Rotavirus and reovirus interaction with mouse peritoneal resident phagocytic cells

Rotaviruses and reoviruses are involved in human and animal diseases. It is known that both viruses penetrate the gastrointestinal tract but their interaction with phagocytic cells is unknown. To study this interaction, peritoneal resident phagocytic cells were used and rotavirus and reovirus replication in peritoneal phagocytic cells was observed. However, rotavirus replication in these cells led to the production of defective particles since MA-104 cells inoculated with rotavirus phagocytic cell lysate did not show any evidence of virus replication. On the basis of these results, we suggest that, although reovirus dissemination may be helped by these phagocytic cells, these cells may control rotavirus infection and probably contribute to the prevention of its dissemination.     

Step-gradient capillary electrochromatography

The analytical benefits of using a step-gradient in capillary electrochromatography (CEC) are demonstrated. The application of step-gradient CEC to the analysis of six diuretics of widely differing lipophilicities was evaluated and shown to result in a marked reduction in the analysis time and an improvement in the peak shape for later-eluting lipophilic components. When the step-gradient approach was performed in an automated mode, the retention time RSD for repeated injections was below 1%.     

A diminished role for hydrogen bonds in antifreeze protein binding to ice

The most abundant isoform (HPLC-6) of type I antifreeze protein (AFP1) in winter flounder is a 37-amino-acid-long, alanine-rich, alpha-helical peptide, containing four Thr spaced 11 amino acids apart. It is generally assumed that HPLC-6 binds ice through a hydrogen-bonding match between the Thr and neighboring Asx residues to oxygens atoms on the {2021} plane of the ice lattice. The result is a lowering of the nonequilibrium freezing point below the melting point (thermal hysteresis). HPLC-6, and two variants in which the central two Thr were replaced with either Ser or Val, were synthesized. The Ser variant was virtually inactive, while only a minor loss of activity was observed in the Val variant. CD, ultracentrifugation, and NMR studies indicated no significant structural changes or aggregation of the variants compared to HPLC-6. These results call into question the role of hydrogen bonds and suggest a much more significant role for entropic effects and van der Waals interactions in binding AFP to ice.     

A single point mutation in the yeast TRP4 gene affects efficiency of mRNA 3' end processing and alters selection of the poly(A) site

The yeast TRP4 mRNA 3' end formation element is a bidirectional element which functions in both orien-tations in an artificial in vivo test system. In this study, the role of 3' end formation was analysed in the context of the entire TRP4 gene. The 3' untranslated region (3'UTR) of TRP4 was altered and changes were analysed for their influence on TRP4 gene expression. The 3'UTR in reverse orientation was fully functional and did not affect TRP4 gene expression. Exchanging the TRP4 3'UTR by the bidirectional ARO4 or the unidirectional GCN4 3' end formation element allowed efficient gene expression. Deletion of the entire TRP4 3'UTR resulted in 70% reduction of TRP4 mRNA and 50% reduced specific Trp4 enzyme activity in comparison to wild-type. A single point mutation within the TRP4 3'UTR caused the same effect on gene expression. This point mutation did not only affect the efficiency of 3' end formation, but also produced new poly(A) sites which were situated upstream of the wild-type poly(A) sites. Therefore this sequence motif in the TRP4 3'UTR acts simultaneously as both an efficiency and positioning element.     

The crystal structure of human procathepsin K

Cathepsin K is a cysteine protease present in human osteoclasts that plays an important role in bone resorption. Cathepsin K is synthesized as an inactive proenzyme and activated under conditions of low pH. Autoproteolytic processing of the N-terminal 99 amino acid propeptide produces the active, mature form of cathepsin K. It is presumed that the activation of procathepsin K in vivo occurs in the bone resorption pit, which has a low-pH environment. We have determined the structure of human procathepsin K at 2.8 A resolution. The structure of the mature enzyme domain within procathepsin K is virtually identical to that of mature cathepsin K. The fold of the propeptide of procathepsin K is similar to that observed in procathepsins B and L despite differences in length and sequence. A portion of the propeptide occupies the active site cleft of cathepsin K. Hydrophobic interactions, salt bridges, and hydrogen-bonding interactions are observed in the structure of the propeptide and between the propeptide and the mature enzyme of procathepsin K. These interactions suggest an explanation for the stability of the proenzyme. The structure of procathepsin K contributes to an understanding of the molecular basis of inhibition by the propeptide portion of the molecule and activation of this important member of the cysteine protease family.