Visualization of the leakage of pancreatic juice using a chymotrypsin-activated fluorescent probe.

BACKGROUND: Pancreatic fistula (PF) remains the most serious complication after digestive surgery. It is difficult to prevent because of the inability to visualize the leakage of pancreatic juice during surgery or to evaluate the protease activity of leaked fluid, which is responsible for PF formation. METHODS: The fluorescence intensities of a chymotrypsin probe (glutaryl-phenylalanine [corrected] hydroxymethyl rhodamine green with added trypsin) in pancreatic juice and in intestinal or abdominal fluids drained after pancreatic resection were evaluated. The chymotrypsin probe was sprayed on to filter papers that had been placed on the resected pancreatic stump in patients undergoing pancreaticoduodenectomy or central pancreatectomy. The ability of this technique to visualize the leakage of pancreatic juice and predict postoperative PF formation was assessed. RESULTS: The fluorescence intensity of the chymotrypsin probe in 76 fluid samples correlated positively with amylase levels (r(s) = 0.678, P < 0.001). The fluorescence patterns of the pancreatic stump were classified grossly into the three types: duct (fluorescence signal visualized only on the stump of the main pancreatic duct, 16 patients), diffuse (ductal stump and surrounding pancreatic parenchyma, 7) and negative (no fluorescence signal, 7). Symptomatic PFs developed in 13 of 23 patients with duct- or diffuse-type fluorescence, but in none of the seven patients with negative-type fluorescence (P = 0.008). CONCLUSION: The chymotrypsin probe enabled determination of the protease activity in drained pancreatic fluid samples and allowed real-time visualization of pancreatic juice leakage during surgery.

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation.

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.

Changing incidence of hepatocellular carcinoma in Japan.

A trend in the incidence of hepatocellular carcinoma (HCC) in Japan was studied from the data of the Osaka Cancer Registry (population, 8,512,351 in 1981) for the period of 1963-1983, the Vital Statistics of Japan, Ministry of Health and Welfare, and the Japan Autopsy Registry which contained 594,132 individually filed cases in the 26-year period from 1958 to 1983. Both cancer registry data and autopsy records showed a more than 2-fold increase in HCC incidence, particularly in the last 10 years or so, among males and a less pronounced increase in females. The same trend was borne out by the cancer registries of Nagasaki City and Miyagi Prefecture and the Vital Statistics. When studied with the autopsy data, it was found that the numbers of autopsies for cirrhosis without HCC and autopsies for HCC (with and without cirrhosis) were about the same in 1958-1961 and that currently (1980-1983) the latter is about 2 times the former. As one of the possible causes of increase in HCC incidence other than prolonged survival of patients with cirrhosis, chronic non-A, non-B hepatitis is discussed.

Interstitial chromosomal deletion within 4q11-q13 in a human hepatoma cell line.

Southern blot analysis revealed the presence of an aberrant albumin gene as well as a normal one in a human hepatoma cell line, HuH-7. A genomic sequence carrying this altered gene was isolated and characterized. This clone contains a 3-kbp 3' segment of the albumin gene linked to a non-albumin sequence at intron 11. The non-albumin sequence is assigned to chromosome 4q12-q13 by in situ hybridization. This indicates an interstitial deletion of a chromosomal segment within 4q11-q13 because the albumin gene is mapped there. The truncated albumin gene is detected in an early passage of HuH-7 cells and has been maintained stably in cell culture.

Novel repetitive sequence families showing size and frequency polymorphism in the genomes of mice.

A middle repetitive sequence, PR1, originally found in mouse rDNA appeared as satellite-like bands when EcoRI and BglII digests of genomic DNA were subjected to Southern blot hybridization using PR1 as probe. The copy number and sizes of PR1-related satellite-like bands, designated as PR1 families, differed remarkably among the subspecies and laboratory strains of mice when the EcoRI digests of genomic DNAs were compared. These bands were not detected in rat and human DNAs. A unit of PR1 sequence was determined by examining cloned EcoRI 3.5 kb (kb, 10(3) bases) fragment and 6.6 kb rDNA by cross-hybridization and sequence analysis: 3.5 kb and 6.6 kb DNAs are composed of homologous PR1 regions and the flanking non-homologous sequences. The results indicate that amplification of different sequences containing PR1 has occurred in different subspecies and strains of mice, and that the segments of satellite-like bands are likely to have been created by recombination of the PR1 sequence with other DNA segments before amplification. The chromosomal distribution of the 3.5 kb PR1 family was studied by back-crossing the female F1 between BALB/c and DDD/1 to male DDD/1. The segregation data strongly suggest that most, if not all, of this family are located on a single chromosome. The stability of these PR1 families in the genomes of cultured cells of a given strain was also examined. An extra band homologous to PR1 appeared in their genomes, but was not detected in other tissues, indicating that some PR1 families may change even during cell propagation.

p53 gene mutations in human skin cancers and precancerous lesions: comparison with immunohistochemical analysis.

Mutations of exons 3 through 9 of the p53 gene in skin lesions were screened in 23 cases of squamous cell carcinoma (SCC), 25 cases of basal cell carcinoma (BCC), two cases of Bowen's disease, 10 cases of solar keratosis, and five cases of keratoacanthoma by polymerase chain reaction--single strand conformation polymorphism analysis. Mutations of the p53 gene were detected in seven of 23 SCCs (30%), three of 25 BCCs (12%), and none in all cases of Bowen's disease, solar keratosis, or keratoacanthoma. Of 23 cases of SCC, mutations were detected in four of 15 SCCs (27%) that originated in the sunlight-exposed skin region, in two of three SCCs (67%) that originated in the scar tissue, and in one of three SCCs (33%) that originated in radiation dermatitis. Mutations of C-->T transition predominated in SCC and BCC that originated in the sunlight-exposed skin region. Mutations of C-->A or CC-->AT observed in tumors that originated in the predisposed conditions, presumably unrelated to UV light, are different from those found in UV light-related SCC or BCC. Twelve cases of SCC were comparatively analyzed with the immunohistochemical staining with anti-p53 antibody. Two of four cases with positive staining had missense mutations, and three of eight cases with negative staining had nonsense mutations. Based on these findings, immunohistochemical results do not necessarily mean the presence or absence of p53 gene mutations in skin tumors, and sequence analysis is essential for determining whether the gene is mutated.

Frequent p53 accumulation in the chronically sun-exposed epidermis and clonal expansion of p53 mutant cells in the epidermis adjacent to basal cell carcinoma.

p53 expression was studied immunohistochemically to identify a precursor lesion of basal cell carcinoma (BCC) in the epidermis adjacent to BCC. With two different anti-p53 antibodies of CM1 and DO7, p53 expression was frequently detected in the epidermis adjacent to BCCs arising on the face and in the normal epidermis with usual sun exposure. In the epidermis adjacent to BCC, stained cells were occasionally clustered in a small area, but no cluster was found in the normal epidermis with usual sun exposure. The expression was less frequent in the normal epidermis with rare sun exposure. Ten cases of normal skin with usual sun exposure, showing CM1 staining in the epidermis, were screened for p53 gene mutations with polymerase chain reaction-single-strand conformation polymorphism analysis using DNAs obtained from the epidermis. No mutation was detected in exons 2 to 10 of the p53 gene in these 10 cases. The epidermis flanking three BCCs that was stained with CM1, on the other hand, carried a missense mutation of C to G transversion at a dipyrimidine site of codon 249. This alteration replaced arginine with threonine. The mutation of codon 249 was not detected in the three BCCs. Our results first suggest that ultraviolet light irradiating the skin in a daily life induces p53 accumulation in the epidermis and secondly that the frequent clonal expansion of p53 mutant cells occurs in the epidermis adjacent to BCCs. This clonal expansion of mutant p53 may provide a molecular basis for high risk of developing subsequent new skin cancers in patients with BCC.

Tandem arrangement of the albumin and alpha-fetoprotein genes in the human genome.

A genomic clone containing human albumin mRNA sequences was isolated from a lambda phage gene library derived from a human hepatoma cell line that produces alpha-fetoprotein (AFP) and albumin. This clone was also found to hybridize with the 5'-flanking region of the human AFP gene. Restriction mapping and nucleotide sequencing revealed that the albumin and AFP genes are present in tandem, in the same transcriptional orientation, with the albumin gene 14.5 kb upstream of the AFP gene. We have also isolated a genomic clone carrying both the albumin and AFP gene sequences from human fibroblasts, which produce neither AFP nor albumin. This DNA showed a restriction map that was indistinguishable from that of the clone obtained from the hepatoma described above, demonstrating that no gross rearrangements of the intergenic DNA sequence are involved in control of expression of the AFP and albumin genes.

Molecular cloning and functional characterization of cDNAs encoding cysteine synthase and serine acetyltransferase that may be responsible for high cellular cysteine content in Allium tuberosum.

The plants belonging to the genus Allium are known to accumulate sulfur-containing secondary compounds that are derived from cysteine. Here, we report on molecular cloning and functional characterization of two cDNAs that encode serine acetyltransferase and cysteine synthase from A. tuberosum (Chinese chive). The cDNA for serine acetyltransferase encodes an open reading frame of 289 amino acids, of which expression could complement the lacking of cysE gene for endogenous serine acetyltransferase in Escherichia coli. The cDNA for cysteine synthase encodes an open reading frame of 325 amino acids, of which expression in the E. coli lacking endogenous cysteine synthase genes could functionally rescue the growth without addition of cysteine. Both deduced proteins seem to be localized in cytosol, judging from their primary structures. Northern blot analysis indicated that both transcripts accumulated in almost equal levels in leaves and root of green and etiolated seedlings of A. tuberosum. The activity of recombinant serine acetyltransferase produced from the cDNA was inhibited by L-cysteine, which is the end-product of the pathway; however, the sensitivity to cysteine (48.7 microM of the concentration for 50% inhibition, IC(50)) was fairly low compared with that of previously reported serine acetyltransferases ( approximately 5 microM IC(50)) from various plants. In A. tuberosum, the cellular content of cysteine was several-fold higher than those in Arabidopsis thaliana and tobacco. This higher concentration of cysteine in A. tuberosum is likely due to the lower sensitivity of feedback inhibition of serine acetyltransferase to cysteine.

Imaging of caspase-3 activation in HeLa cells stimulated with etoposide using a novel fluorescent probe.

Microscopic visualization of intracellular enzyme activity can provide information about the physiological role of the enzyme. Caspases are cysteine proteases that have critical roles in the execution of apoptosis. General fluorometric substrates of caspase-3, such as DEVD-MCA, are unsuitable for imaging because they are excited at short wavelength, so we designed and synthesized novel fluorescent probes that are excited at suitable wavelengths for detecting caspase-3 activity in living cells. Using one of these probes, we succeeded in microscopic visualization of caspase-3-like activity within HeLa cells treated with etoposide. The caspase-3-like activity was increased in the cytosol at first, then expanded to the whole cell.

Development of a time-resolved fluorometric detection system using diffusion-enhanced energy transfer

A novel detection system using both emission energy transfer and time-resolved fluorometry (TRF) was developed, with a europium chelate as the energy donor and a novel fluorophore SNR1, excitable with long-wavelength light corresponding to europium emission, as the energy acceptor. When the donor and acceptor molecules were mixed in solution, energy transfer was observed without direct attachment of the donor and the acceptor, via a diffusion-enhanced energy-transfer mechanism. Thus, the acceptor emission can be detected as a long-lifetime fluorescence in TRF. When the fluorescence properties of the acceptor molecule are changed by interaction with an enzyme or other bioactive molecule, the change can be detected as a long-lived sensitized emission. If we develop or select suitable acceptor molecules, this simple and convenient system should be applicable to a wide variety of bioactive molecules. Since it is based on TRF, it can be used for high-resolution assay.

Vascular smooth muscle cell outgrowth, proliferation, and apoptosis in young and old rats.

We attempted to identify the effects of aging on the response of vascular smooth muscle cells (VSMCs) to injury. Rat aortas were injured by ballooning, and cell outgrowth of the aortic explants, as well as cell proliferation and apoptosis induced by 7-ketocholesterol in the culture system were compared in young (6-7 weeks) and old (40-50 weeks) rats. Explant outgrowth in uninjured rats did not differ between young and old rats. However, 14 days after balloon injury, the number of explants with outgrowth increased by a factor of four (P<0.01) in young rats, while that of the old rats did not change. Cell proliferation in cultured VSMCs also did not differ between young and old uninjured rats, but, in the injured group, proliferation doubled in young rats (P<0.01), but did not change in the older group. The rate of unadhered cells in the presence of 7-ketocholesterol (30 microM) did not differ between young and old uninjured rats. In the injured rats, however, that of young rats was lower (P<0.01) while that of older rats was higher (P<0.01). The extent of DNA fragmentation (%) after the addition of 7-ketocholesterol (30 microM) did not differ between young and old uninjured rats. In the injured groups, DNA fragmentation was lower in the young rats (P<0.01), and higher in the old ones (P<0.01), when compared to their respective controls. These results indicate that the VSMC injury responses of cell outgrowth and proliferation were more pronounced in the young, and that 7-ketocholesterol-induced apoptosis was more extensive in old rats.

Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides.

The aim of this study was to investigate the structure-activity relationship of S-alkyl-L-isothiocitrulline-containing dipeptides towards three partially purified recombinant human nitric oxide synthase (NOS) isozymes, as well as the effects of these compounds on cytokine-induced NO production by human DLD-1 cells. In an in vitro assay, S-methyl-L-isothiocitrulline (L-MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L-amino acid (L-Phe, L-Leu or L-Trp) with L-MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L-MIT-L-Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S-alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS. These results suggest that the human NOS isozymes have different-sized cavities in the binding site near the position to which the C-terminal of L-arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L-MIT-D-Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well-defined direction from the alpha carbon atom. NO production in cytokine-stimulated human DLD-1 cells was measured with a fluorescent indicator, DAF-FM. MILF, L-MIT-L-Trp(-CHO) (MILW) and L-MIT-L-Tyr (MILY) showed more potent activity than L-MIT in this whole-cell assay. Thus, S-alkyl-L-isothiocitrulline-containing dipeptides are selective inhibitors of human iNOS, and work efficiently in cell-based assay.

Lack of somatic mutation in the PTEN gene in squamous cell carcinomas of human skin.

The tumor suppressor gene PTEN is deleted and/or mutated in a variety of tumors and the susceptibility gene for Cowden disease. Loss of heterozygosity of chromosome 10q23, where PTEN resides, in squamous cell carcinomas (SCCs) of human skin and the association of SCC with Cowden disease were reported previously. In the present study, we screened for mutations of PTEN in SCCs by polymerase chain reaction single strand conformation polymorphism analysis to examine whether PTEN is involved in the carcinogenesis of SCC. None of 21 SCCs showed somatic mutations in the coding regions of PTEM. Instead the same allelic variation was detected in two cases without any clinical features of Cowden disease. Our results indicate that inactivation of PTEN does not play an important role in the carcinogenesis of SCC.

The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells.

The effect of cultured normal human dermal papilla cells (DPCs) and conditioned medium prepared with cultured DPCs on chemotactic migration of human hair outer root sheath cells (ORSCs) was examined quantitatively. ORSCs showed significantly increased migration toward both cultured DPCs and the conditioned medium suggesting that DPCs produce and secrete a paracrine factor(s), which attracts hair follicle epithelial cells. Some soluble factors, which are reportedly produced by DPCs, such as insulin-like growth factor-I (IGF-I), hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1), were also examined. ORSCs showed dramatically increased migration toward IGF-I and HGF at concentrations of 1-10 ng/ml. On the other hand, neither VEGF nor TGF-beta1 showed any effect on the chemotaxis of ORSCs. It is interesting that all factors involving mitogenic activity did not always have chemotactic activity for ORSCs. This is the first report to establish that IGF-I and HGF have not only a growth stimulatory but also a chemotactic effect on ORSCs. In addition, the method presented here may help to simplify chemotaxis assays of any type of epithelial keratinocytes with poor mobility.

Culture of human outer root sheath cells from plucked hair follicles in serum-free conditions.

We succeeded in culturing human outer root sheath cells (ORSC) in serum-free conditions without a biological feeder layer. The combination of collagen type IV substrate and modified MCDB 153 medium supplemented with bovine pituitary gland extract (K-GM medium) could support the growth of ORSC. These cells can be serially cultivated for at least 4 passages and stored in liquid nitrogen with good recovery. Thus, a large series of experiments using ORSC may be run simultaneously.

p53 gene mutation analysis in porokeratosis and porokeratosis-associated squamous cell carcinoma.

In this and previous studies, we have shown p53 overexpression immunohistochemically in 14 of 17 porokeratotic specimens obtained from 14 lesions of nine cases, and in all six specimens of squamous cell carcinoma (SCC) arising on porokeratotic lesions of two cases. We screened mutations in exons 5 to 10 of the p53 gene in all these specimens by polymerase chain reaction-single strand conformation polymorphism analysis. Mutations of the p53 gene were detected in two of the six SCCs but not in any of the 17 porokeratotic specimens. These two mutations were C to T transitions at codons 146 and 175 in exon 5, which were a nonsense mutation at a dipyrimidine site and a missense mutation at a CG site, respectively. To our knowledge, neither of these mutations has been identified in skin cancers before. Our observations indicate that mutations of the p53 gene are not the major molecular etiology for porokeratosis, but are related to its skin carcinogenesis, and that p53 overexpression in porokeratosis is not due to p53 gene mutations.

Germline mutations of the PTCH gene in Japanese patients with nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental and skeletal anomalies, palmo-plantar pits, odontogenic keratocysts, ectopic calcification, and occurrence of various types of tumors including basal cell carcinoma. Recent evidence has indicated that the human homologue of a Drosophila segment polarity gene, PTCH, is a NBCCS susceptibility gene. In the study presented here, we detected two novel mutations of the PTCH gene, I805X/2395delC and Y93X/C297A, in two unrelated Japanese patients. Early protection of the skin from the sunlight is important to the prevention of BCC development in NBCCS patients. Genetic analysis of the PTCH gene is essential for the early, definitive diagnosis of NBCCS, especially before the expression of clinical manifestations is complete.

Immunohistological analysis of P53 expression in human skin tumors.

The p53 expression in various skin tumors was immunohistologically evaluated using two mouse monoclonal anti-p53 antibodies, PAb421 and PAb1801. The p53 expression was not detected in the normal epidermal cells. Nuclear staining suggested that the p53 expression was observed in 10 of 26 squamous cell carcinomas (SCCs) from 24 patients, in one undifferentiated carcinoma, one proliferating trichilemmal cyst, one malignant proliferating trichilemmal tumor and in one metastatic carcinoma of breast cancer. None off four cases of Bowen's disease (SCC in situ) showed nuclear staining. In the SCCs, five of 20 primary lesions, three of four recurrent lesions and both of two metastatic lesions had positive nuclei. There was one case of SCC in which a primary lesion was negative but a recurrent lesion was positive. Thus, p53 expression was more frequently observed in SCCs at more clinically advanced stages. This may suggest that p53 has some relevance to progression of SCC. Nuclear staining was not detected in any of the following cases: two cases of seborrheic keratosis, one eccrine poroma, one keratoacanthoma, 11 basal cell epitheliomas, two mammary Paget's disease, three genital Paget's disease, one sebaceous carcinoma, four malignant melanomas, six lymphomas, two leukemia cutis and two angiosarcomas.