Influence of segmental body composition and adiposity hormones on resting metabolic rate and substrate utilization in overweight and obese adults.

PURPOSE: Low resting metabolic rate (RMR) and high carbohydrate reliance at rest are associated with weight gain, but are highly variable in obese individuals. This study determined the relationship of total and segmental body composition and adiposity hormones with RMR and respiratory exchange ratio (RER) in overweight and obese adults. METHODS: In 49 men (n = 23) and premenopausal women (n = 26) [mean ± SD; age = 35.0 ± 8.9 years; body mass index (BMI) = 33.6 ± 5.2 kg·m-2; percent body fat (%fat) = 40.0 ± 8.0%], RMR and RER were evaluated using indirect calorimetry. Total and segmental body composition [fat mass (FM), percent fat (%fat), lean mass (LM), visceral adipose tissue (VAT)] were estimated using dual-energy X-ray absorptiometry. Fasted blood and saliva samples were analyzed for insulin, leptin, estradiol, and cortisol. RESULTS: In men (M) and women (W), RMR significantly correlated (p < 0.05) with FM (M: R = 0.535; W: R = 0.784) and LM (M: R = 0.645; W: R = 0.867). Of the segmental measures, trunk LM (M: R = 0.593; W: R = 0.879; p < 0.05) and leg LM (M: R = 0.664; W: R = 0.821; p < 0.05) had the strongest correlations with RMR. In men, but not women, RER significantly correlated with FM (R = 0.449; p = 0.032), trunk FM (R = 0.501; p = 0.015), and VAT (R = 0.456; p = 0.029). In men, RMR positively correlated with cortisol (R = 0.430, p = 0.040) and estradiol (R = 0.649, p = 0.001) and RER positively correlated with insulin (R = 0.525, p = 0.010). In women, RMR positively correlated with insulin (R = 0.570, p = 0.006), but RER was not significantly correlated with hormones (p > 0.05). CONCLUSIONS: Segmental evaluation of body composition, specifically in the lower extremities and abdomen, may be an effective and efficient way to evaluate metabolic status. Sex-specific evaluations are also imperative.

Parvalbumin- and vasoactive intestinal polypeptide-expressing neocortical interneurons impose differential inhibition on Martinotti cells.

Disinhibition of cortical excitatory cell gate information flow through and between cortical columns. The major contribution of Martinotti cells (MC) is providing dendritic inhibition to excitatory neurons and therefore they are a main component of disinhibitory connections. Here we show by means of optogenetics that MC in layers II/III of the mouse primary somatosensory cortex are inhibited by both parvalbumin (PV)- and vasoactive intestinal polypeptide (VIP)-expressing cells. Paired recordings revealed stronger synaptic input onto MC from PV cells than from VIP cells. Moreover, PV cell input showed frequency-independent depression, whereas VIP cell input facilitated at high frequencies. These differences in the properties of the two unitary connections enable disinhibition with distinct temporal features.

The efficacy of closed reduction in displaced distal radius fractures.

INTRODUCTION: Closed reduction (CR) to restore fracture alignment and subsequent cast fixation is a common practice in the treatment of distal radius fractures. No clear consensus exists about the appropriate indication for CR. This study aims to compare radiological and functional results in patients with moderately to severely displaced distal radius fractures that were immobilised by cast fixation with or without prior CR. PATIENTS AND METHODS: A total of 206 patients with distal radius fractures from one UK hospital were prospectively documented between 2001 and 2002. Patients with moderately to severely displaced fractures that were treated conservatively with or without CR were eligible for this analysis. Fracture displacement was assessed by measurements on injury radiographs and latent class analysis. The radiological and functional results as assessed by range of motion; and the Disabilities of the Arm, Shoulder and Hand (DASH); Gartland and Werley; and SF-36 scores were compared after 6 weeks, 6 months and 1 year. RESULTS: As many as 83 patients (seven males) with a mean age of 62.2 years were included; 62 patients were treated with CR, the remaining 21 did not receive CR. During the follow-up period, no differences were found in the measurements of range of motion, DASH and SF-36 scores between the treatment groups. Fractures that were treated with CR lost anatomical restoration. However, after 1 year, palmar tilt and radial angles had significantly improved compared with the baseline measurements. Although no significant difference of radiological parameters between the treatment groups was found, the Gartland and Werley score resulted in a significantly better outcome for those patients without CR after 1 year. CONCLUSIONS: Although all patients - independent of their treatment - reached a successful level of activities in their daily life, there was no benefit of CR for patients with moderately to severely displaced fractures. The decision to treat with CR should be made carefully, especially in patients with high wrist-function demands.

Macrophage interactions.

B. anthracis virulence is the sum of the contributions of factors involved in toxicity, growth and persistence in the host. Recent data has revealed that the interactions between B. anthracis and macrophage is central to the B. anthracis pathogenesis. This review presents and describes tactics by which B. anthracis not only overcomes and avoids macrophages but also perverts the host defense immune system and defense-related products to its advantage. The understanding of the complex network of such interactions is likely to allow new therapeutic and preventative strategies to be developed.

Fate of germinated Bacillus anthracis spores in primary murine macrophages.

We investigated the fate of germinated Bacillus anthracis spores after their germination in Swiss murine peritoneal macrophages and in the cell line RAW264.7. We found that the lethal toxin and the oedema toxin are germ-associated factors that are essential for the survival of the vegetative form in host cells. We also found that pX02 is not involved in this complex pathogenic process. By transmission electron microscopy, we showed the tight interaction between the exosporium of the spore and the phagosomal membrane of the macrophage. Our data strongly suggest that the B. anthracis toxinogenic, unencapsulated Sterne strain (7702) does not multiply within macrophages. These results contributed to reveal the strategies used by B. anthracis to survive within the host and to reach the external medium where they proliferate.

Distribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressure.

Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species. B. anthracis is a mammal pathogen, B. thuringiensis an entomopathogen and B. cereus a ubiquitous soil bacterium and an occasional human pathogen. In two clinical isolates of B. cereus, in some B. thuringiensis strains and in B. anthracis, an S-layer has been described. We investigated how the S-layer is distributed in B. cereus, and whether phylogeny or ecology could explain its presence on the surface of some but not all strains. We first developed a simple biochemical assay to test for the presence of the S-layer. We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B. cereus and 25 non-B. anthracis of the B. anthracis cluster. When present, the genetic organization of the S-layer locus was analysed further. It was identical in B. cereus and B. anthracis. Nineteen strains harboured an S-layer, 16 of which belonged to the B. anthracis cluster. All 19 were B. cereus clinical isolates or B. thuringiensis, except for one soil and one dairy strain. These findings suggest a common phylogenetic origin for the S-layer at the surface of B. cereus strains and, presumably, ecological pressure on its maintenance.

Toxins of Bacillus anthracis.

Bacillus anthracis, a gram positive bacterium, is the causative agent of anthrax. This organism is capsulogen and toxinogenic. It secretes two toxins which are composed of three proteins: the protective antigen (PA), the lethal factor (LF) and the edema factor (EF). The lethal toxin (PA+LF) provokes a subit death in animals, the edema toxin (PA+EF) induces edema. The edema and the lethal factors are internalised into the eukaryotic target cells via the protective antigen. EF and LF exert a calmoduline dependent adenylate cyclase and a metalloprotease activity respectively. Progress in the structure-function relationship of these three proteins, their regulation mechanisms and their roles in pathogenesis and immunoprotection will be exposed.

Anthrax.

Bacillus anthracis was shown to be the etiological agent of anthrax by R. Koch and L. Pasteur at the end of the nineteenth century. The concepts on which medical microbiology are based arose from their work on this bacterium. The link between plasmids and major virulence factors of B. anthracis was not discovered until the 1980s. The three toxin components are organized in two A-B type toxins, and the bacilli are covered by an antiphagocytic polyglutamic capsule. Structure-function analysis of the toxins indicated that the common B-domain binds to a ubiquitous cell receptor and forms a heptamer after proteolytic activation. One enzyme moiety is an adenylate cyclase and the other is a Zn(2+) metalloprotease, which is able to cleave MAPKKs. The capsule covers an S-layer sequentially composed of two distinct proteins. Knowledge of the toxins facilitates the design of safer veterinary vaccines. Spore-structure analysis could contribute to the improvement of human nonliving vaccines. The phylogeny of B. anthracis within the Bacillus cereus group is also reviewed.

Wegener's granulomatosis: description of a case where cutaneous involvement was correlated with elevation of the c-ANCA titer.

Wegener's granulomatosis is a systemic disease characterized by necrotizing granulomas and vasculitis involving the upper and lower respiratory tract as well as the kidneys. Cutaneous manifestations consist mainly of papules or papulonecrotic lesions. c-ANCA are known to be a valuable adjunct for the diagnosis and follow-up of Wegener's granulomatosis with systemic involvement. We report the case of a 49-year-old man with Wegener's granulomatosis who developed two relapses of the disease with cutaneous manifestation and who presented with concomitant elevation of the c-ANCA and more precisely the subset PR3-ANCA during the acute phase of the disease.

Spatial arrangement of cerebro-pontine terminals.

Understanding the interaction of the cerebral cortex and cerebellum requires knowledge of the highly complex spatial characteristics of cerebro-cerebellar signal transfer. Cerebro-pontine fibers from one neocortical site terminate in several sharply demarcated patches across large parts of the pontine nuclei (PN), and fibers from different neocortical areas terminate in the same pontine region. To determine whether projections from segregated neocortical sites overlap in the PN, we studied double anterograde tracing of cerebro-pontine terminals from large parts of rat neocortex. In none of these experiments, including double injection into two functionally related areas, were we able to demonstrate overlapping patches, although close spatial relationships were always detected. This non-overlapping distribution is consistent with a compartmentalized organization of the cerebro-pontine projection and may be the basis of the fractured type of maps found in the cerebellar granular layer. The critical distance between two sites on the neocortical surface that project to non-overlapping patches in the PN was found to be 600 microm, by using double injection within the whisker representation of the primary somatosensory area. This matches the diameter of dendritic trees of layer 5 projection neurons, indicating that non-overlapping populations of neocortical projection neurons possess non-overlapping patches of pontine terminals. Estimations based on this critical distance and the pontine volume anterogradely labeled by one injection site indicate that the size of the PN may be well suited to accommodate a complete set of non-overlapping pontine patches from all possible neocortical sites.

The incompatibility between the PlcR- and AtxA-controlled regulons may have selected a nonsense mutation in Bacillus anthracis.

Bacillus anthracis, Bacillus thuringiensis and Bacillus cereus are members of the Bacillus cereus group. These bacteria express virulence in diverse ways in mammals and insects. The pathogenic properties of B. cereus and B. thuringiensis in mammals results largely from the secretion of non-specific toxins, including haemolysins, the production of which depends upon a pleiotropic activator PlcR. In B. anthracis, PlcR is inactive because of a nonsense mutation in the plcR gene. This suggests that the phenotypic differences between B. anthracis on the one hand and B. thuringiensis and B. cereus on the other could result at least partly from loss of the PlcR regulon. We expressed a functional PlcR in B. anthracis. This resulted in the transcriptional activation of genes weakly expressed in the absence of PlcR. The transcriptional activation correlated with the induction of enzymatic activities and toxins including haemolysins. The toxicity of a B. anthracis PlcR+ strain was assayed in the mouse subcutaneous and nasal models of infection. It was no greater than that of the parental strain, suggesting that the PlcR regulon has no influence on B. anthracis virulence. The PlcR regulon had dramatic effects on the sporulation of a B. anthracis strain containing the virulence plasmid pXO1. This resulted from incompatible interactions with the major AtxA-controlled virulence regulon. We propose that the PlcR-controlled regulon in B. anthracis has been counterselected on account of its disadvantageous effects.

Characterization of a plasmid region involved in Bacillus anthracis toxin production and pathogenesis.

The germination of spores within the host is the initial step of anthrax infection. We have shown, using immunofluorescence staining, confocal scanning laser microscopy and image cytometry analysis, that the alveolar macrophage is the primary site of B. anthracis germination in a murine inhalation infection model. B. anthracis germinated inside macrophages, in vesicles derived from the phagosomal compartment. We have demonstrated that the toxin genes and their trans-activator, AtxA, are expressed within the macrophages after germination. It was also shown that the pXO1 plasmid strongly enhanced capsule formation and that this influence is mediated by AtxA. This indicates the existence of a regulon where AtxA is the regulatory protein acting on genes located on different plasmids. We identified a tricistronic germination operon gerX located between the pag and atxA genes on the 40-kb toxin-encoding fragment of pXO1 . Analysis of a gerX null mutant indicated that gerX-encoded proteins are involved in the virulence of B. anthracis.

Lethal factor of Bacillus anthracis cleaves the N-terminus of MAPKKs: analysis of the intracellular consequences in macrophages.

The lethal toxin of Bacillus anthracis consists of two proteins, PA and LF, which together induce lethal effects in some animal species and cause macrophage lysis. LF is a zinc-binding protein with metalloprotease activity. With a two-hybrid system approach we identified MAP kinase kinases (MAPKKs) Mekl and Mek2 as proteins interacting with LF. LF was shown to cleave Mek1 and Mek2 and an additional MAPKK family member MKK3, within their N-terminal region. We examined macrophage cell lines and primary peritoneal cells with different sensitivities to LF but did not find a direct correlation between MAPKKs cleavage and cell death. On the other hand, sublytic doses of LF cleave MAPKKs and cause a reduction in the LPS/IFNgamma-induced production of proinflammatory mediators. These findings are discussed with respect to the possible role of LF in the initial phase of infection.

Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor.

The lethal factor (LF) produced by toxigenic strains of Bacillus anthracis is a Zn(2+)-endopeptidase that cleaves the mitogen-activated protein kinase kinases (MAPKKs) MEK1, MEK2 and MKK3. Using genetic and biochemical approaches, we have extended the study of LF proteolytic specificity to all known MAPKK family members and found that LF also cleaves MKK4, MKK6 and MKK7, but not MEK5. The peptide bonds hydrolysed by LF within all MAPKKs were identified. Cleavage invariably occurs within the N-terminal proline-rich region preceding the kinase domain, thus disrupting a sequence involved in directing specific protein-protein interactions necessary for the assembly of signalling complexes. Alignment of the sequences flanking the site of cleavage reveals the occurrence of some consensus motifs: position P2 and P1' are occupied by hydrophobic residues and at least one basic residue is present between P4 and P7. The implications of these findings for the biochemical activity and functional specificity of LF are discussed.

Protective antigen-mediated antibody response against a heterologous protein produced in vivo by Bacillus anthracis.

Bacillus anthracis secretes a lethal toxin composed of two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells. Immunization with attenuated B. anthracis strains induces an antibody response against PA and LF. The LF-specific response is potentiated by the binding of LF to PA. In this study, we investigated the capacity of PA to increase the antibody response against a foreign antigen. We constructed a chimeric gene encoding the PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin (ToxC). The construct was introduced by allelic exchange into the locus encoding LF. Two recombinant B. anthracis strains secreting the hybrid protein LF254-ToxC were generated, one in a PA-producing background and the other in a PA-deficient background. Mice were immunized with spores of the strains, and the humoral response and protection against tetanus toxin were assessed. The B. anthracis strain producing both PA and LF254-ToxC induced significantly higher antibody titers and provided better protection against a lethal challenge with tetanus toxin than did its PA-deficient counterpart. Thus, PA is able to potentiate protective immunity against a heterologous antigen, demonstrating the potential of B. anthracis recombinant strains for use as live vaccine vehicles.

Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation.

Several bacterial proteins are non-covalently anchored to the cell surface via an S-layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non-covalent interaction between the SLH domain and peptidoglycan-associated polymers. Here we report the characterization of a two-gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH-containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan-associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH-containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

Characterization of Bacillus anthracis strains used for vaccination.

Three Bacillus anthracis strains, formerly used as anti-anthrax vaccine strains in Argentina, were characterized from genetic and pathogenic perspectives. Southern blotting and PCR with pXO1 and pXO2 probes and primers, as well as pathogenicity and protection tests in guinea pigs and mice, were performed. Two of the B. anthracis strains contained both pXO1 and pXO2 plasmids, as did the fully virulent strains, while the third was a Sterne-type strain (pXO1+, pXO2-). The three strains were, however, markedly less pathogenic than a wild-type virulent strain. The methodology applied here may be used to characterize other B. anthracis strains.