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Hair cortisol concentration (HCC) might represent a promising marker for retrospective welfare assessment of dairy cows. The objective of the study was to explore the dynamics of HCC in diseased and healthy cows from eight-week ante partum (AP) to eight-week post partum (PP). Twenty-four pregnant cows were followed from drying off to week eight PP. Tail hair was used to measure cortisol at five different time points. The occurrence of peripartum diseases, lameness and the body condition score (BCS) were monitored on a weekly basis. Blood β-hydroxybutyric acid, non-esterified fatty acids, calcium and insulin-like growth factor-1 (IGF-1) concentrations were measured. The temperature-humidity index (THI) was continuously recorded. The median values of HCC in all cows were 0.4, 0.3, 0.6, 0.8 and 0.5 pg/mg at weeks eight, four AP, calving, weeks four, eight PP, respectively. There was no association between HCC and the occurrence of peripartum diseases (P ≥ 0.05). A positive correlation between HCC and BCS loss (P

The goals of this study were to investigate hair cortisol concentration (HCC) in seven different breeds of cows, to establish reference intervals for HCC in Brown Swiss cows and to compare cortisol concentrations of hair collected from four different areas of the body. Three groups of cows were used. Group 1 comprised 70 healthy cows representing four dairy breeds (Brown Swiss, Swiss Fleckvieh, Holstein Friesian, Water Buffalo) and three beef breeds (Raetian Grey, Limousin, Highland). Group 2 consisted of 60 healthy Brown Swiss cows in which two different hair samples were collected from the thoracic region to establish reference intervals; A samples consisted of hair that had grown for one month in a pre-clipped area, and B samples consisted of hair from a previously unshorn area. Group 3 comprised 21 healthy Brown Swiss cows, in which HCCs were measured in A and B samples from four different body regions (neck, shoulder, thorax, rump). Liquid chromatography tandem mass-spectrometry was used for cortisol measurement. In group 1, the highest HCCs were measured in Holstein Friesian cows at 1,75 pg/mg, which was significantly higher than those of the Brown Swiss, the Swiss Fleckvieh and the Water Buffalo cows. Hair cortisol concentration and daily milk yield of the 40 dairy cows were highly correlated (r = 0,57, P0,01). In group 2, the HCCs of 77 % of the A samples and 85 % of the B samp-les were below the laboratory's limit of quantification (LOQ) of 0,50 pg/mg and the results were expressed semiquantitatively as.Das Ziel war es, die Haarcortisolkonzentrationen (HCC) bei 7 Rinderrassen zu untersuchen, Referenzwerte für Braunviehkühe zu bestimmen und 4 Entnahmestellen in Bezug auf die HCC zu vergleichen. Die Untersuchungen wurden an 3 Gruppen von Kühen durchgeführt. In der Gruppe 1 wurden die HCC bei 70 Kühen von 4 Milchvieh- (Brown Swiss, Swiss Fleckvieh, Holstein Friesian, Wasserbüffel) und 3 Mutterkuhrassen (Rätisches Grauvieh, Limousin, Schottisches Hochland­rind) untersucht. In der Gruppe 2 wurden bei 60 gesunden Brown-Swiss-Kühen A- und B-Proben zur Ermittlung von Referenzwerten am Thorax entnommen. Die A-Proben bestanden aus nachgewachsenen Haaren aus einer vor einem Monat geschorenen Fläche und die B-Proben aus nicht vorgeschorenem Haar. Die Gruppe 3 bestand aus 21 gesunden Brown-Swiss-Kühen, bei denen die HCC in A- und B-Proben von 4 verschiedenen Körperstellen (Hals, Schulter, Thorax, Kruppe) untersucht und miteinander verglichen wurden. Die HCC-Bestimmung erfolgte mittels LC-MS/MS (Flüssigkeitschromatographie, gekoppelt mit Massenspektrometrie). In der Gruppe 1 waren die HCC der Holstein-Friesian-Kühe mit 1,75 pg/mg signifikant höher als diejenigen der Brown Swiss-, Swiss Fleckvieh- und Wasserbüffelkühe. Zwischen den HCC und der Tagesmilchleistung der 40 Milchkühe bestand eine signifikante Korrelation (r = 0,57, P0,01). Bei der Gruppe 2 waren die HCC in 77 % der A- und 85 % der B-Proben unterhalb der Bestimmungsgrenze (limit of quantification, LOQ) von 0,50 pg/mg und wurden semiquantitativ alsLOQ angegeben. In den restlichen Proben lagen die HCC zwischen 0,50 und 1,20 pg/mg. Die Werte der A- und B-Proben unterschieden sich nicht signifikant. Bei der Gruppe 3 lagen die HCC-Medianwerte der messbaren Proben für die 4 Lokalisationen zwischen 0,50 und 1,00 pg/mg Haar. Die HCC unterschieden sich weder zwischen den verschiedenen Körperstellen noch zwischen den A- und Proben signifikant. Die Untersuchungen lassen die Vermutung zu, dass Holstein-Friesian-Kühe signifikant höhere HCC als Brown Swiss-, Swiss Fleckvieh- und Wasserbüffelkühe aufweisen und dass dies mindestens teilweise durch die hohe Milchleistung bedingt ist.Les objectifs de cette étude étaient d’étudier la concentration de cortisol dans les poils (hair cortisol concentration (HCC)) chez sept races de vaches différentes, d’établir des intervalles de référence pour le HCC chez les vaches de race Suisse Brune et de comparer les concentrations de cortisol dans les poils prélevés sur quatre zones différentes du corps. Trois groupes de vaches ont été utilisés. Le groupe 1 comprenait 70 vaches saines représentant quatre races laitières (Brown Swiss, Swiss Fleckvieh, Holstein Friesian, Buffle d’eau) et trois races à viande (Grise rhétique, Limousin, Highland). Le groupe 2 était composé de 60 vaches Brown Swiss en bonne santé, pour lesquelles deux échantillons de poils différents ont été prélevés dans la région thoracique afin d’établir des intervalles de référence ; les échantillons A étaient constitués de poils ayant poussé pendant un mois sur une zone précédemment tondue et les échantillons B étaient constitués de poils provenant d’une zone non tondue auparavant. Le groupe 3 comprenait 21 vaches suisses brunes en bonne santé, chez lesquelles les HCC ont été mesurés dans des échantillons A et B provenant de quatre régions corporelles différentes (cou, épaule, thorax, croupe). La chromatographie liquide en tandem avec spectrométrie de masse a été utilisée pour la mesure du cortisol. Dans le groupe 1, les HCC les plus élevés ont été mesurés chez les vaches Holstein Friesian à 1,75 pg/mg, ce qui était significativement plus élevé que ceux des vaches Brown Swiss, Swiss Fleckvieh et Buffle d’eau. La concentration de cortisol dans les poils et le rendement laitier quotidien des 40 vaches laitières étaient fortement corrélés (r = 0,57, P0,01). Dans le groupe 2, les HCC étaient inférieures à la limite de quantification (LOQ) de 0,50 pg/mg dans 77 % des échantillons A et 85 % des échantillons B et ils ont été indiquées de manière semi-quantitative commeLOQ. Dans les échantillons restants, les HCC se situaient entre 0,50 et 1,20 pg/mg. Les valeurs des échantillons A et B n’étaient pas significativement différentes. Dans le groupe 3, les valeurs médianes des HCC des échantillons mesurables pour les 4 localisations se situaient entre 0,50 et 1,00 pg/mg de poils. Les HCC ne différaient pas significativement entre les différentes localisations corporelles ni entre les échantillons A et B. Les analyses permettent de supposer que les vaches Holstein-Friesian présentent des HCC significativement plus élevées que les vaches Brown Swiss, Swiss Fleckvieh et les bufflonnes d’eau et que cela est dû au moins en partie à leur production laitière élevée.L’obiettivo di questo studio è stato quello di analizzare la concentrazione di cortisolo (HCC) nel pelo in sette diverse razze bovine, di stabilire gli intervalli di riferimento per l’HCC nelle vacche di razza Bruna e di confrontare le concentrazioni di cortisolo nei peli raccolti da quattro diverse aree del corpo. Sono stati utilizzati tre gruppi di vacche. Il gruppo 1 comprendeva 70 vacche sane che rappresentavano quattro razze da latte (Bruna svizzera, Swiss Fleckvieh, Frisona, Bufalo d’acqua) e tre razze da carne (Grigia alpina, Limousine, Highlander). Il gruppo 2 era composto da 60 vacche Bruna svizzera sane in cui sono stati raccolti due diversi campioni di pelo dalla regione toracica per stabilire gli intervalli di riferimento. I campioni A erano costituiti da peli cresciuti per un mese in un’area tosata, mentre i campioni B erano costituiti da peli provenienti da un’area precedentemente non tosata. Il gruppo 3 comprendeva 21 vacche Bruna svizzera sane, in cui le HCC sono stati misurate in campioni A e B provenienti da quattro diverse regioni del corpo (collo, spalla, torace, groppa). Per la misurazione del cortisolo è stata utilizzata la cromatografia liquida accoppiata con spettrometria di massa (LC-MS/MS). Nel gruppo 1, le HCC più elevate sono state misurate nelle vacche di razza Frisona con 1,75 pg/mg, significativamente più alte di quelle delle vacche di razza Bruna svizzera, Swiss Fleckvieh e Bufalo d’acqua. La concentrazione di cortisolo nel pelo e la produzione giornaliera di latte delle 40 vacche da latte erano altamente correlate (r = 0,57, P0,01). Nel gruppo 2, le HCC del 77 % dei campioni A e dell’85 % dei campioni B erano inferiori al limite di quantificazione (LOQ) del laboratorio di 0,50 pg/mg e i risultati sono stati espressi semi-quantitativamente come.

Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C. We show that following retinoic acid treatment, human neuroblastoma cells, SiMa and SH-SY5Y, acquire a neuronal phenotype evidenced by axonal growth and expression of neuronal markers. Botulinum neurotoxin type C which cleaves neuron-specific SNAP25 and syntaxin1 caused apoptotic death only in differentiated neuroblastoma cells. Direct comparison of translation-inhibiting enzymes and the type C botulinum protease revealed one order higher cytotoxic potency of the latter suggesting a novel neuroblastoma-targeting pathway. Our mechanistic insights revealed that loss of ubiquitous SNAP23 due to differentiation coupled to SNAP25 cleavage due to botulinum activity may underlie the apoptotic death of human neuroblastoma cells.

Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products. Refined assays for botulinum product testing are urgently needed. Specifically, in vitro replacement assays which can account for all steps of BoNT intoxication are in high demand. Here, we describe a novel SiMa cell-based approach where re-engineering of the VAMP molecule allows detection of all BoNT/B intoxication steps using a luminescent enzymatic reaction with sensitivity comparable to mouse LD50 bioassay. The presented one-step enzyme-linked immunosorbent assay meets 3Rs (replacement, reduction, and refinement of the use of animals) objectives, is user-friendly and will accelerate development of new botulinum drugs. The sensitive enzymatic reporter cell line could also be adapted for the detection of toxin activity during the manufacture of botulinum and tetanus vaccines.

SNAREs (Soluble N-ethylmaleimide sensitive factor attachment protein receptors) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed towards their partner target SNAREs: SNAP25 and syntaxin. SNARE proteins are normally membrane bound but can be cleaved and released by botulinum neurotoxins (BoNTs). We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA-transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of BoNT/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations. This article is protected by copyright. All rights reserved.

Tetanus toxin (TeTx) and the seven structurally related but serologically distinct botulinal neurotoxins, designated BoNT/A to BoNT/G, bind selectively to nerve terminals where they are internalized and sorted by unknown mechanisms. It is generally accepted that toxification is preceeded by a translocation of the L chains from an acid vesicular environment into the cytosole (Niemann, 1991). Despite such homologies, however, the individual toxins display differences in their primary sites of action, their uptake mechanisms (Poulain et al., 1992) and sensitivity to chemical antagonists (Dreyer et al., 1987; Dolly et al., 1990). We have to assume, therefore, that some of the neurotoxins act on different intracellular targets which appear to play key roles in neurotransmitter release from small translucent as well as large dense-core vesicles. Although tetanus patients immediately receive high doses of human tetanus immune globulin to block any nonbound toxin, the internalized toxin evokes its toxic effects for several weeks (Bleck, 1989). This suggests that the toxins act as enzymes. Several enzymatic activities have been proposed over the past years all of which, however, proved to be wrong later on (Niemann, 1991).

mRNAs encoding the light chain of tetanus and botulinum neurotoxins were transcribed, in vitro, from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic identified cholinergic neurons of the buccal ganglia of Aplysia californica. The size of the current response measured in the voltage clamped postsynaptic neuron was taken as indicator of the quantity of acetylcholine released. Depression of neurotransmitter release similar to that observed when native light chains of the two toxins were injected but needing an additional delay of 30 to 40 minutes, demonstrated a successful expression of a foreign mRNA injected into a neuron in situ.