Induction of tumour necrosis factor-alpha (TNF-alpha) mRNA in bladders and spleens of mice after intravesical administration of bacillus Calmette-Guérin

Intravesical bacillus Calmette-Guérin (BCG) therapy is highly effective in the therapy of carcinoma in situ of the bladder, but the mechanism of BCG immunotherapy is not clearly understood. We studied the production of TNF-alpha in spleens and bladders of mice after intravesical BCG or BCG/interferon-gamma (IFN-gamma) instillation. Significant change of TNF-alpha mRNA expression of spleens and bladders of C3H/He mice was observed after intravesical BCG instillation, although intravesical IFN-gamma therapy 3 days after BCG instillation to maintain the activated state of monocyte/macrophage lineage cells did not show a significant change of TNF-alpha mRNA, compared with that of BCG therapy alone. Maximal production of TNF-alpha mRNA in spleens of mice was seen after the first or second intravesical BCG instillation, and production of TNF-alpha mRNA in bladders was also increased after intravesical BCG instillation. The increment of TNF-alpha production by BCG stimulation in HL-60, a promyelocytic leukaemic cell line, and peripheral blood mononuclear cells in vitro may support the in vivo effect of BCG therapy on the bladder. These data show that local production of TNF-alpha as well as systemic production by intravesical BCG treatment may correlate with one of the mechanisms of BCG immunotherapy of superficial bladder cancer.ope

Effects of novel peptides derived from the acidic tail of synuclein (ATS) on the aggregation and stability of fusion proteins

The acidic tail of alpha-synuclein (ATSalpha) has been shown to protect the glutathione S-transferase (GST)-ATSalpha fusion protein from environmental stresses, such as heat, pH and metal ions. In this study, we further demonstrated that the introduction of ATSalpha into other proteins, such as dehydrofolate reductase and adiponectin, renders the fusion proteins resistant to heat-induced aggregation and that the acidic tail of beta- or gamma-synuclein can also protect the fusion proteins from heat-induced aggregation. Interestingly, the heat resistance of GST-ATSalpha deletion mutants, which contain shorter peptides derived from the highly charged regions of ATSalpha, was approximately proportional to the number of added Glu/Asp residues. However, the negative charges in the ATSalpha-derived peptides appear insufficient to explain the extreme heat resistance of the fusion proteins, since polyglutamates appeared to be much less effective than the ATSalpha-derived peptides in conferring heat resistance on the fusion proteins. These results suggest that not only the negatively charged residues but also the specific amino acid sequence of ATSalpha play an important role in conferring extreme heat resistance on the fusion proteins. Furthermore, the heat-induced secondary structural changes and thermal inactivation curves of GST-ATSalpha deletion mutants indicated that the introduction of ATSalpha-derived peptides does not significantly affect the intrinsic stability of the fusion proteinsope

The effect of cytokines and endotoxin on the nitric oxide production and its relation to mitochondrial aconitase activi

ope

Validation of gene expression changes of osteopontin and MMP-1 in primary and metastatic colorectal carcinomas

BACKGROUND: Metastasis is one of the most important characteristics of cancer in terms of its impact on patient survival. Unfortunately, identification of altered genes during tumor metastasis is limited. METHODS: Using high-throughput microarrays containing 19K spotted human oligonucleotides, gene expression of primary and matched metastatic colon cancer were compared in previous study. Although DNA microarray analysis did not demonstrate complete classification of primary and metastatic carcinoma, 80 differentially expressed genes were identified. Among these, expression of osteopontin, matrix metalloproteinase-1 (MMP-1) and serpin A1 was assessed using immunohistochemistry in a validation set containing 43 pairs from tissue microarrays. RESULTS: The expression of osteopontin was significantly higher in metastatic carcinoma than in primary carcinoma, as indicated by mRNA expression. The expression of MMP-1 was significantly lower in metastatic carcinoma. Expression of serpin A1 was not correlated with the microarray results. CONCLUSIONS: Osteopontin and MMP-1 expression successfully classified primary and metastatic colorectal carcinomas and further studies on their clinical application is encouraged

Apoptosis of Chondro cytes and Its Regulation in Pathogenesis of Osteoarthritis

목적 : 골관절염(osteoarthritis)은 관절연골의 점차적인 소실과 이에 따른 활액막과 연골하골의 이차적인 변화를 특징으로 하는 질환으로서 특히 노년층에서 높은 유병율을 보이는데 원인 및 병인에 대해서는 아직 뚜렷하게 규명된 바가 없다. 한편, 세포사멸(apoptosis)은 세포괴사(cell necrosis)와는 상대되는 개념의 세포 사망으로서 염증반응이 없이 진행되는 것이 특징이고 자가면역질환, 종양, 퇴행성 뇌질환 등의 기전연구에서 그 역할이 규명되고 있다. 최근 몇몇 연구에 의하면, 골관절염의 연골세포가 정상관절의 연골세포에 비해 높은 비율의 세포사멸을 보이며, 또한 F a s에 대한 항체를 처치한 경우에도 부분적으로 세포사멸을 일으킨다고 하였다. 본 연구는 골관절염의 연골세포에서 일어나는 세포사멸의 양상을 정상관절의 연골세포와 비교하고, 세포사멸과 관련한 수용체/리간드의 발현을 조사하여 골관절염의 병인에 있어서 세포사멸의 조절기전을 알아보았다. 방법 : 골관절염으로 인공슬관절 치환술을 시행받은 환자의 관절연골과 외상에 의해 슬상절단술을 시행받은 환자의 절단지에서 채취한 관절연골을 재료로, TUNEL assay를 통해 세포사멸의 정도와 위치를 비교하였고 관절연골 조직에서 R N A를 분리하여 역전사중합효소연쇄반응을통해 세포사멸의 조절에 참여를 조사하였으며 면역조직화학염색을 통하여 FasL 단백의 발현을 알아보았다. 결과 : 형광 d U T P를 이용한 TUNEL assay에서 골관절염이 없는 정상 대조군 관절연골 4례 모두에서는 세포사멸을 보이는 세포를 찾아볼 수 없었으나 골관절염의 관절연골 7례의 표본 중 6례에서 전형적인 세포사멸 세포들을 관찰할 수 있었다. 역전사중합효소연쇄반응에서는 caspase-3, caspase-8, Fas 및FasL의 mRNA 발현정도가 골관절염의 연골에서 정상 대조군 연골에 비해 증가하였으며, 특히 FasL는 일부 골관절염에서 현저히 증가된 양상을 보였다. FasL의 유도체로 알려진 IL-18은 FasL가 증가된 환자에서 함께 강한 발현을 보였다. 면역조직화학염색에서는 정상 대조군 연골에서보다 골관절염의 연골에서 FasL 단백이 현저히 강하게 발현됨을 관찰하였다. 결론 : 본 연구는 골관절염 환자의 관절연골에서 의미있게 증가한 세포사멸과 F a s L의 발현을 관찰하였으며, 이는 Fas/FasL 경로를 통한 세포사멸이 골관절염에서 일어나는 관절연골의 퇴화에 기여할 것을시사한다. 또한 F a s L의 유도체 중 하나인 I L - 1 8의 발현도 관찰되었다. 이상의 결과는 차후 골관절염 치료에 있어서 새로운 접근을 제시하는 데 일조할 것으로 기대된다

Expression patterns of alpha-synuclein in human hematopoietic cells and in Drosophila at different developmental stages.

Alpha-synuclein, a presynaptic protein of the central nervous system, has been implicated in the synaptic events such as neuronal plasticity during development and learning, and neuronal degeneration under pathological conditions. As an effort to understand the biological function of alpha-synuclein, we examined the expression patterns of alpha-synuclein in various human hematopoietic cells, and in Drosophila at different developmental stages. The alpha-synuclein was ubiquitously expressed in all the tested hematopoietic cells including T cells, B cells, NK cells, and monocytes, as well as in the lymphoma cell lines, Jurkat and K562. A potential alpha-synuclein homologue was also expressed in Drosophila, and its expression appeared to be temporally and spatially regulated during development. Our data suggest that alpha-synuclein may function in invertebrates as well as in vertebrates and its function may not be restricted to the neuron.ope

Delayed-Onset Anaphylaxis Caused by IgE Response to Influenza Vaccination

Influenza vaccine-associated anaphylaxis is a very rare allergic reaction to vaccines, but the most concerning and life-threatening adverse reaction. Although the safety of influenza vaccines has been well documented, occasional cases of anaphylaxis in vaccinated patients have been reported. In this study, we analyzed the immunoglobulin E (IgE) response to whole influenza vaccines in a pediatric case of delayed-onset anaphylaxis after influenza vaccination. The patient showed elevated specific IgE levels against whole influenza vaccines, especially with split virion from egg-based manufacturing process. Specific IgE levels to influenza vaccines showed decreased over. We evaluated a causal relationship between influenza vaccine and anaphylaxis event by enzyme-linked immunosorbent assay. Delayed-onset anaphylaxis after influenza vaccination can occur in children without predisposing allergic diseases. In addition, the results suggested that formulation and production system of influenza vaccines could affect the probability of severe allergic reaction to vaccines.ope

Troglitazone Enhances the Apoptotic Response of DLD-1 Colon Cancer Cells to Photodynamic Therapy

PURPOSE: The aim of this study was to investigate whether the peroxisomal proliferator-activated receptor gamma (PPARγ) ligand troglitazone in combination with photodynamic therapy (PDT) enhances the apoptotic response of DLD-1 colon cancer cells. MATERIALS AND METHODS: The effects of troglitazone, PDT, and troglitazone in combination with PDT on cell viability and apoptosis were assessed in DLD-1 cells. Cell viability and proliferation were evaluated using the tetrazolium-based MTT assay, and apoptosis was evaluated via cell staining with propidium iodide (PI) and annexin V-FITC. The levels of pro-caspase-3 were measured via Western blot analyses. RESULTS: Treatment of troglitazone and PDT induced the growth retardation and cell death of DLD-1 cells in a dose-dependent manner, respectively. The combination treatment significantly suppressed cell growth and increased the apoptotic response of DLD-1 and resulted in apoptosis rather than necrosis, as shown by PI/annexin V staining and degradation of procaspase-3. CONCLUSION: These results document the anti-proliferative and apoptotic activities of PDT in combination with the PPARγ ligand troglitazone and provide a strong rationale for testing the therapeutic potential of combination treatment in colon cancer.ope

Quantification of tumor suppressor mRNA expression by poly-competitive RT-PCR using a TS-IS that contained multiple internal competitors.

Despite the recent introduction of real-time PCR methods and cDNA microarrays, competitive PCR techniques continue to play an important role in nucleic acid quantification because of the significantly lower cost of equipment and consumables. In this study, we developed a construct, termed tumor suppressor-internal standard (TS-IS) that produced polycompetitive RNA templates as an internal standard to quantify cellular RNA concentration of tumor suppressor genes. This construct is composed of not only sets of primers for detecting the expression of several tumor suppressor genes (such as pRB, p16(INK4A) 15(INK4B), p14(ARF) p53, and p21(WAF1)), but also HPRT as an endogenous marker. Using an internal standard RNA that was synthesized from the TS-IS construct, we were able to establish optimized conditions for the quantification of tumor suppressor genes with minimal amounts (50 ng) of cellular RNA. In addition, the usefulness of this method was confirmed by analyzing the expression levels of tumor suppressor genes in fourteen hepatoma cell lines as a model. The TS-IS assay that we used was inexpensive and a widely applicable method that permitted the reliable and accurate quantification of tumor suppressor genes

Distinct roles of the N-terminal-binding domain and the C-terminal-solubilizing domain of alpha-synuclein, a molecular chaperone

α-Synuclein, an acidic neuronal protein of 140 amino acids, is extremely heat-resistant and is natively unfolded. Recent studies have demonstrated that α-synuclein has chaperone activity both in vitro and in vivo, and that this activity is lost upon removing its C-terminal acidic tail. However, the detailed mechanism of the chaperone action of α-synuclein remains unknown. In this study, we investigated the molecular mechanism of the chaperone action of α-synuclein by analyzing the roles of its N-terminal and C-terminal domains. The N-terminal domain (residues 1–95) was found to bind to substrate proteins to form high molecular weight complexes, whereas the C-terminal acidic tail (residues 96–140) appears to be primarily involved in solubilizing the high molecular weight complexes. Because the substrate-binding domain and the solubilizing domain for chaperone function are well separated in α-synuclein, the N-terminal-binding domain can be substituted by other proteins or peptides. Interestingly, the resultant engineered chaperone proteins appeared to display differential efficiency and specificity in terms of the chaperone function, which depended upon the nature of the binding domain. This finding implies that the C-terminal acidic tail of α-synuclein can be fused with other proteins or peptides to engineer synthetic chaperones for specific purposes.ope