Temporal switching and cell‐to‐cell variability in Ca2+ release activity in mammalian cells

Genetically identical cells in a uniform external environment can exhibit different phenotypes, which are often masked by conventional measurements that average over cell populations. Although most studies on this topic have used microorganisms, differentiated mammalian cells have rarely been explored. Here, we report that only approximately 40% of clonal human embryonic kidney 293 cells respond with an intracellular Ca²⁺ increase when ryanodine receptor Ca²⁺ release channels in the endoplasmic reticulum are maximally activated by caffeine. On the other hand, the expression levels of ryanodine receptor showed a unimodal distribution. We showed that the difference in the caffeine sensitivity depends on a critical balance between Ca²⁺ release and Ca²⁺ uptake activities, which is amplified by the regenerative nature of the Ca²⁺ release mechanism. Furthermore, individual cells switched between the caffeine‐sensitive and caffeine‐insensitive states with an average transition time of approximately 65 h, suggestive of temporal fluctuation in endogenous protein expression levels associated with caffeine response. These results suggest the significance of regenerative mechanisms that amplify protein expression noise and induce cell‐to‐cell phenotypic variation in mammalian cells.     

Genome-Wide Association Study of Plasma Polyunsaturated Fatty Acids in the InCHIANTI Study

Polyunsaturated fatty acids (PUFA) have a role in many physiological processes, including energy production, modulation of inflammation, and maintenance of cell membrane integrity. High plasma PUFA concentrations have been shown to have beneficial effects on cardiovascular disease and mortality. To identify genetic contributors of plasma PUFA concentrations, we conducted a genome-wide association study of plasma levels of six omega-3 and omega-6 fatty acids in 1,075 participants in the InCHIANTI study on aging. The strongest evidence for association was observed in a region of chromosome 11 that encodes three fatty acid desaturases (FADS1, FADS2, FADS3). The SNP with the most significant association was rs174537 near FADS1 in the analysis of arachidonic acid (AA; p=5.95×10⁻⁴⁶). Minor allele homozygotes had lower AA compared to the major allele homozygotes and rs174537 accounted for 18.6% of the additive variance in AA concentrations. This SNP was also associated with levels of eicosadienoic acid (EDA; p=6.78×10⁻⁹) and eicosapentanoic acid (EPA; p=1.07×10⁻¹⁴). Participants carrying the allele associated with higher AA, EDA, and EPA also had higher low-density lipoprotein (LDL-C) and total cholesterol levels. Outside the FADS gene cluster, the strongest region of association mapped to chromosome 6 in the region encoding an elongase of very long fatty acids 2 (ELOVL2). In this region, association was observed with EPA (rs953413; p=1.1×10⁻⁶). The effects of rs174537 were confirmed in an independent sample of 1,076 subjects participating in the GOLDN study. The ELOVL2 SNP was associated with docosapentanoic and DHA but not with EPA in GOLDN. These findings show that polymorphisms of genes encoding enzymes in the metabolism of PUFA contribute to plasma concentrations of fatty acids.     

Structural insight into serine protease Rv3671c that Protects M. tuberculosis from oxidative and acidic stress

Rv3671c, a putative serine protease, is crucial for persistence of Mycobacterium tuberculosis in the hostile environment of the phagosome. We show that Rv3671c is required for M. tuberculosis resistance to oxidative stress in addition to its role in protection from acidification. Structural and biochemical analyses demonstrate that the periplasmic domain of Rv3671c is a functional serine protease of the chymotrypsin family and, remarkably, that its activity increases on oxidation. High-resolution crystal structures of this protease in an active strained state and in an inactive relaxed state reveal that a solvent-exposed disulfide bond controls the protease activity by constraining two distant regions of Rv3671c and stabilizing it in the catalytically active conformation. In?vitro biochemical studies confirm that activation of the protease in an oxidative environment is dependent on this reversible disulfide bond. These results suggest that the disulfide bond modulates activity of Rv3671c depending on the oxidative environment in?vivo.     

Physiological functions of hemocytes newly emerged from the cultured hematopoietic organs in the silkworm, Bombyx mori

Abstract  Cellular immunity is a very important part of insect innate immunity. It is not clear if hemocytes entering the hemolymph require a maturation process to become competent. The establishment of a tissue culture system for the insect hematopoietic organs would enable physiological function assays with hemocytes newly emerged from hematopoietic organs. To this end, we established a hematopoietic organ culture system for the purebred silkworm pnd pS and then studied the physiological functions of the newly emerged hemocytes. We found that Grace's medium supplemented with 10% heated silkworm larval plasma was better for culturing the hematopoietic organs of pnd pS . Newly emerged hemocytes phagocytosed propidium iodide-labeled bacteria and encapsulated the Iml-2 coated nickel beads as well as pupal tissue debris. This culture system is therefore capable of generating physiologically functional hemocytes. These hemocytes can be used to study the mechanisms of the hemocyte immune response among others.     

Evaluation of Pax6 mutant rat as a model for autism

Autism is a highly variable brain developmental disorder and has a strong genetic basis. Pax6 is a pivotal player in brain development and maintenance. It is expressed in embryonic and adult neural stem cells, in astrocytes in the entire central nervous system, and in neurons in the olfactory bulb, amygdala, thalamus, and cerebellum, functioning in highly context-dependent manners. We have recently reported that Pax6 heterozygous mutant (rSey(2)/+) rats with a spontaneous mutation in the Pax6 gene, show impaired prepulse inhibition (PPI). In the present study, we further examined behaviors of rSey(2)/+ rats and revealed that they exhibited abnormality in social interaction (more aggression and withdrawal) in addition to impairment in rearing activity and in fear-conditioned memory. Ultrasonic vocalization (USV) in rSey(2)+ rat pups was normal in male but abnormal in female. Moreover, treatment with clozapine successfully recovered the defects in sensorimotor gating function, but not in fear-conditioned memory. Taken together with our prior human genetic data and results in other literatures, rSey(2)/+ rats likely have some phenotypic components of autism.     

Inhibition of motility and invasiveness of renal cell carcinoma induced by short interfering RNA transfection of beta 1,4GalNAc transferase

Human renal cell carcinoma (RCC) has been characterized by remarkable changes in ganglioside composition. TOS1 cells, typical of metastatic RCC, are characterized by predominance of GM2 as monosialoganglioside, and beta 1,4GalNAc disialyl-Lc(4) (RM2 antigen) as disialoganglioside [J. Biol. Chem. 276 (2001) 16695]. In order to observe the functional role of gangliosides in RCC malignancy, TOS1 cells were transfected with short interfering RNA (siRNA) based on open reading frame sequence of beta 1,4GalNAc transferase (beta 1,4GalNAc-T), and its disordered sequence of siRNA (dsiRNA) as control. In siRNA transfectant, beta 1,4GalNAc-T mRNA level and GM2 expression were greatly reduced, whereby GM3 expression appeared. In contrast, RM2 antigen level was unchanged, even though it has the same beta 1,4GalNAc epitope at the terminus. dsiRNA transfectant showed no change of beta 1,4GalNAc-T mRNA and did not express GM3. Concomitant with reduction of GM2 and appearance of GM3, siRNA transfectant showed greatly reduced motility and invasiveness, although growth rate was unaltered. Both transfectants with siRNA and dsiRNA expressed the same level of tetraspanin CD9. Since CD9/GM3 complex is known to reduce integrin-dependent motility and invasiveness [Biochemistry 40 (2001) 6414], it is plausible that motility and invasiveness of siRNA transfectant of TOS1 cells may be reduced by enhanced formation of such complex.     

Establishment and characterization of a human ovarian granulosa tumor cell line (HSOGT)

We successfully established a novel ovarian granulosa tumor cell line (HSOGT). The tumor tissue of the ovary was derived from a 25 year-old Japanese woman under her consent. The cell line was maintained for over 14 months, subcultured more than 73 times, and had a population doubling time of 18.9 hours. Phase contrast microscopy displayed a pavement-like arrangement without contact inhibition. The chromosome number showed a wide distribution of aneuploidy and the mode was 83; many marker chromosomes were observed. The HSOGT was also successfully xenotransplanted into nude mice. The cell line produced estradiol and has preserved some characters of granulosa cells with stable growth in vitro. We firmly believe that this cell line will be a most useful tool for endocrinological investigation of human granulosa cells.     

Clinicopathologic relevance of asymptomatic endometrial carcinoma

OBJECTIVE: To assess whether screening asymptomatic women is significant for early detection of endometrial carcinoma. STUDY DESIGN: We compared the clinicopathologic findings and prognoses of 21 asymptomatic patients with 427 symptomatic patients with endometrial carcinoma. RESULTS: The incidence of asymptomatic endometrial carcinoma was 4.7%. Nineteen of 21 asymptomatic patients with endometrial carcinoma were found by cytologic screening for endometrial cancer. There was a statistical difference in the histopathology and depth of myometrial invasion between the asymptomatic and symptomatic groups. However, no statistical differences were found in tumor grade, lymph node metastasis, adnexal metastasis, cervical invasion, peritoneal cytology, surgical stage and patient age. Univariate analysis showed that the presence or absence of symptoms was not related to survival. CONCLUSION: The detection of asymptomatic endometrial carcinoma is not related to a reduced mortality rate. Screening asymptomatic women for endometrial carcinoma is not recommended.     

DNA damage response pathway in radioadaptive response

Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to the repair of DSBs and implicated to the current controversial observations on the expression of adaptive response.