Kinetic and structural analysis of a new group of Acyl-CoA carboxylases found in Streptomyces coelicolor A3(2)

Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated alpha subunit, AccA2. The beta and the epsilon subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the beta and epsilon components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding epsilon subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the epsilon subunits were found to specifically interact with their carboxyltransferase component forming a beta-epsilon subcomplex; this appears to facilitate the further interaction of these subunits with the alpha component. The epsilon subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.     

Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila

During animal development, organ size is determined primarily by the amount of cell proliferation, which must be tightly regulated to ensure the generation of properly proportioned organs. However, little is known about the molecular pathways that direct cells to stop proliferating when an organ has attained its proper size. We have identified mutations in a novel gene, shar-pei, that is required for proper termination of cell proliferation during Drosophila imaginal disc development. Clones of shar-pei mutant cells in imaginal discs produce enlarged tissues containing more cells of normal size. We show that this phenotype is the result of both increased cell proliferation and reduced apoptosis. Hence, shar-pei restricts cell proliferation and promotes apoptosis. By contrast, shar-pei is not required for cell differentiation and pattern formation of adult tissue. Shar-pei is also not required for cell cycle exit during terminal differentiation, indicating that the mechanisms directing cell proliferation arrest during organ growth are distinct from those directing cell cycle exit during terminal differentiation. shar-pei encodes a WW-domain-containing protein that has homologs in worms, mice and humans, suggesting that mechanisms of organ growth control are evolutionarily conserved.     

Variability of 6 Colombian strains of Trypanosoma cruzi with restriction fragment length polymorphisms (RFLP) and random amplification of polymorphic DNA (RAPD)

Chagas disease, caused by the hemoflagellate Trypanosoma cruzi, is a public health problem in Colombia. Previous reports have indicated the presence of heterogeneity among parasite populations. Six Colombian T. cruzi strains were obtained that differed by host, geographical region and transmission cycle. The genetic variability of each was compared by random amplified polymorphic DNA (RAPD), and isoenzymes. A restriction fragment length polymorphism (RFLP) was extracted using the 1.2 kb unit encoding the parasite's H2A histone as a probe. Genetic distances between the isolates varied greatly, from 0.611 to 0.99 as determined by RAPD profiles (M13F and M13R primers), between 0 and 0.81 by RFLP profiles (5 endonucleases), and between 0.10 and 0.55 by isoenzymes (13 enzymatic systems). Genetic distance matrixes derived from each of the three methods showed that Colombian strains exhibit a high degree of genetic differentiation. This may account for the broad clinical spectrum of Chagas disease in Colombia.     

Regulator-driven functional diversification of protein phosphatase-1 in eukaryotic evolution

We have used the (nearly) completed eukaryotic genome sequences to trace the evolution of thirteen families of established vertebrate regulators of type-1 protein phosphatases (PP1). Two of these families are present in all lineages of the eukaryotic crown and therefore qualify as candidate primordial regulators that determined the surface of PP1. The set of regulators of PP1 has continued to expand ever since, often in response to functional innovations in different eukaryotic lineages. In particular, the development of metazoan multicellularity was accompanied by an explosive increase in the number of regulators of PP1. The further increase in the functional diversity of PP1 in the vertebrate lineage was mainly achieved by the duplication of genes for regulatory subunits and by the conversion of already existing proteins into regulators of PP1. Unexpectedly, our analysis has also enabled us to classify nine poorly characterized proteins as likely regulators of PP1.     

Molecular mechanisms of phosphate transport in plants

Membrane-spanning transport proteins are responsible for the selective passage of most mineral nutrients and metabolites across cellular and intracellular membranes. This review's focus is on summarising the current state of research covering the molecular regulation and biochemical mechanisms involved in the transport of phosphorus, an often growth-limiting nutrient, in vascular plants. Physiological data illustrating the tight control of Pi homeostasis on the cellular as well as on the organism's level are discussed together with the recent results on molecular transport mechanisms.     

Fat liquefaction: effect of low-level laser energy on adipose tissue

Low-level laser energy has been increasingly used in the treatment of a broad range of conditions and has improved wound healing, reduced edema, and relieved pain of various etiologies. This study examined whether 635-nm low-level lasers had an effect on adipose tissue in vivo and the procedural implementation of lipoplasty/liposuction techniques. The experiment investigated the effect of 635-nm, 10-mW diode laser radiation with exclusive energy dispersing optics. Total energy values of 1.2 J/cm(2), 2.4 J/cm(2), and 3.6 J/cm(2) were applied on human adipose tissue taken from lipectomy samples of 12 healthy women. The tissue samples were irradiated for 0, 2, 4, and 6 minutes with and without tumescent solution and were studied using the protocols of transmission electron microscopy and scanning electron microscopy. Nonirradiated tissue samples were taken for reference. More than 180 images were recorded and professionally evaluated. All microscopic results showed that without laser exposure the normal adipose tissue appeared as a grape-shaped node. After 4 minutes of laser exposure, 80 percent of the fat was released from the adipose cells; at 6 minutes of laser exposure, 99 percent of the fat was released from the adipocyte. The released fat was collected in the interstitial space. Transmission electron microscopic images of the adipose tissue taken at x60,000 showed a transitory pore and complete deflation of the adipocytes. The low-level laser energy affected the adipose cell by causing a transitory pore in the cell membrane to open, which permitted the fat content to go from inside to outside the cell. The cells in the interstitial space and the capillaries remained intact. Low-level laser-assisted lipoplasty has a significant impact on the procedural implementation of lipoplasty techniques.     

A new method to assess air pollution using lichens as bioindicators

Lichens are increasingly used worldwide as air quality biomonitors because they are efficient, easy and cheap, but validation studies of the methodology are scarce. Three foliose lichen biomonitoring methods were compared by field tests (in the tropical urban habitat of San José, Costa Rica) and laboratory simulations: (1) the 100 uniform squares template traditionally used in North America, (2) the European 200 uniform points template and (3) a new computer-generated random points template (10 x 20 cm) in two versions: 100 points and 50 points. Repeated measurement by the same observer causes a variation of 2-14% and the templates' error is 0.2-11%. We recommend the 100 random point template (applied to four sides of trunk) for ecological studies and the 50 random points template (applied to side with greatest lichen cover) for biomonitoring because it reduces time and costs by nearly 50% but still has acceptable reliability values.     

Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release

We have identified mutations in Drosophila endophilin to study its function in vivo. Endophilin is required presynaptically at the neuromuscular junction, and absence of Endophilin dramatically impairs endocytosis in vivo. Mutant larvae that lack Endophilin fail to take up FM1-43 dye in synaptic boutons, indicating an inability to retrieve synaptic membrane. This defect is accompanied by an expansion of the presynaptic membrane, and a depletion of vesicles from the bouton lumen. Interestingly, mutant larvae are still able to sustain release at 15%-20% of the normal rate during high-frequency stimulation. We propose that kiss-and-run maintains neurotransmission at active zones of the larval NMJ in endophilin animals.