Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes

Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials such as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation.     

Isolation and Characterization of Two Types of Xyloglucanases from a Phytopathogenic Fungus,Verticillium dahliae

Xyloglucan is a major hemicellulosic component in plant cell walls. Phytopathogenic fungi secrete cell wall-degrading enzymes on their infection to hosts, while the nature of the cell wall-lytic enzymes of such fungi are yet to be fully understood. Verticillium dahliae is a soil-borne fungus that causes vascular wilt diseases in a variety of commercially important crops worldwide. We purified two types of xyloglucanases, XEG12A and XEG74B, from the culture of naturally isolated Verticillium dahliae strain 2148. XEG12A showed a molecular size of 23 kDa with its maximal activity at pH 7.5. XEG12A specifically hydrolyzed xyloglucan with no activity on other β-glucans. XEG74B had a molecular size of 110 kDa with its optimum pH at 6.0. XEG74B primarily hydrolyzed xyloglucan, with a slight activity on β-1,3-1,4-glucan. Analysis of hydrolytic products of xyloglucanooligasaccharide (XXXGXXXG) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that the both enzymes cleaved β-1,4-glucosidic linkage at the position of unbranched chain, while XEG74B showed a little fluctuation with the cleavage site. Both enzymes did not hydrolyzed xyloglucanoheptasaccharide (XXXG) at all. N-Terminal and internal amino acid sequencing of the enzymes revealed that XEG12A and XEG74B belonged to Glycoside Hydrolase (GH) Families 12 and 74, respectively. Based on these results we concluded that V. dahliae XEG12A and XEG74B were xyloglucan-specific endo-β-1,4-glucanases (EC 3.2.1.151).     

Influence of swelling of noncrystalline regions in silk fibers on modification with methacrylamide

The degrees of swelling of noncrystalline regions of domestic and tussah silk fibers were investigated by measuring the small-angle X-ray scattering intensity of the fibers in wet conditions and analyzing the scattering intensity based on a two-phase model, i.e., crystalline regions and water-swollen noncrystalline regions. The influence of the degree of swelling of noncrystalline regions on the graft treatment of these fibers with methacrylamide was investigated. The changes in the structure caused by the graft treatment were also analyzed using the wide-angle X-ray diffraction measurements. As compared with the tussah silk fibers, the domestic silk fibers showed a larger degree of swelling of the noncrystalline regions, and gained a larger amount of resin by the graft treatment. The crystallites with smaller sizes in the tussah silk fibers were destroyed preferentially by the graft treatment. For the domestic silk fibers, the crystallites were destroyed more seriously and rather homogeneously independent of the crystallite sizes. © 1996 John Wiley & Sons, Inc.     

Preparation of poly(N-isopropylacrylamide)–SiO2 hybrid gels and their thermosensitive properties

The swelling and adsorption behavior of poly(N-isopropylacrylamide)–SiO₂ hybrid gels, derived from copolymerization of N-isopropylacrylamide and 3-methacryloxypropyltrimethoxysilane (MPTMOS) and following hydrolysis and condensation, were investigated. The hybrid gels exhibited temperature dependence of both the swelling ratio and adsorption of solutes, and their temperature dependence of both the swelling ratio and adsorption of solutes, and their temperature dependence was related to the MPTMOS content in the hybrid gels. In addition, the modification of thermosensitive properties of the hybrid gels could be achieved by treatment with other silane compounds. © 1996 John Wiley & Sons, Inc.     

Thermogravimetric behavior of perfluoropolyether

In this article, two kinds of perfluoropolyether (PFPE) with different terminal groups were investigated using thermogravimetric analysis (TGA), infrared (IR) spectroscopy, gel permeation chromatography (GPC), and gas chromatography/gas mass spectrum (GC/MS) analysis. PFPEs with a hydroxyl or carboxylic acid terminal group were more heat stable than was PFPE with carboxylic methyl ester. Perfluoropropylene oxide-type PFPE with a perfluoroethyl terminal group at one end tends to lose weight more rapidly than does copolymer-type PFPE with dihydroxyl or dicarboxyl methyl ester terminal groups at both ends. The residual weight fraction of PFPE with a perfluoroethyl terminal group was dependent on the average molecular weight. The number-average molecular weight of PFPE can be calculated from the peak intensity ratio between the polar group and C F stretching by measuring the IR spectrum of PFPE. The number-average molecular weight of PFPE increased because of the evaporation loss of its low molecular weight fraction and the crosslinking reaction of PFPE with increase in temperature. GC/MS analysis showed that the main product of the pyrolysis of PFPE was hexafluoropylene. We speculated on the PFPE degradation mechanism and the optimum PFPE chemical structure in terms of heat stability. © 1996 John Wiley & Sons, Inc.     

Mutation Analysis of Radioresistant Early-Stage Cervical Cancer

Radiotherapy is a definitive treatment for early-stage cervical cancer; however, a subset of this disease recurs locally, necessitating establishment of predictive biomarkers and treatment strategies. To address this issue, we performed gene panel-based sequencing of 18 stage IB cervical cancers treated with definitive radiotherapy, including two cases of local recurrence, followed by in vitro and in silico analyses. Simultaneous mutations in KRAS and SMAD4 (KRAS^mt/SMAD4^mt) were detected only in a local recurrence case, indicating potential association of this mutation signature with radioresistance. In isogenic cell-based experiments, a combination of activating KRAS mutation and SMAD4 deficiency led to X-ray resistance, whereas either of these factors alone did not. Analysis of genomic data from 55,308 cancers showed a significant trend toward co-occurrence of mutations in KRAS and SMAD4. Gene Set Enrichment Analysis of the Cancer Cell Line Encyclopedia dataset suggested upregulation of the pathways involved in epithelial mesenchymal transition and inflammatory responses in KRAS^mt/SMAD4^mt cancer cells. Notably, irradiation with therapeutic carbon ions led to robust killing of X-ray-resistant KRAS^mt/SMAD4^mt cancer cells. These data indicate that the KRAS^mt/SMAD4^mt signature is a potential predictor of radioresistance, and that carbon ion radiotherapy is a potential option to treat early-stage cervical cancers with the KRAS^mt/SMAD4^mt signature.     

Neuromelanin in Parkinson’s Disease: Tyrosine Hydroxylase and Tyrosinase

Parkinson’s disease (PD) is an aging-related disease and the second most common neurodegenerative disease after Alzheimer’s disease. The main symptoms of PD are movement disorders accompanied with deficiency of neurotransmitter dopamine (DA) in the striatum due to cell death of the nigrostriatal DA neurons. Two main histopathological hallmarks exist in PD: cytosolic inclusion bodies termed Lewy bodies that mainly consist of α-synuclein protein, the oligomers of which produced by misfolding are regarded to be neurotoxic, causing DA cell death; and black pigments termed neuromelanin (NM) that are contained in DA neurons and markedly decrease in PD. The synthesis of human NM is regarded to be similar to that of melanin in melanocytes; melanin synthesis in skin is via DOPAquinone (DQ) by tyrosinase, whereas NM synthesis in DA neurons is via DAquinone (DAQ) by tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC). DA in cytoplasm is highly reactive and is assumed to be oxidized spontaneously or by an unidentified tyrosinase to DAQ and then, synthesized to NM. Intracellular NM accumulation above a specific threshold has been reported to be associated with DA neuron death and PD phenotypes. This review reports recent progress in the biosynthesis and pathophysiology of NM in PD.     

The Complete Loss of p53 Expression Uniquely Predicts Worse Prognosis in Colorectal Cancer

p53 immunohistochemistry is considered an accurate surrogate marker reflecting the underlying TP53 mutation status and has utility in tumor diagnostics. In the present study, 269 primary CRCs were immunohistochemically evaluated for p53 expression to assess its utility in diagnostic pathology and prognostication. p53 expression was wild-type in 59 cases (23%), overexpressed in 143 cases (55%), completely lost in 50 cases (19%), and cytoplasmic in 10 cases (4%). p53 immunoreactivity was associated with tumor size (p = 0.0056), mucus production (p = 0.0015), and mismatch repair (MMR) system status (p < 0.0001). Furthermore, among CRCs with wild-type p53 expression, a significantly higher number of cases had decreased CDX2 than those with p53 overexpression (p = 0.012) or complete p53 loss (p = 0.043). In contrast, among CRCs with p53 overexpression, there were significantly fewer ALCAM-positive cases than p53 wild-type cases (p = 0.0045). However, no significant association was detected between p53 immunoreactivity and the “stem-like” immunophenotype defined by CDX2 downregulation and ALCAM-positivity. Multivariate Cox hazards regression analysis identified tubular-forming histology (hazard ratio [HR] = 0.17, p < 0.0001), younger age (HR = 0.52, p = 0.021), and female sex (HR = 0.55, p = 0.046) as potential favorable factors. The analysis also revealed complete p53 loss (HR = 2.16, p = 0.0087), incomplete resection (HR = 2.65, p = 0.0068), and peritoneal metastasis (HR = 5.32, p < 0.0001) as potential independent risk factors for patients with CRC. The sub-cohort survival analyses classified according to chemotherapy after surgery revealed that CRC patients with wild-type p53 expression tended to have better survival than those with overexpression or complete loss after chemotherapy. Thus, immunohistochemistry for p53 could be used for the prognostication and chemotherapy target selection of patients with CRC.     

High-speed melt spinning of bicomponent fibers: Mechanism of fiber structure development in poly(ethylene terephthalate)/polypropylene system

High-speed bicomponent spinning of poly(ethylene terephthalate)(PET)(core) and poly-propylene (PP) (sheath) was carried out and the structure development in the individual components, PET and PP, was investigated. The orientation and crystallinity development in the PET component was enhanced as compared to that of the single-component spinning while the PP component remained in a low orientation state and had a pseudohexagonal crystal structure even at high take-up speeds. To clarify the mutual interaction between the two components in bicomponent spinning, a semiquantitative numerical simulation was performed. The simulation results obtained using the Newtonian fluid model showed that the solidification stress in the PET component was enhanced while that of the PP component was decreased in comparison with the corresponding single-component spinning. This is due to the difference in the temperature dependence of their elongational viscosity. Simulation with an upper-convected Maxwell model as the constitutive equation suggested that significant stress relaxation of the PP component can occur in the spinline if the PET component solidifies earlier than does PP. Based on the structural characterization results, and the simulation results, it was concluded that the difference in the activation energy of the elongational viscosity and solidification temperature between the two polymers are the main factors influencing the mutual interaction in the bicomponent spinning process. © 1996 John Wiley & Sons, Inc.