Possible autocrine enkephalin regulation of catecholamine release in human pheochromocytoma cells

AIMS: Pheochromocytomas are catecholamine-secreting tumors that also synthesize and secrete several neuropeptides, including opioids. A negative regulation of catecholamine secretion by opioids has been postulated in chromaffin cells. However, results obtained so far are contradictory when referred to human pheochromocytomas. The aim of this study was to define the role of locally produced enkephalins on catecholamine release in human pheochromocytoma cells. MAIN METHODS: Cells obtained from eleven human pheochromocytomas of different genetic origins were cultured for 5 days. Cultures were maintained under basal condition or under enkephalin, dexamethasone and naloxone alone or in combination with enkephalin or dexamethasone-stimulated conditions. Catecholamine and enkephalin levels in the culture medium were measured by HPLC-ED and RIA respectively. KEY FINDINGS: Enkephalin induced a decrease in norepinephrine levels in all tumor cultures. Dexamethasone treatment, which increased enkephalin levels, also decreased catecholamine levels. On the other hand, the addition of naloxone to the cultures reverted to normal the inhibitory action exerted by enkephalin and dexamethasone treatments. SIGNIFICANCE: These results suggest the existence of an autocrine negative regulatory loop exerted by enkephalin on norepinephrine release in human pheochromocytoma cells.     

IL28B Gene Polymorphisms and US Liver Fatty Changes in Patients Who Spontaneously Cleared Hepatitis C Virus Infection

Background

Recent clinical studies have shown that the presence of CC genotype in the rs12979860 region of IL28B gene is associated with an increase in the probability of spontaneous clearance of hepatitis C virus (HCV). Moreover, IL28B polymorphism seems to influence the probability of developing liver steatosis in chronic HCV patients.

Aims

The aims of our clinical study were 1) to verify the distribution of IL28B genotypes (CC, CT or TT) among subjects with spontaneous clearance of HCV infection and 2) to examine the correlation between IL28B polymorphism and hepatic steatosis among these subjects.

Methods and patients

We enrolled 41 subjects with spontaneous resolution of HCV infection (detectable serum anti-HCV but undetectable HCV-RNA) and 134 healthy controls from the same geographical area. The IL28B single-nucleotide polymorphism (SNP) rs12979860 was genotyped by using a Pyrosequencing™ technique. The presence of steatosis was assessed by liver biopsy or ultrasound examination in the 41 study subjects.

Results

CC, CT and TT-genotypes of the SNP rs1979860 were found in 66%, 24% and 10% of the subjects who spontaneously cleared HCV and in 31%, 54% and 15% of controls, respectively (p = 0.0003). Among the study subjects, females with CC-genotype were significantly more represented (p = 0.02). Hepatic steatosis did not correlate with IL28B genotype (p = 0,14) but only with a high body mass index (BMI) value (p = 0.03).

Conclusions

Female subjects carrying IL28B CC-genotype are significantly more represented among Italian patients who spontaneously cleared HCV infection. In addition, among these subjects, the presence of liver steatosis does not correlate with IL28B genotype but is solely related to the occurrence of high BMI. Thus, the association between IL28B polymorphism and steatosis in chronic HCV patients requires the presence of active HCV replication to occur, while in subjects who have cleared the infection, the mechanism(s) inducing liver steatosis are independent from IL28B profile.     

Susceptibility of Murine Norovirus and Hepatitis A Virus to Electron Beam Irradiation in Oysters and Quantifying the Reduction in Potential Infection Risks

Consumption of raw oysters is an exposure route for human norovirus (NoV) and hepatitis A virus (HAV). Therefore, efficient postharvest oyster treatment technology is needed to reduce public health risks. This study evaluated the inactivation of HAV and the NoV research surrogate, murine norovirus-1 (MNV-1), in oysters (Crassostrea virginica) by electron beam (E-beam) irradiation. The reduction of potential infection risks was quantified for E-beam irradiation technology employed on raw oysters at various virus contamination levels. The E-beam dose required to reduce the MNV and HAV titer by 90% (D₁₀ value) in whole oysters was 4.05 (standard deviations [SD], ±0.63) and 4.83 (SD, ±0.08) kGy, respectively. Microbial risk assessment suggests that if a typical serving of 12 raw oysters was contaminated with 10⁵ PFU, a 5-kGy treatment would achieve a 12% reduction (from 4.49 out of 10 persons to 3.95 out of 10 persons) in NoV infection and a 16% reduction (from 9.21 out of 10 persons to 7.76 out of 10 persons) in HAV infections. If the serving size contained only 10² PFU of viruses, a 5-kGy treatment would achieve a 26% reduction (2.74 out of 10 persons to 2.03 out of 10 persons) of NoV and 91% reduction (2.1 out of 10 persons to 1.93 out of 100 persons) of HAV infection risks. This study shows that although E-beam processing cannot completely eliminate the risk of viral illness, infection risks can be reduced.     

Immobilization and stabilization of a bimolecular aggregate of the lipase from Pseudomonas fluorescens by multipoint covalent attachment

The soluble lipase from Pseudomonas fluorescens (PFL) forms bimolecular aggregates in which the hydrophobic active centers of the enzyme monomers are in close contact. This bimolecular aggregate could be immobilized by multipoint covalent linkages on glyoxyl supports at pH 8.5. The monomer of PFL obtained by incubation of the soluble enzyme in the presence of detergent (0.5% TRITON X-100) could not be immobilized under these conditions. The bimolecular aggregate has two amino terminal residues in the same plane. A further incubation of the immobilized derivative under more alkaline conditions (e.g., pH 10.5) allows a further multipoint attachment of lysine (Lys) residues located in the same plane as the amino terminal residues. Monomeric PFL was immobilized at pH 10.5 in the presence of 0.5% TRITON X-100. The properties of both PFL derivatives were compared. In general, the bimolecular derivatives were more active, more selective and more stable both in water and in organic solvents than the monomolecular ones. The bimolecular derivative showed twice the activity and a much higher selectivity (100 versus 20) for the hydrolysis of R,S-2-hydroxy-4-phenylbutyric acid ethyl ester (HPBEt) in aqueous media at pH 5.0 compared to the monomeric derivative. In experiments measuring thermal inactivation at 75 °C, the bimolecular derivative was 5-fold more stable than the monomeric derivative (and 50-fold more stable than a one-point covalently immobilized PFL derivative), and it had a half-life greater than 4 h. In organic solvents (cyclohexane and tert-amyl alcohol), the bimolecular derivative was much more stable and more active than the monomeric derivative in catalyzing the transesterification of olive oil with benzyl alcohol.     

AUXIN UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCF(AUF1/2) provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.     

The extent of whole‐genome copy number alterations predicts aggressive features in primary melanomas

Recent evidence indicates that melanoma comprises distinct types of tumors and suggests that specific morphological features may help predict its clinical behavior. Using a SNP‐array approach, we quantified chromosomal copy number alterations (CNA) across the whole genome in 41 primary melanomas and found a high degree of heterogeneity in their genomic asset. Association analysis correlating the number and relative length of CNA with clinical, morphological, and dermoscopic attributes of melanoma revealed that features of aggressiveness were strongly linked to the overall amount of genomic damage. Furthermore, we observed that melanoma progression and survival were mainly affected by a low number of large chromosome losses and a high number of small gains. We identified the alterations most frequently associated with aggressive melanoma, and by integrating our data with publicly available gene expression profiles, we identified five genes which expression was found to be necessary for melanoma cells proliferation. In conclusion, this work provides new evidence that the phenotypic heterogeneity of melanoma reflects a parallel genetic diversity and lays the basis to define novel strategies for a more precise prognostic stratification of patients.     

Ancient DNA reveals differences in behaviour and sociality between brown bears and extinct cave bears

Ancient DNA studies have revolutionized the study of extinct species and populations, providing insights on phylogeny, phylogeography, admixture and demographic history. However, inferences on behaviour and sociality have been far less frequent. Here, we investigate the complete mitochondrial genomes of extinct Late Pleistocene cave bears and middle Holocene brown bears that each inhabited multiple geographically proximate caves in northern Spain. In cave bears, we find that, although most caves were occupied simultaneously, each cave almost exclusively contains a unique lineage of closely related haplotypes. This remarkable pattern suggests extreme fidelity to their birth site in cave bears, best described as homing behaviour, and that cave bears formed stable maternal social groups at least for hibernation. In contrast, brown bears do not show any strong association of mitochondrial lineage and cave, suggesting that these two closely related species differed in aspects of their behaviour and sociality. This difference is likely to have contributed to cave bear extinction, which occurred at a time in which competition for caves between bears and humans was likely intense and the ability to rapidly colonize new hibernation sites would have been crucial for the survival of a species so dependent on caves for hibernation as cave bears. Our study demonstrates the potential of ancient DNA to uncover patterns of behaviour and sociality in ancient species and populations, even those that went extinct many tens of thousands of years ago.     

Global genetic diversity of Aedes aegypti

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co‐occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub‐Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans‐Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.