Expression of intermediate filaments and other special markers by testicular germ cell tumors. With reference to embryogenesis.

Distribution of intermediate filament proteins (IFs) and several special markers was studied in 39 testicular germ cell tumors and 8 embryos and foetuses. The similarity and difference between development of germ cell tumor and embryogenesis were immunohistochemically investigated. Seminoma and embryonal carcinoma, as tumoral counterparts of undifferentiated germ cells, were characterized by little IF expression. This study revealed that the maturing and differentiating process in germ cell tumor is different from normal embryonal development and the tumor cells showed leaping maturing steps in tumorigenesis. Immunostaining for IFs helped to discover the further differentiation occurring in embryonal carcinoma and to demonstrate heterogeneous elements in non-seminoma germ cell tumors, which sometimes might not be apparent by light microscopical observation of H&E staining section. According to the findings, two patterns in mixed germ cell tumors are suggested; i.e., combined and diffuse types. The mechanism of tumorigenesis of the two types is supposed to be different. Clinically, the prognosis of most patients with testicular germ cell tumor is fairly good because of the improved chemotherapies that are dependent on histological diagnosis.     

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.     

A novel anti-plant viral protein from coelomic fluid of the earthworm Eisenia foetida: purification, characterization and its identification as a serine protease

A novel protein showing strong antiviral activities against cucumber mosaic virus (CMV) and tomato mosaic virus (TMV) was purified from the coelomic fluid of the earthworm Eisenia foetida. The protein was characterized as a cold-adapted serine protease. Its molecular weight was estimated to be 27,000 by SDS-PAGE. The enzyme was most active at pH 9.5 and 40–50 °C. The protease activity at 4 °C was 60% of that obtained at the optimal temperature. The activity was suppressed by various serine protease inhibitors. Partial N-terminal amino acid sequence of the enzyme showed homology with serine proteases of earthworms, E. foetida and Lumbricus rubellus previously studied. Our results suggest that the enzyme can be applicable as a potential antiviral factor against CMV, TMV, and other plant viruses.     

Role of proline residues in conferring thermostability on aqualysin I

To understand the molecular basis of the thermostability of a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1, we introduced mutations at Pro5, Pro7, Pro240 and Pro268, which are located on the surface loops of aqualysin I, by changing these amino acid residues into those found at the corresponding locations in VPR, a psychrophilic serine protease from Vibrio sp. PA-44. All mutants were expressed stably and exhibited essentially the same specific activity as wild-type aqualysin I at 40 degrees C. The P240N mutant protein had similar thermostability to wild-type aqualysin I, but P5N and P268T showed lower thermostability, with a half-life at 90 degrees C of 15 and 30 min, respectively, as compared to 45 min for the wild-type enzyme. The thermostability of P7I was decreased even more markedly, and the mutant protein was rapidly inactivated at 80 degrees C and even at 70 degrees C, with half-lives of 10 and 60 min, respectively. Differential scanning calorimetry analysis showed that the transition temperatures of wild-type enzyme, P5N, P7I, P240N and P268T were 93.99 degrees C, 83.45 degrees C, 75.66 degrees C, 91.78 degrees C and 86.49 degrees C, respectively. These results underscore the importance of the proline residues in the N- and C-terminal regions of aqualysin I in maintaining the integrity of the overall protein structure at elevated temperatures.     

OsSEND-1: a new RAD2 nuclease family member in higher plants

A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H₂O₂, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity.     

Lineage admixture during postglacial range expansion is responsible for the increased gene diversity of Kalopanax septemlobus in a recently colonised territory

We aimed to reveal the effects of range expansion and subsequent lineage admixture from separated glacial refugia on genetic diversity of Kalopanax septemlobus in Japan, by combining nuclear microsatellite data and ecological niche modelling. Allelic richness and gene diversity were compared at the population and regional level. We also statistically examined these indices as a function of population accessibility to the last glacial maximum (LGM) palaeodistribution reconstructed by ecological niche modelling to test a simple range expansion scenario from glacial refugia. Genetic diversity was highest in the populations of southern Japan and gradually decreased towards the north. However, an additional centre of genetic diversity, when measured as gene diversity, was found in northern Honshu Island, where distinct lineages were shown to be in contact. Positive effects of population accessibility to the LGM range were detected in both diversity indices at different spatial scales. The combined data support independent postglacial range expansions towards the north from the edge populations on the exposed coastal shelf of Pacific and Sea of Japan in northern Honshu during the LGM, which subsequently resulted in markedly low genetic diversity in the northernmost extant range, Hokkaido. The regional increase in gene diversity in northern Honshu is likely to be the result of postglacial lineage admixture. Relative difference in the spatial scales best relating population genetic diversity with the LGM distribution can be explained by a higher rate of allelic richness diversity loss during range expansions and stronger effects of lineage admixture on gene diversity.