Comparative statistical mechanics of myosin molecular motors in rat heart, diaphragm and tracheal smooth muscle

Purpose

Statistical mechanics establishes a link between microscopic properties of matter and its bulk properties. A. Huxley's equations (1957) [1] provide the necessary phenomenological formalism to use statistical mechanics.

Methods

We compared statistical mechanics in rat diaphragm in tetanus (tet; n = 10) and twitch (tw; n = 12) modes, in heart in twitch mode (n = 20), and in tracheal smooth muscle in tetanus mode (TSM; n = 10). This powerful tool makes it possible to determine: (i) statistical entropy (S) which is related to the dispersal of energy and represents a measure of the degree of disorder in muscular system; (ii) thermodynamic force A/T (chemical affinity A and temperature T); (iii) thermodynamic flow (υ); (iv) entropy production rate (A/T × υ), which quantifies irreversible chemical processes generated by myosin crossbridge (CB) molecular motors.

Results

All muscles studied operated near equilibrium, i.e., A << 2500 J/mol and in a stationary linear regime, i.e., A/T varied linearly with υ. The heart operated farther from equilibrium than both diaphragm (tet and tw) and TSM, as attested by its high entropy production rate. S was of the same order of magnitude in heart and TSM but lower in diaphragm (tet and tw).

Conclusion

CB kinetics derived from A. Huxley's equations conferred a characteristic profile in terms of statistical mechanics on each muscle type. All studied muscles differed in terms of statistical entropy, chemical affinity, and entropy production rate. Stimulation mode (tet and tw) modulated CB kinetics and statistical mechanics. All muscle types operated near equilibrium and in a stationary linear regime.     

A rapid, homogeneous, fluorescence polarization binding assay for peroxisome proliferator-activated receptors alpha and gamma using a fluorescein-tagged dual PPARalpha/gamma activator

Peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear hormone receptor family are important drug targets for the treatment of metabolic diseases. PPARalpha and PPARgamma play crucial roles in lipid and glucose metabolism, respectively. Therefore, screening methods that help to rapidly identify activators of these receptors should be of considerable value. A homogeneous fluorescence polarization (FP) ligand binding assay capable of rapidly identifying ligands that bind to both PPARalpha and PPARgamma has been developed using purified PPARalpha or PPARgamma ligand binding domains and a fluorescein-labeled analog (FLA) of a potent dual PPARalpha/gamma activator. FLA activator showed good binding affinity toward both PPARalpha (K(i)=0.7microM) and PPARgamma (K(i)=0.4microM). The binding of FLA activator was rapid and reached a plateau within 10 min. The resulting FP signal was stable for at least 18h. The FP binding assay performed robustly in a 384-well format, and the average Z' value was 0.77. There was a good correlation between the binding potency (IC(50) values) and rank order of binding potency for a panel of standard PPAR ligands obtained in FP binding assay and scintillation proximity assay or gel filtration binding assays using (3)H-labeled PPARalpha (r(2)=0.99) and PPARgamma (r(2)=0.99) ligands. There was also a good correlation of IC(50) values obtained by FP binding assay and scintillation proximity assay for the clinically used PPAR activators. Thus, the FP binding assay with a single fluorescein-labeled PPARalpha/gamma dual activator offers a homogeneous nonradioactive, sensitive, robust, and less expensive high-throughput assay for detecting compounds that bind to both PPARgamma and PPARalpha. Using this FP binding assay, we have identified a large number of PPARalpha/gamma dual activators. A similar assay platform may be easily adapted to other members of the nuclear hormone receptor family.     

Increased expression of PPARgamma in high fat diet-induced liver steatosis in mice

The present study was performed to examine a hypothesis that peroxisome proliferator-activated receptor gamma (PPARgamma) is implicated in high fat diet-induced liver steatosis. Mice were fed with control or high fat diet containing approximately 10% or 80% cholesterol, respectively. Macroscopic and microscopic findings demonstrated that lipid accumulation in the liver was observed as early as 2 weeks after high fat diet and that high fat diet for 12 weeks developed a fatty liver phenotype, establishing a novel model of diet-induced liver steatosis. Gene profiling with microarray and real-time PCR studies demonstrated that among genes involved in lipid metabolism, adipogenesis-related genes, PPARgamma and its targeted gene, CD36 mRNA expression was specifically up-regulated in the liver by high fat diet for 2 weeks. Immunohistochemical study revealed that PPARgamma protein expression is increased in the nuclei of hepatocytes by high fat diet. It was also shown that protein expression of cAMP response element-binding protein (CREB), an upstream molecule of PPARgamma, in the liver was drastically suppressed by high fat diet. All these results suggest for the first time that the CREB-PPARgamma signaling pathway may be involved in the high fat diet-induced liver steatosis.     

Arthropod FMRFamide-related peptides modulate muscle activity in helminths

FMRFamide-related peptides are common to a wide variety of invertebrate species, including helminths and arthropods. In arthropods, five distinct FMRFamide-related peptide subfamilies are recognised: the myosuppressins, extended-FLRFamides, -FMRFamides, -RFamides, and sulfakinins, members of which induce potent and diverse myotropic effects. Whilst >80 FMRFamide-related peptides have been identified in nematodes, only four FMRFamide-related peptides have been characterised from flatworms. The Ascaris suum ovijector/body wall bioassay and the Procerodes littoralis muscle fibre bioassay have proved both reliable and sensitive systems for assessing the functional activities of FMRFamide-related peptides in vitro, and data describing the effects of native FMRFamide-related peptides in these systems are rapidly accumulating. This is the first study to determine the cross-phyla activities of non-native FMRFamide-related peptides in both nematode and flatworm species. In the present study, the effects of 10 arthropod FMRFamide-related peptides (leucomyosuppressin [pQDVDHVFLRFamide], schistoFLRFamide [PDVDHVFLRFamide] and truncated analogues [HVFLRFamide and VFLRFamide], lobster peptide I [TNRNFLRFamide], lobster peptide II [SDRNFLRFamide], manducaFLRFamide II [GNSFLRFamide], manducaFLRFamide III [DPSFLRFamide], calliFMRFamide 4 [KPNQDFMRFamide] and perisulfakinin [EQFDDY(SO(3)H)GHMRFamide]), representing the five subfamilies, were examined on the body wall and ovijector of the parasitic porcine nematode, A. suum and dispersed muscle fibres from the free-living turbellarian, P. littoralis. The muscle activity of the ovijector was found to be modulated significantly by each of the arthropod FMRFamide-related peptides tested; the effects were concentration-dependent, reversible and repeatable. All but one (perisulfakinin) of the 10 arthropod FMRFamide-related peptides examined modulated significantly the activity of A. suum body wall muscle. In addition, all of the arthropod FMRFamide-related peptides examined induced potent concentration-dependent contractions of P. littoralis muscle fibres. These results reveal similarities in the ligand requirement(s) between FMRFamide-related peptide receptors within the Phyla Arthropoda, Nematoda and Platyhelminthes, and indicate significant receptor promiscuity, which highlights the potential of FMRFamide-related peptide receptors as legitimate targets for novel endectocidal agents.     

Hematopoietic contribution to skeletal muscle regeneration in acid alpha-glucosidase knockout mice.

Recent studies have shown that cells from bone marrow (BM) can give rise to differentiated skeletal muscle fibers. However, the mechanisms and identities of the cell types involved remain unknown. We performed BM transplantation in acid alpha-glucosidase (GAA) knockout mice, a model of glycogen storage disease type II, and our observations suggested that the BM cells contribute to skeletal muscle fiber formation. Furthermore, we showed that most CD45+:Sca1+ cells have a donor character in regenerating muscle of recipient mice. Based on these findings, CD45+:Sca1+ cells were sorted from regenerating muscles. The cell number was increased with granulocyte colony-stimulating factor after cardiotoxin injury, and the cells were transplanted directly into the tibialis anterior (TA) muscles of GAA knockout mice. Sections of the TA muscles stained with anti-laminin-alpha2 antibody showed that the number of CD45+:Sca1+ cells contributing to muscle fiber formation and glycogen levels were decreased in transplanted muscles. Our results indicated that hematopoietic stem cells, such as CD45+:Sca1+ cells, are involved in skeletal muscle regeneration.     

Phylogenetic relationships of Fusarium poae based on EF-1 alpha and mtSSU sequences

A molecular phylogenetic analysis of Fusarium poae isolates from South America (Argentina) and Europe (mainly England, Germany, Italy) was performed using 98 F. poae, four Fusarium culmorum, two Fusarium sporotrichioides and one Fusarium langsethiae isolates. Phylogenetic analyses were performed using nuclear (translation elongation factor 1-alpha, EF-1 alpha) and mitochondrial (mitochondrial small subunit rDNA, mtSSU) sequences. Partitioned (each dataset separately) and combined (EF-1 alpha+mtSSU) analyses did not reveal any clear correlations from the inferred branching topology, between the distribution of observed haplotypes and the geographic origin and/or host species. Results from the present study confirmed that isolates from F. poae form a monophyletic group, and the low variability within isolates from a broad geographic range suggests a common lineage history. Among F. poae isolates from Argentina, however, some were found to possess an insert within mtSSU with structural similarities to group IC2 introns. F. poae isolates differing by the presence/absence of a mtSSU insertion were characterized further by analysis of a portion of the Tri5 gene, but this sequence was unable to reveal variability. The presence of this insert only within isolates from Argentina suggests that evolutionary events (insertions/deletions) are probably taking place within the Argentinian F. poae isolates, and that the acquisition of this insert occurred after geographic isolation of the Argentinian and European populations.     

Brain metabolic markers reflect susceptibility status in cytokine gene knockout mice with murine cerebral malaria

Treatment of cerebral malaria, a complication of the world's most significant parasitic disease, remains problematic due to lack of understanding of its pathogenesis. Metabolic changes, along with cytokine expression alterations and blood cell sequestration in the brain, have previously been reported during severe disease in human infection and mouse models leading to the "cytopathic hypoxia" and "sequestration" theories of pathogenesis. Here, to determine the robustness of the metabolic changes and their relationship to disease development, we investigated changes in cerebral metabolic markers in a mouse model of cerebral malaria (CM) in wildtype (C57BL/6) and cytokine knockout (TNF(-/-), IFNgamma(-/-) and LTalpha(-/-)) mice using multinuclear magnetic resonance spectroscopy. Mice susceptible to CM (wildtype, TNF(-/-)) showed decreased cerebral glucose use, decreased Krebs cycle metabolism and decreased high-energy phosphates. Conversely, mice resistant to CM (IFNgamma(-/-), LTalpha(-/-)) showed little sign of these effects, despite identical levels of parasitemia. Previously reported changes in lactate were shown to be strain dependent. Elevated glutamine and decreased phosphorylation potential emerged as robust metabolic markers of susceptibility, further implicating the trytophan/NAD(+) pathway in disease development. Thus these metabolic changes are firmly linked both to the immune system response to malaria and to the occurrence of pathogenic changes in experimental CM.     

Enhanced pro-inflammatory cytokine production in Galphai2-deficient mice on colitis prone and colitis resistant 129Sv genetic backgrounds

Mice deficient in G-protein subunit alphai2 develop colitis closely resembling human ulcerative colitis when raised on 129SvEv background. When backcrossing the Galphai2-deficiency into a 129SvJBom genetic background, surprisingly, mice did not develop colitis. In vitro stimulation of splenocytes with formalin-killed Staphylococcus aureus resulted in significantly increased production of interleukin-1beta, tumor necrosis factor, and interleukin-12p40 in Galphai2(-/-) as compared to control mice. The enhanced production of pro-inflammatory cytokines was seen in colitis prone as well as in colitis resistant genetic background. A similar outcome was seen upon stimulation with toxic shock syndrome toxin-1, a T cell superantigen, except that Galphai2(-/-) colitis resistant 129SvJBom splenocytes did not show increased production of IL-12p40 as compared to their controls.     

Cost-effective production of Bacillus thuringiensis by solid-state fermentation

Production of Bacillus thuringiensis (Bt) was standardized on wheat bran based media in 250 ml Erlenmeyer flasks. Scale-up of Bt production on the best medium in plastic tubs with aeration at 8 h intervals starting 16 h after incubation yielded a significant increase in spore count and toxin content of the product. Maximum lysis of Bt cells was obtained by 60 h of incubation at 30 degrees C. This protocol was suitable for production of Bt strains and local isolates. The Bt produced proved highly effective at 0.1% concentration against larvae of castor semilooper, Achaea janata L, resulting in complete mortality by three days in laboratory bioassays. In field trials, the population of castor semilooper larvae on the castor bean crop was reduced significantly by three days after application. The cost for material production of 1 kg of Bt was approximately US dollars 0.70.     

Calcineurin regulates enteric muscle contraction through EXP-1, excitatory GABA-gated channel, in C. elegans

The enteric muscle contraction (EMC) is the last step of the defecation behavior which occurs every 50 s in Caenorhabditis elegans. This EMC is regulated by intestinal and anal depressor muscles, which are innervated by GABA motor neurons. Our data show that calcineurin (tax-6) is expressed in intestinal muscle and anal depressor muscle, and the gain-of-function mutant of calcineurin, tax-6(jh107), shows defects in enteric muscle contractions. In addition, the intracellular region of EXP-1, an excitatory GABA receptor, specifically binds to calcineurin A. This interaction between TAX-6 and EXP-1 appears to be independent of both calcium and CNB, which is the calcium-binding regulatory subunit. Genetic evidence of epistasis between cnb-1(jh103) and exp-1(sa6) suggests that calcineurin functions as a negative regulator of excitatory GABA receptor in GABA signaling in C.elegans.     

5alpha, 8alpha-Epidioxysterol sulfate from a diatom Odontella aurita

A 5alpha,8alpha-epidioxysterol sulfate was isolated from the cultured diatom Odontella aurita (NIES 589), and its structure was elucidated by spectroscopic methods.     

Sclerotomal origin of vascular smooth muscle cells and pericytes in the embryo

We previously demonstrated that progenitors of both endothelium and smooth muscle cells in the aortic wall originated from the somite in the trunk of the embryo. However whether the contribution to vascular Smooth Muscle Cells (vSMC) is restricted to the aorta or encompasses other vessels of the trunk is not known. Moreover, the somitic compartment that gives rise to vSMC is yet to be defined. Quail-chick orthotopic transplantations of either the segmental plate or the dorsal or ventral halves from single somites demonstrate that 1° vSMC of the body wall including those of the limbs originate from the somite. 2° Like vSMC, aortic pericytes originate from the somite. 3° The sclerotome is the somite compartment that gives rise to vSMC and pericytes. PAX1 and FOXC2, two molecular markers of the sclerotomal compartment, are expressed by vSMC and pericytes during the earliest phases of vascular wall formation. Later on, PDGFR-β and MYOCARDIN are also expressed by these cells. In contrast, the dermomyotome gives rise to endothelium but never to cells in the vascular wall. Taken together, out data point out to the critical role of the somite in vessel formation and demonstrate that vSMC and endothelial cells originate from two independent somitic compartments.     

Secondary production of Calanus helgolandicus in the Western English Channel

The objective of this study was to investigate the production of Calanus helgolandicus off Plymouth, Western English Channel, from April to August 1998 comparing a range of different methods. The abundance of all developmental stages from NI to adult was determined. Body carbon and nitrogen and length were measured on individual NVI to adults. Female egg production was also monitored. All developmental stages were generally represented but four main periods of recruitment were determined. Two periods of low egg production were observed on the 13th and on the 27th of July: (i) the first was due to the arrival of newly moulted or not yet fertilised females in the population and (ii) the second low period was possibly due to the senescence of the females. Finally, the total production of C. helgolandicus was estimated using four different methods: the weight increment, the egg production, Huntley and Lopez and Hirst and Lampitt methods. Depending on the methods used, the production accumulated over the period from May 14 to August 10 ranged between 12 and 45 mg C m⁻³.     

Misexpression of acetylcholinesterases in the C. elegans pha-2 mutant accompanies ultrastructural defects in pharyngeal muscle cells

pha-2 is the Caenorhabditis elegans homolog of the vertebrate homeobox gene Hex. Embryonic expression of pha-2 is mostly pharyngeal and the only described mutant allele of pha-2 results in a severe pharyngeal defect in which certain muscle cells (pm5 cells) and neurons are grossly deformed. Here, we performed a detailed characterization of the pha-2 phenotype using cell-type-specific reporters, physical manipulation of the nuclei in pharyngeal muscle cells using "optical tweezers", electron microscopy, staining of the actin cytoskeleton as well as phenotypic rescue and ectopic expression experiments. The main findings of the present study are (i) the pha-2 (ad472) mutation specifically impairs the pharyngeal expression of pha-2; (ii) in the pha-2 mutant, the cytoskeleton of the pm5 cells is measurably weaker than in normal cells and is severely disrupted by large tubular structures and organelles; (iii) the pm5 cells of the pha-2 mutant fail to express the acetylcholinesterase genes ace-1 and ace-2; (iv) ectopic expression of pha-2 can induce ectopic expression of ace-1 and ace-2; and (v) the anc-1 mutant with mislocalized pm5 cell nuclei occasionally shows an isthmus phenotype similar to that of pha-2 worms.