Cold adaptation of tropomyosin

The conformational stability of unphosphorylated and phosphorylated α,α-striated tropomyosins from rabbit and shark (95% identical sequences) has been investigated. Three additional core positions are occupied by atypical amino acids in the protein from shark: Thr179(d), Ser190(a), and Ser211(a). These changes are thought to have further destabilized most, if not all, of the carboxyl-terminal half of the molecule. Heat-induced unfolding of shark tropomyosin (2 mg/mL, 0.1 M salt, pH 7) as monitored by far-UV circular dichroism is biphasic [T(m1) ～ 33 °C (main), and T(m2) ～ 54 °C] and takes place over a wider temperature span than that of the mammalian protein. The relationship between ellipticity (and excess heat) and temperature is insensitive to the presence in either tropomyosin of covalently bound phosphate. At ～10 mg/mL, the minor endotherm of shark tropomyosin is shifted to ～60 °C and T(m2) - T(m1) is increased to 25 °C; otherwise, the results of calorimetry are in agreement with those of circular dichroism. Analyses of cyanogen bromide fragments by far-UV circular dichroism and intact protein by near-UV circular dichroism (T(m) ～ 32 °C) show that the most stable sizable portion of shark tropomyosin is located within the amino-terminal half of the molecule. These findings illuminate those regions in tropomyosin where flexibility is critical and show that substitutions predicted to be unfavorable in one temperature regime are desirable in another.     

Dissociation of conditioned taste avoidance from conditioned disgust reactions induced by wheel running in rats

It is well established that wheel running in rats produces conditioned taste avoidance; that is, rats that run in wheels after consuming a novel-tasting solution later consume less of that solution than rats that do not run. In experiment 1, we found that wheel running also produces conditioned disgust reactions, indicated by gapes elicited by both the taste and context that were experienced before running. Experiment 2 showed that the conditioned disgust reactions were likely not due to running itself but to a by-product of running, the rocking of the wheel that occurs when the running stops. When rocking was reduced, the disgust reactions were also reduced, but consumption of the taste solution was not changed, showing dissociation of conditioned taste avoidance and disgust. These findings indicate that the taste avoidance induced by wheel running itself is more like the taste avoidance produced by rewarding drugs than that produced by nausea-inducing drugs.     

Effect of phosphorylation on the interaction and functional properties of rabbit striated muscle alpha alpha-tropomyosin

Phosphorylated rabbit cardiac alpha alpha-tropomyosin has been prepared either enzymatically (Montgomery, K., and Mak, A.S. (1984) J. Biol. Chem. 259, 5555-5560) or by fractionation of the phosphorylated and nonphosphorylated forms on a Mono Q column in 9 M urea, 50 mM Tris, pH 8.0. Although the phosphorylated and nonphosphorylated forms showed no difference in their F-actin binding properties, the phosphorylated protein had substantially higher viscosities at low ionic strengths, indicating a greater propensity for head-to-tail interaction. Similar measurements showed the strengthening of this interaction by whole troponin to be substantially reduced by phosphorylation even though the binding of whole troponin and troponin T to tropomyosin was demonstrated by affinity chromatography to be, if anything, strengthened by phosphorylation. In a reconstituted actin (4 microM) plus myosin subfragment 1 ATPase assay (50 mM ionic strength), significantly higher activities over a range (1 to 8 microM) of subfragment 1 concentrations were observed with phosphorylated tropomyosin compared with the nonphosphorylated protein. In the fully reconstituted system with troponin, there was no significant difference in the inhibition of ATPase in the absence of Ca2+. However, in its presence, the activities were appreciably increased with the phosphorylated tropomyosin compared to those with the nonphosphorylated form. These differences were eliminated by treatment of the phosphorylated tropomyosin with alkaline phosphatase. This is the first demonstration of an effect of phosphorylation on the functional properties of tropomyosin.     

Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.     

Strain persistence of invasive Candida albicans in chronic hyperplastic candidosis that underwent malignant change

Objectives: The aim of this study was to assess persistence and tissue invasion of Candida albicans strains isolated from a 65 year‐old patient with chronic hyperplastic candidosis (CHC), that subsequently developed into squamous cell carcinoma (SCC). Materials and Methods: C. albicans (n=7) were recovered from the oral cavity of the patient over seven years. Confirmation of CHC and SCC in this patient was achieved by histopathological examination of incisional biopsy tissue. DNA fingerprinting was performed on the seven isolates from the CHC patient together with a further eight isolates from patients with normal oral mucosa (n=2), chronic atrophic candidosis (n=1), SCC (n=1) and CHC (n=4). Genotyping involved the use of inter‐repeat PCR using the eukaryotic repeat primer 1251. Characterisation of the tissue invasive abilities of the isolates was achieved by infecting a commercially available reconstituted human oral epithelium (RHE; SkinEthic, Nice, France). After 24 h. C. albicans tissue invasion was assessed by histopathological examination. Results: DNA fingerprinting demonstrated strain persistence of C. albicans in the CHC patient over a seven year period despite provision of systemic antifungal therapy. The strain of C. albicans isolated from this patient was categorised as a high invader within the RHE compared to other isolates. Conclusions: Candidal strain persistence was evident in a patient with CHC over seven years. This persistence may be due to incomplete eradication from the oral cavity following antifungal therapy or subsequent recolonisation from other body sites or separate exogenous sources. The demonstration of enhanced in vitro tissue invasion by this particular strain may, in part, explain the progression to carcinoma.     

A comparison of the molecular species compositions of mammalian lung surfactant phospholipids

While dipalmitoyl phosphatidylcholine (PC16:0/16:0) is essential for pulmonary surfactant function, roles for other individual molecular species of surfactant phospholipids have not been established. If any phospholipid species other than PC16:0/16:0 is important for surfactant function, then it may be conserved across animal species. Consequently, we have quantified, by electrospray ionisation mass spectrometry, molecular species compositions of phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) in surfactants from human, rabbit, rat and guinea pig lungs. While PC compositions displayed only relatively minor variations across the animal species studied, there were wide variations of PG and PI concentrations and compositions. Human surfactant PG and PI were enriched in the same three monounsaturated species (PG16:0/18:1, PG18:1/18:1 and PG18:0/18:1) with minimal amounts of PG16:0/16:0 or polyunsaturated species, while all animal surfactant PG contained increased concentrations of PG16:0/16:0 and PG16:0/18:2. Animal surfactant PIs were essentially monounsaturated except for a high content of PI18:0/20:4 (29%) in the rat. As these four surfactants all maintain appropriate lung function of the respective animal species, then all their varied compositions of acidic phospholipids must be adequate at promoting the processes of adsorption, film refinement, respreading and collapse characteristic of surfactant. We conclude that this effectively monounsaturated composition of anionic phospholipid molecular species is a common characteristic of mammalian surfactants.     

A vertebrate slow skeletal muscle actin isoform

Salmonids utilize a unique, class II isoactin in slow skeletal muscle. This actin contains 12 replacements when compared with those from salmonid fast skeletal muscle, salmonid cardiac muscle and rabbit skeletal muscle. Substitutions are confined to subdomains 1 and 3, and most occur after residue 100. Depending on the pairing, the 'fast', 'cardiac' and rabbit actins share four, or fewer, substitutions. The two salmonid skeletal actins differ nonconservatively at six positions, residues 103, 155, 278, 281, 310 and 360, the latter involving a change in charge. The heterogeneity has altered the biochemical properties of the molecule. Slow skeletal muscle actin can be distinguished on the basis of mass, hydroxylamine cleavage and electrophoretic mobility at alkaline pH in the presence of 8 m urea. Further, compared with its counterpart in fast muscle, slow muscle actin displays lower activation of myosin in the presence of regulatory proteins, and weakened affinity for nucleotide. It is also less resistant to urea- and heat-induced denaturation. The midpoints of the change in far-UV ellipticity of G-actin versus temperature are approximately 45 degrees C ('slow' actin) and approximately 56 degrees C ('fast' actin). Similar melting temperatures are observed when thermal unfolding is monitored in the aromatic region, and is suggestive of differential stability within subdomain 1. The changes in nucleotide affinity and stability correlate with substitutions at the nucleotide binding cleft (residue 155), and in the C-terminal region, two parts of actin which are allosterically coupled. Actin is concluded to be a source of skeletal muscle plasticity.     

Mechanism of Regulation of Native Cardiac Muscle Thin Filaments by Rigor Cardiac Myosin-S1 and Calcium

We have studied the mechanism of activation of native cardiac thin filaments by calcium and rigor myosin. The acceleration of the rate of 2′-deoxy-3′-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from cardiac myosin-S1-mdADP-P_i and cardiac myosin-S1-mdADP by native cardiac muscle thin filaments was measured using double mixing stopped-flow fluorescence. Relative to inhibited thin filaments (no bound calcium or rigor S1), fully activated thin filaments (with both calcium and rigor-S1 bound) increase the rate of product dissociation from the physiologically important pre-power stroke myosin-mdADP-P_i by a factor of ∼75. This can be compared with only an ∼6-fold increase in the rate of nucleotide diphosphate dissociation from nonphysiological myosin-mdADP by the fully activated thin filaments relative to the fully inhibited thin filaments. These results show that physiological levels of regulation are not only dependent on the state of the thin filament but also on the conformation of the myosin. Less than 2-fold regulation is due to a change in affinity of myosin-ADP-P_i for thin filaments such as would be expected by a simple “steric blocking” of the myosin-binding site of the thin filament by tropomyosin. Although maximal activation requires both calcium and rigor myosin-S1 bound to the cardiac filament, association with a single ligand produces ∼70% maximal activation. This can be contrasted with skeletal thin filaments in which calcium alone only activated the rate of product dissociation ∼20% of maximum, and rigor myosin produces ∼30% maximal activation.