Effect of benzalkonium chloride on viability and energy metabolism in exponential- and stationary-growth-phase cells of Listeria monocytogenes.

The difference in killing exponential- and stationary-phase cells of Listeria monocytogenes by benzalkonium chloride (BAC) was investigated by plate counting and linked to relevant bioenergetic parameters. At a low concentration of BAC (8 mg liter(-1)), a similar reduction in viable cell numbers was observed for stationary-phase cells and exponential-phase cells (an approximately 0.22-log unit reduction), although their membrane potential and pH gradient were dissipated. However, at higher concentrations of BAC, exponential-phase cells were more susceptible than stationary-phase cells. At 25 mg liter(-1), the difference in survival on plates was more than 3 log units. For both types of cells, killing, i.e., more than 1-log unit reduction in survival on plates, coincided with complete inhibition of acidification and respiration and total depletion of ATP pools. Killing efficiency was not influenced by the presence of glucose, brain heart infusion medium, or oxygen. Our results suggest that growth phase is one of the major factors that determine the susceptibility of L. monocytogenes to BAC.

AgRP(83-132) acts as an inverse agonist on the human-melanocortin-4 receptor.

The central melanocortin (MC) system has been demonstrated to act downstream of leptin in the regulation of body weight. The system comprises alpha-MSH, which acts as agonist, and agouti-related protein (AgRP), which acts as antagonist at the MC3 and MC4 receptors (MC3R and MC4R). This property suggests that MCR activity is tightly regulated and that opposing signals are integrated at the receptor level. We here propose another level of regulation within the melanocortin system by showing that the human (h) MC4R displays constitutive activity in vitro as assayed by adenylyl cyclase (AC) activity. Furthermore, human AgRP(83-132) acts as an inverse agonist for the hMC4R since it was able to suppress constitutive activity of the hMC4R both in intact B16/G4F melanoma cells and membrane preparations. The effect of AgRP(83-132) on the hMC4R was blocked by the MC4R ligand SHU9119. Also the hMC3R and the mouse(m)MC5R were shown to be constitutively active. AgRP(83-132) acted as an inverse agonist on the hMC3R but not on the mMC5R. Thus, AgRP is able to regulate MCR activity independently of alpha-MSH. These findings form a basis to further investigate the relevance of constitutive activity of the MC4R and of inverse agonism of AgRP for the regulation of body weight.

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein.

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.

Characterization of melanocortin receptor ligands on cloned brain melanocortin receptors and on grooming behavior in the rat.

Since the melanocortin MC3 and melanocortin MC4 receptors are the main melanocortin receptor subtypes expressed in rat brain, we characterized the activity and affinity of nine melanocortin receptor ligands using these receptors in vitro, as well as their activity in a well-defined melanocortin-induced behavior in the rat: grooming behavior. We report here that [D-Tyr4]melanotan-II and RMI-2001 (Ac-cyclo-[Cys4, Gly5, D-Phe7, Cys10]alpha-MSH-NH2) have significantly higher affinity and potency on the rat melanocortin MC4 receptor as compared to the rat melanocortin MC3 receptor. Nle-gamma-MSH (melanocyte-stimulating hormone) was the only ligand with higher affinity and potency on the rat melanocortin MC3 receptor. The potency order of melanocortin MC4 receptor agonists, but not that of melanocortin MC3 receptor agonists, fitted with the potency of these ligands to stimulate grooming behavior, when administered intracerebroventricularly. SHU9119 (Ac-cyclo-[Nle4, Asp5, D-Nal(2)7, Lys10]alpha-MSH-(4-10)-NH2) and RMI-2005 (Ac-cyclo-[Cys4, Gly5, D-Na](2)7, Nal(2)9, Cys10]alpha-MSH-(4-10)-NH2) were able to inhibit alpha-MSH-induced melanocortin receptor activity in vitro, as well as alpha-MSH-induced grooming behavior. Melanotan-II, [Nle4-D-Phe7]alpha-MSH and RMI-2001 were also effective in inducing grooming behavior when administered intravenously. In the absence of purely selective melanocortin MC(3/4) receptor ligands, we demonstrated that careful comparison of ligand potencies in vitro with ligand potencies in vivo, could identify which melanocortin receptor subtype mediated alpha-MSH-induced grooming behavior. Furthermore, blockade of novelty-induced grooming behavior by SHU9119 demonstrated that this physiological stress response is mediated via activation of the melanocortin system.

Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.

Asp10 in Lys-gamma2-MSH determines selective activation of the melanocortin MC3 receptor.

The melanocortin MC3 and MC4 receptors are the main melanocortin receptors expressed in brain. Of the endogenous melanocortins, gamma2-melanocortin stimulating hormone (MSH) selectively activates the melanocortin MC3 receptor, whereas alpha- and beta-MSH activate all melanocortin receptors. The aim was to gain an insight into the contribution of amino acids in positions 5 and 10 of melanocortins to the selectivity of [Nle4]Lys-gamma2-MSH for the melanocortin MC3 receptor versus the melanocortin MC4 receptor. Introduction of Asp10 into [Nle4]alpha-MSH as in [Nle4,Gly5,Asp10]alpha-MSH selectively increased the EC50 value for the melanocortin MC4 receptor. Conversely, removal of Asp10, as in [Nle4,Gly10]Lys-gamma2-MSH, selectively decreased the EC50 value for the melanocortin MC4 receptor. Thus, Asp10 in Lys-gamma2-MSH determined selectivity for the melanocortin MC3 receptor versus the melanocortin MC4 receptor.

Molecular pharmacology of neural melanocortin receptors.

The cloning of melanocortin receptors opened new avenues to identify selective ligands for this receptor family. gamma-MSH was characterized as a melanocortin-3 receptor selective agonist, [D-Arg8]ACTH-(4-10) and [Pro8,10, Gly9]ACTH-(4-10) were characterized as melanocortin-4 receptor antagonists. The application of these ligands in vivo revealed that melanocortin-4 receptors mediate melanocortin-induced grooming behaviour in the rat. Since we still lack potent and selective melanocortin receptor ligands, we performed homology modelling and site directed mutagenesis of the melanocortin-4 receptor, in order to understand how melanocortins bind melanocortin receptors. A histidine at position 260 in the melanocortin-4 receptor is important for normal receptor function. However this residue is not forming a salt bridge with a glutamate at position 92 to keep the receptor in an inactive conformation, nor with the glutamate in the melanocortin peptides as had been suggested before.

Modified suspension Ames test for testing proteinaceous substances: an initial step.

The basic Salmonella/microsome assay (Ames test) is a valuable primary tool by which to discriminate mutagens from non-mutagens. For a variety of chemical test substances this test is easily conducted according to international guidelines for genotoxicity testing. However, the testing of proteinaceous substances in the basic Ames test may generate false positives owing to the presence of growth-promoting constituents in the test sample, such as histidine or its precursors. It was hypothesized that the growth-promoting capacities of biological test samples might be overcome by testing according to the 'suspension variant' of the Ames test, which uses very rich growth conditions thereby overwhelming any growth-enhancing constituents present in a biological test sample. This hypothesis appeared to be correct, although several important modifications had to be made to the suspension assay. The most important aspect of this 'new suspension Ames test' appeared to be the plating of overnight regrown bacteria in the poorest way possible (by omitting histidine and nutrient broth from the overlay agar). This study may comprise an initial step in the development of a modified suspension Ames test for testing proteinaceous substances.

Identification of antagonists for melanocortin MC3, MC4 and MC5 receptors.

Antagonists for the melanocortin receptor family were identified by analysis of the effects of four melanocortin analogues on alpha-MSH(alpha-melanocyte-stimulating hormone)-induced cAMP accumulation in 293 human embryonal kidney (HEK) cells that expressed either the rat melanocortin MC3 receptor, the human melanocortin MC4 receptor or the ovine melanocortin MC5 receptor. Two peptides, [D-Arg8]ACTH(adrenocorticotrope hormone)-(4-10) and [Pro8,10,Gly9]ACTH-(4-10), antagonized the action of alpha-MSH on the melanocortin MC4 and MC5 receptors, but not the melanocortin MC3 receptor. [Ala6]ACTH-(4-10) inhibited the alpha-MSH activation of the melanocortin MC3 and MC5, but only weakly antagonized the activation of the melanocortin MC4 receptor. [Phe-I7]ACTH-(4-10) antagonized the melanocortin MC3, MC4 and MC5 receptors equally well. These antagonists were also tested to block a behavioral response induced by alpha-MSH. alpha-MSH-induced excessive grooming behavior in rats was inhibited by [Phe-I7]ACTH-(4-10), [D-Arg8]ACTH-(4-10) and [Pro8,10,Gly9]ACTH-(4-10), but not by [Ala6]ACTH-(4-10). This suggests that alpha-MSH-induced excessive grooming behavior is mediated by melanocortin MC4 receptors.

Campylobacter: pathogenicity and significance in foods.

In the last 10 years Campylobacter jejuni has emerged as the most frequent cause of bacterial gastroenteritis in man. Acute enterocolitis, the most common presentation of C. jejuni infection, can affect persons of all ages. C. jejuni has been found in virtually every country where investigations have been carried out. The frequent finding of dysenteric stools suggests that mucosal damage due to an invasive process analogous to that seen in shigellosis is important in the pathogenesis. Campylobacteriosis in man is mainly a foodborne infection in which foods of animal origin, particularly poultry, play an important role. Epidemiological investigations have demonstrated a significant correlation between the handling and consumption of poultry meat and the occurrence of Campylobacter enteritis. Barbecues appear to present special hazards for infection, because they permit easy transfer of bacteria from raw meats to hands and other foods and from these to the mouth. Milk is sometimes found to be contaminated and consumption of raw milk has caused several outbreaks of campylobacteriosis. Campylobacter can remain viable in fresh cheese for only a short period of time. The organism is also found in shellfish, such as clams. Campylobacter is probably very vulnerable to factors such as high temperatures and dry environments, and also to the presence of oxygen in atmospheric concentrations. Therefore, it is assumed that the organism does not persist in products like pelleted feed, meals, egg powder and spices, which are often contaminated by Salmonella. A number of preventive measures on different levels, taken simultaneously, are needed to reduce the incidence of campylobacteriosis in man.

Epidemiological studies and proposed preventive measures in the fight against human salmonellosis.

A large number of countries annually report high numbers of Salmonella infections in man. Since it is estimated that these reported cases only represent 1 to 10% of the real incidence of this disease, it must be concluded that human salmonellosis is a serious problem all over the world. The major source for human salmonellosis is caused by farm animals, which may frequently be intestinal carriers of the organism. Particularly pigs and poultry are incriminated in this respect, and, to a lesser degree, cattle and sheep. Because generally no symptoms of disease can be observed, these animals usually pass veterinary slaughterhouse inspection without restrictions. During slaughter however, intestinal material, often containing Salmonella bacteria, pollutes the surface of carcasses, which in later stages may lead to extensive Salmonella contamination of meat and meat products. Additionally, milk may also be contaminated by faecal material during collection, while eggs may be externally contaminated by faecal material or internally infected by way of transovarial transmission. Cross-contamination in slaughterhouses, in butchers' shops and in the kitchen may add to the problem. Human infection occurs when animal products are improperly handled during final preparation. This may happen at home, but also in large kitchens of restaurants, hospitals, institutions and factories. In most instances infection is related to cross-contamination from meat to foods that are to be consumed without further treatment, such as bread, salads and fruits, to improper heating of the animal products themselves, as for instance with eggs, or even to the consumption of raw animal products, as may be the case with meat and milk. The faecal excretion of Salmonella by human patients, wild and domesticated animal carriers, as well as the disposal of slaughter offal, sludge, slurry and manure contributes to an overall Salmonella spread in the environment. This may lead, by way of contamination of surface waters and colonisation of birds, rodents and insects, to the contamination of animal feeds or directly contribute to the re-colonisation of farm animals. In order to solve the problem of human salmonellosis, measures should be taken simultaneously on several levels. Since there are so many transmission ways, particularly in the environment and on the farm, isolated actions, such as the decontamination of animal feed, will never give lasting results. Likewise, the sole responsibility for the problem should never lie with the consumer.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidemiological studies on Salmonella and Campylobacter jejuni.

An overview is given of investigations concerning the epidemiology of Salmonella, carried out at the National Institute of Public Health and Environmental Hygiene in Bilthoven, The Netherlands, during the last thirty years. It is made clear that Salmonella, because of its ubiquitous occurrence and its large variety in sero- and phage-types, is the organism of choice to study the epidemiological pathways of pathogens between man, animals and the environment. It is demonstrated that these are in fact the pathways of faecal contamination, and therefore have validity for a larger number of bacterial, and perhaps even parasitic and viral, micro-organisms. This last statement is illustrated by the presentation of studies on the epidemiology of Campylobacter jejuni.

Kinetics of anti-Campylobacter jejuni monomeric and polymeric immunoglobulin A1 and A2 responses in serum during acute enteritis.

The intensity and kinetics of the serum polymeric and monomeric immunoglobulin A1 (IgA1) and IgA2 antibody responses to Campylobacter jejuni were analyzed. A rapid and marked serum IgA antibody response involving both the monomeric and polymeric components of IgA was observed after C. jejuni infections. IgA antibodies reached a peak of activity in serum during week 2 after the first symptoms of enteritis, about 10 days before the peak of IgG activity. Polymeric IgA accounted for most of the anti-C. jejuni activity at the peak of the IgA response (median, 90%; range, 44 to 98%) but rapidly disappeared from serum over a few weeks. In contrast, the serum monomeric IgA antibody response was low and was maintained over a prolonged period of time. Anti-C. jejuni IgA detected in the serum of healthy blood donors was mainly monomeric (median, 83%; range, 17 to 94%). In both the patients and the positive controls, IgA1 was the predominant (greater than 85%) subclass involved, even when the IgA antibody response was mainly polymeric. Our results suggest that polymeric IgA antibody responses are linked to a strong or persisting antigenic stimulation or both. Polymeric IgA antibodies appear to be a potential marker of acute C. jejuni infections, and their determination could provide a useful tool for the serological diagnosis of recent C. jejuni infections.

[The hygienic disposal and rendering of dead animals and animal waste]. / De onschadelijkmaking en verdere verwerking van dode dieren en dierlijke afvallen.

In this publication an overview is made of both the fundamental and practical approaches laid down in the 'WHO Guidelines on the hygienic disposal and rendering of dead animals and animal wastes to protect human and animal health'. It describes methods that can be used both in developed and developing countries for the hygienic disposal and rendering of contaminated animal materials. In view of the various veterinary-hygienic measures that might be taken a distinction is made between low-risk materials (for instance slaughter offal of healthy animals) and high-risk materials (for instance animals that died because of infectious diseases). The hygienic disposal of low-risk materials is usually done by sterilisation in autoclaves or by cooking, so that proteins and fat may be re-used, mostly in the form of animal feed components. High-risk materials should at the least be sterilised or otherwise be burnt. Attention is also paid to the hygiene in rendering plants, particularly concerning the avoidance of cross-contamination from raw to decontaminated materials. Finally aspects of environmental hygiene are discussed and attention is paid to the protection of the personnel that is involved in the hygienic disposal and rendering of dead animals and animal wastes.

[Hygiene in poultry processing]. / Pluimveeslachthygiëne.

Investigations during the last two decades directed to measures designed to improve hygiene in poultry slaughtering are reviewed. Attention is paid to investigations on the degree and origin of bacterial contamination as well as on the mechanisms of attachment. Finally, future developments with regard to hygiene in poultry processing are discussed.

[Bacterial cycles]. / Bacteriële kringlopen.

As a result of several epidemiological investigations on Salmonella it was realized that bacterial cycles occur in the environment which are of direct importance in bacterial contamination of man, animals and food. The manner in which knowledge of these bacterial cycles may help in identifying the most important routes of transmission and designing adequate measures to reduce the hazards to public health wherever possible are described.

[Incidence of Clostridium botulinum on cattle farms]. / Voorkomen van Clostridium botulinum op rundveehouderij bedrijven.

Clostridium botulinum, mainly type B, was constantly found to be present on cattle farms. The organism was isolated both from samples of the soil of pastures and from the faeces of cattle during the winter housing period. The number of C. botulinum type B in samples of soil varied from 10 to 300 organisms per 100 grams. Contamination with C. botulinum was found to be of a similar order of magnitude on farms on which pastures are regularly dressed with sewage sludge. C. botulinum was detected in 13 per cent of the faecal samples (420 samples of one gram each). Particularly grass silage pits prepared with wilted grass were found to provide a possible link between contamination of pastures and cattle.

Prevalence of Campylobacter jejuni and Salmonella during pig slaughtering.

It was found that 79% of healthy pigs, slaughtered in three different slaughterhouses in the Netherlands, were intestinal carriers of Campylobacter jejuni (mean number 4000 cfu per g), and 21% of the same pigs had Salmonella in the intestinal tract (mean number 10 cfu per g). Immediately after slaughter, Campylobacter was swabbed from 9% of the carcasses and Salmonella from 13%. It is concluded from these data that most of the contamination on carcasses does not originate directly from the intestinal tracts of the animals but rather from surfaces, equipment, and utensils in the slaughter hall. It was demonstrated that Salmonella could survive in the slaughter hall, whereas Campylobacter died off, probably due to its vulnerability to drying conditions and its inability to grow at temperatures below 30 degrees C. Campylobacter was not isolated from the carcasses after cooling. It had been shown earlier that this again was caused by dry conditions, brought about by the use of forced ventilation in the cooling rooms. In an additional investigation, Campylobacter was not isolated from 248 samples of minced pork (10 g each), whereas Salmonella was found in 13% of these samples.