Calcium montmorillonite clay reduces urinary biomarkers of fumonisin B1 exposure in rats and humans.

Fumonisin B1 (FB1) is often a co-contaminant with aflatoxin (AF) in grains and may enhance AF's carcinogenicity by acting as a cancer promoter. Calcium montmorillonite (i.e. NovaSil, NS) is a possible dietary intervention to help decrease chronic aflatoxin exposure where populations are at risk. Previous studies show that an oral dose of NS clay was able to reduce AF exposure in a Ghanaian population. In vitro analyses from our laboratory indicated that FB1 (like aflatoxin) could also be sorbed onto the surfaces of NS. Hence, our objectives were to evaluate the efficacy of NS clay to reduce urinary FB1 in a rodent model and then in a human population highly exposed to AF. In the rodent model, male Fisher rats were randomly assigned to either FB1 control, FB1 + 2% NS or absolute control group. FB1 alone or with clay was given as a single dose by gavage. For the human trial, participants received NS (1.5 or 3 g day⁻¹) or placebo (1.5 g day⁻¹) for 3 months. Urines from weeks 8 and 10 were collected from the study participants for analysis. In rats, NS significantly reduced urinary FB1 biomarker by 20% in 24 h and 50% after 48 h compared to controls. In the humans, 56% of the urine samples analysed (n = 186) had detectable levels of FB1. Median urinary FB1 levels were significantly (p < 0.05) decreased by >90% in the high dose NS group (3 g day⁻¹) compared to the placebo. This work indicates that our study participants in Ghana were exposed to FB1 (in addition to AFs) from the diet. Moreover, earlier studies have shown conclusively that NS reduces the bioavailability of AF and the findings from this study suggest that NS clay also reduces the bioavailability FB1. This is important since AF is a proven dietary risk factor for hepatocellular carcinoma (HCC) in humans and FB1 is suspected to be a dietary risk factor for HCC and oesophageal cancer in humans.

Comparison of insect kinin analogs with cis-peptide bond, type VI-turn motifs identifies optimal stereochemistry for interaction with a recombinant arthropod kinin receptor from the southern cattle tick Boophilus microplus.

The multifunctional 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1=His, Asn, Ser, or Tyr and X2=Ser, Pro, or Ala; and are associated with the regulation of diuresis in a variety of species of insects. We previously reported the functional expression of a southern cattle tick (Boophilus microplus) G protein-coupled receptor that is activated by insect kinins. Four different stereochemical variants of each of the 4-aminopyroglutamic acid (APy) and tetrazole moieties, mimics of a cis-peptide bond, type VI beta-turn in insect kinins were now evaluated on the expressed tick receptor using a calcium bioluminescence plate assay. This study represents the first investigation of the interaction of restricted-conformation analogs incorporating components that mimic specific conformations and/or peptide bond orientations in an expressed arthropod neuropeptide receptor. Analog Ac-RF[APy]WGa (2R,4S) was at least 10-fold more active than the other analogs, thus identifying the optimal stereochemistry for tick receptor interaction. The optimal stereochemistry for the tetrazole insect kinin analogs in the tick receptor assay was identified as (D,L). The APy is superior to the tetrazole as a scaffold for the design of mimetic insect kinin analogs. These biostable analogs provide new tools for arthropod endocrinologists and potential leads in the development of selective, environmentally friendly arthropod pest control agents capable of disrupting insect kinin regulated processes.

Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices.

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist-stimulated human neutrophils show significantly enhanced migration in 3-D collagen lattices. PAR2 agonist stimulation also induced down-regulation of L-selectin display and up-regulation of membrane-activated complex-1 very late antigen-4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)-1beta, IL-8, and IL-6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up-regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.

Endothelial intracellular Ca2+ release following monocyte adhesion is required for the transendothelial migration of monocytes.

Although molecular changes accompanying leukocyte extravasation have been investigated intensively, the particular events following leukocyte adhesion and leading to the actual transendothelial migration process remain largely unknown. To characterize intraendothelial signals elicited by leukocyte adhesion and functionally required for their transmigration, we recorded endothelial free cytosolic intracellular Ca(2+)levels ([Ca(2+)]i) during the course of leukocyte adhesion. We show that monocyte and granulocyte adhesion induced Ca(2+)transients in either untreated or TNF-alpha-stimulated microvascular endothelial cells (HMEC-1). The functional significance of these [Ca(2+)]i rises was demonstrated by treating filter-grown endothelial monolayers with BAPTA/AM. This in traendothelial Ca(2+)chelation left monocyte adhesion basically unaffected, but caused a significant and dose-dependent reduction of the transendothelial migration of monocytes. Granulocyte diapedesis, on the other hand, was hardly modified. Thapsigargin-treatment of endothelial cells almost completely inhibited the transmigration of monocytes suggesting that the necessary Ca(2+)transients depended on a release from intracellular Ca(2+)stores. Our results thus show that the transmigration of monocytes through endothelial monolayers of microvascular origin is favoured by an increase of the intraendothelial [Ca(2+)]i induced by leukocyte adhesion to the endothelial cells.

Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis.

OBJECTIVE: To analyze which physiologic stimuli induce secretion of myeloid-related protein 8 (MRP8) and MRP14, two S100 proteins expressed in neutrophils and monocytes, and to determine whether serum concentrations of these proteins are reliable parameters for monitoring inflammatory activity in pauciarticular juvenile rheumatoid arthritis (JRA). METHODS: Secretion of MRP8 and MRP14 was analyzed using a coculture system of endothelial cells and monocytes. Concentrations of MRP8/MRP14 in the serum and synovial fluid of JRA patients or culture medium were determined by enzyme-linked immunosorbent assay. The expression of MRP8 and MRP14 by leukocytes in synovial tissue or fluid was investigated using immunohistochemistry. RESULTS: MRP8 and MRP14 were specifically released during interaction of activated monocytes with tumor necrosis factor-stimulated endothelial cells. Secretion was mediated via an increase in intracellular calcium levels in monocytes. In contrast, contact with resting endothelium inhibited protein kinase C-induced secretion of the proteins by monocytes. In JRA patients, MRP8 and MRP14 were strongly expressed in infiltrating neutrophils and monocytes within the inflamed joints and could be found in significantly higher concentrations in synovial fluid (mean 42,800 ng/ml) compared with serum (2,060 ng/ml). Concentrations of MRP8/MRP14 in serum correlated well with those in synovial fluid (r = 0.78) and showed a strong correlation with disease activity (r = 0.62). After intraarticular triamcinolone therapy, the serum concentrations of MRP8/MRP14 decreased significantly in therapy responders, whereas no differences were found in patients who showed no clinical benefit. CONCLUSION: MRP8 and MRP14 are specifically released during the interaction of monocytes with inflammatory activated endothelium, probably at sites of local inflammation. Their serum concentrations represent a useful marker for monitoring local inflammation in JRA.

S100A12 is expressed exclusively by granulocytes and acts independently from MRP8 and MRP14.

Changes in cytosolic calcium concentrations regulate a wide variety of cellular processes, and calcium-binding proteins are the key molecules in signal transduction, differentiation, and cell cycle control. S100A12, a recently described member of the S100 protein family, has been shown to be coexpressed in granulocytes and monocytes together with two other S100 proteins, MRP8 (S100A8) and MRP14 (S100A9), and a functional relationship between these three S100 proteins has been suggested. Using Western blotting, calcium overlays, intracellular flow cytometry, and cytospin preparations, we demonstrate that S100A12 expression in leukocytes is specifically restricted to granulocytes and that S100A12 represents one of the major calcium-binding proteins in these cells. S100A12, MRP8, and MRP14 translocate simultaneously from the cytosol to cytoskeletal and membrane structures in a calcium-dependent manner. However, no evidence for direct protein-protein interactions of S100A12 with either MRP8 or MRP14 or the heterodimer was found by chemical cross-linking, density gradient centrifugation, mass spectrometric measurements, or yeast two hybrid detection. Thus, S100A12 acts individually during calcium-dependent signaling, independent of MRP8, MRP14, and the heterodimer MRP8/MRP14. This granulocyte-specific signal transduction pathway may offer attractive targets for therapeutic intervention with exaggerated granulocyte activity in pathological states.

Hydrolysis of insect neuropeptides by an angiotensin-converting enzyme from the housefly, Musca domestica.

The presence in insect tissues of peptides with structural similarities to angiotensin I and to bradykinin, the two best known substrates of mammalian angiotensin-converting enzyme, has not been reported. As part of our study to identify potential substrates for insect angiotensin-converting enzyme, we have investigated the susceptibility of a number of known insect peptide hormones and neurotransmitters to hydrolysis by Musca domestica angiotensin-converting enzyme. Insect peptides belonging to the red pigment-concentrating hormone, leucokinin, locust tachykinin, and depolarizing peptide families were hydrolyzed by housefly angiotensin-converting enzyme, whereas proctolin and crustacean cardioactive peptide were not substrates. Cus-DP II, LK I, LK II, and Lom-TK I were all cleaved at the penultimate C-terminal peptide bond to release a dipeptide amide as a major fragment with Km values of 94 +/- 11, 634 +/- 8, and 296 +/- 35 microM for Cus-DP II, LK I, and Lom-TK I, respectively. The ability of insect angiotensin-converting enzyme to hydrolyze C-terminally amidated peptides in vitro might be of functional significance because the enzyme has been localized to neuropile regions of the insect brain and is present in the hemolymph of houseflies.

Identification and partial characterization of receptors for allatostatins in brain and corpora allata of the cockroach Diploptera punctata using a binding assay and photoaffinity labeling.

We have developed both an in vitro binding assay and a photoaffinity labeling assay to demonstrate and partially characterize putative receptors for allatostatins in brain and in corpora allata of Diploptera punctata. Isolated brain membranes were photoaffinity labeled with 125I-RYBPA (photoaffinity analogue of dip-allatostatin 5). Following labeling with 125I-RYBPA, SDS-PAGE and autoradiography revealed the presence of a putative receptor (37 kDa) for dip-allatostatin 5 and dip-allatostatin 7. Specific labeling was demonstrated by dose-dependent competition with either dip-allatostatin 5 or dip-allatostatin 7. The in vitro binding assay indicated that the receptor for dip-allatostatin 5 had a Kd of (9.0 +/- 0.9).10(-10) M and Bmax of 2.2 +/- 0.3 pmol/mg membrane protein. For dip-allatostatin 7, two Kd values of (1.5 +/- 0.1).10(-9) M and (3.8 +/- 0.3).10(-9) M were obtained, with Bmax values of 7.2 +/- 0.7 pmol/mg membrane protein and 11.4 +/- 1.0 pmol/mg membrane protein respectively. This indicates that there were probably two putative receptor sites for dip-allatostatin 7 although only one band was observable following photoaffinity labeling. Binding was saturable, specific and reversible. Using the in vitro binding assay, the Kd of the putative receptor in CA for dip-allatostatin 7 was shown to be (7.2 +/- 0.9).10(-10) M.

Culekinin depolarizing peptide: a mosquito leucokinin-like peptide that influences insect Malpighian tubule ion transport.

A peptide termed culekinin depolarizing peptide (CDP) was isolated from approximately 1.2 million mosquitos (94% Culex salinarius). The peptide was isolated on the basis of a rapid myotropic assay that utilized a hindgut preparation from Leucophaea maderae and a transepithelial voltage assay that used mosquito Malpighian tubules from Aedes aegypti. A 15% trifluoroacetic acid extraction from the mosquitos, two solid phase extraction steps, and six HPLC steps resulted in the isolation of 9.7 nmol of CDP. This value corresponds to approximately 8 fmol/mosquito. Edman degradation indicated the following sequence for CDP: Asn-Pro-Phe-His-Ser-Trp-Gly-NH2. The sequence was confirmed as the suspected C-terminal amide form of the peptide, since native and synthetic CDP had identical chemical and biological properties. CDP is a member of the leucokinin family of neuropeptides. The leucokinins have been found in three other insect species (Leucophaea maderae, Acheta domesticus and Locusta migratoria) where these peptides were isolated by their myotropic properties alone. CDP shares a C-terminal sequence homology (i.e., Phe-X-Ser-Trp-Gly-NH2) with the rest of the leucokinins. CDP corresponds to the strongest tubule depolarizing activity in the C. salinarius extract. These findings agree with previous structure-activity studies that suggest that mosquitos would contain a leucokinin-like factor that had Phe-His-Ser-Trp-Gly-NH2 as the C-terminal pentapeptide. This is the first leucokinin isolated from blood feeding or holometabolous insects.

Structure-activity studies of allatostatin 4 on the inhibition of juvenile hormone biosynthesis by corpora allata: the importance of individual side chains and stereochemistry.

The production of juvenile hormone III (JH III) by the corpora allata of the cockroach Diploptera punctata is regulated in part by peptides originating from the brain. One group of these peptides, termed allatostatins, reversibly inhibits the biosynthesis of JH in vitro. Allatostatin 4 (AST4: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-amide) is the smallest member of the AST family yet defined and was used as the benchmark peptide for these initial structure-activity studies. Two initial analog series of AST4 were examined for the ability of each analog to inhibit JH biosynthesis by corpora allata in vitro. Each analog series consisted of analogs that contained a single amino acid change from the native AST4 sequence. The first series contained Ala replacement analogs and the second contained analogs with D-amino acid replacements. The first analog series used Ala replacements to help indicate which amino acid side chains were most important for inhibition of JH biosynthesis. The most important side chain appeared to be Leu8 followed by Phe6 and Tyr4. Additionally, the D-amino acid series suggested that a secondary structural element(s) at the C-terminus of AST4 could be important to the biological activity.

[Stimulation of Brucella clearance in rats after treatment with BCG suspensions]. / Steigerung der Brucella-Clearance bei der Ratte nach Behandlung mit BCG-Suspensionen.

Intraperitoneal application of BCG induced a significantly increased clearance of Brucella in rats. The effect starts three days after application of 7 mg BCG to the rat and continued for eight weeks. This effect could be used for estimation of efficiency of BCG for nonspecific resistance stimulation. Comparative investigation of seven different batches reveals that three BCG preparations induce an unspecific resistance more than eight weeks, one for two weeks and three batches exerted no effect. Addition of Gelafusal as a stabilizer prolonged durability for about two weeks.

Influenza virus A/swine-outbreaks in domestic pigs and antibody findings in human sera.

In the last years, the occurrence of influenza viruses A/H1N1 (Hsw1N1) in pig stocks of different countries has been increasingly reported. In general, the isolated viruses were related to the influenza virus A/New Jersey/8/76 H1N1 (Hsw1N1). Human infections were not reported in these outbreaks. Since March 1981, very limited influenza outbreaks in several pig stocks of the GDR with high morbidity and very low lethality have been observed. The illness took an uncomplicated path and generally subsided after 3 days. Some of the virus isolates were examined and typed as influenza virus A/swine/Potsdam/81/H1N1 (Hsw1N1). By serological examinations of convalescent pigs the aetiologic importance of the isolates was confirmed. Infection of the contact persons by the influenza virus A/swine/Potsdam/81 is to be regarded as likely according to the serological results.

Toxicological tests on the safety of fermosin for pigs, a novel microbial feed from petroleum distillate.

The dog fodder yeast fermosin did not influence the health of pigs in long-term feeding experiments. It is recommended to use fermosin in concentrations up to 7.5% in mixed feed for pigs.