Pathological change of articular cartilage in the mandibular head treated with immunosuppressant FK 506

While several reports have documented immunosuppressant-induced osteoporosis, the exact mechanism of the pathological change of the joint remains to be clarified. In the present study, we have demonstrated the pathological change of the articular cartilage in the mandibular head of five Sprague-Dawley rats administered with the immunosuppressant FK 506 for 28 days. Three-dimensional micro-computed tomography of the mandibular heads in treated rats showed a significant decrease in trabecular bone volume compared to control rats. Histological observation revealed atrophic change of the articular cartilage. Immunohistological observation using anti-proliferative cell nuclear antibody (PCNA), type I, II, and type X collagen antibodies showed significantly decreased proliferation and differentiation of chondrocytes in the articular cartilage compared with the control group (p<0.05). Tartrate-resistant acid phosphatase (TRAP) staining revealed no significant difference in the numbers of osteoclasts at the chondro-osseous junction. Thus, FK 506 administration inhibited chondrogenic cell proliferation and differentiation and might cause osteoporotic change of subcartilage trabecular bone that subsequently forms in the mandibular head.     

Defining the position of the switches between replicative and bypass DNA polymerases

Cells contain specialized DNA polymerases that are able to copy past lesions with an associated risk of generating mutations, the major cause of cancer. Here, we reconstitute translesion synthesis (TLS) using the replicative (Pol III) and major bypass (Pol V) DNA polymerases from Escherichia coli in the presence of accessory factors. When the replicative polymerase disconnects from the template in the vicinity of a lesion, Pol V binds the blocked replication intermediate and forms a stable complex by means of a dual interaction with the tip of the RecA filament and the beta-clamp, the processivity factor donated by the blocked Pol III holoenzyme. Both interactions are required to confer to Pol V the processivity that will allow it synthesize, in a single binding event, a TLS patch long enough to support further extension by Pol III. In the absence of these accessory factors, the patch synthesized by Pol V is too short, being degraded by the Pol III-associated exonuclease activity that senses the distortion induced by the lesion, thus leading to an aborted bypass process.     

Complete nucleotide sequence and gene rearrangement of the mitochondrial genome of the bell-ring frog, Buergeria buergeri (family Rhacophoridae)

In this study we determined the complete nucleotide sequence (19,959 bp) of the mitochondrial DNA of the rhacophorid frog Buergeria buergeri. The gene content, nucleotide composition, and codon usage of B. buergeri conformed to those of typical vertebrate patterns. However, due to an accumulation of lengthy repetitive sequences in the D-loop region, this species possesses the largest mitochondrial genome among all the vertebrates examined so far. Comparison of the gene organizations among amphibian species (Rana, Xenopus, salamanders and caecilians) revealed that the positioning of four tRNA genes and the ND5 gene in the mtDNA of B. buergeri diverged from the common vertebrate gene arrangement shared by Xenopus, salamanders and caecilians. The unique positions of the tRNA genes in B. buergeri are shared by ranid frogs, indicating that the rearrangements of the tRNA genes occurred in a common ancestral lineage of ranids and rhacophorids. On the other hand, the novel position of the ND5 gene seems to have arisen in a lineage leading to rhacophorids (and other closely related taxa) after ranid divergence. Phylogenetic analysis based on nucleotide sequence data of all mitochondrial genes also supported the gene rearrangement pathway.     

Phenotypic variability in human embryonic holoprosencephaly in the Kyoto Collection

BACKGROUND: Holoprosencephaly (HPE) is one of the most common developmental disorders of the brain associated with specific craniofacial dysmorphogenesis. Although numerous postnatal cases have been reported, early phases of its pathogenesis are not well understood. We examined over 200 cases of HPE human embryos both grossly and histologically, and studied their phenotypic variability and stage-specific characteristics. METHODS: Among over 44,000 human embryos in the Kyoto Collection of Human Embryos, 221 embryos have been diagnosed as HPE. Their developmental stages ranged from Carnegie stage (CS) 13 to CS 23. They were examined grossly and were also serially sectioned for detailed histological analysis. RESULTS: HPE embryos after CS 18 were classified into complete (true) cyclopia, synophthalmia (partially fused eyes in a single eye fissure), closely apposed separate eyes (possible forerunners of ethmocephaly and cebocephaly), and milder HPE with median cleft lip (premaxillary agenesis). At CS 13-17, when facial morphogenesis is not completed, HPE embryos had some facial characteristics that are specific to these stages and different from those in older HPE embryos. The midline structures of the brain, including the pituitary gland, were lacking or seriously hypoplastic in HPE embryos. Complete cyclopia was found in two cases after CS 18 but none at earlier stages. CONCLUSIONS: The early development of HPE in human embryos was systematically studied for the first time. The pathogenesis of craniofacial abnormalities, especially eye anomalies, in HPE was discussed in the light of recent studies with mutant laboratory animals.     

Geographic variability in expression of the sex-linked AAT-1 gene of the bell-ring frog, Buergeria buergeri

Cytological observation and artificial crossing experiments were used to examine the geographic differences in the sex-determining mechanism and mode of inheritance of the sex-linked AAT-1 gene in the bell-ring frog, Buergeria buergeri. The AAT-1 phenotypes were also examined by allozyme analysis using field-caught females and males collected from 19 populations from the Honshu, Shikoku, and Kyushu islands of Japan, in order to comprehensively elucidate the geographic variability in the expression of the sex-linked AAT-1 gene of B. buergeri. The results showed that the Aomori population of B. buergeri from the northern end of Honshu was female heterogametic in sex determination, that chromosome No. VII was a sex chromosome of the ZZ/ZW type, and that the sex-linked AAT-1 gene was expressed on both the Z and W chromosomes. This mode of AAT-1 expression in the Aomori population was different from that in the Hiroshima population from western Honshu, in which the AAT-1 gene was expressed on the Z chromosome but not on the W chromosome. The results also showed that there was no differentiation among populations in the expression of the AAT-1 genes on the Z chromosome, whereas two populations, the Hiroshima and Aomori frogs, exhibited distinct modes of expression of the AAT-1 gene on the W chromosome. These two modes of expression may be widely distributed in western and eastern Japan, and coexist in the central part of Honshu.     

Liquid chromatographic determination of 1-adamantanamine and 2-adamantanamine in human plasma after pre-column derivatization with o-phthalaldehyde and 1-thio-beta-D-glucose

We investigated high-performance liquid chromatographic (HPLC) determination of 1-adamantanamine hydrochloride (1-ADA) and 2-adamantanamine hydrochloride (2-ADA) in human plasma after the derivatization with o-phthalaldehyde (OPA) and 1-thio-beta-D-glucose (TG). Extracted human plasma samples were mixed with OPA and TG at room temperature for 6 min and injected onto HPLC. Retention times of 1-ADA and 2-ADA derivatives were 12.6 and 14.1 min, respectively. The lower limits of detection of 1-ADA and 2-ADA were 0.02 and 0.008 microg/ml, and the lower limits of quantitation of 1-ADA and 2-ADA were 0.025 and 0.01 microg/ml, respectively. The coefficients of variation for intra-day and inter-day assay of 1-ADA and 2-ADA were less than 4.4 and 6.0%, respectively. L-Dopa and dopamine were not found to interfere with the peaks of 1-ADA and 2-ADA derivatives. Human plasma unbound fraction (f(p)) values of 1-ADA varied between 0.32 and 0.48, while those of 2-ADA varied between 0.38 and 0.68. These results indicate that HPLC assay of 1-ADA and 2-ADA by derivatization with OPA and TG is simple, rapid, sensitive and reproducible for determining 1-ADA and 2-ADA in human plasma.     

Proteomic signature of human cancer cells

We assessed proteomic profiles as biomarkers for monitoring cell phenotypes. Protein expression profiles were obtained by fluorescence two-dimensional difference gel electrophoresis (2-D-DIGE), in which quantitative ability is improved by labeling proteins with fluorescent dyes prior to electrophoresis. Integrated protein spot intensities were analyzed by a statistical approach. The proteomic data of two groups of cell lines: (1) adenocarcinoma (AC) cell lines derived from lung, pancreas and colon tissues and (2) lung cancer cell lines with different histological backgrounds, including AC, squamous cell carcinoma and small cell carcinoma, were assessed on the basis of prior biological information. Hierarchical clustering analysis and principal component analysis were used to divide the cell lines into subgroups on the basis of similarities between their protein expression profiles. The majority of cell lines were grouped according to their organ of origin or histological background. A machine-learning algorithm selected 32 protein spots that were responsible for the classification. The results indicate that proteomic data generated by 2-D-DIGE can provide a signature of essential cell phenotypes, suggesting that it might be possible to apply this technique to developing tumor markers that could identify the organ of origin of metastatic tumors and contribute to the differential diagnosis of lung cancer.     

Spinnokinetic analyses of blood disposition and biliary excretion of nitric oxide (NO)-Fe(II)-N-(dithiocarboxy)sarcosine complex in rats: BCM-ESR and BEM-ESR studies

Nitric oxide (NO) is well known to have a wide variety of biological and physiological functions in animals. On the basis of the fact that Fe(II)-dithiocarbamates react with NO, a Fe(II)-N-(dithiocarboxy)sarcosine complex (Fe(II)-DTCS) was proposed as a trapping agent for endogenous NO. However, quantitative pharmacokinetic investigation for NO-Fe(II)-dithiocarbamate complexes in experimental animals has been quite limited. This paper describes the results on the quantitative pharmacokinetic features of a NO-Fe(II)-N-DTCS in both the blood and bile of rats following intravenous (i.v.) administration of the complex. For this purpose, we applied two in vivo methods, i.e. (1) in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) which previously developed, and (2) in vivo biliary excretion monitoring-electron spin resonance (BEM-ESR). We monitored real-time ESR signals due to nitrosyl-iron species in the circulating blood and bile flow. The ESR signal due to NO-Fe(II)-DTCS was stable in biological systems such as the fresh blood and bile. In in vivo BCM- and BEM-ESR, the pharmacokinetic parameters were calculated on the basis of the two-compartment and hepatobiliary transport models. The studies also revealed that the compound is widely distributed in the peripheral organs and partially excreted into the bile. We named a kinetic method to follow spin concentrations as spinnokinetics and this method will be useful for detecting and quantifying the endogenously generated NO in Fe(II)-DTCS administered animals.     

Contribution of IL-1 beta and TNF-alpha to the initiation of the peripheral lung response to atmospheric particulates (PM10)

Alveolar macrophages (AM) play a key role in clearing atmospheric particulates from the lung surface and stimulating epithelial cells to produce proinflammatory mediators. The present study examines the role of "acute response" cytokines TNF-alpha and IL-1 beta released by AM exposed to ambient particulate matter with a diameter of <10 microm (PM(10)) in amplifying the proinflammatory mediator expression by A549 cells and human bronchial epithelial cells (HBEC). The results showed that supernatants from human AM incubated 24 h with PM(10) (100 microg/ml) contained more TNF-alpha, IL-1 beta, granulocyte-macrophage colony stimulating factor, IL-6, and IL-8 than nonexposed AM supernatants. The 3-h treatment of A549 cells with PM(10)-exposed AM supernatants increased TNF-alpha, IL-1 beta, IL-8, regulated on activation normal T-cells expressed and secreted (RANTES), and leukemia inhibitory factor mRNA compared with the treatment with nonexposed AM supernatants and, compared with untreated A549 cells, additionally increased ICAM-1 and monocyte chemotactic protein-1 mRNA. Preincubating PM(10)-exposed AM supernatants with anti-IL-1 beta antibodies reduced all the above mediators as well as VEGF mRNA expression (P < 0.05), while anti-TNF-alpha antibodies were less effective (P > 0.05), and the combination of the two antibodies most effective. When HBEC were treated similarly, anti-TNF-alpha antibodies had the greatest effect. In A549 cells PM(10)-exposed AM supernatants increased NF-kappa B, activator protein (AP)-1 and specificity protein 1 binding, while anti-TNF-alpha and anti-IL-1 beta antibodies reduced NF-kappa B and AP-1 binding. We conclude that AM-derived TNF-alpha and IL-1 beta provide a major stimulus for the production of proinflammatory mediators by lung epithelial cells and that their relative importance may depend on the type of epithelial cell target.     

Changes in oligosaccharide expression on plasma membrane of the mouse oocyte during fertilisation and early cleavage

It has been reported that various structural and functional changes occur on the surface of the plasma membrane of the ovum and embryo during fertilisation and cleavage in preparation for implantation. Glycoproteins are thought to be one of the factors in cell attachment. Thus, we investigated the changes in glycoprotein expression on the cell surface membrane of the mouse embryo by using lectins. Among seven types of lectin (ConA, WGA, UEA-I, MPA, LCA, DBA and PNA), the fluorescent intensities of ConA and WGA markedly increased from unfertilised ova to blastocysts. By quantitative analysis using immuno-scanning electron microscopy, the numbers of ConA-gold particles were small until 4-cell cleavage, but increased significantly at the blastocyst stage. In contrast, an increased number of WGA-gold particles was detected even at the 4-cell stage, and this increase continued to the blastocyst stage. From the above observations, we conclude that the numbers of sugar chains bound to both ConA andWGA increases with blastocyst formation and earlier expression is observed with WGA. The present study dearly shows that glycoproteins on the cell membrane surface of the mouse embryo quantitatively increase at the time of implantation, and the possibility has been indicated that glycoproteins are involved in intercellular recognition and adhesion between the embryo and endometrial epithelium.