BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26(Sox10Cre) mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10-Cre line. Cx26(Sox10Cre) mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26(Sox10Cre) mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.     

Minimal cells: relevance and interplay of physical and biochemical factors

Research on the construction of minimal cell-like systems is continuously progressing. The aim is to assemble a synthetic or semi-synthetic cell by encapsulating the minimal set of different macromolecules into a lipid vesicle (liposome). Synthetic cells have their relevance as new biotechnological tool for use in synthetic biology and in research into the origin of life. In recent years, several technical advances have been reported and reviewed, but most deal with the biochemical and molecular biology of protein synthesis inside liposomes, whereas a discussion on the importance and the interplay of some physical factors has not been discussed. In this short review, we comment on physical aspects, such as compartment formation and solute entrapment, and on the nature of lipid membrane. Emphasis is given to their relevance for the technology of construction of synthetic cells, and for new aspects of vesicle population studies.     

Human influence on distribution and extinctions of the late Pleistocene Eurasian megafauna

Late Pleistocene extinctions are of interest to paleontological and anthropological research. In North America and Australia, human occupation occurred during a short period of time and overexploitation may have led to the extinction of mammalian megafauna. In northern Eurasia megafaunal extinctions are believed to have occurred over a relatively longer period of time, perhaps as a result of changing environmental conditions, but the picture is much less clear. To consider megafaunal extinction in Eurasia, we compare differences in the geographical distribution and commonness of extinct and extant species between paleontological and archaeological localities from the late middle Pleistocene to Holocene. Purely paleontological localities, as well as most extinct species, were distributed north of archaeological sites and of the extant species, suggesting that apart from possible differences in adaptations between humans and other species, humans could also have a detrimental effect on large mammal distribution. However, evidence for human overexploitation applies only to the extinct steppe bison Bison priscus. Other human-preferred species survive into the Holocene, including Rangifer tarandus, Equus ferus, Capreolus capreolus, Cervus elaphus, Equus hemionus, Saiga tatarica, and Sus scrofa. Mammuthus primigenius and Megaloceros giganteus were rare in archaeological sites. Carnivores appear little influenced by human presence, although they become rarer in Holocene archaeological sites. Overall, the data are consistent with the conclusion that humans acted as efficient hunters selecting for the most abundant species. Our study supports the idea that the late Pleistocene extinctions were environmentally driven by climatic changes that triggered habitat fragmentation, species range reduction, and population decrease, after which human interference either by direct hunting or via indirect activities probably became critical.     

The role of CXCR2 activity in the contact hypersensitivity response in mice

The recruitment of polymorphonuclear neutrophil leukocytes (PMN) into a challenge site, and their subsequent activation, are thought to play a role in the elicitation of the contact hypersensitivity (CHS) response. The present study investigated the role played by CXCR2 activity in tissue PMN infiltration and subsequent triggering of CHS. Our results show that the cutaneous infiltration by PMN, induced by hapten challenge was dramatically inhibited in sensitized, CXCR2-deficient (CXCR2(-/-)) mice. Inhibition of PMN recruitment into the hapten-challenged ears of CXCR2(-/-) mice was associated with a consistent reduction of the CHS response (ear swelling) in CXCR2(-/-) mice as compared with that observed in neutropenic, wild-type (CXCR2(+/+)) mice. Prevention of skin PMN infiltration and the ear swelling response by the absence of functional CXCR2 was observed regardless of the hapten used. These data clearly suggest that CXCR2 activity plays an essential role in mediating cutaneous recruitment and activation of PMN, and thus indirectly regulates recruitment of hapten-primed T cells into challenge sites, with the subsequent elicitation of the CHS response. The role played by CXCR2 activity in the CHS response provides the rationale for testing CXCR2 inhibitors as a new therapeutic approach to skin diseases.     

Endothelin-1 response to mental stress in early ischemic lesions of the extremities due to systemic sclerosis

We studied circulating levels of endothelin-1, catecholamines and nitric oxide after a mental arithmetic test in 14 patients with early ischemic lesions of the extremities due to systemic sclerosis and slightly impaired peripheral vascular flow. The test induced an increase (P < 0.01) in blood pressure, heart rate, endothelin-1 and catecholamine levels, whereas it did not change the low basal levels of nitric oxide. In healthy subjects (n = 20) the test significantly (P < 0.01) decreased endothelin-1 without affecting nitric oxide. The low basal levels of nitric oxide and the high plasma concentration of endothelin-1 after psychological stress cannot be explained by an impaired release from the limited ischemic lesions alone. This suggests a diffuse microvascular derangement that aggravates the course of peripheral microvascular ischemic lesions.     

Nicking activity on pBR322 DNA of ribosome inactivating proteins from Phytolacca dioica L. leaves

Ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adult P . dioica plants, produces both free 3'-OH and 5'-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [alpha- 32P]dCTP and [gamma- 32 P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity with E . coli HB101 competent cells in comparison to untreated control plasmid DNA.     

Inhibition of integrin-mediated cell adhesion but not directional cell migration requires catalytic activity of EphB3 receptor tyrosine kinase. Role of Rho family small GTPases

Genetic studies have shown that Eph receptor tyrosine kinases have both kinase-dependent and kinase-independent functions through incompletely understood mechanisms. We report here that ephrin-B1 stimulation of endogenous EphB kinases in LS174T colorectal epithelial cells inhibited integrin-mediated adhesion and HGF/SF-induced directional cell migration. Using 293 cells stably transfected with wild type (WT)- or kinase-deficient (KD-EphB3), we found that inhibition of integrin-mediated cell adhesion and induction of cell rounding was kinase-dependent. Unexpectedly, in two independent assays, both KD- and WT-EphB3 significantly inhibited directional cell migration. Upon ephrin-B1 stimulation, the activities of Rac1 and Cdc42 were reduced in both WT- and KD-EphB3-expressing cells that were induced to migrate. Pharmacological evidence demonstrates that a relative increase in RhoA signaling as a result of decreased Rac1/Cdc42 activities contributes to the inhibitory effects. Furthermore, EphB3-mediated inhibitory effect on cell adhesion but not migration was abolished by the integrin activating antibodies, suggesting that the inhibition of cell migration is not because of down-regulation of integrin function. These results uncover a differential requirement for EphB3 catalytic activity in the regulation of cell adhesion and migration, and suggest that while catalytic activity of EphB3 is required for inhibition of integrin-mediated cell adhesion, a distinct signaling pathway to Rho GTPases shared by WT- and KD-EphB3 receptor mediates inhibition of directional cell migration.     

Mechanisms of airway smooth muscle relaxation during maturation

Greater airway responsiveness in healthy juveniles is considered a factor in the higher asthma prevalence at a young age compared with adults. We have developed a guinea pig maturational model that utilizes tracheal strips from 1-week-, 3-week-, and 3-month-old guinea pigs to study the role of airway smooth muscle (ASM) in juvenile airway hyperresponsiveness. Because a reduced ability of ASM to spontaneously relax may contribute to airway hyperresponsiveness by maintaining bronchospasm and thus high airway resistance, we have employed this model to study ASM spontaneous relaxation during electrical field stimulation (EFS). Since relaxation during EFS had been neither described nor quantified during maturation, we developed new indices that allowed an appropriate comparison of the relaxing response from strips of different age animals. Using these indices we found that, whereas strips from adult animals relax to a level of tension similar to that found in the absence of stimulation, this ability to spontaneously relax is essentially absent in trachealis from infant animals. These results confirmed that maturation of ASM relaxation may play a role in juvenile airway hyperresponsiveness and that our maturational model is suitable to study the mechanisms regulating spontaneous relaxation in physiological conditions. We investigated the role of prostanoids in ASM relaxation and showed that cyclooxygenase inhibition increases relaxation in infant ASM to levels similar to adults. These results suggest that prostanoids regulate the ability of ASM to spontaneously relax, i.e., they reduce relaxation. We have produced preliminary data suggesting a maturational change in the level of prostanoids. Moreover, the possible action of acetylcholinesterase on maturation of ASM relaxation is discussed here on the basis of a preliminary study. We suggest that impairment of ASM relaxation likely contributes to increased airway responsiveness.