Multiple mechanisms compensate to enhance tumor-protective CD8(+) T cell response in the long-term despite poor CD8(+) T cell priming initially: comparison between an acute versus a chronic intracellular bacterium expressing a model antigen

We evaluated CD8(+) T cell responses against the dominant CTL epitope, OVA(257-264), expressed by an acute (Listeria monocytogenes (LM) OVA) vs a chronic pathogen (Mycobacterium bovis bacillus Calmette-Guérin (BCG) OVA) to reveal the influence on CD8(+) T cell memory and consequent protection against a challenge with OVA-expressing tumor cells. Infection with lower doses of both pathogens resulted in stronger bacterial growth but weaker T cell memory indicating that memory correlates with pathogen dose but not with bacterial expansion. The CD8(+) T cell response induced by LM-OVA was helper T cell-independent and was characterized by a rapid effector response followed by a rapid, but massive, attrition. In contrast, BCG-OVA induced a delayed and weak response that was compensated for by a longer effector phase and reduced attrition. This response was partly dependent on CD4(+) T cells. CD8(+) T cell response induced by BCG-OVA, but not LM-OVA, was highly dependent on pathogen persistence to compensate for the weak initial CD8(+) T cell priming. Despite a stronger initial T cell response with LM-OVA, BCG-OVA provided more effective tumor (B16OVA) control at both local and distal sites due to the induction of a persistently activated acquired, and a more potent innate, immunity.     

Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release

Defective Cu,Zn-superoxide dismutase (SOD1) is responsible for some types of amyotrophic lateral sclerosis, and ventral horn motor neurons (VMN) have been shown to die through a mitochondria-dependent apoptotic pathway after chronic exposure to high levels of reactive oxygen species (ROS). VMN are also selectively vulnerable to mild spinal cord injury (SCI); however, the involvement of SOD1, ROS, and apoptosis in their death has not been clarified. Mild compression SCI was induced in SOD1-overexpressing transgenic rats and wild-type littermates. Superoxide production, mitochondrial release of cytochrome c, and activation of caspase-9 were examined, and apoptotic DNA injury was also characterized. In the wild-type animals, increased superoxide production, mitochondrial release of cytochrome c, and cleaved caspase-9 were observed exclusively in VMN after SCI. Subsequently, a majority of VMN (75%) selectively underwent delayed apoptotic cell death. Transgenic animals showed less superoxide production, mitochondrial cytochrome c release, and caspase-9 activation, resulting in death of only 45% of the VMN. These results suggest that the ROS-initiated mitochondrial signaling pathway possibly plays a pivotal role in apoptotic VMN death after SCI and that increased levels of SOD1 in VMN reduce oxidative stress, thereby attenuating the activation of the pathway and delayed cell death.     

Enhancement of the inducible NO synthase activation by retinoic acid is mimicked by RARalpha agonist in vivo

We have previously shown that all-trans retinoic acid (atRA), the active metabolite of vitamin A, enhances the activation of the inducible nitric oxide synthase (NOS II) pathway, a component of innate immunity, in rats in vivo. We investigated the relative contribution of retinoic acid receptor-alpha (RARalpha) and retinoid X receptors (RXRs) to NOS II activation triggered by LPS. Five-day supplementation with 10 mg/kg of either atRA or the RARalpha selective agonist Ro-40-6055, but not with 10 mg/kg of the pan-RXR agonist Ro-25-7386, enhanced the LPS-induced NOS II mRNA, protein expression in liver, and plasma nitrite/nitrate concentration. Both atRA and the RARalpha agonist (but not the RXR agonist) increased the number of peripheral T helper lymphocytes and plasma interferon-gamma concentration. Synergism between retinoids and LPS on NOS II activation within an organ coincided with synergism on interferon regulatory factor-1 mRNA expression but not with the level of expression of the RARalpha protein. These results suggest that, in vivo, atRA activates NOS II through RARalpha and contributes to characterizing the complex effect of retinoids on the host inflammatory/immune response.     

Cortisol secretion in the elderly. Influence of age,sex and cardiovascular disease in a Chinese population

Adrenal function and aging have been the object of intense interest in recent years. In this study we analyzed morning (08:00 h) serum cortisol concentrations from a sample of Chinese subjects aged from 31 to 110 years. These levels differed according to age, health status and sex, although the sex difference was confirmed only among the healthy elderly. These results suggest that age (older than 60 years), disease and male sex are associated with increased morning serum cortisol levels in a Chinese population.     

Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins

Hydrophobic proteins are difficult to analyze by two-dimensional electrophoresis (2-DE) because of their intrinsic tendency to self-aggregate during the first dimension (isoelectric focusing, IEF) or the equilibration steps. This aggregation renders their redissolution for the second dimension uncertain and results in the reduction of the number and intensity of protein spots, and in undesirable vertical and horizontal streaks across gels. Trifluoroethanol (TFE) is traditionally used at high concentration to solubilize peptides and proteins for NMR studies. Depending upon its concentration, TFE strongly affects the three-dimensional structure of proteins. We report here a phase separation system based on TFE/CHCl(3), which is able to extract a number of intrinsic membrane proteins. The addition of TFE in the in-gel sample rehydration buffer to improve membrane protein IEF separation is also presented. The procedure using urea, thiourea, and sulfobetaine as chaotropic agents was modified by the addition of TFE and removing of sulfobetaine at an optimized concentration in the solubilization medium used for the first dimension. When using membrane fractions isolated from Escherichia coli, the intensity and the number of spots detected from 2-DE gels that used TFE in the solubilization medium were significantly increased. The majority of the proteins identified using peptide mass fingerprinting and tandem mass spectrometry (MS/MS) were intrinsic membrane proteins, proteins of beta barrel structure or transmembrane proteins.     

A new mouse limb mutation identifies a <Emphasis Type="Italic">Twist</Emphasis> allele that requires interacting loci on Chromosome 4 for its phenotypic expression

Pluridigite (Pdt) is a semi-dominant mutation obtained after a mutagenesis experiment with ethyl-nitroso-urea (ENU). The mutant exhibits abnormal skeletal pattern formation characterized by the formation of extra digits (polydactyly) in the preaxial (anterior) part of the hindlimbs. The phenotype shows incomplete penetrance, depending on the genetic background. In an F₂ cross with C57BL/6, the phenotype could not be associated with a single locus. Strong linkage was observed with markers located on Chromosome (Chr) 12, in a 2-cM interval between D12Mit136 and D12Mit153. This region contains the Twist gene, and we show that the [Pdt] phenotype is dependent upon a new allele of Twist. We further identified that the whole Chr 4 is associated with the [Pdt] phenotype. The Pluridigite phenotype thus results from the combination of a Twist mutant allele and at least two additional loci.     

<Emphasis Type="Italic">Hox</Emphasis> gene survey in the chaetognath<Emphasis Type="Italic"> Spadella cephaloptera</Emphasis>: evolutionary implications

We present the isolation of six Hox genes in the chaetognath Spadella cephaloptera. We identified one member of the paralogy group 3, four median genes and a mosaic gene that shares features of both median and posterior classes ( SceMedPost). Several hypotheses may account for the presence of a mosaic Hox gene in this animal. Here we propose that SceMedPost may represent an ancestral gene, which has not diverged totally into a posterior or a median one. This hypothesis has interesting implications for the reconstruction of the evolutionary history of Hox genes and suggests that Chaetognatha lineage divergence could predate the deuterostome/protostome split. Such a phylogenetic position is considered in the light of their embryological and morphological characters.     

Vasa expression and germ-cell specification in the spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

The specification of germ cells is an important process during the development of all animals. Expression of an evolutionarily conserved gene such as vasa can be used as a marker for germ cell fate. We have isolated a vasa-related gene from the two-spotted spider mite (Tetranychus urticae) and used it to examine the segregation of germ cells in this animal. In spider mites, vasa expression first appears in a group of cells that do not join the initial blastoderm surface. Instead, these cells remain in the interior of the blastoderm and then migrate to posterior regions of the embryo, where they form a cluster that appears in regions of the embryo consistent with the gonads. The expression pattern of this spider mite vasa homologue implies a novel process acts to specify germ cells in this species and that the specification of germ cells is an evolutionarily labile process.     

Artificially increased ascorbate content affects zeaxanthin formation but not thermal energy dissipation or degradation of antioxidants during cold-induced photooxidative stress in maize leaves

Infiltrating detached maize (Zeamays L.) leaves with L-galactono-1,4-lactone (L-GAL) resulted in a 4-fold increase in the content of leaf ascorbate. Upon exposure to high irradiance (1000 μmol photons m⁻² s⁻¹) at 5 °C, L-GAL leaves de-epoxidized the xanthophyll-cycle pigments faster than the control leaves; the maximal ratio of de-epoxidized xanthophyll-cycle pigments to the whole xanthophyll-cycle pool was the same in both leaf types. The elevated ascorbate content, together with the faster violaxanthin de-epoxidation, did not affect the degree of photoinhibition and the kinetics of the recovery from photoinhibition, assayed by monitoring the maximum quantum efficiency of photosystem II primary photochemistry (F_v/F_m). Under the experimental conditions, the thermal energy dissipation seems to be zeaxanthin-independent since, in contrast to the de-epoxidation, the decrease in the efficiency of excitation-energy capture by open photosystem II reaction centers (F_v′/F_m′) during the high-irradiance treatment at low temperature showed the same kinetic in both leaf types. This was also observed for the recovery of the maximal fluorescence after stress. Furthermore, the elevated ascorbate content did not diminish the degradation of pigments or α-tocopherol when leaves were exposed for up to 24 h to high irradiance at low temperature. Moreover, a higher content of ascorbate appeared to increase the requirement for reduced glutathione. Received: 20 May 1999 / Accepted: 29 October 1999