Heterogenous induction of carcinoma-associated fibroblast-like differentiation in normal human prostatic fibroblasts by co-culturing with prostate cancer cells

In the tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are considered to play a critical role in the promotion of tumorigenesis. However, the mechanisms that generate CAFs are not well elucidated. To understand how CAFs are generated during primary cancer progression, we investigated the biochemical characteristics of normal human prostate stromal cells (PrSC) co-cultured with human prostate cancer (PCa) cells in vitro. In primary cultures of human PCa-derived stromal cells (PCaSC-8 and PCaSC-9), expression of TNC, ACTA2, EGF, FGF7, and IGF1 mRNA was generally higher than PrSC but gene expression patterns were not uniform between PCaSC-8 and PCaSC-9 cells. Transforming growth factor β (TGFβ) and vascular endothelial growth factor (VEGF) protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than PrSC but levels of both secreted proteins were not same. When PrSCs were co-cultured with androgen-sensitive LNCaP cells or its sublines, androgen-low-sensitive E9 cells and androgen-insensitive AIDL cells, mRNA expression of IGF1 was significantly increased in all combinations. In contrast, expression of COL1A1, TNC, and ACTA2 mRNA was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Protein production of VEGF was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Increase of TGFβ protein was observed only in E9 + PrSC co-cultures. These biochemical characteristics of PrSC were partially recapitulated in TGFβ-treated PrSC. We have demonstrated that normal fibroblasts co-cultured with cancer cells become activated and exhibit biochemical characteristics of CAFs in a heterogenous manner. Our results suggest that heterogenous induction of CAF-like differentiation might be strongly dependent on biochemical characteristics of adjacent cancer cells.     

Paramagnetic NMR study of Cu(2+)-IDA complex localization on a protein surface and its application to elucidate long distance information

The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information.     

Early Archean planktonic mode of life: Implications from fluid dynamics of lenticular microfossils

Lenticular, and commonly flanged, microfossils in 3.0–3.4 Ga sedimentary deposits in Western Australia and South Africa are unusually large (20–80 μm across), robust, and widespread in space and time. To gain insight into the ecology of these organisms, we performed simulations of fluid dynamics of virtual cells mimicking lenticular forms of variable sizes, oblateness, flange presence, and flange thickness. Results demonstrate that (a) the flange reduces sedimentation velocity, (b) this flange function works more effectively in larger cells, and (c) modest oblateness lowers sedimentation rate. These observations support interpretations that the lenticular microbes were planktonic—a lifestyle that could have been advantageous in an early Earth harsh environment including violent volcanic activities, repeated asteroid impacts, and relatively high UV‐radiation. Although the robustness of these organisms could have provided additional protection on the early Earth, this architecture may have impeded a planktonic lifestyle by increasing cell density. However, our data suggest that this disadvantage could have been compensated by enlargement of cell volume, which could have enhanced the ability of the flange to slow sedimentation rate, especially if coupled with vacuolation. The results of this simulation study may help to explain the unique morphology and unusually large size of these Archean microfossils.     

Effectiveness of Trivalent Inactivated Influenza Vaccine in Children Estimated by a Test-Negative Case-Control Design Study Based on Influenza Rapid Diagnostic Test Results

We assessed vaccine effectiveness (VE) against medically attended, laboratory-confirmed influenza in children 6 months to 15 years of age in 22 hospitals in Japan during the 2013–14 season. Our study was conducted according to a test-negative case-control design based on influenza rapid diagnostic test (IRDT) results. Outpatients who came to our clinics with a fever of 38°C or over and had undergone an IRDT were enrolled in this study. Patients with positive IRDT results were recorded as cases, and patients with negative results were recorded as controls. Between November 2013 and March 2014, a total of 4727 pediatric patients (6 months to 15 years of age) were enrolled: 876 were positive for influenza A, 66 for A(H1N1)pdm09 and in the other 810 the subtype was unknown; 1405 were positive for influenza B; and 2445 were negative for influenza. Overall VE was 46% (95% confidence interval [CI], 39–52). Adjusted VE against influenza A, influenza A(H1N1)pdm09, and influenza B was 63% (95% CI, 56–69), 77% (95% CI, 59–87), and 26% (95% CI, 14–36), respectively. Influenza vaccine was not effective against either influenza A or influenza B in infants 6 to 11 months of age. Two doses of influenza vaccine provided better protection against influenza A infection than a single dose did. VE against hospitalization influenza A infection was 76%. Influenza vaccine was effective against influenza A, especially against influenza A(H1N1)pdm09, but was much less effective against influenza B.     

Microarray analysis of tonsils in immunoglobulin A nephropathy patients

Background

Recently, combination of tonsillectomy and steroid pulse therapy was reported to be effective as the treatment of the immunoglobulin A nephropathy (IgAN). However, the gene expression difference between the tonsils in patients with IgAN and those in control patients is not established.

Methods

We performed tonsillectomy combined with steroid pulse as a treatment to IgAN, analyzed the gene expression in the tonsils (N = 23) using microarray, compared with those with patients suffering from chronic tonsillitis (N = 22). From some candidate genes related with IgAN, we confirmed the apolipoprotein B messenger RNA-editing enzyme catalytic polypeptides 2 (APOBEC2) gene expression in the tonsil and we also analyzed its expression levels and clinical features.

Results

Up-regulated genes seem to be categorized into two groups. One group belongs to the muscle related genes which might be caused by structural differences. The other group includes the immune system-related genes, such as APOBEC2, CALB2, DUSP27, and CXCL11. APOBEC2 was positively stained in the epithelium and the peripheral region of the germinal center in both tonsils. APOBEC2 expression level was negatively related with serum igg level, but did not correlate with clinical course after tonsillectomy.

Conclusion

We confirmed gene expression differences related with immune system and muscle structure. The APOBEC2 was confirmed to be elevated in the tonsils with IgAN patients, and the gene expression level was negatively related with serum igg level in overall patients. These results might be helpful to reveal the mechanism of IgAN.     

Growth-active peptides are produced from alpha-lactalbumin and lysozyme

We determined the growth-active domains of milk-growth factor (MGF), human alpha-lactalbumin (HMLA) and human lysozyme (HMLZ), and their sequences. Fetal calf serum (FCS) showed inhibitors against proteases. The growth-stimulation of IMR90 cells in CG medium (free-serum) without FCS was induced in a dose-dependent manner up to 400 ng/ml of HMLA, HMLZ or chicken lysozyme (ChLZ), and also in a time-dependent manner until 48 h but was induced gradually until 1000 ng/ml of bovine alpha-lactalbumin (BVLA). The HMLAL6-peptide (HMLAL6), a cleaved product from HMLA by Endpeptidase Lys C, was growth-stimulative. The sequence of HMLAL6 was matched to 35 amino-acid residues (from No. 59 to No. 93 of HMLA), owing to the sequences of HMLAL6R3, HMLAL6R5 and HMLAL6R7 after the reduction of HMLAL6. The sequences of the reduced peptides from MGFL7-peptide (MGFL7: a cleaved product from MGF by Endpeptidase lysine C matched to those of the peptides from HMLAL6, and were similarly identified as the partial sequence of HMLA (59-93, H(2)N-L.W.C.?.K./S.S.Q.V.P.Q.S.R.N.I.?.D.I.S.?.D.K./F.L. D.D.D.I.T.D.D.I.M.?.A.-COOH). The sequence of HMLZ is similar to that of HMLA. HMLZT7-peptide (HMLZT7), a cleaved product of HMLZ by trypsin, was confirmed to have growth-stimulating activity and it's sequence was partially identified as Y. W.?.N.D.G.K.T.P.G.A.V.N.A.?.H.L. -, owing to the results of HMLZT7R1 (reduction of HMLZT7) and HMLZA7R2 (reduction of HMLZA7-peptide (HMLZA7) cleaved product of HMLZ by Endpeptidase Arg C) and is accordingly the sequence from No. 63 to No. 97 of HMLZ. Therefore, the peptides produced from LA and LZ by proteolysis may play a role of growth-stimulation.     

Inhibition of p38 mitogen-activated protein kinase-induced apoptosis in cultured mature oligodendrocytes using SB202190 and SB203580

p38 Mitogen-activated protein kinase (p38 MAPK) is expressed in the oligodendrocyte lineage, and its activity has been implicated in the proliferation and transition of early progenitors into late progenitors. Although p38 MAPK expression has been found in the myelin sheath, however, its role in mature oligodendrocytes remains unknown. In the present study, in order to address the role of p38 MAPK in mature oligodendrocytes, selective inhibitors of p38 MAPK, SB202190, and SB203580 were added to primary cultures of mature oligodendrocytes. After 24h of exposure to the inhibitors, the appearance, and number of A2B5-positive progenitors were unchanged. However, the 2',3'-cyclic nucleotide-3'-phosphohydrolase-positive mature oligodendrocytes disappeared, and the numbers of living cells decreased in comparison to the control cells treated with SB202474, a negative analog of SB203580. Increases in the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive nuclei and in the activity of caspase-3/7 were detected 16 h after exposure to the inhibitors, thus causing the mature oligodendrocytes to die due to apoptosis. Similar results were obtained using a differentiated rat oligodendrocyte precursor cell (OPC) line, central glia-4 (CG-4). These findings indicate that p38 MAPK is vital for mature oligodendrocyte survival.     

Convergent synthesis and in vivo inhibitory effect on fat accumulation of (-)-ternatin, a highly N-methylated cyclic peptide

(-)-Ternatin (1), a highly N-methylated cyclic heptapeptide, is a potent inhibitor of fat accumulation against 3T3-L1 murine adipocytes (EC50 = 0.14 microg/mL) [Shimokawa, K.; Mashima, I.; Asai, A.; Yamada, K.; Kita, M.; Uemura, D. Tetrahedron Lett. 2006, 47, 4445]. Compound 1 was synthesized from Boc-protected amino acids in solution. Upon treatment with 1 at 5 mg/kg/day, increases in body weight and fat accumulation in high-fat-fed mice were both significantly suppressed.