Cutting edge: activity of human adult microglia in response to CC chemokine ligand 21

The approximately 50 known chemokines are classified in distinct subfamilies: CXC, CC, CX3C, and C. Although the signaling of chemokines often is promiscuous, signaling events between members of these distinct chemokine classes are hardly observed. The only known exception so far is the murine CC chemokine ligand (CCL)21 (secondary lymphoid tissue chemokine, Exodus-2, 6Ckine), which binds and activates the murine CXC chemokine receptor CXCR3. However, this exception has not been found in humans. In this study, we provide evidence that human CCL21 is a functional ligand for endogenously expressed CXCR3 in human adult microglia. In absence of CCR7 expression, CCL21 induced chemotaxis of human microglia with efficiency similar to the CXCR3 ligands CXC chemokine ligand 9 (monokine induced by IFN-gamma) and CXC chemokine ligand 10 (IFN-gamma-inducible protein-10). Because human CCL21 did not show any effects in CXCR3-transfected HEK293 cells, it is indicated that CXCR3 signaling depends on the cellular background in which the CXCR3 is expressed.     

Agonist‐Independent,High Constitutive Activity of the Human Melanocortin 1 Receptor

The melanocortins (α‐melanocyte‐stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain‐of‐function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss‐of‐function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss‐of‐function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist‐independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist‐independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over‐expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E^so‐3J allele. Stable or transient expression of wild‐type MC1R, but not of loss‐of‐function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs‐coupled receptors. Therefore, human MC1R displays a strong agonist‐independent constitutive activity.     

Bacteroides xylanolyticus sp. nov., a xylanolytic bacterium from methane producing cattle manure

As part of a study of the biogas production from cattle waste, xylanolytic bacteria were isolated from enrichments of fermenting cattle manure. From 34 isolates, mostly Gram-negative rods, a typical strain was investigated in more detail. It was an anaerobic non-sporeforming, Gramnegative rod, which was motile with peritrichous flagella. This organism fermented xylan and many soluble sugars (glucose, cellobiose, mannose, xylose, arabinose). Other hemicelluloses such as gum xanthan, laminaran, locust bean gum, and gum arabic were not utilized. It also could not use cellulose. Fermentation products were carbon dioxide, hydrogen, acetate and ethanol. The bacterium produced carboxymethylcellulase and xylanase, especially when growing on xylan. Growth was optimal between 25°C and 40°C and between pH 6.5 and 7.5. The guanine plus cytosine content of the DNA was 34.8±0.8%. The isolate was identified as a member of the genus Bacteroides, and a new species is proposed: Bacteroides xylanolyticus (xylan dissolving). The type strain of B. xylanolyticus is strain X5-1 (DSM 3808).     

Propagation of tomato spotted wilt virus in Frankliniella occidentalis does neither result in pathological effects nor in transovarial passage of the virus

The effect of tomato spotted wilt virus (TSWV) on Frankliniella occidentalis Pergande (Thysanoptera; Thripidae) following a 6-hour acquisition access period on infected plants was investigated. No statistically significant differences were observed among viruliferous, non-viruliferous and control thrips with respect to developmental time, reproduction rate and survival. Thrips larvae, exposed or non-exposed to TSWV, developed from egg to adult in 13.1 and 13.2 days, respectively. Exposed females produced an average of 28.3 larvae whereas control thrips produced 22.3 larvae and longevity was 13.4 and 12.5 days, respectively. None of these values were significantly different. Population reproductive statistics, net reproductive rate (R ₀), mean generation time (T) and intrinsic rate of increase (r _m) were calculated from the life fertility tables. R ₀ and r _m were higher for viruliferous thrips as compared to non-viruliferous and non-exposed thrips. Virus transmission studies revealed that viruliferous thrips were able to transmit virus until death and that TSWV was not transovarially transmitted.     

An electron microscopical study of the development of peroxisomes during formation and germination of ascospores in the methylotrophic yeast Hansenula polymorpha

Ascospore formation was studied in liquid cultures of the yeast Hansenula polymorpha, previously grown under conditions in which the synthesis of alcohol oxidase was repressed (glucose as growth substrate) or derepressed (methanol, glycerol and dihydroxyacetone as growth substrates and after growth on malt agar plates). In ascospores obtained from repressed cells, generally one small peroxisome was present. The organelle probably originated from the small peroxisome, originally present in the vegetative cells. They had no crystalline inclusions and cytochemical experiments indicated the presence of catalase, urate oxidase and amino acid oxidase activities in these organelles. In ascospores obtained from derepressed cells, generally 1–3 crystalline peroxisomes were observed. These organelles also originated from the peroxisomes originally present in the vegetative cells by means of fragmentation or division. They contained, in addition to the enzymes characteristic for peroxisomes in spores from repressed cells, also alcohol oxidase. The latter enzyme is probably responsible for the crystalline substructure of these peroxisomes.Peroxisomes had no apparent physiological function in the process of ascosporogenesis. A glyoxysomal function of the organelles during germination of the ascospores was also not observed. Germination of mature ascospores in media containing different sources of carbon and nitrogen showed that the function of the peroxisomes present in ascospores of Hansenula polymorpha is probably identical to that in vegetative haploid cells. They are involved in the oxidative metabolism of different carbon and nitrogen sources. Their enzyme profile is a reflection of that of peroxisomes of vegetative cells and their presence may enable the formation of cells which are optimally adapted to environmental conditions extant during spore germination.     

Enzymes of nitrogen assimilation in maize roots

The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH₄)₂SO₄ gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed. New address: Institut für Pflanzenphysiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a. D-1000 Berlin 33     

Energy metabolism of Plantago major ssp. major as dependent on the supply of mineral nutrients

Plantago major L. ssp. major , a grassland species from a relatively nutrient-rich habitat, was grown in nutrient-rich and nutrient-poor culture solutions. Half of the plants were transferred from high to low or from low to high nutrient conditions. The rate of dry matter accumulation in both shoots and roots decreased slowly upon transfer of plants to low nutrient conditions and the shoot to root ratio was unaffected. The rate of structural growth of both roots and shoots increased upon transfer from low to high nutrient conditions and the shoot to root ratio, if calculated from non-structural-carbohydrate-free dry weights, increased.
Photosynthesis was largely independent of the nutrient supply. Root respiration, particularly the activity of the alternative oxidative pathway, decreased with increasing age. This decrease was ascribed to a decreased shoot to root ratio, which reduced the relative amount of carbohydrates translocated to the roots and thus the amount available for the alternative pathway. It is calculated that in young as well as in old plants grown in full nutrient solution 48% of the daily produced photosynthates was translocated to the roots.
This is at variance with data on P. lanceolata , where a decreasing proportion of the daily produced photosynthates was translocated to the roots when the plants grew older. It is concluded that shoot growth plus shoot respiration consumed a constant amount of the daily produced photosynthates in P. major and that the rest was left for translocation. It is further calculated that in P. major plants grown in full nutrient solution c . 25% and c . 2% of the daily produced photosynthates in young and old plants, respectively, was respired in a way that is not involved in production of energy that is utilized in growth and maintenance ('inefficient root respiration').
The results are discussed in comparison with those of P. lanceolata , a species from a relatively nutrient-poor habitat.     

Folding of CFTR is predominantly cotranslational

The folding process for newly synthesized, multispanning membrane proteins in the endoplasmic reticulum (ER) is largely unknown. Here, we describe early folding events of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC-transporter family. In vitro translation of CFTR in the presence of semipermeabilized cells allowed us to investigate this protein during nascent chain elongation. We found that CFTR folds mostly during synthesis as determined by protease susceptibility. C-terminally truncated constructs showed that individual CFTR domains formed well-defined structures independent of C-terminal parts. We conclude that the multidomain protein CFTR folds mostly cotranslationally, domain by domain.     

In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation

Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) alpha (HP1alpha) and HP1beta on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1alpha and HP1beta in vivo targeting. HP1alpha targeting causes the recruitment of endogenous HP1beta to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1alpha and HP1beta targeting is sufficient to induce heterochromatin formation.     

Genotype-phenotype correlation in patients suspected of having Sotos syndrome

BACKGROUND: Deletions and mutations in the NSD1 gene are the major cause of Sotos syndrome. We wanted to evaluate the genotype-phenotype correlation in patients suspected of having Sotos syndrome and determine the best discriminating parameters for the presence of a NSD1 gene alteration. METHODS: Mutation and fluorescence in situ hybridization analysis was performed on blood samples of 59 patients who were clinically scored into 3 groups. Clinical data were compared between patients with and without NSD1 alterations. With logistic regression analysis the best combination of predictive variables was obtained. RESULTS: In the groups of typical, dubious and atypical Sotos syndrome, 81, 36 and 0% of the patients, respectively, showed NSD1 gene alterations. Four deletions were detected. In 23 patients (2 families) 19 mutations were detected (1 splicing defect, 3 non-sense, 7 frameshift and 8 missense mutations). The best predictive parameters for a NSD1 gene alteration were frontal bossing, down-slanted palpebral fissures, pointed chin and overgrowth. Higher incidences of feeding problems and cardiac anomalies were found. The parameters, delayed development and advanced bone age, did not differ between the 2 subgroups. CONCLUSIONS: In our patients suspected of having Sotos syndrome, facial features and overgrowth were highly predictive of a NSD1 gene aberration, whereas developmental delay and advanced bone age were not.