Microtiter plate cellular assay for human steroid sulfatase with fluorescence readout

Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay.     

Distal side tryptophan, tyrosine and methionine in catalase-peroxidases are covalently linked in solution

Distal side tryptophan and tyrosine have been shown to be essential in the catalase but not the peroxidase activity of bifunctional catalase-peroxidases (KatGs). Recently published crystal structures suggest that both residues could be part of a novel adduct including in addition a conserved methionine. A mass spectrometric analysis of the tryptic peptides from recombinant wild-type Synechocystis KatG and the variants Trp122Phe, Tyr249Phe and Met275Ile confirms that this novel adduct really exists in solution and thus may be common to all KatGs. Exchange of either Trp122 or Tyr249 prevents cross-linking, whereas exchange of Met275 still allowed bond formation between Trp122 and Tyr249. It is proposed that the covalent bond between Trp and Tyr may form before that between Tyr and Met. The findings are discussed with respect to the mechanism of cross-linking and its role in KatG catalysis.     

Interactions of valerian extracts and a fixed valerian-hop extract combination with adenosine receptors

Phytopharmaceuticals and dietary supplements containing valerian are used as mild sleep-inducing agents. An in vitro radioligand binding assay at A(1) and A(2A) adenosine receptors (ARs) was conducted with a fixed extract combination of valerian and hop (Ze 91019) to investigate a possible mechanism for the pharmacological activity of the extract. Component extracts of valerian and hop were also individually investigated. The fixed combination Ze 91019 as well as the valerian extracts therein exhibited selective affinity to A(1)ARs (K(i) = 0.15-0.37 mg/mL vs [(3)H]CCPA). The same extracts exhibited partial agonist activity at the A(1) adenosine receptor as indicated by a lower degree of stimulation of [35S]GTP gamma S binding in membrane preparations of CHO-hA(1) cells as compared to the full A(1) AR agonist N(6)-cyclopentyladenosine (CPA). In addition valerian extract inhibited cAMP accumulation in CHO-hA(1) cell membranes. The partial agonistic activity at A(1)ARs may thus play a role in the sleep inducing effect of Ze 91019 and the valerian extract therein.     

Evidence for segment-nonspecific packaging of the influenza a virus genome

The influenza A virus genome is composed of eight negative-sense RNA segments (called vRNAs), all of which must be packaged to produce an infectious virion. It is not clear whether individual vRNAs are packaged specifically or at random, however, and the total vRNA capacity of the virion is unknown. We have created modified forms of the viral nucleoprotein (NP), neuraminidase (NA), and nonstructural (NS) vRNAs that encode green or yellow fluorescent proteins and studied the efficiency with which these are packaged by using a plasmid-based influenza A virus assembly system. Packaging was assessed precisely and quantitatively by scoring transduction of the fluorescent markers in a single-round infectivity assay with a flow cytometer. We found that, under conditions in which virions are limiting, pairs of alternatively tagged vRNAs compete for packaging but do so in a nonspecific manner. Reporters representing different vRNAs were not packaged additively, as would be expected under specific packaging, but instead appeared to compete for a common niche in the virion. Moreover, 3 to 5% of transduction-competent viruses were found to incorporate two alternative reporters, regardless of whether those reporters represented the same or different vRNAs - a finding compatible with random, but not with specific, packaging. Probabilistic estimates suggest that in order to achieve this level of dual transduction by chance alone, each influenza A virus virion must package an average of 9 to 11 vRNAs.     

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.     

Hydroxylated 2-amino-3H-phenoxazin-3-one derivatives as products of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) biotransformation by Chaetosphaeria sp., an endophytic fungus from Aphelandra tetragona

The biotransformation of the phytoanticipin HBOA and its major degradation metabolites 2-hydroxy-N-(2-hydroxyphenyl)acetamide (7) and N-(2-hydroxyphenyl)acetamide (8) by Chaetosphaeria sp., an endophytic fungus isolated from Aphelandra tetragona, was studied. Three new metabolites could be identified as 2-amino-7-hydroxy-3H-phenoxazin-3-one (12), 2-acetylamino-7-hydroxy-3H-phenoxazin-3-one (13) and 7-hydroxy-2-(2-hydroxyacetyl)-amino-3H-phenoxazin-3-one (14). Structure elucidation of 12 and 13 was performed by MS, 1H, 13C NMR and 2D NMR techniques and confirmed by chemical transformation.     

Non-cell autonomous requirement for the bloodless gene in primitive hematopoiesis of zebrafish

Vertebrate hematopoiesis occurs in two distinct phases, primitive (embryonic) and definitive (adult). Genes that are required specifically for the definitive program, or for both phases of hematopoiesis, have been described. However, a specific regulator of primitive hematopoiesis has yet to be reported. The zebrafish bloodless (bls) mutation causes absence of embryonic erythrocytes in a dominant but incompletely penetrant manner. Primitive macrophages appear to develop normally in bls mutants. Although the thymic epithelium forms normally in bls mutants, lymphoid precursors are absent. Nonetheless, the bloodless mutants can progress through embryogenesis, where red cells begin to accumulate after 5 days post-fertilization (dpf). Lymphocytes also begin to populate the thymic organs by 7.5 dpf. Expression analysis of hematopoietic genes suggests that formation of primitive hematopoietic precursors is deficient in bls mutants and those few blood precursors that are specified fail to differentiate and undergo apoptosis. Overexpression of scl, but not bmp4 or gata1, can lead to partial rescue of embryonic blood cells in bls. Cell transplantation experiments show that cells derived from bls mutant donors can differentiate into blood cells in a wild-type host, but wild-type donor cells fail to form blood in the mutant host. These observations demonstrate that the bls gene product is uniquely required in a non-cell autonomous manner for primitive hematopoiesis, potentially acting via regulation of scl.     

Diversity and abundance of Crenarchaeota in terrestrial habitats studied by 16S RNA surveys and real time PCR

Novel phylogenetic lineages of as yet uncultivated crenarchaeota have been frequently detected in low to moderate-temperature, marine and terrestrial environments. In order to gain a more comprehensive view on the distribution and diversity of Crenarchaeota in moderate habitats, we have studied 18 different terrestrial and freshwater samples by 16S rDNA-based phylogenetic surveys. In seven different soil samples of diverse geographic areas in Europe (forest, grassland, ruderal) and Asia (permafrost, ruderal) as well as in two microbial mats, we have consistently found one particular lineage of crenarchaeota. The diversity of Crenarchaeota in freshwater sediments was considerably higher with respresentative 16S rDNA sequences distributed over four different groups within the moderate crenarchaeota. Systematic analysis of a 16S rDNA universal library from a sandy ecosystem containing 800 clones exclusively revealed the presence of the soil-specific crenarchaeotal cluster. With primers specific for non-thermophilic crenarchaeota we established a rapid method to quantify archaeal 16S rDNA in real time PCR. The relative abundance of crenarchaeotal rDNA was 0.5-3% in the bulk soil sample and only 0.16% in the rhizosphere of the sandy ecosystem. A nearby agricultural setting yielded a relative abundance of 0.17% crenarchaeotal rDNA. In total our data suggest that soil crenarchaeota represent a stable and specific component of the microbiota in terrestrial habitats.     

The telomeric region of BTA18 containing a potential QTL region for health in cattle exhibits high similarity to the HSA19q region in humans

We have applied a targeted physical mapping approach, based on the isolation of bovine region-specific large-insert clones using homologous human sequences and chromosome microdissection, to enhance the physical gene map of the telomeric region of BTA18 and to prove its evolutionary conservation. The latter is a prerequisite to exploit the dense human gene map for future positional cloning approaches. Partial sequencing and homology search were used to characterize 20 BACs targeted to the BTA18q2.4-q2.6 region. We used fluorescence in situ hybridization (FISH) to create physical maps of 11 BACs containing 15 gene loci; these BACs served as anchor loci. Using these approaches, 12 new gene loci (CKM, STK13, PSCD2, IRF3, VASP, ACTN4, ITPKC, CYP2B6, FOSB, DMPK, MIA, SIX5) were assigned on BTA18 in the bovine cytogenetic map. A resolved physical map of BTA18q2.4-q2.6 was developed, which encompasses 28 marker loci and a comparative cytogenetic map that contains 15 genes. The mapping results demonstrate the high evolutionary conservation between the telomeric region of BTA18q and HSA19q.     

Intra-specific variation in social organization of gorillas: implications for their social evolution

We analysed intra-specific variation in the social organization of gorillas and ecological and social factors influencing them, based on recent data on diet, day journey length, home range size, group size and proportion of multi-male groups in three subspecies [western lowland gorillas (WLG); eastern lowland gorillas (ELG); mountain gorillas (MG)]. Median group size was similar across subspecies and across habitats, but the extraordinarily large group including >30 gorillas was only found in habitat with dense terrestrial herbaceous vegetation. Within-group competition may determine the upper limit of group size in frugivorous WLGs and ELGs in lowland habitats with scarce undergrowth. A frugivorous diet may be a causal factor of subgrouping in multi-male groups of WLGs and ELGs, while a folivorous diet may prevent subgrouping in multi-male groups of MGs. Social factors, rather than ecological factors, may play an important role in the formation of multi-male groups and their cohesiveness in MGs. High gregariousness of female gorillas and their prolonged association with a protector male are explained by their vulnerability to both infanticide (MGs) and predators (ELGs). Comparison of long-term changes in group composition and individual movements between ELGs in Kahuzi and MGs in the Virungas suggest that the occurrence of infanticide may promote kin-male association within a group. Threat of infanticide may stimulate MG females to transfer into multi-male groups to seek reliable protection and maturing MG males to stay in their natal groups after maturity. By contrast, the absence of infanticide may facilitate ELG females to associate with infants and other females at transfer and ELG males to establish large groups in a short period by taking females from their natal groups, by luring females from neighbouring groups, or by takeover of a widow group after the death of its leading male. These conditions may prevent ELG and WLG maturing males from remaining to reproduce in their natal groups and possibly result in a rare occurrence of multi-male groups in their habitats. Similar reproductive features of MG and ELG females suggest both female strategies have been adaptive in their evolutionary history.