Kentaro Horiuchi

Collagen types in normal and prematurely ruptured amniotic membranes

Collagen content in preterm amnions with premature rupture of the membranes was significantly lower than that of preterm amnions without premature rupture of the membranes. Collagen types were studied through sodium dodecyl sulfate-polyacrylamide gel electrophoresis in human amnions from pregnant women with or without premature rupture of the membranes. Collagen types I, III, and V were recognized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in all samples. In samples taken from preterm patients with premature rupture of the membranes, the ratios of III/I, III/V, and III/total collagen were significantly lower than those from ones without premature rupture of the membranes. The ratios of I/V, I/total collagen, and V/total collagen showed no change in gestations with and without premature rupture of the membranes, respectively. In term samples there was no significant difference in the ratios of all collagen types between those with and those without premature rupture of the membranes. Elastins were not demonstrable in amnion with and without premature rupture of the membranes. These studies suggest that the reduction of type III collagen content in amnion is related to the cause of premature rupture of the membranes, particularly in preterm samples.

Isotrityrosine, a new crosslinking amino acid isolated from Ascaris cuticle collagen.

Abstract A new amino acid was isolated from the cuticle collagen of Ascaris lumbricoides and characterized by ultraviolet, mass and nmr spectroscopies and chemical degradation. The results indicate that the compound is an isomer of trityrosine, having an ether linkage. The name “isotrityrosine” is proposed. Its structure suggests that it serves as a crosslink and plays a role in the organization of the collagen structure.

Emerging and entraining patterns of the sleep–wake rhythm in preterm and term infants

It has been repeatedly reported that the sleep-wake rhythm in infants entrains around 3-4 months of age after a transient free-run rhythm. To clarify the emerging and entraining patterns of the sleep-wake rhythm, the sleep and wakefulness of 84 infants (44 preterm and 40 term infants) were longitudinally recorded at home for more than 16 weeks by the day-by-day plot method. Our results showed that the entrained sleep-wake rhythm emerged after transient manifestation of either ultradian or irregular sleep-wake patterns for 3-4 weeks in 75% of the infants. Only 7% of the infants showed a free-running sleep-wake rhythm before the entrainment. These facts suggest that most infants would be entrained to an ordinary daily schedule of mothers without expression of overt free-running rhythm of the biological clock. The mean age of the entrainment was 44.8 postconceptional weeks. There were no significant differences in either frequency of each pattern or the mean age of the entrainment, between preterm and term infants. In conclusion, the entrained sleep-wake rhythm emerges around 1 corrected month, after ultradian patterns in the majority of infants.

Use of phospho-cellulose paper disks for the assay of histone acetyltransferase

Abstract A modified method for the assay of histone acetyltransferase is presented. Previously reported methods depended upon the determination of the incorporation of radioactivity from [ 14 C]acetyl coenzyme A into trichloroacetic acid (TCA)-precipitable material. However, as shown in this paper, [ 14 C]acetylated histone cannot be precipitated quantitatively by TCA. In the method described in this paper, phospho-cellulose (P-cellulose) paper disks are used as an adsorbent for [ 14 C]acetylated histone and 0.05 m carbonate buffer, pH 9.2, is used as a washing medium. This P-cellulose disk method allows more quantitative determination of [ 14 C]acetylated histone than the TCA-precipitation methods.

Promotion of collagen production by human fibroblasts with gastric cancer cells in vitro

SummaryTo elucidate the histogenesis of gastric scirrhous cancer, the promotion of collagen production by normal human skin fibroblasts (HSF-1) with human gastric cancer cells (KATO-III, MKN-45 and MKN-28) was investigated by direct coculture and parabiotic culture.Argyrophilic collagenous fibers were demonstrated among fibroblasts on both direct cocultures and parabiotic cultures of the fibroblasts with gastric cancer cells. Microscopic examination showed that these fibers appeared earlier and were more abundant and thicker in direct cocultures and parabiotic cultures than in single cultures of fibroblasts. Gastric cancer cells in single or parabiotic culture did not form argyrophilic fibers.For quantitative proof of the promotion of collagen production by fibroblasts with gastric cancer cells, hydroxyproline produced by fibroblasts was measured. Much higher fibroblast hydroxyproline values were obtained in parabiotic cultures with gastric cancer cell lines than in single cultures of HSF-1. Moreover, the rate of collagen synthesis by HSF-1 was much higher than that of any gastric cancer cell line tested.These results demonstrate that gastric cancer cells enhance collagen production by fibroblasts in vitro. This finding suggests that they may produce a factor promoting fibroblast collagen synthesis and that this may contribute to the formation of stromal collagen in human gastric scirrhous cancer.

The changes in crosslink contents in tissues after formalin fixation

The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage. Pyridinoline (Pyr) is a stable and nonreducible crosslink of collagen. Pentosidine (Pen) is a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety of tissues. We used HPLC to compare the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with their concentrations in the frozen tissues. Pyr and Pen were detected in both the formalin-fixed yellow ligament and the cartilage, and their concentrations were not significantly affected by or related to the duration of formalin fixation. Des and Isodes were detected in the formalin-fixed yellow ligament but in significantly lower amounts compared to the frozen samples. We concluded that crosslinks of collagen were preserved in formalin, but crosslinks of elastin were not preserved in it. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered elastin crosslinks.

The relationship between trypsin activity in amniotic fluid and premature rupture of membranes

Trypsin activity and concentration of alpha 1-antitrypsin in amniotic fluid were measured in patients with and without premature rupture of membranes. Trypsin activity in amniotic fluid with premature rupture of membranes (127 +/- 57 U/L) was found to be significantly higher than in cases without premature rupture of membranes (14 +/- 20 U/L) (p less than 0.01). The concentration of alpha 1-antitrypsin in amniotic fluid with premature rupture of membranes (6.8 +/- 5.5 mg/dl) was significantly lower than in cases without premature rupture (25.2 +/- 20.2 mg/dl) (p less than 0.01). Trypsin activity and concentration of alpha 1-antitrypsin in amniotic fluid were not influenced by rupture of membranes and labor. These studies suggest that high trypsin activity and low concentration of alpha 1-antitrypsin in amniotic fluid is one cause of premature rupture of membranes.

Detection of the cross-linking amino acid, histidinoalanine, in human brown cataractous lens protein

In human nuclear cataract, with its progression, high polymers protein mass, which is aggregated and strengthened by disulfide cross-links and other covalent cross-links, accumulates in the nuclear region to cause hardening of the lens nucleus. However, the structure of non-disulfide covalent cross-links has not been clearly confirmed yet. Recently a cross-linking amino acid, histidinoalanine, was discovered in connective tissue and is thought to cause hardening of the tissue with aging. The content of this amino acid in the lens nucleus was studied. Nuclear cataract was classified on the basis of nuclear color as described by Pirie (1986). The content of histidinoalanine was found to be 0.05 +/- 0.03 nmol mg-1 in normal nucleus; 0.06 +/- 0.03 nmol mg-1 in grade I (uniform pale yellow); 0.05 +/- 0.03 nmol mg-1 in grade II (deep yellow nucleus); 0.38 +/- 0.07 nmol mg-1 (P less than 0.01) in grade III (hazel-brown nucleus) and 3.35 +/- 2.76 nmol mg-1 (P less than 0.01) in grade IV (deep brown nucleus). The result suggests that histidinoalanine is one of the non-disulfide cross-links of lens proteins in nuclear cataract and is closely related to the grade of cataract.

The relationship of oxidative DNA damage marker 8-hydroxydeoxyguanosine and glycoxidative damage marker pentosidine.

OBJECTIVES 8-hydroxydeoxyguanosine (8-OHdG) is a biomarker of oxidative DNA damage. Pentosidine is a biomarker of glycoxidation reaction. In this study, we investigated relationships among 8-OHdG, pentosidine and age. DESIGN AND METHODS We determined the urinary concentrations of 8-OHdG and pentosidine in adults with mild hypercholesterolemia or/and mild hypertension (hypercholesterolemia group, n = 31; hypertension group, n = 25; hypercholesterolemia and hypertension group, n = 7). RESULTS The strength of the relationship between 8-OHdG and age was the same as that between pentosidine and age (the correlation coefficient between 8-OHdG and age was 0.33, pentosidine and age was 0.37). In addition, there was a positive and significant correlation between 8-OHdG and pentosidine. On the other hand, mean values of 8-OHdG and pentosidine showed no significant difference among the three groups. CONCLUSION The results of the present study indicate that both 8-OHdG and pentosidine levels increase similarly in degenerative pathologic conditions.

Immunohistochemical detection of meconium in the fetal membrane, placenta and umbilical cord

OBJECTIVE To develop the immunohistochemistry specific for meconium in the placenta, fetal membrane and umbilical cord. STUDY DESIGN We previously reported the specific presence of zinc coproporphyrin I (ZnCP-I) in human meconium and demonstrated the possible diagnostic use of an elevation in maternal plasma ZnCP-I levels in cases of amniotic fluid embolism. In this study, we developed a new specific monoclonal antibody for ZnCP-I and applied it to the immunostaining of meconium in the placenta, fetal membrane, and umbilical cord. RESULTS Immunoreactivity of ZnCP-I clearly and specifically identified meconium in the placenta, fetal membrane, and umbilical cord. It was especially useful in cases of severe chorioamnionitis to detect meconium in the macrophages surrounded by numerous neutrophils. In more than half of the cases, meconium was detected in clear amniotic fluid at delivery, suggesting previous exposure. CONCLUSIONS Immunohistochemical detection of ZnCP-I is a highly sensitive histological diagnosis of meconium.