Possible involvement of keratinocyte growth factor and its receptor in enhanced epithelial-cell proliferation and acquired recurrence of middle-ear cholesteatoma

Middle-ear cholesteatoma is characterized by enhanced proliferation of epithelial cells and granular tissue formation. However, the molecular mechanism underlying these pathological changes is largely unknown. Keratinocyte growth factor (KGF) is a mesenchymal cell-derived paracrine growth factor that specifically stimulates epithelial cell proliferation. In the present study, we investigated the possible involvement of KGF and its receptor, KGFR, in the pathogenesis of cholesteatoma using in situ hybridization and immunohistochemistry, respectively. We examined 56 cholesteatoma specimens, and 8 normal skin areas as control. KGF and KGFR expression was examined by immunohistochemistry using rabbit anti-human KGF and anti-human KGFR polyclonal antisera raised in our laboratories against synthetic peptides corresponding to parts of human KGF and KGFR, respectively. KGF protein and mRNA were detected exclusively in stromal fibroblasts and infiltrating T lymphocytes in 80% of cholesteatoma cases, whereas KGFR protein and mRNA were localized in the epithelium in 72% of cases. Assessment of the proliferative activity of cholesteatoma using the labeling index for Ki-67 showed a significantly higher Ki-67 labeling index (66%) in KGF+/KGFR+ cases than other cases. There was a significant correlation between KGF+/KGFR+ expression and recurrence. Our results indicate the possible involvement of both KGF and KGFR in enhanced epithelial cell proliferative activity and recurrence of cholesteatoma.     

Diagnostic Limitations to Accurate Diagnosis of Cholera

The treatment regimen for diarrhea depends greatly on correct diagnosis of its etiology. Recent diarrhea outbreaks in Bangladesh showed Vibrio cholerae to be the predominant cause, although more than 40% of the suspected cases failed to show cholera etiology by conventional culture methods (CMs). In the present study, suspected cholera stools collected from every 50th patient during an acute diarrheal outbreak were analyzed extensively using different microbiological and molecular tools to determine their etiology. Of 135 stools tested, 86 (64%) produced V. cholerae O1 by CMs, while 119 (88%) tested positive for V. cholerae O1 by rapid cholera dipstick (DS) assay; all but three samples positive for V. cholerae O1 by CMs were also positive for V. cholerae O1 by DS assay. Of 49 stools that lacked CM-based cholera etiology despite most being positive for V. cholerae O1 by DS assay, 25 (51%) had coccoid V. cholerae O1 cells as confirmed by direct fluorescent antibody (DFA) assay, 36 (73%) amplified primers for the genes wbe O1 and ctxA by multiplex-PCR (M-PCR), and 31 (63%) showed El Tor-specific lytic phage on plaque assay (PA). Each of these methods allowed the cholera etiology to be confirmed for 97% of the stool samples. The results suggest that suspected cholera stools that fail to show etiology by CMs during acute diarrhea outbreaks may be due to the inactivation of V. cholerae by in vivo vibriolytic action of the phage and/or nonculturability induced as a host response.Cholera is a harsh disease, the fundamental clinical feature of which is severe dehydrating diarrhea that can lead to rapidly progressing dehydration and death. The recent cholera epidemics that occurred in South America (7), Asia (8), and sub-Saharan Africa (18) affected millions of people, with a high mortality rate. The World Health Organization (WHO) annual figures on global cholera incidence (26), which are based on official cases reported by affected countries, are believed to be underestimated due to limitations related to a lack of adequate surveillance systems. In addition, the actual number of cholera cases globally is estimated to be much higher than officially reported (22) because outbreaks are often not reported to avoid the risk of travel and trade embargoes on the affected country.Prompt and accurate diagnosis of Vibrio cholerae is a key step in cholera outbreak surveillance that can greatly influence rapid intervention and prevention to minimize disease spread and mortality. Conventional culture methods (CMs) currently used for diagnosis of V. cholerae remain the gold standard, but this procedure is not precise and requires highly skilled technicians and laboratory infrastructure. In remote settings where cholera is endemic and modern laboratory facilities are often nonexistent, simple dark-field microscopy to detect cells showing characteristic darting motility is used to identify V. cholerae in stool specimens. Diagnostic tests known as cholera dipstick (DS) assays, which involve either cholera toxin (3) or lipopolysaccharide (LPS) antigens (15, 21), have been introduced for rapid bedside detection of V. cholerae in cholera stools. DS tests produce results comparable to those from the CMs, perhaps because both require the physical presence of V. cholerae cells, although only readily culturable cells respond when CMs are used. Fluorescent monoclonal antibody (6) and PCR-based (13) methods have been proposed for detecting the cholera bacterium, including nonculturable cells present in stools.In recent diarrheal outbreaks in Bangladesh, analysis of acute diarrhea cases showed V. cholerae to be the most commonly identified causative agent, even when approximately half of the stool samples from suspected cholera patients did not have any defined etiology (4). In the present study, stool samples collected from suspected cholera patients who had fallen ill during recent seasonal outbreaks in Bangladesh were subjected to extensive analyses by CMs, multiplex PCR (M-PCR), and direct fluorescent antibody (DFA) tests to determine etiology. The stool samples were also analyzed by plaque assay (PA) for the presence of vibriolytic phages in the stools.     

Anisotropic Photomechanical Response of Stretched Blend Film Made of Polycaprolactone‐Polyvinyl Ether with Azobenzene Group as Side Chain

A rapid and reversible anisotropic photomechanical response of polymer film was achieved by using uniaxially stretched blend film composed of polycaprolactone and poly(vinyl ether) with azobenzene moiety (Azo‐PVE) as a side chain. The photomechanical response behavior of the film was studied under a constant tensile stress. The deformation or recovery was rapidly completed within at most 0.1 min of UV + Vis light irradiation being switched on or off, respectively. The anisotropy of deformation was confirmed on a stretched film subjected to light irradiation. The deformation was observed as a contraction in a direction parallel to the stretching direction and as an elongation in the perpendicular direction. The anisotropy of the photoresponse was remarkably observed with increase in the stretching ratio, reflecting the binding between amorphous areas by Azo‐PVE long chains.

     

OspE2 of Shigella sonnei is required for the maintenance of cell architecture of bacterium-infected cells

The OspE2 product of Shigella spp., the expression of which is regulated by the mxiE gene, is secreted through a type III secretion system into host cells. We investigated the function of OspE2 of Shigella sonnei by using cultured epithelial cells. Cells invaded by an ospE2 deletion mutant altered their morphology into the rounding shape, which was not due to cell death, whereas cells invaded by the wild-type strain kept their cell shape intact. The ospE2 mutation did not affect initial cell entry and multiplication in cells, but the mutant formed smaller-than-normal plaques on cell monolayers, indicating a deficiency in cell-to-cell spread by the bacteria. An mxiE deletion mutant also showed changes in cell morphology and deficiency in bacterial spread to adjacent cells. In cells invaded by the ospE2 mutant, disturbance of actin stress fibers was prominent at 3 h after invasion. Analysis of OspE2 localization indicated that the OspE2 protein accumulated on focal contact-like structures in the infected host cells. These results suggest that colocalization of the OspE2 protein in the focal contacts of infected cells may function to maintain an intact cell morphology. The morphological change induced by invasion of the ospE2 mutant may affect secondary bacterial transmission.     

Comprehensive genetic analysis of relevant four genes in 49 patients with Marfan syndrome or Marfan-related phenotypes

In order to evaluate the contribution of FBN1, FBN2, TGFBR1, and TGFBR2 mutations to the Marfan syndrome (MFS) phenotype, the four genes were analyzed by direct sequencing in 49 patients with MFS or suspected MFS as a cohort study. A total of 27 FBN1 mutations (22 novel) in 27 patients (55%, 27/49), 1 novel TGFBR1 mutation in 1 (2%, 1/49), and 2 recurrent TGFBR2 mutations in 2 (4%, 2/49) were identified. No FBN2 mutation was found. Three patients with either TGFBR1 or TGFBR2 abnormality did not fulfill the Ghent criteria, but expressed some overlapping features of MFS and Loeys-Dietz syndrome (LDS). In the remaining 19 patients, either of the genes did not show any abnormalities. This study indicated that FBN1 mutations were predominant in MFS but TGFBRs defects may account for approximately 5-10% of patients with the syndrome.     

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

The sodium-potassium pump (Na⁺,K⁺-ATPase) is responsible for establishing Na⁺ and K⁺ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na⁺,K⁺-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K⁺·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K⁺, in marked contrast to previous models. Due to antagonism between ouabain and K⁺, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.     

Cultivation and preparation of <Emphasis Type="Italic">Lithospermum</Emphasis> roots quantification of total pigment in slender lateral roots

Development of a new preparation method of Lithospermi Radix enabled almost all the slender lateral roots (diameter smaller than 2 mm) to be preserved. The weight ratio of slender lateral roots was about 5.5% in the whole root. The amount of acetylshikonin in the slender lateral roots was two-fold greater than in its tap and thick lateral roots (diameter larger than 2 mm). The acid-insoluble ash content in slender lateral roots was 1.16%.     

Structural determination of a new mutagenic heterocydic amine, 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), present in beef extract

We previously found two new mutagens, compounds I and II, inbacteriological-grade beef extract by monitoring the mutagenicityto a new Salmonella strain, YG1024; compound I was identifiedas 2-amino-4-hydroxymethyl-3,8-dimethyli-midazo[4,5-f]quinoxaline(4-CH₂OH-8-MeIQX) In the present study, we isolated compoundII from the beef extract, which accounted for 2% of the totalmutagenicity of materials adsorbed on blue cotton. Further,we found that a large quantity of compound II was produced byheating a mixture of creatine, threonine and glucose (1:1:0.5)at 200^°C for 5 h, the level being 860-fold of that in thebeef extract. The structure of this compound was determinedto be 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx)by X-ray crystallography. The amount of 7,9-DiMeIgQx in bacteriological-gradebeef extract was estimated to be 53 ng/g. This compound induced13 800 and 670 revertants of S.typhimurium YG1024 and TA98 respectively,per µg in the presence of S9 mix.     

Interaction of Interleukin-1 and Interferon-γ on Fibroblast Growth Factor-induced Angiogenesis

The interaction of interleukin-1 (IL-1) and interferon-γ (IFN-γ) actions on several aspects of angiogenesis in vitro and in vivo was studied. The proliferation and migration of human umbilical vein endothelial cells cultured with basic fibroblast growth factor (bFGF) were synergistically inhibited by cotreatment with IL-1 and IFN-γ. Endothelial cell adhesion to collagen was suppressed by IL-1 and the effect was slightly enhanced by the combination of IL-1 and IFN-γ. Local administration of IL-1 (10,000 U) and IFN-γ (1,000 U) inhibited bFGF-induced angiogenesis in the skin of mice, and synergistic inhibitory activity of the combination was demonstrated. Expression of FGF receptors was strongly downregulated by the combination, whereas expressions of epidermal growth factor (EGF) receptors, integrin β₁ and integrin β₃ were not. EGF partially abrogated the growth-inhibitory effects of IL-1 and IFN-γ. These findings indicate that IL-1 and IFN-γ are each able to act an angiogenesis inhibitor in a situation where FGF plays a major role in angiogenesis, and the activity is synergistically enhanced when they are used in combination.