Matrilin-2, a large, oligomeric matrix protein, is expressed by a great variety of cells and forms fibrillar networks

Matrilin-2 is a member of the protein superfamily with von Willebrand factor type A-like modules. Mouse matrilin-2 cDNA fragments were expressed in 293-EBNA cells, and the protein was purified, characterized, and used to immunize rabbits. The affinity-purified antiserum detects matrilin-2 in dense and loose connective tissue structures, subepithelial connective tissue of the skin and digestive tract, specialized cartilages, and blood vessel walls. In situ hybridization of 35S-labeled riboprobes localizes the matrilin-2 mRNA to fibroblasts of dermis, tendon, ligaments, perichondrium, and periosteum; connective tissue elements in the heart; smooth muscle cells; and epithelia and loose connective tissue cells of the alimentary canal and respiratory tract. RNA blot hybridization and immunoblotting revealed both matrilin-2 mRNA and protein in cultures of a variety of cell types, confirming the tissue distribution. Alternative splicing affects a module unique for matrilin-2 in all of the above RNA sources. SDS-polyacrylamide gel electrophoresis and electron microscopy reveals matrilin-2 from tissue extracts and cell line cultures as a mixture of mono-, di-, tri-, and tetramers. Matrilin-2 is substituted with N-linked oligosaccharides but not with glycosaminoglycans. Because of other, yet unidentified, cell-type dependent posttranslational modifications, the monomer is heterogeneous in size. Immunofluorescence showed that matrilin-2 functions by forming an extracellular, filamentous network.     

Phosphorylated aspartate in the structure of a response regulator protein

Phosphorylation of aspartic acid residues is the hallmark of two- component signal transduction systems that orchestrate the adaptive responses of micro-organisms to changes in their surroundings. Two-component systems consist of a sensor kinase that interprets environmental signals and a response regulator that activates the appropriate physiological response. Although structures of response regulators are known, little is understood about their activated phosphorylated forms, due to the intrinsic instability of the acid phosphate linkage. Here, we report the phosphorylated structure of the receiver/phosphoacceptor domain of Spo0A, the master regulator of sporulation, from Bacillus stearothermophilus. The phosphoryl group is covalently bonded to the invariant aspartate 55, and co-ordinated to a nearby divalent metal cation, with both species fulfilling their electrostatic potential through interactions with solvent water molecules, the protein main chain, and with side-chains of amino acid residues strongly conserved across the response regulator family. This is the first direct visualisation of a phosphoryl group covalently linked to an aspartic acid residue in any protein, with implications for signalling within the response regulator family.     

Effect of killer toxin K1 on yeast membrane potential reported by the diS-C3(3) probe reflects strain-and physiological state-dependent variations

The rate and extent of uptake of the fluorescent probe diS-C₃(3) reporting on membrane potential inS. cerevisiae is affected by the strain under study, cell-growth phase, starvation and by the concentration of glucose both in the growth medium and in the monitored cell suspension under non-growth conditions. Killer toxin K1 brings about changes in membrane potential. In all types of cells tested,viz. in glucose-supplied stationary or exponential cells of the killer-sensitive strain S6/1 or a conventional strain RXII, or in glucose-free exponential cells of both strains, both active and heat-inactivated toxin slow down the potential-dependent uptake of diS-C₃(3) into the cells. This may reflect “clogging” of pores in the cell wall that hinders, but does not prevent, probe passage to the plasma membrane and its equilibration. The clogging effect of heat-inactivated toxin is stronger than that exerted by active toxin. In susceptible cells,i.e. in exponential-phase glucose-supplied cells of the sensitive strain S6/1, this phase of probe uptake retardation is followed by an irreversible red shift in probe fluorescence maximumλ _max indicating damage to membrane integrity and cell permeabilization. A similar fast red shift inλ _max signifying lethal cell damage was found in heat-killed or nystatin-treated cells.     

Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1

In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is the only phage protein present. However, 100% excision is observed when the phage protein Orf7 is provided as well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the family of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (orf7) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1 attP site which are integrated into attB on the chromosome of Lactococcus lactis. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a method for positive selection of loss of genetic material based upon the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10(-5) per cell could easily be measured. The described selection principle may be of general use for many organisms and also for types of deletion events other than excision.     

Characterization of activity and expression of isocitrate lyase in Mycobacterium avium and Mycobacterium tuberculosis

Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians. Only marginal similarity was observed between this open reading frame (ORF) (termed icl) and a second distinct ORF (named aceA) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.     

Characterization of TCR gene rearrangements during adult murine T cell development

Development of the alphabeta and gammadelta T cell lineages is dependent upon the rearrangement and expression of the TCRalpha and beta or gamma and delta genes, respectively. Although the timing and sequence of rearrangements of the TCRalpha and TCRbeta loci in adult murine thymic precursors has been characterized, no similar information is available for the TCRgamma and TCRdelta loci. In this report, we show that approximately half of the total TCRdelta alleles initiate rearrangements at the CD44highCD25+ stage, whereas the TCRbeta locus is mainly in germline configuration. In the subsequent CD44lowCD25+ stage, most TCRdelta alleles are fully recombined, whereas TCRbeta rearrangements are only complete on 10-30% of alleles. These results indicate that rearrangement at the TCRdelta locus can precede that of TCRbeta locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCRdelta and TCRgamma genes among CD44highCD25+ thymocytes, suggesting that functional gammadelta TCR complexes can be formed before the rearrangement of TCRbeta. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence alphabeta/gammadelta lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44highCD25+ stage of development.     

Numerical simulations of stochastic circulatory models

Properties of two of the stochastic circulatory models theoretically introduced by Smith et al., 1997, Bull. Math. Biol. 59, 1–22 were investigated. The models assumed the gamma distribution of the cycle time under either the geometric or Poisson elimination scheme. The reason for selecting these models was the fact that the probability density functions of the residence time of these models are formally similar to those of the Bateman and gamma-like function models, i.e., the two common deterministic models. Using published data, the analytical forms of the probability density functions of the residence time and the distributions of the simulated values of the residence time were determined on the basis of the deterministic models and the stochastic circulatory models, respectively. The Kolmogorov-Smirnov test revealed that even for 1000 xenobiotic particles, i.e., a relatively small number if the particles imply drug molecules, the probability density functions of the residence time based on the deterministic models closely matched the distributions of the simulated values of the residence time obtained on the basis of the stochastic circulatory models, provided that parameters of the latter models fulfilled selected conditions.     

Membrane skeleton detachment in spherical and cylindrical microexovesicles

We observed that amphiphile-induced microexovesicles may be spherical or cylindrical, depending on the species of the added amphiphile. The spherical microexovesicle corresponds to an extreme local difference between the two monolayer areas of the membrane segment with a fixed area, while the cylindrical microexovesicle corresponds to an extreme local area difference if the area of the budding segment is increased due to lateral influx of anisotropic membrane constituents. Protein analysis showed that both types of vesicles are highly depleted in the membrane skeleton. It is suggested that a partial detachment of the skeleton in the budding region is favoured due to accumulated skeleton shear deformations in this region.     

Taxonomic study of Ascocotyle (Phagicola) longa Ransom, 1920 (Digenea: Heterophyidae) and related taxa

Species of Ascocotyle Looss, 1899 (subgenus Phagicola Faust, 1920) (Digenea: Heterophyidae) related to A. (P.) longa Ransom, 1920, i.e. those with a bipartite gonotyl and a single crown of 16 circumoral spines, were revised on the basis of examination of type and voucher specimens. The species A. (P.) arnaldoi Travassos, 1929, A. (P.) byrdi (Robinson, 1956) and A. (P.) longicollis (Kuntz & Chandler, 1956), all possessing 16 circumoral spines, are synonymised with A. (P.) longa. A. (P.) ascolonga Witenberg, 1929, the types of which were not available, is provisionally retained as a valid species despite its close similarity to A. (P.) longa (identical number of circumoral spines and morphology of the ventrogenital sac). It differs from A. (P.) longa in the presence of uterine loops posterior to the testes and intestinal caeca ending anterior to the ovary (versus the pretesticular uterus and intestinal caeca reaching to the posterior margin of the ovary in A. (P.) longa). A. (P.) inglei (Hutton & Sogandares-Bernal, 1959), found only once in a single specimen, is also tentatively considered a valid species because it differs in number of circumoral spines (19), slightly larger eggs (23–26×13–16 m) and the distribution of refractile bodies of the gonotyl arranged in four separate groups from A. (P.) longa and A. (P.) ascolonga (16 circumoral spines; eggs 15–20×–12 m; refractile bodies in two groups).     

Molecular properties of 4-substituted indole-3-acetic acids affecting pea pericarp elongation

Pea (Pisum sativum L.) fruit naturally contain the auxins, indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA). However, only 4-Cl-IAA can substitute for the seeds in maintaining pea fruit growth in planta. The importance of the substituent at the 4-position of the indole ring was tested by comparing the molecular properties of 4-X-IAA (X = H, Me, Et, F, or Cl) and their effect on the elongation of pea pericarps in planta. Structure-activity is discussed in terms of structural data derived from X-ray analysis, computed conformations in solution, semiempirical shape and bulk parameters, and experimentally determined lipophilicities and NH-acidities. The size of the 4-substituent, and its lipophilicity are associated with growth promoting activity of pea pericarp, while there was no obvious relationship with electromeric effects.