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The family Picornaviridae consists of a large group of plus-strand RNA viruses that share a similar genome organization. The nomenclature of the picornavirus proteins is based on their position in the viral RNA genome but does not necessarily imply a conserved function of proteins of different genera. The enterovirus 2B protein is a small hydrophobic protein that, upon individual expression, is localized to the endoplasmic reticulum (ER) and the Golgi complex, reduces ER and Golgi complex Ca 2؉ levels, most likely by forming transmembrane pores, and inhibits protein trafficking through the Golgi complex. At present, little is known about the function of the other picornavirus 2B proteins. Here we show that rhinovirus 2B, which is phylogenetically closely related to enterovirus 2B, shows a similar subcellular localization and function to those of enterovirus 2B. In contrast, 2B proteins of hepatitis A virus, foot-and-mouth disease virus, and encephalomyocarditis virus, all of which are more distantly related to enteroviruses, show a different localization and have little, if any, effects on Ca 2؉ homeostasis and intracellular protein trafficking. Our data suggest that the 2B proteins of enterovirus and rhinovirus share the same function in virus replication, while the other picornavirus 2B proteins support the viral life cycle in a different manner. Moreover, we show that an enterovirus 2B protein that is retained in the ER is unable to modify Ca 2؉ homeostasis and inhibit protein trafficking, demonstrating the importance of Golgi complex localization for its functioning.The family Picornaviridae is a group of small, nonenveloped cytolytic viruses that include a number of important human and animal pathogens. The picornavirus family consists of nine genera, including enterovirus (e.g., coxsackievirus [CBV] and poliovirus [PV]), rhinovirus (e.g., human rhinovirus [HRV]), cardiovirus (e.g., encephalomyocarditis virus [EMCV]), aphthovirus (e.g., foot-and-mouth disease virus [FMDV]), hepatovirus (hepatitis A virus [HAV]), teschovirus (e.g., porcine teschovirus), erbovirus (e.g., equine rhinitis B virus), parechovirus (e.g., parechovirus 2), and kobuvirus (e.g., aichivirus). In addition, the picornavirus family contains a number of unassigned viruses. All picornaviruses have a similar genome organization. The viral genome typically consists of a positivestranded RNA molecule of approximately 7,500 to 8,000 nucleotides that contains one single large open reading frame preceded by a long 5Ј-untranslated region and followed by a much smaller 3Ј-untranslated region and a genetically encoded poly(A) tail. A small viral protein, VPg, is covalently linked to the 5Ј end of the viral genome. Translation of the RNA genome yields a polyprotein of approximately 2,200 amino acids (aa) that is divided into the P1, P2, and P3 regions. The polyprotein is processed by virus-encoded proteases to generate the individual structural and nonstructural proteins. Processing of the P1 region yields the structural capsid proteins 1A (VP4), 1B...

In the present study, we investigated DNA barcoding effectiveness to characterize honeybee pollen pellets, a food supplement largely used for human nutrition due to its therapeutic properties. We collected pollen pellets using modified beehives placed in three zones within an alpine protected area (Grigna Settentrionale Regional Park, Italy). A DNA barcoding reference database, including rbcL and trnH-psbA sequences from 693 plant species (104 sequenced in this study) was assembled. The database was used to identify pollen collected from the hives. Fifty-two plant species were identified at the molecular level. Results suggested rbcL alone could not distinguish among congeneric plants; however, psbA-trnH identified most of the pollen samples at the species level. Substantial variability in pollen composition was observed between the highest elevation locality (Alpe Moconodeno), characterized by arid grasslands and a rocky substrate, and the other two sites (Cornisella and Ortanella) at lower altitudes. Pollen from Ortanella and Cornisella showed the presence of typical deciduous forest species; however in samples collected at Ortanella, pollen of the invasive Lonicera japonica, and the ornamental Pelargonium x hortorum were observed. Our results indicated pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species. Therefore, pollen molecular characterization based on DNA barcoding might serve useful to beekeepers in obtaining honeybee products with specific nutritional or therapeutic characteristics desired by food market demands.

We investigated the effects of 1 and 10 mg L(-1) AgNPs on germinating Triticum aestivum L. seedlings. The exposure to 10 mg L(-1) AgNPs adversely affected the seedling growth and induced morphological modifications in root tip cells. TEM analysis suggests that the observed effects were due primarily to the release of Ag ions from AgNPs. To gain an increased understanding of the molecular response to AgNP exposure, we analyzed the genomic and proteomic changes induced by AgNPs in wheat seedlings. At the DNA level, we applied the AFLP technique and we found that both treatments did not induce any significant DNA polymorphisms. 2DE profiling of roots and shoots treated with 10 mg L(-1) of AgNPs revealed an altered expression of several proteins mainly involved in primary metabolism and cell defense.

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DNA barcoding is a recent and widely used molecular-based identification system that aims to identify biological specimens, and to assign them to a given species. However, DNA barcoding is even more than this, and besides many practical uses, it can be considered the core of an integrated taxonomic system, where bioinformatics plays a key role. DNA barcoding data could be interpreted in different ways depending on the examined taxa but the technique relies on standardized approaches, methods and analyses. The existing reference towards a common way to treat DNA barcoding data, analyses and results is the Barcode of Life Data Systems. However, the scientific community has produced in the recent years a number of alternative methods to manage barcoding data. The present work starts from this point, because users should be aware of the consequences their choices produce on the results. Despite the fact that a strict standardization is the essence of DNA barcoding, we propose a tour of six questions to improve the users' awareness about the method, the correct use of concepts and alternative tools provided by scientific community.

Abstract. Mutations in the Aquaporin-2 gene, which encodes a renal water channel, have been shown to cause autosomal nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Most AQP2 missense mutants in recessive NDI are retained in the endoplasmic reticulum (ER), but AQP2-T125M and AQP2-G175R were reported to be nonfunctional channels unimpaired in their routing to the plasma membrane. In five families, seven novel AQP2 gene mutations were identified and their cell-biologic basis for causing recessive NDI was analyzed. The patients in four families were homozygous for mutations, encoding AQP2-L28P, AQP2-A47V, AQP2-V71M, or AQP2-P185A. Expression in oocytes revealed that all these mutants, and also AQP2-T125M and AQP2-G175R, conferred a reduced water permeability compared with wt-AQP2, which was due to ER retardation. The patient in the fifth family had a GϾA nucleotide substitution in the splice donor site of one allele that results in an out-of-frame protein.The other allele has a nucleotide deletion (c652delC) and a missense mutation (V194I). The routing and function of AQP2-V194I in oocytes was not different from wt-AQP2; it was therefore concluded that c652delC, which leads to an out-of-frame protein, is the NDI-causing mutation of the second allele. This study indicates that misfolding and ER retention is the main, and possibly only, cell-biologic basis for recessive NDI caused by missense AQP2 proteins. In addition, the reduced single channel water permeability of AQP2-A47V (40%) and AQP2-T125M (25%) might become of therapeutic value when chemical chaperones can be found that restore their routing to the plasma membrane.The aquaporin-2 (AQP2) water channel plays an important role in reabsorption of water in the kidney collecting duct and consequently in concentrating urine (1). Binding of arginine vasopressin (AVP) to its V2 receptor (AVPR2) at the basolateral side of principal cells of collecting ducts leads to an increase of intracellular cAMP levels, resulting in phosphorylation of AQP2 and possibly other proteins, by protein kinase A and subsequent redistribution of AQP2 from subapical storage vesicles to the apical plasma membrane. Driven by the interstitial hypertonicity, water reabsorption and urine concentration is thereby initiated. This process is reversed after dissociation of AVP from its receptor (2,3).Several mutations in the AVPR2 and AQP2 genes have been reported to cause congenital nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to AVP. Mutations in the AVPR2 gene result in NDI that is inherited as an X-linked recessive trait, whereas mutations in the AQP2 gene cause NDI that is inherited as either an autosomal recessive or a dominant trait (1,4 -6,7). Expression studies in oocytes showed that an AQP2 mutant in dominant NDI, AQP2-E258K, was a functional water channel but was retained in the region of the Golgi complex (7). In coexpression studies with wild-...

Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca 2؉ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca 2؉ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca 2؉ into the stores. Measurement of intra-organelle-free Ca 2؉ in permeabilized cells revealed that the ability of 2B to reduce the Ca 2؉ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cellfree system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca 2؉ -ATPase that transports Ca 2؉ from the cytosol into the stores. To investigate whether 2B specifically affects Ca 2؉ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H ؉ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.Enteroviruses (e.g. poliovirus, coxsackievirus, ECHOvirus) belong to the family of Picornaviridea, a large family of nonenveloped, cytolytic viruses that contain a single-stranded RNA genome of positive polarity. Upon infection, enteroviruses induce a number of dramatic alterations in their host cell, which serve to create the appropriate conditions for viral RNA replication and/or prevent antiviral host cell responses. One of these alterations is the modification of intracellular Ca 2ϩ homeostasis. We have previously shown that infection of HeLa cells with coxsackievirus results in a reduction of the amount of Ca 2ϩ that can be released from the intracellular stores using thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), 2 the Ca 2ϩ -ATPase that transports Ca 2ϩ from the cytosol into the stores. In addition, a gradual increase in the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] cyt ) was observed due to the influx of extracellular Ca 2ϩ (1). The enterovirus 2B protein, one of the nonstructural proteins involved in viral RNA replication, plays a major role in the alterations in intracellular Ca 2ϩ homeostasis that take place in enterovirus-infected cells (1, 2). The mechanism by which 2B, or its precursor 2BC, exerts its effects is largely unknown. Ca 2ϩ homeostasis in the intracellular stores (i.e. endoplasmic reticulum (ER) and Golgi) is the net result of the activity of the SERCA on the one hand and the continuous passive Ca 2ϩ leak from these organelles that exists under normal conditions on the other hand (3). Thus, the reductions in the Ca 2ϩ filling state of the stores ...