Hepatocyte growth factor stimulates root growth during the development of mouse molar teeth.

BACKGROUND AND OBJECTIVE: It is well known that tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS). However, relatively little is known about the regulatory mechanisms involved in root development. As hepatocyte growth factor (HGF) is one of the mediators of epithelial-mesenchymal interactions in rodent tooth, the objective of this study was to examine the effects of HGF on the root development of mouse molars. MATERIAL AND METHODS: The HERS of mouse molars and HERS01a, a cell line originated from HERS, were used in this study. For detection of HGF receptors in vivo and in vitro, we used immunochemical procedures. Root development was assessed by implanting molar tooth germs along with HGF-soaked beads into kidney capsules, by counting cell numbers in HERS01a cell cultures and by performing a 5'-bromo-2'-deoxyuridine (BrdU) assay in an organ-culture system. RESULTS: HGF receptors were expressed in the enamel epithelium of molar germs as well as in HERS cells. HGF stimulated root development in the transplanted tooth germs, the proliferation of HERS01a cells in culture and HERS elongation in the organ-culture system. Examination using BrdU revealed that cell proliferation in HERS was increased by treatment with HGF, especially that in the outer layer of HERS. This effect was down-regulated when antibody against HGF receptor was present in the culture medium. CONCLUSION: Our results raise the possibility that HGF signaling controls root formation via the development of HERS. This study is the first to show that HGF is one of the stimulators of root development.

Sensitivities of peripheral lymphocytes from healthy humans to induction of sister chromatid exchanges by chemicals.

The spontaneous sister chromatid exchanges (SCEs) and SCEs induced by 50 microM methyl methanesulfonate, 1 microM 4-nitroquinoline 1-oxide, and 5 microM 3-amino-1-methyl-5H-pyrido[4,3-b]indole were examined in phytohemagglutinin-activated peripheral lymphocytes from 53 healthy adult donors. The means of the mean spontaneous SCEs and SCEs induced by methyl methanesulfonate, 4-nitroquinoline 1-oxide, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole in cells of these donors had coefficients of variability of 12, 14, 24, and 28%, respectively. Thus, it was possible to estimate the SCE-inducing activities of chemicals in peripheral lymphocytes from a population of healthy humans under the defined experimental conditions. These facts provide one of the useful parameters for evaluating the genotoxicities of chemicals to human beings, a genetically heterogeneous species. Cells from two of the donors were considerably more sensitive to particular chemicals than were those from other donors, consistently giving higher numbers of induced SCEs in two repeat examinations.

A greatly simplified method of establishing B-lymphoblastoid cell lines.

Ten lymphoblastoid cell lines were established by Epstein-Barr virus-induced transformation directly from 0.04 to 0.15 ml of peripheral whole blood of one patient with xeroderma pigmentosum and four normal healthy adults. All these lines expressed B-lymphocyte characteristics. The advantages of this method are: (a) only a few drops of blood are required for establishing a permanent line; (b) damage and loss of cells in separation procedures are minimal; and (c) the method is simple, reliable, and applicable, if desired, to any patient, even babies.

Epstein-Barr virus-transformed human lymphoblastoid cells for study of sister chromatid exchange and their evaluation as a test system.

Epstein-Barr virus-transformed human lymphoblastoid cell lines are suitable for detection of sister chromatid exchange (SCE) induced by mutagens-carcinogens because they have shown a stable chromosome number and stable frequency of spontaneous SCE for more than two years in culture. Their spontaneous and induced SCE frequencies were practically the same as those of phytohemagglutinin-stimulated lymphocytes from the same blood donors. The SCE responses of one established cell line, NL3, to 13 typical mutagens and five nonmutagens were examined. This cell line responded to all the mutagens tested but not to the nonmutagens. The SCE-inducing activities of these chemicals were well correlated with their mutagenic activities assayed with the Salmonella system by Ames' and Sugimura's groups, although there were a few but significant deviations.

Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines.

1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links.

A convenient method of establishing permanent lines of xeroderma pigmentosum cells.

Nine lymphoblastoid cell lines were established after transformation by Epstein-Barr virus of peripheral lymphocytes from four xeroderma pigmentosum (XP) patients, the parents of one XP patient, and three normal donors. All these cell lines proliferate as suspension in Roswell Park Memorial Institute Medium 1640 supplemented with 20% fetal bovine serum, without detectable release of infectious Epstein-Barr virus. Some characteristics of these cell lines, such as growth rates, chromosome numbers, UV sensitivities, and activities of unscheduled DNA syntheses induced by UV, 4-nitroquinoline 1-oxide, and N-methyl-N'-nitro-N-nitrosoguanidine, were determined. Results confirm that the properties related to XP are not altered by transformation with Epstein-Barr virus and are the same in degrees of defect as are those of dermal fibroblasts from the respective individuals. These XP and normal lymphoblastoid cell lines should be especially useful for biochemical studies on the mechanism of DNA repair, because they are easy to grow in mass culture.

Identification of cellular defect in UVS1, a UV-sensitive Chinese hamster ovary mutant cell line.

UVS1 is an intermediately UV-sensitive Chinese hamster ovary mutant originally isolated by its hypersensitivity to an anticancer drug, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride. By cell fusion analysis, UVS1 complemented the UV sensitivity of the mouse lymphoma cell line US31 from the eighth complementation group of UV-sensitive rodent cell lines. By enzyme-linked immunosorbent assay we found that within 3 h after UV irradiation both pyrimidine dimers and (6-4)photoproducts in UVS1 were not removed from chromosomal DNA in UVS1 at all. Twenty-four h after UV irradiation the removal rate of (6-4)photoproducts was intermediate between CHO9, the parental cell line, and 43-3B, a UV-hypersensitive Chinese hamster ovary mutant of the complementation group 1, whereas the pyrimidine dimers in UVS1 were removed less efficiently as 43-3B. Alkaline elution assay showed that the incising activity to damaged DNA after UV irradiation of UVS1 was as low as that of 43-3B. The number of 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride-induced DNA interstrand cross-links of UVS1 was almost equal to that of 43-3B and about 1.5 times more than that of CHO9, suggesting that the gene products defective in UVS1 and 43-3B are essential for the excision repair of DNA damages produced by 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride.

Novel responses of peripheral lymphocytes of cancer patients to chemical induction of sister chromatid exchanges.

Sensitivities to sister chromatid exchange (SCE) induction by chemicals of peripheral lymphocytes from 26 cancer patients were estimated under conditions identical to those for healthy humans which had been reported (Cancer Res., 43: 439-442, 1983). The sensitive individual was defined as one whose cells give a mean induced SCE frequency more than 2 standard deviation units above the population mean of induced SCEs in cells from the healthy humans. When cells were treated with 3-amino-1-methyl-5H-pyrido[4, 3-b]indole in the presence of rat liver S9 mix, 8 in 10 stomach cancer patients, 4 in 4 colon cancer patients, 3 in 9 lung cancer patients, 0 in 3 patients bearing other cancers, and 0 in 9 non-cancerous individuals were sensitive. The corresponding frequency of individuals in the healthy population, reported previously, was 1 in 33 persons. Thus, the frequency of sensitive individuals in the combined group of stomach and colon cancer patients was very significantly higher than were frequencies in control groups. Three in 10 patients with stomach cancer and 4 in 16 patients with other cancers were sensitive to induction of SCE by methyl methanesulfonate. Six in these 7 methyl methanesulfonate-sensitive patients were also 3-amino-1-methyl-5H-pyrido[4,3-b]indole sensitive. The frequency of methyl methanesulfonate-sensitive individuals in the healthy populations was 2 in 50. There was no patient who was sensitive to SCE induction by 4-nitroquinoline 1-oxide. The frequency was not significantly different from the healthy population, in which 3 in 50 persons were sensitive. These results suggest that a particular cancer correlates with the sensitivity of peripheral lymphocytes to SCE induction by particular chemicals.

Design, fabrication and perivascular implantation of bioactive scaffolds engineered with human adventitial progenitor cells for stimulation of arteriogenesis in peripheral ischemia.

Cell therapy represents a promising option for revascularization of ischemic tissues. However, injection of dispersed cells is not optimal to ensure precise homing into the recipient's vasculature. Implantation of cell-engineered scaffolds around the occluded artery may obviate these limitations. Here, we employed the synthetic polymer polycaprolactone for fabrication of 3D woodpile- or channel-shaped scaffolds by a computer-assisted writing system (pressure assisted micro-syringe square), followed by deposition of gelatin (GL) nanofibers by electro-spinning. Scaffolds were then cross-linked with natural (genipin, GP) or synthetic (3-glycidyloxy-propyl-trimethoxy-silane, GPTMS) agents to improve mechanical properties and durability in vivo. The composite scaffolds were next fixed by crown inserts in each well of a multi-well plate and seeded with adventitial progenitor cells (APCs, 3 cell lines in duplicate), which were isolated/expanded from human saphenous vein surgical leftovers. Cell density, alignment, proliferation and viability were assessed 1 week later. Data from in vitro assays showed channel-shaped/GPTMS-crosslinked scaffolds confer APCs with best alignment and survival/growth characteristics. Based on these results, channel-shaped/GPTMS-crosslinked scaffolds with or without APCs were implanted around the femoral artery of mice with unilateral limb ischemia. Perivascular implantation of scaffolds accelerated limb blood flow recovery, as assessed by laser Doppler or fluorescent microspheres, and increased arterial collaterals around the femoral artery and in limb muscles compared with non-implanted controls. Blood flow recovery and perivascular arteriogenesis were additionally incremented by APC-engineered scaffolds. In conclusion, perivascular application of human APC-engineered scaffolds may represent a novel option for targeted delivery of therapeutic cells in patients with critical limb ischemia.

Actions of human DNA glycosylases on uracil-containing DNA, methylated DNA and their reconstituted chromatins.

Extracts of human lymphoblastoid cells catalyzed complete release of uracil (Ura) from PBS1 DNA, which contains Ura instead of thymine as a normal component (Ura-DNA), and 3-methyladenine (3-MeAde) from DNA methylated with methyl methanesulfonate (Me-DNA). These two activities, Ura-DNA glycosylase and 3-MeAde-DNA glycosylase, differed in heat stability. Cell extracts released Ura more rapidly and 3-MeAde more slowly from alkali-denatured preparations of Ura- and Me-DNA, respectively, than from native DNA's. On incubation with reconstituted chromatins, prepared from Ura-DNA and Me-DNA, respectively, with calf thymus chromosomal protein by salt gradient dialysis, cell extracts released all the Ura but only about half of the 3-MeAde residues, although both these chromatins were degraded by micrococcal nuclease until about half of the nucleotides became acid soluble. The activities of Ura-DNA and 3-MeAde-DNA glycosylase of xeroderma pigmentosum cells were similar to those of normal cells.

Chromatin structure interferes with excision of abnormal bases from DNA.

Cell-free extracts of human lymphoblastoid cells NL3 excised almost all uracil residues from free DNA with misincorporated dUMP, but only about half the uracil residues from nuclei, chromatin and reconstituted chromatin with dUMP-misincorporated DNA. This difference in susceptibility to uracil-DNA glycosylase of free and complexed DNAs was similar to the difference in susceptibility of free and complexed methylated DNAs to 3-methyladenine-DNA glycosylase. Methylated poly(dA-dT) was also protected by formation of complexes with calf thymus chromosomal proteins. It seems that the nucleosome structure prevents the action of DNA glycosylases. The very high sensitivity of PBS1 phage DNA, which contains uracil as a natural component, in complexes with calf thymus chromosomal proteins as well as in the free form [1] was confirmed. This high sensitivity seems ascribable to the high uracil content of PBS1 DNA. Methylated nucleosome monomers and dimers, and reconstituted nucleosome monomers containing methylated DNA of about 150 bp length, were considerably more resistant to 3-methyladenine-DNA glycosylase than chromatin reconstituted from methylated DNA of longer chain length. This may be due to the lower proportion of linker regions of free form stretches of the DNA chain in nucleosome oligomers.

Stimulation by ionophores of tyrosinase activity of mouse melanoma cells in culture.

The hypothesis that the melanosome is an acidic vesicle in which the tyrosinase action is suppressed under the ordinary culture conditions was examined with a variety of ionophores added in cultures of mouse melanoma cell line B16-C2M. In the presence of monensin or nigericin, which exchange H+ for Na+ or K+, respectively, through bio-membrane, the tyrosinase activity of cells in culture was more than 10 times that in the control culture. This stimulation was observed without delay after addition of the chemicals and was not inhibited by cycloheximide. The enzyme activity of sonicated cell-free extracts, in which melanosomes were disrupted, was not stimulated by these ionophores. The tyrosinase activity was stimulated to a lesser extent by a proton ionophore, p-trifluoromethoxyphenylhydrazone (FCCP). The activity was also stimulated by kryptofix 221, valinomycin (Na+ and K+ carrier, respectively), and tetraethylammonium ions (permeant cations) but only in the presence of a limited concentration of FCCP. N-Ethylmaleimide and N, N'-dicyclohexylcarbodiimide, inhibitors of lysosomal proton pump, stimulated tyrosinase activity of cells in the presence of FCCP. These facts are consistent with the hypothesis described above.

Sister chromatid exchange-related characteristics of excision repair-proficient xeroderma pigmentosum cells.

A high incidence of skin cancer characterizes patients with xeroderma pigmentosum (XP). XP patients have hereditary defects in repair mechanisms of ultraviolet light (UV)-induced damage to DNA. Progress in elucidating the pathogenesis of cutaneous cancers can be expected by analysis of the biologic defects of cultured cells from XP patients. Such information may also contribute, at least in part, to an understanding of carcinogenesis in general.

Induced accumulation of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) in maize leaves.

Accumulation of 2-(2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (HDMBOA-Glc) was induced in maize leaves by treatment with CuCl2, chitopentaose, penta-N-acetylchitopentaose, or jasmonic acid (JA). The accumulation of HDMBOA-Glc was accompanied by a decrease in level of 2-(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (DIMBOA-Glc). When the leaf segments were treated with JA in the presence of [Me-2H3]L-methionine, the label was efficiently incorporated into HDMBOA-Glc, while no incorporation into DIMBOA-Glc or HMBOA-Glc was detected, suggesting the conversion of constitutive DIMBOA-Glc to HDMBOA-Glc by methylation at the 4-position. Levels of endogenous JA and its leucine conjugate transiently increased prior to the accumulation of HDMBOA-Glc in leaf segments treated with CuCl2 and chitopentaose. The lipoxygenase inhibitor ibuprofen suppressed the accumulation of HDMBOA-Glc induced by CuCl2 treatment, and the reduced accumulation of HDMBOA-Glc was recovered by addition of JA. These findings suggested that JA functions as a signal transducer in the induction of HDMBOA-Glc accumulation.

Intracranial arteriovenous fistula manifesting as progressive neurological deterioration in an infant: case report.

This 1-year-old male infant had been diagnosed with cerebral degenerative disease because he developed psychomotor regression, and brain atrophy was demonstrated on computed tomography. He underwent magnetic resonance imaging, which suggested a cerebrovascular malformation. Cerebral angiography disclosed an arteriovenous fistula, fed by an anterior cerebral artery and directly draining into a cortical vein. The occlusion of the feeding artery was followed by a satisfactory recovery. The mechanism of the neurological symptoms in this patient is thought to be caused by a steal phenomenon and compression of the brain stem by venous engorgement in the posterior fossa.

Spontaneous regeneration of the foramen magnum after decompressive suboccipital craniectomy in Chiari malformation: case report.

A 21-year-old woman, who had undergone foramen magnum decompression for her symptomatic Chiari malformation (Type 1) 7 years before, presented with recurrence of the symptoms. Neuroradiological examinations demonstrated regeneration of the foramen magnum that was caused by new bone formation, as confirmed at the second surgery. Neurological improvement was obtained after the removal of the regenerated foramen magnum. This observation, though rare, deserves to be kept in mind during the postoperative follow-up period in young patients with Chiari malformation.

Expression of an Anacystis nidulans photolyase gene in Escherichia coli; functional complementation and modified action spectrum of photoreactivation.

The Anacystis nidulans photolyase gene inserted in an expression vector plasmid was introduced into Escherichia coli cells and the production of Anacystis photolyase protein was confirmed by reaction with antibodies raised against photolyase purified from A. nidulans cells. The Anacystis photolyase functioned in photoreactivation repair defective E. coli cells. The E. coli transformants exhibited an action spectrum with a maximum around 380 nm similar to that of E. coli photolyase in contrast with the action spectrum of A. nidulans cells which has a maximum at 437 nm. These results indicate that the Anacystis photolyase produced in E. coli cells has enzymatic activity in spite of the apparent lack of its intrinsic 8-hydroxy-5-deazaflavin cofactor.

Clinical and biological studies of 26 cases of xeroderma pigmentosum in northeast district of Japan.

Twenty-six patients with xeroderma pigmentosum (XP), who live in the Northeast (Tohoku) District of Japan, were examined for the clinical characteristics of UV-induced DNA synthesis (unscheduled DNA synthesis, UDS) and UV sensitivity of skin fibroblasts or lymphoblastoid cells, or both. A history of consanguineous marriage within two generations was found in 19 of 26 cases (73%). Two pairs of siblings showed similar manifestations and almost the same levels of UDS and of UV sensitivity. Squamous cell carcinoma, basal cell carcinoma, or both were observed on the exposed skin in 14 patients, but no malignant melanoma was found. Cancer had developed in approximately 71% (10/14) of the cancer-bearing patients by the age of 20, and 8 of them belonged to the UDS-deficient group. Neurological manifestations were associated with nine patients, including 3 with typical de Sanctis-Cacchione syndrome (DSC), and most of the cells derived from these patients had a UDS level less than 10% of that of the normal cells. A clear correlation between the levels of UDS and UV sensitivity, on the one hand, and the severity of clinical manifestations on the other could not be detected, but it seems that the UDS-deficient group is generally much more sensitive to UV in terms of cell killing and the induction of sister chromatid exchange (SCE) than the UDS-proficient group. After a photosensitivity test, one patient with mild skin manifestations showed distinct skin tanning without preceding erythema.

Arteriovenous malformation presenting as acute subdural haematoma.

A case of acute subdural haematoma caused by ruptured arteriovenous malformation is reported. At surgery, there was no association with intracerebral haematoma or definite subarachnoid haemorrhage. The mechanism of acute subdural haematoma in the present case was considered to be rupture of an arteriolized bridging vein drained by arteriovenous malformation.

Serial neuroimaging studies in Sotos syndrome (cerebral gigantism syndrome).

To elucidate the mechanism of excessive size of the head in Soto syndrome, serial neuroimaging features from birth were reviewed in two patients. Macrocephaly shortly after birth was attributed to increased volume of the cerebral parenchyma itself (megalencephalon). Subsequent excessive size of the head was related to retention of cerebrospinal fluid in the ventricles and the subarachnoid spaces. Thus, macrocephaly in Sotos syndrome reflects two different mechanisms. The value of serial evaluation of intracranial structures is emphasized.