Structure and spectroscopy of oxyluciferin, the light emitter of the firefly bioluminescence.

The crystal structures of the pure, unsubstituted firefly emitter oxyluciferin (OxyLH(2)) and its 5-methyl analogue (MOxyLH(2)) were determined for the first time to reveal that both molecules exist as pure trans-enol forms, enol-OxyLH(2) and enol-MOxyLH(2), assembled as head-to-tail hydrogen-bonded dimers. Their steady-state absorption and emission spectra (in solution and in the solid state) and nanosecond time-resolved fluorescence decays (in solution) were recorded and assigned to the six possible trans chemical forms of the emitter and its anions. The spectra of the pure emitter were compared to its bioluminescence and fluorescence spectra when it is complexed with luciferase from the Japanese firefly (Luciola cruciata) and interpreted in terms of the intermolecular interactions based on the structure of the emitter in the luciferase active site. The wavelengths of the emission spectral maxima of the six chemical forms of OxyLH(2) are generally in good agreement with the theoretically predicted energies of the S(0)-S(1) transitions and range from the blue to the red regions, while the respective absorption maxima range from the ultraviolet to the green regions. It was confirmed that both neutral forms, phenol-enol and phenol-keto, are blue emitters, whereas the phenolate-enol form is yellow-green emitter. The phenol-enolate form, which probably only exists as a mixture with other species, and the phenolate-enolate dianion are yellow or orange emitters with close position of their emission bands. The phenolate-keto form always emits in the red region. The concentration ratio of the different chemical species in solutions of OxyLH(2) is determined by several factors which affect the intricate triple chemical equilibrium, most notably the pH, solvent polarity, hydrogen bonding, presence of additional ions, and pi-pi stacking. Due to the stabilization of the enol group of the 4-hydroxythiazole ring by hydrogen bonding to the proximate adenosine monophosphate, which according to the density functional calculations is similar to that due to the dimerization of two enol molecules observed in the crystal, the phenolate ion of the enol tautomer, which is the predominant ground-state species within the narrow pH interval 7.44-8.14 in buffered aqueous solutions, is the most probable emitter of the yellow-green bioluminescence common for most wild-type luciferases. This conclusion is supported by the bioluminescence/fluorescence spectra and the NMR data, as well the crystal structures of OxyLH(2) and MOxyLH(2), where the conjugated acid (phenol) of the emitter exists as pure enol tautomer.

Species identification of animal cells by nested PCR targeted to mitochondrial DNA.

We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.

Chromosomal instability in human mesenchymal stem cells immortalized with human papilloma virus E6, E7, and hTERT genes.

Human mesenchymal stem cells (hMSCs) are expected to be an enormous potential source for future cell therapy, because of their self-renewing divisions and also because of their multiple-lineage differentiation. The finite lifespan of these cells, however, is a hurdle for clinical application. Recently, several hMSC lines have been established by immortalized human telomerase reverse transcriptase gene (hTERT) alone or with hTERT in combination with human papillomavirus type 16 E6/E7 genes (E6/E7) and human proto-oncogene, Bmi-1, but have not so much been characterized their karyotypic stability in detail during extended lifespan under in vitro conditions. In this report, the cells immortalized with the hTERT gene alone exhibited little change in karyotype, whereas the cells immortalized with E6/E7 plus hTERT genes or Bmi-1, E6 plus hTERT genes were unstable regarding chromosome numbers, which altered markedly during prolonged culture. Interestingly, one unique chromosomal alteration was the preferential loss of chromosome 13 in three cell lines, observed by fluorescence in situ hybridization (FISH) and comparative-genomic hybridization (CGH) analysis. The four cell lines all maintained the ability to differentiate into both osteogenic and adipogenic lineages, and two cell lines underwent neuroblastic differentiation. Thus, our results were able to provide a step forward toward fulfilling the need for a sufficient number of cells for new therapeutic applications, and substantiate that these cell lines are a useful model for understanding the mechanisms of chromosomal instability and differentiation of hMSCs.

Contrast-enhanced sonography of pancreatic carcinoma: correlations with pathological findings.

BACKGROUND: We examined contrast-enhanced harmonic gray-scale sonographic findings of pancreatic carcinoma in relation to the pathological findings in resected specimens to evaluate correlations between observations made by this modality and the pathological findings. METHODS: The pathological findings of surgical specimens obtained from 16 patients were examined in relation to the contrast-enhanced harmonic gray-scale sonography findings. Lesion vascularity was examined by contrast-enhanced harmonic gray-scale sonography from 20 to 50 s after the injection of Levovist (Schering, Berlin, Germany) (early phase), and lesion enhancement was also monitored at approximately 90 s after injection (delayed phase). RESULTS: Contrast-enhanced harmonic gray-scale sonography showed positive enhancement in 12 of the 16 lesions (peripheral tumor region alone, n = 9; entire tumor, n = 3), while the other 4 lesions showed no contrast enhancement in any region. Twelve enhanced regions (9 peripheral tumor region and 3 entire tumor regions) detected by contrast-enhanced harmonic gray-scale sonography showed: (1) mild fibrosis with inflammation, in 10 regions (83%); (2) the presence of both carcinoma cells and residual acinar cells in 8 (67%); and (3) presence of relatively large arteries in 2 (17%). In contrast, 13 non-enhanced regions (4 entire tumor regions and 9 central regions) showed: (1) severe fibrosis in 10 regions (77%); (2) necrosis in 7 (54%); and (3) mucin in 4 (31%). CONCLUSIONS: Contrast-enhanced harmonic gray-scale sonographic findings of pancreatic carcinoma are influenced by interstitial histological features associated with tumor growth.

Early effects of lafutidine or rabeprazole on intragastric acidity: which drug is more suitable for on-demand use?

BACKGROUND: Medication for the relief of heartburn should have the rapid onset of action required for on-demand use. We studied the inhibition of gastric acid secretion by lafutidine and rabeprazole, given in single doses to fasting and postprandial subjects. METHODS: A total of 22 healthy male, Helicobacter pylori-negative volunteers participated in this randomized, two-way crossover study. They were randomly assigned to receive a single oral dose of 10 mg lafutidine or 20 mg rabeprazole after fasting overnight (12 subjects, fasting study) or after eating a test meal (noodles, 364 kcal; protein, 10.1 g; fat, 16 g; carbohydrates, 44.9 g; NaCl, 1.1 g; 10 subjects, postprandial study). Intragastric pH was monitored continuously for 6 h after treatment. The other drug was given after a washout period of at least 7 days, and intragastric pH was similarly monitored. RESULTS: In the fasting study, lafutidine sustained pH at >3 and >4 during the second, third, fourth, fifth, and sixth hours of the study for significantly longer than rabeprazole. During the first 6 h after treatment, lafutidine sustained pH at more than 2, 3, 3.5, 4, 5, 6, and 7 longer than rabeprazole. In the postprandial study, lafutidine sustained pH >3 and >4 for longer periods than rabeprazole during the third, fourth, fifth, and sixth hours of the study. During the first 6 h after treatment, lafutidine sustained pH at more than 2, 3, 3.5, 4, 5, 6, and 7 longer than rabeprazole. CONCLUSIONS: Lafutidine 10 mg produces a prompter rise in intragastric pH than rabeprazole 20 mg in fasting and postprandial Helicobacter pylori-negative male subjects.

Aneuploidy in immortalized human mesenchymal stem cells with non-random loss of chromosome 13 in culture.

Aneuploidy (an abnormal number of chromosomes) is commonly observed in most human cancer cells, highlighting the need to examine chromosomal instability in tumorigenesis. Previously, the immortalized human mesenchymal stem cell line UE6E7T-3 was shown to undergo a preferential loss of one copy of chromosome 13 after prolonged culture. Here, the loss of chromosome 13 was found to be caused by chromosome missegregation during mitosis, which involved unequal segregation, exclusion of the misaligned chromosome 13 on the metaphase plate, and trapping of chromosome 13 in the midbody region, as observed by fluorescence in situ hybridization. Near-diploid aneuploidy, not tetraploidy, was the direct result. The loss of chromosome 13 was non-random, and was detected by analysis of microsatellites and single nucleotide polymorphism-based loss of heterozygosity (LOH). Of the five microsatellite loci on chromosome 13, four loci showed microsatellite instability at an early stage in culture, and LOH was apparent at a late stage in culture. These results suggest that the microsatellite mutations cause changes in centromere integrity provoking loss of this chromosome in the UE6E7T-3 cell line. Thus, these results support the use of this cell line as a useful model for understanding the mechanism of aneuploid formation in cell cultures.

Structural analysis of a p44/msp2 expression site of Anaplasma phagocytophilum in naturally infected ticks in Japan.

Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, infects neutrophils and causes an emerging tickborne febrile disease. The genome of this bacterium contains a large number of p44/msp2-related genes encoding 44 kDa major outer-membrane proteins, and it is known that a specific p44/msp2 gene is predominantly transcribed from a single expression locus. This study successfully characterized the genomic expression site for p44/msp2 (3.8 kb) in uncultured A. phagocytophilum from Ixodes persulcatus ticks inhabiting a northern part of Japan. Comparative analysis of the sequences revealed that the structures of the expression sites in Japanese A. phagocytophilum were similar to those of US strains from human patients and European strains from a dog and sheep, but omp-1N (upstream from p44/msp2) and a truncated recA (downstream from p44/msp2) in the p44/msp2 expression site seemed to share similarities with those of US and European strains. The central hypervariable region sequences of Japanese p44/msp2 were found to be quite diverse (24.4-100 % amino acid similarities) and distinct from their closest relatives from US human patients or animal host origins (56.3-97.6 % amino acid similarities) with some exceptions. Thus, this study provides significant information about the molecular characteristics of A. phagocytophilum in East Asia, as well as the global diversity of p44/msp2.

Contrast-enhanced sonography of autoimmune pancreatitis: comparison with pathologic findings.

OBJECTIVE: We evaluated the vascularity of autoimmune pancreatitis lesions on contrast-enhanced harmonic gray scale sonographic images in comparison with the pathologic findings. METHODS: Six patients with autoimmune pancreatitis were examined. All patients held their breath from 20 to 50 seconds after the injection of a contrast agent while the vascularity of the lesion was examined by contrast-enhanced harmonic gray scale sonography (early phase), and lesion enhancement was monitored at about 90 seconds after the injection while the patients held their breath for a few seconds (delayed phase). We then compared the vascularity on the contrast-enhanced harmonic gray scale sonographic images with the pathologic findings (fibrosis and inflammation) in all lesions. The vascularity of 3 of the 6 lesions was also evaluated by contrast-enhanced harmonic gray scale sonography before and after treatment with corticosteroids. RESULTS: The autoimmune pancreatitis lesions exhibited mild (n = 1), moderate (n = 3), or marked (n = 2) enhancement throughout almost the entire lesions in both the early and delayed phases. The grade of lesion vascularity on the contrast-enhanced harmonic gray scale sonographic images correlated with the pathologic grade of inflammation and inversely correlated with the grade of fibrosis associated with autoimmune pancreatitis. The vascularity of all 3 lesions had decreased on the contrast-enhanced harmonic gray scale sonographic images after steroid therapy. CONCLUSIONS: Contrast-enhanced harmonic gray scale sonography may be useful for evaluating the vascularity of autoimmune pancreatitis lesions and the therapeutic efficacy of steroid therapy.

Percutaneous ablation therapy guided by contrast-enhanced sonography for patients with hepatocellular carcinoma.

OBJECTIVE: The newly developed contrast-enhanced harmonic gray-scale sonography technique enables us to improve the real-time detectability of viable tumor tissue in hepatocellular carcinoma lesions. We evaluated the usefulness of real-time percutaneous ablation therapy under guidance with this method for patients with hepatocellular carcinoma that is not depicted on conventional sonography. SUBJECTS AND METHODS: We examined 30 patients with 56 hepatocellular carcinomas using real-time contrast-enhanced harmonic gray-scale sonography after injection of a galactose-palmitic acid contrast agent and compared the results with the findings of contrast-enhanced helical CT. We performed percutaneous ablation therapy guided by this modality for treatment of viable hepatocellular carcinoma lesions that could not be detected using conventional sonography. RESULTS: High detection rates of viable hepatocellular carcinoma lesions were obtained using real-time contrast-enhanced harmonic gray-scale sonography (52/56 lesions, 93%); these rates were comparable to those of helical CT (54/56 lesions, 96%). Nine (90%) of the 10 lesions that were not detected on conventional sonography but were depicted on real-time contrast-enhanced harmonic gray-scale sonography (incomplete local treatment, n = 4; small new lesion, n = 6) were successfully treated with percutaneous ablation therapy guided by this method. CONCLUSION: Real-time contrast-enhanced harmonic gray-scale sonography improved the sensitivity for the detection of viable hepatocellular carcinoma lesions. Percutaneous ablation therapy guided by this modality may be useful in patients with hypervascular hepatocellular carcinoma lesions that cannot be detected using conventional sonography.

Contrast-enhanced sonography of small pancreatic mass lesions.

OBJECTIVE: To evaluate the usefulness of contrast-enhanced wideband harmonic gray scale sonography in assessing the vascularity of small pancreatic mass lesions. METHODS: Twenty-five patients with 25 pancreatic mass lesions (20 pancreatic carcinomas, 1 islet cell tumor, 1 malignant lymphoma, and 3 focal inflammatory pancreatic masses due to chronic pancreatitis) were examined. All patients held their breath for 20 to 50 seconds after injection of a contrast agent while the vascularity of the tumor was observed on contrast-enhanced wideband harmonic gray scale sonography (early phase). We then monitored the tumor enhancement 60 to 120 seconds after the injection while the patients held their breath for a few seconds (delayed phase). RESULTS: All 20 (100%) of the pancreatic carcinomas showed no contrast enhancement in the early phase. Fifteen (75%) of the 20 pancreatic carcinomas also showed no contrast enhancement in the delayed phase. The remaining 5 (25%) pancreatic carcinomas showed mild enhancement in the peripheral regions of the tumor in the delayed phase. The other pancreatic masses showed mild or pronounced enhancement throughout the entire lesions in both the early and delayed phases. CONCLUSIONS: Contrast-enhanced wideband harmonic gray scale sonography is a useful tool for differentiating pancreatic carcinomas from focal inflammatory pancreatic masses or hypervascular pancreatic tumors.