Penetration of analogues of H2O and CO2 in proteins studied by room temperature phosphorescence of tryptophan.

The influence of the protein matrix on the reactivity of external molecules with a species buried within the protein interior is considered in two general ways: (1) there may be structural fluctuations that allow for the diffusive penetration of the small molecules and/or (2) the external molecule may react over a distance. As a means to study the protein matrix, a reactive species within the protein can be formed by exciting tryptophan to the triplet state, and then the reaction of the triplet-state molecule with an external molecule can be monitored by a decrease in phosphorescence. In this work, the quenching ability (i.e., reactivity) was examined for H2S, CS2, and NO2- acting on tryptophan phosphorescence in parvalbumin, azurin, horse liver alcohol dehydrogenase, and alkaline phosphatase. A comparison of charged versus uncharged quenchers (H2S vs SH- and CS2 vs NO2-) reveals that the uncharged molecules are much more effective than charged species in quenching the phosphorescence of fully buried tryptophan, whereas the quenching for exposed tryptophan is relatively independent of the charge of the quencher. This is consistent with the view that uncharged triatomic molecules can penetrate the protein matrix to some extent. The energies of activation of the quenching reaction are low for the charged quenchers and higher for the uncharged CS2. A model is presented in which the quenchability of a buried tryptophan is inversely related to the distance from the surface when diffusion through the protein is the rate-limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interacting Effects of Discharge and Channel Morphology on Transport of Semibuoyant Fish Eggs in Large,Altered River Systems

Habitat fragmentation and flow regulation are significant factors related to the decline and extinction of freshwater biota. Pelagic-broadcast spawning cyprinids require moving water and some length of unfragmented stream to complete their life cycle. However, it is unknown how discharge and habitat features interact at multiple spatial scales to alter the transport of semi-buoyant fish eggs. Our objective was to assess the relationship between downstream drift of semi-buoyant egg surrogates (gellan beads) and discharge and habitat complexity. We quantified transport time of a known quantity of beads using 2–3 sampling devices at each of seven locations on the North Canadian and Canadian rivers. Transport time was assessed based on median capture time (time at which 50% of beads were captured) and sampling period (time period when 2.5% and 97.5% of beads were captured). Habitat complexity was assessed by calculating width∶depth ratios at each site, and several habitat metrics determined using analyses of aerial photographs. Median time of egg capture was negatively correlated to site discharge. The temporal extent of the sampling period at each site was negatively correlated to both site discharge and habitat-patch dispersion. Our results highlight the role of discharge in driving transport times, but also indicate that higher dispersion of habitat patches relates to increased retention of beads within the river. These results could be used to target restoration activities or prioritize water use to create and maintain habitat complexity within large, fragmented river systems.     

Sequence requirements for Hid binding and apoptosis regulation in the baculovirus inhibitor of apoptosis Op-IAP. Hid binds Op-IAP in a manner similar to Smac binding of XIAP.

It has been suggested that the Drosophila Hid protein interacts with the baculovirus Op-IAP protein in a manner similar to that of human Smac binding to XIAP, based largely on amino acid sequence homology. However, there is little direct experimental evidence in support of this hypothesis; indeed, evidence exists from previous studies suggesting that the mode of binding is not similar. We have now precisely mapped the interaction between Hid and Op-IAP, and we show clearly for the first time that the biochemical interactions between the amino terminus of Hid and BIR2 of Op-IAP are highly similar to those found between the processed amino terminus of Smac and BIR3 of XIAP. Also similar to Smac, the amino terminus of Hid must be processed to bind Op-IAP. In addition, our data also suggest that a second interaction between Hid and Op-IAP exists that does not involve the amino terminus of Hid, which may explain some of the earlier contradictory results. The evolutionary conservation of this mechanism of binding underscores its importance in apoptotic regulation. Nevertheless, interaction with Hid is not sufficient for Op-IAP to inhibit apoptosis induced by Hid overexpression or by treatment with actinomycin D, indicating that additional sequence elements are required for the anti-apoptotic function of Op-IAP.     

Chromosomal assignment of amplified genes in hydroxyurea-resistant hamster cells

We have shown previously that cDNAs for the M1 and M2 subunits of ribonucleotide reductase, ornithine decarboxylase (ODC), and p5-8, a 55,000-Dalton protein, hybridize to amplified genomic sequences in a highly hydroxyurea-resistant hamster cell line. We have extended these observations to include two additional, independently isolated, hydroxyurea-resistant cell lines: SC8, a single-step hamster ovary cell line, and KH450, a multistep human myeloid leukemic cell line, have also undergone genomic amplification for sequences homologous to ODC and p5-8 cDNAs. However, neither SC8 nor KH450 contains amplified genomic sequences homologous to an M1 cDNA probe. A panel of mouse-hamster somatic cell hybrids was used to map sequences homologous to M1, M2, ODC, and 5-8 cDNAs in the hamster genome. The M2, ODC, and p5-8 cDNAs hybridized to DNA fragments that segregated with hamster chromosome 7. In contrast, M1 cDNA hybridized to DNA fragments that segregated with hamster chromosome 3. These data suggest that the genes RRM2, (M2), ODC, and p5-8, but not RRMI (M1), are linked and may have been co-amplified in the selection of the hydroxyurea-resistant hamster and human cell lines.