AT-101 simultaneously triggers apoptosis and a cytoprotective type of autophagy irrespective of expression levels and the subcellular localization of Bcl-xL and Bcl-2 in MCF7 cells.

The effects of autophagy on cell death are highly contextual and either beneficial or deleterious. One prime example for this dual function of autophagy is evidenced by the cell responses to the BH3 mimetic AT-101 that is known to induce either apoptotic or autophagy-dependent cell death in different settings. Based on previous reports, we hypothesized that the expression levels of pro-survival Bcl-2 family members may be key determinants for the respective death mode induced by AT-101. Here we investigated the role of autophagy in the response of MCF7 breast cancer cells to AT-101. AT-101 treatment induced a prominent conversion of LC3-I to LC3-II and apoptotic cell death characterized by the appearance of Annexin-positive/PI-negative early apoptotic cells and PARP cleavage. Inhibition of the autophagy pathway, either through application of 3-MA or by lentiviral knockdown of ATG5, strongly potentiated cell death, indicating a pro-survival function of autophagy. Overexpression of wild type Bcl-xL significantly diminished the net amount of AT-101-induced cell death, but failed to alter the death-enhancing effects of the ATG5 knockdown. This was also observed with the organelle-specific variants Bcl-xL-ActA and Bcl-2-ActA (mitochondrial) as well as Bcl-xL-cb5 and Bcl-2-cb5 (ER) which all reduced AT-101-induced cell death, but did not affect the death-enhancing effects of 3-MA. Collectively, our data indicate that in apoptosis-proficient MCF7 cells, AT-101 triggers Bcl-2- and Bcl-xL-dependent apoptosis and a cytoprotective autophagy response that is independent of the expression and subcellular localization of Bcl-xL and Bcl-2.

Presence of Clostridium botulinum and botulinum toxin in milk and udder tissue of dairy cows with suspected botulism.

Clostridium botulinum is an anaerobic spore-forming bacterium prevalent in the environment, and causes botulism in man and animals via toxins. Dairy cattle may be contaminated or infected by feed, water or other environmental factors. Milk may also carry the pathogen. Hence, milk and udder samples need to be tested. The number of clinical cases of bovine botulism in Germany has been increasing since the mid-1990s. Besides routine samples, additional 99 milk samples from 37 farms, and 51 udder samples from 51 farms from sick animals presumably affected by botulism were tested microbiologically by the mouse bioassay. Milk from three farms (8.1 per cent) contained botulinum toxin, and from two (5.4 per cent) bacterial states of C botulinum. Ten udder samples (19.6 per cent) contained toxin, and 7 (13.7 per cent) bacterial forms, including one case where both toxin and bacteria were found. The findings are discussed. Positive milk samples containing botulinum toxin or bacteria raise concern of food safety for the human consumer. Pathological udder samples may show either infection prior to, or contamination after death.

Investigation of serology for diagnosis of outbreaks of botulism in cattle.

Serology has been used to diagnose retrospectively types C and D outbreaks of botulism in cattle in Australia and this study has investigated whether the approach would be applicable in England and Wales. Three hundred sera from routine surveillance submissions in England and Wales were used as a negative control population. Some stored sera were available from a small number of clinical cases of botulism and 125 samples were collected from cohort groups of clinical cases in four new outbreaks of botulism. Three of these outbreaks were identified as being caused by type D Clostridium botulinum toxin. Sera were tested by antibody ELISA in laboratories in Australia and Germany. There was no increase in the proportion of animals seropositive to type C or D antibody in the botulism-associated cattle. The proportion of samples which were seropositive to type D antibodies was <2% in both the negative control and outbreak populations. It was concluded that single time serology is unlikely to be helpful for retrospective diagnosis of outbreaks of type D botulism in England and Wales.

The catalase gene differentiates between some strains of Staphylococcus aureus ssp. anaerobius.

Staphylococcus aureus ssp anaerobius strain S10 was isolated from an outbreak of sheep abscess disease. Sequence of the catalase gene of this strain showed 99% identity to the catalase gene (katB) sequence of the reference strain (S. aureus ssp. anaerobius strain MVF213) with mismatching of three base pairs. An important substitution located 1036 nucleotides upstream of the initiation codon from "C" in katB to "T" in the catalase gene of strain S10 originated a stop codon. The deduced protein (345 amino acids) is 105 amino acids shorter than that of katB. Partial sequence of the catalase gene of other 8 local isolates in addition to another reference strain (DSM 20714/ATCC 35844) revealed the same mutations in all local (African) strains, whereas the sequence of the reference (European) strain was typical to that of katB. Sequence of the catalase gene of S. aureus ssp. anaerobius strain S10 was deposited in GenBank under accession no. EU281993.

Inhibition of tissue factor/protease-activated receptor-2 signaling limits proliferation, migration and invasion of malignant glioma cells.

Tissue factor (TF) is upregulated in several malignant diseases, including gliomas. Here, we demonstrate pronounced differences in the expression of TF and its interactors factor VII and protease-activated receptor 2 (PAR-2) in nine human glioma cell lines (U87, U251, U343, U373, MZ-18, MZ-54, MZ-256, MZ-304, Hs 683) as detected by RT-PCR and Western blot analysis. Inhibition of TF signaling by a neutralizing monoclonal antibody (mAb TF9-10H10) led to significantly reduced proliferation in high-grade astroglial (MZ-18 and MZ-304) and oligodendroglial (Hs 683) cell lines abundantly expressing TF, but not in U373 cells expressing low amounts of TF. Scratch migration assays and Boyden chamber assays indicated that mAb TF9-10H10 and lentiviral knockdown of TF significantly reduced cell migration and invasion of MZ-18, MZ-304 and Hs 683 cells, both under normoxic and hypoxic conditions. Of note, all three cell lines displayed increased cell migration and invasion under hypoxic conditions (1% O(2)), which was associated with enhanced expression of TF and increased phosphorylation of p44/42 mitogen-activated protein kinase (ERK1/2). Silencing of TF blocked activation of the ERK pathway, induction of TF expression and the potentiating effect of hypoxia on cell migration and invasion. RNA interference against PAR-2 abrogated the autocrine effects of TF on cell proliferation, migration and invasion, indicating that TF signals via PAR-2 in glioma cells. Our results suggest an important role for the TF/FVIIa/PAR-2/ERK axis in tumor growth and invasion of glioma and suggest that TF may be a suitable target for the development of novel therapies against high-grade glioma.

Tonsils--place of botulinum toxin production: results of routine laboratory diagnosis in farm animals.

Since a case of a veterinarian was reported, who was likely to be infected/intoxicated by Clostridium botulinum during the handling of a diseased animal, tonsils in animals were tested for botulinum neurotoxin and bacterial forms of neurotoxic Clostridium botulinum during routine botulism laboratory examinations including standard samples (intestinal tract and liver) from 48 cattle, 11 horses, and 14 goats. Ten out of 60 samples from tonsils contained free botulinum toxin, and 12 out of 59 were positive for live toxin producing bacteria. In 32 out of 162 intestinal samples toxin was detected. Toxin producing bacteria were found in 37 samples. Eight of 56 liver samples contained free toxin, and 15 out of 43 toxigenic bacteria. Samples from 10 slaughter pigs were all negative, whereas from slaughter cattle tonsils had a high incidence of toxin (7 of 10) or toxigenic bacteria (2 of 8). The results are discussed in the context of effects on animal health and botulism as zoonosis.

Botulinum toxins--cause of botulism and systemic diseases?

Toxins of Clostridium botulinum (types A-G) are known as 'neurotoxins', causing the clinically well-known picture of flaccid muscular paralysis. The molecular biological background is the blocking of acetylcholine secretion in neuromuscular junctions by enzymatic cleavage of molecules forming the machinery of exocytosis. Two 'non-neurotoxins' (types C2, C3) are produced by some strains of C. botulinum types C and D. These affect the cytoskeleton by ribosylating actin filaments. All these toxins are used as cell biological tools for the study of specific actions and effects in different eukaryotic cells. Pharmaceutical and molecular biological research has shown their influence on several crucial organs (or cell cultures thereof) of humans and animals (brain and spinal cord, cerebellum, hippocampus, hypophysis, pancreas, adrenal glands, salivary glands and others). Under natural conditions, botulinum toxins may pass the intestinal barrier and circulate in the bloodstream for a certain time. Carriers occurring naturally in food, such as wheat germ agglutinin, digitonin or saponin, and bacterial toxins such as streptolysin O, perfringolysins, C2 toxin or botulinolysin may also form pores in cell walls. They facilitate the entry of botulinum toxins into cells that may not have natural binding receptors. It is concluded that in vivo actions of different botulinum toxins after their entry into the organism may contribute to the onset of different diseases of hitherto cryptogenic origin. Some examples are given and future problems are discussed.

Two cases of equine grass sickness with evidence for soil-borne origin involving botulinum neurotoxin.

Botulism is caused by different types of Clostridium botulinum, a soil bacterium. Equine grass sickness (equine dysautonomia) is suspected of being a clinical form of this disease. On a stud where this disease occurred twice within 8 months, grass and soil samples and necropsy specimens of one horse were tested for the presence of bacterial forms and toxin of C. botulinum. Different types and type mixtures (A-E) of C. botulinum and botulinum neurotoxin were found. For the first time, it has been shown that green grass blades contain botulinum toxin. The results support the hypothesis that equine grass sickness is a clinical form of botulism, a soil-borne disease.

Is there a link between infant botulism and sudden infant death? Bacteriological results obtained in central Germany.

UNLABELLED: Despite the fact that botulism was described in Germany for the first time by Kerner in 1820, the disease is almost forgotten in this country. Only about 10-20 cases of classical botulism (intoxication) are recorded every year, including 1-2 cases of clinical infant botulism. As we assumed a high incidence of botulism to be connected with cases of sudden infant death (SID), we undertook the research work presented here. From every case of unexpected infant death up to 12 months of age, standardised specimens (blood, liver and intestine) were taken at autopsy. They were tested for the presence of botulinum neurotoxin (BoNT) and/or bacterial forms of Clostridium botulinum with subsequent BoNT neutralisation tests by the international standard mouse bioassay. Age, sex, pathological findings and season were recorded. Over a 5-year period, 75 samples including 57 SID cases were tested. Free toxin was found in nine and bacterial forms were detected in six samples. Toxin neutralisation revealed the definite presence of BoNT/BoNT producing bacteria (mainly type E), whereas another 11 toxin tests were inconclusive. According to international literature, these 15 cases are to be interpreted as infant botulism. CONCLUSION: the results show a remarkable incidence of infant botulism without any known previous medical history, partly hidden as sudden infant death. We propose to systematically search for botulism in connection with sudden infant death.

Visceral botulism--a new form of bovine Clostridium botulinum toxication.

There are reports of a hitherto unknown bovine disease in Germany. The symptoms are, in general, indigestion (constipation alternating with diarrhoea), non-infectious chronic laminitis, engorged veins, oedemas, retracted abdomen, emaciation and apathy. Most cases occur during the peripartal period and often result in unexpected death. In addition, there are findings of delayed growth and wasting in heifers, as well as decreasing milk yield. Clinical and standard laboratory examinations leave the origin undisclosed. Bioassays for Clostridium botulinum, its spores and toxins in animals of affected farms revealed the presence of free botulinum toxin in the contents of the lower sections of the intestine. In two control farms without signs of the disease, the tests remained negative. This seems to support our hypothesis that long-lasting absorption of low quantities of botulinum toxin may interfere with the neurological control of intestinal physiology. The authors propose to name this disease complex 'visceral botulism'.

Production and purification of Clostridium botulinum type C and D neurotoxin.

Neurotoxins of Clostridium botulinum are needed in basic neurologic research, but as therapeutic agent for certain neuromuscular disorders like strabism as well. A method for the production and purification of botulinum neurotoxins C and D is reported using a two-step hollow-fiber cross flow filtration and a newly developed chromatographic purification procedure. Hollow-fiber filtration proved to be a rapid and safe concentration and pre-purification step, which can easily be scaled up. The chromatographic purification included hydrophobic interaction, anion exchange and size exclusion chromatography runs. Botulinum neurotoxins C and D could be recovered with an overall yield of 12.6% and 10.6%, respectively. A specific toxicity of 1.86 x 10(7) minimal lethal dose mg(-1) (type C) and 5.26 x 10(7) minimal lethal dose mg(-1) (type D) was determined in the mouse bioassay.

[Oral long-term administration of probiotic B. cereus--an alternative for the the prevention of enterotoxemia?]. / Orale Dauerapplikation des probiotischen B. cereus--eine Alternative zur Verhütung der Enterotoxämie?

Aetiology and epizootiology of enterotoxaemia, especially the connection between development of the disease and the management of feeding are described and newer knowledge about the physiopathology of the disease is presented. A comparative description of prophylactic schemes which include immuno- and chemoprophylaxis follows. The principles of the application and the effect of probiotic organisms as a new approach to prevent the disease are explained. The probiotic effect of apathogenic non-haemolysing and non-toxic B. cereus strains which have proven to be able to suppress enteropathies and also enterotoxaemia is described.