Time-resolved spectroscopy (TRS-20) measures tissue oxygen saturation (%) by evaluating the absolute concentrations of oxygenated, deoxygenated and total haemoglobin based on measurement of the transit time of individual photons through a tissue of interest. We measured tissue oxygen saturation in the prefrontal lobes of the brain by TRS-20 in eighteen pregnant women during caesarean section. In a case of placenta previa, massive bleeding immediately decreased cerebral oxygen saturation from 67·2% to 54·2%, but did not alter peripheral tissue oxygenation as measured by pulse oximetry. Four cases of pre-eclampsia revealed chronic changes in elevated base levels of cerebral oxygen saturation, though peripheral oxygen saturation was similar to that in normotensive pregnant women. Average cerebral oxygen saturation in the cases of pre-eclampsia before the introduction of anaesthesia was 73·6 ± 4·4 (SD)% (n = 4), significantly higher than in normotensive pregnant women, 67·2 ± 4·3% (n = 13, P<0·05). Z-scores of cerebral oxygen saturation prior to anaesthesia positively correlated with those of systolic or diastolic blood pressure. TRS-20 could detect acute as well as chronic changes in brain oxygen saturation in response to pregnancy-associated complications.
The Drosophila melanogaster Schneider 2 (S2) cell line was established in 1972. Many studies have indicated that generation of recombinant proteins with S2 cells is more desirable than using other methods, since native proteins derived from S2 cells do not usually interact with those derived from mammalian cells. In order to minimize the duration for selections, we established an all-in-one single plasmid pMT-PURO, which enables to express the gene of interest as well as a selection gene ''pac''. However, there is a weak point in the system. In order to verify the hallmark of the transformed cells, puromycin selection as well as verification of the gene of interests is still necessary. To improve this situation, we generated pMT-PURO2G and pMT-PURO2R, which enable to verify the hallmark of the transformed cells during the selections by the detection of enhanced green fluorescent protein (EGFP) or DsRED2. This new system gives reliable and reproductive results for recombinant protein synthesis and gets rid of some degree of uncertainty for the outcome of the transfection.
Serpinopathy is characterised as abnormal accumulation of serine protease inhibitors (SERPINs) in cells and results in clinical symptoms owing to lack of SERPIN function or excessive accumulation of abnormal SERPIN. We recently identified a patient with functional deficiency of plasminogen activator inhibitor-1 (PAI-1), a member of the SERPIN superfamily. The patient exhibited life-threatening bleeding tendencies, which have also been observed in patients with a complete deficiency in PAI-1. Sequence analysis revealed a homozygous single-nucleotide substitution from guanine to cytosine at exon 9, which changed amino acid residue 397 from glycine to arginine (c.1189G>C; p.Gly397Arg). This glycine was located in strand 5B and was well conserved in other serpins. The mutant PAI-1 was polymerised in the cells, interfering with PAI-1 secretion. The corresponding mutations in SERPINC1 (anti-thrombin III) at position 456 (Gly456Arg) and SERPINI1 (neuroserpin) at position 392 (Gly392Glu) caused an anti-thrombin deficiency and severe dementia due to intracellular retention of the polymers. Glycine is the smallest amino acid, and these mutated amino acids were larger and charged. To determine which factors were important, further mutagenesis of PAI-1 was performed. Although the G397A, C, I, L, S, T, and V were secreted, the G397D, E, F, H, K, M, N, P, Q, W, and Y were not secreted. The results revealed that the size was likely triggered by the polymerisation of SEPRINs at this position. Structural analyses of this mutated PAI-1 would be useful to develop a novel PAI-1 inhibitor, which may be applicable in the context of several pathological states.
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