Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability

Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.     

Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast

The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.     

Blood leptin homeostasis: sex-associated differences in circulating leptin levels in rats are independent of tissue leptin expression

Circulating leptin levels are higher in women than in men. The aim of the study has been to determine in rats the putative existence of sex-associated differences in leptin expression in different adipose tissue depots (gonadal, retroperitoneal, mesenteric and inguinal white adipose tissue and interscapular brown adipose tissue) and the relationship with circulating leptin levels. Adult male and female Wistar rats acclimated to 22 degrees C or to 28 degrees C were used. Leptin mRNA expression was assessed by northern blot and serum leptin levels by enzyme-linked immunosorbent assay (ELISA). Contrary to what happens in humans, we report here that male rats acclimated to standard animal house conditions (22 degrees C) have a higher leptin concentration in blood than female rats. This situation cannot be explained by a greater size of the fat depots in males, because the adiposity index is similar in both genders, but are rather associated to higher leptin specific mRNA expression by the white adipose tissue. Around thermoneutral conditions (28 degrees C), sex related differences in leptin mRNA expression disappear, but the gender difference in circulating leptin levels remains. In addition, leptin mRNA expression is higher in both genders in thermoneutral conditions but this is not reflected in changes in the circulating leptin levels. In conclusion, this study shows that rat circulating leptin levels are finely regulated, and not exclusively dependent on leptin mRNA expression, but other mechanisms are also involved, possibly regarding leptin rate of degradation.     

Evaluation of Candida species' susceptibility to fluconazole with the disk diffusion method

Infections caused by yeasts belonging to the genus Candida have increased dramatically in the last decades, especially in hospital settings. Concomittantly, antimycotic resistance has emerged, as well as the appearance of non-Candida albicans isolates. To standardize in vitro antifungal susceptibility tests, the agar diffusion test was developed using disks impregnated with the antimycotic compound. Electronic recording of the inhibition zone (BIOMIC), furnishes objective values for the minimal inhibitory concentration (MIC). The fluconazole susceptibility patterns were determined for Candida species isolated from 2.139 patients seen in outpatient clinics or in health-care centers in Colombia, Ecuador and Venezuela. Candida albicans was the species most frequently isolated (62%), followed at a distance by Candida parapsilosis (11%), Candida tropicalis (8.5%), Candida glabata (3.5%) and Candida krusei (2.2%). MIC determinations showed that 88.1% of these isolates were susceptible to fluconazole, 5.1% were susceptible-dose-dependant and 6.8% resistant. An important proportion (92.1%) of the C. albicans isolates proved susceptible while resistance predominated in the remaining species. These results indicate that the BIOMIC method is rapid and simple, constituting a suitable tool for the epidemiologic surveillance of resistance in Candida species.     

Resistin expression in different adipose tissue depots during rat development

Resistin is a hormonal factor synthesised by adipocytes that was first thought to be related with the resistance to insulin in obesity, but whose function is not yet completely established. Here we have studied the ontogenic pattern of resistin mRNA expression in different white adipose tissue depots (WAT) – epididymal, inguinal, mesenteric and retroperitoneal – and in brown adipose tissue (BAT), as well as the circulating resistin levels, in rats of different ages (from the suckling period to one year of age). Resistin mRNA was determined by Northern blotting, and serum levels by enzyme immunoassay. In WAT, resistin expression remains almost constant with age, except in early development, where there is a peak of expression in the epididymal and retroperitoneal depots, and a decrease in the inguinal one, while the expression remains constant for the mesenteric depot. Moreover, there is a site-specific difference regarding resistin expression: all the depots express characteristic levels of mRNA, especially at the age of 2 months, the moment when resistin mRNA levels are significantly higher in the epididymal and the retroperitoneal than in the inguinal and mesenteric WAT and than in the BAT. The transient increased resistin expression in the epididymal and the retroperitoneal WAT at a period of time in which there is a change in diet (from milk to chow) suggests a common nutritional regulation of the resistin gene. Circulating resistin levels increase with age probably reflecting the increase in the body fat content.     

G protein-coupled receptor kinase 2 (GRK2) in migration and inflammation

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors and other plasma membrane receptors stimulated by chemotactic messengers. On top of that, GRK2 has been reported to interact with a variety of signal transduction proteins related to cell migration such as MEK, Akt, PI3Kgamma or GIT. Interestingly, the levels of expression and activity of this kinase are altered in a number of inflammatory disorders (as rheumatoid arthritis or multiple sclerosis), thus suggesting that it may play an important role in the onset or development of these pathologies. This review summarizes the mechanisms involved in the control of GRK2 expression and function and highlights novel functional interactions of this protein that might help to explain how altered GRK2 levels affects cell migration in different cell types and pathological settings.     

Human gamma oscillations during slow wave sleep

Neocortical local field potentials have shown that gamma oscillations occur spontaneously during slow-wave sleep (SWS). At the macroscopic EEG level in the human brain, no evidences were reported so far. In this study, by using simultaneous scalp and intracranial EEG recordings in 20 epileptic subjects, we examined gamma oscillations in cerebral cortex during SWS. We report that gamma oscillations in low (30-50 Hz) and high (60-120 Hz) frequency bands recurrently emerged in all investigated regions and their amplitudes coincided with specific phases of the cortical slow wave. In most of the cases, multiple oscillatory bursts in different frequency bands from 30 to 120 Hz were correlated with positive peaks of scalp slow waves ("IN-phase" pattern), confirming previous animal findings. In addition, we report another gamma pattern that appears preferentially during the negative phase of the slow wave ("ANTI-phase" pattern). This new pattern presented dominant peaks in the high gamma range and was preferentially expressed in the temporal cortex. Finally, we found that the spatial coherence between cortical sites exhibiting gamma activities was local and fell off quickly when computed between distant sites. Overall, these results provide the first human evidences that gamma oscillations can be observed in macroscopic EEG recordings during sleep. They support the concept that these high-frequency activities might be associated with phasic increases of neural activity during slow oscillations. Such patterned activity in the sleeping brain could play a role in off-line processing of cortical networks.     

Targeting of proConA to the plant vacuole depends on its nine amino-acid C-terminal propeptide

Concanavalin A (ConA) is a well characterized and extensively used lectin accumulated in the protein bodies of jack bean cotyledons. ConA is synthesized as an inactive precursor proConA. The maturation of inactive proConA into biologically active ConA is a complex process including the removal of an internal glycopeptide and a C-terminal propeptide (CTPP), followed by a head-to-tail ligation of the two largest polypeptides. The cDNA encoding proConA was cloned and expressed in tobacco BY-2 cells. ProConA was slowly transported to the vacuole where its maturation into ConA was similar to that in jack bean cotyledons, apart from an incomplete final ligation. To investigate the role of the nine amino acid CTPP, a truncated form lacking the propeptide (proConADelta9) was expressed in BY-2 cells. In contrast to proConA, proConADelta9 was rapidly chased out of the endoplasmic reticulum (ER) and secreted into the culture medium. The CTPP was then fused to the C-terminal end of a secreted form of green fluorescent protein (secGFP). When expressed in tobacco BY-2 cells and leaf protoplasts, the chimaeric protein was located in the vacuole whereas secGFP was located in the culture medium and in the vacuole. Altogether, our results show we have isolated a new C-terminal vacuolar sorting determinant.