Alan Carne

An exo-β-(1,3)-glucanase of Candida albicans : purification of the enzyme and molecular cloning of the gene

A nucleotide sequence encoding an exo-beta-(1,3)-glucanase was cloned from a library of genomic DNA of Candida albicans ATCC 10261. The sequenced gene encodes a protein of 438 amino acid residues. The amino terminal and an internal peptide sequence of the enzyme matched with deduced sequences within the cloned gene. Analysis of the sequence indicated that the nascent protein is processed during secretion by the signal peptidase and a Kex2-like proteinase, yielding a predicted mature enzyme of 400 residues. There is 58% identity and 85% similarity between the amino acid sequences of this exoglucanase and the homologous enzyme of Saccharomyces cerevisiae. An antiserum to the purified exoglucanase cross-reacted with the S. cerevisiae exoglucanase and a similar protein secreted by other C. albicans strains and Candida species. There are no sites for N-linked glycosylation in the sequence and this is consistent with the carbohydrate content of the secreted enzyme. Putative upstream promoter elements are associated with the gene. Southern analysis of the gene indicated that it was present at one copy per genome and that the diploid genome of C. albicans ATCC 10261 is heterozygous at this locus for a BglII RFLP. A 2.5 kb mRNA transcript was detected by Northern analysis and gene expression, as monitored by Northern and Western blots, reflected the growth rates of the cultures.

Slaughterhouse Blood: An Emerging Source of Bioactive Compounds

Slaughterhouse blood is an inevitable part of the meat production food chain and represents a rich source of protein. The physicochemical characteristics and utilization of animal blood in various food and industrial applications has been well explored. However, in recent years much attention has been paid to the generation of peptides with biological activities from food by-products including blood. This review examines the angiotensin-converting enzyme inhibitory, antioxidant, antimicrobial, and other bioactive peptides derived from various slaughterhouse animal blood sources. Furthermore, the effect of enzyme choice, degree of hydrolysis, and peptide sequence or size on the potency of these bioactivity is discussed.

Amino acid sequence of atrial natriuretic peptides in human coronary sinus plasma

Two atrial natriuretic peptides were purified from pooled human coronary sinus plasma by Sep-Pak extraction, immunoaffinity chromatography and reverse phase HPLC. The amino acid sequences of the two peptides were homologous with 99-126 human atrial natriuretic peptide (hANP) and 106-126 hANP, the latter being most probably linked to 99-105 ANP by the disulphide bond. The molar ratio of the peptides in plasma, as assessed by radioimmunoassay was 10:3.

The amino-acid sequences of sculpin islet somatostatin-28 and peptide YY

Two pancreatic peptides, somatostatin‐28 and peptide YY, have been isolated from the Brockmann bodies of the teleost fish Cottus scorpius (daddy sculpin). Following purification by reverse‐phase HPLC, each peptide was sequenced completely through to the carboxyl‐terminus by gas‐phase Edman degradation. Somatostatin‐28 was the major form of somatostatin detected and is similar to the gene II product from angler‐fish. Peptide YY (36 amino acids) more closely resembles porcine neuropeptide YY and intestinal peptide YY than it does the pancreatic polypeptides.

Physical Interventions to Manipulate Texture and Tenderness of Fresh Meat: A Review

Meat tenderness is a major eating quality attribute that ensures consumer satisfaction and repeat purchase of red meat. The variability in meat tenderness is related to several factors that are spread across the production chain (biological, on farm, processing, and consumer factors), which can lead to inconsistent tenderness in fresh red meat products. The tenderization process is dictated by physical and biochemical factors, which appear to affect the proteases involved in protein degradation and, consequently, they regulate the rate and extend of tenderization in meat. Several physical, chemical, and biochemical interventions have been investigated to improve the tenderness of meat. The present review discusses the physical interventions used to manipulate the texture of meat and their mechanism of action, optimal tenderizing conditions, and their effects on other meat quality attributes (colour stability, lipid oxidation, and water holding capacity). Attention should be paid to other quality attributes for full evaluation of the differing interventions.

Isolation, Cloning, and Characterisation of a trp Homologue from Squid (Loligo forbesi) Photoreceptor Membranes

Abstract: The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92‐kDa component isolated from a detergent‐insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full‐length cDNA, encoding the 92‐kDa component, using both cDNA library screening and 5′‐rapid amplification of cDNA ends (5′‐RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl‐terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl‐terminal domain has been shown to be in the regulation of several ligand‐gated channels. The carboxyl‐terminal domain has been expressed and shown to interact with calmodulin in a calcium‐dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.

Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases.

Five commercially available food-grade microbial protease preparations were evaluated for their ability to hydrolyse meat myofibrillar and connective tissue protein extracts to produce bioactive peptides. A bacterial-derived protease (HT) extensively hydrolysed both meat protein extracts, producing peptide hydrolysates with significant in vitro antioxidant and ACE inhibitor activities. The hydrolysates retained bioactivity after simulated gastrointestinal hydrolysis challenge. Gel permeation chromatography sub-fractionation of the crude protein hydrolysates showed that the smaller peptide fractions exhibited the highest antioxidant and ACE inhibitor activities. OFFGEL electrophoresis of the small peptides of both hydrolysates showed that low isoelectric point peptides had antioxidant activity; however, no consistent relationship was observed between isoelectric point and ACE inhibition. Cell-based assays indicated that the hydrolysates present no significant cytotoxicity towards Vero cells. The results indicate that HT protease hydrolysis of meat myofibrillar and connective tissue protein extracts produces bioactive peptides that are non-cytotoxic, should be stable in the gastrointestinal tract and may contain novel bioactive peptide sequences.

L-asparaginase from developing seeds of Lupinus arboreus

Asparaginase (EC 3.5.1.1) activity reached a maximum 40 days post anthesis in developing seeds of Lupinus arboreus and this correlated with the appearance of other ammonia assimilatory enzymes. Asparaginase, purified from these developing seeds, was resolved into three isoforms, designated asparaginases A, B and C. A major protein species in asparaginase A preparations co-focussed with enzyme activity on an isoelectric focussing gel. When analysed by SDS-PAGE, asparaginase isoforms A and B each yielded several polypeptides with M(r)s in the 14,000 to 19,000 ranged. These peptides are fragmentation products of an M(r) 36,000 asparaginase subunit. Polyclonal antibodies raised against asparaginase isoforms A and B precipitated asparaginase activity from a partially purified L. arboreus seed extract. Immunoaffinity chromatography recovered polypeptides with M(r)s between 14,000 and 19,000. Partial protein sequences were obtained for these asparaginase polypeptides.

Identification and Characterization of a Bacitracin Resistance Network in Enterococcus faecalis

ABSTRACT Resistance of Enterococcus faecalis against antimicrobial peptides, both of host origin and produced by other bacteria of the gut microflora, is likely to be an important factor in the bacteriums success as an intestinal commensal. The aim of this study was to identify proteins with a role in resistance against the model antimicrobial peptide bacitracin. Proteome analysis of bacitracin-treated and untreated cells showed that bacitracin stress induced the expression of cell wall-biosynthetic proteins and caused metabolic rearrangements. Among the proteins with increased production, an ATP-binding cassette (ABC) transporter with similarity to known peptide antibiotic resistance systems was identified and shown to mediate resistance against bacitracin. Expression of the transporter was dependent on a two-component regulatory system and a second ABC transporter, which were identified by genome analysis. Both resistance and the regulatory pathway could be functionally transferred to Bacillus subtilis, proving the function and sufficiency of these components for bacitracin resistance. Our data therefore show that the two ABC transporters and the two-component system form a resistance network against antimicrobial peptides in E. faecalis, where one transporter acts as the sensor that activates the TCS to induce production of the second transporter, which mediates the actual resistance.

Effect of pulsed electric field on the proteolysis of cold boned beef M. Longissimus lumborum and M. Semimembranosus.

The effects of pulsed electric field (PEF) and ageing (3, 7, 14 and 21days) on the shear force, protein profile, and post-mortem proteolysis of beef loins (M. Longissimus lumborum, LL) and topsides (M. Semimembranosus, SM) were investigated using a range of pulsed electric field treatments [voltages (5 and 10kV) and frequencies (20, 50, and 90Hz)]. PEF treatment decreased the shear force of beef LL and SM muscles by up to 19%. The reduction in the shear force in the LL was not affected by the treatment intensity whereas the reduction in the SM was dependent on PEF frequency. PEF treated beef loins showed increased proteolysis, both early post-mortem and during subsequent post-mortem storage reflected by increased degradation of troponin-T and desmin. The most prominent troponin-T degradation was found in samples treated with 5kV-90Hz, 10kV-20Hz at day 3 and day 7 post-treatment in addition to 10kV-50Hz in subsequent post-treatment times. The degradation of desmin in PEF treated beef loins increased with ageing time.