Long-term exercise prior to brain ischemia enhances the activities of antioxidant enzymes and leads to a significant reduction in brain damage and neurological deficits in rats subjected to transient middle cerebral artery occlusion. However, it has not been established whether relatively short-term exercise generates similar results following middle cerebral artery occlusion. We aimed to determine whether short-term exercise could reduce oxidative damage and prevent sensori-motor dysfunction. Male Wistar rats were subjected to perform daily exercise on a treadmill for 30 min at a speed of 15 m/min for 3 weeks, followed by a 90-min middle cerebral artery occlusion. Animals were assessed after middle cerebral artery occlusion for neurological deficits and sensori-motor function. Brain tissues were processed to evaluate infarct volume and oxidative damage. Oxidative stress was assessed using immunohistochemistry for 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2'-deoxyguanosine. Antioxidant enzymes were evaluated using immunohistochemistry for thioredoxin and activity assay for superoxide dismutase. Exercise for 3 weeks decreased the severity of paralysis and impairment in forelimb motor coordination. Furthermore, exercise had effect on superoxide dismutase and reduced the infarct volume and the number of cells immunopositive for 4-hydroxy-2-nonenal-modified proteins and 8-hydroxy-2'-deoxyguanosine. Our results suggest that pre-conditioning treadmill exercise for 3 weeks is useful for ameliorating ischemia-induced brain injury.
middle cerebral artery occlusion; pre-conditioning exercise; oxidative stress; sensori-motor function; rat
Sulfur mustard (SM) is a chemical warfare agent that causes extensive skin injury. Previously we reported that SM exposure resulted in suppression of inducible nitric oxide synthase (iNOS) expression to inhibit the healing of scratch wounds in a cultured normal human epidermal keratinocyte (NHEK) model. Based on this finding, the present study was to use adenovirus-mediated gene transfer of iNOS to restore the nitric oxide (NO) supply depleted by exposure to SM and to evaluate the effect of NO on wound healing inhibited by SM in NHEKs. The effect of the iNOS gene transfer on iNOS protein expression and NO generation were monitored by Western blot and flow cytometry, respectively. Wound healing with or without the iNOS gene transfer after SM exposure was assessed by light and confocal microscopy. The iNOS gene transfer via adenovirus resulted in overexpression of the iNOS and an increase in NO production regardless of SM exposure in the NHEK model. The gene transfer was also effective in overcoming the inhibition of wound healing due to SM exposure leading to the promotion of wound closure. The findings in this study suggest that the iNOS gene transfer is a promising therapeutic strategy for SM-induced skin injury.
We report the case of a Japanese family suffering from familial juvenile hyperuricemic nephropathy (FJHN) due to a rare missense mutation of the uromodulin (UMOD) gene. An 18-year-old male presented with gout, hyperuricemia, and stage 3 chronic kidney disease. Mostly, FJHN is caused by a mutation altering the cystine residue of UMOD/Tamm-Horsfall protein. However, in the present case, a T688C mutation was identified in exon 4, resulting in amino acid substitution with arginine replacing tryptophan at position 230 (Trp230Arg). This mutation was also found in his brother and father with the same phenotype, indicating autosomal dominant inheritance. The affected amino acid was conserved in 200 healthy Japanese controls. Therefore, mutation T688C most likely causes rare structural and/or functional abnormalities in UMOD/Tamm-Horsfall protein.
Chronic kidney disease; Familial juvenile hyperuricemic nephropathy; Gene; mutation; Uromodulin
We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.
The child’s brain is more malleable or plastic than that of adults and this accounts for the ability of children to learn new skills quickly or recovery from brain injuries. Several mechanisms contribute to this ability including overproduction and deletion of neurons and synapses, and activity-dependent stabilization of synapses. The molecular mechanisms for activity dependent synaptic plasticity are being discovered and this is leading to a better understanding of the pathogenesis of several disorders including neurofibromatosis, tuberous sclerosis, Fragile X syndrome and Rett syndrome. Many of the same pathways involved in synaptic plasticity, such as glutamate-mediated excitation, can also mediate brain injury when the brain is exposed to stress or energy failure such as hypoxia-ischemia. Recent evidence indicates that cell death pathways activated by injury differ between males and females. This new information about the molecular pathways involved in brain plasticity and injury are leading to insights that will provide better therapies for pediatric neurological disorders.
Plasticity; Injury; Fragile X Syndrome; Rett Syndrome; Hypoxia-Ischemia; NMDA; AMPA; Periventricular Leukomalacia
Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this “first mycotoxin from lower fungi” is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.
Thrombin and angiotensin II (angII) have trophic properties as mediators of vascular remodeling. Focal adhesions and actin cytoskeleton are involved in cell growth, shape, and movement and may be important in vascular remodeling. To characterize mechanisms by which thrombin and angII modulate vessel structure, we studied the effects of these G protein–coupled receptor ligands on focal adhesions in vascular smooth muscle cells (VSMCs). Both thrombin and angII stimulated bundling of actin filaments to form stress fibers, assembly of focal adhesions, and protein tyrosine phosphorylation at focal adhesions, such as p130Cas, paxillin, and tensin. To test whether c-Src plays a critical role in focal adhesion rearrangement, we analyzed cells with altered c-Src activity by retroviral transduction of wild-type (WT) and kinase-inactive (KI) c-Src into rat VSMCs, and by use of VSMCs from WT (src+/+) and Src-deficient (src–/–) mice. Tyrosine phosphorylation of Cas, paxillin, and tensin were markedly decreased in VSMCs expressing KI-Src and in src–/– VSMCs. Expression of KI-Src did not inhibit stress fiber formation by thrombin. Surprisingly, actin bundling was markedly decreased in VSMCs from src–/– mice both basally and after thrombin stimulation, compared with src+/+ mice. We also studied the effect of KI-Src and WT-Src on VSMC spreading. Expression of KI-Src reduced the rate of VSMC spreading on collagen, whereas WT-Src enhanced cell spreading. In conclusion, c-Src plays a critical role in agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in VSMCs. c-Src kinase activity is required for the cytoskeletal turnover that occurs in cell spreading, whereas c-Src appears to regulate actin bundling via a kinase-independent mechanism.
Interactions between biofilm cells of Pseudomonas aeruginosa and levofloxacin were studied. P. aeruginosa incubated for 6 days with Teflon sheets formed a biofilm on its surface. Against the biofilm bacteria, levofloxacin at an MIC determined by the standard method for the strain was highly bactericidal whereas gentamicin, ceftazidime, and ciprofloxacin showed no significant killing activity. Levofloxacin, ciprofloxacin, and gentamicin, but not ceftazidime, exhibited killing activity against nongrowing cells of the strain incubated in phosphate buffer. In addition, levofloxacin, ciprofloxacin, and ceftazidime, but not gentamicin, showed the ability to penetrate an agar containing alginate. These findings may explain the efficacy of levofloxacin and the ineffectiveness of gentamicin and ceftazidime against biofilm bacteria; however, the cause of the ineffectiveness of ciprofloxacin still remains to be determined. In experimental pneumonia in guinea pigs, in which the biofilm mode of growth of the strain was observed in the lung, only levofloxacin exhibited substantial therapeutic efficacy. These findings suggest the significant role of levofloxacin in therapy of biofilm bacterium-associated infectious diseases.
Contemporary hunter–gatherer groups are often thought to serve as models of an ancient lifestyle that was typical of human populations prior to the development of agriculture. Patterns of genetic variation in hunter–gatherer groups such as the !Kung and African Pygmies are consistent with this view, as they exhibit low genetic diversity coupled with high frequencies of divergent mtDNA types not found in surrounding agricultural groups, suggesting long-term isolation and small population sizes. We report here genetic evidence concerning the origins of the Mlabri, an enigmatic hunter–gatherer group from northern Thailand. The Mlabri have no mtDNA diversity, and the genetic diversity at Y-chromosome and autosomal loci are also extraordinarily reduced in the Mlabri. Genetic, linguistic, and cultural data all suggest that the Mlabri were recently founded, 500–800 y ago, from a very small number of individuals. Moreover, the Mlabri appear to have originated from an agricultural group and then adopted a hunting–gathering subsistence mode. This example of cultural reversion from agriculture to a hunting–gathering lifestyle indicates that contemporary hunter–gatherer groups do not necessarily reflect a pre-agricultural lifestyle.
Genes, language and culture reveal that the Mlabri reverted from an agricultural to a hunter-gatherer lifestyle, suggesting that hunter-gatherer groups might not always represent the pre- agricultural lifestyle of humans
Primary colorectal choriocarcinoma is an extremely rare neoplasm and is usually associated with a poor prognosis. Only 13 cases of colorectal choriocarcinoma have previously been reported. There is no standard chemotherapeutic regimen for this tumor type. A 68-year-old man presented with melena and was diagnosed with sigmoid colonic adenocarcinoma with multiple liver metastases. He underwent a laparoscopic sigmoidectomy. Pathology revealed choriocarcinoma with a focal component of moderately differentiated adenocarcinoma of colon origin. Based on the collagen gel droplet-embedded culture drug sensitivity test (CD-DST) results, mFOLFOX6 and bevacizumab were administered, which suppressed aggressive tumor growth for 4 mo. The patient died 9 mo after the initial diagnosis. Our study results suggest that the standard chemotherapy regimen for colorectal cancer might have suppressive effects against primary colorectal choriocarcinoma. Moreover, CD-DST may provide, at least in part, therapeutic insight for the selection of appropriate antitumor agents for such patients.
Colon cancer; Colorectal cancer; Chemotherapy; Culture drug sensitivity test; Choriocarcinoma
Small changes – big effect A new aureothin derivative, aureopyran, that features an unusual pyran backbone was generated by simply altering the enzymatic methylation topology. The α-pyrone ring hampers the correct placement of the polyketide backbone in the multifunctional cytochrome P450 monooxygenase AurH. Instead of a THF ring an oxo intermediate is formed that readily undergoes a rare electrocyclization reaction.
Aureothin; AurH; Combinatorial biosynthesis; Cytochrome P450 monooxygenase; Polyketide
Cancer is a leading cause of death accounting for 15-20% of global mortality. Although advancements in diagnostic and therapeutic technologies have improved cancer survival statistics, 75% of the world population live in underdeveloped regions and have poor access to the advanced medical remedies. Natural therapies hence become an alternative choice of treatment. Ashwagandha, a tropical herb used in Indian Ayurvedic medicine, has a long history of its health promoting and therapeutic effects. In the present study, we have investigated an anticancer activity in the water extract of Ashwagandha leaves (ASH-WEX).
Anticancer activity in the water extract of Ashwagandha leaves (ASH-WEX) was detected by in
vitro and in
vivo assays. Bioactivity-based size fractionation and NMR analysis were performed to identify the active anticancer component(s). Mechanism of anticancer activity in the extract and its purified component was investigated by biochemical assays. We report that the ASH-WEX is cytotoxic to cancer cells selectively, and causes tumor suppression in
vivo. Its active anticancer component was identified as triethylene glycol (TEG). Molecular analysis revealed activation of tumor suppressor proteins p53 and pRB by ASH-WEX and TEG in cancer cells. In contrast to the hypophosphorylation of pRB, decrease in cyclin B1 and increase in cyclin D1 in ASH-WEX and TEG-treated cancer cells (undergoing growth arrest), normal cells showed increase in pRB phosphorylation and cyclin B1, and decrease in cyclin D1 (signifying their cell cycle progression). We also found that the MMP-3 and MMP-9 that regulate metastasis were down regulated in ASH-WEX and TEG-treated cancer cells; normal cells remained unaffected.
We provide the first molecular evidence that the ASH-WEX and TEG have selective cancer cell growth arrest activity and hence may offer natural and economic resources for anticancer medicine.
Multilevel and multimodal interventions have been suggested for suicide prevention. However, few studies have reported the outcomes of such interventions for suicidal behaviours.
We examined the effectiveness of a community-based multimodal intervention for suicide prevention in rural areas with high suicide rates, compared with a parallel prevention-as-usual control group, covering a total of 631,133 persons. The effectiveness was also examined in highly populated areas near metropolitan cities (1,319,972 persons). The intervention started in July 2006, and continued for 3.5 years. The primary outcome was the incidence of composite outcome, consisting of completed suicides and suicide attempts requiring admission to an emergency ward for critical care. We compared the rate ratios (RRs) of the outcomes adjusted by sex, age group, region, period and interaction terms. Analyses were performed on an intention-to-treat basis and stratified by sex and age groups.
In the rural areas, the overall median adherence of the intervention was significantly higher. The RR of the composite outcome in the intervention group decreased 7% compared with that of the control group. Subgroup analyses demonstrated heterogeneous effects among subpopulations: the RR of the composite outcome in the intervention group was significantly lower in males (RR = 0.77, 95% CI 0.59–0.998, p = 0.0485) and the RR of suicide attempts was significantly lower in males (RR = 0.39, 95% CI 0.22–0.68, p = 0.001) and the elderly (RR = 0.35, 95% CI 0.17–0.71, p = 0.004). The intervention had no effect on the RR of the composite outcome in the highly populated areas.
Our findings suggest that this community-based multimodal intervention for suicide prevention could be implemented in rural areas, but not in highly populated areas. The effectiveness of the intervention was shown for males and for the elderly in rural areas.
ClinicalTrials.gov NCT00737165 UMIN Clinical Trials Registry UMIN000000460
Chafuroside B was recently isolated as a new polyphenolic constituent of oolong tea leaves. However, the effects of chafuroside B on skin function have not been examined. In this study, we investigated the protective effects of chafuroside B against UVB-induced DNA damage, apoptosis and generation of photo-immunosuppression related mediators in cultured normal human epidermal keratinocytes (NHEK). Chafuroside B at 1 µM attenuated both UVB-induced apoptosis, evaluated in terms of caspase-3/7 activity, and UVB-induced DNA damage, evaluated in terms of formation of cyclobutane pyrimidine dimers (CPD), in NHEK exposed to UVB (20 mJ/cm2). In addition, chafuroside B at 0.3 or 1 µM suppressed the UVB-induced production of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2), as determined by ELISA, and conversely enhanced IL-12 mRNA expression and production, as measured by RT-PCR and ELISA. Further, chafuroside B at 1 µM also suppressed UVB-induced expression of receptor activator of nuclear factor κB ligand (RANKL) mRNA. These results indicate that chafuroside B promotes repair of UVB-induced DNA damage and ameliorates the generation of IL-10, TNF-α, PGE2, and RANKL, all of which are UVB-induced immunosuppression related mediators. These effects of chafuroside B may be mediated at least in part through induction of IL-12 synthesis in human keratinocytes. Because chafuroside B might have practical value as a photoprotective agent, a further study of the in vivo effects of chafuroside B seems warranted.
ADVANCE® Medial-Pivot (MP) (Wright Medical Technology, Arlington, TN, USA) total knee arthroplasty (TKA) was developed to replicate normal tibiofemoral knee joint kinematics, allowing medial-pivot knee motion. The design concept of the prosthesis is unique; therefore, the influence on the patellofemoral knee joint remains unclear at present. The purpose of this study was to determine the in vivo patellofemoral kinematics with ADVANCE® MP TKA and compare them with the pre-operative conditions.
ADVANCE® MP TKA was performed in ten subjects with osteoarthritis (OA). At before and one month after surgery, lateral radiographs with weight-bearing at maximum extension, 30, 60 and 90° were taken, and patella flexion angle (PF), tibiopatellar angle (TP) and estimated patellofemoral contact point (PC) were evaluated, according to a previously reported method.
In PF and TP, there was no statistically significant change between pre-operative and postoperative values. Pre-operative PC reached its peak at 90°; however, its peak was at 60° at one month after surgery. Postoperative PC at maximum extension was significantly higher compared to before surgery.
The results in this study indicated that ADVANCE® MP TKA changed patellofemoral joint kinematics compared to before surgery. Early postoperative evaluation is the limitation of this study; however, we consider that the results in this study might be one of the keys to resolving the kinematic features of this prosthesis, helping clinicians to comprehend this prosthesis.
The human kinome comprises over 800 individual kinases. These contribute in multiple ways to regulation of cellular metabolism and may have direct and indirect effects on virus replication. Kinases are tempting therapeutic targets for drug development, but achieving sufficient specificity is often a challenge for chemical inhibitors. While using inhibitors to assess whether c-Jun N-terminal (JNK) kinases regulate hepatitis C virus (HCV) replication, we encountered unexpected off-target effects that led us to discover a role for a mitogen-activated protein kinase (MAPK)-related kinase, MAPK interacting serine/threonine kinase 1 (MKNK1), in viral entry. Two JNK inhibitors, AS601245 and SP600125, as well as RNA interference (RNAi)-mediated knockdown of JNK1 and JNK2, enhanced replication of HCV replicon RNAs as well as infectious genome-length RNA transfected into Huh-7 cells. JNK knockdown also enhanced replication following infection with cell-free virus, suggesting that JNK actively restricts HCV replication. Despite this, AS601245 and SP600125 both inhibited viral entry. Screening of a panel of inhibitors targeting kinases that may be modulated by off-target effects of AS601245 and SP600125 led us to identify MKNK1 as a host factor involved in HCV entry. Chemical inhibition or siRNA knockdown of MKNK1 significantly impaired entry of genotype 1a HCV and HCV-pseudotyped lentiviral particles (HCVpp) in Huh-7 cells but had only minimal impact on viral RNA replication or cell proliferation and viability. We propose a model by which MKNK1 acts to facilitate viral entry downstream of the epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), both of which have been implicated in the entry process.
A 46-year-old man underwent living-donor liver transplantation and splenectomy for primary biliary cirrhosis. On postoperative day 22, cloudiness of ascites increased, and triglyceride concentration in ascites was as high as 1046 mg/dL. With a diagnosis of chylous ascites, total parenteral nutrition was started. Nine days after starting total parenteral nutrition, cloudiness of ascites decreased, and triglycerides in ascites decreased to 93 mg/dL. Oral intake was restarted, and the patient was discharged on postoperative day 46. Chylous ascites is a rare complication after living-donor liver transplantation for which total parenteral nutrition may be useful.
Chylous ascites; Living-donor liver transplantation; Primary biliary cirrhosis
We report a case of necrosis of a reconstructed gastric tube in a 77-year-old male patient who had undergone esophagectomy. At the time of admission, the patient had active gastric ulcers, but these were resolved by treatment with a proton pump inhibitor. Subtotal esophagectomy with gastric tube reconstruction was performed. Visually, the reconstructed gastric tube appeared to be well perfused with blood. Using indocyanine green (ICG) fluorescence imaging the gastroepiploic vessels were well enhanced and no enhancement was visable 3 to 4 cm from the tip of the gastric tube. Four days after esophagectomy, gastric tube necrosis was confirmed, necessitating a second operation. The necrosis of the gastric tube matched the area that had been shown to lack blood perfusion by ICG angiography imaging. It seems that ICG angiography is useful for the evaluation of perfusion in a reconstructed gastric tube.
Ischemia; Necrosis; ICG angiography; Esophageal cancer; Gastric tube; Esophagectomy
The crystal structure of human-heart-type fatty-acid-binding protein in complex with anilinonaphthalene-8-sulfonate was solved at 2.15 Å resolution revealing the detailed binding mechanism of the fluorescent probe 1-anilinonaphthalene-8-sulfonate.
Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3–ANS and FABP4–ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.
X-ray structure; FABP3–ANS complex; human-heart fatty-acid-binding protein
The incidence of cutaneous melanoma is increasing worldwide and early diagnosis is essential since the prognosis is poor in advanced stages of disease. Dermoscopy emerged as an additional and important diagnostic procedure for the early diagnosis of cutaneous melanoma.
We report a case of a 52-year-old man, who developed a pigmented lesion in the right pretibial region. Dermoscopy highly suggestive of melanoma. The initial histopathological evaluation suggested a benign lesion. Since dermoscopy was very suspicious, a more extensive histopathological study of the excised skin fragment was performed. This led to a change of diagnosis to a melanoma with partial regression.
The present case shows that occasionally dermoscopy may be more informative for diagnosis of melanoma than an initial histopathology result.
dermoscopy; dermatoscopy; diagnosis; melanoma; nevus
Fluorescence microscopy-based affinity assay could enable highly sensitive and selective detection of airborne asbestos, an inorganic environmental pollutant that can cause mesothelioma and lung cancer. We have selected an Escherichia coli histone-like nucleoid structuring protein, H-NS, as a promising candidate for an amphibole asbestos bioprobe. H-NS has high affinity to amphibole asbestos, but also binds to an increasingly common asbestos substitute, wollastonite. To develop a highly specific Bioprobe for amphibole asbestos, we first identified a specific but low-affinity amosite-binding sequence by slicing H-NS into several fragments. Second, we constructed a streptavidin tetramer complex displaying four amosite-binding fragments, resulting in the 250-fold increase in the probe affinity as compared to the single fragment. The tetramer probe had sufficient affinity and specificity for detecting all the five types of asbestos in the amphibole group, and could be used to distinguish them from wollastonite. In order to clarify the binding mechanism and identify the amino acid residues contributing to the probe’s affinity to amosite fibers, we constructed a number of shorter and substituted peptides. We found that the probable binding mechanism is electrostatic interaction, with positively charged side chains of lysine residues being primarily responsible for the probe’s affinity to asbestos.
To evaluate the postmortem changes of the heart wall on postmortem (PM) computed tomography (CT) in comparison with those on ante mortem CT (AMCT), and in comparison with the pathological findings, obtained in the same patients.
Materials and Methods
We studied 57 consecutive patients who had undergone AMCT, PMCT, and pathological autopsy in our tertiary care hospital between April 2009 and December 2010. PMCT was performed within 20 hours after death, followed by pathological autopsy. The cardiac chambers were measured at five sites on both AMCT and PMCT by two board-certified radiologists who were not provided with clinical information. The differences in heart wall thickness between AMCT with and without contrast medium, between AMCT and PMCT, and between PMCT and pathological anatomy were evaluated statistically. Confounding factors of postmortem change such as gender, presence of arteriosclerosis, the organ related to cause of death, age, and elapsed time since death were examined statistically.
No significant differences were observed on AMCT in comparison of contrasted and non-contrasted images. The heart wall was significantly thicker on PMCT than on AMCT (p < 0.0001) at all five measurement sites. The heart wall was significantly thicker on PMCT than on pathology specimens when measured in accordance with pathological standard mensuration. However, no significant difference was observed between PMCT measurements and those of pathology specimens at any site when the papillary muscles and epicardial fat were included. No significant association was found between postmortem change in heart wall thickness and gender, presence of arteriosclerosis, the organ related to cause of death, age, or elapsed time since death.
This is the first longitudinal study to confirm greater thickness of heart wall on postmortem images compared with ante mortem images, in the same patients. Furthermore, the postmortem changes on CT were supported by the pathological findings.
The crystallization-phase-diagram-guided method is effective for growing large protein crystals for neutron protein crystallography.
Neutron protein crystallography (NPC) is a powerful tool for determining the hydrogen position and water orientation in proteins, but a much larger protein crystal is needed for NPC than for X-ray crystallography, and thus crystal preparation is a bottleneck. To obtain large protein crystals, it is necessary to know the properties of the target protein in the crystallization solution. Here, a crystal preparation method of fungal cellulase PcCel45A is reported, guided by the phase diagram. Nucleation and precipitation conditions were determined by sitting-drop vapor diffusion. Saturation and unsaturation conditions were evaluated by monitoring crystal dissolution, and a crystallization phase diagram was obtained. To obtain a large crystal, crystallization solution was prepared on a sitting bridge (diameter = 5 mm). Initial crystallization conditions were 40 µl of crystallization solution (40 mg ml−1 protein with 30.5% 3-methyl-1,5-pentanediol in 50 mM tris-HCl pH 8.0) with a 1000 µl reservoir (61% 3-methyl-1,5,-pentanediol in 50 mM tris-HCl pH 8.0) at 293 K. After the first crystal appeared, the concentration of precipitant in the reservoir solution was reduced to 60% to prevent formation of further crystals. Finally, we obtained a crystal of 6 mm3 volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction.
cellulase; neutron protein crystallography; crystallization phase diagram
Pandemic (H1N1) 2009 influenza virus spread throughout the world since most people did not have immunity against the virus. In the post pandemic phase when many humans might possess immunity against the pandemic virus, one of the concerns is infection in immunocompromised people. Therefore, we used an immunosuppressed macaque model to examine pathogenicity of the pandemic (H1N1) 2009 virus under an immunocompromised condition. The virus in nasal samples of immunosuppressed macaques infected with the pandemic (H1N1) 2009 virus was detected longer after infection than was the virus in nasal samples of immunocompetent macaques. As expected, not only virus amounts but also virus propagation sites in the immunosuppressed macaques were larger than those in lungs of the immunocompetent macaques when they were infected with the pandemic virus. Immunosuppressed macaques possessed low levels of immune cells producing cytokines and chemokines, but levels of inflammatory cytokines/chemokine interleukin (IL)-6, IL-18, and monocyte chemotactic protein (MCP)-1 in lungs of the immunosuppressed macaques were higher than those in lungs of the immunocompetent macaques, though the differences were not statistically significant. Therefore, under an immunosuppressive condition, the pandemic influenza (H1N1) 2009 virus might cause more severe morbidity with high cytokine/chemokine production by the host innate immune system than that seen in macaques under the immunocompetent condition.