Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.     

Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 μM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of β-oxidation might be correlated with the lower level of acetylcholine synthesis.     

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery

EF-hand Ca²⁺-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca²⁺. Upon binding Ca²⁺, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca²⁺-binding affinity of CaM and most S100s in the absence of target is weak (^CaK_D > 1 μM). However, upon effector protein binding, the Ca²⁺ affinity of these proteins increases via heterotropic allostery (^CaK_D < 1 μM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca²⁺ homeostasis and (ii) strict maintenance of Ca²⁺-signaling within a narrow dynamic range of free Ca²⁺ ion concentrations, [Ca²⁺]^free. In this review, mechanisms of allostery are coalesced into an empirical “binding and functional folding (BFF)” physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca²⁺ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.     

Developmental status of the French consumer home product information database

Summary The purpose of this study is, first, to perform a national data bank cellecting as many data as possible for various household products with attention to the frequency of their use and, secondly, to determine their volatile organic compound (VOC) emissions.     

Anti-salmonella properties of kefir yeast isolates: An in vitro screening for potential infection control

The rise of antibiotic resistance has increased the need for alternative ways of preventing and treating enteropathogenic bacterial infection. Various probiotic bacteria have been used in animal and human. However, Saccharomyces boulardii is the only yeast currently used in humans as probiotic. There is scarce research conducted on yeast species commonly found in kefir despite its claimed potential preventative and curative effects. This work focused on adhesion properties, and antibacterial metabolites produced by Kluyveromyces lactis and Saccharomyces unisporus isolated from traditional kefir grains compared to Saccharomyces boulardii strains. Adhesion and sedimentation assay, slide agglutination, microscopy and turbidimetry assay were used to analyze adhesion of Salmonella Arizonae and Salmonella Typhimurium onto yeast cells. Salmonella growth inhibition due to the antimicrobial metabolites produced by yeasts in killer toxin medium was analyzed by slab on the lawn, turbidimetry, tube dilution and solid agar plating assays. Alcohol and antimicrobial proteins production by yeasts in killer toxin medium were analyzed using gas chromatography and shotgun proteomics, respectively. Salmonella adhered onto viable and non-viable yeast isolates cell wall. Adhesion was visualized using scanning electron microscope. Yeasts-fermented killer toxin medium showed Salmonella growth inhibition. The highest alcohol concentration detected was 1.55%, and proteins with known antimicrobial properties including cathelicidin, xanthine dehydrogenase, mucin-1, lactadherin, lactoperoxidase, serum amyloid A protein and lactotransferrin were detected in yeasts fermented killer medium. These proteins are suggested to be responsible for the observed growth inhibition effect of yeasts-fermented killer toxin medium. Kluyveromyces lactis and Saccharomyces unisporus have anti-salmonella effect comparable to Saccharomyces boulardii strains, and therefore have potential to control Salmonella infection.     

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling^1,2, while LRRK2 is implicated in the pathogenesis of Parkinson''s disease^3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with ³²P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing ³²P-orthophosphate. The ³²P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (³²P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.     

Drought,high temperature,and their combination affect ultrastructure of chloroplasts and mitochondria in wheat (Triticum aestivum L.) leaves

Plants experience a number of limiting factors, as drought and heat, which are often coinciding stress factors in natural environment. This study evaluated the changes in mesophyll cell ultrastructure in the leaves of two varieties of winter wheat (Triticum aestivum L.), differing in their drought tolerance, under individual or combined drought and heat treatment. Although the individual stress factors affected leaf ultrastructure, the damaging effect of the combined drought and heat was more pronounced and manifested certain differences between genotypes. Chloroplasts and mitochondria were affected in a variety-specific manner under all adverse treatments. The organelles of the drought-tolerant Katya were better preserved than those in the sensitive variety Sadovo. Leaf ultrastructure can be considered as one of the important characteristics in the evaluation of the drought susceptibility of different wheat varieties.     

Dietary practices,site-specific tumors,and age-at-death relationships

When rats are given freedom of dietary choice, the quantity and composition of the diet selected varies from animal to animal, but the risk to the individual of developing a variety of neoplasms is greatly increased over that of rats fed fixed diets. The increase in frequency of tumors cannot be explained on the basis of the amount or the composition of the diet selected. However, the application of “variable selection” statistical procedures shows that there are combinations of temporal-specific interacting dietary and dietary-dependent variables that are closely linked with cancer mortality. The multi-variable statistical model for animals dying with one type of tumor is not applicable to those dying with tumors of other types or sites. In every case, the only data required to characterize the dates of death of tumor-bearing individuals are those describing the dietary practices and growth responses prior to maturity. The model for animals with pancreatic tumors is used to illustrate the extent to which dietary practices of early life are linked with the animals’ survival.