Controlling Listeria monocytogenes in Cold Smoked Salmon with the Antimicrobial Peptide Salmine.

Listeria monocytogenes (LM) is a major safety concern for smoked salmon producers, as it can survive both the brining and smoking process in cold smoked salmon production. Salmine is a cationic antimicrobial peptide derived from the milt of salmon that has been shown to inhibit the growth of LM in vitro. Commercialization of this peptide would add value to a waste product produced when raising salmon. The purpose of this study was to determine the anti-listeria activity of salmine in smoked salmon by measuring the viable counts of LM over time. Cold smoked salmon was treated with a salmine solution or coated with agar or k-carrageenan films incorporating salmine to maintain a high surface concentration of the antimicrobial. Samples were then inoculated with approximately 1.0 × 10(3) cells of LM. The viable counts were then enumerated throughout 4 wk at 4 °C storage. It was found that 5 mg/g salmine delayed the growth of LM on smoked salmon. These samples had significantly (P < 0.05) lower LM counts than on the untreated samples on days 13 and 22. Edible films did not significantly (P > 0.05) improve the antimicrobial efficacy of salmine. The peptide combined with biopolymers also had lower antimicrobial activity in vitro when compared to salmine alone. These results suggest there is potential for salmine to be used as a natural hurdle to inhibit growth of LM due to post process contamination; however, future investigations for extending this effect throughout the shelf life of smoked salmon products are warranted.

Comparative structure of vertebrate sperm chromatin.

A consistent feature of sperm nuclei is its exceptionally compact state in comparison with somatic nuclei. Here, we have examined the structural organization of sperm chromatin from representatives of three vertebrate lineages, bony fish (Danio rerio), birds (Gallus gallus domesticus) and mammals (Mus musculus) using light and transmission electron microscopy (TEM). Although the three sperm nuclei are all highly compact, they differ in morphology and in the complement of compaction-inducing proteins. Whereas zebrafish sperm retain somatic histones and a nucleosomal organization, in the rooster and mouse, histones are largely replaced by small, arginine-rich protamines. In contrast to the mouse, the rooster protamine contains no cysteine residues and lacks the potential stabilizing effects of S-S bonds. Protamine driven chromatin compaction results in a stable, highly condensed chromatin, markedly different from the somatic nucleosome-based beads-on-a-string architecture, but its structure remains poorly understood. When prepared gently for whole mount TEM, the rooster and mouse sperm chromatin reveal striking rod-like units 40-50 nm in width. Also present in the mouse, which has very flattened sperm nuclei, but not rooster, where nuclei take the form of elongated cylinders, are toroidal shaped structures, with an external diameter of about 90 nm. In contrast, similarly prepared zebrafish sperm exhibit nucleosomal chromatin. We also examined the early stages in the binding of salmine (the salmon protamine) to defined sequence DNA. These images suggest an initial side-by-side binding of linear DNA-protamine complexes leading to the nucleation of thin, flexible rods with the potential to bend, allowing the ends to come into contact and fuse to form toroidal structures. We discuss the relationship between these in vitro observations and the rods and toroids seen in nuclei, and suggest an explanation for the apparent absence of these structures in TEM images of fully condensed sperm nuclei.

Salmine and the homeotic integrity of early embryos of Norway spruce.

Salmine, an arginine-rich protamine, is explored for its concentration-dependent potential to restructure the genome and remodel the homeotic development of Norway spruce embryos expressing monozygotic cleavage polyembryony (MCP). In controls and at low salmine, two protein fractions on SDS-PAGE gels were associated with cells responsible for generating the basal plan for early embryogenesis. With high salmine, embryonal initials no longer differentiated into embryonal tubes. Embryos having embryonal tubes no longer enucleated and differentiated into embryonal suspensors. Biomass and amino acid N declined. Nuclear and cytoplasmic organization was disrupted and nucleoli were highly vacuolated. The transcription of the two protein fractions, PCNA (cyclin) activity and MCP were blocked. Cellular proteins were turned over by proteasomal ubiquitination and others released into the culture medium. Biomass loss and gluconeogenesis of amino acids led to the accumulation of free arginine N. No evidence was obtained with salmine for the remodeling of cells into gametes.

The preferential binding of histone H1 to DNA scaffold-associated regions is determined by its C-terminal domain.

Histone H1 preferentially binds and aggregates scaffold-associated regions (SARs) via the numerous homopolymeric oligo(dA).oligo(dT) tracts present within these sequences. Here we show that the mammalian somatic subtypes H1a,b,c,d,e and H1 degrees and the male germline-specific subtype H1t, all preferentially bind to the Drosophila histone SAR. Experiments with the isolated domains show that whilst the C-terminal domain maintains strong and preferential binding, the N-terminal and globular domains show weak binding and poor specificity for the SAR. The preferential binding of SAR by the H1 molecule thus appears to be determined by its highly basic C-terminal domain. Salmine, a typical fish protamine, which could have its evolutionary origin in histone H1, also shows preferential binding to the SAR. The interaction of distamycin, a minor groove binder with high affinity for homopolymeric oligo(dA).oligo(dT) tracts, abolishes preferential binding of the C-terminal domain of histone H1 and protamine to the SAR, suggesting the involvement of the DNA minor groove in the interaction.

DNA condensation by protamine and arginine-rich peptides: analysis of toroid stability using single DNA molecules.

Both somatic cells and sperm have been shown to take up exogenous DNA, but the frequency of its integration is usually low. Scanning probe microscopy studies of sperm chromatin and synthetic DNA-protamine complexes indicate that the coiling of DNA into toroidal subunits, a process initiated in the maturing spermatid to prepare its genome for delivery into the egg, can be mimicked by simply adding protamine to DNA in vitro. The increased resistance of DNA-protamine complexes to nuclease digestion and their structural similarity to native sperm chromatin suggest that the packaging of DNA by protamine might offer a new approach for improving the efficiency of DNA uptake by sperm. Decondensation experiments performed with individual DNA molecules have provided a direct measure of the stability of toroids produced using salmon protamine and smaller arginine-rich peptides. These experiments show that the arginine content of protamine-related sequences can have a dramatic effect on their rate of dissociation from DNA. This technique and the information it provides can be used to identify protamine analogs that can be bound to DNA to increase the efficiency of its uptake by sperm and other cells.

Conformation of nucleoplasmin and its interaction with DNA-protamine complex as a simple model of fish sperm nuclei.

Nucleoplasmin was isolated from Xenopus laevis eggs and purified by an improved method using an open column. Its conformation was investigated spectrophotometrically by UV, CD and fluorescence. It was shown that alpha-helix content of nucleoplasmin was 30-40%, and one of the two tryptophan residues in nucleoplasmin located in the hydrophobic surroundings and the other in the relatively hydrophilic surroundings. The isolated nucleoplasmin was found to decondense sperm nuclei of salmon also, suggesting a possibility of the existence of nucleoplasmin-like protein in fish as well. Collapse of the protamine (salmine)-DNA complex as a simple model for fish sperm nuclei by nucleoplasmin was directly observed by measuring OD320 of aqueous protamine-DNA mixtures. This is a molecular level observation for the removal of protamine from DNA-protamine complex.

N(omega)-phosphoarginine phosphatase (17 kDa) and alkaline phosphatase as protein arginine phosphatases.

Seven synthetic polymers, (Glu4, Tyr)n, (Arg)n, (Arg, Pro, Thr)n, (Arg-Gly-Glu)6, (Arg-Gly-Phe)6, (Glu-Arg-Gly-Phe)5, and (Ala-Leu-Arg-Arg-Ile-Arg-Gly-Glu-Arg)2, were treated with phosphoryl chloride to phosphorylate their Tyr, Thr, and Arg residues. Protamines and histones were phosphorylated similarly. These phosphorylated peptides were examined as to whether or not they serve as substrates for intestinal alkaline phosphatase [EC 3.1.3.1] and liver N(omega)-phosphoarginine phosphatase [Kuba, M., Ohmori, H., and Kumon, A. (1992) Eur. J. Biochem. 208, 747-752]. Phosphorylated polyarginine was hydrolyzed with a lower Km with alkaline phosphatase than with N(omega)-phosphoarginine phosphatase, while the phosphorylated forms of (Arg-Gly-Phe)6 and culpeine were better substrates for N(omega)-phosphoarginine phosphatase. When (Arg, Pro, Thr)n and culpeine were phosphorylated chemically after treatment with phenylglyoxal, these phosphorylated peptides were worse substrates for N(omega)-phosphoarginine phosphatase than for alkaline phosphatase. Moreover, the results of proton-decoupled 31P NMR analysis indicated that N(omega)-phosphoarginine phosphatase released Pi from N(omega)-phosphoarginine residues of phosphopeptides. These results indicate that both phosphatases function as protein arginine phosphatases in different manners, and that N(omega)-phosphoarginine phosphatase is useful for selectively detecting N(omega)-phosphoarginine residue in peptides containing various kinds of phosphorylated amino acids.

Evidence of novel secondary structure in DNA-bound protamine is revealed by Raman spectroscopy.

Raman spectroscopy studies of protamine-DNA complexes are reported for samples in the solid state at 98% relative humidity. Previous reports utilizing other physical techniques have indicated the presence of B-form DNA in protamine-DNA complexes. The present Raman data support the assignment of a modified B-form which is characterized by appreciable unstacking of the bases. The quality of the present spectra has made it possible, for the first time, to obtain the Raman spectrum of DNA-bound protamine by digital spectral subtraction. The difference spectrum indicates that protamine adopts an unusual secondary structure upon binding to DNA. A dominant amide I band is observed at 1683 cm-1 which is indicative of neither an alpha-helix or beta-sheet conformation. An amide I band at this position has been associated with the 1-->3 hydrogen bond that occurs within a gamma-turn [Bandekar, J., & Krimm, S. (1985) Int. J. Pept. Protein Res. 26, 158-165]. On the basis of this assignment, as well as preliminary results obtained by computer modeling, we propose a new model for the secondary structure of DNA-bound protamine that is rich in 1-->3 hydrogen bonding. Spectral data demonstrate that this structure is absent in protamine molecules in solution. Analyses of spectra of polyarginine-DNA complexes suggest that polyarginine, although similar to protamine in primary structure, assumes a conformation when bound to DNA that is distinct from that adopted by protamine.

[3 types of sperm proteins in eukaryotes]. / Tri tipa spermal'nykh belkov éukariot.

Basic spermal proteins of various species of hydrobionts attributed to Pisces and Cephalopoda are studied. It is established that chromatin of nine species referring to two Cypriniformes families includes the somatic histones. Histone H1 of Cypriniformes is attributed to the lysine-rich type histones and contains 35% mol. of lysine and 0.7% mol. of tyrosine. Chromatin of 14 species of fish referring to nine families of the percoid fish superorder includes protamines similar to salmin, a typical protamine of salmon. The amino acidic analysis of protamine from the sandre sperma has shown that it contains 59% mol. of arginine and no tyrosine. Chromatin of three species from squid superorder referring to Cephalopoda includes gametones -- proteins differing from histones and protamines both in the electrophoretic mobility and amino acidic composition (75% mol. of arginine, 3% mol. of tyrosine).

Factors affecting nucleosome disassembly by protamines in vitro. Histone hyperacetylation and chromatin structure, time dependence, and the size of the sperm nuclear proteins.

Histone displaced in vitro from nuclei by protamine competition display a higher degree of hyperacetylation than the residual histones. In addition, hyperacetylated core particle pools are disassembled in vitro with a higher efficiency than control or nonacetylated core particles and when analyzed by electron microscopy display an elongated shape (length/width ratio = 1.52 +/- 0.19) instead of the round compact shape of control nucleosomes (length/width ratio = 1.06 +/- 0.06). In the absence of histone hyperacetylation, the fish protamines, salmine and iridine (32-33 residues), are relatively inefficient in disassembling nucleosomal core particles in vitro as compared to the large (65-70 residues), tyrosine-containing protamines from rooster (galline), squid, and cuttlefish which disassemble nucleosomes in a range of protamine concentrations close to physiological. The fact that an artificially cross-linked salmine dimer acquires the ability of the large protamines from rooster, squid, and cuttlefish to disassemble core particles in vitro and also binds more tightly to the DNA, suggests that the size of the sperm nuclear protamines is a critical factor in this process. Even when the core histones of spermatid chromatin are hyperacetylated in the trout testis, the replacement process by iridine or salmine is slow and time-dependent in vitro. However, since spermiogenesis in trout occurs over several weeks, the slow in vitro nucleosome disassembly process by salmine is sufficient to allow complete displacement, thus supporting the hypothesis that a protamine-mediated displacement of the histones from DNA in vivo may take place in the salmonid fishes by a mechanism similar to that in the rooster, squid, and cuttlefish.

Effect of exogenous histone H5 on integration of histone H1 in rat liver chromatin. Correlations with aberrant epsilon-N-methylation of histone H1.

Binding modes of histones H1 and H5, and their competition for chromatin-binding sites in rat liver nuclei, were correlated with aberrant N-methylation of H1 histone lysine residues, induced by chicken erythrocyte histone H5, in order to gain more insight into the integration of lysine-rich histones in chromatin. Addition of approx. 2.5 molecules of histone H5 per nucleosome to rat liver nuclei increases the ratio of total basic residues in histones to DNA nucleotides (BR/NT) in the nuclear chromatin from 1.0 to 1.5. At this concentration, approx. 0.7 molecule of histone H5 is bound per nucleosome, and there is no displacement of histone H1 from the nuclear chromatin. If S-adenosyl[Me-3H]methionine is present in the incubation mixture, the aberrant incorporation of labeled methyl groups into histone H1 reaches a maximum at this concentration of histone H5. The radioactivity present in histone H1 from nuclei incubated with labeled AdoMet at a total BR/NT ratio of 1.5: resides mainly in a histone H1 subfraction tentatively identified by Bio-Rex 70 chromatography and acrylamide gel electrophoresis as histone H1c; presents as a single spot upon peptide mapping of tryptic hydrolysates by means of two-dimensional thin-layer chromatography; and elutes in the position of mono-N-methyllysine upon ion-exchange chromatography of histone H1 hydrolysates. Upon further increase of the BR/NT ratio, the following changes are produced: a gradual decrease in radioactive methyl uptake into histone H1; a gradual displacement of histone H1 from the chromatin; increased binding of histone H5 in chromatin, up to a maximum of 3.4 residues per nucleosome; and a slowly increasing uptake of label into histone H5. The combined data from histone H1/H5 binding and histone H1 methylation studies suggest that upon addition of exogenous histone H5 to rat liver nuclei the binding of two lysine-rich histones per nucleosome plays a significant role in the induction of specific changes in chromatin structure, which in vivo may have important functional implications in terms of chromatin condensation and suppression of transcription.

High-performance liquid chromatography using spherical aggregates of hydroxyapatite micro-crystals as adsorbent.

High-performance liquid chromatography (HPLC) using spherical aggregates of hydroxyapatite (HA) microcrystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. In comparison with previously developed plate-like HA packed columns for HPLC, spherical HA packed columns show considerably high chromatographic resolutions in spite of extremely reduced column lengths of 0.5-3 cm. The pressure generated by the latter columns is much higher than that generated by the former, however.

Nuclear magnetic resonance of protamines. A 1H-NMR study of the interaction of clupeine fractions with mononucleotides.

The interaction of the three clupeine fractions, YI, YII, Z, and salmine fraction AI with mononucleotides has been examined by means of 1H nuclear magnetic resonance. The results obtained are interpreted in terms of electrostatic interactions between positive arginine guanidinyl groups and negative nucleotide phosphates. In addition, clupeine fraction YI and salmine fraction AI exhibit with guanine and adenine nucleotides a more specific interaction that leads to the formation of large aggregates in solution. The experimental data presented in this work demonstrate that the strength of interaction between clupeine YI and salmine AI with mononucleotides follows the order: 5'-dTMP approximately equal to 5'-dCMP much less than 5'-dAMP less than 5'-dGMP approximately equal to 5'-GMP.

Polycations as outer membrane-disorganizing agents.

The outer membrane-disorganizing effect of a short (10-min) treatment with polycationic agents was studied with smooth Salmonella typhimurium used as a test organism. The polycationic agents were the protamine salmine, a lysine polymer with 20 lysine residues (lysine20), and the deacylated polymyxin B derivative polymyxin B nonapeptide. Two different types of outer membrane-disorganizing were found. Protamine and lysine20 released 20 to 30% of the lipopolysaccharide from the outer membrane and sensitized the bacteria to the anionic detergent sodium dodecyl sulfate but did not (under these conditions) make the bacteria permeable to the hydrophobic probes fusidic acid and actinomycin D. In contrast, polymyxin B nonapeptide did not release lipopolysaccharide or sensitize the bacteria to sodium dodecyl sulfate but made the outer membrane permeable to the hydrophobic probes. None of the agents was bactericidal under the conditions used or caused any leakage of periplasmic beta-lactamase. Polymyxin B was used as a reference and showed characteristic outer membrane-disorganizing action. In thin-section electron microscopy, polymyxin B nonapeptide caused the appearance of long, narrow, finger-like projections on the outer membrane. Protamine and lysine20 caused a distinctly wrinkled appearance of the outer membrane but no projections.

Heat stability of milk: influence of cationic detergents on pH sensitivity.

The heat stability--pH profile of milk is shifted to more acidic values by anionic compounds such as SDS, lysolecithin and beta-lactoglobulin and to more alkaline values by cationic compounds such as quaternary ammonium compounds. Proline, which reduces the hydrophobicity of proteins, destabilized milk slightly. The results suggest that the heat stability of milk and the shape of the HCT-pH curve may be dependent on micellar charge effects.

Lethal effect of protamine and histone on competent Bacillus subtilis cells. Inhibition of genetic transformation by protamine in sublethal concentration.

Under experimental conditions of genetic transformation, protamine and total histone were bactericidal for Bacillus subtilis cells. The abilities to cause lethality were very similar for both, either protamine or histone, with no antagonistic effects amongst these natural polycations. With both basic proteins acting simultaneously the enhancement was higher than a summation of the separate lethal effects. Sublethal concentration of protamine added at the beginning of transformation time, produced a strong inhibition of transforming efficiency. The same concentration added later than 10 min from the start of transformation had no inhibitory effect. These facts together with the absence of inhibition by simple pretreatment of DNA alone as well as the cell protection by protamine against lytic activity of lysozyme, suggest a protamine-cell surface interaction which impedes DNA uptake events.