these lakes are structured by bottom-up forces, differences in plankton distributions could be related to the physicochemical characteristics of each lake. The effect of lake evolution (legacy) and present-day climate change on planktonic dynamics is discussed.     

Streptococcus alactolyticus is the dominating culturable lactic acid bacterium species in canine jejunum and feces of four fistulated dogs

Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was cultured and enumerated using MRS agar. LAB levels ranging from 1.4x10(6) to 1.5x10(7) CFU ml(-1) were obtained in jejunal chyme. In the fecal samples 7.0x10(7) and 2.0x10(8) CFU g(-1) were detected. Thirty randomly selected isolates growing in the highest sample dilutions were identified to species level using numerical analysis of 16S and 23S rDNA restriction fragment length polymorphism patterns (ribotyping) and 16S rDNA sequence analysis. According to these results, Streptococcus alactolyticus was the dominant culturable LAB species in both feces and jejunal chyme. In addition, Lactobacillus murinus and Lactobacillus reuteri were detected.