Kinetic analysis of protein production after DNA transfection

The production of an exogenous protein by the transfection of a plasmid DNA encoding the protein was kinetically analyzed, to determine the efficiency of the transfection. Cultured NIH3T3 or HeLa cells, and the luciferase protein were used as a model system in this experiment. The findings indicate that at least a 8x10(4)- and 4x10(3)-fold molar amounts of luciferase protein was produced from one copy of the plasmid DNA molecule in NIH3T3 and HeLa cells, respectively. The rate of elimination of luciferase activity upon DNA transfection was smaller than that for the luciferase protein itself (k(el) for DNA transfection相似文献   

Development of efficient packaging method of oligodeoxynucleotides by a condensed nano particle in lipid envelope structure

An efficient delivery system is required if antisense oligodeoxynucleotides (ODN) are to be utilized for gene therapy. We report herein on the development of a novel ODN delivery system, ODN-encapsulated nano particles (ODN-ENP) using an efficient and simple packaging method. The ODN-ENP consists of a condensed ODN particle and a lipid envelope, which can be equipped with various functional devices for the efficient delivery of ODN with a small diameter (150 nm). The encapsulation efficiency and ODN recovery of ODN-ENP were significantly higher than those of other packaging methods, such as a stabilized antisense-lipid particles method or a freeze-thaw method. Furthermore, the time required for the preparation of the ODN-ENP was shorter than the other methods. The method developed in this study is a simple and efficient packaging method for ODN with a condensed nano particle in lipid-envelope structure.     

Deficiency of hMLH1 and hMSH2 expression is a poor prognostic factor in esophageal squamous cell carcinoma

BACKGROUND: The human Mut-L-Homologon-1 (MLH1) and Mut-S-Homologon-2 (MSH2) are post replication mismatch repair (MMR) genes. METHODS: We examined the correlation of the clinical features of 122 patients with esophageal squamous cell carcinoma (ESCC) with the expression of MLH1 and MSH2 by immunohistochemical analysis. RESULTS: According to our criteria, 34 and 25 cases did not express MLH1 and MSH2, respectively. Expression of both the MLH1 and MSH2 gene products was observed in 73 (59.8%) cases; loss of MLH1 or MSH2 expression was detected in 35(28.7%) cases. Fourteen (11.5%) cases demonstrated loss of both MLH1 and MSH2 expression in ESCC. Loss of MLH1 and/or MSH2 gene expression significantly correlated with increases in malignancy, as evidenced by increases in the existence of metastatic lymph nodes (P = 0.0056), extensive invasion (P = 0.0007), and poor differentiation (P = 0.0992). The MLH1-negative patients had a significantly poorer prognosis than those in the MLH1-positive group (P = 0.0043). Similar results were observed for MSH2 expression (P = 0.0002). Patients both MLH1 and MSH2 negative exhibited the most poor clinical outcome than other patients (P < 0.0001). CONCLUSION: We conclude that MMR protein expression, detected by immunohistochemistry, is a useful marker providing information necessary to decide appropriate therapeutic strategies in patients with ESCC.     

Non-close-packed arrangement of soft elastomer microspheres on solid substrates

Unlike rigid microparticles, soft and deformable elastomer (rubber) microspheres were found to exhibit a non-close-packed arrangement on solid substrates after the evaporation of water from their dispersions. The microscopic observation revealed that individual microspheres are ordered in regular intervals at the air/water interface of a sessile droplet and remain fixed on the substrate without being affected by the capillary forces during evaporation due to their deformability. Moreover, using the Langmuir–Blodgett method, thin films of non-close-packed structures could be successfully generated over large areas. Our findings may potentially help to control the arranged structures of elastomer microspheres, which can be expected to improve the nano-science and technology for the precise control for e.g. surface patterning.

Unlike rigid microparticles, deformable elastomer microspheres were found to exhibit a non-close-packed arrangement on solid substrates after evaporating water from their dispersions. The underlying mechanism of their unique ordering is discussed.     

Cancer‐associated fibroblasts educate normal fibroblasts to facilitate cancer cell spreading and T‐cell suppression

In some tumors, a small number of cancer cells are scattered in a large fibrotic stroma. Here, we demonstrate a novel mechanism for expansion of pro‐tumor fibroblasts via cancer‐associated fibroblast (CAF)‐mediated education of normal fibroblasts (NFs). When NFs were incubated with conditioned medium from CAFs, the resulting CAF‐educated fibroblasts (CEFs) generated reactive oxygen species, which induced NF‐κB‐mediated expression of inflammatory cytokines and the extracellular matrix protein asporin (ASPN), while expression of a common CAF marker gene, α‐SMA, was not increased. ASPN further increased CEF expression of downstream molecules, including indoleamine 2,3‐dioxygenase 1 (IDO‐1), kynureninase (KYNU), and pregnancy‐associated plasma protein‐A (PAPP‐A). These CEFs induce cytocidal effects against CD8⁺ T cells and IGF‐I activation in cancer cells. CEFs were generated without cancer cells by the direct mixture of NFs and CAFs in mouse xenografts, and once CEFs were generated, they sequentially educated NFs, leading to continuous generation of CEFs. In diffuse‐type gastric cancers, ASPN^high/IDO‐1^high/KYNU^high/α‐SMA⁻ CEFs were located at the distal invading front. These CEFs expanded in the fibrotic stroma and caused dissemination of cancer cells. ASPN may therefore be a key molecule in facilitating tumor spreading and T‐cell suppression.     

Clinicopathological significance of CD28 overexpression in adult T‐cell leukemia/lymphoma

CD28, one of the costimulatory molecules, has a pivotal role in T‐cell activation, and its expression is strictly regulated in normal T cells. Gain‐of‐function genetic alterations involving CD28 have been frequently observed in adult T‐cell leukemia/lymphoma (ATLL). These abnormalities, such as CD28 fusions and copy number variations, may not only confer continuous, prolonged, and enhanced CD28 signaling to downstream pathways but also induce overexpression of the CD28 protein. In this study, 120 ATLL cases were examined by immunohistochemistry for CD28 and its ligands CD80 and CD86, and their expression on tumor cells was semiquantitatively evaluated. CD28 was overexpressed in 55 (46%) cases, and CD80 or CD86 (CD80/CD86) was infrequently overexpressed in 12 (11%). Compared with non‐overexpressers, CD28 overexpressers showed a higher frequency of CD28 genetic alterations and had an increased number of CD80/CD86‐positive non‐neoplastic cells infiltrating tumor microenvironment. In the entire ATLL patient cohort, CD28 overexpressers showed a significantly poorer overall survival (OS) compared with non‐overexpressers (P = .001). The same was true for a subgroup who were treated with multidrug regimens with or without mogamulizumab. CD28 overexpression had no prognostic impact in the group who received allogeneic hematopoietic stem cell transplantation. In the multivariate analysis for OS, CD28 overexpression was selected as an independent risk factor. These results suggest ATLL patients with CD28 overexpression have more aggressive clinical course and are more refractory to treatment with multidrug chemotherapy. CD28 overexpression appears to be a novel unfavorable prognostic marker in ATLL patients, and further prospective studies are warranted to establish its prognostic significance.     

The inhibitory effect of TU-100 on hepatic stellate cell activation in the tumor microenvironment

Introduction: The tumor microenvironment is involved in acquiring tumor malignancies of colorectal liver metastasis (CRLM). We have reported that TU-100 (Daikenchuto) suppresses hepatic stellate cell (HSC) activation in obstructive jaundice. In this study, we report new findings as the direct and indirect inhibitory effects of TU-100 on cancer cell growth through the suppression of HSC activation.Materials and Methods: The HSCs (LX2) were cultured in colon cancer cells (HCT116 and HT29)-conditioned medium (CM) with or without TU-100 treatment (90, 270, 900 μg/ml). Activated HSCs (aHSCs) were detected by α-SMA and IL-6 mRNA expressions and cytokine arrays of HSC’s culture supernatants. Cancer cell growth was analyzed for proliferation and migration ability, compared with TU-100 treatment. To investigate the direct anti-tumor effect of TU-100, cancer cells were cultured in the presence of aHSC-CM and TU-100 (90, 270, 900) or aHSC-CM alone, and assessed autophagosomes, conversion to LC3-II protein, and Beclin-1 mRNA expression.Results: Colon cancer-CM significantly increased α-SMA and IL-6 mRNA expressions of aHSC. α-SMA and IL-6 mRNA expressions of aHSC, and IL-6 secretions from aHSCs were significantly decreased with TU-100 (270, 900) treatment, compared to colon cancer-CM alone. Compared with normal culture medium, aHSC-CM led to a significantly increased cell number and modified HSC-CM (TU-100; 270, 900) significantly suppressed cancer cell growth and migration. TU-100 (900) treatment induced autophagy and significantly promoted the autophagic cell death.Conclusions: TU-100 inhibited colon cancer cell malignant potential by both suppressing HSC activation and inducing directly autophagy of cancer cells.     

Correlations between alder specific IgE and alder-related tree pollen specific IgE by RAST method.

BACKGROUND: Wild birch trees grow in limited areas in Japan and are not a common aero-allergen. However, many patients who do not live in the area show positive birch pollen Radioallergosorbent Test (RAST). Therefore, being sensitized by another tree pollen which is closely related to birch may result in showing a specific IgE antibody to birch. Alder is a one of these trees and in the past it grew widely in Japan. However, there is no available RAST data as to the correlations between alder and alder-related trees. METHODS: We measured the alder specific IgE (CAP-RAST, Phadea) in stored sera which was positive in birch RAST (228 samples), beech RAST (36 samples), oak RAST (152 samples) and cedar RAST (411 samples) and examined correlations between the RAST of alder and other trees. RESULTS: The correlation coefficient value of birch was very high (0.971). The other coefficient values of beech and oak were high (0.884 in beech and 0.895 in oak) but were slightly lower than that of the birch. This means that in terms of allergenicities, birch pollen is almost the same as alder and beech and oak are partly different from the alder. CONCLUSIONS: The Japanese respond to alder pollen just same as they do to birch pollen in forming specific IgE antibody. In clinical practice, positive alder RAST has the same meaning as positive birch RAST.