Abundant expression and purification of biologically active mitochondrial citrate carrier in baculovirus-infected insect cells

Heterologous expression of recombinant proteins is an essential technology for protein characterization. A major obstacle to investigating the biochemical properties of membrane proteins is the difficulty in obtaining sufficient amounts of functional protein. Here we report the successful expression of the tricarboxylate (or citrate) carrier (CIC) of eel (Anguilla anguilla) from Spodoptera frugiperda (Sf9) cells using the baculovirus expression system. The recombinant CIC was purified by affinity chromatography on Ni²⁺-NTA agarose; the yield of the purified active protein was 0.4–0.5 mg/l of culture. The transport characteristics of the recombinant CIC and the effects of inhibitors on transport are similar to those determined for eel liver mitochondrial CIC. Because the CIC is one member of an extensive family of mitochondrial transport proteins, it is likely that the procedure used in this study to express and purify this carrier can be successfully applied to other mitochondrial transport proteins, thus providing sufficient protein for functional characterization. Marianna Madeo and Chiara Carrisi contributed equally to this work.     

Glutathionylation of the Pro-apoptotic Protein p53 in Alzheimer’s Disease Brain: Implications for AD Pathogenesis

Neurochemical Research -     

Glutathionylation of the Pro-apoptotic Protein p53 in Alzheimer’s Disease Brain: Implications for AD Pathogenesis

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder. The exact mechanism for the AD pathogenesis is not clearly understood. However, a number of hypotheses have been proposed to explain the pathogenesis of AD. One the hypotheses is the oxidative stress hypothesis that is supported by a number of studies which reported an increase in the levels of reactive oxygen/reactive nitrogen species and their products with a concomitant decrease in the levels of antioxidant enzymes in AD brain. In the present study, we measured in AD brain the expression levels of different forms (monomer, dimer and tetramer) of the pro-apoptotic protein, p53, and observed greater levels of p53 monomer and dimer in AD brain compared to control. In addition, we also showed the selective glutathionylation of monomeric and dimeric form of p53 in AD brain. We propose that glutathionylation of p53 may prevent the formation of tetramer, an aggregate form required for effective action of p53, and may be involved in oxidative stress conditions and neurodegeneration observed in this dementing disorder. Special issue article in Honour of Dr. Akitane Mori. Fabio Di Domenico and Giovanna Cenini contributed equally. An erratum to this article can be found at     

A New Species of Rhaponticoides (Asteraceae) from Southern Italy

A new diploid (2n?=?30) species, Rhaponticoides calabrica, is described from Calabria and Basilicata (southern Italy). This species differs from the closely related R. centaurium – an endemic of Apulia and northern Basilicata – by different flower colour (white in the former, purplish in the latter), width of membranous margin of phyllaries (1.8?±?0.4 mm vs 0.8?±?0.3 mm) and pappus length (6.8?±?0.9 mm vs 9.1?±?1.0 mm). The two species are completely allopatric and seem to have also different chromosome numbers.     

Surgical treatment of a giant tibial high-grade mixofibrosarcoma with preservation of limb function: a case report

Myxofibrosarcoma is one of the most common sarcomas in elderly patients showing a slight male prevalence. The tumor is mainly located in lower and upper extremities and rarely in trunk, neck and feet. We present a case of a 84-year-old man referred to our tumour center with a giant and neglected high-grade tibial myxofibrosarcoma in the anteromedial side of tibial mid-diaphysis. Large size lesions in association with older age may jeopardise the maintenance of limb vitality, vascularity and stability.Authors performed a complete tumour resection, followed by reconstruction of bone and soft tissue defects with cement, plate and a musculocutaneous gastrocnemius flap, in order to cover the underlying bone and promote uneventful healing and perfusion of the operated extremity.At 2 years postoperatively, limb salvage, good functional outcome and no tumour recurrence were reported while the patient was able to effectively perform the majority of the daily activities.     

Hepatic FoxO1 Integrates Glucose Utilization and Lipid Synthesis through Regulation of Chrebp O-Glycosylation

In liver, glucose utilization and lipid synthesis are inextricably intertwined. When glucose availability exceeds its utilization, lipogenesis increases, leading to increased intrahepatic lipid content and lipoprotein secretion. Although the fate of three-carbon metabolites is largely determined by flux rate through the relevant enzymes, insulin plays a permissive role in this process. But the mechanism integrating insulin receptor signaling to glucose utilization with lipogenesis is unknown. Forkhead box O1 (FoxO1), a downstream effector of insulin signaling, plays a central role in hepatic glucose metabolism through the regulation of hepatic glucose production. In this study, we investigated the mechanism by which FoxO1 integrates hepatic glucose utilization with lipid synthesis. We show that FoxO1 overexpression in hepatocytes reduces activity of carbohydrate response element binding protein (Chrebp), a key regulator of lipogenesis, by suppressing O-linked glycosylation and reducing the protein stability. FoxO1 inhibits high glucose- or O-GlcNAc transferase (OGT)-induced liver-pyruvate kinase (L-PK) promoter activity by decreasing Chrebp recruitment to the L-PK promoter. Conversely, FoxO1 ablation in liver leads to the enhanced O-glycosylation and increased protein level of Chrebp owing to decreased its ubiquitination. We propose that FoxO1 regulation of Chrebp O-glycosylation is a mechanism linking hepatic glucose utilization with lipid synthesis.     

Size Distribution of Sperm Whales Acoustically Identified during Long Term Deep-Sea Monitoring in the Ionian Sea

The sperm whale (Physeter macrocephalus) emits a typical short acoustic signal, defined as a “click”, almost continuously while diving. It is produced in different time patterns to acoustically explore the environment and communicate with conspecifics. Each emitted click has a multi-pulse structure, resulting from the production of the sound within the sperm whale’s head. A Stable Inter Pulse Interval (Stable IPI) can be identified among the pulses that compose a single click. Applying specific algorithms, the measurement of this interval provides useful information to assess the total length of the animal recorded. In January 2005, a cabled hydrophone array was deployed at a depth of 2,100 m in the Central Mediterranean Sea, 25 km offshore Catania (Ionian Sea). The acoustic antenna, named OνDE (Ocean noise Detection Experiment), was in operation until November 2006. OνDE provided real time acoustic data used to perform Passive Acoustic Monitoring (PAM) of cetacean sound emissions. In this work, an innovative approach was applied to automatically measure the Stable IPI of the clicks, performing a cepstrum analysis to the energy (square amplitude) of the signals. About 2,100 five-minute recordings were processed to study the size distribution of the sperm whales detected during the OνDE long term deep-sea acoustic monitoring. Stable IPIs were measured in the range between 2.1 ms and 6.4 ms. The equations of Gordon (1991) and of Growcott (2011) were used to convert the IPIs into measures of size. The results revealed that the sperm whales recorded were distributed in length from about 7.5 m to 14 m. The size category most represented was from 9 m to 12 m (adult females or juvenile males) and specimens longer than 14 m (old males) seemed to be absent.