Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family

Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)avidin-like protein, named rhizavidin. The amino acid sequence identity of rhizavidin in relation to other known avidin-like proteins is 20-30%. The amino acid residues involved in the (strept)avidin-biotin interaction are well conserved in rhizavidin. The structural and functional properties of rhizavidin were carefully studied, and we found that rhizavidin shares characteristics with bradavidin, streptavidin and avidin. However, we found that it is the first naturally occurring dimeric protein in the avidin protein family, in contrast with tetrameric (strept)avidin and bradavidin. Moreover, it possesses a proline residue after a flexible loop (GGSG) in a position close to Trp-110 in avidin, which is an important biotin-binding residue. [3H]Biotin dissociation and ITC (isothermal titration calorimetry) experiments showed dimeric rhizavidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of avidin; although similar to streptavidin, it was insensitive to proteinase K. The immunological cross-reactivity of rhizavidin was tested with human serum samples obtained from cancer patients exposed to (strept)avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by SPECT (single-photon emission computed tomography) imaging in rats. Similarly to avidin, rhizavidin was observed to accumulate rapidly, mainly in the liver. Evidently, rhizavidin could be used as a complement to (strept)avidin in (strept)avidin-biotin technology.     

Immunohistochemical detection of c-fos proteins in cultured human glial cells--induction by cyclic AMP and phorbol ester

The expression of c-fos protein in cultured human glial cells derived from the brain and spinal cord was investigated immunocytochemically. Primary cultures of fetal glial cells were maintained in culture for three weeks and deprived of animal sera for 22 h. The glial cell nature of the cells was ascertained by GFAP-immunoreactivity. Incubations with phorbol dibutyrate, 8-Br-cAMP and sodium nitroprusside representing signal transduction pathways of PKC, PKA and cyclic GMP kinase, respectively, were carried out for 60 and 120 min. The control serum-deprived cultures did not display c-fos protein immunoreactivity (c-fos-IR), whereas phorbol dibutyrate incubation for 120 min induced strong c-fos-IR in the nuclei of both brain and spinal cord derived glial cells. Semiquantitative intensity measurements revealed a slight c-fos-IR induction after 8-Br-cAMP as well, but not after sodium nitroprusside. The observations suggest that c-fos protein is involved in PKC and PKA signal transduction in cultured human glial cells.     

DOC and N<Subscript>2</Subscript>O dynamics in upland and peatland forest soils after clear-cutting and soil preparation

Forest clear-cutting followed by soil preparation means disturbance for soil microorganisms and disruption of N and C cycles. We measured fluxes of N₂O and dissolved organic carbon (DOC) in upland soil (podzol) and adjacent peat within a clear-cut forest catchment. Both soil types behaved in a similar way, showing net uptake of N₂O in the first year after the clear-cutting, and turning to net release in the second. The N₂O flux dynamics were similar to those of N content in logging residues, as reported from a nearby site. As organic matter is used in the food web of the decomposers, we attempted to explain the dynamics of N₂O uptake and release by measuring the concurrent dynamics of the low molecular weight (LMW) fraction and the aromaticity of DOC in a soil solution. The labile and most readily available LMW fractions of DOC were nearly absent in the year following the clear-cutting, but rose after two years. The more refractory high molecular weight (HMW) fraction of DOC decreased two years after the clear-cutting. The first year’s net uptake of N₂O could be accounted for by the growth of decomposer biomass in the logging residues and detritus from the degenerating ground vegetation, resulting in immobilization of nitrogen. Simultaneously, the labile, LMW fraction of DOC became almost completely exhausted. The low availability of the LMW fraction could retard the growth and cause the accumulated decomposer biomass to collapse. During the following winter and summer the fraction of LMW clearly increased, followed by increased N₂O emissions. The presence of LMW DOC fractions, not the concentration of DOC, seems to be an important controller for N₂O liberation after a major disturbance such as clear-cutting and site preparation. The complex connection between DOC characteristics, nitrification or denitrification merits further studies.     

Enzymatic transfer of a {beta}1,6-linked N-acetylglucosamine to the {alpha}-galactose of globo-N-tetraose: in vitro synthesis of a novel hybrid pentasaccharide of lacto-globo type

A novel saccharide was synthesized by incubating globo-N-tetraose,GalNAcß1–3Gal     

Experimentally Constrained Virulence is Costly for Common Cuckoo Chicks

Chicks of some avian brood parasites show high virulence by eliminating all host progeny in the nest whereas others develop in the presence of host nestmates. Common cuckoo ( Cuculus canorus ) chicks are typically highly virulent parasites as they attempt to evict all host eggs and chicks soon after hatching. However, several features of nest design, including steep walls and/or cavity nests, may effectively prevent cuckoo hatchlings from evicting nestmates. A previous observational study showed low success of cuckoo chicks in evicting progeny of a cavity nester host, the redstart ( Phoenicurus phoenicurus ) but cuckoo chicks showed low survival both when reared alone or in mixed broods with host nestmates. Whether poor cuckoo performance was caused by eviction costs and/or by the effect of presence of host chicks per se remains unclear. We experimentally cancelled any potential eviction costs by removing host eggs immediately after the cuckoo hatched and creating mixed broods 5 days later when the eviction instinct of the cuckoo already ceased. Cuckoos that were forced to compete with host nestlings experienced lower provisioning rates, poorer growth, and lower fledging success than control lone cuckoos. Cuckoos in mixed broods that survived until fledging fledged later, and at lower masses, than those in the sole cuckoo group. Thus, the cuckoo gens specializing on redstarts is similar to other cuckoo gentes, whose chicks are more successful in evicting host nestmates, and it does not benefit from the presence of host brood. Cohabitation with host nestlings then should be viewed as a maladaptive by-product of host cavity nest design.     

Immunohistochemical detection of c-fos proteins in cultured human glial cells — induction by cyclic AMP and phorbol ester

Summary The expression of c-fos protein in cultured human glial cells derived from the brain and spinal cord was investigated immunocytochemically. Primary cultures of fetal glial cells were maintained in culture for three weeks and deprived of animal sera for 22 h. The glial cell nature of the cells was ascertained by GFAP-immunoreactivity. Incubations with phorbol dibutyrate, 8-Br-cAMP and sodium nitroprusside representing signal transduction pathways of PKC, PKA and cyclic GMP kinase, respectively, were carried out for 60 and 120 min. The control serum-deprived cultures did not display c-fos protein immunoreactivity (c-fos-IR), whereas phobol dibutyrate incubation for 120 min induced strong c-fos-IR in the nuclei of both brain and spinal cord derived glial cells. Semiquantitative intensity measurements revealed a slight c-fos-IR induction after 8-Br-cAMP as well, but not after sodium nitroprusside. The observations suggest that c-fos protein is involved in PKC and PKA signal transduction in cultured human glial cells.