Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Thus far, three isoforms of SCD (SCD1, SCD2, and SCD3) have been identified and characterized. Regulation of the SCD1 isoform has been shown to be an important component of the metabolic actions of leptin in liver, but the effects of leptin on SCD isoforms in other tissues have not been investigated. We found that although the mRNA levels of SCD1 and SCD2 were not affected by leptin deficiency in the hearts of ob/ob mice, the SCD activity and levels of monounsaturated fatty acids were increased, implying the existence of another SCD isoform. This observation has led to the cDNA cloning and characterization of a fourth SCD isoform (SCD4) that is expressed exclusively in the heart. SCD4 encodes a 352-amino acid protein that shares 79% sequence identity with the SCD1, SCD2, and SCD3 isoforms. Liver X receptor ␣ (LXR␣) agonists and a high carbohydrate fat-free diet induced SCD4 expression, but unlike SCD1, SCD4 expression was not repressed by dietary polyunsaturated fatty acids. SCD4 mRNA levels were elevated 5-fold in the hearts of leptin-deficient ob/ob mice relative to wild type controls. Treatment of ob/ob mice with leptin decreased mRNA levels of SCD4, whereas levels of SCD1 and SCD2 were not affected. Furthermore, in the hearts of SCD1-deficient mice, SCD4 mRNA levels were induced 3-fold, whereas the levels of SCD2 were not altered. The current studies identify a novel heart-specific SCD isoform that demonstrates tissue-specific regulation by leptin and dietary factors.

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA ؊ Orfx ؉ A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA ؊ Orfx ؉ A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.

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The genus Clostridium comprises a heterogeneous group of organisms for which the phylogeny and evolutionary relationships are poorly understood. The elucidation of these evolutionary relationships necessitates the use of experimental methods that can distinguish Clostridium lineages that are time and cost effective, and can be accurately and reproducibly employed in different laboratories. Multi-locus sequence typing (MLST) has been successfully used as a reproducible and discriminating system in the study of eukaryotic and prokaryotic evolutionary biology, and for strain typing of various bacteria. In this study, MLST was applied to evaluate the evolutionary lineages in the serotype A group of Clostridium botulinum. C. botulinum type A has recently been shown to produce multiple subtypes, suggesting that it is not monophyletic as previously reported, but comprises distinct lineages. For MLST analysis, we initially evaluated 14 housekeeping genes (gapdh, tuf, sod, oppB, hsp60, dnaE, aroE, pta, 23S rDNA, aceK, rpoB, 16S rDNA, mdh and recA) for amplification and sequence analysis. In the first phase of the analysis, 30 C. botulinum type A strains producing botulinum neurotoxin subtypes A1-A4 were examined. Results of this pilot study suggested that seven of the genes (mdh, aceK, rpoB, aroE, hsp60, oppB and recA) could be used for elucidation of evolutionary lineages and strain typing. These seven housekeeping genes were successfully applied for the elucidation of lineages for 73 C. botulinum type A strains, which resulted in 24 distinct sequence types. This strategy should be applicable to phylogenetic studies and typing of other C. botulinum serotypes and Clostridium species.

Botulinum toxins (BoNT) are zinc proteases (serotypes A-G) which cause flaccid paralysis through the cleavage of SNARE proteins within motor neurons. BoNT/A was originally organized into two subtypes: BoNT/A1 and BoNT/A2, which are ~ 95 % homologous and possess similar catalytic activities. Subsequently, two additional subtypes were identified; BoNT/A3 (Loch Maree), and BoNT/A4 (657Ba), which have 81 and 88% homology with BoNT/A1, respectively. Alignment studies predicted that BoNT/A3 and BoNT/A4 were sufficiently different to BoNT/A1 to affect SNAP25 binding and cleavage. Recombinant Light Chain (LC) of BoNT/A3 (LC/A3) and BoNT/A4 (LC/A4) were subjected to biochemical analysis. LC/A3 cleaved SNAP25 at 50% the rate of LC/A1, but cleaved SNAPtide® at a faster rate than LC/A1, while LC/A4 cleaved SNAP25 and SNAPtide® at slower rates than LC/A1. LC/A3 and LC/A4 had similar Kms for SNAP25 relative to LC/A1, while the kcat for LC/A4 was 10- fold slower than LC/A1, suggesting a defect in substrate cleavage. Neither LC/A3 nor LC/A4 possessed autocatalytic activity, a property of LC/A1 and LC/A2. Thus, the four subtypes of BoNT/A bind SNAP25 with similar affinity but have different catalytic capacities for SNAP25 cleavage, SNAPtide® cleavage, and autocatalysis. The catalytic properties identified among the subtypes of LC/A may influence strategies for the development of small molecule- or peptide- inhibitors as therapies against botulism.

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Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.

A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences-specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the most potent neurotoxin known. BoNTs are characterized as category A select agents because of their potential as a bioterrorism threat as listed by the Centers for Disease Control and Prevention (1). BoNTs can be immunologically distinguished by homologous antitoxins into seven primary serotypes, designated A to G. Among these serotype distinctions, there is considerable genetic variation, as demonstrated by the recognition of at least 24 subtypes (3,8,11,17). These subtypes have been distinguished based on their degree of genetic variation, with subtypes having a minimum of 2.6% divergence at the amino acid level (3), but except for BoNT subtypes A1 (BoNT/A1) and -A2, they have not been purified and analyzed at the protein level, which is important to delineate functional differences between the subtypes (15). The purification and characterization of the biochemical, toxicological, and molecular mechanisms of the subtype toxins of various serotypes will provide valuable information as to their biochemical, immunological, and cell biology properties.Recently, a new subtype of BoNT/A was identified and named "BoNT/A5"; there are five strains known to possess the gene encoding BoNT/A5 (3,8). Among these five strains, four of them have neurotoxin sequences that are identical, and the fifth strain has a neurotoxin sequence that is 99.8% identical to the others at the amino acid level. The subtype features both a high degree of similarity to BoNT/A1 and a hemagglutinin (HA)-type gene cluster which is present in only BoNT/A1 clusters and none of the other BoNT/A subtypes. The Eric A. Johnson (E.A.J.) laboratory identified an additional A5 strain of C. botulinum, A661222, which possessed a gene for BoNT/A5 identical to that of strain IBCA94-0216 (8).To investigate the BoNT/A5 of this strain, the neurotoxin gene and its associated genes were completely sequenced and analyzed at both the nucleotide and amino acid level. Thirty-six amino acid differences were observed between BoNT/A1 and BoNT/A5, with most of them in the h...