Spatial control of protein phosphatase 2A (de)methylation

Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A(C)(1)) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A(C) antibodies revealed a good correlation with the methylation status of PP2A(C), demethylated PP2A(C) being substantially nuclear. Throughout mitosis, demethylated PP2A(C) is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A(C) in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1.     

The influence of indoor lighting with low blue light dose on urine 6-sulphatoxymelatonin concentrations and sleep efficiency of healthy volunteers

Light, especially its blue component, is the main synchronizer of circadian rhythms. We investigated effects of suppressed blue band of the spectrum on melatonin production and sleep efficiency in 18 young volunteers. During control days, participants lived in their home environment, and next five days in a room lit only by daylight with windows equipped with a filter blocking the blue band of the light spectrum. Light intensity, circadian stimulus and light irradiance were monitored. No significant changes in the daily pattern and total urinary 6-sulphatoxymelatonin excretion were found between control and experimental conditions. Parameters of sleep efficiency measured by wrist actigraphy were not worsened, but neutral chronotypes exhibited shortened sleep duration under light-modified conditions. We conclude that young healthy people can compensate for negative effects of transitory-worsened lighting conditions on their daily rhythms, but chronotypes and other personal characteristics may modify biological responses and should be considered.     

Mössbauer studies of the non-heme iron and cytochrome b559 in a Chlamydomonas reinhardtii PSI- mutant and their interactions with alpha-tocopherol quinone

Spin and valence states of the non-heme iron and the heme iron of cytochrome b559, as well as their interactions with alpha-tocopherol quinone (alpha-TQ) in photosystem II (PSII) thylakoid membranes prepared from the Chlamydomonas reinhardtii PSI- mutant have been studied using M?ssbauer spectroscopy. Both of the iron atoms are in low spin ferrous states. The Debye temperature of the non-heme is 194 K and of the heme iron is 182 K. The treatment of alpha-TQ does not change the spin and the valence states of the non-heme iron but enhances the covalence of its bonds. alpha-TQ oxidizes the heme iron into the high spin Fe3+ state. A possible role of the non-heme iron and alpha-TQ in electron flow through the PSII is discussed.     

Functional Characterization of the Short Neuropeptide F Receptor in the Desert Locust,Schistocerca gregaria

Whereas short neuropeptide F (sNPF) has already been reported to stimulate feeding behaviour in a variety of insect species, the opposite effect was observed in the desert locust. In the present study, we cloned a G protein-coupled receptor (GPCR) cDNA from the desert locust, Schistocerca gregaria. Cell-based functional analysis of this receptor indicated that it is activated by both known isoforms of Schgr-sNPF in a concentration dependent manner, with EC₅₀ values in the nanomolar range. This Schgr-sNPF receptor constitutes the first functionally characterized peptide GPCR in locusts. The in vivo effects of the sNPF signalling pathway on the regulation of feeding in locusts were further studied by knocking down the newly identified Schgr-sNPF receptor by means of RNA interference, as well as by means of peptide injection studies. While injection of sNPF caused an inhibitory effect on food uptake in the desert locust, knocking down the corresponding peptide receptor resulted in an increase of total food uptake when compared to control animals. This is the first comprehensive study in which a clearly negative correlation is described between the sNPF signalling pathway and feeding, prompting a reconsideration of the diverse roles of sNPFs in the physiology of insects.     

Localization and distribution patterns of nicotinamide adenine dinucleotide phosphate diaphorase exhibiting axons in the white matter of the spinal cord of the rabbit

The funicular distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-exhibiting axons was examined in the white matter of the rabbit spinal cord by using horizontal, parasaggital, and transverse sections. Four morphologically distinct kinds of NADPHd-exhibiting axons (2.5–3.5 m in diameter) were identified in the sulcomarginal fasciculus as a part of the ventral column in the cervical and upper thoracic segments and in the long propriospinal bundle of the ventral column in Th3–L3 segments. Varicose NADPHd-exhibiting axons of the sympathetic preganglionic neurons, characterized by widely spaced varicosities, were found in the ventral column of Th2–L3 segments. A third kind of NADPHd-positive ultrafine axons, 0.3–0.5 m in diameter with numerous varicosities mostly spherical in shape, was identified in large number within Lissauer's tract. The last group of NADPHd-exhibiting axons (1.0–1.5 m in diameter) occurred in the Lissauer tract. Most of these axons were traceable for considerable distances and generated varicosities varying in shape from spherical to elliptical forms. The majority of NADPHd-exhibiting axons identified in the cuneate and gracile fascicles were concentrated in the deep portion of the dorsal column. An extremely reduced number of NADPHd-exhibiting axons, confirmed by a computer-assisted image-processing system, was found in the dorsal half of the gracile fascicle. Axonal NADPHd positivity could not be detected in a wide area of the lateral column consistent with the location of the dorsal spinocerebellar tract. Numerous, mostly thin NADPHd-positive axonal profiles were detected in the dorsolateral funiculus in all the segments studied and in a juxtagriseal portion of the lateral column as far as the cervical and lumbar enlargements. A massive occurrence of axonal NADPHd positivity was detected in the juxtagriseal layer of the ventral column all along the rostrocaudal axis of the spinal cord. The prominent NADPHd-exhibiting bundles containing thick, smooth, nonvaricose axons were identified in the mediobasal and central portion of the ventral column. First, the sulcomarginal fasciculus was found in the basal and medial portion of the ventral column in all cervical and upper thoracic segments. Second, more caudally, a long propriospinal bundle displaying prominent NADPHd positivity was localized in the central portion of the ventral column throughout the Th3–L3 segments.     

Humus Form Development during Forest Restoration in Exclosures of the Tigray Highlands, Northern Ethiopia

Forest restoration in protected exclosures has become a common practice to fight land degradation in the highlands of northern Ethiopia. Insights into ecosystem processes governing restoration in these formerly degraded areas are gained through the study of humus forms and factors influencing humus formation during vegetation recovery. Humus forms of 135 sample plots located in different land use types were morphologically described. The subsequent classification into six humus form types was based on principal component analysis and cluster analysis. Where areas are closed for a longer time, humus profiles are commonly more developed and higher organic matter accumulation is noticed as well as increased nutrient stocks. The combined effects of seasonal drought conditions and low fresh litter quality account for an overall slow decomposition, which explains the high importance of litter input for organic matter accumulation. Based on a correlation analysis, vegetation cover, litter production, litter quality, soil nutrient content, soil moisture, and topography were identified as important factors influencing humus formation. It is inferred that humus formation leads to improvements in soil fertility and structure, microclimate development, and soil protection and therefore forms part of the restoration processes taking place in exclosures.     

Effects of heme oxygenase-1 on induction and development of chemically induced squamous cell carcinoma in mice

Heme oxygenase-1 (HO-1) is an antioxidative and cytoprotective enzyme, which may protect neoplastic cells against anticancer therapies, thereby promoting the progression of growing tumors. Our aim was to investigate the role of HO-1 in cancer induction. Experiments were performed in HO-1^+/+, HO-1^+/−, and HO-1^−/− mice subjected to chemical induction of squamous cell carcinoma with 7,12-dimethylbenz[a]anthracene and phorbol 12-myristate 13-acetate. Measurements of cytoprotective genes in the livers evidenced systemic oxidative stress in the mice of all the HO-1 genotypes. Carcinogen-induced lesions appeared earlier in HO-1^−/− and HO-1^+/− than in wild-type animals. They also contained much higher concentrations of vascular endothelial growth factor and keratinocyte chemoattractant, but lower levels of tumor necrosis factor-α and interleukin-12. Furthermore, tumors grew much larger in HO-1 knockouts than in the other groups, which was accompanied by an increased rate of animal mortality. However, pathomorphological analysis indicated that HO-1^−/− lesions were mainly large but benign papillomas. In contrast, in mice expressing HO-1, most lesions displayed dysplastic features and developed to invasive carcinoma. Thus, HO-1 may protect healthy tissues against carcinogen-induced injury, but in already growing tumors it seems to favor their progression toward more malignant forms.     

Evolution of linear chromosomes and multipartite genomes in yeast mitochondria

Mitochondrial genome diversity in closely related species provides an excellent platform for investigation of chromosome architecture and its evolution by means of comparative genomics. In this study, we determined the complete mitochondrial DNA sequences of eight Candida species and analyzed their molecular architectures. Our survey revealed a puzzling variability of genome architecture, including circular- and linear-mapping and multipartite linear forms. We propose that the arrangement of large inverted repeats identified in these genomes plays a crucial role in alterations of their molecular architectures. In specific arrangements, the inverted repeats appear to function as resolution elements, allowing genome conversion among different topologies, eventually leading to genome fragmentation into multiple linear DNA molecules. We suggest that molecular transactions generating linear mitochondrial DNA molecules with defined telomeric structures may parallel the evolutionary emergence of linear chromosomes and multipartite genomes in general and may provide clues for the origin of telomeres and pathways implicated in their maintenance.     

Roles of the ubiquitin/proteasome pathway in pollen tube growth with emphasis on MG132-induced alterations in ultrastructure, cytoskeleton, and cell wall components

The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner, while hardly similar effects were detected when cysteine-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanate-phalloidin and beta-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.