Recurrent immediate type hypersensitivity reaction induced by macrogol in a 3-year-old Boy

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Phylogenetic analysis of methanogens in sheep rumen ecosystem and detection of Methanomicrobium mobile By fluorescence in situ hybridization.

The population of methanogens in the sheep rumen microbial ecosystem was studied by using 16S rDNA cloning analysis, epifluorescence microscopy (which detects autofluorescence of a specific cofactor F420 in methanogens) and the 16S rRNA-targeted in situ hybridization technique. The 16S rDNA clone libraries were constructed by PCR amplification with an Archaea-specific primer set and partial sequencing of the clonal 16S rDNAs was done. Phylogenetic analysis indicated that the clones were affiliated with Methanomicrobium ruminantium and mobile, Methanobrevibacter smithii. Epifluorescence microscopy (F420 autofluorescence) and in situ hybridization by using a newly designed M. mobile-specific 16S rRNA-targeted oligonucleotide probe found that methanogens accounted for approximately 3.6% of total ruminal microorganisms and approximately 54% of the total methanogens were M. mobile.

Marked enhancement in the reductive dehalogenation of hexachloroethane by a Thr319Ala mutation of cytochrome P450 1A2.

Mutation of the conserved Thr319 residue to Ala of cytochrome P4501A2 (CYP1A2) increased the value of Vmax 9-fold for reductive dehalogenation of hexachloroethane in the reconstituted system under anaerobic conditions. The Thr319Ala mutation also increased the elimination over substitution product ratio by 5-fold. The addition of aliphatic alcohols increased by 22-fold the activity obtained with the wild type and varied the elimination over substitution product ratio. Increasing pH increased the ratio of elimination over substitution by primarily affecting the rate of elimination.

Distal site and surface mutations of cytochrome P450 1A2 markedly enhance dehalogenation of chlorinated hydrocarbons.

Chlorinated compounds such as chlorinated ethylenes and ethanes are serious environmental pollutants. In the present study, we examined whether or not a recombinant strain of Saccharomyces cerevisiae that expresses rat liver cytochrome P450 1A2 (P450 1A2) wild-type and mutant proteins can efficiently catalyze oxidative and reductive dehalogenations of trichloroethylene, pentachloroethane, and hexachloroethane. Mutations at putative heme distal and protein surface sites of P450 1A2 greatly enhanced turnover values toward those substrates under both aerobic and anaerobic conditions. For example, a Thr319Ala mutation at the putative heme distal site enhanced the degradation rate of trichloroethylene and pentachloroethane by 2- and 2.7-fold, respectively, under aerobic conditions. The Thr319Ala mutation also strongly facilitated the reaction with hexachloroethane up to 13- and 4.5-fold under aerobic and anaerobic conditions, respectively. The Thr319Ala mutation increased dechlorinated over protonated product ratios by 3-fold as well when either pentachloroethane or hexachloroethane was used as a substrate. A Lys250Leu mutation on the putative protein surface site enhanced the dehalogenation rate of hexachloroethane up to 4.8-fold under anaerobic conditions. In contrast, a Glu318Ala mutation at the putative distal site markedly decreased the activities with trichloroethylene and pentachloroethane substrates under aerobic conditions. Conserved amino acids Thr319 and Glu318 at the heme distal site have been suggested to be important in the O2 activation during monooxidation reactions of P450s. However, the present study indicates that Thr319 is likely to be an inhibitor of dechlorination of trichloroethylene and penta- and hexachloroethanes. The roles of Thr319, Glu318, and Lys250 in the catalysis with chlorinated hydrocarbons are discussed in association with reaction mechanisms.

Undectable expression of hMLH1 protein in sporadic colorectal cancer with replication error phenotype.

PURPOSE: Four DNA mismatch repair genes have been identified as being susceptible genes for hereditary nonpolyposis colorectal cancer. Deficiency of one of the mismatch repair genes causes the replication error phenotype in more than 80 percent of patients with hereditary nonpolyposis colorectal cancer and in 10 to 30 percent of patients with sporadic colorectal cancer. To determine which mismatch repair gene is lacking the function in patients with replication error-positive colorectal cancer, several approaches have been used at the nucleic acid and protein levels. We studied replication error in 40 samples of randomly selected colorectal cancers and expression of hMSH2 and hMLH1 proteins analyzed by immunoblot in the tumor and normal tissues of the replication error-positive and replication error-negative samples. MATERIALS AND METHODS: Frozen tumor and normal tissues were obtained from 40 Japanese patients who had colorectal cancer. According to the Amsterdam criteria, those patients were classified as having 39 sporadic and 1 unknown colorectal cancers. Genomic DNA was extracted from tumor and normal tissues for determining replication error with eight microsatellite markers. Expression of hMSH2 and hMLH1 proteins in cell lysates of tumor and normal tissues of 16 patients was analyzed by immunoblot. RESULTS: The replication error phenotype was found in 6 (15 percent) of the 39 sporadic cases. hMLH1 protein was not detected in two of the six replication error-positive tumor tissues and not in the normal tissues, indicating that the tumor cells of the two patients had severe mutations in both alleles of the hMLH1 gene. Another four replication error-positive and ten replication error-negative tumors and normal tissues expressed hMLH1 protein. hMSH2 protein was detected in all samples. CONCLUSION: hMLH1 protein was undetectable in the two tumor tissues of the six replication error-positive samples of sporadic colorectal cancer. The detection procedure used here may have potential use for determining a dysfunctional mismatch repair gene product.

Effects of fetus weight, dam strain, dam weight, and litter size on the craniofacial morphogenesis of CL/Fr mouse fetuses affected with cleft lip and palate.

OBJECTIVE: This study examined the factors related to the morphogenesis of the craniofacial complex of the CL/Fr mouse fetus affected with CLP based on the findings of a lateral cephalogram. DESIGN: Embryo transfer experiments were performed to determine the effect of the fetus weight, dam strain, dam weight, and litter size on the Intra-uterine craniofacial morphogenesis of CL/Fr mouse fetuses. On the 18th gestational day, each pregnant dam that had received CL/Fr mouse embryos was laparotomized to remove the transferred fetuses that had developed in the uteri of the cleft lip and palate (CLP)-susceptible CL/Fr strain dam and the CLP-resistant C57BL strain dam. A cephalometric observation of the craniofacial morphology of each fetus was subsequently performed. RESULTS: Based on a multiple regression analysis, the standardized partial regression coefficients of the affected fetus weight, the dam weight, and the litter size on the maxillary size of the affected CL/Fr fetus were 0.71 (p < .01), 0.03, and -0.07. According to a least-squares analysis of variance, the dam strain effect in addition to the effect of the affected fetus weight on the maxillary size and the cranial size of the affected fetuses was significant (p < .01 for cranial size, p < .05 for maxillary size) and close to a significant level (p = .09) for the mandibular size of the affected fetuses. The adjusted maxillary size and cranial size after statistically eliminating the effects of the affected fetus weight, dam weight, and litter size on each original craniofacial size of the affected fetuses that had developed in the CL/Fr dam strain were also significantly smaller than those of the affected fetuses that had developed in the C57BL dam strain. CONCLUSIONS: The present results indicate that the craniofacial growth of the CL/Fr mouse fetus affected with CLP increased in proportion to the fetus weight. The dam strain effect, in addition to the effect of the affected fetus weight, could thus not be ignored when the etiology of the spontaneous CLP was examined, while the uterine environment, provided by the CL/Fr strain dam, retarded the intra-uterine craniofacial growth of the affected fetuses. It was therefore concluded that the dam strain effect, as well as the effect of the affected fetus weight, both play an important role on the craniofacial morphogenesis of the CL/Fr strain of the affected fetuses that developed in both strain dams.

The effect of dam strain on the craniofacial morphogenesis of CL/Fr mouse fetuses.

The embryo transfer technique and cephalometry were used to investigate the effect of dam strain in intrauterine craniofacial growth and the severity of cleft lip and palate (CLP) in a CLP-susceptible CL/Fr strain of embryos. The CL/Fr strain of embryos at early blastocyst stage was transferred to the same dam strain and to the CLP-resistant C57BL dam strain. On the 18th gestational day, each dam was laparotomized to take out the fetuses. The spontaneous incidence of CLP in the fetuses was checked and a cephalometric observation of the craniofacial complex of each fetus was done just after laparotomy. The dorsoventral craniofacial size of the unaffected fetuses and the severity of CLP i the affected ones were compared between both dam strains. The following results were obtained: 1) The overall craniofacial sizes of the unaffected fetuses observed in the CL/Fr dam strain were significantly smaller than those seen in the C57/BL dam strain. Those of the affected fetuses observed in the CL/Fr dam strain were smaller than those seen in the C57BL dam strain although the interstrain difference was not significant. 20 The dam strain had a highly significant effect on the craniofacial size of the unaffected fetuses. 3) The CLP frequency in the CL/Fr dam strain was significantly higher than that in the C57BL dam strain. 4) The severity of CLP in the affected fetuses observed in the CL/Fr dam strain was significantly more serious than that seen in the C57BL dam strain. These results indicated that the CLP-susceptible CL/Fr dam strain retarded the intrauterine craniofacial growth of the fetuses and that the cleft condition in the affected fetuses observed in the CL/Fr dam strain was more seriously affected than that seen in the CLP-resistant C57BL dam strain. Thus, it can be concluded that the effect of the dam strain played an important role in the craniofacial morphogenesis of the CL/Fr strain of mouse fetuses that developed from the embryo transferred to the CL/Fr and C57BL dam strains along with the genotype of embryos.

[An immunohistochemical study of the relationship between p53 expression and the DNA ploidy pattern using image cytometry in colorectal carcinoma].

We studied p53 expression in 46 colorectal carcinomas by immunohistochemistry. We examined the relationship between p53 expression and the pathological findings, the prognosis and tumor proliferative activity by Ki-67 staining or DNA ploidy pattern using an image cytometer. Expression of p53 was observed in 41.3% of carcinomas. No correlation was found between p53 expression and the depth of invasion, lymph node metastasis or Dukes' stage. No difference in survival was found between p53 positive and negative patients after curative surgery. We also studied the DNA ploidy pattern using an image cytometer in 28 carcinomas. Aneuploidy was found more frequently in the p53 positive areas than in the negative ones, but there was no significant difference. The DNA index showed no difference between the p53 positive and negative areas. We examined proliferating activity using Ki-67. There was no difference in the Ki-67 labeling index between p53 positive and negative areas. In the present study, there was no correlation between p53 expression and pathological findings, prognosis, tumor proliferative activity using Ki-67 staining or DNA ploidy pattern. Thus, p53 expression was suggested to have little relationship to grade of malignancy.

The effect of dam's strain on the intrauterine craniofacial growth of mouse fetuses.

PURPOSE: The maternal effect is one of the important factors in mammalian growth in conjunction with the genetic effect. The present study investigated the prenatal maternal effect of a dam on the intrauterine craniofacial growth of a mouse fetus using embryo transfer and cephalometry. DDD/Qdj strain mouse embryos were transferred to four strains of recipient female mice (DDD/Qdj, C3H/Qdj, C57BL/Qdj, and DBA/1J Sea). Just after parturition cephalometric observation of the newborn offspring, which developed in the uteri of the four strains of dams, was performed and then the craniofacial size of the newborn offspring was calculated on the lateral cephalogram. Statistical analyses were performed to examine the correlation between the dam's weight and the craniofacial size of the newborn offspring, to test the significance of the effect of the dam's strain on the craniofacial size of the newborn offspring, and to evaluate the interstrain difference of the intrauterine craniofacial growth of the mouse fetuses. RESULTS: It was disclosed that there were a direct relation between the dam's weight and the craniofacial size of the newborn offspring, a significant effect of the dam's strain on the craniofacial size of the newborn offspring, and a significant interstrain difference in the craniofacial size of the newborn offspring after eliminating the effects of litter size and gestation period on the craniofacial size of the newborn offspring (DDD/Qdj > C3H/Qdj = C57BL/Qdj > DBA/1J Sea). CONCLUSION: Thus, it could be concluded that the four strains of dams affected differently the intrauterine craniofacial growth of the DDD/Qdj strain fetuses through each uterine condition, indicating that the dam's weight played an important role as one of the prenatal maternal effects on the intrauterine craniofacial growth of mouse fetuses.

[Study on relationship between intratumoral DNA heterogeneity and clinicopathological findings in colorectal cancer].

We studied the relationship between intratumoral DNA heterogeneity and clinicopathological findings in 85 colorectal cancers. DNA ploidy was analyzed in 5 specimens from different sites of each tumor. When the difference in the DNA index (D.I) between several peaks in the same tumor was more than 0.1, the tumor was considered to consist of heterogeneous subpopulations with different DNA clones. DNA heterogeneity was found in 34 cases (40.0%). There was no relationship between DNA heterogeneity and depth of tumor invasion, lymph node metastasis or stage. The incidence of heterogeneity was significantly higher in the tumor with lower differentiation, liver metastasis or vessel invasion.

Hepatocyte growth factor may act as a pulmotrophic factor on lung regeneration after acute lung injury.

Hepatocyte growth factor (HGF) has been shown to have hepatotrophic and renotropic functions for regeneration of the liver and kidney through its mitogenic, motogenic, and morphogenic properties. To examine the involvement of HGF in lung regeneration after acute injury, we analyzed changes of HGF mRNA, HGF activity, and HGF receptors in the rat lung after lung insult and measured HGF concentration in sera of patients with various lung diseases. Following the onset of acute lung injury induced by intratracheal hydrochloride injection, a compensatory DNA synthesis occurred in the bronchial epithelium with a peak at 24 h and in the alveolar epithelium with a peak at 48 h. Expression of HGF mRNA in the rat lung remarkably increased only 3 h after the treatment and HGF activity in the lung also increased to about 3-fold at 6 h later. HGF receptors in the lung but not in the other noninjured organs were down-regulated 12 h later. These marked increases in HGF mRNA and HGF activity and the concomitant down-regulation of HGF receptor occurred before the marked compensatory DNA synthesis in bronchial and alveolar epithelial cells. HGF concentration in sera of patients with various lung diseases, as measured by radioimmunoassay, was much higher than that in healthy donors. These results suggest that HGF is newly produced in the lung after acute lung injury and may have a role in regeneration of the lung.

Intrauterine effect of dam on prenatal development of craniofacial complex of mouse embryo.

Embryo transfer effect and intrauterine effect of the dam on prenatal development of the craniofacial complex of mice embryos were investigated with the use of embryo transfer and cephalostat. DDD strain embryos were transferred to the three strains of recipients (DDD, C57BL, and DBA). The cephalometric observation of newborn offspring developed from transferred embryos was performed just after parturition. Dorso-ventral craniofacial size of newborn offspring was calculated using values of X- and Y-coordinates on a dorsoventral cephalogram. Statistical analysis showed that a significant intergroup difference in craniofacial size between transferred and nontransferred groups as well as a significant inter-strain difference among those of the three strains of recipients were observed. Thus, it was disclosed that embryo transfer technique might retard the prenatal development of craniofacial complex of transferred embryo and that the three strains of recipients contributed unequally to the prenatal development of craniofacial complex of transferred embryo through each of their intrauterine environments as a prenatal maternal effect. These results indicated that the intrauterine environment of the recipient played an important role in the prenatal development of the craniofacial complex of the mice embryo.

Genetic and environmental effects on preimplantation development of mouse embryo in vitro.

To examine the degree of the relative contribution of genetic and environmental effects to preimplantation development of mouse embryo in vitro, two-cell stage embryos collected from oviducts of five strains of inbred mice (DDD/Qdj, C3H/Qdj, C57BL/6J Sea, DBA/1J Sea, and BALB/C Sea) were cultivated up to the stage of expanded blastocyst for 72 hr. Both fertility and survival rates were calculated in each recipient and also in each strain. A Student-Newman-Keuls test for both rates showed a significant inter-strain difference and an analysis of variance done to estimate the relative importance of genetic and environmental effects on the preimplantation development of embryo showed a low degree of genetic determination for both rates. It was thus possible to conclude that the environmental effect played a more important role on preimplantation development of mouse embryo in vitro.

[Intermittent intra-arterial chemotherapy using subcutaneously implanted reservoir for hepatocellular carcinoma and liver metastasis of colorectal carcinoma].

Hepatectomy has been performed as a treatment for hepatocellular carcinoma (HCC) and metastatic liver carcinoma. The prognosis has improved, but it is not sufficient. In our department hepatic arterial infusion chemotherapy using subcutaneous implanted reservoir has been undertaken in 21 of 66 patients who underwent hepatectomy in hepatocellular carcinoma and 13 of 30 in liver metastasis of colorectal carcinoma since 1986. There was no significant difference between the group with and without arterial infusion chemotherapy in HCC but there was a significant difference in metastasis of colorectal carcinoma. In unresectable cases, intra-arterial chemotherapy was undertaken, but there was no significant difference. On the other hand, 26 of 58 cases receiving arterial infusion chemotherapy have shown complications.

Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs.

Hepatocyte growth factor (HGF) is a potent growth factor for various epithelial cells including mature hepatocytes and renal tubular cells. When 70% of the rat liver was excised, HGF mRNA in the intact lung markedly increased at 6 h later, then decrease to normal levels at 24 h. A similar marked increase of HGF mRNA was found in the lung of rats with hepatitis induced by CCl4. Moreover HGF mRNA in the intact lung also increased to about a 5 times higher level than the normal, within 12 h after unilateral nephrectomy. Isolated alveolar macrophages significantly expressed HGF mRNA, yet the amount remained unchanged after injury of the liver. The marked increase of HGF mRNA in lungs of partially hepatectomized rats remained even after removal of alveolar macrophages. In situ hybridization showed a marked increase of HGF mRNA signal found in endothelial cells in the lung after partial hepatectomy. We postulate that endothelial cells in the lung recognize damage of distal organs through a mediator and that lung-derived HGF may contribute to tissue repair or regeneration of injured organs, through endocrine-related mechanisms.

Genetic study of the craniofacial growth in male rats by factor scores.

The craniofacial growth of F1 male offspring produced by a complete diallel cross of five strains of rats was observed with the use of cephalometry. Factor analysis was applied to several dimensions of dorsoventral and lateral cephalograms to assess the integration of the craniofacial complex with growth. The first three factors were chosen in each cephalogram, resulting in an explanation of 50.7% and 59.3% of the variation in the dorsoventral and lateral cephalograms, respectively, on an average throughout the period of the experiment, and disclosing that Factor I was associated most closely with the change in the craniofacial growth of rats. A further genetic analysis of the factor scores of each offspring lead to the conclusion that the growth changes of craniofacial width and length were more strongly influenced by the genetic effects; while those of upper jaw, nasal cavity, and pharyngeal part were more strongly influenced by the environmental effects.

[Biochemical and histopathological studies on a mouse brain ischemic model induced by bilateral carotid artery occlusion: comparison with the carbon monoxide inhalation method and other ischemic or anoxic models].

Bilateral occlusion of the carotid arteries (BCAO) killed 52% of the male ddY mice (N = 86) and 77% of the ICR mice (N = 96) within 10 min, and the mean survival time of the ddY strain recorded for the 10 min was significantly longer than the time of the ICR strain. Among animals that survived longer than 1 hr after BCAO, some (5 of ddY and 3 of ICR) were able to survive for more than 24 hr. All of the neurobehavioral and histopathological signs developed by BCAO and in most cases followed by death were found to be also inducible by unilateral occlusion alone, although this was in a small fraction of mice. The brain levels of ATP, glucose and acetylcholine significantly decreased in mice that died within 10 min after BCAO, while none of these changes were detectable in mice surviving BCAO for 1 hr, just as in mice that died by carbon monoxide or ether inhalation. The results obtained herein indicate that mice may not be homogeneous in the functional level of the collateral route of blood supply to the brain tissue and/or in the sensitivity toward the ischemia-inducible lethality.