Introduction of macromolecules into synaptosomes using electroporation

Synaptic terminals are sites of high metabolic activity and thus are particularly vulnerable to oxidative stress. Oxidative damage to proteins can be toxic to neurons and may cause irreversible cell damage and neurodegeneration. A neuroprotective mechanism used by cells to combat oxidative damage is to selectively degrade damaged proteins. Therefore, it is of interest to study the mechanism of degradation of oxidatively damaged proteins in synaptosomes. One way of oxidizing synaptosomal proteins in vitro is by incubating intact synaptosomes in the presence of an oxidizing agent. A problem with this approach is that it may also cause oxidative damage to the machinery required to recognize and degrade oxidized proteins. We have, therefore, introduced a fluorescent macromolecule into synaptosomes to assess the feasibility of using this technique to study how oxidized proteins are degraded and removed from synaptic terminals. Synaptosomes were subjected to electroporation in the presence of FITC labelled-dextran with an average molecular weight of 70000 (FD-70) and non-specific binding was determined by running parallel experiments in lysed synaptosomes. Following extensive washing, synaptosomes were assayed for the presence of intra-synaptosomal FD-70 by measuring fluorescence in a microplate fluorescence reader. Significant differences in fluorescence were found between intact and lysed synaptosomes with maximal uptake at 100 V/ 1500 microF (approx. 36 pmol/mg protein). To determine if membrane transport was compromised by electroporation, uptake of 3H-arginine was compared in control and electroporated synaptosomes. While untreated electroporated synaptosomes showed a loss of 22% in the ability to transport arginine, preincubation in the presence of 1 mM ATP resulted in a complete restoration of arginine transport. These results show that electroporation is a potentially useful technique for introducing a specific oxidized protein, into synaptic terminals so its metabolic fate can be examined.     

Quality of sleep and related factors during chemotherapy in patients with stage I/II breast cancer.

BACKGROUND: Insomnia causes severe distress in patients with breast cancer who receive chemotherapy. Few studies have focused on using objective methods to assess sleep. This study explored the quality of sleep and related factors in patients with breast cancer during chemotherapy. METHODS: The participants were 16 women with stage I or II breast cancer receiving their third cycle of chemotherapy with cyclophosphamide, epirubicin and fluorouracil, or cyclophosphamide, methotrexate and fluorouracil. The effects of chemotherapy on sleep were assessed on the 8th and 9th days of the third cycle, i.e. the active phase in terms of side effects, and the last 2 days before the start of the fourth cycle for comparison. Instruments used to assess sleep quality and related factors included actigraphy, the Hospital Anxiety and Depression Scale (HADS), the Symptom Distress Scale (SDS), the Fatigue Visual Analogue Scale (FVAS), the Epworth Sleepiness Scale (ESS), and sleep logs. RESULTS: During the active phase, patients showed an anxiety tendency with an average HADS score of 7.8 +/- 3.8. The average FVAS score was 4 +/- 2, indicative of mild fatigue, and SDS score (1.8 +/- 0.3) also indicated mild symptom distress. The number of awakenings each night was 2.2 +/- 1.6 by sleep logs, and the total time spent awake during these episodes was 47.8 +/- 26.1 minutes by Actiwatch. Sleep efficiency measured by Actiwatch in the active phase was 82.1 +/- 9.4% below the normal limit. Daytime sleepiness assessed by ESS showed mild sleepiness (6.0 +/- 3.5) in the active phase. CONCLUSION: The study showed poor sleep quality and daytime sleepiness in patients with breast cancer during the active phase of chemotherapy. Chemotherapy may bring symptom distress to patients and adversely influence sleep quality.     

Successful treatment of imported cerebral malaria with artesunate-mefloquine combination therapy.

Treatment of cerebral malaria with intravenous quinine is frequently associated with life-threatening cardiotoxicity. We report a case of imported cerebral malaria successfully treated with artesunate-mefloquine combination therapy. The 27-year-old woman presented with fever, sudden onset of binocular blindness and altered consciousness 10 days after a short stay in Indonesia. Hyperparasitemia with Plasmodium falciparum and P. vivax in more than 5% of red blood cells was demonstrated on peripheral blood smear. She was admitted to the intensive care unit due to shock, jaundice and acute renal failure. Because of a shortage of intravenous quinine, intravenous artesunate was given as an alternative. Her condition stabilized on the 3rd day of therapy, with resolution of fever and disappearance of parasitemia. Consolidation therapy with oral mefloquine and primaquine was then given to prevent recrudescence and relapse. The only adverse event associated with artesunate was transient reticulocytopenia, which resolved after discontinuation of therapy. Her vision completely recovered, along with renal and liver function.     

Development and psychometric properties of the dialysis module of the WHOQOL-BREF Taiwan version.

BACKGROUND: Quality of life (QOL) is now considered to be an important part of the assessment of dialysis patients. The aim of this study was to develop and assess the reliability, validity and sensitivity of the dialysis module of the World Health Organization Quality of Life - Brief (WHOQOL-BREF) Taiwan version [WHOQOL-BREF(TW)] in patients undergoing regular hemodialysis (HD). METHODS: QOL survey was administered to 283 regular HD patients in metropolitan Taipei. The instruments used included: (1) the proposed module - composed of the core part, the WHOQOL-BREF(TW), and the six specific items; (2) the symptom/problem (S/P) scale - composed of 12 items specific for dialysis patients; (3) the utility measure, which was performed with standard gamble (SG) methods; and (4) the rating scale (RS). RESULTS: Based on the six criteria of validity, reliability and variance of the items, four HD-specific items were selected. Reliability study showed that Cronbach's alphas, composite reliability, and test-retest reliability (intraclass correlation at an average retest interval of 4-8 weeks) of the four domains of physical, psychological, social relationship and environment, ranged from 0.74-0.82, 0.79-0.84 and 0.61-0.79, respectively. Validity study showed that all the correlations between an item and its corresponding domain were highly significant (r>0.4, p<0.01) and larger than the correlations between the item and other domains. SG and psychometric measures showed relatively low correlations (0.12-0.26). The module showed the same construct as the WHOQOL-BREF(TW) under confirmatory factor analysis, whereas the exploratory factor analysis showed mild variation. Convergent and discriminant validity were good. Global QOL, physical, psychological and environment domains had some sensitivity to differentiate the severity of the condition of patients receiving HD. Clinical validity was demonstrated in global QOL, physical and psychological domains to have significant correlations with S/P scores. CONCLUSION: Besides broader coverage than the core WHOQOL-BREF(TW), the dialysis module of the WHOQOL-BREF(TW) is a valid, reliable and sensitive QOL instrument for the assessment of HD patients in Taiwan.     

In vitro and in vivo antifungal activities of the eight steroid saponins from Tribulus terrestris L. with potent activity against fluconazole-resistant fungal pathogens

Antifungal activity of natural products is being studied widely. Saponins are known to be antifungal and antibacterial. We have isolated eight steroid saponins from Tribulus terrestris L. , namely TTS-8, TTS-9, TTS-10, TTS-11, TTS-12, TTS-13, TTS-14 and TTS-15. TTS-12 and TTS-15 were identified as tigogenin-3-O-β-D-xylopyranosyl（1→ 2）-[-β-D-xylopyranosyl（ 1 → 3 ） 3-β- D-glucopyranosyl （ 1 → 4 ）- 1- α-L-rhamnopyranosyl （ 1 → 2 ） 3-β-D-galactopyranoside and tigogenin-3-O-β-D-glucopyranpyranosyl（1→2）-[-β-D-xylopyranosyl（1→ 3）3-β-D-glucopyranosyl（1→4）-β-D-galactopyranoside, respectively. The in vitro antifungal activities of the eight saponins against six fluconazole-resistant yeasts, Candida albicans, Candida glabrata, Candida para psilosis , Candida tropicalis , Candida krusei , and Cryptococcus neo f ormans were studied using microbroth dilution assay. The results showed that TTS-12 and TTS-15 were very effective against several pathogenic candidal species and C. neoformans in vitro. It is noteworthy that TTS-12 and TTS-15 were very active against fluconazole-resistant C. albicans （MIC80 = 4.4, 9.4 mg/ml）, C. neoformans （MIC80 =10.7, 18.7 mg/ml） and inherently resistant C. krusei （MIC80 =8.8, 18.4 mg/ml）. So in vivo activity of TTS-12 in a vaginal infection model with fluconazole-resistant C. albicans was studied in particular. Our studies revealed TTS-12 also showed in vivo activities against fluconazole-resistant yeasts. In conclusion, steroid saponins TTS-12 and TTS-15 from Tribulus terrestris L. have significant in vitro antifungal activity against fluconazole-resistant fungi, especially TTS-12 also showed in vivo activity against fluconazole-resistant C. albicans.     

The antiproliferative activity of prodelphinidin B-2 3'-O-gallate from green tea leaf is through cell cycle arrest and Fas-mediated apoptotic pathway in A549 cells.

Prodelphinidin B-2 3'-O-gallate, a proanthocyanidin gallate isolated from green tea leaf, was investigated for its anti-proliferative activity in human non-small cell lung cancer A549 cells. The results showed that prodelphinidin B-2 3'-O-gallate inhibited the proliferation of A549 cells with no detectable toxic effects on normal WI-38 cells as measured by the XTT assay. Flow cytometric analysis showed that prodelphinidin B-2 3'-O-gallate blocked cell cycle progression in the G0/G1 phase. In addition, prodelphinidin B-2 3'-O-gallate effectively induced A549 cell apoptosis as determined by assessing the nucleosome level in cytoplasm. Enzyme-linked immunosorbent assay showed that the G0/G1 phase arrest is due to p53-independent induction of p21/WAF1. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by prodelphinidin B-2 3'-O-gallate. We suggested that prodelphinidin B-2 3'-O-gallate's activities might be potentially contribute to its overall chemopreventive effects against lung cancer, and can possibly be considered for future therapeutic application.     

Dynamic contrast-enhanced subtraction and delayed MRI of gastric tumors: radiologic-pathologic correlation

PURPOSE: Our goal was to determine whether dynamic MR subtraction images could be used to detect and stage gastric tumors. METHOD: Dynamic MR subtraction images were prospectively performed in 20 patients without gastric lesions and in 39 patients with gastric tumors. The flat- or depressed-type early gastric cancers were excluded. The MR findings were assessed for layered pattern of the normal gastric wall, detectability of tumors, enhanced pattern of tumor, and depth of the tumor invasion. Surgical specimens were obtained from 30 of the patients with tumors, and histopathologic sections were made in the dynamic MR scanning direction. RESULTS: The three-layered structure of the normal gastric wall was apparent in more of the dynamic MR subtraction images (60%) than of the nonsubtraction images (30%) in the control group. All 39 gastric tumors were detected by MRI. The intact inner layers overlying stromal tumors and outer layers interrupted by advanced gastric cancers were clear on the subtracted images. MRI accurately T-staged 88% of the gastric cancers. CONCLUSION: Dynamic MR subtraction images can be used to identify gastric tumors and to stage gastric cancers.     

Phase II study of gefitinib, fluorouracil, leucovorin, and oxaliplatin therapy in previously treated patients with metastatic colorectal cancer.

PURPOSE: To investigate the gefitinib, fluorouracil (FU), leucovorin, and oxaliplatin regimen (IFOX) in previously treated patients with metastatic colorectal cancer. PATIENTS AND METHODS: Eligible patients had stage IV colorectal adenocarcinoma and had demonstrated progression or intolerance to a prior chemotherapy regimen not including oxaliplatin. Each cycle consisted of 14 days. Cycle 1 consisted of oxaliplatin 85 mg/m2 intravenously (IV) during 2 hours on day 1, hours 0 to 2; leucovorin 200 mg/m2 IV on days 1 and 2, hours 0 to 2; FU 400 mg/m2 IV push on days 1 and 2; and FU 600 mg/m2 IV on days 1 and 2, hours 2 to 24 (FOLFOX-4). All subsequent cycles consisted of FOLFOX-4 with gefitinib at 500 mg/d administered orally throughout the 14-day cycle. RESULTS: Twenty-seven patients were enrolled onto the study. The median number of prior chemotherapy regimens was two, and 74% of all patients received prior irinotecan. Nine of the 27 patients (33%) and six of the 20 patients (30%) who had prior FU and irinotecan had a partial response by Response Evaluation Criteria in Solid Tumors Group criteria. Median overall survival was 12.0 months. Median event-free survival was 5.4 months. Grade 3 to 4 toxicities included neutropenia (48%), diarrhea (48%), nausea (22%), and vomiting (15%). CONCLUSION: IFOX is an active regimen in patients with previously treated metastatic colorectal adenocarcinoma, demonstrating higher response rates than those reported with FOLFOX-4 alone in a similar patient population.     

Cyr61 induces gastric cancer cell motility/invasion via activation of the integrin/nuclear factor-kappaB/cyclooxygenase-2 signaling pathway.

PURPOSE: Cysteine-rich 61 (Cyr61/CCN1) is involved in many different types of tumor development and progression. Nonetheless, the role of Cyr61 in human gastric cancer has not yet been fully characterized.Experimental design: We addressed the issue by immunohistochemical staining of 81 gastric adenocarcinoma specimens. Liposome-mediated transfection was used to introduce a Cyr61 expression vector into gastric cancer AGS cell lines. Transfectants were tested in invasion assay by a Boyden chamber. Furthermore, a cyclooxygenase-2 (COX-2) reporter assay and gel mobility shift assay were done to investigate the potential signal pathway of Cyr61. RESULTS: Patients with gastric adenocarcinoma whose tumor displayed high expression of Cyr61 correlated well with aggressive lymph node metastasis, more advanced tumor stage, histologic diffuse type, and early recurrence. Stable transfection of Cyr61 into the AGS cell line strongly enhanced its invasive activity. The overexpression of Cyr61 into AGS cells significantly increased the expression of COX-2 mRNA, protein, and enzymatic activity. Gel mobility shift assays further showed that the nuclear factor-kappaB (NF-kappaB) pathway was evidently activated in Cyr61-expressing AGS cells. Function-neutralizing antibody to alphavbeta3 but not alphavbeta5 effectively suppressed Cyr61-mediated NF-kappaB activation, COX-2 gene expression, and cell invasiveness. CONCLUSIONS: Cyr61 may contribute to the malignant progression of gastric cancer by promoting tumor cell motility/invasion through up-regulation of the functional COX-2 via an integrin alphavbeta3/NF-kappaB-dependent pathway.