Transient up-regulation of biglycan during skeletal muscle regeneration: delayed fiber growth along with decorin increase in biglycan-deficient mice

The onset and progression of skeletal muscle regeneration are controlled by a complex set of interactions between muscle precursor cells and their environment. Decorin is the main proteoglycan present in the extracellular matrix (ECM) of adult muscle while biglycan expression is lower, but both are increased in mdx mice dystrophic muscle. Both of these small leucine-rich proteoglycans (SLRPs) can bind other matrix proteins and to the three TGF-beta isoforms, acting as modulators of their biological activity. We evaluated biglycan and decorin expression in skeletal muscle during barium chloride-induced skeletal muscle regeneration in mice. A transient and dramatic up-regulation of biglycan was associated with newly formed myotubes, whereas decorin presented only minor variations. Studies both in vitro and in intact developing newborn mice showed that biglycan expression is initially high and then decreases during skeletal muscle differentiation and maturation. To further evaluate the role of biglycan during the regenerative process, skeletal muscle regeneration was studied in biglycan-null mice. Skeletal muscle maintains its regenerative capacity in the absence of biglycan, but a delay in regenerated fiber growth and a decreased expression of embryonic myosin were observed despite to normal expression of MyoD and myogenin. Transient up-regulation of decorin during muscle regeneration in these mice may possibly obscure further roles of SLRPs in this process.     

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 3₁₀-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 3₁₀-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.     

Tissue distribution of L-fucokinase in rodents

L-fucose (fucose) is a monosaccharide normally present in mammals and is unique in being the only levorotatory sugar that can be synthesized and utilized by mammals. The metabolism of fucose is incompletely understood, but fucose can be synthesized de novo or salvaged. The utilization of fucose in the salvage pathway begins with phosphorylation by fucokinase. As part of an investigation of fucose metabolism in normal and disease states, we began an investigation of this enzyme. In this report, we present the tissue distribution of the enzyme in rat and mouse. The highest amount of activity was present in brain of both species. Some activity was found in all tissues examined (liver, kidney, heart, lung, spleen, brain, muscle, thymus, white adipose, testes, eye, aorta, small intestine, and submaxillary gland). Very low levels were found in small intestine. Varying levels in the tissues seems most likely to be the result of varying amounts of fucokinase protein as no difference in the Km values of crude enzyme could be shown. Protein-bound fucose levels were determined using the L-cysteine-phenol-sulfuric acid (CPS) assay. There is not a good correlation between fucokinase activity and protein-bound fucose, suggesting some tissues are more active in synthesis of fucose than others.     

Ultrastructure of the spermatozoa of the flatworms Phaenocelis peleca and Boninia divae (Platyhelminthes, Polycladida)

The ultrastructure of spermatozoa of the acotylean Phaenocelis peleca and the cotylean Boninia divae is described. All spermatozoa are filiform and biflagellate with a 9+"1" microtubular pattern in the axoneme. Sperm characters in P. peleca follow the morphologies described for other acotyleans, with axonemes exiting the sperm shaft at the distal end and remaining in close contact with the sperm membrane. The nucleus occupies the proximal region of the shaft, and two types of dense bodies and mitochondria are located at the distal end. Unlike other members of the Cotylea, the axonemes of B. divae spermatozoa are incorporated into the sperm shaft, leaving the shaft at some distance from the distal end and then remaining free. This type of morphology is characteristic for acotyleans. Additionally, the spermatozoa of B. divae contain only one type of dense bodies plus a unique structure, which we call a central core. The nucleus in this species is unique as well; it shows periodic constrictions and rings of electron-dense granules, characters that further contribute to the distinct status of Boniniidae.     

New body mass estimates for Omomys carteri,a middle Eocene primate from North America

We report new body mass estimates for the North American Eocene primate Omomys carteri. These estimates are based on postcranial measurements and a variety of analytical methods, including bivariate regression, multiple regression, and principal components analysis (PCA). All body mass estimation equations show high coefficients of determination (R²), and some equations exhibit low prediction errors in accuracy tests involving extant species of body size similar to O. carteri. Equations derived from PCA-summarized data and multiple regression generally perform better than those based on single variables. The consensus of estimates and their statistics suggests a body mass range of 170–290 g. This range is similar to previous estimates for this species based on first molar area (Gingerich, J Hum Evol 10:345–374, 1981; Conroy, Int J Primatol 8:115–137, 1987). Am J Phys Anthropol 109:41–52, 1999. © 1999 Wiley-Liss, Inc.     

Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca²⁺ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca²⁺ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca²⁺ but was activated upon incubation with Ca²⁺ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ~5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a K_i of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.     

Mouse hepatitis coronavirus replication induces host translational shutoff and mRNA decay, with concomitant formation of stress granules and processing bodies

Many viruses, including coronaviruses, induce host translational shutoff, while maintaining synthesis of their own gene products. In this study we performed genome-wide microarray analyses of the expression patterns of mouse hepatitis coronavirus (MHV)-infected cells. At the time of MHV-induced host translational shutoff, downregulation of numerous mRNAs, many of which encode protein translation-related factors, was observed. This downregulation, which is reminiscent of a cellular stress response, was dependent on viral replication and caused by mRNA decay. Concomitantly, phosphorylation of the eukaryotic translation initiation factor 2alpha was increased in MHV-infected cells. In addition, stress granules and processing bodies appeared, which are sites for mRNA stalling and degradation respectively. We propose that MHV replication induces host translational shutoff by triggering an integrated stress response. However, MHV replication per se does not appear to benefit from the inhibition of host protein synthesis, at least in vitro, since viral replication was not negatively affected but rather enhanced in cells with impaired translational shutoff.     

Reptilian transferrins: evolution of disulphide bridges and conservation of iron-binding center

Transferrins, found in invertebrates and vertebrates, form a physiologically important family of proteins playing a major role in iron acquisition and transport, defense against microbial pathogens, growth and differentiation. These proteins are bilobal in structure and each lobe is composed of two domains divided by a cleft harboring an iron atom. Vertebrate transferrins comprise of serotransferrins, lactoferrins and ovotransferrins. In mammals serotransferrins transport iron in physiological fluids and deliver it to cells, while lactoferrins scavenge iron, limiting its availability to invading microbes. In oviparous vertebrates there is only one transferrin gene, expressed either in the liver to be delivered to physiological fluids as serotransferrin, or in the oviduct with a final localization in egg white as ovotransferrin. Being products of one gene sero- and ovotransferrin are identical at the amino-acid sequence level but with different, cell specific glycosylation patterns. Our knowledge of the mechanisms of transferrin iron binding and release is based on sequence and structural data obtained for human serotransferrin and hen and duck ovotransferrins. No sequence information about other ovotransferrins was available until our recent publication of turkey, ostrich, and red-eared turtle (TtrF) ovotransferrin mRNA sequences [Ciuraszkiewicz, J., Olczak, M., Watorek, W., 2006. Isolation, cloning and sequencing of transferrins from red-eared turtle, African ostrich and turkey. Comp. Biochem. Physiol. 143 B, 301-310]. In the present paper, ten new reptilian mRNA transferrin sequences obtained from the Nile crocodile (NtrF), bearded dragon (BtrF), Cuban brown anole (AtrF), veiled and Mediterranean chameleons (VtrF and KtrF), sand lizard (StrF), leopard gecko (LtrF), Burmese python (PtrF), African house snake (HtrF), and grass snake (GtrF) are presented and analyzed. Nile crocodile and red-eared turtle transferrins have a disulphide bridge pattern identical to known bird homologues. A partially different disulphide bridge pattern was found in the Squamata (snakes and lizards). The possibility of a unique interdomain disulphide bridge was predicted for LtrF. Differences were found in iron-binding centers from those of previously known transferrins. Substitutions were found in the iron-chelating residues of StrF and TtrF and in the synergistic anion-binding residues of NtrF. In snakes, the transferrin (PtrF, HtrF and GtrF) N-lobe "dilysine trigger" occurring in all other known transferrins was not found, which indicates a different mechanism of iron release.     

The genus Cabomba (Cabombaceae)–a taxonomic study

The genus Cabomba (Cabombaceae) is revised. Five species and three varieties are recognized: C. aquatica, C. palaeformis, C. furcata, C. haynesii and C. caroliniana including var. caroliniana , var. pulcherrima , and var. flavida var. nov. A basic chromosome number of x = 13 is proposed. Special emphasis has been given to pollen and seed morphology as revealed by SEM studies.