UNC-108/RAB-2 and its effector RIC-19 are involved in dense core vesicle maturation in Caenorhabditis elegans

Small guanosine triphosphatases of the Rab family regulate intracellular vesicular trafficking. Rab2 is highly expressed in the nervous system, yet its function in neurons is unknown. In Caenorhabditis elegans, unc-108/rab-2 mutants have been isolated based on their locomotory defects. We show that the locomotion defects of rab-2 mutants are not caused by defects in synaptic vesicle release but by defects in dense core vesicle (DCV) signaling. DCVs in rab-2 mutants are often enlarged and heterogeneous in size; however, their number and distribution are not affected. This implicates Rab2 in the biogenesis of DCVs at the Golgi complex. We demonstrate that Rab2 is required to prevent DCV cargo from inappropriately entering late endosomal compartments during DCV maturation. Finally, we show that RIC-19, the C. elegans orthologue of the human diabetes autoantigen ICA69, is also involved in DCV maturation and is recruited to Golgi membranes by activated RAB-2. Thus, we propose that RAB-2 and its effector RIC-19 are required for neuronal DCV maturation.     

Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A(1-17) as a non-substrate inhibitor

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V(max) = 39 micromol/min/10(6) cells, vs 3.7 micromol/min/10(6) cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A(1-17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A(1-17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A(1-17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A(1-17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A(1-17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A(1-17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.     

Spatial and temporal traction response in human airway smooth muscle cells

Tractions that cells exert on theirsubstrates are essential in cell spreading, migration, and contraction.These tractions can be determined by plating the cells on a flexiblegel and measuring the deformation of the gel by using fluorescent beadsembedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we usethe recently developed method of Fourier transform traction cytometry(FTTC). The ICM and FTTC methods are applied to human airway smoothmuscle cells during stimulation with the contractile agonist histamineor the relaxing agonist isoproterenol. The overall intensity of thecell contraction (the median traction magnitude, the energy transferredfrom the cell to the gel, and the net contractile moment) increasedafter activation with histamine, and decreased after treatment withisoproterenol. Cells exhibited regional differences in the time courseof traction during the treatment. Both temporal evolution and magnitudeof traction increase induced by histamine varied markedly amongdifferent cell protrusions, whereas the nuclear region showed thesmallest response. These results suggest that intracellular mediatorsof cell adhesion and contraction respond to contractile stimuli withdifferent rates and intensities in different regions of the cell.

     

Production of hairy root cultures of lettuce (Lactuca sativa L.)

Hairy root cultures of lettuce (Lactuca sativa L.) were obtained by inoculation of cotyledonary leaves of in vitro lettuce seedlings (cvs. Nansen and Ljubljanska ledenka) with Agrobacterium rhizogenes A4M70GUS. Approximately in 96.7% cvs. Nansen and in 91.2% Ljubljanska ledenka inoculated explants produced hairy root when they were incubated on Murashige and Skoog (MS) half-strength medium without plant growth regulators. A total of 54% of all hairy root cultures expressed GUS activity. Every hairy root represented an independent transformation event. Line Ljubljanska ledenka 18 showed the highest biomass (5.5 times the biomass of control root). A PCR analysis of the genomic DNA confirmed the presence of marker and target genes in 15 hairy roots examined.     

Antitumor activity of amidino-substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives tested in 2D and 3D cell culture systems

Due to a poor clinical predictive power of 2D cell cultures, standard tool for in vitro assays in drug discovery process, there is increasing interest in developing 3D in vitro cell cultures, biologically relevant assay feasible for the development of robust preclinical anti-cancer drug screening platforms. Herein, we tested amidino-substituted benzimidazoles and benzimidazo[1,2-a]quinolines as a small platform for comparison of antitumor activity in 2D and 3D cell culture systems and correlation with structure–activity relationship. 3D cell culture method was applied on a human cancer breast (SK-BR-3, MDA-MB-231, T-47D) and pancreatic cancer cells (MIA PaCa-2, PANC-1). Results obtained in 2D and 3D models were highly comparable, but in some cases we have observed significant disagreement indicating that some prominent compounds can be discarded in early phase of researching because of compounds with false positive result. To confirm which of cell culture systems is more accurate, in vivo profiling is needed.     

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split,Croatia

International Microbiology - Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally...     

Fitting a geometric graph to a protein-protein interaction network

Motivation: Finding a good network null model for protein–proteininteraction (PPI) networks is a fundamental issue. Such a modelwould provide insights into the interplay between network structureand biological function as well as into evolution. Also, network(graph) models are used to guide biological experiments anddiscover new biological features. It has been proposed thatgeometric random graphs are a good model for PPI networks. Ina geometric random graph, nodes correspond to uniformly randomlydistributed points in a metric space and edges (links) existbetween pairs of nodes for which the corresponding points inthe metric space are close enough according to some distancenorm. Computational experiments have revealed close matchesbetween key topological properties of PPI networks and geometricrandom graph models. In this work, we push the comparison furtherby exploiting the fact that the geometric property can be testedfor directly. To this end, we develop an algorithm that takesPPI interaction data and embeds proteins into a low-dimensionalEuclidean space, under the premise that connectivity informationcorresponds to Euclidean proximity, as in geometric-random graphs.We judge the sensitivity and specificity of the fit by computingthe area under the Receiver Operator Characteristic (ROC) curve.The network embedding algorithm is based on multi-dimensionalscaling, with the square root of the path length in a networkplaying the role of the Euclidean distance in the Euclideanspace. The algorithm exploits sparsity for computational efficiency,and requires only a few sparse matrix multiplications, givinga complexity of O(N²) where N is the number of proteins. Results: The algorithm has been verified in the sense that itsuccessfully rediscovers the geometric structure in artificiallyconstructed geometric networks, even when noise is added byre-wiring some links. Applying the algorithm to 19 publiclyavailable PPI networks of various organisms indicated that:(a) geometric effects are present and (b) two-dimensional Euclideanspace is generally as effective as higher dimensional Euclideanspace for explaining the connectivity. Testing on a high-confidenceyeast data set produced a very strong indication of geometricstructure (area under the ROC curve of 0.89), with this networkbeing essentially indistinguishable from a noisy geometric network.Overall, the results add support to the hypothesis that PPInetworks have a geometric structure. Availability: MATLAB code implementing the algorithm is availableupon request. Contact: natasha{at}ics.uci.edu Associate Editor: Olga Troyanskaya     

Developmental and transplacental genotoxicology: fluconazole

Over the last 40 years mankind has been facing new types of radiochemical environmental settings with every decade. During the last decade, biomonitoring was additionally focused on assessing associations between environmental exposure(s) and both early and late biological effects in children. Despite efforts to control and avoid child exposure to genotoxic agents the incidence of childhood cancers is increasing. Some cancers in adulthood may be the consequence of a multi-step process which starts with intrauterine and childhood exposure. This highlights the importance of a comprehensive interpretation of multiple health effects, especially considering recent studies suggesting that most health disorders are related to DNA changes. When exposed to genotoxic agents, a developing organism (fetus or child) is constantly being forced to reorganize into new equilibriums in order to adjust to a xenobiotic environment. In addition, the influence of sex hormones on radiochemical sensitivity is still unknown. For this reason special attention should be paid to puberty. The results of recent studies on animal models and follow up studies on children after nuclear accidents show long-lasting cytogenetic damage even after low dose exposures and their transgenerational persistance. To evaluate age-related difference and transplacental genotoxic potency fluconazole (FC) was investigated by in vivo micronucleus (MN) assay in adult mice, young mice and in transplacentally exposed newborn pups. Compared to the baseline values, FC caused no detectable genome damage in adult animals, but there was a significant increase in MN frequency in young animals and in newborn pups. Our study thus exemplifies an age-related chemosensitivity, and argues that cancer-promoting disturbances of complex prenatal developmental mechanisms and maturation during childhood require a new approach using systems biology.