Calf thymus ribonuclease H IIa activity lacks ribonuclease H specificity

Summary Less purified fractions of ribonuclease H IIa activity of calf thymus display divalent cation-dependent ribonuclease H activity and divalent cation-independent ribonuclease activity. Because the ratio of the two enzyme activities does not change during successive chromatographic procedures, we suggest that ribonuclease H IIa activity is indeed able to degrade both ssRNA and the RNA moiety of RNA·DNA-hybrids. Ribonuclease H IIa activity can therefore be differentiated from calf thymus ribonuclease H I and H IIb by its lack of ribonuclease H specificity. The native molecular mass of ribonuclease H IIa activity is between 23 and 28 kDa. Under denaturing conditions a 23 kDa-protein band copurifies with the enzyme activity suggesting that this enzyme is monomeric.     

The contribution of noncatalytic phosphate-binding subsites to the mechanism of bovine pancreatic ribonuclease A

The enzymatic catalysis of polymeric substrates such as proteins, polysaccharides or nucleic acids requires precise alignment between the enzyme and the substrate regions flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be directed by electrostatic interactions between the phosphate groups of the RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The substrate-binding subsites of ribonuclease A have been characterized by structural and kinetic studies. In addition to the active site (p₁ ), the role of other noncatalytic phosphate-binding subsites in the correct alignment of the polymeric substrate has been proposed. p₂ and p₀ have been described as phosphate-binding subsites that bind the phosphate group adjacent to the 3′ side and 5′ side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p₂ , and Lys-66 in p₀ ) are involved in binding. However, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p₂ are important both in catalysis and in the endonuclease activity of the enzyme, whilst the p₀ electrostatic interaction contributes only to binding of the RNA.     

The cytotoxicity of eosinophil cationic protein/ribonuclease 3 on eukaryotic cell lines takes place through its aggregation on the cell membrane

Human eosinophil cationic protein (ECP)/ ribonuclease 3 (RNase 3) is a protein secreted from the secondary granules of activated eosinophils. Specific properties of ECP contribute to its cytotoxic activities associated with defense mechanisms. In this work the ECP cytotoxic activity on eukaryotic cell lines is analyzed. The ECP effects begin with its binding and aggregation to the cell surface, altering the cell membrane permeability and modifying the cell ionic equilibrium. No internalization of the protein is observed. These signals induce cell-specific morphological and biochemical changes such as chromatin condensation, reversion of membrane asymmetry, reactive oxygen species production and activation of caspase-3-like activity and, eventually, cell death. However, the ribonuclease activity component of ECP is not involved in this process as no RNA degradation is observed. In summary, the cytotoxic effect of ECP is attained through a mechanism different from that of other cytotoxic RNases and may be related with the ECP accumulation associated with the inflammatory processes, in which eosinophils are present. Received 26 October 2007; accepted 23 November 2007     

Ribonuclease 4, an evolutionarily highly conserved member of the superfamily

The structural and enzymatic properties of RNase 4 are reviewed. This RNase shows a much higher interspecies similarity (approximately 90%) than the other members of the RNase A superfamily. The enzyme is ubiquitous, with the highest amounts present in liver and lung. Its unique uridine specificity results from alterations in and around the pyrimidine-binding site. In particular, the shortened C-terminus and the side chains of Phe-42, Asp-80 and Arg-101 appear to be involved. RNase 4 binds tightly to the intracellular RNase inhibitor, with a K _d of 4 × 10⁻¹⁵ M.     

Introduction: molecular and biomechanical basis of osteoarthritis

Osteoarthritis has developed into the most common chronic disease in the highly industrialized nations. Moreover, because of the prevalence of the disease in the elderly, this trend occurs worldwide as a consequence of increasing longevity due to the overall improvement in living conditions and health status. In contrast, research on osteoarthritis is still financially marginalized within biomedical research, so that the molecular and biophysical bases for disease initiation and progression are largely unmapped. The following sequence of five reviews highlights a remarkable change in that body of knowledge taking place at the beginning of the World Health Organization (WHO) 'Bone and Joint Decade 2001-2010'. The data and ideas presented in these articles reflect to some extent the guidelines set up by the WHO and by the National Institutes of Health of the USA and therefore allow a glimpse into the directions that research in osteoarthritis will take in the future.     

Correction of the anomalous electrophoretic behavior of ribonuclease A

Ribonuclease A behaves anomalously on polyacrylamide gel electrophoresis at acid pH. The distance traveled by the protein is a function of the amount of enzyme added at the lower range of detectable activity (10 pg to 100 ng). Addition of myoglobin (1 mg/ml) abolishes the anomaly. The observations are consistent with the known affinity of RNase for anions. Caution is warranted in the interpretation of apparent electrophoretic variants of RNase observed at low concentrations of enzyme.     

Correction of the anomalous electrophoretic behavior of ribonuclease A

Summary Ribonuclease A behaves anomalously on polyacrylamide gel electrophoresis at acid pH. The distance traveled by the protein is a function of the amount of enzyme added at the lower range of detectable activity (10 pg to 100 ng). Addition of myoglobin (1 mg/ml) abolishes the anomaly. The observations are consistent with the known affinity of RNase for anions. Caution is warranted in the interpretation of apparent electrophoretic variants of RNase observed at low concentrations of enzyme.Acknowledgment. This is publication No. 80-33 from the Department of Medical Genetics and was supported in part by the Indiana University Human Genetics Center, PHS GM 21054.     

Peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts

Cyclosporine A therapy for prophylaxis against graft rejection revolutionized human organ transplantation. The immunosuppressant drugs cyclosporin A (CsA), FK506 and rapamycin block T-cell activation by interfering with the signal transduction pathway. The target proteins for CsA and FK506 were found to be cyclophilins and FK506-binding proteins, (FKBPs), respectively. They are unrelated in primary sequence, although both are peptidyl-prolyl cis-trans isomerases catalyzing the interconversion of peptidyl-prolyl imide bonds in peptide and protein substrates. However, the prolyl isomerase activity of these proteins is not essential for their immunosuppressive effects. Instead, the specific surfaces of the cyclophilin-CsA and FKBP-FK506 complexes mediate the immunosuppressive action. Moreover, the natural cellular functions of all but a few remain elusive. In some cases it could be demonstrated that prolyl isomerization is the rate-limiting step in protein folding in vitro, but many knockout mutants of single and multiple prolyl isomerases were viable with no detectable phenotype. Even though a direct requirement for in vivo protein folding could not be demonstrated, some important natural substrates of the prolyl isomerases are now known, and they demonstrate the great variety of prolyl isomerization functions in the living cell: (i) A human cyclophilin binds to the Gag polyprotein of the human immunodeficiency virus-1 (HIV-1) virion and was found to be essential for infection with HIV to occur, probably by removal of the virion coat. (ii) Together with heat shock protein (HSP) 90, a member of the chaperone family, high molecular weight cyclophilins and FKBPs bind and activate steroid receptors. This example also demonstrates that prolyl isomerases act together with other folding enzymes, for example the chaperones, and protein disulfide isomerases. (iii) An FKBP was found to act as a modulator of an intracellular calcium release channel. (iv) Along with the cyclophilins and FKBPs, a third class of prolyl isomerases exist, the parvulins. The human parvulin homologue Pin1 is a mitotic regulator essential for the G2/M transition of the eukaryotic cell cycle. These findings place proline isomerases at the intersection of protein folding, signal transduction, trafficking, assembly and cell cycle regulation. Received 18 September 1998; received after revision 4 November 1998; accepted 23 November 1998     

Unique gene structure and paralogy define the 7D-cadherin family

Cadherins are Ca²⁺-dependent transmembrane glycoproteins crucial for cell-cell adhesion in vertebrates and invertebrates. Classification of this superfamily due to their phylogenetic relationship is currently restricted to three major subfamilies: classical, desmosomal and protocadherins. Here we report evidence for a common phylogenetic origin of the kidney-specific Ksp- (Cdh16) and the intestine-specific LI-cadherin (Cdh17). Both genes consist of 18 exons and the positions of their exon-intron boundaries as well as their intron phases are perfectly conserved. We found an extensive paralogy of more than 40 megabases in mammals as well as teleost fish species encompassing the Ksp- and LI-cadherin genes. A comparable paralogy was not detected for other cadherin gene loci. These findings suggest that the Ksp- and LI-cadherin genes originated by chromosomal duplication early during vertebrate evolution and support our assumption that both proteins are paralogues within a separate cadherin family that we have termed 7D-cadherins. Received 16 January 2006; received after revision 18 April 2006; accepted 11 May 2006     

Introduction: integrins, dynamic cell receptors

Integrins are a family of adhesive receptors consisting of α- and β-subunits which attach cells together via adhesive protein ligands or bind cells to extracellular matrix. They are found on virtually all cell types and link the external ligand to the cytoskeleton of the cell. Integrins also act as signal transducers both from the outside of the cell to the interior and also inside-out. Their main functions are in recognition and in tight but regulated binding. The series of reviews presented here cover both basic aspects of integrin function, including signal transduction, snake disintegrins and structure and function of I-domains in some integrin α-subunits, as well as the role of integrins in diseases, cancer, inflammation and cardiovascular diseases. The search for suitable inhibitors of integrins for treatment of these diseases and future prospects for their use are also discussed.     

Copernicus: A Very Short Introduction

The reception of Buffon's Histoire Naturelle in the Enlightenment has not received the historical attention it deserves. Drawing primarily on archival sources, this paper examines Aberdeen reactions to the Histoire during the period c. 1750–1800. As pedagogues, the Aberdonians endeavoured to maintain intellectual orthodoxy, and hence they attacked Buffon for his apparent materialism and atheism. Moreover, the Aberdonians rejected Buffon's critique of taxonomy because they based their natural history courses on classifications of the three kingdoms of nature, and because they attempted to use classification systems in nosology and the study of the human mind. Finally, in the 1790s Aberdeen readings of the Histoire were profoundly affected by the fears aroused by the French Revolution.     

Introduction: genetic determinants of mid- and late-life dementias

We introduce a series of papers dealing with genetic aspects of a subset of dementias of mid-life and late-life in order to illustrate four principles. First, there appear to be many genetic loci with the potential to modulate susceptibility to such dementias. Second, most of those so far discovered are autosomal dominants and none are autosomal recessives. Third, the autosomal dominant mutations are individually rare. Their frequencies in a given population are likely to be functions of genetic drift. Fourth, despite their rarity, they may inform us about the pathogenesis of more common late-life dementias, notably dementias of the Alzheimer type, which have polygenic determinants. The most important such modulation so far discovered involves polymorphic forms of the APOE locus.     

Theories that narrate the world: Ronald A. Fisher's mass selection and Sewall Wright's shifting balance

Theories are composed of multiple interacting components. I argue that some theories have narratives as essential components, and that narratives function as integrative devices of the mathematical components of theories. Narratives represent complex processes unfolding in time as a sequence of stages, and hold the mathematical elements together as pieces in the investigation of a given process. I present two case studies from population genetics: R. A. Fisher's “mas selection” theory, and Sewall Wright's shifting balance theory. I apply my analysis to an early episode of the “R. A. Fisher – Sewall Wright controversy.”     

tRNase Z: the end is not in sight

Although the enzyme tRNase Z has only recently been isolated, a plethora of data has already been acquired concerning the enzyme. tRNase Z is the endonuclease that catalyzes the removal of the tRNA 3′ trailer, yielding the mature tRNA 3′ end ready for CCA addition and aminoacylation. Another substrate cleaved by tRNase Z is the small chromogenic phosphodiester bis(p-nitrophenyl)phosphate (bpNPP), which is the smallest tRNase Z substrate known so far. Hitherto the biological function as tRNA 3′-end processing enzyme has been shown only in one prokaryotic and one eukaryotic organism, respectively. This review summarizes the present information concerning the two tRNase Z substrates pre-tRNA and bpNPP, as well as the metal requirements of tRNase Z enzymes. Received 29 March 2007; received after revision 15 May 2007; accepted 21 May 2007     

Introduction: the selenium conundrum

Selenium was first suspected of being an essential dietary trace element in the 1950s. We now know that indeed it is an essential biological element that serves as an integral component of several enzymes, including those in the families of deiodinases and glutathione peroxidases as well as selenoproteins P and W. The multi-author review that follows this introduction concentrates on the important biological role of selenium in enzymes as well as some of the physiological aspects of selenium as either a potential anticarcinogenic agent or insulin mimetic. What should become clear from these contributed articles is the complex and dynamic role that selenium plays in many biological processes and that the investigations in these areas are at the edge of exciting new frontiers.     

The histone deacetylase inhibitor trichostatin A influences the development of Drosophila melanogaster

We examined the consequences of the deacetylase inhibitor trichostatin A (TSA) on the development of Drosophila melanogaster. When fed to flies, TSA caused lethality and delayed development at concentrations as low as 5 μM, had stronger effects on males than females, and acted synergistically with mutations in the gene encoding the RPD3 deacetylase to cause notched wings, but did not appear to affect a SINA signaling pathway that is normally repressed by the SIN3 corepressor. These findings suggest that deacetylated histones play an important role in normal developmental progression and establish parameters for genetic screens to dissect the role of deacetylases in this process. Received 14 June 2001; received after revision 31 July 2001; accepted 21 August 2001