Oxidation-reduction potentials in the cecal contents of rats and mice.

The oxidation-reduction potential (ORP) in the cecal contents of conventional rats, germ-free mice, and mice with a Colonization Resistance Factor flora (CRF-mice) was investigated. By using animals that were anaesthetized for a longer period of time, we attempted to eliminate the disturbing influence of oxygen. In addition, measurements were made under anaerobic conditions. For the rats, the ORP values reached a more or less constant level after about 30 min following the insertion of the electrodes. The mean ORP at that time was -458 mV (SD = 45 mV). The mean ORP values for the mice showed a more gradual reduction than was found in the rats. The curves leveled off at about 100 min following the insertion of the electrodes. The mean ORP values 100 min after electrode insertion were: germ-free mice, + 3 mV (SD = 39 mV); CRF-mice, -554 mV (SD = 29 mV). In rats, the ORP value decreased after death; no decrease was observed in mice. No difference was found in the values obtained when measuring under anaerobic or aerobic conditions after death.     

The effects of LPS on the cellular composition of the splenic white pulp in responder C3H/He and non-responder C3H/HeJ mice

The aim of the present study was to compare the effects of LPS on the cellular composition of the splenic white pulp in responder C3H/He and non-responder C3H/HeJ mice. The present results show that an intravenous injection of LPS in C3H/He mice results in a number of prominent changes in the histology of the spleen, but none of these histological changes could be demonstrated in the unresponsive C3H/HeJ mice. However, the present study shows that LPS administration resulted in the disappearance of previously trapped immune complexes from the follicles in both responder C3H/He and non-responder C3H/HeJ mice. The significance of this phenomenon is discussed. The localization of intravenously injected LPS in both mouse strains was compared using an immunoperoxidase technique. Most of the injected LPS was taken up by marginal zone macrophages at 2 h after administration. No major differences could be detected in the localization pattern of LPS between C3H/He and C3H/HeJ mice. The present results support the suggestion that the genetically based unresponsiveness of C3H/HeJ mice could be due to an intracellular defect in their response to LPS.     

Influence of prolonged endurance cycling and recovery diet on intramuscular triglyceride content in trained males

Intramuscular triglycerides (IMTG) are assumed to form an important substrate source during prolonged endurance exercise in trained males. This study investigated the effects of endurance exercise and recovery diet on IMTG content in vastus lateralis muscle. Nine male cyclists were provided with a standardized diet for 3 days, after which they performed a 3-h exercise trial at a 55% maximum workload. Before and immediately after exercise and after 24 and 48 h of recovery, magnetic resonance spectroscopy (MRS) was performed to quantitate IMTG content. Muscle biopsies were taken after 48 h of recovery to determine IMTG content by using quantitative fluorescence microscopy. The entire procedure was performed two times; in one trial, a normal diet containing 39% energy (En%) as fat was provided (NF) and in the other a typical carbohydrate-rich athlete's diet (LF: 24 En% fat) was provided. During exercise, IMTG content decreased by 21.4 +/- 3.1%. During recovery, IMTG content increased significantly in the NF trial only, reaching preexercise levels within 48 h. In accord with MRS, fluorescence microscopy showed significantly higher IMTG content in the NF compared with the LF trial, with differences restricted to the type I muscle fibers (2.1 +/- 0.2 vs. 1.4 +/- 0.2% area lipid staining, respectively). In conclusion, IMTG content in the vastus lateralis muscle declines significantly during prolonged endurance exercise in male cyclists. When a normal diet is used, IMTG contents are subsequently repleted within 48 h of postexercise recovery. In contrast, IMTG repletion is impaired substantially when a typical, carbohydrate-rich athlete's diet is used. Data obtained by quantitative fluorescence microscopy correspond well with MRS results, implying that both are valid methods to quantify IMTG content.     

Pharmacological targeting of mitochondrial complex I deficiency: the cellular level and beyond

Complex I (CI) represents a major entry point of electrons in the mitochondrial electron transport chain (ETC). It consists of 45 different subunits, encoded by the mitochondrial (mtDNA) and nuclear DNA (nDNA). In humans, mutations in nDNA-encoded subunits cause severe neurodegenerative disorders like Leigh Syndrome with onset in early childhood. The pathophysiological mechanism of these disorders is still poorly understood. Here we summarize the current knowledge concerning the consequences of nDNA-encoded CI mutations in patient-derived cells, present mouse models for human CI deficiency, and discuss potential treatment strategies for CI deficiency.     

Subunits of mitochondrial complex I exist as part of matrix- and membrane-associated subcomplexes in living cells

Mitochondrial complex I (CI) is a large assembly of 45 different subunits, and defects in its biogenesis are the most frequent cause of mitochondrial disorders. In vitro evidence suggests a stepwise assembly process involving pre-assembled modules. However, whether these modules also exist in vivo is as yet unresolved. To answer this question, we here applied submitochondrial fluorescence recovery after photobleaching to HEK293 cells expressing 6 GFP-tagged subunits selected on the basis of current CI assembly models. We established that each subunit was partially present in a virtually immobile fraction, possibly representing the holo-enzyme. Four subunits (NDUFV1, NDUFV2, NDUFA2, and NDUFA12) were also present as highly mobile matrix-soluble monomers, whereas, in sharp contrast, the other two subunits (NDUFB6 and NDUFS3) were additionally present in a slowly mobile fraction. In the case of the integral membrane protein NDUFB6, this fraction most likely represented one or more membrane-bound subassemblies, whereas biochemical evidence suggested that for the NDUFS3 protein this fraction most probably corresponded to a matrix-soluble subassembly. Our results provide first time evidence for the existence of CI subassemblies in mitochondria of living cells.     

Noisy-threshold control of cell death

Background  

Cellular responses to death-promoting stimuli typically proceed through a differentiated multistage process, involving a lag phase, extensive death, and potential adaptation. Deregulation of this chain of events is at the root of many diseases. Improper adaptation is particularly important because it allows cell sub-populations to survive even in the continuous presence of death conditions, which results, among others, in the eventual failure of many targeted anticancer therapies.     

Numerical analysis of morphology of natural hybrids between Carex hostiana and the members of Carex flava agg. (Cyperaceae)

Uni‐variate and multi‐variate statistical methods, based on data taken from dried specimens, were used to determine the morphological variance of Carex hostiana 3 Carex flava agg. hybrids and to establish their parents among members of the C. flava complex. The following hybrids were found: C. demissa 3 hostiana [C. 3 fulva], C. hostiana 3 lepidocarpa [C. ×leutzii] and C. flava 3 hostiana [C. 3 xanthocarpa]. The least variable traits, namely beak length, utricle length, ratio of beak length to the overall utricle length, female spike width, and width of the lowest bract, proved to be the most useful in delimiting the hybrids. Carex flava 3 hostiana specimens usually have long utricles and beaks, wide male and female spikes, as well as wide bracts and leaf blades. Carex hostiana 3 lepidocarpa specimens are characterized by relatively short beaks (with low ratio of beak length to the overall utricle length) and narrow bracts. Common features of C. demissa 3 hostiana specimens, on the other hand, are male spikes with long peduncles, usually longly parted female spikes and a long beak compared to the overall utricle length.     

Rapid Screening of Gene Function by Systemic Delivery of Morpholino Oligonucleotides to Live Mouse Embryos

Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis.