Phosphorylation of Subunit Proteins of Intermediate Filaments from Chicken Muscle and Nonmuscle Cells

The phosphorylation of the subunit proteins of intermediate (10-nm) filaments has been investigated in chicken muscle and nonmuscle cells by using a two-dimensional gel electrophoresis system. Desmin, the 50,000-dalton subunit protein of the intermediate filaments of muscle, had previously been shown to exist as two major isoelectric variants--alpha and ß --in smooth, skeletal, and cardiac chicken muscle. Incubation of skeletal and smooth muscle tissue with 32PO4{}3- reveals that the acidic variant, alpha -desmin, and three other desmin variants are phosphorylated in vivo and in vitro. Under the same conditions, minor components of alpha - and ß -tropomyosin from skeletal muscle, but not smooth muscle, are also phosphorylated. Both the phosphorylated desmin variants and the nonphosphorylated ß -desmin variant remain insoluble under conditions that solubilize actin and myosin filaments, but leave Z-discs and intermediate filaments insoluble. Primary cultures of embryonic chicken muscle labeled with 32PO4{}3- possess, in addition to the desmin variants described above, a major nonphosphorylated and multiple phosphorylated variants of the 52,000-dalton, fibroblast-type intermediate filament protein (IFP). Filamentous cytoskeletons, prepared from primary myogenic cultures by Triton X-100 extraction, contain actin and all of the phosphorylated and nonphosphorylated variants of both desmin and the IFP. Similarly, these proteins are the major components of the caps of aggregated 10-nm filaments isolated from the same cell cultures previously exposed to Colcemid. These results demonstrate that a nonphosphorylated and several phosphorylated variants of desmin and IFP are present in assembled structures in muscle and nonmuscle cells

Critical Temperature and Amplitude Ratios from a Finite-Temperature Renormalization Group

We study \l\f^4 theory using an environmentally friendly finite-temperature renormalization group. We derive flow equations, using a fiducial temperature as flow parameter, develop them perturbatively in an expansion free from ultraviolet and infrared divergences, then integrate them numerically from zero to temperatures above the critical temperature. The critical temperature, at which the mass vanishes, is obtained by integrating the flow equations and is determined as a function of the zero-temperature mass and coupling. We calculate the field expectation value and minimum of the effective potential as functions of temperature and derive some universal amplitude ratios which connect the broken and symmetric phases of the theory. The latter are found to be in good agreement with those of the three-dimensional Ising model obtained from high- and low-temperature series expansions.Comment: 14 pages of LaTeX. Postscript figures available upon request form [email protected]

Antimicrobial antagonists against food pathogens; a bacteriocin perspective

peer-reviewedEfforts are continuing to find novel bacteriocins with enhanced specificity and potency. Traditional plating techniques are still being used for bacteriocin screening studies, however, the availability of ever more bacterial genome sequences and the use of in silico gene mining tools have revealed novel bacteriocin gene clusters that would otherwise have been overlooked. Furthermore, synthetic biology and bioengineering-based approaches are allowing scientists to harness existing and novel bacteriocin gene clusters through expression in different hosts and by enhancing functionalities. The same principles apply to bacteriocin producing probiotic cultures and their application to control pathogens in the gut. We can expect that the recent developments on bacteriocins from Lactic Acid Bacteria (LAB) described here will contribute greatly to increased commercialisation of bacteriocins in food systems.This work was funded by the Alimentary Pharmabiotic Centre, a research centre funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. The authors and their work were supported by SFI (grant no. 12/RC/2273

Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

peer-reviewedIt is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.This work was supported by the Irish Government under the National Development Plan; by the Irish Research Council for Science Engineering and Technology (IRCSET); by Enterprise Ireland; and by Science Foundation Ireland (SFI), through the Alimentary Pharmabiotic Centre (APC) at University College Cork, Ireland, which is supported by the SFI-funded Centre for Science, Engineering and Technology (SFI-CSET) and provided P.D.C., C.H. and R.P.R. with SFI Principal Investigator funding

The Specific Heat of a Ferromagnetic Film.

We analyze the specific heat for the $O(N)$ vector model on a $d$-dimensional film geometry of thickness $L$ using ``environmentally friendly'' renormalization. We consider periodic, Dirichlet and antiperiodic boundary conditions, deriving expressions for the specific heat and an effective specific heat exponent, \alpha\ef. In the case of $d=3$, for $N=1$, by matching to the exact exponent of the two dimensional Ising model we capture the crossover for \xi_L\ra\infty between power law behaviour in the limit {L\over\xi_L}\ra\infty and logarithmic behaviour in the limit {L\over\xi_L}\ra0 for fixed $L$, where $\xi_L$ is the correlation length in the transverse dimensions.Comment: 21 pages of Plain TeX. Postscript figures available upon request from [email protected]

Efficacy of nisin A and nisin V semi-purified preparations alone and in combination with plant essential oils to control Listeria monocytogenes

peer-reviewedThe foodborne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates reaching up to 25-30%. Listeria is a hardy organism and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semi-purified preparations (spp) containing either nisin A or an enhanced bioengineered derivative nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol and trans-cinnamaldehyde, to control L. monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing spp (25 μg/ml) with thymol (0.02%), carvacrol (0.02%) or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil/nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V + carvacrol or nisin V + cinnamaldehyde combinations was twice that of the equivalent nisin A + essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural and effective means to enhance food safety by inhibiting foodborne pathogens such as L. monocytogenes.This work was supported by the Irish Government under the National Development Plan, through Science Foundation Ireland Investigator awards to C.H. and R.P.R. (10/IN.1/B3027), and C.H., R.P.R. and P.D.C. (06/IN.1/B98)

Camera trap arrays improve detection probability of wildlife: Investigating study design considerations using an empirical dataset.

Camera trapping is a standard tool in ecological research and wildlife conservation. Study designs, particularly for small-bodied or cryptic wildlife species often attempt to boost low detection probabilities by using non-random camera placement or baited cameras, which may bias data, or incorrectly estimate detection and occupancy. We investigated the ability of non-baited, multi-camera arrays to increase detection probabilities of wildlife. Study design components were evaluated for their influence on wildlife detectability by iteratively parsing an empirical dataset (1) by different sizes of camera arrays deployed (1-10 cameras), and (2) by total season length (1-365 days). Four species from our dataset that represented a range of body sizes and differing degrees of presumed detectability based on life history traits were investigated: white-tailed deer (Odocoileus virginianus), bobcat (Lynx rufus), raccoon (Procyon lotor), and Virginia opossum (Didelphis virginiana). For all species, increasing from a single camera to a multi-camera array significantly improved detection probability across the range of season lengths and number of study sites evaluated. The use of a two camera array increased survey detection an average of 80% (range 40-128%) from the detection probability of a single camera across the four species. Species that were detected infrequently benefited most from a multiple-camera array, where the addition of up to eight cameras produced significant increases in detectability. However, for species detected at high frequencies, single cameras produced a season-long (i.e, the length of time over which cameras are deployed and actively monitored) detectability greater than 0.75. These results highlight the need for researchers to be critical about camera trap study designs based on their intended target species, as detectability for each focal species responded differently to array size and season length. We suggest that researchers a priori identify target species for which inference will be made, and then design camera trapping studies around the most difficult to detect of those species

Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives

peer-reviewedNisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.This work was financed by a grant from the Irish Department of Agriculture, Food and the Marine through the Food Institutional Research Measure (08/RD/C/691) and with Science Foundation Investigator award (10/IN.1/B3027)

Impact of Environmental Factors on Bacteriocin Promoter Activity in Gut-Derived Lactobacillus salivarius

peer-reviewedBacteriocin production is regarded as a desirable probiotic trait that aids in colonization and persistence in the gastrointestinal tract (GIT). Strains of Lactobacillus salivarius, a species associated with the GIT, are regarded as promising probiotic candidates and have a number of associated bacteriocins documented to date. These include multiple class IIb bacteriocins (salivaricin T, salivaricin P, and ABP-118) and the class IId bacteriocin bactofencin A, which show activity against medically important pathogens. However, the production of a bacteriocin in laboratory media does not ensure production under stressful environmental conditions, such as those encountered within the GIT. To allow this issue to be addressed, the promoter regions located upstream of the structural genes encoding the L. salivarius bacteriocins mentioned above were fused to a number of reporter proteins (green fluorescent protein [GFP], red fluorescent protein [RFP], and luciferase [Lux]). Of these, only transcriptional fusions to GFP generated signals of sufficient strength to enable the study of promoter activity in L. salivarius. While analysis of the class IIb bacteriocin promoter regions indicated relatively weak GFP expression, assessment of the promoter of the antistaphylococcal bacteriocin bactofencin A revealed a strong promoter that is most active in the absence of the antimicrobial peptide and is positively induced in the presence of mild environmental stresses, including simulated gastric fluid. Taken together, these data provide information on factors that influence bacteriocin production, which will assist in the development of strategies to optimize in vivo and in vitro production of these antimicrobials.This work was funded by a SFI PI award “Obesibiotics” (11/PI/1137) to PD

Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

peer-reviewedNisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.This work was supported by the Irish Government under the National Development Plan, through Science Foundation Ireland Investigator awards (10/IN.1/B3027) and (06/IN.1/B98) (http://www.sfi.ie)