The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.     

The Eating Inventory and Body Adiposity from Leanness to Massive Obesity: a Study of 2509 Adults

Objective: To examine how chronic dietary restraint, disinhibition, and hunger, as assessed by the Eating Inventory, vary over a broad range of BMI values from leanness to massive obesity, in subjects with family obesity. Eating Inventory factors were also studied as a function of personal weight history. Research Methods and Procedures: Subjects were 2509 participants in a genetic study of obesity. BMIs ranged from 15 to 87 kg/m². Six BMI groups were formed (<27, 27 to 30, 30 to 35, 35 to 40, 40 to 45, and >45). Results: Multivariate analyses showed that restraint and disinhibition were significantly associated with BMI in men, whereas only disinhibition was in women. Disinhibition scores correlated strongly with hunger scores in both genders in all BMI categories; dietary restraint tended to correlate with the other two factors positively in leaner subjects and negatively in the highest BMI categories. Highly restrained normal‐weight subjects were likely to exhibit disinhibition and hunger, whereas massively obese persons with very high disinhibition scores showed high hunger but little restraint. The highest restraint scores were observed in nonobese adult women with previous obesity in childhood and/or adolescence. Discussion: The factor most strongly associated with BMI in this large population was disinhibition, suggesting that obesity treatment should target behaviors associated with disinhibition, especially in individuals showing a low level of dietary restraint. High restraint scores in formerly obese normal‐weight women suggest that dietary restraint may exert a beneficial influence on body weight control under conditions that deserve further investigation.     

Control of Lipid Accumulation in the Yeast Yarrowia lipolytica

A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This Δgut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to β-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal β-oxidation. Additional deletion of the POX1 to POX6 genes in the Δgut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.     

The Adipose Tissue Phenotype of Hormone‐Sensitive Lipase Deficiency in Mice

Objective: To directly ascertain the physiological roles in adipocytes of hormone‐sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes. Research Methods and Procedures: We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes. Results: Homozygous HSL^?/? mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild‐type and deficient littermates. HSL‐deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL^?/? mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL^?/? adipocytes, lipolysis is not significantly increased by β₃‐adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL^?/? adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48‐hour period at 4 °C was similar in HSL^?/? mice and controls. Overnight fasting was well‐tolerated clinically by HSL^?/? mice, but after fasting, liver triglyceride content was significantly lower in HSL^?/? mice compared with wild‐type controls. Conclusions: In isolated fat cells, the lipolytic rate after β‐adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL^?/? adipocytes suggests that HSL‐independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.     

A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis

Background  

Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies.     

Parkin and CASK/LIN-2 associate via a PDZ-mediated interaction and are co-localized in lipid rafts and postsynaptic densities in brain.

Mutations in the gene encoding parkin cause an autosomal recessive juvenile-onset form of Parkinson's disease. Parkin functions as a RING-type E3 ubiquitin-ligase, coordinating the transfer of ubiquitin to substrate proteins and thereby targeting them for degradation by the proteasome. We now report that the extreme C terminus of parkin, which is selectively truncated by a Parkinson's disease-causing mutation, functions as a class II PDZ-binding motif that binds CASK, the mammalian homolog of Caenorhabditis elegans Lin-2, but not other PDZ proteins in brain extracts. Importantly, parkin co-localizes with CASK at synapses in cultured cortical neurons as well as in postsynaptic densities and lipid rafts in brain. Further, parkin associates not only with CASK but also with other postsynaptic proteins in the N-methyl d-aspartate (NMDA) receptor-signaling complex, in rat brain in vivo. Finally, despite exhibiting E2-dependent ubiquitin ligase activity, rat brain parkin does not ubiquitinate CASK, suggesting that CASK may function in targeting or scaffolding parkin within the postsynaptic complex rather than as a direct substrate for parkin-mediated ubiquitination. These data implicate for the first time a PDZ-mediated interaction between parkin and CASK in neurodegeneration and possibly in ubiquitination of proteins involved in synaptic transmission and plasticity.     

Ontogeny of the epicranial portion of the dorsal fin in Solea solea and Scophthalmus maximus (Teleostei, Pleuronectiformes)

A study of laboratory-reared larvae of Solea solea (Soleidae) and Scophthalmus maximus (Scophthalmidae) indicated that the epicranial portion of the dorsal fin results from the anterior displacement of proximal pterygiophores during ontogeny. The adult epicranial formula is attained early during ontogeny, and the anterior displacement is finalized after the passage of the migrating eye. In both species, the first two proximal pterygiophores fuse to form an erisma that is particularly long and well-developed in Solea. Moreover, in Solea, the neural spine of the second abdominal vertebra curves over the otic region, and the neural arch of the first vertebra remains incomplete. Received: August 3, 2000 / Revised: August 10, 2001 / Accepted: October 12, 2001     

Mixed arterial concentration of O2 in a global pulmonary gas exchange model

In a global pulmonary gas exchange model where global diffusion and ventilation are given, the effects of variations of local conductance?perfusion and ventilation?perfusion ratios on O₂ concentration in arterial blood are studied. We give a special regard to the ability to get local maxima for this O₂ concentration when the repartition of our parameters is heterogeneous.