Search

Introduction: In patients with limited disease small cell lung cancer (LD-SCLC) treated with concurrent chemoradiotherapy (CCRT), long-term survival data have not been fully evaluated. Moreover, the association between long-term prognosis and prognostic factors has not been sufficiently investigated. Methods: In this retrospective study, we evaluated the efficacy of CCRT in 120 patients with LD-SCLC with a plan for curative CRT using concurrent accelerated hyperfractionated radiotherapy. Results: The patients had a median age of 65.5 years, predominantly male (73%), and had clinical stage III disease (80%). The median follow-up time for overall survival (OS) was 72.2 months, median OS was 42.5 months, and the 3-year and 5-year survival rates were 52.4% and 41.8%, respectively. The median progression-free survival (PFS) was 12.5 months, and the 3-year and 5-year PFS rates were 37.6% and 33.6%, respectively. The 5-year OS rates of patients who achieved PFS at each time point were 70.9%, 83.6%, and 91.9% at 12, 24, and 36 months, respectively. The gradual increase in the 5-year OS rate following PFS extension and initial depression of the Kaplan–Meier curve showed disease progression frequently occurred in the first 2 years after initiation of CCRT. The Cox proportional hazards model showed no significant factors correlated with long-term survival through univariate and multivariate analyses. Although the prognostic factors associated with long-term prognosis in LD-SCLC were not identified, the 5-year survival rate was 41.8%, and among patients without disease progression at 2 years, the 5-year survival rate was 83.6%. Conclusion: These data suggested that the prognosis of patients with LD-SCLC was improving.

Background Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) are currently the primary treatment option for patients with EGFR‐mutant non‐small cell lung cancer (NSCLC). However, the effect of EGFR‐TKIs are eventually weakened due to resistance, and there is also a differential efficacy based on EGFR mutation subtypes. The combination of angiogenesis inhibitor (AI) with EGFR‐TKI has shown better efficacy than EGFR‐TKI monotherapy, regardless of the mutation subtypes. Nevertheless, the effect of AI eligibility on overall survival (OS) and progression‐free survival (PFS) remains to be elucidated. Thus, we assessed this impact on patients with NSCLC harboring EGFR mutation. Methods In this study, the data for 450 patients with EGFR‐mutant NSCLC, who were treated with EGFR‐TKI monotherapy, were retrospectively analyzed for AI eligibility. The patients were categorized into AI‐eligible (AI fit) and ineligible groups (AI unfit). Results The median PFS of the AI fit group was 12.9 months, compared to 9.6 months in the unfit group (p = 0.007), and OS was also significantly longer in the AI fit group (median OS = 33.0 months) compared to that in the unfit group (18.5 months, p, We assessed the impact of angiogenesis inhibitor (AI) eligibility on epidermal growth factor receptor mutant non‐small cell lung cancer. AI eligibility was associated with longer progression‐free survival and overall survival. Selection bias may affect previous data on the efficacy of AI combination therapy.

Biofunctional peptide FNIII14, which is derived from the 14th fibronectin (FN) type III-like (FN-III) repeat of FN molecule, is capable of inhibiting cell adhesion to the extracellular matrix (ECM). This functional site is usually buried within the molecular structure of FN, but can be exposed by conformational changes and proteolytic cleavage. Peptide FNIII14 can induce a conformational change in β1-integrin from the active to the inactive form, causing functional inactivation. Based on this anti-adhesive activity, peptide FNIII14 exhibits therapeutic potential for several diseases such as metabolic diseases, organ fibrosis, and malignant tumors. Peptide FNIII14 blocks integrin-mediated signaling by a mechanism entirely distinct from that of conventional antagonisitic peptides, including Arg-Gly-Asp peptides that competitively inhibit the ECM binding of integrin.

The expression of programmed cell death-ligand 1 (PD-L1) is a biomarker for administering immune check point inhibitors in patients with advanced stage non-small cell lung cancer. Although the consolidation therapy of durvalumab after definitive chemoradiotherapy has become the new standard of care for patients with unresectable stage III non-small cell lung cancer, the prevalence and prognostic role of PD-L1 expression in this population remain unclear. We retrospectively reviewed data from patients with unresectable stage III non-small cell lung cancer who received definitive chemoradiotherapy at our institution between 2012 and 2017. Levels of PD-L1 were assessed using 22C3 antibody, and associations of progression-free and overall survival rates with PD-L1 statuses at a tumor proportion score cutoff of 1% were analyzed. Among the 104 patients enrolled, PD-L1 statuses were as follows: tumor proportion score

Objectives Pembrolizumab is recommended for patients with previously untreated non-small cell lung cancer (NSCLC) with a programmed death ligand 1 (PD-L1) tumor proportion score (TPS) of ≥1%. The KEYNOTE-024 study described the efficacy of pembrolizumab in patients with previously untreated NSCLC who had a PD-L1 TPS of at least 50 %. However, patients with untreated brain metastasis (BM) were excluded from many clinical trials. Therefore, we assessed the efficacy of pembrolizumab against BM of NSCLC with high tumor PD-L1 expression. Materials and Methods We retrospectively reviewed patients who received pembrolizumab as first-line treatment against NSCLC with PD-L1 TPS ≥ 50 % between March 2017 and September 2019. Treatment efficacy was compared between patients with (BM group) and without BM (non-BM group). In addition, the BM group was divided into patients who previously received treatment for BM before pembrolizumab (BM-T group) and those with no prior treatment for BM (BM-not T group). Results Eighty-seven patients (23 BM group and 64 non-BM group) were assessable for efficacy. No significant differences in patient characteristics were found between the BM and non-BM groups, but proportion of patients with stage IV at diagnosis was significantly higher in the BM group. Median progression-free survival (PFS) (6.5 months vs. 7.0 months) and overall survival (OS) (21.6 months vs. 24.6 months) did not significantly differ between the two groups. The response rate of BM was 70 %. The BM group was subdivided into 13 patients in the BM-T group and 10 patients in the BM-not T group. No significant differences in patient characteristics were found between the two groups, but maximum diameter of BM and proportion of patients with symptomatic BM were significantly greater in the BM-T group. PFS and OS did not significantly differ between the two groups. The median PFS of BM was 13.6 months in the BM-T group and 18.6 months in the BM-not T group. Conclusion Pembrolizumab may be effective for BM caused by previously untreated NSCLC with high PD-L1 tumor expression.

Background Quantitative skeletal muscle mass loss has the potential to predict the therapeutic effects of immune checkpoint inhibitors. This study aimed to assess the impact of muscular quality on the abovementioned outcomes. Methods This study retrospectively reviewed the medical records of patients with advanced non‐small cell lung cancer (NSCLC) who had received PD‐1/PD‐L1 inhibitor monotherapy between March 2016 and February 2018. High muscle quality was stipulated as a skeletal muscle density ≥41 and ≥33 Hounsfield units in patients with a body mass index (BMI), High muscle quality correlates with higher objective response rate and longer progression‐free survival duration in treatment with immune checkpoint inhibitors. On the other hand, there is no association between muscle quantity and the efficacy of immune checkpoint inhibitors.

Background Local ablative therapy (LAT) may be beneficial for patients with epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC) with oligo-residual disease after treatment with EGFR tyrosine kinase inhibitor (EGFR-TKI). However, this has not been fully established. This study aimed to evaluate the predominant progressive disease (PD) pattern limited to residual sites of disease after treatment with EGFR-TKI. Methods Patients with advanced EGFR-mutated NSCLC treated with EGFR-TKIs as first-line therapy were retrospectively analysed during a 7-year period. Oligo-residual disease was defined as the presence of 1 – 4 lesions (including the primary site) at 3 months from the start of EGFR-TKI treatment. The predictive factors of PD patterns after EGFR-TKI treatment were evaluated. Results A total of 207 patients were included. Three months after the start of EGFR-TKI treatment, 66 patients (32%) had oligo-residual disease. A total of 191 patients had PD, 60 with oligo-residual disease and 131 with non-oligo-residual disease. Regarding the pattern, 44 patients (73%) with oligo-residual disease and 37 patients (28%) with non-oligo-residual disease had PD limited to the residual sites. Multivariate logistic regression analysis at 3 months from the start of EGFR-TKI treatment revealed that oligo-residual disease (P P = 0.032), and initial treatment with osimertinib (P = 0.028) were independent predictors of PD limited to residual disease sites. Conclusions This study provided a rationale for LAT to all sites of residual disease in patients with oligo-residual disease during EGFR-TKI treatment.

Prognostic data on Japanese patients receiving durvalumab after chemoradiotherapy (CRT) for locally advanced non-small cell lung cancer (LA-NSCLC) are insufficient. Whether pneumonitis has prognostic implications in patients with LA-NSCLC who have received durvalumab also remains unclear.We retrospectively assessed the data of 82 consecutive patients who had received durvalumab after CRT at our institution between May 2018 and August 2020. A multi-state model was used to establish the associations between co-variables and progression-free survival (PFS).The median observation period for all the censored cases was 14.5 months (5.7-28.9 months), the median PFS was 22.7 months, and the 12-month PFS rate was 62.3% (95% CI: 50.2%-72.3%). The median percentage of the lung volume receiving a radiation dose in excess of 20 Gray (V20) was 22% (4%-35%). Thirteen patients (16%) had Grade 1 pneumonitis before receiving durvalumab, and 62 patients developed pneumonitis after durvalumab (Grades 1, 2, and 3 in 25 [30%], 32 [39%], and 4 [5%], respectively). Twenty-four patients (29%) completed the 1-year durvalumab treatment period, 16 patients (20%) were continuing to receive treatment, and 42 (51%) had discontinued treatment. In a multi-state analysis, patients with pneumonitis before durvalumab therapy had a poorer PFS than those without pneumonitis (HR: 4.29, p = 0.002). The development of Grade 2 or higher pneumonitis after durvalumab was not a significant prognostic factor for PFS (HR: 0.71, p = 0.852).Grade 2 or higher pneumonitis after durvalumab was not a prognostic factor of PFS in LA-NSCLC patients received durvalumab.

SummaryBackground. Prognostic data on Japanese patients receiving durvalumab after chemoradiotherapy (CRT) for locally advanced non-small cell lung cancer (LA-NSCLC) are insufficient. Whether pneumonitis has prognostic implications in patients with LA-NSCLC who have received durvalumab also remains unclear. Methods. We retrospectively assessed the data of 82 consecutive patients who had received durvalumab after CRT at our institution between May 2018 and August 2020. A multi-state model was used to establish the associations between co-variables and progression-free survival (PFS). Results. The median observation period for all the censored cases was 14.5 months (5.7–28.9 months), the median PFS was 22.7 months, and the 12-month PFS rate was 62.3% (95% CI: 50.2%-72.3%). The median percentage of the lung volume receiving a radiation dose in excess of 20 Gray (V20) was 22% (4%-35%). Thirteen patients (16%) had Grade 1 pneumonitis before receiving durvalumab, and 62 patients developed pneumonitis after durvalumab (Grades 1, 2, and 3 in 25 [30%], 32 [39%], and 4 [5%], respectively). Twenty-four patients (29%) completed the 1-year durvalumab treatment period, 16 patients (20%) were continuing to receive treatment, and 42 (51%) had discontinued treatment. In a multi-state analysis, patients with pneumonitis before durvalumab therapy had a poorer PFS than those without pneumonitis (HR: 4.29, p = 0.002). The development of Grade 2 or higher pneumonitis after durvalumab was not a significant prognostic factor for PFS (HR: 0.71, p = 0.852). Conclusion. Grade 2 or higher pneumonitis after durvalumab was not a prognostic factor of PFS in LA-NSCLC patients received durvalumab.

Background. Long-term survival data of patients with limited-disease small-cell lung cancer (LD-SCLC) treated with concurrent chemoradiotherapy (CRT) have not been fully evaluated. Furthermore, the association between long-term prognosis and prognostic factors has not been sufficiently investigated.Methods. The efficacy of concurrent CRT was retrospectively evaluated in 120 patients with LD-SCLC who were planned for curative CRT using concurrent accelerated hyperfractionated radiotherapy.Results. The median patient age was 65.5 years. The majority of patients were males (73%) and had clinical stage III disease (80%). The median follow-up time for overall survival (OS) was 71.3 months. The median OS was 34.3 months; the 3- and 5-year OS rates were 52.4% and 41.8%, respectively. The median progression-free survival (PFS) was 12.2 months; the 3- and 5-year PFS rates were 37.6% and 33.6%, respectively. The 5-year OS rates of patients who achieved PFS at 12, 24, and 36 months were 70.9%, 83.6%, and 91.9%, respectively. The gradual increase in the 5-year OS rate following PFS extension and initial depression of Kaplan–Meier curves showed that disease progression frequently occurred in the first 2 years after concurrent CRT initiation. The Cox proportional hazards model revealed no significant factors that correlated with long-term survival through univariate and multivariate analyses.Conclusions. Prognostic factors associated with long-term prognosis in LD-SCLC were not identified. Nonetheless, the 5-year OS rate was 41.8%, whereas the 5-year survival rate among patients without disease progression at 2 years was 83.6%. These data suggest that the prognosis of patients with LD-SCLC was improving.

Tenascin-C is a member of the matricellular protein family, and its expression level is correlated to poor prognosis in cancer, including glioblastoma, whereas its substantial role in tumor formation and malignant progression remains controversial. We reported previously that peptide TNIIIA2 derived from the cancer-associated alternative splicing domain of tenascin-C molecule has an ability to activate β1-integrin strongly and to maintain it for a long time. Here, we demonstrate that β1-integrin activation by TNIIIA2 causes acquisition of aggressive behavior, dysregulated proliferation, and migration, characteristic of glioblastoma cells. TNIIIA2 hyperstimulated the platelet-derived growth factor–dependent cell survival and proliferation in an anchorage-independent as well as -dependent manner in glioblastoma cells. TNIIIA2 also strongly promoted glioblastoma multiforme cell migration, which was accompanied by an epithelial–mesenchymal transition–like morphologic change on the fibronectin substrate. Notably, acquisition of these aggressive properties by TNIIIA2 in glioblastoma cells was abrogated by peptide FNIII14 that is capable of inducing inactivation in β1-integrin activation. Moreover, FNIII14 significantly inhibited tumor growth in a mouse xenograft glioblastoma model. More importantly, FNIII14 sensitized glioblastoma cells to temozolomide via downregulation of O6-methylguanine-DNA methyltransferase expression. Consequently, FNIII14 augmented the antitumor activity of temozolomide in a mouse xenograft glioblastoma model. Taken altogether, the present study provides not only an interpretation for the critical role of tenascin-C/TNIIIA2 in aggressive behavior of glioblastoma cells, but also an important strategy for glioblastoma chemotherapy. Inhibition of the tenascin-C/β1-integrin axis may be a therapeutic target for glioblastoma, and peptide FNIII14 may represent a new approach for glioblastoma chemotherapy. Significance: These findings provide a proposal of new strategy for glioblastoma chemotherapy based on integrin inactivation.

The MYC family oncogenes (MYC, MYCN, and MYCL) contribute to the genesis of many human cancers. Among them, amplification of the MYCN gene and over-expression of N-Myc protein are the most reliable risk factors in neuroblastoma patients. On the other hand, we previously found that a peptide derived from fibronectin, termed FNIII14, is capable of inducing functional inactivation in β1-integrins. Here, we demonstrate that inactivation of β1-integrin by FNIII14 induced proteasomal degradation in N-Myc of neuroblastoma cells with MYCN amplification. This N-Myc degradation by FNIII14 reduced the malignant properties, including the anchorage-independent proliferation and invasive migration, of neuroblastoma cells. An in vivo experiment using a mouse xenograft model showed that the administration of FNIII14 can inhibit tumor growth, and concomitantly a remarkable decrease in N-Myc levels in tumor tissues. Of note, the activation of proteasomal degradation based on β1-integrin inactivation is applicable to another Myc family oncoprotein, c-myc, which also reverses cancer-associated properties in pancreatic cancer cells. Collectively, β1-integrin inactivation could be a new chemotherapeutic strategy for cancers with highly expressed Myc. FNIII14, which is a unique pharmacological agent able to induce β1-integrin inactivation, may be a promising drug targeting Myc oncoproteins for cancer chemotherapy.

BACKGROUND/AIM Despite intensive chemotherapy, the survival rates for high-risk neuroblastoma, most of which have MYCN amplification, remain low. Overexpression of N-myc oncoprotein promotes expression of cancer-associated properties. We recently found that combination of all-trans retinoic acid (ATRA) with the β1-integrin-activating peptide TNIIIA2 attenuated cancer-associated properties of neuroblastoma cells through N-Myc degradation. However, ATRA has serious side-effects and there are concerns about late adverse effects. The aim of this study was to examine the effects of the combination of acyclic retinoid (ACR) with TNIIIA2 on neuroblastoma. MATERIALS AND METHODS The effects of ACR and TNIIIA2 were examined by neuroblastoma cell proliferation and survival assays as well as by using a neuroblastoma xenograft model. The levels of N-Myc and cancer-associated malignant properties were assayed by western blot and colony formation assay, respectively. RESULTS Combining ACR, which is clinically safe, with TNIIIA2 induced proteasomal degradation of N-Myc and reduction of neuroblastoma cell malignant properties. An in vivo experiment showed therapeutic potential. CONCLUSION ACR-TNIIIA2 combination treatment may be efficacious and clinical safe chemotherapy for high-risk neuroblastoma.

Objective Immune checkpoint inhibitors (ICIs) combined with chemotherapy have been approved as first-line treatment for patients with untreated extensive disease-small cell lung cancer (ED-SCLC). However, there are few reports about the long-term outcomes in patients with ED-SCLC treated without ICIs. Thus, we analyzed the long-term outcomes in patients with ED-SCLC. Methods We retrospectively examined the medical records of patients with SCLC who were treated at our hospital between September 2002 and September 2019. The main inclusion criteria were as follows: (i) histological or cytological confirmation of SCLC, (ii) diagnosed with ED-SCLC and (iii) received chemotherapy, not including ICIs, as the first-line treatment. To assess the trends of treatment outcomes, we compared the survival outcomes between 2002–2010 (early) and 2011–2019 (late) groups. Results A total of 314 patients were included in this study. Patient characteristics at the time of first-line treatment were as follows: median age was 69 years; 82% of the patients were male and 70% had a performance status of 0 or 1. The median follow-up time of overall survival (OS) was 7.4 years, and 89% of the patients died. The median progression-free survival and survival time were 4.9 and 12.1 months, respectively. Five-year survival rate was 2%. There was no significant difference in survival between the early and late groups. Conclusions We found that the long-term outcomes in ED-SCLC patients treated without ICIs were poor. Prior to the approval of ICI treatment for ED-SCLC, there was no improvement in the OS for ~20 years.

PurposeProgrammed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors plus chemotherapy has become the standard first-line treatment in patients with advanced non-small-cell lung cancer (NSCLC). However, few studies have explicitly focused on the impact of cancer cachexia on the efficacy of PD-1/PD-L1 inhibitors plus chemotherapy. Thus, we evaluated the clinical implications of cancer cachexia on the survival outcomes in patients who received this treatment.MethodsWe conducted a retrospective review of medical records of patients with advanced NSCLC treated with PD-1/PD-L1 inhibitors plus chemotherapy from December 2018 to December 2020. Cancer cachexia was diagnosed as an unintentional weight loss of 5% or more over six months. We evaluated the progression-free survival (PFS) and overall survival (OS) for patients with or without cancer cachexia who received PD-1/PD-L1 inhibitors plus chemotherapy.ResultsAmong the 80 included patients, 37 (46%) had cancer cachexia. Cachectic patients had a lower objective response rate (30 vs 51%, P P P

Programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors plus chemotherapy have become the standard first line of treatment in patients with advanced non-small-cell lung cancer (NSCLC). However, few studies have explicitly focused on the impact of weight loss on the efficacy of PD-1/PD-L1 inhibitors plus chemotherapy. Thus, we evaluated the clinical implications of weight loss on the survival outcomes in patients who received this treatment. We conducted a retrospective review of medical records of patients with advanced NSCLC who were treated with PD-1/PD-L1 inhibitors plus chemotherapy from December 2018 to December 2020. Significant weight loss was defined as an unintentional weight loss of 5% or more over 6 months. We evaluated the progression-free survival (PFS) and overall survival (OS) of patients with or without weight loss. Among the 80 included patients, 37 (46%) had weight loss, and were associated with a lower objective response rate (30 vs 51%, P

Objectives: In EGFR-mutated non-small cell lung cancer (NSCLC) patients, approximately 80–90% of leptomeningeal metastasis (LM) develops after failed initial epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI) treatment. However, the efficacy of rechallenging previously administered EGFR-TKIs in patients with EGFR-mutated NSCLC and LM that develops after EGFR-TKI treatment failure remains unknown.Materials and Methods: We retrospectively reviewed the medical records of patients with EGFR-mutated NSCLC and LM from November 2011 to August 2019. Patients were classified into two groups according to LM treatment type: switched to previously unadministered EGFR-TKIs (Switch-TKI) and rechallenge with previously administered EGFR-TKIs (Rechallenge-TKI).Results: In total, 50 patients treated with EGFR-TKI after LM diagnosis were included; 35 were treated with Switch-TKI, and 15 with Rechallenge-TKI. The median overall survival (OS) from the time of LM diagnosis was 6.2 months in all study patients. According to the treatment type, the median OS from the time of LM diagnosis was 6.9 months in Switch-TKI patients and 4.9 months in Rechallenge-TKI patients. There was no significant difference in the OS between the Switch-TKI and Rechallenge-TKI groups (P = 0.864). Thirty-five patients were treated with erlotinib and 15 with osimertinib; Regardless of the type for EGFR-TKI, there was no significant difference in OS between patients treated with Switch-TKI and those treated with Rechallenge-TKI. Conclusion: Our findings suggest that rechallenge of previously administered EGFR-TKIs may be a therapeutic option for LM development after EGFR-TKI treatment failure in patients with EGFR-mutated NSCLC, not only switching to previously unadministered EGFR-TKIs.

Background Combination immune checkpoint inhibitor (ICI) therapy has become the mainstay in cancer treatment, and the various antitumor effects of ICIs are being observed. Synchronous multiple primary lung cancers (SMPLCs), which simultaneously involve tumors of different histologies, are often encountered in clinical settings. In standard lung cancer treatment, an anticancer drug, usually a platinum-based drug, is administered, and this first treatment provides some antitumor effect. Thus, the initial administration of platinum-based anticancer agent may mask the detection of SMPLCs. The following case represents different antitumor effects on two different primary lung lesions during treatment with ICIs. Case presentation A 72-year-old man was referred to our hospital for an abnormal chest shadow, and computed tomography showed masses in the left lower and right upper lungs. Transbronchial lung biopsy from the left lung tumor revealed an adenocarcinoma. Following the administration of pembrolizumab (200 mg/body over 3 weeks) as monotherapy, the tumor in the left lung rapidly reduced in size. However, the tumor in the right upper lung continued to grow. Finally, his disease was diagnosed as SMPLCs of adenocarcinoma and small cell lung cancer. Conclusion Bilateral lung lesions considered to be intrapulmonary metastases have completely different responses to ICI treatment. It is necessary to consider a diagnosis of SMPLCs if lesions with different responses to antitumor therapy are observed.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that has exhibited efficacy in patients with EGFR-mutant non-small cell lung cancer (NSCLC). Interstitial lung disease (ILD) is a fatal adverse event of osimertinib treatment, and it requires treatment discontinuation. There are few reports regarding the safety and efficacy of osimertinib re-challenge in patients who experienced osimertinib-induced ILD. This retrospective study assessed this treatment option. We retrospectively collected data for patients treated with osimertinib who developed ILD at Shizuoka Cancer Center from April 2016 to March 2020. ILD was diagnosed by two doctors based on imaging tests and blood tests to exclude other causes. Among 215 patients treated with osimertinib, 28 developed ILD. The median age of patients with ILD was 69.5 years (range, 39.0–80.0). In addition, 29% of patients were men, and 46% had a history of smoking. Eleven patients were re-administered EGFR TKIs, including eight patients treated with osimertinib and three patients treated with alternative EGFR TKIs. Among patients re-challenged with osimertinib, none who previously experienced grade 1 ILD exhibited ILD relapse, even with the same osimertinib dose and without the concurrent administration of systemic steroids. Meanwhile, one of the four patients who previously exhibited grade 2 ILD experienced despite a dose reduction for osimertinib and systemic steroid administration. For patients with EGFR-mutant NSCLC who experience grade 1 ILD during osimertinib therapy, osimertinib re-challenge may be suitable when no other treatments are available.

Expression level of tenascin-C is closely correlated to poor prognosis in glioblastoma patients, while the substantial role of tenascin-C responsible for aggressive progression in glioblastoma cells has not been clarified. We previously found that peptide TNIIIA2, which is derived from the tumor-associated tenascin-C variants, has the ability to promote cell adhesion by activating &beta, 1-integrins. Our recent study demonstrated that potentiated activation of integrin &alpha, 5&beta, 1 by TNIIIA2 causes not only a dysregulated proliferation in a platelet-derived growth factor (PDGF)-dependent manner, but also disseminative migration in glioblastoma cells. Here, we show that TNIIIA2 enhances the proliferation in glioblastoma cells expressing PDGF-receptor&beta, even without exogenous PDGF. Mechanistically, TNIIIA2 induced upregulated expression of PDGF, which in turn stimulated the expression of tenascin-C, a parental molecule of TNIIIA2. Moreover, in glioblastoma cells and rat brain-derived fibroblasts, tenascin-C upregulated matrix metalloproteinase-2, which has the potential to release TNIIIA2 from tenascin-C. Thus, it was shown that autocrine production of PDGF triggered by TNIIIA2 functions to continuously generate a functional amount of PDGF through a positive spiral loop, which might contribute to hyper-proliferation in glioblastoma cells. TNIIIA2 also enhanced in vitro disseminative migration of glioblastoma cells via the PKC&alpha, signaling. Collectively, the tenascin-C/TNIIIA2 could be a potential therapeutic target for glioblastoma.

e21113 Background: In phase Ⅲ trial comparing crizotinib with chemotherapy in patients with ALK-positive non-small-cell lung cancer (NSCLC) patients, it suggests that pemetrexed is highly effective against ALK-positive NSCLC. However, it is unclear that pemetrexed-based chemotherapy is effective in patients previously treated with ALK-tyrosine kinase inhibitor (ALK-TKI) as well as ALK-TKI naïve patients. Therefore, we investigated the impact of prior ALK-TKI therapy on pemetrexed-based chemotherapy. Methods: We retrospectively reviewed 28 ALK positive NSCLC patients who had received pemetrexed-based chemotherapy at Shizuoka Cancer Center, Japan, from January 2011 to October 2020. The efficacies of pemetrexed-based chemotherapy after or before ALK-TKI was evaluated. Results: Eight patients (29 %) were treated with pemetrexed-based chemotherapy before ALK-TKI (ALK-TKI treated group), and 20 (71%) with this regimen after the failure of ALK-TKI (ALK-TKI naïve group). Patients characteristics (ALK-TKI naïve/ALK-TKI treated) were as follows: median age (range) 66 (27-72)/57 (35-73); male 100/45%; PS0-1 100/85%; adenocarcinoma 100/90%; stageⅣ 88/90%; with CNS metastasis 12/40%. The pemetrexed-based chemotherapy was administered in 5 (18%) patients as the 1st line, 11 (39%) as 2nd line, and 12 (43%) as 3rd or latter line. Twenty-five patients received the combination of platinum with pemetrexed and 3 done pemetrexed monotherapy. Regarding previous ALK-TKI, 17 patients received only alectinib and 8 patients did both alectinib and crizotinib. Progression-free survival (PFS) in ALK-TKI treated group was shorter than in ALK-TKI naïve group (median PFS 3.8 months vs 12.4 months Hazard ratio = 0.41; 95%CI 0.16-1.01 by wald test, p = 0.047 by log-rank test). In overall survivals (OS) from first line treatment between two groups, and ALK-TKI treated group tended to be shorter than ALK-TKI naïve group (median OS 29.5 months vs. 48.8 months; Hazard ratio 0.47, 95%CI 0.13-1.7, p = 0.248). OS from pemetrexed-based chemotherapy of ALK-TKI treated group was also significantly shorter than ALK-naïve group (median OS 10.1 months vs. not reached; Hazard ratio 0.20, 95%CI 0.05-0.75, p = 0.01). Overall response rate (ORR) of pemetrexed-based chemotherapy was lower in ALK-TKI treated group than ALK-TKI naïve group (35% vs 50%) and ORR of ALK-TKI was 85% vs 63%. There was no difference in PFS of pemetrexed-based chemotherapy regardless of efficacy of prior ALK-TKI (PFS ≥24 months vs < 24 months; median PFS 4.5 months vs 7.2 months, Hazard ratio 0.99, 95%CI 0.42-2.31, p = 0.99). Conclusions: Pemetrexed-based chemotherapy might be less effective in ALK-positive NSCLC patients previously treated with ALK-TKI. This retrospective study results suggested that ALK-TKI sequence can be a better treatment option in ALK-positive NSCLC patients.

Redox imbalance induces oxidative damage and causes age-related pathologies. Mice lacking the antioxidant enzyme SOD1 (Sod1-/-) exhibit various aging-like phenotypes throughout the body and are used as aging model mice. Recent reports suggested that age-related changes in the intestinal environment are involved in various diseases. We investigated cecal microbiota profiles and gastrointestinal metabolites in wild-type (Sod1+/+) and Sod1-/- mice. Firmicutes and Bacteroidetes were dominant in Sod1+/+ mice, and most of the detected bacterial species belong to these two phyla. Meanwhile, the Sod1-/- mice had an altered Firmicutes and Bacteroidetes ratio compared to Sod1+/+ mice. Among the identified genera, Paraprevotella, Prevotella, Ruminococcus, and Bacteroides were significantly increased, but Lactobacillus was significantly decreased in Sod1-/- mice compared to Sod1+/+ mice. The correlation analyses between cecal microbiota and liver metabolites showed that Bacteroides and Prevotella spp. were grouped into the same cluster, and Paraprevotella and Ruminococcus spp. were also grouped as another cluster. These four genera showed a positive and a negative correlation with increased and decreased liver metabolites in Sod1-/- mice, respectively. In contrast, Lactobacillus spp. showed a negative correlation with increased liver metabolites and a positive correlation with decreased liver metabolites in Sod1-/- mice. These results suggest that the redox imbalance induced by Sod1 loss alters gastrointestinal microflora and metabolites.

We found that Au(I) complexed with 2,3-bis(tert-butyl(methyl)phosphino) quinoxaline (10) was a potent anti-tumor agent (half-maximal growth inhibitory concentration, GI50=0.87μM) with broad anti-tumor activity. In particular, the activity of complex 10 was high in tumor cell lines derived from the colon and ovary. Treatment with complex 10 resulted in the apoptosis of HL-60 cells. The ligand for the preparation of complex 10 is commercially available implying that complex 10 might be a good drug candidate for cancer therapy.

Inflammatory bowel diseases increase the risk of colorectal cancer and colitis-associated colorectal cancer (CAC). Tenascin-C, a matricellular protein, is highly expressed in inflammatory bowel diseases, especially colorectal cancer. However, the role of tenascin-C in the development of CAC is not yet fully understood. We previously showed that a peptide derived from tenascin-C, peptide TNIIIA2, induces potent and sustained activation of &beta, 1-integrin. Moreover, we recently reported that peptide TNIIIA2 promotes invasion and metastasis in colon cancer cells. Here, we show the pathological relevance of TNIIIA2-related functional site for the development of CAC. First, expression of the TNIIIA2-containing TNC peptides/fragments was detected in dysplastic lesions of an azoxymethane/dextran sodium sulfate (AOM/DSS) mouse model. In vitro experiments demonstrated that conditioned medium from peptide TNIIIA2-stimulated human WI-38 fibroblasts induced malignant transformation in preneoplastic epithelial HaCaT cells. Indeed, these pro-proliferative effects stimulated by peptide TNIIIA2 were abrogated by peptide FNIII14, which has the ability to inactivate &beta, 1-integrin. Importantly, peptide FNIII14 was capable of suppressing polyp formation in the AOM/DSS model. Therefore, tenascin-C-derived peptide TNIIIA2 may contribute to the formation of CAC via activation of stromal fibroblasts based on &beta, 1-integrin activation. Peptide FNIII14 could represent a potential prophylactic treatment for CAC.

The extract of compost from fermented marine animals and thermophiles, including Bacillaceae, confers health benefits as a feed additive for fish and pigs. However, little research has explored how such compost extracts affect the physiological functions of the animals. In this study, the physiological effects of oral administration of the compost extract on the liver and muscle of rats are evaluated. After long-term administration of the compost extract in rats fed with either a normal diet or a high-fat diet over 3 months, accumulation of lipid peroxide and malondialdehyde, a marker of peroxidation, in the livers was reduced. Under such conditions, the unsaturated fatty acid composition in the liver was not significantly different in the rats fed either with or without the compost extract. In contrast, analyzes of 2,2-diphenyl-1-picrylhydrazyl (DPPH) revealed that free-radical-scavenging activity was increased in the livers of rats fed with the compost extract, although the extract itself had little of this activity. Glutathione, an antioxidant, was slightly increased following compost exposure. In addition, the levels of glutamate and glutamine, sources of glutathione, were slightly raised. Such a tendency was also observed in the muscle. Thus, thermophile-fermented compost can be a fermented feed additive to prevent peroxidation in the liver and muscle, and the effects of this additive may, in part, be associated with the retention of antioxidants and free amino acids within the organs.

The acquisition of drug resistance mediated by the interaction of tumor cells with the extracellular matrix (ECM), commonly referred to as cell adhesion-mediated drug resistance (CAM-DR), has been observed not only in hematopoietic tumor cells but also in solid tumor cells. We have previously demonstrated that a 22-mer peptide derived from fibronectin, FNIII14, can inhibit cell adhesion through the inactivation of β1 integrin; when coadministered with cytarabine, FNIII14 completely eradicates acute myelogenous leukemia by suppressing CAM-DR. In this study, we show that our FNIII14 peptide also enhances chemotherapy efficacy in solid tumors. Coadministration of FNIII14 synergistically enhances the cytotoxicity of doxorubicin and aclarubicin in mammary tumor and melanoma cells, respectively. The solid tumor cell chemosensitization induced by FNIII14 is dependent upon the upregulation and activation of the pro-apoptotic protein, Bim. Furthermore, the metastasis of tumor cells derived from ventrally transplanted mammary tumor grafts is suppressed by the coadministration of FNIII14 and doxorubicin. These results suggest that the coadministration of our FNIII14 peptide with chemotherapy could achieve efficient solid tumor eradication by increasing chemosensitivity and decreasing metastasis. The major causes of tumor recurrence are the existence of chemotherapy-resistant primary tumor cells and the establishment of secondary metastatic lesions. As such, coadministering FNIII14 with anti-cancer drugs could provide a promising new approach to improve the prognosis of patients with solid tumors.

Oral administration of an extract consisting of compost fermented with thermophiles to pigs reduces the incidence of stillbirth and promotes piglet growth. However, the mechanism by which the compost extract modulates the physiological conditions of the animals remains largely unknown. Here, we investigate the effects of compost extract on the physiological responses in the intestine of a mammalian rat model. The level of fecal immunoglobulin A (IgA), which provides protection against pathogens and is secreted from the small intestine, was significantly higher in rats treated with continuous administration of the compost extract than in untreated rats after 2 months, but not after 1 month. However, the fecal IgA level was not significantly different in rats that received the filtered compost extract compared with the untreated rats or the rats that received the compost extract. Gene expression analyses of the small intestine indicated that several immune-related genes were upregulated following compost exposure. Specifically, the expression levels of lymphocyte chemoattractant chemokine CXCL13 and Granzyme B, which is released within cytotoxic T lymphocytes and natural killer cells, increased in the small intestinal tract following compost exposure. Based on these observations, it was postulated that the increased level of fecal IgA following compost exposure was associated with the expression of CXCL13 and Granzyme B in the intestinal tract. Thus, thermophile-fermented compost could contain microbes or substances that activate the rat's gut mucosal immune response.

Food produced via fermentation with mesophilic bacteria has been used to confer health benefits. In contrast, mammalian physiological responses to the intake of thermophile-fermented products have not been thoroughly investigated. We examined the effects of administering a compost extract consisting of fermented marine animals with thermophiles, including Bacillaceae, to pregnant sows and piglets. Retrospective studies were performed on two different swine farms (n=330-1050 sows). The rate of stillbirth was markedly lower in all parities of the compost extract-fed group compared to those of the control group (p≦0.001). Additionally, the birth to weaning period of newborns was significantly shorter (p

The widespread natural sources-derived cationic peptides have been reported to reveal bacterial killing and/or growth-inhibiting properties. Correspondingly, a number of artificial peptides have been designed to understand antibacterial mechanism of the cationic peptides. These peptides are expected to be an alternative antibiotic against drug-resistant pathogenic bacteria because major antimicrobial mechanism of cationic peptides involves bacterial membrane disorder, although those availabilities have not been well evaluated. In this study, cationic peptides containing Aib were prepared to evaluate the availability as an antimicrobial agent, especially against representative pathogenic bacteria. Among them, BRBA20, consisting of five repeated Aib-Arg-Aib-Ala sequences, showed strong antibacterial activity against both Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Additionally, growth of Serratia marcescens and multidrug-resistant Pseudomonas aeruginosa, known as proteases-secreting pathogenic bacteria, were also completely inhibited by BRBA20 under 20 µg/ml peptide concentrations. Our results suggested availabilities of Aib-derived amphiphilicity and protease resistance in the design of artificial antimicrobial peptides. Comparing BRBA20 with BKBA20, it was also concluded that Arg residue is the preferred cationic source than Lys for antimicrobial action of amphiphilic helices.

After landing on the stigma, the pollen grain germinates and elongates a tube to deliver its generative nuclei to the egg cell of the ovule. The molecular mechanisms involved in the drastic morphological changes in the pollen grain during this fertilization process remain largely unknown. In this study, the expression of 732 randomly selected genes in petunia pollen and pollen tubes was analyzed by microarray and quantitative PCR analyses. We found no evidence for up-regulation of any of these genes in the pollen tube. Our findings provide support at the gene level for the longstanding hypothesis that pollen germination and tube growth are not dependent on new RNA synthesis and that the large number of RNAs required for germination and tube growth are stored in mature pollen grains.

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. The mercaptoacetoamide-based inhibitors are reported to be less toxic than hydroxamate and are worthy of further consideration. Therefore, we have designed a series of analogs as potential inhibitors of HDACs, in which the mercaptoacetamide group was replaced by (mercaptomethyl)fluoroalkene, and their HDAC inhibitory activity was evaluated. Subnanomolar inhibition was observed for all synthetic compounds.

Osteoclast deficiency in op/op mice is cured by a single injection of 5 micrograms recombinant human macrophage colony-stimulating factor (rhM-CSF). In this study, we found that mouse osteoclasts are positive for Mac-2 antigen, but not for F4/80, MOMA-2, Mac-1, or BM8 antigen. By using F4/80 and MOMA-2 monoclonal antibodies, we confirmed the absence of mature macrophages in the femora of op/op mice and found that multiple injections of rhM-CSF are required for the recruitment of macrophages in the bones. After a single rhM-CSF injection, we found Mac-2 positive mononuclear cells in the femora of op/op mice. The time course of the appearance of Mac-2-positive cells was very similar to that of tartrate-resistant acid phosphatase (TRAP)-positive cells. In bone sections prepared from the mutant mice that received rhM-CSF 3 days earlier, 91% of the TRAP-positive mononuclear cells were also positive for Mac-2 antigen. These results demonstrate the expression of Mac-2 antigen in preosteoclasts. The antigen was detected on the plasma membrane of preosteoclasts, as well as in their cytoplasm and nucleus, and in the extracellular matrix in the space between the cells and bone. Since Mac-2 is a galactose-specific lectin, a potential role of the lectin in cell-cell and cell-matrix adhesion during osteoclast differentiation is suggested.

A tryptic [EC 3.4.21.4] digestion assay of 2-aminoisobutyric acid (Aib)-containing peptides was carried out to investigate the effect of alpha,alpha-dialkyl amino acid residues on the protease resistance. The introduction of Aib residues to the P1' positions exhibited a 19-fold higher protease resistance than the peptide with Aib residues introduced to the P2 position or the non-Aib peptide. The peptide having Aib residues introduced to the P1' and P2 positions resulted in complete resistance.

We investigated five cases of cardiac myxoma and one case of cardiac undifferentiated sarcoma by light and electron microscopy, in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction for cardiomyocyte-specific transcription factors, Nkx2.5/Csx, GATA-4, MEF2, and eHAND. Conventional light microscopy revealed that cardiac myxoma and sarcoma cells presented variable cellular arrangements and different histological characteristics. Ultrastructurally, some of the myxoma cells exhibited endothelium-like or immature mesenchymal cell differentiation. Immunohistochemistry for Nkx2.5/Csx, GATA-4, and eHAND was slightly to intensely positive in all myxoma cases. MEF2 immunoreactivity was observed in all cases including the case of sarcoma, thus suggesting myogenic differentiation of myxoma or sarcoma cells. In situ hybridization for Nkx2.5/Csx also revealed that all myxoma cells, but not sarcoma cells, expressed mRNA of the cardiac homeobox gene, Nkx2.5/Csx. Furthermore, nested reverse transcriptase-polymerase chain reaction from formalin-fixed, paraffin-embedded tissue was performed and demonstrated that the Nkx2.5/Csx and eHAND gene product to be detected in all cases, and in three of six cases, respectively. In conclusion, cardiac myxoma cells were found to express various amounts of cardiomyocyte-specific transcription factor gene products at the mRNA and protein levels, thus suggesting cardiomyogenic differentiation. These results support the concept that cardiac myxoma might arise from mesenchymal cardiomyocyte progenitor cells.

OBJECTIVESWe sought to determine whether potassium (K+) channel blockers (KBs) can activate extracellular signal-regulated kinase (ERK) and to characterize the upstream signals leading to ERK activation in cardiomyocytes.BACKGROUNDBecause KBs attenuate K+outward current, they may possibly prolong the duration of action potentials, leading to an increase in calcium (Ca2+) transient ([Ca2+]i) in cardiomyocytes. Elevation of intracellular Ca2+levels can trigger various signaling events. Influx of Ca2+through L-type Ca2+channels after membrane depolarization induced activation of MEK and ERK through activation of Ras in neurons. Although KBs are frequently used to treat cardiac arrhythmias, their effect on signaling pathways remains unknown.METHODSPrimary cultured rat cardiomyocytes were stimulated with four different KBs—4-aminopyridine (4-AP), E-4031, tetra-ethylammonium and quinidine—and phosphorylation of ERK, proline-rich tyrosine kinase 2 (Pyk2) and epidermal growth factor receptor (EGFR) was detected. Action potentials were recorded by use of a conventional microelectrode. (Ca2+)iwas monitored by the fluorescent calcium indicator Fluo-4.RESULTSE-4031, 4-AP, tetra-ethylammonium and quinidine induced phosphorylation of ERK. 4-Aminopyridine prolonged the duration of action potentials by 37% and increased (Ca2+)iby 52% at 1 mmol/l. Pre-incubation of ethyleneglycoltetraacetic acid, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis and diltiazem completely blocked this phosphorylation, whereas flufenamic acid and benzamil did not. 4-Aminopyridine induced tyrosine phosphorylation of Pyk2 and EGFR, which peaked at 5 and 10 min, respectively. Cytochalasin D, AG1478 and dominant-negative EGFR strongly inhibited the phosphorylation of ERK, whereas calphostin C, calmidazolium and KN62 did not.CONCLUSIONSThese findings indicate that KBs induce ERK activation, which starts with Ca2+entry through the L-type Ca2+channel in cardiomyocytes, and that EGFR and Pyk2 are involved in this activation.

We demonstrated previously that a single injection of recombinant human macrophage colony-stimulating factor (rhM-CSF) is sufficient for osteoclast recruitment and survival in osteopetrotic (op/op) mice with a deficiency in osteoclasts resulting from a mutation in M-CSF gene. In this study, we show that a single injection of recombinant human vascular endothelial growth factor (rhVEGF) can similarly induce osteoclast recruitment in op/op mice. Osteoclasts predominantly expressed VEGF receptor 1 (VEGFR-1), and activity of recombinant human placenta growth factor 1 on osteoclast recruitment was comparable to that of rhVEGF, showing that the VEGF signal is mediated through VEGFR-1. The rhM-CSF–induced osteoclasts died after injections of VEGFR-1/Fc chimeric protein, and its effect was abrogated by concomitant injections of rhM-CSF. Osteoclasts supported by rhM-CSF or endogenous VEGF showed no significant difference in the bone-resorbing activity. op/op mice undergo an age-related resolution of osteopetrosis accompanied by an increase in osteoclast number. Most of the osteoclasts disappeared after injections of anti-VEGF antibody, demonstrating that endogenously produced VEGF is responsible for the appearance of osteoclasts in the mutant mice. In addition, rhVEGF replaced rhM-CSF in the support of in vitro osteoclast differentiation. These results demonstrate that M-CSF and VEGF have overlapping functions in the support of osteoclastic bone resorption.

Abstract —This study was designed to determine whether mechanical stretch activates the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in cardiomyocytes and, if so, by what mechanism. Neonatal rat/murine cardiomyocytes were cultured on malleable silicone dishes and were stretched by 20%. Mechanical stretch induced rapid phosphorylation of JAK1, JAK2, Tyk2, STAT1, STAT3, and glycoprotein 130 as early as 2 minutes and peaked at 5 to 15 minutes. It also caused gel mobility shift of sis-inducing element, which was supershifted by preincubation with anti-STAT3 antibody. Preincubation with CV11974 (AT 1 blocker) partially inhibited the phosphorylation of STAT1, but not that of STAT3. Preincubation with TAK044 (endothelin-1-type A/B-receptor blocker) did not attenuate this pathway. RX435 (anti–glycoprotein 130 blocking antibody) inhibited the phosphorylation of STAT3 and partially inhibited that of STAT1. Phosphorylation of STAT1 and STAT3 was strongly inhibited by HOE642 (Na + /H + exchanger inhibitor) and BAPTA-AM (intracellular calcium chelator), but not by gadolinium (stretch-activated ion channel inhibitor), EGTA (extracellular Ca 2+ chelator), or KN62 (Ca 2+ /calmodulin kinase II inhibitor). Chelerythrine (protein kinase C inhibitor) partially inhibited the phosphorylation of STAT1 and STAT3. Mechanical stretch also augmented the mRNA expression of cardiotrophin-1, interleukin-6, and leukemia inhibitory factor at 60 to 120 minutes. These results indicated that the JAK/STAT pathway was activated by mechanical stretch, and that this activation was partially dependent on autocrine/paracrine–secreted angiotensin II and was mainly dependent on the interleukin-6 family of cytokines but was independent of endothelin-1. Moreover, certain levels of intracellular Ca 2+ were necessary for stretch-induced activation of this pathway, and protein kinase C was also partially involved in this activation.

Previously, we showed that the JAK/STAT pathway was activated in pressure-overloaded rat heart, and that angiotensin II was partially involved in this activation. The present study was designed to investigate whether gp130-mediated signaling is involved in this activation, and if so, which interleukin (IL)-6 family cytokine is involved. Pressure overload was produced by ligation of the abdominal aorta of Wistar rats or ICR mice. IP-Western blot was performed to detect tyrosine phosphorylation of STATs, gp130, and the association of gp130 with JAK kinases. The serum concentration of IL-6 was measured by enzyme-linked immunosorbent assay. Expression of IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M (OSM), and cardiotrophin-1 (CT-1) mRNA was quantitated. After pressure overload, rapid phosphorylation of STAT1 and STAT3 was observed at 5 min, STAT1 was rephosphorylated at 60 min, and intense phosphorylation of STAT3 was observed at 60 min. Both the phosphorylation of gp130 and the association of gp130 with JAK1 and JAK2 were increased after pressure overload. IL-6 was significantly increased by two-fold in the pressure-overloaded rats. Only CT-1 mRNA expression could be detected by Northern blot, and it increased after pressure overload. Reverse transcription-polymerase chain reaction revealed that IL-6 mRNA expression was increased 9.5-fold. IL-11, LIF, CNTF, and OSM expression were unaffected by pressure overload. These results suggested that gp130-mediated signaling was involved in the pressure overload-induced activation of the JAK/STAT pathway, and that IL-6 and CT-1 might be involved in this activation.

The interaction and orientation of a membrane protein ion channel model, an alpha-aminoisobutyric acid analogue of gramicidin B (GBA), in egg yolk phosphatidylcholine vesicles was studied by means of fluorescence spectroscopic techniques. GBA helices form stable ion-conducting pores in membranes [Jelokhani-Niaraki et al. (1995) J. Chem. Soc. Perkin Trans. 2, 801-808]. In an alpha-helical model for the peptide, all Trp residues (intrinsic fluorophores) are distributed near the C-terminus. Fluorescence quenching experiments revealed the exposure of the helical peptides' C-termini to aqueous environments. Dansyl-labeled vesicles were used to investigate the GBA dynamism of the interaction with membranes. It was shown that considerable amounts of peptide reside on and in the vicinity of the outer surface of lipid bilayers. The transmembrane transfer to the inner layer is slow due to the high affinity of Trp residues for bilayer interfaces which anchor the peptide to the outer surface. A structural-functional interpretation of the GBA interaction with membranes is presented.

Abstract This study was designed to determine whether the JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) pathway is activated in cardiac hypertrophy induced in vivo by pressure overload in rats and to demonstrate whether angiotensin II is involved in the activation of the JAK/STAT pathway. Acute pressure overload was produced by constricting the abdominal aorta of Wistar rats. Immunoprecipitation–Western blot analysis revealed that pressure overload activated JAK1, JAK2, and Tyk2 as early as 5 minutes and that STAT1, STAT2, and STAT3 were tyrosine-phosphorylated rapidly after exposure to the pressure overload. Phosphorylation of STAT1 and STAT2 peaked in the early stage at 5 to 15 minutes, whereas that of STAT3 peaked in the late stage at 60 minutes. Gel mobility shift of the interferon gamma activation site/interferon alpha–stimulating response element was observed immediately after the aortic banding, whereas the band of sis -inducing element was shifted in the late stage at 60 minutes. Both cilazapril (angiotensin II–converting enzyme inhibitor) and E4177 (angiotensin II type 1 [AT 1 ] receptor antagonist) significantly suppressed the phosphorylation of Tyk2 and partially inhibited the phosphorylation of JAK2, but neither affected JAK1. Coimmunoprecipitation of the AT 1 receptor with JAK2 or Tyk2 was clearly observed at 5 minutes and peaked at 15 minutes (20-fold the control value). These results indicate that the JAK/STAT pathway is activated by acute pressure overload in rats and that angiotensin II is involved in activating Tyk2, and partially activating JAK2, via the AT 1 receptor. Both angiotensin II–dependent and –independent pathways take part in activating the JAK/STAT pathway in the pressure-overloaded rat heart.

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1-0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 μg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.