Immunohistochemical analysis of 14-3-3 sigma and related proteins in hyperplastic and neoplastic breast lesions, with particular reference to early carcinogenesis

In order to confirm the role of 14-3-3 sigma (sigma) as a tumor suppressor in breast carcinogenesis, we have studied the expression of 14-3-3sigma immunohistochemically in usual ductal hyperplasia (UDH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) breast lesions. Immunostaining for estrogen receptor alpha (ERalpha), p53 and estrogen-responsive RING finger protein (Efp) was also carried out. Immunohistochemically, expression of 14-3-3sigma was seen in 92% UDH lesions and gradually decreased from 65% in DCIS to 23% in IDC. The expression of ERalpha decreased gradually from UDH to DCIS to IDC, while p53 showed an inverse staining pattern to that of ERalpha. The expression of Efp showed no significant difference among the three breast lesions. Hence, the present immunohistochemical study confirmed 14-3-3sigma as a tumor suppressor in breast carcinogenesis. A similar immunohistochemical analysis was then carried out on columnar cell hyperplasia with atypia (CCHA), in which the expression pattern of tumor suppressor 14-3-3sigma, ERalpha and p53 suggested that it might be possible that CCHA is a precancerous lesion.     

A new gene encoding tRNAPro (GGG) is present in the chloroplast genome of black pine: a compilation of 32 tRNA genes from black pine chloroplasts

The chloroplast genome of black pine (Pinus thumbergii), a gymnosperm, contains 32 different tRNA genes, 30 of which correspond to those previously identified in tobacco and rice chloroplast genomes. Two additional genes encode tRNA^Pro (GGG) and tRNA^Arg (CCG); the former is newly identified while the latter is present in liverwort, Physcomitrella patens and Angiopteris lygodiifolia, chloroplast genomes. Moreover, a partial copy of the split tRNA^Gly (UCC) gene and full copies of tRNA^His (GUG), tRNA^Thr (GGU) and tRNA^Ser (GCU) genes are present in the large single-copy region of the genome, suggesting extensive rearrangements of the chloroplast genome during evolutio. No tRNA genes whose tRNA products can recognize codons CUU/C (Leu) and GCU/C (Ala) have been found. We propose that the 32 tRNAs are sufficient to read all the 61 sense codons in the black pine system using the two-out-of-three and the U:N wobble mechanisms.     

Tumor necrosis factor alpha-induced interleukin-8 production via NF-kappaB and phosphatidylinositol 3-kinase/Akt pathways inhibits cell apoptosis in human hepatocytes

Tumor necrosis factor alpha (TNF-alpha) not only induces apoptotic signals but also causes antiapoptotic and regenerative responses in the liver. However, the molecular mechanism(s) of the latter events remains unclear. In the present study, we examined TNF-alpha-induced genes in Hc human normal (unsensitized) hepatocytes by cDNA microarray analysis. Interleukin-8 (IL-8) induction was the most pronounced of the upregulated genes. The IL-8 protein level was also increased. IL-8 belongs to the ELR-CXC chemokine family and appears to exert mitogenic and antiapoptotic functions in other cell systems. IL-8 expression by TNF-alpha was inhibited when two survival signals, nuclear factor kappaB (NF-kappaB) and phosphatidylinositol 3-kinase (PI3K)/Akt, were inhibited by a mutant form of inhibitor of NF-kappaB (IkappaB); by dominant negative (kinase-dead) Akt; or by treatment with LY 294002, an inhibitor of PI3K. TNF-alpha induced apoptosis in Hc cells that were sensitized by inhibition of NF-kappaB and PI3K activation. IL-8 administration protected mice against concanavalin A-induced hepatitis in vivo. IL-8 also rescued the sensitized Hc cells, at least in part, from TNF-alpha-induced apoptosis in vitro. TNF-alpha inhibited DNA synthesis in unsensitized Hc cells in the absence of serum. Exogenous IL-8 reversed, though anti-IL-8 neutralization antibody enhanced, growth inhibition by TNF-alpha. These results indicate that IL-8, the production of which is stimulated by TNF-alpha, inhibits apoptosis of sensitized hepatocytes and releases normal (unsensitized) hepatocytes from growth inhibition induced by TNF-alpha.     

Novel mutations in TNFRSF1A in patients with typical tumor necrosis factor receptor-associated periodic syndrome and with systemic lupus erythematosus in Japanese

Molecular defects of TNFRSF1A was investigated in members of a family presenting with typical phenotypes of tumor necrosis factor receptor-associated periodic syndrome (TRAPS) and in patients with the autoimmune disorders, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Genomic DNA from the members of a family with typical TRAPS, as well as from 100 patients with SLE, 100 patients with RA and 100 healthy individuals, was studied for mutations in exons 2, 3 and 4 of the TNFRSF1A gene. All individuals were Japanese. Three novel missense mutations were identified in the TNFRSF1A. The C70G mutation was identified in family members with typical TRAPS, which was the second case in eastern Asian population. In addition, the T61I and R104Q mutations were each identified in 2 of the 100 SLE patients. The T61I mutation was identified in one of the 100 healthy individuals. No mutations were identified in the 100 RA patients. Functional analysis revealed that PMA-induced shedding of TNFRSF1A from PBMCs was impaired in a patient carrying T61I. A larger scale of study will clarify whether these two mutations, T61I and R104Q, are associated with chronic inflammatory disorders, such as SLE, or not.     

Evaluation of the test method “activated sludge, respiration inhibition test” proposed by the OECD

The test method of "activated sludge, respiration inhibition test" proposed by the OECD was critically carried out and compared with other test methods. Investigation of test conditions showed that the moderate deviation from the test conditions defined by the OECD Test Guidelines did not have much effect on the result, and some modifications were proposed to improve the method. This method had a poor detection limit compared with the LC50 test with Oryzias latipes and EC50 of the growth inhibition test with Tetrahymena pyriformis. The susceptivity of the method was particularly poor for the chemicals which were highly toxic in the other two tests.     

Prognostic significance of the Ki67 index and programmed death-ligand 1 expression after radical cystectomy in patients with muscle-invasive bladder cancer

ObjectivesTo investigate the association between Ki67 index and programmed death-ligand 1 (PD-L1) expression in muscle-invasive bladder cancer (MIBC) patients after RC.Materials and MethodsWe retrospectively evaluated 262 MIBC patients treated with RC between April 2004 and April 2020. The impact of Ki67 index and PD-L1 expression on prognosis was evaluated by univariate Cox regression analysis. In addition, a pathomolecular risk score, including Ki67 and PD-L1, was developed to predict prognosis and pathological factors. We also evaluated the link between the Ki67 index and PD-L1 under the IL-6 stimulation in the bladder cancer cell lines of T24 and 5637 cells.ResultsThe median age and follow-up period was 69 years and 52 months, respectively. Ki67 index and PD-L1 expression were significantly associated with tumor recurrence. Univariate Cox regression analysis showed that pT3–4, mixed histology, lymphovascular invasion positive (LVI+), pN+, Ki67-high (>17%), and PD-L1+ were significantly associated with recurrence-free survival (RFS). The pathomolecular risk score was developed using resection margin+ (1 point), mixed histology (1 point), LVI+ (1 point), pN+ (1 point), and Ki67-high (1 point). RFS and overall survival were significantly shorter in patients with higher pathomolecular risk scores (>1) than in those with lower risk scores (≤1). Cell proliferation was significantly increased in the T24 and 5637 cells under the IL-6 stimulation, while PD-L1 expression was not.ConclusionsA significant effect of Ki67-high and PD-L1 expression on poor prognosis was observed in patients with MIBC. Further studies are necessary to elucidate the precise mechanisms of cell proliferation and PD-L1 expression in patients with MIBC.