Roles of diet and the acid tolerance response in survival of common Salmonella serotypes in feces of finishing pigs

The persistence of Salmonella in the environment is an important factor influencing the transmission of infection in pig production. This study evaluated the effects of acid tolerance response (ATR), organic acid supplementation, and physical properties of feed on the survival of a five-strain Salmonella mixture in porcine feces held at 4 and 22°C for 88 days. Acid-adapted or non-acid-adapted nalidixic acid-resistant Salmonella strains were used to inoculate feces of pigs fed four different diets, which consisted of a nonpelleted, finely ground meal feed or a finely ground, pelleted feed that was left unsupplemented or was supplemented with K-diformate. Organic acid supplementation and physical properties of feed markedly influenced Salmonella survival, but the effects were highly dependent on storage temperature; survival was unaffected by ATR. The most pronounced effects were observed at 22°C, a temperature similar to that of finishing pig houses. The supplementation of meal diets with K-diformate significantly reduced the duration of survival (P < 0.1) and increased rates of decline (P < 0.0001) of salmonellae in feces compared to survival in feces of pigs fed unsupplemented meal. The pelleting of feed, compared to feeding meal, significantly reduced (P < 0.1) the duration of survival in feces held at 22°C. Only minor effects of feed form and acid supplementation on survivor numbers were observed at 4°C. Differences in the fecal survival of Salmonella could not be related to diet-induced changes in fecal physiochemical parameters. The predominant survival of S. enterica serovar Typhimurium DT193 and serotype 4,[5],12:i:- in porcine feces demonstrates the superior ability of these serotypes to survive in this environment. Fecal survival and transmission of Salmonella in pig herds may be reduced by dietary approaches, but effects are highly dependent on environmental temperature.     

Chronic acetyl-L-carnitine alters brain energy metabolism and increases noradrenaline and serotonin content in healthy mice

Acetyl-L-carnitine (ALCAR), the short-chain ester of carnitine, is a common dietary supplement readily available in health food stores, claimed to improve energy levels and muscle strength. ALCAR has numerous effects on brain and muscle metabolism, protects against neurotoxic insults and may be an effective treatment for certain forms of depression. However, little is known about the effect of chronic ALCAR supplementation on the brain metabolism of healthy mice. Here, we investigated ALCAR's effect on cerebral energy and neurotransmitter metabolism after supplementing the drinking water of mice with ALCAR for 25 days, providing a daily dose of about 0.5 g/kg. Thereafter the animals were injected with [1-(13)C]glucose, and (13)C incorporation into and levels of various metabolites were quantified in extracts of the hippocampal formation (HF) and cortex using (1)H- and (13)C-nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC). Increased glucose levels were detected in both regions together with a decreased amount of [3-(13)C]lactate, but no alterations in incorporation of (13)C derived from [1-(13)C]glucose into the amino acids glutamate, GABA and glutamine. These findings are consistent with decreased metabolism of glucose to lactate but not via the TCA cycle. Higher amounts of the sum of adenosine nucleotides, phosphocreatine and the phosphocreatine/creatine ratio found in the cortex of ALCAR-treated mice are indicative of increased energy levels. Furthermore, ALCAR supplementation increased the levels of the neurotransmitters noradrenaline in the HF and serotonin in cortex, consistent with ALCAR's potential efficacy for depressive symptoms. Other ALCAR-induced changes observed included reduced amounts of GABA in the HF and increased myo-inositol. In conclusion, chronic ALCAR supplementation decreased glucose metabolism to lactate, resulted in increased energy metabolite and altered monoamine neurotransmitter levels in the mouse brain.     

A novel functional assay for fungal histidine kinases group III reveals the role of HAMP domains for fungicide sensitivity

Signal transduction systems comprising histidine kinases are suggested as new molecular targets of antibiotics. The important human fungal pathogen Candida albicans possesses three histidine kinases, one of which is the type III histidine kinase CaNik1, which activates the MAP kinase Hog1. We established a screening system for inhibitors of this class of histidine kinases by functional expression of the CaNIK1 gene in S. cerevisiae. This transformant was susceptible to fungicides to which the wild type strain was resistant, such as fludioxonil and ambruticin. Growth inhibition correlated with phosphorylation of Hog1 and was dependent on an intact Hog1 pathway. At the N-terminus the histidine kinase CaNik1 has four amino acid repeats of 92 amino acids each and one truncated repeat of 72 amino acids. Within these repeats we identified 9 HAMP domains with a paired structure. We constructed mutants in which one or two pairs of these domains were deleted. S. cerevisiae transformants expressing the full-length CaNIK1 showed the highest sensitivity to the fungicides, any truncation reduced the susceptibility of the transformants to the fungicides. This indicates that the HAMP domains are decisive for the mode of action of the antifungal compounds.     

An operational concept for long-term cinemicrography of cells in mono- and co-culture under highly controlled conditions--the SlideObserver

Cell morphology, proliferation and motility, as well as mono- and heterotypic cell-to-cell interactions, are of increasing interest for in vitro experiments. However, tightly controlling culture conditions whilst simultaneously monitoring the same set of cells is complicated. Moreover, video-microscopy of distinct cells or areas of cells over a prolonged period of time represents a technical challenge. The SlideObserver was designed for cinemicrography of cells in co-and monoculture. The core elements of the system are the SlideReactors, miniaturised hollow fibre-based bioreactors operated in closed perfusion loops. Within the SlideReactors, cells can be cultured under adaptable conditions as well as in direct- and indirect co-culture. The independent perfusion loops enable controlled variation of parameters such as medium, pH, and oxygenation. A combined automated microscope stage and camera set-up allows for micrograph acquisition of multiple user-defined regions of interest within the bioreactor units. For proof of concept, primary cells (HUVEC, human hepatocytes) and cell lines (HuH7, THP-1) were cultured under stable and varying culture conditions, as well as in mono- and co-culture. The operational system enabled non-stop imaging and automated control of process parameters as well as elective manipulation of either reactor. As opposed to non-perfused culture systems or comparable devices for cinemicrographic analysis, the SlideObserver allows simultaneous morphological monitoring of an entire culture of cells in multiple bioreactors.     

Kidney pathology and parasite intensity in rainbow trout Oncorhynchus mykiss surviving proliferative kidney disease: time course and influence of temperature

Proliferative kidney disease (PKD) is an endoparasitic disease of salmonids caused by the myxozoan parasite Tetracapsuloides bryosalmonae. We recently described the development of the disease from initial infection until manifestation of clinical disease signs in rainbow trout held at 2 water temperatures, 12 and 18°C. The aim of the present study is to investigate whether (1) infected fish surviving the clinical phase would recover from renal pathological changes, (2) whether they would be able to reduce the parasite load in the kidneys, and (3) whether water temperatures would influence renal recovery and parasite clearance. At 18°C, fish showed a gradual recovery of normal kidney morphology which was associated with a decline in parasite numbers and infection prevalence. Fish kept at 12°C initially showed an enhancement of kidney lesions before recovery of normal kidney morphology took place. The decrease in renal parasite load was retarded compared to 18°C. The results from the present study provide evidence that rainbow trout surviving the clinical phase of PKD are able to (1) fully restore renal structure, and (2) significantly reduce renal parasite loads, although 100% clearance was not achieved within the experimental period of this study. Water temperature influences the rate but not the outcome of the recovery process.     

Rescue of tropomyosin deficiency in Drosophila and human cancer cells by synaptopodin reveals a role of tropomyosin α in RhoA stabilization

Tropomyosins are widespread actin-binding proteins that influence numerous cellular functions including actin dynamics, cell migration, tumour suppression, and Drosophila oocyte development. Synaptopodin is another actin-binding protein with a more restricted expression pattern in highly dynamic cell compartments such as kidney podocyte foot processes, where it promotes RhoA signalling by blocking the Smurf1-mediated ubiquitination of RhoA. Here, we show that synaptopodin has a shorter half-life but shares functional properties with the highly stable tropomyosin. Transgenic expression of synaptopodin restores oskar mRNA localization in Drosophila oocytes mutant for TmII, thereby rescuing germline differentiation and fertility. Synaptopodin restores stress fibres in tropomyosin-deficient human MDA-MB 231 breast cancer cells and TPMα-depleted fibroblasts. Gene silencing of TPMα but not TPMβ causes loss of stress fibres by promoting Smurf1-mediated ubiquitination and proteasomal degradation of RhoA. Functionally, overexpression of synaptopodin or RhoA(K6,7R) significantly reduces MDA-MB 231 cell migration. Our findings elucidate RhoA stabilization by structurally unrelated actin-binding proteins as a conserved mechanism for regulation of stress fibre dynamics and cell motility in a cell type-specific fashion.     

SUMO-1 controls the protein stability and the biological function of phosducin

Phosducin regulates Gbetagamma-stimulated signaling by binding to Gbetagamma subunits of heterotrimeric G-proteins. Control of phosducin activity by phosphorylation is well established. However, little is known about other mechanisms that may control phosducin activity. Here we report that phosducin is regulated at the posttranslational level by modification with the small ubiquitin-related modifier, SUMO. We demonstrate modification with SUMO for phosducin in vitro expressed in cells and for native phosducin purified from retina and the heart. A consensus motif for SUMOylation was identified in phosducin at amino acid positions 32-35. Mutation of the conserved lysine 33 to arginine in this motif abolished SUMOylation of phosducin, indicating that SUMO is attached to lysine 33 of phosducin. In transfected cells the steady-state levels of the K33R mutant protein were much lower compared with wild-type phosducin. The investigation of the stability of wild-type phosducin and of phosducinK33R showed a decreased protein stability of the SUMOylation-deficient mutant. The decreased protein stability correlated with increased ubiquitinylation of the SUMOylation-deficient mutant. These findings indicate that SUMOylation protects phosducin from proteasomal degradation. SUMOylation of phosducin decreased its ability to bind Gbetagamma. PhlP, a closely related member of the phosducin family, was not a target for SUMOylation, but its SUMOylation can be achieved by a single amino acid insertion in the conserved N terminus of PhlP. Together, these findings show that phosducin is a previously unrecognized target of SUMO modification and that SUMOylation controls phosducin stability in cells as well as its functional properties.     

Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes

A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms.     

NADP-dependent mannitol dehydrogenase, a major allergen of Cladosporium herbarum

Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.     

Test of the Lamendin aging method on two historic skeletal samples

The Lamendin aging method involves the quantification of root translucency and the attachment position of the periodontal membrane. It was developed using recent medical-examiner specimens, and was tested on modern skeletal samples such as the Terry Collection (Lamendin et al. [1992] J. Forensic Sci. 37:1373-1379; Prince and Ubelaker [2002] J. Forensic Sci. 47:107-116). The method may be one of the most useful for estimating age after the mid-30s. The current study is an evaluation of the Lamendin criteria on two historic skeletal samples from Britain. Both the Christ's Church Spitalfields and St. Bride's Church collections represent documented skeletal samples that were interred in the 18th and 19th centuries. In total, 1,188 teeth from 220 adult individuals were examined from these two collections. The Lamendin method requires measuring total root length (cementoenamel junction to apex), gingival regression (cementoenamel junction to periodontal ligament attachment), and root translucency (root apex to maximum level of root translucency) on the labial surface of single-rooted teeth. Our results indicate that postmortem factors affect the applicability of the Lamendin technique to archaeological and historical samples. In particular, root translucency disappears with time, or is obscured by unknown postmortem taphonomic effects related to the length of interment or postmortem environment. Thirty-five percent of our sample showed no root translucency, indicating that caution is required when applying this method to archaeological or historical remains. The mean error of age estimates for Spitalfields and St. Bride's was higher than in the original study of Lamendin et al. ([1992] J. Forensic Sci. 37: 1373-1379), and higher than in the test by Prince and Ubelaker ([2002] J. Forensic Sci. 47:107-116) of the Lamendin method on the Terry Collection.