Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate.

An automated assay of plasma prekallikrein is described. Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the hydrolytic activity of kallikrein to H-D-Pro-Phe-Arg-paranitroanilide subsequently measured.

The conversion was complete within 8 minutes and the amidolytic activity remained stable at least another 10 min at 37 ° C. This method worked in plasma deficient in Hageman factor (blood coagulation factor XII). Using anti-prekallikrein antibody and plasma deficient in prekallikrein, the amidolytic activity generated in normal plasma was identified as due to kallikrein. With plasma samples, the coefficients of variation (CV) for multiple measurements within run (n = 10) and between run (n = 10) were as low as 5.0% and 6.6%, respectively, and the minimum measurable concentration of prekallikrein in plasma was 10% of the normal level.     

Endovascular Treatment of Aneurysmal Subarachnoid Hemorrhage in Japanese Registry of Neuroendovascular Therapy (JR-NET) 1 and 2

To distinguish the characteristics of ruptured cerebral aneurysm that are suitable for endovascular treatment from those that are not, we evaluated factors that influenced the results of aneurysm embolization in patients with ruptured cerebral aneurysm, based on data from the Japanese Registry of Neuroendovascular Therapy (JR-NET) 1 and 2. The multivariate analysis revealed that young patients, patients with low modified Rankin Scale (mRS) scores before onset, and patients with low World Federation of Neurosurgical Societies (WFNS) grades had good outcome. Compared to proximal internal carotid artery (ICA) aneurysms, the odds ratio of middle cerebral artery (MCA) aneurysms was 1.67, indicating poorer outcome for MCA aneurysms, and patients with small, wide-neck cerebral aneurysms had poor outcome. Patients treated after 15 days had better outcome than during other periods. The timing of treatment, however, did not influence the outcome in patients treated within 14 days. The outcome was poorer when the responsible doctor for the treatment was a specialist or a non-specialist than a supervisory doctor. The outcome of patients treated with bare platinum coils, and three dimensional (3D) rotational angiography was better, and the outcome of patients who completed treatment with body filling was poorer than in patients with complete occlusion. Perioperative hemorrhagic complications, all ischemic complications, and rebleeding occurred in 4.5%, 6.4%, and 1.4% of patients, respectively. All these complications had poor outcome factors on day 30, with odds ratios of 2.72, 2.96, and 25.49, respectively. We must be fully aware of these risk factors and determine indications for the treatment when endovascular treatment is performed as the treatment of choice for ruptured cerebral aneurysm.     

Conversion of Aβ43 to Aβ40 by the successive action of angiotensin‐converting enzyme 2 and angiotensin‐converting enzyme

The longer and neurotoxic species of amyloid‐β protein (Aβ), Aβ42 and Aβ43, contribute to Aβ accumulation in Alzheimer's disease (AD) pathogenesis and are considered to be the primary cause of the disease. In contrast, the predominant secreted form of Aβ, Aβ40, inhibits amyloid deposition and may have neuroprotective effects. We have reported that angiotensin‐converting enzyme (ACE) converts Aβ42 to Aβ40 and that Aβ43 is the earliest‐depositing Aβ species in the amyloid precursor protein transgenic mouse brain. Here we found that Aβ43 can be converted to Aβ42 and to Aβ40 in mouse brain lysate. We further identified the brain Aβ43‐to‐Aβ42‐converting enzyme as ACE2. The purified human ACE2 converted Aβ43 to Aβ42, and this activity was inhibited by a specific ACE2 inhibitor, DX600. Notably, the combination of ACE2 and ACE could convert Aβ43 to Aβ40. Our results indicate that the longer, neurotoxic forms of Aβ can be converted to the shorter, less toxic or neuroprotective forms of Aβ by ACE2 and ACE. Moreover, we found that ACE2 activity showed a tendency to decrease in the serum of AD patients compared with normal controls, suggesting an association between lower ACE2 activity and AD. Thus, maintaining brain ACE2 and ACE activities may be important for preventing brain amyloid neurotoxicity and deposition in Alzheimer's disease. © 2014 Wiley Periodicals, Inc.     

Activin signaling through type IB activin receptor stimulates aromatase activity in the ovarian granulosa cell-like human granulosa (KGN) cells

In addition to a stimulatory effect on FSH production by the pituitary gland, activin is thought to have a paracrine or autocrine role in follicular development in the ovary, where it is produced. Recently, we established a human ovarian granulosa tumor cell line, KGN, which possesses in vivo characteristics of granulosa cells, namely the expression of functional FSH receptors and cytochrome P-450 aromatase. Here, we have demonstrated the activin signaling pathway and its role in KGN cells. A series of transient transfection experiments revealed that activin type IB receptor (ActRIB) is an essential component of the activin signaling pathway in KGN cells. Smad2 was found to act downstream of ActRIB as an intracellular signal transmitter. Smad7, but not Smad6, was an inhibitory Smad in the pathway. Finally, we show that FSH receptor expression and cytochrome P-450 (P-450) aromatase activity was up-regulated by activin stimulation through ActRIB in KGN cells. These results show that we have clarified the signaling mechanisms and the roles of activin in the human granulosa cell line, KGN. Activin signaling mediated by ActRIB-Smad2 system in the ovary may thus be essential for the regulation of follicular differentiation.     

Leukemic relapse in the central nervous system after allogeneic stem cell transplantation with complete remission in the bone marrow and donor-type chimerism: report of two cases

We studied two cases with leukemia that relapsed in the central nervous system (CNS) after allogeneic stem cell transplantation. One patient underwent peripheral blood stem cell transplantation (SCT) from a related, yet haplotype-mismatched, donor for chronic myelomonocytic leukemia. She was kept in complete remission (CR) in the bone marrow (BM) for 7 months, until relapse in the cerebrospinal fluid (CSF) was evident. In the other patient, with acute lymphoblastic leukemia, systemic relapse occurred when he was still on immunosuppression 6 months after SCT from an unrelated donor. After induction chemotherapy following cessation of immunosuppression, the BM examination proved CR. During consolidation chemotherapy, however, he developed leukemic dissemination in the CSF, despite the fact that the BM was in CR. Chimerism status in the BM mononuclear cells and fractionated peripheral blood (PB) cells (granulocytes, T-lymphocytes, and the others) was assessed by short tandem repeat analysis. In both patients, the BM cells and all the fractions of the PB cells proved donor-type chimeras. These results seem to suggest that the graft-versus-leukemia effects might not be as effective in the CNS as in the BM, even when complete T-lymphoid chimerism is achieved.     

The latest wire technique for chronic total occlusion.

If patency is restored after chronic total occlusion (CTO), it can be maintained over the long term by implanting drug-eluting stents. The cause of unsuccessful percutaneous coronary intervention is usually failure of the wire to cross the site of CTO. The objective of this article is to describe the latest wire techniques for CTO. As for wire selection, CTO should generally be treated with hard-tipped spring wires, preferably Conquest Pro series (Asahi Intec). According to the penetrating strategy, the course of a blood vessel with CTO is established preoperatively and the wire is advanced based on the imaging data with minimum rotation (a torque of +/- 90 degrees or less). If the operator encounters divergence between the preoperative CTO image and the actual course of the coronary artery, the parallel wire technique should be used. With this method, a wire which enters the subintimal space is left there, and a second wire is inserted along it to find a new channel. When this technique is successful, the following findings are often noted: 1) the second wire crosses over the first one in the CTO; 2) the second wire shows more acute curve than the first wire; 3) the second wire penetrates the lesion from the outer curvature of the coronary artery and then is advanced along the same curvature of the vessel. Indeed, the second wire should be operated intentionally to achieve these findings so that the probability of success increases and the duration of the procedure is shortened.     

Molecular phylogeny of a newfound hantavirus in the Japanese shrew mole (Urotrichus talpoides)

Recent molecular evidence of genetically distinct hantaviruses in shrews, captured in widely separated geographical regions, corroborates decades-old reports of hantavirus antigens in shrew tissues. Apart from challenging the conventional view that rodents are the principal reservoir hosts, the recently identified soricid-borne hantaviruses raise the possibility that other soricomorphs, notably talpids, similarly harbor hantaviruses. In analyzing RNA extracts from lung tissues of the Japanese shrew mole (Urotrichus talpoides), captured in Japan between February and April 2008, a hantavirus genome, designated Asama virus (ASAV), was detected by RT-PCR. Pairwise alignment and comparison of the S-, M-, and L-segment nucleotide and amino acid sequences indicated that ASAV was genetically more similar to hantaviruses harbored by shrews than by rodents. However, the predicted secondary structure of the ASAV nucleocapsid protein was similar to that of rodent- and shrew-borne hantaviruses, exhibiting the same coiled-coil helix at the amino terminus. Phylogenetic analyses, using the maximum-likelihood method and other algorithms, consistently placed ASAV with recently identified soricine shrew-borne hantaviruses, suggesting a possible host-switching event in the distant past. The discovery of a mole-borne hantavirus enlarges our concepts about the complex evolutionary history of hantaviruses.     

Copper deficiency in the mitochondria of cultured skin fibroblasts from patients with menkes syndrome

Summary The mitochondrial copper concentrations and cytochrome C oxidase activity of the fibroblasts from the patients with Menkes syndrome were investigated. Both the mitochondrial copper concentrations and cytochrome C oxidase activity of fibroblasts from patients with Menkes syndrome were lower than those of the control fibroblasts. These data indicate that the mitochondria of fibroblasts from patients with Menkes syndrome are in a state of copper deficiency. The activity decline of cytochrome C oxidase, a mitochondrial cuproenzyme, seems to be caused by copper deficiency in the mitochondria.