Mutation of residues 423 (Met/Ile), 444 (Thr/Met), and 506 (Asn/Ser) confer cholesteryl esterase activity on rat lung carboxylesterase. Ser-506 is required for activation by cAMP-dependent protein kinase.

Site-directed mutagenesis is used to identify amino acid residues that dictate reported differences in substrate specificity between rat hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) and rat lung carboxylesterase (LCE), proteins differing by only 4 residues in their primary sequences. Beginning with LCE, the substitution Met(423) --> Ile(423) alone or in combination with other mutations increased activity with p-nitrophenylcaprylate (PNPC) relative to more hydrophilic p-nitrophenylacetate (PNPA), typical of hncCEH. The substitution Thr(444) --> Met(444) was necessary but not sufficient for expression of cholesteryl esterase activity in COS-7 cells. The substitution Asn(506) --> Ser(506), creating a potential phosphorylation site, uniformly increased activity with both PNPA and PNPC, was necessary but not sufficient for expression of cholesteryl esterase activity and conferred susceptibility to activation by cAMP-dependent protein kinase, a property of hncCEH. The 3 mutations in combination were necessary and sufficient for expression of cholesteryl esterase activity by the mutated LCE. The substitution Gln(186) --> Arg(186) selectively reduced esterase activity with PNPA and PNPC but was not required for cholesteryl esterase activity. Homology modeling from x-ray structures of acetylcholinesterases is used to propose three-dimensional models for hncCEH and LCE that provide insight into the effects of these mutations on substrate specificity.     

Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.     

Influence of carbon dioxide supply on the chloroplast structure of Chlorella pyrenoidosa

Summary Electron microscopic investigations show that the structure of the lamellar system of Chlorella pyrenoidosa depends on the carbon dioxide supply in the culture medium. Chloroplasts of C. pyrenoidosa when grown with 0.03% CO₂, show typical grana structure whereas with 3% CO₂ the chloroplast structure is typical of that of the Chlorophyceae.     

A complex containing at least one zinc dependent HeLa nuclear protein binds to the intronic (gaa)n block of the frataxin gene

We analyzed HeLa nuclear proteins binding to the (gaa)_n harbouring intron 1 of nine frataxin alleles and characterized the structures of the repeats. Fragments with blocks longer than (gaa)₉ form spontaneously different intramolecular H-y topoisomeres in linear state. The observed triplexes depend on the length of the repeat. Interruption of the perfectly repeated (gaa)_n block entails two structural regions. At least two HeLa nuclear proteins bind to the (gaa)_n fragments resulting in a distinct major retarded complex as revealed by EMSA. One of these proteins is zinc dependent. Importantly, the fragment harbouring (gan)₁₂₁ binds additional proteins. Protein binding appears to be locus specific, and the binding affinity was found to be not random. The affinities of the different target fragments varied by a factor of four. Binding affinities of the fragments were not obviously correlated to differences in the composition of the repeats. DNase I footprinting revealed only weakly protected binding regions, but multiple HS sites in the repeat regions of the fragments. These findings and the fact, that DNA conformers observed in EMSA and electron microscopical experiments bind proteins, lead to the assumption that the proteins recognize, both, B-DNA and triple helical structures, but with different affinity. Possible functions of the proteins are discussed in the context of transformation of triple helical structures into B-DNA and the pathogenesis of FRDA.     

The relationship between morningness-eveningness preference and online learning

This study explored whether circadian preference is related to students' attitudes and choices to attend lectures or watch them online, and whether these variables relate to course performance. The subjects were 847 students enrolled in an introductory psychology course who completed an online survey that contained the Morningness - Eveningness Questionnaire and that ascertained their attitudes towards online lectures and the extent to which they attended lectures or watched them online; course performance was also recorded. The results revealed that evening-type students were significantly more likely to have a positive attitude toward online lectures and to choose to watch lectures online. Course performance was not linked to morningness - eveningness preference, lecture mode choice, or their interaction. The results suggest that online lectures appeal differentially to students with a morning or evening orientation, but that watching lectures in a modality that does not accommodate a student's circadian preference does not handicap performance.     

Kinetic basis for activation of CDK2/cyclin A by phosphorylation

The activation of most protein kinases requires phosphorylation at a conserved site within a structurally defined segment termed the activation loop. A classic example is the regulation of the cell cycle control enzyme, CDK2/cyclin A, in which catalytic activation depends on phosphorylation at Thr(160) in CDK2. The structural consequences of phosphorylation have been revealed by x-ray crystallographic studies on CDK2/cyclin A and include changes in conformation, mainly of the activation loop. Here, we describe the kinetic basis for activation by phosphorylation in CDK2/cyclin A. Phosphorylation results in a 100,000-fold increase in catalytic efficiency and an approximate 1,000-fold increase in the overall turnover rate. The effects of phosphorylation on the individual steps in the catalytic reaction pathway were determined using solvent viscosometric techniques. It was found that the increase in catalytic power arises mainly from a 3,000-fold increase in the rate of the phosphoryl group transfer step with a more moderate increase in substrate binding affinity. In contrast, the rate of phosphoryl group transfer in the ATPase pathway was unaffected by phosphorylation, demonstrating that phosphorylation at Thr(160) does not serve to stabilize ATP in the ATPase reaction. Thus, we hypothesize that the role of phosphorylation in the kinase reaction may be to specifically stabilize the peptide phosphoacceptor group.     

Evidence for an RNA chaperone function of polypyrimidine tract-binding protein in picornavirus translation

The cellular polypyrimidine tract-binding protein (PTB) is recruited by the genomic RNAs of picornaviruses to stimulate translation initiation at their internal ribosome entry site (IRES) elements. We investigated the contribution of the individual RNA recognition motif (RRM) domains of PTB to its interaction with the IRES of foot-and-mouth disease virus (FMDV). Using a native gel system, we found that PTB is a monomer, confirming recent reports that challenged the previous view that PTB is a dimer. Mapping the spatial orientation of PTB relative to the bound IRES RNA, we found that the two C-terminal RRM domains III and IV of PTB bind in an oriented way to the IRES. Domain III contacts the IRES stem-loop 2, while domain IV contacts the separate IRES 3' region. PTB domain I appears not to be involved directly in RNA binding, but domain II stabilizes the RNA binding conferred by domains III and IV. A PTB protein containing only these two C-terminal PTB domains is sufficient to enhance the entry of initiation factor eIF4G to the IRES and stimulate IRES activity, and the long-lived PTB-IRES interaction stabilized by domain II is not a prerequisite for this function. Thus, PTB most likely acts as an RNA chaperone to stabilize IRES structure and, in that way, augment IRES activity.     

The presence of microbodies in three strains of Chlorella

Summary Electron microscopic investigations show that cells of three strains of Chlorella contain microbodies. The organelles have a dense granular matrix and are surrounded by a single membrane.     

Interaction of translation initiation factor eIF4B with the poliovirus internal ribosome entry site

Poliovirus translation is initiated at the internal ribosome entry site (IRES). Most likely involving the action of standard initiation factors, this highly structured cis element in the 5" noncoding region of the viral RNA guides the ribosome to an internal silent AUG. The actual start codon for viral protein synthesis further downstream is then reached by ribosomal scanning. In this study we show that two of the secondary structure elements of the poliovirus IRES, domain V and, to a minor extent, domain VI, are the determinants for binding of the eukaryotic initiation factor eIF4B. Several mutations in domain V which are known to greatly affect poliovirus growth also seriously impair the binding of eIF4B. The interaction of eIF4B with the IRES is not dependent on the presence of the polypyrimidine tract-binding protein, which also binds to the poliovirus IRES. In contrast to its weak interaction with cellular mRNAs, eIF4B remains tightly associated with the poliovirus IRES during the formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. These results indicate that the interaction of eIF4B with the 3" region of the poliovirus IRES may be directly involved in translation initiation.