Phospholipid base exchange enzyme activity in sarcolemmal membranes from the heart of cardiomyopathic hamsters

The activity of phospholipid base exchange enzymes has been evaluated in cardiac sarcolemmal membranes from Syrian Golden hamsters and from a hamster strain (UM-X7.1) characterized by a genetic form of hypertrophic cardiomyopathy. No choline base exchange activity and only a little serine base exchange activity were detected, whereas the ethanolamine base exchange enzyme was found highly active in membranes from both strains. For this reason, the present study is focussed on the ethanolamine base exchange enzyme. The apparent Km for ethanolamine of ethanolamine base exchange enzyme from Syrian Golden membranes and from UM-X7.1 strain membranes are 18 and 32 μM, respectively. The specific activity of the sarcolemmal ethanolamine base exchange enzyme is lower in the UM-X7.1 strain than in Syrian Golden hamsters. The calcium-dependence of the enzyme appears different when the membranes from the two strains are compared. Indeed, after removal of the membrane-bound divalent cations, comparable activities are found in both membrane preparations, whereas, upon addition of Ca²⁺ to the incubation mixtures, the activity of the enzyme is enhanced in the membranes from Syrian Golden strain more than in those from UM-X7.1 strain. The cholesterol content of sarcolemmal membranes is higher in the cardiomyopathic strain than in the Syrian Golden hamsters. A possible relation between changes of the membrane lipid composition and of the ethanolamine base exchange activity is discussed.     

PI-3-kinase/NF-kappaB mediated response of Jurkat T leukemic cells to two different chemotherapeutic drugs, etoposide and TRAIL

Jurkat T leukemic cells respond to Etoposide, antineoplastic agent which targets the DNA unwinding enzyme, Topoisomerase II, and TNF-Related-Apoptosis-Inducing-Ligand (TRAIL), 34 kDa transmembrane protein, which displays minimal or no toxicity on normal cells and tissues, not only disclosing the occurrence of apoptosis but also a kind of resistance. A similar rate of viability upon the exposure to these two drugs up to 24 h has been evidenced, followed by the occurrence of a rescue process against TRAIL, not performed against Etoposide, along with an higher number of dead cells upon Etoposide exposure, in comparison with TRAIL treatment. These preliminary results let us to speculate on the possible involvement of PI-3-kinase in TRAIL resistance disclosed by surviving cells (20%), may be phosphorylating Akt-1 and, in parallel, IkappaB alpha on both serine and tyrosine residues. On the other hand, in Etoposide Jurkat exposed cells Ser 32-36 phosphorylation of IkappaB alpha is not sufficient to overbalance the apoptotic fate of the cells, since Bax increase, IAP decrease, and caspase-3 activation determine the persistence of the apoptotic state along with the occurrence of cell death by necrosis. Thus, the existence of a balance between apoptotic and rescue response in 20% of cells surviving to TRAIL suggests the possibility of pushing it in favor of cell death in order to improve the yield of pharmacological strategies.     

A molecular beacon, bead-based assay for the detection of nucleic acids by flow cytometry

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.     

Peptide receptor radionuclide therapy (PRRT) with 177Lu-DOTATATE in individuals with neck or mediastinal paraganglioma (PGL)

Paragangliomas (PGLs) are neuroendocrine tum-ors that arise embryologically from the neural crest. Sympathetic PGLs can be located in the thoracic-abdominal region while parasympathetic PGLs are mainly situated in the head and neck region. Most PGLs are sporadic, but in 30% of cases they are hereditary (associated with mutations of SDHB, SDHC, SDHD, SDHAF2, SDHA, TMEM, MAX, and VHL); they can be classified into 4 different paraganglioma syndromes: PGL1, PGL2, PGL3, and PGL4. Surgery is the treatment of choice for both sympathetic and parasympathetic PGLs. Other types of treatment include medical agents (such as gemcitabine, cisplatin, or sunitinib) and radiotherapy (external-beam radiotherapy or stereotactic surgery). Surgery and radiotherapy, however, can cause important side effects such as vascular complications and peripheral nerve damage (hypoglossal, recurrent laryngeal, glossopharyngeal, and vagus). Another possible treatment option is the use of peptide receptor radionuclide therapy (PRRT), including PRRT with 177Lu-DOTATATE. We studied 4 patients with hereditary nonmetastatic paraganglioma syndrome type 1 (PGL1), with progressive disease, in whom surgical excision was not possible. They were treated with 177Lu-DOTATATE (3-5 cycles) and all had a partial response (PR) or a stable disease (SD) to the treatment. In conclusion, a good alternative treatment when surgical or radiation therapy are contraindicated could be radiometabolic therapy with 177Lu-DOTATATE.     

Subcellular localization of APE1/Ref-1 in human hepatocellular carcinoma: possible prognostic significance

APE1/Ref-1, normally localized in the nucleus, is a regulator of the cellular response to oxidative stress. Cytoplasmic localization has been observed in several tumors and correlates with a poor prognosis. Because no data are available on liver tumors, we investigated APE1/Ref-1 subcellular localization and its correlation with survival in 47 consecutive patients undergoing hepatocellular carcinoma (HCC) resection. APE1/Ref-1 expression was determined by immunohistochemistry in HCC and surrounding liver cirrhosis (SLC) and compared with normal liver tissue. Survival probability was evaluated using Kaplan-Meier curves (log-rank test) and Cox regression. Cytoplasmic expression of APE1/Ref-1 was significantly higher in HCC than in SLC (P = 0.00001); normal liver showed only nuclear reactivity. Patients with poorly differentiated HCC showed a cytoplasmic expression three times higher than those with well-differentiated HCC (P = 0.03). Cytoplasmic localization was associated with a median survival time shorter than those with negative cytoplasmic reactivity (0.44 compared with 1.64 years, P = 0.003), and multivariable analysis confirmed that cytoplasmic APE1/Ref-1 localization is a predictor of survival. Cytoplasmic expression of APE1/Ref-1 is increased in HCC and is associated with a lower degree of differentiation and a shorter survival time, pointing to the use of the cytoplasmic localization of APE1/Ref-1 as a prognostic marker for HCC.     

Secretion of alpha-amylase from Pseudoalteromonas haloplanktis TAB23: two different pathways in different hosts

Secretion of cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria. We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for alpha-amylase secretion in Escherichia coli. The occurrence of two different secretion pathways in different hosts is proposed.     

海拔对辽东栎林地土壤微生物群落的影响

以北京东灵山辽东栎林地土壤为对象,运用氯仿熏蒸-浸提法及磷脂脂肪酸分析(PLFA)法,研究林木生长季节土壤微生物群落随海拔梯度的变化特征.结果表明:随着海拔升高,辽东栎林土壤微生物生物量碳、氮,以及微生物各类群含量均有差异但不显著;土壤细菌/真菌升高,而革兰氏阳性菌(G+)/革兰氏阴性菌(G-)降低.土壤微生物生物量碳、氮以及细菌、真菌、G+细菌、G-细菌的含量与土壤含水量、有机碳、全氮呈显著正相关,土壤真菌含量与土壤碳氮比值呈正相关.土壤微生物群落组成结构(细菌/真菌和G+细菌/G-细菌)的变化主要受土壤温度和土壤含水量的显著影响,说明土壤微生物群落结构对环境条件的变化敏感.随着全球变暖的加剧,暖温带辽东栎林地土壤真菌和G+细菌的比例有升高的趋势.     

The Vitamin D Receptor Agonist BXL-01-0029 as a Potential New Pharmacological Tool for the Treatment of Inflammatory Myopathies

Objective

This study aims to investigate in vitro the effect of the VDR agonist BXL-01-0029 onto IFNγ/TNFα-induced CXCL10 secretion by human skeletal muscle cells compared to elocalcitol (VDR agonist), methylprednisolone, methotrexate, cyclosporin A, infliximab and leflunomide; to assess in vivo circulating CXCL10 level in subjects at time of diagnosis with IMs, before therapy, together with TNFα, IFNγ, IL-8, IL-6, MCP-1, MIP-1β and IL-10, vs. healthy subjects.

Methods

Human fetal skeletal muscle cells were used for in vitro studies; ELISA and Bio-Plex were used to measure cell supernatant and IC₅₀ determination or serum cytokines; Western blot and Bio-Plex were for cell signaling analysis.

Results

BXL-01-0029 decreased with the highest potency IFNγ/TNFα-induced CXCL10 protein secretion and targeted cell signaling downstream of TNFα in human skeletal muscle cells; CXCL10 level was the highest in sera of subjects diagnosed with IMs before therapy and the only one significantly different vs. healthy controls.

Conclusions

Our in vitro and in vivo data, while confirm the relevance of CXCL10 in IMs, suggested BXL-01-0029 as a novel pharmacological tool for IM treatment, hypothetically to be used in combination with the current immunosuppressants to minimize side effects.