Indigestible dextrin stimulates glucoamylase production in submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis>

Submerged batch cultures of Aspergillus kawachii grown on indigestible dextrin were investigated for potential improvements in glucoamylase (GA) production. In flask culture, specific GA productivities per dry weight biomass using dextrin and indigestible dextrin were 11.0 and 56.1 mU/mg-DW, respectively. Indigestible dextrin was a poor substrate for enzymatic hydrolysis. Rates of glucose formation from dextrin and indigestible dextrin by enzymatic hydrolysis were 0.477 and 0.100 mg-glucose/ml/h, respectively. For this reason, residual glucose concentrations in batch cultures grown on indigestible dextrin remained below 1.32 mg/ml where glucose-limiting conditions were easily maintained. Batch culture using indigestible dextrin had the same residual glucose profile as dextrin fed-batch culture, and nearly the same GA activity was obtained after 42.5 h of growth. However, between 42.5 and 66 h, the GA production rate of the indigestible dextrin batch culture (11.5 mU/ml/h) was higher than that of the dextrin fed-batch culture (6.5 mU/ml/h). During this period, a high amount of residual maltooligosaccharide was detected in the culture supernatant grown on indigestible dextrin. The high GA productivity observed in the indigestible dextrin batch culture may have resulted from the absence of glucose and the simultaneous presence of maltooligosaccharides throughout growth.     

Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site

Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and β are phosphorylated, site‐specific phosphorylation of topo IIβ is poorly characterized. Using LC‐MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80‐Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H₃PO₄ (?98 Da) in the CID spectra and on differences in 2‐D‐phosphopeptide maps of ³²P‐labeled wild‐type (WT) and S1395A or T1426A/S1545A mutant topo IIβ. However, phosphorylation at tyr656 could not be verified by 2‐D‐phosphopeptide mapping of ³²P‐labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine‐specific antibodies. Since the +80‐Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re‐evaluation of the CID spectra identified +78/+80‐Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIβ activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.     

In vivo tracking of transplanted mononuclear cells using manganese-enhanced magnetic resonance imaging (MEMRI)

Background

Transplantation of mononuclear cells (MNCs) has previously been tested as a method to induce therapeutic angiogenesis to treat limb ischemia in clinical trials. Non-invasive high resolution imaging is required to track the cells and evaluate clinical relevance after cell transplantation. The hypothesis that MRI can provide in vivo detection and long-term observation of MNCs labeled with manganese contrast-agent was investigated in ischemic rat legs.

Methods and Findings

The Mn-labeled MNCs were evaluated using 7-tesla high-field magnetic resonance imaging (MRI). Intramuscular transplanted Mn-labeled MNCs were visualized with MRI for at least 7 and up to 21 days after transplantation in the ischemic leg. The distribution of Mn-labeled MNCs was similar to that of ¹¹¹In-labeled MNCs measured with single-photon emission computed tomography (SPECT) and DiI-dyed MNCs with fluorescence microscopy. In addition, at 1–2 days after transplantation the volume of the site injected with intact Mn-labeled MNCs was significantly larger than that injected with dead MNCs, although the dead Mn-labeled MNCs were also found for approximately 2 weeks in the ischemic legs. The area covered by CD31-positive cells (as a marker of capillary endothelial cells) in the intact Mn-MNCs implanted site at 43 days was significantly larger than that at a site implanted with dead Mn-MNCs.

Conclusions

The present Mn-enhanced MRI method enabled visualization of the transplanted area with a 150–175 µm in-plane spatial resolution and allowed the migration of labeled-MNCs to be observed for long periods in the same subject. After further optimization, MRI-based Mn-enhanced cell-tracking could be a useful technique for evaluation of cell therapy both in research and clinical applications.     

A new method for measuring torsional deformity in scoliosis

Background

This study evaluates the outcome and complications of decompressive cervical Laminectomy and lateral mass screw fixation in 110 cases treated for variable cervical spine pathologies that included; degenerative disease, trauma, neoplasms, metabolic-inflammatory disorders and congenital anomalies.

Methods

A retrospective review of total 785 lateral mass screws were placed in patients ages 16-68 years (40 females and 70 males). All cases were performed with a polyaxial screw-rod construct and screws were placed by using Anderson-Sekhon trajectory. Most patients had 12-14-mm length and 3.5 mm diameter screws placed for subaxial and 28-30 for C1 lateral mass. Screw location was assessed by post operative plain x-ray and computed tomography can (CT), besides that; the facet joint, nerve root foramen and foramen transversarium violation were also appraised.

Results

No patients experienced neural or vascular injury as a result of screw position. Only one patient needed screw repositioning. Six patients experienced superficial wound infection. Fifteen patients had pain around the shoulder of C5 distribution that subsided over the time. No patients developed screw pullouts or symptomatic adjacent segment disease within the period of follow up.

Conclusion

decompressive cervical spine laminectomy and Lateral mass screw stabilization is a technique that can be used for a variety of cervical spine pathologies with safety and efficiency.     

Gamma-ray exposure from neutron-induced radionuclides in soil in Hiroshima and Nagasaki based on DS02 calculations

As a result of joint efforts by Japanese, US and German scientists, the Dosimetry System 2002 (DS02) was developed as a new dosimetry system, to evaluate individual radiation dose to atomic bomb survivors in Hiroshima and Nagasaki. Although the atomic bomb radiation consisted of initial radiation and residual radiation, only initial radiation was reevaluated in DS02 because, for most survivors in the life span study group, the residual dose was negligible compared to the initial dose. It was reported, however, that there were individuals who entered the city at the early stage after the explosion and experienced hemorrhage, diarrhea, etc., which were symptoms of acute radiation syndrome. In this study, external exposure due to radionuclides induced in soil by atomic bomb neutrons was reevaluated based on DS02 calculations, as a function of both the distance from the hypocenters and the elapsed time after the explosions. As a result, exposure rates of 6 and 4 Gy h(-1) were estimated at the hypocenter at 1 min after the explosion in Hiroshima and Nagasaki, respectively. These exposure rates decreased rapidly by a factor of 1,000 1 day later, and by a factor of 1 million 1 week later. Maximum cumulative exposure from the time of explosion was 1.2 and 0.6 Gy at the hypocenters in Hiroshima and Nagasaki, respectively. Induced radiation decreased also with distance from the hypocenters, by a factor of about 10 at 500 m and a factor of three to four hundreds at 1,000 m. Consequently, a significant exposure due to induced radiation is considered feasible to those who entered the area closer to a distance of 1,000 m from the hypocenters, within one week after the bombing.     

Physical association of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) immune receptors in Arabidopsis

Plants possess two distinct types of immune receptor. The first type, pattern recognition receptors (PRRs), recognizes microbe-associated molecular patterns (MAMPs) and initiates pattern-triggered immunity (PTI) on recognition. FLS2 is a PRR, which recognizes a part of bacterial flagellin. The second type, resistance (R) proteins, recognizes pathogen effectors and initiates effector-triggered immunity (ETI) on recognition. RPM1, RPS2 and RPS5 are R proteins. Here, we provide evidence that FLS2 is physically associated with all three R proteins. Our findings suggest that signalling interactions occur between PTI and ETI at very early stages and/or that FLS2 forms a PTI signalling complex, some components of which are guarded by R proteins.     

N-type voltage-dependent Ca channel in non-excitable microglial cells in mice is involved in the pathophysiology of neuropathic pain

Peripheral nerve injury induces neuropathic pain which is characterized by tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca²⁺ channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Ca_v2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the Ca_v2.2 knockout (KO) spinal cord after spinal nerve ligation (SNL)-injury revealed altered expression of genes known to be expressed in microglia, raising an odd idea that N-type VDCC may function in not only excitable (neurons) but also non-excitable (microglia) cells in neuropathic pain state. In the present study, we have tested this idea by using a transgenic mouse line, in which suppression of Ca_v2.2 expression can be achieved specifically in microglia/macrophage by the application of tamoxifen. We found SNL-operated transgenic mice exhibited greatly reduced signs of tactile allodynia, whereas the degree of thermal hyperalgesia was almost the same as that of control. Immunohistochemical analysis of the transgenic lumbar spinal cord revealed reduced accumulation of Iba1-positive cells (microglia/macrophage) around the injured neurons, indicating microglial N-type VDCC is important for accumulation of microglia at the lesion sites. Although the mechanism of its activation is not clear at present, activation of N-type VDCC expressed in non-excitable microglial cells contributes to the pathophysiology of neuropathic pain.     

Casein kinase I δ/ɛ phosphorylates topoisomerase IIα at serine-1106 and modulates DNA cleavage activity

We previously reported that phosphorylation of topoisomerase (topo) IIα at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Iδ and/or CKI, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II–DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II–DNA cleavable complex formation. Since, IC261 specifically targets the Ca²⁺-regulated isozymes, CKIδ and CKI, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIα and α′ did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIδ/ homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIα, was enhanced following expression of human CKI. Down-regulation of CKIδ and CKI also led to reduced formation of etoposide stabilized topo II–DNA cleavable complex. These results provide strong support for an essential role of CKIδ/ in phosphorylating Ser-1106 in human topo IIα and in regulating enzyme function.