Morphological comparison of axenic amastigogenesis of trypomastigotes and metacyclic forms of Trypanosoma cruzi

Amastigogenesis occurs first when metacyclic trypomastigotes from triatomine urine differentiate into amastigotes inside mammalian host cells and a secondary process when tissue-derived trypomastigotes invade new cells and differentiate newly to amastigotes. Using scanning electron microscopy, we compared the morphological patterns manifested by trypomastigotes and metacyclic forms of Trypanosoma cruzi during their axenic-transformation to amastigotes in acidic medium at 37 C. We show here that in culture MEMTAU medium, secondary and primary axenic amastigogenesis display different morphologies. As already described, we also observed a high differentiation rate of trypomastigotes into amastigotes. Conversely, the transformation rate of in vitro-induced-metacyclic trypomastigotes to amastigotes was significantly slower and displayed distinct patterns of transformation that seem environment-dependent. Morphological comparisons of extracelullar and intracellular amastigotes showed marked similarities, albeit some differences were also detected. SDS-PAGE analyses of protein and glycoprotein from primary and axenic extracelullar amastigotes showed similarities in glycopeptide profiles, but variations between their proteins demonstrated differences in their respective macromolecular constitutions. The data indicate that primary and axenic secondary amastigogenesis of T. cruzi may be the result of different developmental processes and suggest that the respective intracellular mechanisms driving amastigogenesis may not be the same.     

Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells

The canonical condensin complex (henceforth condensin I) plays an essential role in mitotic chromosome assembly and segregation from yeast to humans. We report here the identification of a second condensin complex (condensin II) from vertebrate cells. Condensins I and II share the same pair of structural maintenance of chromosomes (SMC) subunits but contain different sets of non-SMC subunits. siRNA-mediated depletion of condensin I- or condensin II-specific subunits in HeLa cells produces a distinct, highly characteristic defect in chromosome morphology. Simultaneous depletion of both complexes causes the severest defect. In Xenopus egg extracts, condensin I function is predominant, but lack of condensin II results in the formation of irregularly shaped chromosomes. Condensins I and II show different distributions along the axis of chromosomes assembled in vivo and in vitro. We propose that the two condensin complexes make distinct mechanistic contributions to mitotic chromosome architecture in vertebrate cells.     

Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris

Cell adhesion and communication are interdependent aspects of cell behavior that are critical for morphogenesis and tissue architecture. In the skin, epidermal adhesion is mediated in part by specialized cell-cell junctions known as desmosomes, which are characterized by the presence of desmosomal cadherins, known as desmogleins and desmocollins. We identified a cadherin family member, desmoglein 4, which is expressed in the suprabasal epidermis and hair follicle. The essential role of desmoglein 4 in skin was established by identifying mutations in families with inherited hypotrichosis, as well as in the lanceolate hair mouse. We also show that DSG4 is an autoantigen in pemphigus vulgaris. Characterization of the phenotype of naturally occurring mutant mice revealed disruption of desmosomal adhesion and perturbations in keratinocyte behavior. We provide evidence that desmoglein 4 is a key mediator of keratinocyte cell adhesion in the hair follicle, where it coordinates the transition from proliferation to differentiation.     

Analysis of the argK-tox gene cluster in nontoxigenic strains of Pseudomonas syringae pv. phaseolicola

The analysis of 46 isolates obtained directly from different and distant common bean fields from the northwestern part of Spain revealed that they do not produce phaseolotoxin. The isolates were classified as race 5, and their analysis revealed that they do not carry the argK-tox gene cluster involved in the biosynthesis of the phaseolotoxin.     

Galactose induces the expression of penicillin acylase under control of the lac promoter in recombinant Escherichia coli

The expression of penicillin acylase (PA), cloned in the pPA102 plasmid under control of the wild-type lac promoter and using galactose as inducer in Escherichia coli JM101, JM103 and JM105 transformant cells, was analyzed. The E. coli JM101/pPA102 cultures attained the highest specific activity of PA. For large scale PA production based on E. coli JM101/pPA102 a culture media with galactose instead of isopropyl-thio-galactopyranoside as inducer would be as successful and less expensive.     

Thermodynamics of glutathione binding to the tyrosine 7 to phenylalanine mutant of glutathione S-transferase from Schistosoma japonicum

The binding of glutathione (GSH) to the tyrosine 7 to phenylalanine mutant of Schistosoma japonicum glutathione S-transferase (SjGST-Y7F) has been studied by isothermal titration calorimetry (ITC). At pH 6.5 and 25 °C this mutant shows a higher affinity for glutathione than wild type enzyme despite an almost complete loss of activity in the presence of 1-chloro-2,4-dinitrobenzene (CDNB) as second substrate. The enthalpy change upon binding of GSH is more negative for the mutant than for the wild type GST (SjGST). Changes in accessible solvent areas (ASA) have been calculated based on enthalpy and heat capacity changes. ASA values indicated the burial of apolar surfaces of protein and ligand upon binding. A more negative ΔC_p value has been obtained for the mutant enzyme, suggesting a more hydrophobic interaction, as may be expected from the change of a tyrosine residue to phenylalanine.     

The structure of the FMO protein from Chlorobium tepidum at 2.2 Å resolution

The bacteriochlorophyll protein, or FMO protein, from Chlorobium tepidum, which serves as a light-harvesting complex and directs light energy from the chlorosomes attached to the cell membrane to the reaction center has been crystallized in a new space group. The crystals belong to the cubic space group P4₃32 and the structure has been refined to a resolution 2.2 Å with a R factor of 19.7%. The electron density maps show that the structure is composed of two sheets that surround seven bacteriochlorophylls as previously reported (Li et al. (1997) J Mol Biol 271: 456–471). The availability of the new data allows a more accurate refinement of the pigment–protein complex including identification of bound solvent molecules. Several structural differences probably contribute to the observed spectroscopic differences between the FMO proteins from Cb. tepidum and Prosthecochloris aestuarii, including differences in the planarity of corresponding tetrapyrroles. A citrate molecule is found on the surface of each protein subunit of the trimer from Cb. tepidum. However, the citrate molecule is over 15 Å from any bacteriochlorophyll. The presence of the citrate probably does not contribute to the function of the protein although it does contribute to the crystallization as it interacts with a crystallographically related trimer. Among the 236 water molecules found in the protein are four that appear to play a special role in the properties of bacteriochlorophyll 2, as this tetrapyrrole is coordinated by one of these water molecules and the waters form a hydrogen-bonded network that leads to the surface of the protein.This revised version was published online in October 2005 with corrections to the Cover Date.     

Synchrony in human,mouse and bacterial cell cultures--a comparison

Growth characteristics of synchronous human MOLT-4, human U-937 and mouse L1210 cultures produced with a new minimally-disturbing technology were compared to each other and to synchronous Escherichia coli B/r. Based on measurements of cell concentrations during synchronous growth, synchrony persisted in similar fashion for all cells. Cell size and DNA distributions in the mammalian cultures also progressed synchronously and reproducibly for multiple cell cycles. The results demonstrate that unambiguous multi-cycle synchrony, critical for verifying the absence of significant growth imbalances induced by the synchronization procedure, is feasible with these cell lines, and possibly others.     

Yeast communities associated with stingless bees

The yeast communities associated with the stingless bees Tetragonisca angustula, Melipona quadrifasciata and Frieseomelitta varia were studied. The bees T. angustula and F. varia showed a strong association with the yeast Starmerella meliponinorum. M. quadrifasciata more frequently carried a species related to Candida apicola, but also vectored low numbers of S. meliponinorum. Some of the yeasts isolated from adult bees were typical of species known to occur in flowers. Other yeast species found in adult bees were more typical of those found in the phylloplane. S. meliponinorum and the species in the C. apicola complex, also part of the Starmerella clade, may have a mutualistic relationship with the bees studied. Many yeasts in that group are often found in bees or substrates visited by bees, suggesting that a mutually beneficial interaction exists between them.