Identification of thyroid hormone response elements in?vivo using mice expressing a tagged thyroid hormone receptor α1

TRα1 (thyroid hormone receptor α1) is well recognized for its importance in brain development. However, due to the difficulties in predicting TREs (thyroid hormone response elements) in silico and the lack of suitable antibodies against TRα1 for ChIP (chromatin immunoprecipitation), only a few direct TRα1 target genes have been identified in the brain. Here we demonstrate that mice expressing a TRα1–GFP (green fluorescent protein) fusion protein from the endogenous TRα locus provide a valuable animal model to identify TRα1 target genes. To this end, we analysed DNA–TRα1 interactions in vivo using ChIP with an anti-GFP antibody. We validated our system using established TREs from neurogranin and hairless, and by verifying additional TREs from known TRα1 target genes in brain and heart. Moreover, our model system enabled the identification of novel TRα1 target genes such as RNF166 (ring finger protein 166). Our results demonstrate that transgenic mice expressing a tagged nuclear receptor constitute a feasible approach to study receptor–DNA interactions in vivo, circumventing the need for specific antibodies. Models like the TRα1–GFP mice may thus pave the way for genome-wide mapping of nuclear receptor-binding sites, and advance the identification of novel target genes in vivo.     

Estimation of beta-structure content of proteins by means of deconvolved FTIR spectra

Fourier self-deconvolution was applied to the infrared spectra of five globular proteins with a high beta-structure content and to the essentially alpha-helical protein hemoglobin. The featureless amide I' bands around 1650 cm-1 were thereby resolved into six to nine components, depending on the protein. Specific components were assigned to the beta-structure segments in each protein. The frequencies and the number of 'beta-bands' differ from one protein to another. The areas of the components were evaluated by means of a Gauss-Newton iteration procedure. It appears that the total area of the beta-bands, as a fraction of the total amide I' band area, reflects the relative beta-structure content of each protein studied.     

Apoptotic-like Leishmania exploit the host′s autophagy machinery to reduce T-cell-mediated parasite elimination

Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed—in contrast to viable parasites—that apoptotic-like parasites enter an LC3⁺, autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4⁺ T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferation-reducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´ autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.     

Application of computerized infrared and Raman spectroscopy to conformation studies of casein and other food proteins

Summary The ability of modern biotechnology to produce new or modified proteins has outpaced current understanding of the relationship between protein structure and protein function. Resolution-enhanced infrared spectroscopy and Raman spectroscopy are excellent non-destructive techniques for investigating the secondary structure of proteins under a wide variety of conditions. The techniques yield rapid, reliable estimates of the proportion of helical structure, -strands, and turns of proteins in solution, as gels, or as solids. These methodologies can also detect subtle variations in protein conformation that frequently occur upon change of the biomolecular environment. In particular, it is possible to study structural changes which arise from alterations in pH, ionic strength, nature of solvent, and from interactions with other molecules or ions, such as another protein or Ca²⁺ ions. The first part of this paper will briefly review various important aspects of the techniques. The subsequent part describes application to structural problems of casein and other food proteins.     

Sleep-waking cycle and behaviour after diethyldithiocarbamate in the rat

Young adult Louis rats were implanted for chronic sleep recording to test the effect of diethyldithiocarbamate (DDC) on sleep. Recordings of EEG and EMG were done continuously for 12 h during the 12 consecutive days. There were 2 days of baseline recording, 3 days of recording with a single daily injection of placebo, 3 days of recording with a single daily injection of DDC (500 mg/kg i.p.), and 3 days of DDC withdrawal recording with placebo injection. Placebo injections did not change the proportion of time spent in different behavioural states. With daily injection of DDC there was an increase in wakefulness, no change in slow-wave sleep and elimination or drastic reduction in paradoxical sleep (PS). There was no PS rebound during the DDC withdrawal days. These results suggest that the reduction of PS produced by DDC and the absence of PS rebound may be due to a lowering in norepinephrine in the brain. In other experiments rats were injected with DDC (500 mg/kg i.p.) daily for 3 days and whole brains were analysed chemically. Norepinephrine was significantly decreased, while 5-hydroxytryptamine, 5-hydroxyindolacetic acid, dopamine and homovanilic acid were unchanged. Seizure activity appeared during relaxed wakefulness in all rats treated with DDC. Taken together it seems that lowering of brain NE is responsible for the appearance of seizure activity and also, for PS reduction. PS reduction might, per se, produce seizure activity.     

Effects of restricted sleep with different exercise loads upon subsequent sleep

Seven male cats were adapted to different schedules of restricted sleep. The cat was permitted to go to sleep either 2, 4 or 8 hours per day with the balance to 24-h periode spent in wakefulness enforced by means of a treadmill. Two experiments were run and the same cats served in both runs. The experiments and schedules were separated by at least two weeks during which time cats were maintained under ordinary laboratory conditions. Our experiment used treadmill speed of 2.6 m/min which was easily tolerated and effective in eliminating sleep. Another experiment used treadmill speed of 4.6 m/min which produced more physical exercise. As available sleep time become progressively shorter, REM sleep increased while SWS decreased. If restriction in sleep time was associated with more physical exercise then the composition of the subsequent sleep was different : SWS increased while REM sleep decreased. The functional significance of these opposite effects are presumably different. The immediate SWS response to the prior muscular exercise is suggestive of its recovery function.     

mtDNA variability in two Bantu‐speaking populations (Shona and Hutu) from Eastern Africa: Implications for peopling and migration patterns in sub‐Saharan Africa

In this study, we report novel data on mitochondrial DNA in two of the largest eastern Bantu‐speaking populations, the Shona from Zimbabwe and the Hutu from Rwanda. The goal is to evaluate the genetic relationships of these two ethnic groups with other Bantu‐speaking populations. Moreover, by comparing our data with those from other Niger‐Congo speaking populations, we aim to clarify some aspects of evolutionary and demographic processes accompanying the spread of Bantu languages in sub‐Saharan Africa and to test if patterns of genetic variation fit with models of population expansion based on linguistic and archeological data. The results indicate that the Shona and Hutu are closely related to the other Bantu‐speaking populations. However, there are some differences in haplogroup composition between the two populations, mainly due to different genetic contributions from neighboring populations. This result is confirmed by estimates of migration rates which show high levels of gene flow not only between pairs of Bantu‐speaking populations, but also between Bantu and non‐Bantu speakers. The observed pattern of genetic variability (high genetic homogeneity and high levels of gene flow) supports a linguistic model suggesting a gradual spread of Bantu‐speakers, with strong interactions between the different lines of Bantu‐speaker descent, and is also in agreement with recent archeological findings. In conclusion, our data emphasize the role that population admixture has played at different times and to varying degrees in the dispersal of Bantu languages. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.