Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.     

Medium composition and methyl jasmonate influence the amount and spectrum of secondary metabolites in callus cultures of Zanthoxylum stenophyllum Hemsl.

Callus cultures of Zanthoxylum stenophyllum were initiated in vitro and the effect of growth regulators and elicitors was tested both upon callus growth and secondary metabolite production. On a medium containing naphthaleneacetic acid, kinetin, and 2,4-dichlorophenoxyacetic acid, a yellowish and friable callus was obtained from 90% of cotyledon explants. Callus growth and secondary metabolite accumulation was followed after sub-culturing the established callus culture on different media containing various hormonal combinations. Results indicate that medium containing naphthaleneacetic acid and a higher concentration of 2,4-dichlorophenoxyacetic acid gave the highest stimulation of growth. Addition of an organic nitrogen source also had a positive effect on growth. Rapid HPLC screening of methanol extractable secondary metabolites from calluses showed that phytohormones and nutrients were able to modify the chromatographic pattern of compounds. Calluses grown on the medium giving the highest stimulation of growth show a reduced accumulation of some secondary products, but not all. In response to elicitation by methyl jasmonate, metabolite production was different for the different classes of compounds, and hormonal composition of the culture medium influenced the response. Thus, results confirm the importance of the reciprocal interactions between hormones, nutrients, and elicitors when attempts are made to enhance secondary metabolite accumulation in in vitro cultures.     

How formalin affects the outcome of routine and special stains

The discovery of formaldehyde for preserving tissue structures produced a new dimension in microscopy. Preserving structure and morphology became important; therefore, identifying a proper fixing agent for particular structures, chemical entities, and tissues, also became important. The methods for demonstrating tissue structures evolved and were implemented with careful observation and documentation of the results and outcomes. Formalin was incorporated into many techniques, and provided helpful results in many cases and hindrances in others. The effects of formalin on the outcomes of routine and special staining techniques are reported here.     

Interfacial Behavior and Antioxidant Efficiency of Olive Phenolic Compounds in O/W Olive oil Emulsions as Affected by Surface Active Agent Type

The aim of this study was to evaluate the interfacial behavior of syringic acid, tyrosol and oleuropein, phenolic antioxidant compounds naturally present in olives and olive oils, and their ability to influence the stability to lipid oxidation of olive oil O/W emulsions. To test also the interactions of these molecules with other components and the effects on their activity, two different surfactants were used to prepare the olive oil emulsions, Tween 20, and a whey protein concentrate (WPC). All the antioxidants affected the olive oil/water interfacial tension; among them, oleuropein showed the highest interfacial activity and, thus, is supposed to locate at the interface. In emulsified state, the presence of the phenolic compounds in WPC emulsions did not cause any significant effect on the dispersion degree if compared to the control whilst a general improvement was observed in Tween 20-emulsions, in particular when oleuropein was added systems. The antioxidants were thus proven not to impair the dispersed structure but rather to improve it. As regards the oxidative stability, the antioxidants under investigation caused the occurrence of similar induction phases in the hydroperoxides production not observed in the control emulsions. In the case of secondary oxidation products, the highest inhibition was achieved in both the emulsified systems by oleuropein. In general, however, a lower amount of both primary and secondary oxidation products were observed in WPC emulsions than in Tween 20-emulsions likely due to the antioxidative effect of whey proteins.     

Identification of Candidate Children for Maturity-Onset Diabetes of the Young Type 2 (MODY2) Gene Testing: A Seven-Item Clinical Flowchart (7-iF)

MODY2 is the most prevalent monogenic form of diabetes in Italy with an estimated prevalence of about 0.5–1.5%. MODY2 is potentially indistinguishable from other forms of diabetes, however, its identification impacts on patients'' quality of life and healthcare resources. Unfortunately, DNA direct sequencing as diagnostic test is not readily accessible and expensive. In addition current guidelines, aiming to establish when the test should be performed, proved a poor detection rate. Aim of this study is to propose a reliable and easy-to-use tool to identify candidate patients for MODY2 genetic testing. We designed and validated a diagnostic flowchart in the attempt to improve the detection rate and to increase the number of properly requested tests. The flowchart, called 7-iF, consists of 7 binary “yes or no” questions and its unequivocal output is an indication for whether testing or not. We tested the 7-iF to estimate its clinical utility in comparison to the clinical suspicion alone. The 7-iF, in a prospective 2-year study (921 diabetic children) showed a precision of about the 76%. Using retrospective data, the 7-iF showed a precision in identifying MODY2 patients of about 80% compared to the 40% of the clinical suspicion. On the other hand, despite a relatively high number of missing MODY2 patients, the 7-iF would not suggest the test for 90% of the non-MODY2 patients, demonstrating that a wide application of this method might 1) help less experienced clinicians in suspecting MODY2 patients and 2) reducing the number of unnecessary tests. With the 7-iF, a clinician can feel confident of identifying a potential case of MODY2 and suggest the molecular test without fear of wasting time and money. A Qaly-type analysis estimated an increase in the patients'' quality of life and savings for the health care system of about 9 million euros per year.     

Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in cardiac hypoxia/reoxygenation-related damage?

Nitric oxide (NO) has been reported to act both as a destructive and a protective agent in the pathogenesis of the injuries that occur during hypoxia/reoxygenation (H/R). It has been suggested that this dual role of NO depends directly on the isoform of NO synthase (NOS) involved. In this work, we investigate the role that NO derived from endothelial NOS (eNOS) plays in cardiac H/R-induced injury. Wistar rats were submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 12 h and 5 days), with or without prior treatment using the selective eNOS inhibitor l-NIO (20 mg/kg). Lipid peroxidation, apoptosis and protein nitration, as well as NO production (NOx), were analysed. The results showed that l-NIO administration lowered NOx levels in all the experimental groups. However, no change was found in the lipid peroxidation level, the percentage of apoptotic cells or nitrated protein expression, implying that eNOS-derived NO may not be involved in the injuries occurring during H/R in the heart. We conclude that l-NIO would not be useful in alleviating the adverse effects of cardiac H/R.     

Involvement of cerebellum in emotional behavior

In the last decade a growing body of data revealed that the cerebellum is involved in the regulation of the affective reactions as well as in forming the association between sensory stimuli and their emotional values. In humans, cerebellar areas around the vermis are activated during mental recall of emotional personal episodes and during learning of a CS-US association. Lesions of the cerebellar vermis may affect retention of a fear memory without altering baseline motor/autonomic responses to the frightening stimuli in both human and animal models. Reversible inactivation of the vermis during the consolidation period impairs retention of fear memory in rodents. Recent findings demonstrate that long-term potentiation (LTP) of synapses in the cerebellar cortex occurs in relation to associative fear learning similar to previously reported data in the hippocampus and amygdala. Plastic changes affect both excitatory and inhibitory synapses. This concomitant potentiation allows the cerebellar cortical network to detect coincident inputs, presumably conveying sensorial stimuli, with better efficacy by keeping the time resolution of the system unchanged. Collectively, these data suggest that the vermis participates in forming new CS-US association and translate an emotional state elaborated elsewhere into autonomic and motor responses.     

Muscle fibre conduction velocity and cardiorespiratory response during incremental cycling exercise in young and older individuals with different training status

We investigated the effect of ageing and training on muscle fibre conduction velocity (MFCV) and cardiorespiratory response during incremental cycling exercise. Eight young (YT; 24 ± 5 yrs) and eight older (OT; 64 ± 3 yrs) cyclists, together with eight young (YU; 27 ± 4 yrs) and eight older (OU; 63 ± 2 yrs) untrained individuals underwent to an incremental maximal test on a cycle ergometer. Ventilatory threshold (VT), respiratory compensation point (RCP) and maximal oxygen uptake (VO₂max) were identified and MFCV recorded from the vastus lateralis muscle using surface electromyography with linear arrays electrodes.In YT MFCV increased with the exercise intensity, reaching a peak of 4.99 ± 1.02 [m/s] at VT. Thereafter, and up to VO₂max, MFCV declined. In YU MFCV showed a similar trend although the peak [4.55 ± 0.53 m/s] was observed, at 75% of VO₂max an intensity higher than VT (66% of VO₂max). In both YT and YU MFCV did not decline until RPC, which occurred at 78% VO₂max in YU and at 92% VO₂max (P < 0.01) in YT. Differently from young individuals, MFCV in older subjects did not increase with exercise intensity. Moreover, maximal MFCV in OU was significantly lower [3.53 ± 0.40 m/s;] than that of YT (P < 0.005) and YU (P < 0.05).The present study shows that, especially in young individuals, MFCV reflects cardiorespiratory response during incremental dynamic cyclic exercise and hence can be used to investigate motor unit recruitment strategies.     

The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.