Yukio Iwamura

Induction of a particular deletion in mitochondrial DNA by X rays depends on the inherent radiosensitivity of the cells

We examined whether X radiation induces a particular deletion in the mitochondrial DNA (mtDNA) of the cells of two human squamous cell carcinoma lines with different sensitivity to radiation and in a radiosensitive ataxia telangiectasia (AT) cell line. We used polymerase chain reaction (PCR) to quantify the accumulation of a particular 4977-bp deletion (delta mtDNA4977). PCR products of delta mtDNA4977 were detectable after exposure to 10 Gy in the radioresistant squamous cell carcinoma cells, 2 Gy in the radiosensitive squamous cell carcinoma cells and 1 Gy in the radiosensitive AT cells. These observations suggest that ionizing radiation induces the delta mtDNA4977 in human cells and that the radiation doses required to induce this deletion reflect the sensitivity of cells to radiation.

Identification and differentiation of human schistosomes by polymerase chain reaction.

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.

Acute exercise causes mitochondrial DNA deletion in rat skeletal muscle

The present study was conducted to determine the effects of acute overload exercise on mitochondrial DNA and the structure of skeletal muscles. Rats were forced to run for 20 min until reaching complete exhaustion. We detected the large‐scale deletion (7052 bp) of mitochondrial DNA by the nested polymerase chain reaction, and also observed mitochondrial ultrastructural changes in the soleus muscle.

Horizontal and vertical transmission of mouse class I MHC sequence in Schistosoma mansoni.

The mouse major histocompatibility complex (MHC) class I sequence was detected in 8-week-old Schistosoma mansoni by in situ polymerase chain reaction (in situ PCR). The signals to the mouse class I MHC sequence were observed in the nuclei of the mesenchymal and reproductive cells of S. mansoni. Signals were also observed in the cytoplasm of the tegumental tubercles. This finding suggested the possibility of MHC gene transfer from the host to schistosomes. Furthermore, the class I MHC sequence was detected in the DNA extracted from the cercariae of S. mansoni by nested PCR. Neither the nucleotide sequence of class I MHC detected in adult worm DNA nor that of class I MHC detected in the host (mouse) DNA was identical with that of class I MHC detected in the cercarial DNA. From the data we assumed that S. mansoni may have retained their own mouse class I MHC sequence in their genome throughout their life-cycle.

Existence of host DNA sequences in schistosomes—horizontal and vertical transmission

The localization of repetitive DNA sequences in the mouse genome such as mouse type 2 Alu sequence (B2) and mouse retrovirus-related sequences was shown in the body of adult Schistosoma japonicum and Schistosoma mansoni by applying an in situ PCR and hybridization technique. Using the same method, mouse major histocompatibility complex (MHC) class I sequence was also found in schistosomes. Furthermore, mouse MHC class I sequence and type A retroviral sequence were detected in S. japonicum and S. mansoni cercarial DNA by blot hybridization. These findings indicated that horizontal and vertical transmission of host DNA sequences occurred in schistosomes. The incorporation and propagation of host sequences in schistosomes and the roles played by such host sequences form the focus of this brief review.

Stimulation of 125I-3-iodo-α-methyl-l-tyrosine uptake in Chinese hamster ovary (CHO-K1) cells by tyrosine esters

INTRODUCTION Transport of the amino acid analog (123)I-3-iodo-alpha-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of (125)I-3-iodo-alpha-methyl-L-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. METHODS Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. L-Gly, L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of L-Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of L- and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of L- and D-Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37 degrees C or under ice-cold conditions. RESULTS Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of l-Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. CONCLUSIONS The L-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and L-Tyr ethyl or methyl esters to examine LAT1 function in tumor cells or tissues in vivo.

Finite Size Stability Analysis for Stochastic Cellular Automata

Real simulations are performed on a finite size of lattice. It is therefore very difficult to predict a phase diagram on an infinitely large lattice. Here, we present a Finite Size Stability Analysis (FSSA) to know whether the phase is sustainable or not. Although this analysis is a hypothesis, it enables us to determine the boundary of phase diagram. We apply FSSA to multi-state system. For example we study ten-species system in ecology. From computer simulations on various sizes of lattices, we obtain the waiting time i¾?to extinction. The system is found to have two phases: the coexistence of all species is either unstable or marginally (neutrally) stable. In the latter case, i¾?diverges on a power law with the increase of lattice size.

Heterogeneity of class I and class II MHC sequences in Schistosoma mansoni

We investigated the genetic variations in class I and class II major histocompatibility complex (MHC) genes of Schistosoma mansoni and the effects of host MHC genotypes. S. mansoni was maintained in combinations of two mouse strains with different MHC genotypes, and the MHC gene sequences of the cercariae were investigated. The detected class I MHC gene sequences were variable, with high similarity between the H-2Db murine host and the parasite. For other combinations, however, the parasite sequence was homologous to those of anthropoids. All class II MHC sequences detected in S. mansoni were homologous to those of anthropoids. Our results suggest that the genetic variation in the MHC sequences of S. mansoni is derived in part from the current host, indicating horizontal transfer of the sequences from mammal to parasite.

Horizontal and vertical transfer of mouse endogenous retroviral DNA sequences in schistosomes.

The in situ polymerase chain reaction (PCR) results revealed that mouse type A and type C retroviral sequences were transmitted horizontally from the host to schistosomes. The signals to these retroviral sequences were observed in the nuclei of the mesenchymal and reproductive cells of 8-week Schistosoma japonicum. These signals were also detected in the nuclei of the mesenchymal and reproductive cells and in the cytoplasm of the tegumental tubercles of 24-week S. mansoni. Furthermore, mouse type A retroviral sequence was detected in the DNA extracted from the cercariae of both species. However, mouse type C retroviral sequence and mouse type 2 Alu sequence (B2) were difficult to detect in the cercarial DNA of either species. These findings may indicate that some host sequences are propagated in the schistosome progeny, that is to say, not only horizontal but also vertical transfer of the host gene may occur in schistosomes.

Localization of mouse type 2 Alu sequence in schistosomes.

Localization of the type 2 Alu sequence (B2), a highly repetitive DNA sequence in the mouse genome, was examined by in situ polymerase chain reaction (in situ PCR) in schistosomes. The signals to the B2 sequence were detected in the cytoplasm of the tegumental membrane and in the nuclei of the mesenchymal, testicular, ovarian and vitelline cells of 8-week Schistosoma japonicum. In contrast, it was difficult to detect any signals of this sequence in 8-week S. mansoni, whereas in 24-week male S. mansoni the signals were observed in the cytoplasm of the tegumental tubercles and in the nuclei of the mesenchymal and testicular cells. On the other hand, in 24-week female S. mansoni the signals were found in the nuclei of the mesenchymal, ovarian and vitelline cells but not found in the tegument. On the contrary, no hybridization band of the B2 sequence was detected in the amplified DNA of 3-week schistosomula of either species. These observations proved that the host DNA sequences existed in restricted schistosome cells and were accumulated in the schistosome body during their development.