Mutations of the transforming growth factor-beta type II receptor gene are strongly related to sporadic proximal colon carcinomas with microsatellite instability

BACKGROUND: Mutations of the transforming growth factor-beta type II receptor gene (TGF-beta RII) have been found in several replication error-positive sporadic colorectal carcinomas and hereditary nonpolyposis colorectal carcinoma cell lines. The aim of this study was to clarify the role of TGF-beta RII in sporadic colorectal carcinogenesis. METHODS: The authors screened for mutations at simple repeated sequences in the TGF-beta RII gene by polymerase chain reaction-single strand conformation polymorphism. They also examined genomic instability, using five microsatellite DNA markers in 69 sporadic colorectal carcinomas. When the carcinomas exhibited the TGF-beta RII mutations, the authors screened further for mutations in two DNA mismatch repair genes, hMSH2 and hMLH1. RESULTS: Seven of the 69 cancers (10%) showed one or two A deletions in TGF-beta RII and resultant frameshift mutations in nucleotide positions 709-718 containing a (A) 10 repeated sequence; but none of these appeared in the corresponding normal DNA, indicating a somatic mutation. All of the seven cancers were located in the proximal colon; there were none in the distal colon (P < 0.01). On the other hand, 22 of the 69 carcinomas (32%) showed the replication error-positive phenotype. The frequency of replication errors in proximal colon carcinomas was higher than that in distal colon carcinomas (P < 0.05). All 7 cancers with TGF-beta RII mutations showed replication errors. One of them revealed a nonsense mutation at codon 413, and 1 revealed a loss of heterozygosity in hMSH2. CONCLUSIONS: These data indicate that mutations of TGF-beta RII are strongly related to proximal colon carcinomas with microsatellite instability and that the mechanism of carcinogenesis in some proximal colon carcinomas is similar to that in hereditary nonpolyposis colorectal carcinoma.     

A peptidyl-prolyl cis/trans-isomerase (cyclophilin G) in regulated secretory granules

A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a beta-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned. The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolyl cis/trans-isomerase. p27 exhibits isomerase activity with a kcat/Km of 0.18 microM-1 s-1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506. Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion. Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation. These results indicate that p27 is a new peptidyl-prolyl cis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules.     

cDNA cloning, tissue distribution, and subcellular localization of horseshoe crab big defensin

A full-length cDNA for horseshoe crab big defensin with a strong antimicrobial activity was obtained from a hemocyte cDNA library. The open reading frame of the cDNA coded for an NH2-terminal signal sequence followed by a propeptide and the mature big defensin. The propeptide is linked to the mature protein through an -Arg-X-Lys/Arg-Arg- motif, the processing site for Kex2-like proteases. Northern blot analysis revealed that big defensin is expressed in all the tissues tested, suggesting that big defensin plays an important role not only in hemocytes but also in other tissues for host defense. The subcellular localization, determined by immunocytochemistry at ultrastructural level, confirmed the previous findings obtained by biochemical analysis that big defensin locates in both small and large granules in hemocytes. Big defensin is the first example to demonstrate the existence of broad tissue distribution in horseshoe crab.     

Fabrication of three‐dimensionally ordered microporous membrane by wet phase separation

We report a novel porous fluorinated polyimide membrane with a cylinder structure fabricated by a wet phase inversion process, which is formed by a ternary system, polyimide/solvent/water. The porous polyimide membranes consisted of a thin top porous layer and three‐dimensionally ordered cylinder micropores. The porous membrane‐forming solvents were N‐methylpyrrolidone containing nonsolvent additives such as alcohol, and the height and width of the cylinder structure were controlled by the solvents. Water fluxes through the porous polyimide membranes were measured using a stirred dead‐end filtration cell, and the fluxes of the porous membrane with the cylinder‐type structure were approximately three times greater than those of the membrane with the finger‐type structure. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3016–3021, 2004     

Effects of sample orientation on nonpiloted ignition of thin poly(methyl methacrylate) sheets by a laser: 2. Experimental results

The effect of the sample orientation angle on frontside (irradiated surface) ignition and subsequent backside (nonirradiated surface) flame appearance over thin poly(methyl methacrylate) (PMMA) sheets having thicknesses of 0.2 and 0.5 mm has been experimentally investigated, using a CO₂ laser as an external radiant source in quiescent normal gravity. The sample orientation angle was varied from θ=−90° (ceiling configuration) to +90° (floor configuration) at intervals of 15° under three different laser powers of 16.0, 17.3, and 26.1 W. The shortest frontside ignition delay time was observed for the ceiling configuration (θ=−90°) and frontside ignition delay time significantly varied with increase in sample orientation angle at a laser power of 16.0 W. As the laser power was increased, frontside ignition was observed at all angles and its delay time became less dependent on the sample orientation angle. The appearance of a backside flame was achieved after the formation of an open hole (due to local consumption of the sample) by two different processes: the onset of laser induced ignition over the backside sample (backside ignition) and a flame traveling from the frontside through an open hole to the backside (backside flame). The former process was observed for a limited number of cases only around the vertical configurations (−30°?θ?30°). The delay time for the appearance of backside flame tended to be longer for sample surfaces facing downward (θ°<0) than for the sample surface facing upward (θ?0°) regardless of the laser power. When the duration of laser irradiation was shortened from 10 to 4 s, as soon as the laser was shut off, the flame on the frontside immediately shrank, moved close to the sample surface, and then traveled rapidly to the backside. Therefore, the delay time of backside flame appearance (about 6 s) became longer with longer duration of laser irradiation after the onset of a frontside flame. The size of the hole (about 4 mm diameter) was large enough for the flame to travel through it, even after 4 s of laser irradiation to sample. These results indicate that the size of the hole appears to be not a critical parameter for the appearance of the backside flame.     

Stabilizing the optimal carrier concentration for high thermoelectric efficiency

The band structure of PbTe can be manipulated by alloying with MgTe to control the band degeneracy. This is used to stabilize the optimal carrier concentration, making it less temperature dependent, demonstrating a new strategy to improve overall thermoelectric efficiency over a broad temperature range.     

Nanotrap and mass analysis of aromatic molecules by phenyl group-modified nanoparticle

To functionalize the surface of nanoparticles with phenyl groups for subsequent cross-linking with aromatic molecules by mutual interactions, we prepared functional nanoparticles (d = 3 nm) by silanization with phenyl-triethoxysilane. The nanoparticles had Fe(2)O(3) cores conjugated to phenyl groups; this was confirmed by Fourier transform infrared (FT-IR) spectroscopy and absorption spectrophotometry. The typical C-H and C-C peaks and the absorption at 240 nm, which corresponds to aromatic rings, were detected in the spectroscopic results for the phenyl group-modified nanoparticles. The nanoparticles could ionize aromatic (colchicine, reserpine, and bradykinin peptide) and nonaromatic (L-α-phosphatidylethanolamine,dioleoyl, and polyethylene glycol) molecules by nanoparticle-assisted laser desorption/ionization mass spectrometry. The nanoparticles worked as a selective trap and an ionization-assisting reagent in mass spectrometry for the aromatic molecular targets.     

Noninvasive subsurface analysis using multiple miniaturized Raman probes, part I: basic study of thin-layered transparent models of biomedical tissues

This study describes a basic theory for reconstructing pure Raman signals of materials composing a multilayer sample from Raman spectra obtained using two types of miniaturized Raman probes. An illustrative example is demonstrated using a multilayer system of samples composed of the transparent plastics polymethylmethacrylate (PMMA) and polyethylene (PE) as a model of thin-layered biomedical tissues. When the same region of an object is measured using Raman probes with different focal properties, the Raman spectra provide different depth profile information depending on the level of light penetration. Thus, a detailed comparison of the spectra can provide an interesting opportunity to probe the differences between the layers. A simple analytic form is presented for reconstructing the pure Raman spectra of the embedded layer. The method applies an understanding of the Raman sampling volume in layered transparent materials to the interpretation of Raman spectra experimentally measured by multiple probes. The basic theory described here is necessary for the expansion of the technique to turbid media, such as biological samples, where light-scattering effects must be considered. The potential applications of the proposed method include material and catalyst subsurface probing through different embedded materials, such as assessment of silicon wafers, effective noninvasive screening for catalyst synthesis, and biomedical tissue research.     

Significance of the detection of staphylococcal enterotoxin A gene in low fat milk which caused a serious outbreak of food poisoning

In June 2000, there was a large-scale outbreak of food poisoning after consumption of Snow Brand low fat milk. In the evening of a day the incident made public, some cartons of low fat milk were brought to our laboratory for examination. Next day, we detected only staphylococcal enterotoxin (SE) A gene among SE (A-E) genes by PCR in left-over milk samples or samples from the same lots that patients had consumed. We presumed that the outbreak was caused by the intake of SEA. We subsequently confirmed the presence of SEA in these samples. To investigate the existence of SE (A-E) genes in milk, we examined 100 samples of commercial low fat milk and milk by PCR, but none of the genes was detected. We estimated the detection limit of SEA gene in low fat milk by PCR. Four strains of SEA-producing Staphylococcus aureus cultures were serially diluted in low fat milk. The SEA gene was detected at levels of 5.5 x 10(2) to 1.6 x 10(4) cfu/mL of S. aureus. These amounts of S. aureus are higher than the values in raw milk reported previously. Therefore we consider that SE genes in low fat milk should usually be undetectable by our PCR. This study shows that quick detection of SE genes by PCR is very helpful to analyze outbreaks, especially if no significant bacterium can be cultured.