Globin genes are present in Ciona intestinalis

The key position of the Ciona intestinalis basal to the vertebrate phylogenetic tree brings up the question of which respiratory proteins are used by the tunicate to facilitate oxygen transport and storage. The publication of the Ciona draft genome sequence suggests that globin genes are completely missing and that-like some molluscs and arthropods-the sea squirt uses hemocyanin instead of hemoglobin for respiration. However, we report here the presence and expression of at least four distinct globin gene/protein sequences in Ciona. This finding is in agreement with the ancestral phylogeny of the vertebrate globins. Moreover, it seems likely that the Ciona hemocyanin-like sequences have enzymatic instead of respiratory functions.     

Survival and growth of Francisella tularensis in Acanthamoeba castellanii

Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.     

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.     

The A1Ao ATPase from Methanosarcina mazei: Cloning of the 5′ End of the aha Operon Encoding the Membrane Domain and Expression of the Proteolipid in a Membrane-Bound Form in Escherichia coli

Three additional ATPase genes, clustered in the order ahaH, ahaI, and ahaK, were found upstream of the previously characterized genes ahaECFABDG coding for the archaeal A₁A_o ATPase from Methanosarcina mazei. ahaH, the first gene in the cluster, is preceded by a conserved promoter sequence. Northern blot analysis revealed that the clusters ahaHIK and ahaECFABDG are transcribed as one message. AhaH is a hydrophilic polypeptide and is similar to peptides of previously unassigned function encoded by genes preceding postulated ATPase genes in Methanobacterium thermoautotrophicum and Methanococcus jannaschii. AhaI has a two-domain structure with a hydrophilic domain of 39 kDa and a hydrophobic domain with seven predicted transmembrane α helices. It is similar to the 100-kDa polypeptide of V₁V_o ATPases and is therefore suggested to participate in proton transport. AhaK is a hydrophobic polypeptide with two predicted transmembrane α helices and, on the basis of sequence comparisons and immunological studies, is identified as the proteolipid, a polypeptide which is essential for proton translocation. However, it is only one-half and one-third the size of the proteolipids from M. thermoautotrophicum and M. jannaschii, respectively. ahaK is expressed in Escherichia coli, and it is incorporated into the cytoplasmic membrane despite the different chemical natures of lipids from archaea and bacteria. This is the first report on the expression and incorporation into E. coli lipids of a membrane integral enzyme from a methanogens, which will facilitate analysis of the structure and function of the membrane domain of the methanoarchaeal ATPase.     

Respiratory changes associated with rapid eye movements in normo- and hypercapnia during sleep

Rapid eyemovements during rapid-eye-movement (REM) sleep are associated withrapid, shallow breathing. We wanted to know whether thiseffect persisted during increased respiratory drive byCO₂. In eight healthy subjects, werecorded electroencephalographic, electrooculographic, andelectromyographic signals, ventilation, and end-tidalPCO₂ during the night. InspiratoryPCO₂ was changed to increaseend-tidal PCO₂ by 3 and 6 Torr. During normocapnia, rapid eye movements were associated with a decreasein total breath time by 0.71 ± 0.19 (SE) s(P < 0.05) because of shortenedexpiratory time (0.52 ± 0.08 s,P < 0.001) and with a reduced tidalvolume (89 ± 27 ml, P < 0.05) because of decreased rib cage contribution (75 ± 18 ml, P < 0.05). Abdominal (11 ± 16 ml, P = 0.52) and minuteventilation (0.09 ± 0.21 ml/min, P = 0.66) did not change. Inhypercapnia, however, rapid eye movements were associated with afurther shortening of total breath time. Abdominal breathing was alsoinhibited (79 ± 23 ml, P < 0.05), leading to a stronger inhibition of tidal volume and minuteventilation (1.84 ± 0.54 l/min,P < 0.05). We conclude thatREM-associated respiratory changes are even more pronounced duringhypercapnia because of additional inhibition of abdominal breathing.This may contribute to the reduction of the hypercapnic ventilatory response during REM sleep.

     

The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 g peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (−81%) and bone formation (−80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations.KEY WORDS: Whole-body vibration, LMHFV, Fracture healing, Oestrogen receptor signalling, Wnt signalling     

Genomics of Divergence along a Continuum of Parapatric Population Differentiation

The patterns of genomic divergence during ecological speciation are shaped by a combination of evolutionary forces. Processes such as genetic drift, local reduction of gene flow around genes causing reproductive isolation, hitchhiking around selected variants, variation in recombination and mutation rates are all factors that can contribute to the heterogeneity of genomic divergence. On the basis of 60 fully sequenced three-spined stickleback genomes, we explore these different mechanisms explaining the heterogeneity of genomic divergence across five parapatric lake and river population pairs varying in their degree of genetic differentiation. We find that divergent regions of the genome are mostly specific for each population pair, while their size and abundance are not correlated with the extent of genome-wide population differentiation. In each pair-wise comparison, an analysis of allele frequency spectra reveals that 25–55% of the divergent regions are consistent with a local restriction of gene flow. Another large proportion of divergent regions (38–75%) appears to be mainly shaped by hitchhiking effects around positively selected variants. We provide empirical evidence that alternative mechanisms determining the evolution of genomic patterns of divergence are not mutually exclusive, but rather act in concert to shape the genome during population differentiation, a first necessary step towards ecological speciation.     

Nerve Sheath Tumors in Neurofibromatosis Type 1: Assessment of Whole-Body Metabolic Tumor Burden Using F-18-FDG PET/CT

Purpose

To determine the metabolically active whole-body tumor volume (WB-MTV) on F-18-fluorodeoxyglucose positron emission tomography/computed tomography (F-18-FDG PET/CT) in individuals with neurofibromatosis type 1 (NF1) using a three-dimensional (3D) segmentation and computerized volumetry technique, and to compare PET WB-MTV between patients with benign and malignant peripheral nerve sheath tumors (PNSTs).

Patients and Methods

Thirty-six NF1 patients (18 patients with malignant PNSTs and 18 age- and sex-matched controls with benign PNSTs) were examined by F-18-FDG PET/CT. WB-MTV, whole-body total lesion glycolysis (WB-TLG) and a set of semi-quantitative imaging-based parameters were analyzed both on a per-patient and a per-lesion basis.

Results

On a per-lesion basis, malignant PNSTs demonstrated both a significantly higher MTV and TLG than benign PNSTs (p < 0.0001). On a per-patient basis, WB-MTV and WB-TLG were significantly higher in patients with malignant PNSTs compared to patients with benign PNSTs (p < 0.001). ROC analysis showed that MTV and TLG could be used to differentiate between benign and malignant tumors.

Conclusions

WB-MTV and WB-TLG may identify malignant change and may have the potential to provide a basis for investigating molecular biomarkers that correlate with metabolically active disease manifestations. Further evaluation will determine the potential clinical impact of these PET-based parameters in NF1.