Treatment of nonalbumin rats by transplantation of immortalized hepatocytes using artificial human chromosome.

The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes, however, could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, rat hepatocyte clones were developed by transduction with the gene encoding the simian virus 40 T antigen (SVLT) using the new technique of human artificial mini chromosome (HAC). Immortalized rat hepatocyte clones were developed by transduction with the gene encoding the SV40 using HAC. Many clones were obtained using this technique. From comparison of the properties of all these clones using the normal hepatocytes and reverse transcription-polymerase chain reaction (RT-PCR), the characteristics of the cell clones (at least partially characterized, and assayed for albumin, glucose-6-phosphate and dipeptidyl peptidase-4, gamma-glutamyltranspeptidase, SVLT and beta-actin expression by RT-PCR) showed no differences other than the immortalization. We compared the albumin bands of the first-day (0-day) and 30-day cells by RT-PCR, showing conditions to be stable for at least 1 month. Three experimental animal model groups were used for albumin analysis: nonalbumin rats with 2/3 hepatectomy only (R-NARs; n = 4); R-NARs with intrasplenic transplantation of 3 x 10(7) primary hepatocytes (pHTx; n = 4); and R-NARs with intrasplenic transplantation of 3 x 10(7) immortalized hepatocytes (iHTx; n = 4). All HTx groups produced albumin, but the immortalized hepatocyte group did not generate significantly elevated albumin levels compared with primary hepatocytes. The results presented herein have demonstrated an initial step toward the development of immortalized hepatocytes for transplantable cells or artificial organs using HAC technology.

Construction of YAC-based mammalian artificial chromosomes.

To construct a mammalian artificial chromosome (MAC), telomere repeats and selectable markers were introduced into a 100 kb yeast artificial chromosome (YAC) containing human centromeric DNA. This YAC, which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E. The MACs appear to be about 1-5 Mb in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism. The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.

Comparison of human mesenchymal stem cells derived from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous tooth pulp.

Populations of pluripotent stem cells were isolated from bone marrow, synovial fluid, adult dental pulp, and exfoliated deciduous teeth and their multipotentiality properties compared. Osteogenic, chondrogenic, adipogenic, and neurogenic differentiation potentials were examined. Bone marrow mesenchymal stem cells (BMMSCs) and synovial fluid-derived cells (SFCs) showed the highest levels of osteogenesis as expressed by alkaline phosphatase (ALP) activity (0.54±0.094 U/mg protein and 0.57±0.039 U/mg protein, respectively; P=0.60) and by osteocalcin (BGLAP; determined by real-time RT-PCR). SFCs showed the highest levels of chondrogenesis as expressed by ALP activity (1.75±0.097 U/mg protein) and of COL2A1 and COL10A1 by real-time PCR. In terms of adipogenesis, lipid vesicles were observed in the BMMSCs and SFCs. Dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) exhibited neurogenesis potential, as shown by increases in expression of class III ß-tubulin (TUBB3) and microtubule-associated protein 2 (MAP2) on RT-PCR. Variability was found in the differentiation potential corresponding to the tendency of the original tissue to differentiate. It is suggested that the cell type should be selected depending on the regenerative treatment regimen.

Studies on the ileum-contracting mechanisms and identification as a complement C3a receptor agonist of oryzatensin, a bioactive peptide derived from rice albumin.

Oryzatensin (Gly-Tyr-Pro-Met-Tyr-Pro-Leu-Pro-Arg) is an ileum-contracting and immunostimulating peptide derived from rice albumin. The mechanisms for the ileal contraction that it induces, consisting of rapid and slow components, were examined. The rapid contraction was mediated by histamine release and the slow contraction by a prostaglandin E2-like substance, judging from the effects of various pharmacological inhibitors and antagonists on ileal contraction and titration of histamine release. The contractile profile was very similar to that of human complement C3a(70-77), which is the COOH-terminal octapeptide of C3a and has, although less potent, qualitatively the same biological activities as C3a. Oryzatensin showed homology with C3a(70-77) and affinity for C3a receptors (IC50 = 44 microM) by radioreceptor assay. This is the first report of a food-derived bioactive peptide acting through complement C3a receptors.

Minor desacylsaponins from the corms of Crocosmia masoniorum.

Two novel triterpene saponins, named desacylmasonosides 4 and 5, and a new oxygenated fatty acid have been isolated, together with a known saponin, desacylcrocosmioside A, from the weak alkaline hydrolysate of the crude saponin obtained from the corms of Crocosmia masoniorum. On the basis of spectral and chemical evidence, the structures of desacylmasonosides 4 and 5 were elucidated as 3-O-[beta-D-fucopyranosyl-(1-->6)-beta-D-glucopyranosyl]-28-O-(2-O-[beta - D-apiofuranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)-alpha-L- rhamnopyranosyl]-3-O-(beta-D-glucopyranosyl)-beta-D-fucopyranosyl)-po lygalaci c acid and 3-O-[alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranosyl]-28-O- [2-O-(alpha-L-rhamnopyranosyl)-3-O-(beta-D-glucopyranosyl)-beta-D- fucopyranosyl]-polygalacic acid, respectively. An oxygenated fatty acid derived from the acyl moiety of the esterified saponins was identified as 2,16-dihydroxy-9-oxohexadecanoic acid.

Acylated saponins, masonosides A-C, from the corms of Crocosmia masoniorum.

Three novel triterpenoid saponins, named masonosides A-C, were isolated from the corms of Crocosmia masoniorum. On the basis of spectral and chemical evidence, the structures of masonosides A-C were elucidated as 3-O-[alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranosyl]-28-O- (2-O- [beta-D-apiofuranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- alpha-L-rhamnopyranosyl]-3-O-(beta-D-glucopyranosyl)-4-O-(2-hydroxy-9-ox o-16- alpha-L-rhamnopyranosyloxyhexadecanoyl)- beta-D-fucopyranosyl)-polygalacic acid, 3-O-[alpha-L-arabinopyranosyl-(1-->6)- beta-D-glucopyranosyl]-28-O-(2-O-[beta-D-apiofuranosyl-(1-->4)-bet a-D- xylopranosyl-(1-->4)-alpha-L-rhamnopyranosyl]- 3-O-(beta-D-glucopyranosyl)-4-O-(2,16-dihydroxy- 9-oxohexadecanoyl)-beta-D-fucopyranosyl)-polygalacic acid and 3-O-[alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranosyl]- 28-O-(2-O-[beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl]-3-O- (beta-D-glucopyranosyl)-4-O-(2,16-dihydroxy- 9-oxohexadecanoyl)-beta-D-fucopyranosyl)-polygalacic acid, respectively.

4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl semipiperazide as a fluorescent labelling reagent for determination of carboxylic acids in precolumn high-performance liquid chromatography.

A fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl semipiperazide, was designed for the determination of carboxylic acids by precolumn HPLC. The reagent was reacted with carboxylic acids at 100 degrees C for 15 min in the presence of an activating reagent (a mixture of triphenylphosphine, 2,2'-dipyridyldisulfide and pyridine) and produced highly fluorescent derivatives, which were separated on a reversed-phase column by fluorescence measurement at 317 nm (excitation) and 380 nm (emission). The detection limits were 4-12 fmol/injection. The reagent was used for HPLC assays of long chain fatty acids in human serum by isocratic elution.

Mutagenicity and dominant lethal test of kojic acid--Ames test, forward mutation test in cultured Chinese hamster cells and dominant lethal test in mice.

The mutagenicity of kojic acid was studied by means of reversion mutation test in bacteria (Ames test), forward mutation test in cultured Chinese hamster cells and dominant lethal test in mice. A positive result was obtained only in Ames test (TA 98, 1535 and 100) which was not modified by the presence of S-9 fraction. Thus, it is concluded that although kojic acid is a weak mutagen in bacteria, it is nonmutagenic in eukaryotic systems either in vivo or in vitro.

Effects of breastfeeding on the risk factors for metabolic syndrome in preterm infants.

Evidence suggests that breastfeeding during infancy lowers the risk of metabolic syndrome (MS) and its attendant risk factors in adult life. To investigate the influence of feeding type on the risk factors of MS, we assessed insulin sensitivity and lipid and apolipoprotein metabolism in preterm infants. Blood samples were collected from preterm infants at the time of discharge. Infants were separated into two groups: a breast milk (BM) group receiving ⩾90% of their intake from BM, and a mixed-fed (MF) group receiving ⩾50% of their intake from formula. The following indices were then compared between the two groups. Blood glucose and serum insulin levels were used to calculate the quantitative insulin sensitivity check index (QUICKI). We also measured serum total cholesterol (TC), low-density lipoprotein cholesterol (LDLc), high-density lipoprotein cholesterol (HDLc), apolipoprotein-A1 (apoA1) and apolipoprotein-B (apoB) levels, and the ratios of TC/HDLc, LDLc/HDLc and apoB/apoA1. The mean gestational age was 32.9 weeks at birth, and blood samples were collected at a mean corrected age of 37.4 weeks. There were 22 infants in the BM group and 19 in the MF group. QUICKI was significantly higher in the BM group. TC, HDLc and apoA1 were not significantly different between the groups, but LDLc and apoB levels were significantly higher in the BM group. The TC/HDLc, LDLc/HDLc and apoB/apoA1 ratios were significantly higher in the BM group. In preterm infants, the type of feeding exposure in the early postnatal period may influence glucose, lipid and apolipoprotein metabolism, and affect markers of MS.

Nuclear resonant scattering measurements on (57)Fe by multichannel scaling with a 64-pixel silicon avalanche photodiode linear-array detector.

We developed a silicon avalanche photodiode (Si-APD) linear-array detector for use in nuclear resonant scattering experiments using synchrotron X-rays. The Si-APD linear array consists of 64 pixels (pixel size: 100 × 200 µm(2)) with a pixel pitch of 150 µm and depletion depth of 10 µm. An ultrafast frontend circuit allows the X-ray detector to obtain a high output rate of >10(7) cps per pixel. High-performance integrated circuits achieve multichannel scaling over 1024 continuous time bins with a 1 ns resolution for each pixel without dead time. The multichannel scaling method enabled us to record a time spectrum of the 14.4 keV nuclear radiation at each pixel with a time resolution of 1.4 ns (FWHM). This method was successfully applied to nuclear forward scattering and nuclear small-angle scattering on (57)Fe.

Outbreak of Legionnaires' disease on a cruise ship linked to spa-bath filter stones contaminated with Legionella pneumophila serogroup 5.

In January 2003, two cases of Legionnaires' disease associated with a ship's cruise were registered in the database of National Epidemiological Surveillance of Infectious Diseases. A 70-year-old male heavy smoker with mild emphysema contracted the disease during a cruise. Legionella pneumophila serogroup (sg) 5 was isolated from the patient's sputum and the ship's indoor spa. The isolate from the spa matched the patient's isolate by genotyping performed by pulsed-field gel electrophoresis (PFGE). The second case was in a 73-year-old female. During epidemiological investigation, a third case of Legionnaire's disease in a 71-year-old male was subsequently diagnosed among passengers on the same ship on the following cruise. Environmental investigation revealed that porous natural stones (Maifanshi) in the filters of the spas had harboured L. pneumophila, a phenomenon which has not been reported except in Japan. This is the first documented evidence of L. pneumophila sg 5 infection on a ship and of porous stones as a source of Legionella infection.

Distribution of CENP-B boxes reflected in CREST centromere antigenic sites on long-range alpha-satellite DNA arrays of human chromosome 21.

The long-range organization of alphoid DNA arrays of human chromosome 21 was investigated using a mouse-human somatic cell hybrid. Two distinct long alphoid DNA arrays, the loci alpha 21-I and alpha 21-II, were identified in the centromere region of human chromosome 21. The alpha 21-I locus, composed of an array of 11 monomer repeat units (the 11 mer), was estimated to have a total length of 1.3 Mbp. CENP-B boxes, the binding sites of the centromere protein B (CENP-B), appeared in every other monomer unit in the 11 mer except for one place where two monomer units were repeated without any CENP-B box. The other locus, alpha 21-II, was found to be composed of alphoid subfamilies with low homology to the components of alpha 21-I locus. Five different alphoid clones presenting 32 monomer units in total were isolated from the alpha 21-II locus. Sequences of these monomer units diverged between 71-89% and no unit containing a CENP-B box was found. By analysis using two color FISH, the alpha 21-I was localized to the primary constriction, whereas the alpha 21-II site was located slightly to the short arm side. Furthermore, a combination of FISH and immunofluorescent staining indicated that the alpha 21-I site was co-localized and overlapped with the CREST centromere antigenic site on mitotic chromosomes and in interphase nuclei, while alpha 21-II was distributed broadly. Our data suggest that the locus alpha 21-I containing regularly spaced CENP-B boxes at high-frequency and the assembly site of the centromere antigens may be involved in common centromere function in both human and mouse cells.

N omega-hydroxyagmatine: a novel substance causing endothelium-dependent vasorelaxation.

Agmatine (Agm), a decarboxylated L-arginine, has been suggested to be an endogenous clonidine-displacing substance in the brain. We hypothesed that Agm enzymatically produces nitric oxide (NO) through N omega-hydroxyagmatine (OHAgm) similar to the well-known endogenous NO generation from L-arginine through N omega-hydroxy-L-arginine, because Agm possesses a guanidyl function in its molecule. OHAgm was originally synthesized from delta-aminopentanoic acid in 36% overall yield. Agm and the synthetic OHAgm were examined using rat aortic rings whether they could cause endothelium-dependent vasorelaxation or not. These substances equally elicited vasorelaxations. The relaxations were completely abolished by a NO synthase inhibitor, N omega-nitro-L-arginine methyl ester, or endothelium denudation. These results suggested that Agm and OHAgm are alternative precursors for NO generated by NOS and that OHAgm may be an endogenous substance distributable in the brain.

Specific characters of serum MAO in dogs.

The effect of Tris-hydroxymethylaminomethane (Tris) on MAO activity in dog serum was studied by a modification of the method of McEwen and Cohen. Partially purified MAO from dog serum was inhibited 75% and 31% by 10 mM and 1 nM Tris, respecitively. The inhibitory effect of Tris was 1/10 of that by nialamide and 1/1000 of that by catron. Tris also slightly inhibited MAO activity in rabbit serum, but did not affect MAO from bovine or human serum, or from dog liver, kidney or brain. The extent of inhibition of dog serum MAO by Tris remained constant during incubation for 3 hrs. The inhibition was found to be reversible and noncompetitive. Benzylamine was most rapidly oxidized by MAO of dog serum followed in order by amylamine, beta- phenylethylamine and tyramine, while tryptamine and serotonin were not oxidized. Tris inhibited MAO activity with benzylamine or butylamine as substrates, but it was scarcely inhibitory with tyramine or beta-phenylethylamine as substrate. This work shows that Tris is a specific inhibitor of dog serum MAO and suggests that dog serum MAO may differ in enzymic properties from MAO in other animals.

Assay of centromere function using a human artificial chromosome.

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1-5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.

Genomic organization, chromosomal localization, and the complete 22 kb DNA sequence of the human GCMa/GCM1, a placenta-specific transcription factor gene.

The genomic sequence of the human GCMa/GCM1 gene, a mammalian homologue of Drosophila melanogaster GCM, was determined. Drosophila GCM is a neural transcription factor that regulates glial cell fate. The mammalian homolog however, is a placenta-specific transcription factor that is necessary for placental development. The 22 kb DNA sequence spanning the GCMa gene contains six exons and five introns, encoding a 2.8 kb cDNA. Overall genomic organization is similar for the human and mouse. Several potential binding sites for transcription factors like GATA, Oct-1, and bHLH proteins were found in the 5'-flanking region of the human gene. A DNA motif for GCM protein binding exists in the 5'-flanking region that is highly homologous with that of the mouse gene. The location of this gene was mapped to chromosome 6 using fluorescence in situ hybridization.

Incidence of arthro-osteitis in patients with pustulosis palmaris et plantaris.

Arthro-osteitis at the anterior chest wall was found in 12 (9.4%) out of 128 consecutive patients with pustulosis palmaris et plantaris. This finding indicates that the concomitance of arthro-osteitis with PPP is not incidental but is based on some common aetiological factor. We propose a term ;pustulotic arthro-osteitis' for this condition.