Mutation induction by heavy ion irradiation of gpt delta transgenic mice.

Using the new transgenic mice produced by mating gpt delta with p53 knockout, mutation induction by heavy-ion irradiation and the effect of p53 background on such induction were studied. After the whole body irradiation with 10 Gy of 135 MeV/u carbon-ion beam, the genomic DNA was isolated from the different organs and the lambda DNA was rescued as a lambda phage. Mutations in the transgene on the lambda DNA were determined by the spi(-) selection (deletion assay). The spi(-) mutation was induced by the above irradiation, but enhancement of the mutant frequency by the knockout of p53 gene was found not in the phages recovered from liver but in those from kidney. We are now making an effort to determine the nature of spi(-) mutation to confirm such p53 effect.     

Cell shape controls rheotaxis in small parasitic bacteria

Mycoplasmas, a group of small parasitic bacteria, adhere to and move across host cell surfaces. The role of motility across host cell surfaces in pathogenesis remains unclear. Here, we used optical microscopy to visualize rheotactic behavior in three phylogenetically distant species of Mycoplasma using a microfluidic chamber that enabled the application of precisely controlled fluid flow. We show that directional movements against fluid flow occur synchronously with the polarized cell orienting itself to be parallel against the direction of flow. Analysis of depolarized cells revealed that morphology itself functions as a sensor to recognize rheological properties that mimic those found on host-cell surfaces. These results demonstrate the vital role of cell morphology and motility in responding to mechanical forces encountered in the native environment.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

Clinicopathological assessment of cancer/testis antigens NY-ESO-1 and MAGE-A4 in osteosarcoma

The cancer/testis antigens (CTAs), New York esophageal squamous cell carcinoma-1 (NY-ESO-1) and melanoma antigen gene (MAGE)-A4 are normally restricted to male germ cells but are aberrantly expressed in several cancers. Considering the limited information regarding their significance in osteosarcoma (OS), the purpose of this study was to determine the clinical significance of NY-ESO-1 and MAGE-A4 expression in OS. Nine patients with OS treated at Kindai University Hospital were included in the study. The median age was 27 years, and median follow-up period was 40 months. The specimens obtained at the time of biopsy were used to perform immunostaining for NY-ESO, MAGE-A4, p53, and Ki-67. The positive cell rates and positive case rates of NY-ESO, MAGE-A4, p53, and Ki-67 were calculated. The correlation between the positive cell rate of immunohistochemical markers was also calculated. The correlation between the positive cell rate of NY-ESO-1 or MAGE-A4 and tumor size or maximum standardized uptake (SUV-max) was also determined. The positive cell rates of NY-ESO-1 or MAGE-A4 in continuous disease-free (CDF) cases were also compared with those in alive with disease (AWD) or dead of disease (DOD) cases. The average positive cell rates of NY-ESO, MAGEA4, p53, and Ki-67 were 71.7%, 85.1%, 16.2%, and 14.7%, and their positive case rates were 33.3%, 100%, 44.4%, and 100%, respectively. The positivity rates of NY-ESO-1 and p53 were strongly correlated, whereas those of NY-ESO-1 and Ki-67 were moderately correlated. The MAGE-A4 and p53 positivity rates and the MAGE-A4 and Ki-67 positive cell rates were both strongly correlated. The NY-ESO-1 and MAGE-A4 positivity rates were moderately correlated. The positive correlation between the NY-ESO-1 positive cell rate and tumor size was medium, and that between the MAGE-A4 positivity rate and SUV-max was very strong. There was no significant difference in the positive cell rates of NY-ESO-1 or MAGE-A4 between CDF cases and AWD or DOD cases. Overall, our results suggest that NY-ESO-1 and MAGE-A4 may be involved in the aggressiveness of OS.Key words: New York esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma antigen gene (MAGE)- A4, osteosarcoma, prognosis, cancer/testis antigen (CTA), immunohistochemistry     

Oxidative stress‐induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson''s disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4‐TRPML1‐ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca²⁺ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4‐TRPML1‐ALG2 pathway was also activated by H₂O₂, indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation‐induced lysosomal retrograde transport involved both the TMEM55B‐JIP4 and TRPML1‐ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy‐inducing signal.     

An activated S6 kinase in regenerating rat liver

S6 kinase activity was increased in the regenerating liver 5 h after partial hepatectomy compared with sham-operated liver. The protein kinase activity was eluted from DE-52 column at approximately 250 mM NaCl and was not affected by known regulators of protein kinases. The S6 kinase was further purified by chromatography on peptide R1A13-Sepharose 4B and Sephadex G-150. The molecular weight of the enzyme was estimated to be 4.5 X 10(4) by gel filtration. The enzyme catalyzes the phosphorylation of whole histone, mainly H2B histone, at 75 mM Mg2+. These properties are similar to those of a proteolytically modified Ca2+/phospholipid-independent form of protein kinase C.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan.

Vibrio cholerae O1 strains isolated from various sources in Japan over the years 1977 through 1987 were examined to confirm the presence or absence of the cholera enterotoxin (CT) gene and production of CT and to determine the kappa-phage type. The CT gene was detected in none of 225 isolates from natural waters but was present in all of the 10 isolates from environmental waters implicated in domestic cholera cases, in 64 strains (26.6%) of the 241 isolates from imported seafoods, in 43 strains (95.6%) of the 45 isolates from domestic cholera cases, and in 119 strains (93.7%) of the 127 isolates from imported cholera cases. The results suggest that the CT gene-positive strains of V. cholerae O1 have been imported into Japan through seafoods and/or by travelers. Sporadic cholera cases have resulted in contamination of the surrounding environment, but the CT gene-positive strains may not have persisted in natural waters to serve as a reservoir for epidemic cholera. The commercially available VET-RPLA kit (a latex agglutination kit for immunological detection of CT) detected production of CT in all of the CT gene-positive strains, indicating that there was no silent CT gene in the test strains. There was a strong correlation between the kappa-phage type and the presence or absence of the CT gene, suggesting a significant clonal difference between CT gene-positive and -negative strains. Five CT gene-negative strains isolated from imported cholera cases (travelers with mild diarrhea) induced a considerable amount of fluid accumulation in rabbit and/or suckling mouse intestines, indicating production of an enterotoxic factor(s) other than CT.(ABSTRACT TRUNCATED AT 250 WORDS)