Expression of voltage-dependent sodium and transient potassium currents in an identified sub-population of dorsal root ganglion cells acutely isolated from 12-day-old mouse embryos

The electrophysiological properties of a subset of dorsal root ganglion (DRG) neurons microdissected from 12-day-old (E12) mouse embryos and acutely isolated were analyzed as soon as 3 after their isolation. Two classes of neurons were defined according to their mean diameter. The larger diameter class was examined in this study. They display uniform cytoskeletal properties with co-expression of vimentin and neurofilament triplet proteins. Patch-clamp methods also revealed a homogeneous and limited repertoire of ionic channels that included (1) a TTX-sensitive Na⁺ current whose properties are similar to that reported in mature mammalian neurons, and (2) two types of K⁺ currents that can be compared with the delayed rectifier (I _k ) and the transient (I _a) potassium currents found in other mammalian preparations. It may be possible to use this in vitro model to examine the development of new types of currents, such as Ca²⁺ currents during neuronal growth and differentiation.     

Noninvasive fluorescent study in situ and in real time of glucose effects on the pharmacokinetic of calcein

This study was undertaken to compare the effect of glucose injection on the pharmacokinetic behavior of a soluble dye in normal and tumoral tissues. The measurements were done using a noninvasive fluorescent spectroscopy in situ and in real time. The experiments were performed on three groups of animals with calcein as a soluble pH-insensitive fluorescent dye combined or not with glucose. Glucose solution was injected 5 or 30 min before calcein. Fluorescence emission intensity was recorded on normal and tumor tissues with an optical multichannel analyzer. Calcein concentration was also measured in blood using repetitive blood sampling. In the control group (without glucose injection), calcein is rapidly cleared from the blood, with a slow tissue clearance. Fluorescence of normal tissue was higher than fluorescence measured in tumor tissue. When glucose is injected 5 min before calcein, there was a rapid increase of tissue fluorescence followed by a plateau remaining during the whole experiment. No difference between tumor and normal tissue fluorescence intensity was observed. When glucose was injected 30 min before calcein, the plateau phase was reduced to 50 min in normal tissue. Tumor tissue fluorescence displays no distinct plateau phase. These results clearly showed the effect of glucose injection in situ and in real time, by a noninvasive method, on the pharmacokinetic of a soluble dye in a tumor tissue compared to a normal tissue. Differences between blood compartment and tissues kinetic profiles were also clearly demonstrated.     

Cloning,sequencing and analysis of the yeastS. uvarum ERG10 gene encoding acetoacetyl CoA thiolase

Summary TheERG10 gene specific toS. uvarum, a brewing yeast, has been cloned by complementation of anS. cerevisiae erg10 mutant.S. uvarum contains two differentERG10 genes. One of these is similar to theS. cerevisiae ERG10 gene; they are structurally different, but functionally homologous. The clonedERG10 gene has been located on chromosome XVI, and we have shown that it is allelic to the previously isolatedtsm0115 mutants. Northern blot and sequence analysis indicate that theERG10 gene is highly expressed, and biochemical and genetic evidence show that it encodes the cytoplasmic acetoacetyl CoA thiolase.     

Use of type 1 /type 2 chimeric polioviruses to study determinants of poliovirus type 1 neurovirulence in a mouse model

We previously described the characteristics of a type 1/type 2 (PV-1/PV-2) chimeric poliovirus, v510, which contains the six amino acids specific for PV-2 in the B-C loop of VP1. This virus was found to be mouse-adapted, as PV-2 and in contrast with PV-1. Determinants of host range were studied in detail and are reported here. PV-1/PV-2 chimeras containing partial PV-1----PV-2 substitutions in the B-C loop of VP1 were obtained by making use of a mutagenesis cartridge on PV-1 cDNA. Analysis of mouse neurovirulence of these chimeras, when correlated with the three-dimensional structure of the v510 capsid, revealed that PV-2 residues important for mouse tropism are those which determine the particular conformation of the B-C loop of VP1 in v510. The mutation of the adenine residue at position 480 of the 5' noncoding region into a guanine residue has been shown to be an important determinant of PV-1 attenuation in monkeys. We show that introduction of this mutation in the v510 genome results in a virus which is partially attenuated for mice. This suggests that analysis of genomic determinants important for PV-1 neurovirulence could be carried out in a mouse model by making use of a mouse-adapted PV-1/PV-2 chimera.     

Effects of Various Environmental Stress Paradigms and Adrenalectomy on the Expression of Autoimmune Type 1 Diabetes in the Non-obese Diabetic (NOD) Mouse

The effects of long-term chronic stress (induced by repeated restraint, overcrowding or both), short-term chronic stress (induced by a triad of stressors over a short period of time early in life) and adrenalectomy were investigated on the prevalence, on the degree of insulitis and various physiological and immunological parameters in the NOD mouse, a spontaneous model of type I-insulin-dependent diabetes mellitus (IDDM). Long-term chronic stress, obtained by restraint once a week or overcrowding, significantly protected NOD females, while both applied concomitantly had only a tendency to protect against diabetes. In contrast, short-term chronic stress had no significant effect on diabetes expression, whereas adrenalectomy resulted in a trend toward accelerated diabetes onset. The various long-term chronic stress paradigms exerted different effects on the progression of insulitis: repeated restraint tended to protect against insulitis, overcrowding had no effect but, when associated with restraint, significantly counteracted the beneficial effect of restraint alone. Adrenalectomy and short-term chronic stress had no significant effect on the development of insulitis. Various parameters, such as body, thymus and spleen weights, thymus and spleen cellularities, mitogen-induced spleen cell proliferation and serum corticosterone levels were also studied under the various experimental conditions. Taken together, the observations suggest that stressors modulate the expression of spontaneous autoimmune diabetes by exerting pleiotropic effects on immune and/or inflammatory components at the pancreas level and on peripheral glucose metabolism.     

Skinfold thickness versus isotope dilution for body fat assessment during simulated microgravity: results from three bed-rest campaigns in men and women with and without countermeasures

Because body composition is altered during head-down bed rest (HDBR), body mass can not be used as an index of energy balance. Consequently diet allowances should not be based on body mass evolution but on fat mass changes. Though criticized, skinfold thickness (ST) is the costless, easiest and fastest method to use for such an objective. The aim of this study was to compare the percentage of body fat (%BF) estimated by ST with the isotope dilution of H₂¹⁸O. We compiled data from three HDBR campaigns, one on women (n=8) in November 1998 and two on the same men (n=8) in December 1997 (without countermeasure) and January 1998 (with thigh-cuffs countermeasure), according to a crossover design. Body composition was assessed before and after 6 days of HDBR. %BF was derived from the biceps, triceps, sub-scapular and sup-iliac ST according to Durnin and Wormersly (1974). Fat-free mass was measured on the same day by H₂¹⁸O dilution and fat mass was calculated by the difference with body mass and expressed as a percentage. Based on precision tests, the minimum measurable change by ST was 1.1%BF for single measurement point. Both intercepts (F _4,30=0.89, P=0.45) and slopes (F _4,30=0.74; P=0.57) of the ST versus dilution relationships were not affected by the periods (December vs January), experimental conditions (control vs HDBR vs HDBR + thigh cuffs) or sex allowing the derivation of a common relationship %BF_st=0.94 × %BF_dil (F _1,47=97.9, P<0.0001; non-significant intercept excluded) with a bias between methods of −1.7±2.0 %BF (95% CI: −5.8, 2.4 %BF). ST can be used to measure %BF during HDBR provided great care is placed on training and changes are higher than 1.1 %BF. If the method can be applied for in-flight energy balance monitoring given the high observed energy deficit, a tight monitoring of the individual nutritional status as needed during simulation appears, however, dubious based on this solely method.     

TNF-alpha impairs peripheral tolerance towards beta-cells,and local costimulation by B7.1 enhances the effector function of diabetogenic T cells

Maintenance of peripheral tolerance and inactivation of autoreactive T cells is based on a delicate balance between pro-inflammatory and protective cytokines that is poorly understood. We have here addressed how the local expression of the inflammatory cytokine TNF-alpha can impair peripheral tolerance and lead to autoreactivity. After transplantation of pancreata that are immunogenic due to beta-cell expression of B7.1 and TNF-alpha, into thymectomized and euthymic syngeneic mice, we found that only euthymic mice rejected the grafts. This result suggests that under normal circumstances autoreactive T cells are functionally inactivated, and initiation of an autoreactive response requires de-novo generation of T cells. By contrast, thymectomized mice expressing TNF-alpha on the endogenous islets rejected the grafts, showing that expression of TNF-alpha prevents functional silencing of the autoreactive T cells. Thus, this study provides a mechanism by which TNF-alpha and possibly chronic inflammatory responses may promote autoimmune diseases. Furthermore, we have investigated whether B7.1 can enhance T cell responses of already activated T cells leading to islet rejection. By transplantation of wild-type and B7.1-expressing islets into overtly diabetic mice we found that only the wild-type islets could restore normoglycemia, suggesting that costimulation by B7.1 is required in the expansion or effector phase of the response.     

On the various manifestations of spontaneous autoimmune diabetes in rodent models

Autoimmune (type 1) diabetes mellitus in mouse, rat, and humans shares several features, including T lymphocyte infiltration into pancreatic islets and a dependence on permissive class II major histocompatibility complex (MHC) alleles. We report here on an experimental model involving mice that express influenza hemagglutinin (HA) under the control of the insulin promoter and, at the same time, a transgenic class II MHC-restricted T cell receptor (TcR) specific for an HA peptide. These mice spontaneously develop islet infiltrates resembling those found in NOD mice and most animals become diabetic within 8 weeks of age. Because of the availability of a clonotypic TcR antibody, we can be confident that the Ins-HA transgene does not induce any measurable alterations in the vast majority of T cells with the transgenic TcR in primary and secondary lymphoid organs. Continuous export of large numbers of HA-specific lymphocytes from the thymus was not required for the manifestation of the disease since mice thymectomized at 3 days after birth still developed the disease albeit with smaller infiltrates.