Studies of an active site mutant of the selenoprotein thioredoxin reductase: the Ser-Cys-Cys-Ser motif of the insect orthologue is not sufficient to replace the Cys-Sec dyad in the mammalian enzyme

We have mutated the redox active C-terminal motif, Gly-Cys-Sec-Gly, of the mammalian selenoprotein thioredoxin reductase (TrxR) to mimic the C-terminal Ser-Cys-Cys-Ser motif of the non-selenoprotein orthologue of Drosophila melanogaster (DmTrxR). The activity of DmTrxR is almost equal to that of mammalian TrxR, which is surprising, because Cys mutants of selenoproteins are normally 1-2 orders of magnitude less active than their selenocysteine (Sec) containing counterparts. It was shown earlier that the flanking Ser residues were important for activating the Cys residues in DmTrxR (Gromer, et.al. (2003) PNAS 100, 12618-12623). However, the "Drosophila mimic" mutant of the mammalian enzyme studied herein had <0.5% activity compared to wild-type. Rapid kinetic studies revealed that all of the redox centers of the mutant were active, but that the C-terminal dithiols were not effective reductants of thioredoxin. The charge-transfer complex of the two-electron reduced enzyme slowly disappeared as the N-terminal dithiols reduced the C-terminal disulfide. In wild-type enzyme, the selenenylsulfide is more difficult to reduce and the charge-transfer complex is more stable. These findings suggest that features in addition to the flanking Ser residues are important for facilitating the high activity of the insect enzyme and that the corresponding features are absent in mammalian TrxR.     

Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, α-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.     

Aquatic Habitat Dynamics along a Braided Alpine River Ecosystem (Tagliamento River, Northeast Italy)

Aquatic habitat change caused by flooding was quantified along the Fiume Tagliamento, a morphologically intact gravel-bed river ecosystem in northeast Italy. Five different geomorphic reaches (each around 1.5 km), ranging from near the headwaters at 800 m above sea level (a.s.l.) to near the mouth at 5 m a.s.l., were studied over a 1-year period. All floodplain water bodies in each reach were delineated in August 1999 using a differential global positioning system. Each reach was remapped twice (in December 1999 and August 2000) to investigate the impact of autumn and spring flood seasons on aquatic habitat composition and configuration. A high degree (nearly 62%) of aquatic habitat turnover was documented in a braided headwater floodplain. The degree of aquatic habitat turnover decreased with decreasing elevation to approximately 20% turnover in a meandering reach at 5 m a.s.l. In contrast to turnover, braiding, sinuosity, and aquatic habitat composition changed little in response to flooding in all reaches. Location of aquatic habitats in floodplains changed considerably (turnover), whereas habitat configuration and composition remained relatively stable. These results support the applicability of the shifting mosaic steady-state model to riverine floodplain environments.     

Oestrus synchronization in sheep with progesterone-impregnated (MAP) intravaginal sponges and a prostagland in analogue

A double injection of PGF2α at two different dosage levels, 5 mg and 10 mg, eleven days apart, were compared with a combination of MAP-sponges and 5 mg or 10 mg PGF2α.The combination of MAP-sponges plus 10 mg PGF2α gave the best syn=chronization results (93,4%), as well as the best conception rate (84,9%) when ewes were inseminated on a fixed time basis 68 and 80 hours after treatment.