Two cases of single-stage lip and nostril reconstruction in holoprosencephaly.

Holoprosencephaly is a rare disorder of embryological development that presents with midline clefting of the lip. The incidence has been estimated at approximately 1 in 15,000. Previously, infants with severe holoprosencephaly were thought to die within 1-2 years of birth and seldom to benefit from surgery. Survival has increased with improved perinatal care and support services. Parents often request complete cheiloplasty, because the presence of a columella greatly influences aesthetic outcome. The authors report two cases of simultaneous columella reconstruction with cheiloplasty.

Robust NADH-regenerator: improved alpha-haloketone-resistant formate dehydrogenase.

Formate dehydrogenases (FDH) are useful for the regeneration of NADH, which is required for asymmetric reduction by several dehydrogenases and reductases. FDHs have relatively low activity and are labile, especially to alpha-haloketones, thus FDH cannot be applied to the industrial manufacture of optically active alpha-haloalcohols. To stabilize a FDH from Mycobacterium vaccae (McFDH) against the alpha-haloketone ethyl 4-chloroacetoacetate (ECAA), a set of cysteine-mutant enzymes was constructed. Sensitivity to ECAA of mutant C6S was similar to that of the wild-type enzyme, and mutants C249S and C355S showed little activity. In contrast, mutant C256S exhibited remarkable tolerance to ECAA. Surprisingly, mutant C146S was activated by several organic compounds such as ethyl acetate. An optimized mutant, C6A/C146S/C256V (McFDH-26), was obtained by combining several effective mutations. Ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-ECHB] was synthesized from ECAA to 49.9 g/l with an optical purity of more than 99% e.e. using recombinant Escherichia coli cells coexpressing McFDH-26 and a carbonyl reductase (KaCR1) from Kluyveromyces aestuarii.

Site-dependent modulating effects of conjugated fatty acids from safflower oil in a rat two-stage carcinogenesis model in female Sprague-Dawley rats.

Modifying effects of dietary administration of conjugated fatty acids from safflower oil (CFA-S), rich in conjugated linoleic acid, on major organs were examined in the post-initiation stage of a two-stage carcinogenesis model in female rats. Groups of 21 or 22 F344 female rats were treated sequentially with 2,2'-dihydroxy-di-n-propylnitosamine (intragastrically, i.g.), 7,12-dimethylbenz[a]anthracene (i.g.), 1,2-dimethylhydrazine (subcutaneously) and N-butyl-N-(4-hydroxybutyl)nitrosamine (in drinking water) during the first 3 weeks for initiation, and then administered diet containing 1 or 0.1% CFA-S for 33 weeks. Further groups of animals were treated with carcinogens or 1% CFA-S alone, or maintained as non-treated controls. All surviving animals were killed at week 36, and major organs were examined histopathologically for development of pre-neoplastic and neoplastic lesions. The 1 and 0.1% CFA-S treatment significantly decreased the incidence and multiplicity of mammary carcinomas, though a clear dose response was not observed. In the urinary bladder, the incidence of papillary or nodular hyperplasia but not tumors was significantly increased in the 1% CFA-S-treated group. The results indicate that low dose CFA-S may find application as a potent chemopreventor of mammary carcinogenesis.

Enhancing effects of 2-amino-4,5-diphenylthiazole-induced polycystic kidneys on renal carcinogenesis in rats treated with dimethylnitrosamine.

The effects of the polycystic kidney environment on dimethylnitrosamine (DMN)-induced renal carcinogenesis were investigated in rats. In Experiment 1, male Wistar rats were given 25 or 10 ppm DMN in their drinking water and simultaneously administered 1% 2-amino-4,5-diphenylthiazole (DPT) in the diet for 30 weeks. DPT-induced polycystic kidney was associated with a significant increase in the number of renal cell tumors and incidence of mesenchymal tumors in the 25 ppm DMN + DPT group and the incidence of atypical tubules in the 10 ppm DMN + DPT group. PCNA labeling indices of cystic renal tubules in DPT-treated rats were significantly higher than for corresponding noncystic tubules. In Experiment 2, PCNA indices of renal tubules in 10 ppm + DPT rats and immunohistochemically CYP2E1-positive renal tubules in DPT-treated rats were demonstrated to be significantly increased on day 14. CYP2E1 mRNA expression in the kidneys of DPT-treated rats showed a fivefold increase over constitutive levels. The results thus indicate that DPT induction of polycystic kidneys enhances DMN-induced renal carcinogenesis in rats, with DPT-induced elevated cell proliferation and CYP2E1 expression in renal tubules as possible underlying mechanisms.

Effects of arctiin on PhIP-induced mammary, colon and pancreatic carcinogenesis in female Sprague-Dawley rats and MeIQx-induced hepatocarcinogenesis in male F344 rats.

Chemopreventive effects of arctiin, a lignan isolated from Arctium lappa (burdock) seeds, on the initiation or post initiation period of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced mammary carcinogenesis in female rats and on 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-associated hepatocarcinogenesis in male rats were examined. In experiment 1, female Sprague-Dawley (SD) rats were given intragastric doses of 100 mg/kg body wt of PhIP once a week for 8 weeks as initiation. Groups of 20 rats each were treated with 0.2 or 0.02% arctiin during or after PhIP initiation. Control rats were fed 0.2 or 0.02% arctiin, or basal diet alone during the experimental period. Animals were killed at the end of week 48. Although the incidence of mammary carcinomas did not significantly differ among the PhIP-treated groups, multiplicity was significantly decreased in rats given 0.2 (0.7+/-0.7, P<0.05) or 0.02% (1.0+/-1.1, P<0.05) arctiin after PhIP initiation as compared with the PhIP alone controls (2.1+/-2.5). The average number of colon aberrant crypt foci was also significantly decreased in these two groups. Pancreas acidophilic foci were induced in PhIP treated animals with slight decrease in the multiplicity with arctiin during the initiation phase. For liver carcinogenesis, groups of 15 male F344 rats were given a single intraperitoneal injection of diethylnitrosamine (DEN) and starting 2 weeks later, they were administered 0.03% MeIQx in the diet, MeIQx together with 0.5% arctiin, 0.1% arctiin or basal diet for 6 weeks. They were subjected to two-third partial hepatectomy 3 weeks after DEN initiation and killed at the end of week 8 for glutathione S-transferase placental form (GST-P) immunohistochemistry. The numbers and areas of preneoplastic GST-P positive foci were elevated by the treatment with MeIQx, and further increased by the simultaneous treatment with arctiin. These results indicate that arctiin has a protective effect on PhIP-induced carcinogenesis particularly in the mammary gland in the promotion period. On the other hand, it may have a weak co-carcinogenic influence on MeIQx-induced hepatocarcinogenesis. In addition, the results suggested that PhIP is a weak pancreatic carcinogen in female SD rats, targeting acinar cells.

Modifying effects of propolis on MeIQx promotion of rat hepatocarcinogenesis and in a female rat two-stage carcinogenesis model after multiple carcinogen initiation.

The modifying effects of the dietary administration of water- and ethanol-extracted propolis produced in Brazil (WB and EB, respectively) on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) promotion of rat hepatocarcinogenesis were investigated in a medium-term liver bioassay system with use of male Fischer 344 rats. The number and area of glutathione S-transferase placental form (GST-P)-positive foci in rats given 0.5% WB were significantly increased compared with the group given MeIQx alone. Furthermore, the numbers of GST-P-positive foci were higher in rats given 0.1% WB or EB than in those given the basal diet alone. The modifying effects of propolis on other organs were also examined in female Fischer 344 rats given multiple carcinogens for initiation. Rats received water- and ethanol-extracted propolis produced in Brazil and Uruguay (WB, EB, WU, and EU, respectively) in the diet after exposure to three different carcinogens. The incidence of total mammary tumors was significantly lower in rats given EU than in the control group. These results indicate that a water extract of propolis exerts a cocarcinogenic effect on MeIQx hepatocarcinogenesis while promoting the effect at low dose in a two-stage hepatocarcinogenesis model. Moreover, they suggest that ethanol-extracted propolis may be an inhibitor of mammary gland carcinogenesis.

Aberrant expression of p27(Kip1) is associated with malignant transformation of the rat urinary bladder epithelium.

Alteration in cell cycle regulators is considered to play an important role in carcinogenesis. In order to cast light on changes in reversible hyperplastic and irreversible tumorigenic lesions in the rat urinary bladder, expression of p27(Kip1), cyclin D1 and cyclin E proteins was sequentially compared. In the first study, 3% uracil was fed for 4 weeks to cause urinary calculi and consequent hyperplasia and papillomatosis, both regressing after withdrawal of the insult. Compared with normal bladder epithelium, in papillomatosis at week 4, the BrdU index and immunohistochemical positivities for cyclin D1 and cyclin E were significantly elevated, whereas values for p27(Kip1) tended to be reduced. One week after withdrawal of uracil, the BrdU index and positivities for cyclin D1 and cyclin E were decreased to below the control levels, while positivity for p27(Kip1) was dramatically increased, with a strong staining intensity. In a second study, rats were initiated with a bladder carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine for 4 weeks, then fed 3% uracil for 8 weeks. During this latter period, expression of cyclin D1, cyclin E and p27(Kip1) in hyperplastic urothelium were comparable with those in the first study. One week after withdrawal of uracil, most urothelial lesions regressed, showing high p27(Kip1) and low cyclin D1 and cyclin E staining. Two weeks after uracil withdrawal, transitional cell carcinomas, with a low p27(Kip1) and high cyclin D1 and cyclin E staining pattern, could be easily distinguished from surrounding regressing epithelium. These data indicate that during regression of papillomatosis after cessation of a proliferative stimulus, expression of p27(Kip1)is elevated, accompanied by a lowering of cyclin D1 and cyclin E. In irreversible tumorous bladder lesions, on the other hand, persistent low expression of p27(Kip1) and elevated cyclin D1 and cyclin E are characteristic.

Mutation induction by mechanical irritation caused by uracil-induced urolithiasis in Big Blue rats.

Some chronic mechanical irritations induce cancers, and it is speculated that mutations are induced by increased rate of cell proliferation caused by the irritation. In this study, it was investigated using chronic mechanical irritation to urothelium caused by urolithiasis, whether mutations are really induced by such cell proliferation or not. Male rats transgenic for lacI (Big Blue(R) rats), in which lacI mutations accumulated in tissue can be measured, were fed 3% uracil, a component of RNA, to induce urolithiasis associated with papillomatosis, and eventually with bladder cancers. The frequency of independent mutations in the bladders of the treated rats showed 3-5 fold increases at weeks 10, 20, and 51 (P=0.01 at week 51) while the frequency was not elevated at week 2. The mutation frequencies in the control bladders ranged from 3 to 9x10(-6). In both groups, G to A transitions at CpG sites, indicative of spontaneous mutations, constituted the most prevalent mutations. Mechanical irritation caused by uracil was shown to induce a 3-5 fold increase of mutations, possibly through an elevation of spontaneous mutations by vigorous cell proliferation.

Post-initiation effects of a super critical extract of propolis in a rat two-stage carcinogenesis model in female F344 rats.

Post-initiation modifying effects of dietary administration of a super critical extract of propolis on major organs were examined using a two-stage carcinogenesis model. Groups of 21 or 22 F344 female rats were treated sequentially with 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN, i.g.), 7,12-dimethylbenz[a]anthracene (DMBA, i.g.), 1,2-dimethylhydrazine (DMH, s.c.) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN, in drinking water) during the first 3 weeks for initiation, and then administered diet containing 0.1 or 0.01% propolis for 33 weeks. Further groups were treated with the carcinogens alone, 0.1% propolis alone or basal diet alone. All surviving animals were killed at week 36, and major organs were examined histopathologically for development of preneoplastic and neoplastic lesions. The incidence and multiplicity of mammary carcinomas were significantly decreased by the 0.1 and 0.01% propolis treatments. In the urinary bladder, the incidence of PN hyperplasia but not tumors was, in contrast, significantly increased by 0.1% propolis. Similarly, the number and area of glutathione S-transferase placental form (GST-P)-positive liver foci were significantly elevated with this high dose. The results indicate that a low dose of a super critical extract of propolis may find application as a potent chemopreventor of mammary carcinogenesis.

Immunohistochemical demonstration of carcinogen-DNA adducts in tissues of rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP): detection in paraffin-embedded sections and tissue distribution.

A polyclonal antibody against 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA adducts was raised for their immunohistochemical demonstration in paraffin-embedded sections. Specificity of this antibody was confirmed by competitive ELISA. Positive signals were immunohistochemically detected in acetone-fixed but not in formalin- or ethanol-fixed sections from F344 rats treated by gavage with a single dose of PhIP at 37.5-300 mg/kg and killed at 1, 2, and 7 days thereafter. Dose-dependent positive staining was observed in almost all organs of both sexes, including the colon, prostate, and mammary gland but largely independent of the tumor response. Repair activity, judged by disappearance of adducts with time, differed according to the organ or cell type. One exception was hepatocytes, the liver incidentally being a nontarget organ. The results suggest that the generated antibody is applicable for detection of cells targeted by PhIP in paraffin-embedded sections and also for the investigation of the mechanisms of PhIP-carcinogenesis.

Strong promoting activity of phenylethyl isothiocyanate and benzyl isothiocyanate on urinary bladder carcinogenesis in F344 male rats.

Post-initiation effects of phenylethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC) on hepatocarcinogenesis and urinary bladder carcinogenesis were examined in rats pretreated with diethylnitrosamine (DEN) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Groups of 21 rats received a single intraperitoneal injection of 200 mg/kg body weight of DEN. Starting 2 days thereafter, they were administered 0.05% BBN in the drinking water for 4 weeks. Three days after completion of the carcinogen treatment, they were placed on a diet containing PEITC or BITC at a dose of 0.1%, or a basal diet alone for 32 weeks and then killed for autopsy. Further groups of 6 rats each were similarly treated with PEITC, BITC or basal diet alone for 32 weeks without prior DEN and BBN exposure. In the liver, although the incidences of liver tumors were not significantly affected, the multiplicity of foci larger than 0.5 cm in diameter was slightly increased by PEITC. In the urinary bladder, the incidences of papillary or nodular (PN) hyperplasias and carcinomas were significantly elevated by PEITC or BITC after DEN and BBN initiation. In the groups without initiation, PN hyperplasia was found in all rats of both PEITC and BITC groups, along with papillomas and carcinomas in some animals. Tumors and PN hyperplasias in the groups treated with PEITC and BITC are characterized by downward growth. Our results thus showed PEITC and BITC to be strong promoters of urinary bladder carcinogenesis with some complete carcinogenic potential.

Relationship between CD44 expression and differentiation of human prostate adenocarcinomas.

It is well documented that CD44 plays an important role in tumor metastasis. We investigated whether there is a correlation between the expression of its isoforms in prostate cancer cells and patient prognosis using 72 cases with biopsy specimens. Immunohistochemistry demonstrated expression of CD44H (68.1%), v6 (36.1%) and v9 (68.1%) to be relatively more frequent than that of other isoforms. A positive correlation between CD44H expression and tumor differentiation was found but this did not extend to clinical staging or prognosis. Likewise, results for CD44v6 or v9 expression suggest that they may be useful markers for prostate adenocarcinoma differentiation but not prognosis.

Identification of a neural alpha-catenin as a key regulator of cadherin function and multicellular organization.

The function of cadherin cell adhesion molecules is thought to be regulated by a group of cytoplasmic proteins, including alpha-catenin. We identified a subtype of alpha-catenin, termed alpha N-catenin, which is associated with N-cadherin and expressed mainly in the nervous system. cDNA transfection experiments showed that alpha N-catenin can also bind with E-cadherin. To investigate the role of alpha N-catenin, we transfected lung carcinoma PC9 cells, which express E-cadherin and beta-catenin but neither alpha- nor alpha N-catenin, with alpha N-catenin cDNA. While parental PC9 grew as isolated cells, the transfectant lines formed aggregates in which cells were tightly adhered to each other, showing epithelial arrangements, and they occasionally gave rise to cystic spheres. These results suggest that alpha N-catenin is crucial not only for cadherin function but also for organization of multicellular structures.