RNase P from bacteria. Substrate recognition and function of the protein subunit

RNase P recognizes many different precursor tRNAs as well as other substrates and cleaves all of them accurately at the expected position. RNase P recognizes the tRNA structure of the precursor tRNA by a set of interactions between the catalytic RNA subunit and the T- and acceptor-stems mainly, although residues in the 5-leader sequence as well as the 3-terminal CCA are important. These conclusions have been reached by several studies on mutant precursor tRNAs as well as cross-linking studies between RNase P RNA and precursor tRNAs. The protein subunit of RNase P seems also to affect the way that the substrate is recognized as well as the range of substrates that can be used by RNase P, although the protein does not seem to interact directly with the substrates. The interaction between the protein and RNA subunits of RNase P has been extensively studiedin vitro. The protein subunit sequence is not highly conserved among bacteria, however different proteins are functionally equivalent as heterologous reconstitution of the RNase P holoenzyme can be achieved in many cases.Abbreviations C5 protein protein subunit fromE. coli RNase P - EGS external guide sequence - M1 RNA RNA subunit formE. coli RNase P - ptRNA precursor tRNA - RNase P ribonuclease P     

Precursors of the Cocoa-Specific Aroma Components are Derived from the Vicilin-Class (7S) Globulin of the Cocoa Seeds by Proteolytic Processing

Essential precursors of the cocoa-specific aroma components are formed during fermentation of cocoa seeds in the tropics. During the past decades, indications have accumulated that these aroma precursors are derived from seed proteins by acid-induced proteolysis. This has been recently corroborated by in vitro studies on the formation of the aroma-related components. It has been shown that the essential proteolysis products are derived from the vicilin-class (7S) globulins of the cocoa seeds by the cooperative action of an endogenous aspartic endoprotease and a carboxypeptidase.     

Radical Formation from the Reaction of Combustion Smoke with Diphenylamine

We have attempted to examine the effects of radical scavengers, such as amines and phenols, to trap gasphase radicals produced from the combustion of Poly (methyl methacrylate)(PMMA), which might cause damage to a living body, using an electron spin resonance (ESR) spin-trapping technique.

As a result, diphenylamine did not decrease the amount of radicals but rather increased it. It indicates that under the conditions of this study, gas-phase radicals were hardly trapped by radical scavengers and that the precursors to produce other kinds of radicals can exist.

It was suggested that from the experiments using several peroxides, the precursors should be diacylperoxides produced from the combustion of PMMA.     

Physico-chemical principles of cAMP-dependent protein phosphorylation. Catalysis of phosphoryl group transfer to nucleophilic agents

The specificity of pig brain protein kinase towards high- and low-Mr 'analogs' of protein substrate was studied. Solvolysis of the phosphoenzyme intermediate by various nucleophilic agents is shown. The transition state structure of the phosphoryl transfer reaction is discussed.     

The Deoxynucleoside Triphosphate Triphosphohydrolase Activity of SAMHD1 Protein Contributes to the Mitochondrial DNA Depletion Associated with Genetic Deficiency of Deoxyguanosine Kinase

The dNTP triphosphohydrolase SAMHD1 is a nuclear antiviral host restriction factor limiting HIV-1 infection in macrophages and a major regulator of dNTP concentrations in human cells. In normal human fibroblasts its expression increases during quiescence, contributing to the small dNTP pool sizes of these cells. Down-regulation of SAMHD1 by siRNA expands all four dNTP pools, with dGTP undergoing the largest relative increase. The deoxyguanosine released by SAMHD1 from dGTP can be phosphorylated inside mitochondria by deoxyguanosine kinase (dGK) or degraded in the cytosol by purine nucleoside phosphorylase. Genetic mutations of dGK cause mitochondrial (mt) DNA depletion in noncycling cells and hepato-cerebral mtDNA depletion syndrome in humans. We studied if SAMHD1 and dGK interact in the regulation of the dGTP pool during quiescence employing dGK-mutated skin fibroblasts derived from three unrelated patients. In the presence of SAMHD1 quiescent mutant fibroblasts manifested mt dNTP pool imbalance and mtDNA depletion. When SAMHD1 was silenced by siRNA transfection the composition of the mt dNTP pool approached that of the controls, and mtDNA copy number increased, compensating the depletion to various degrees in the different mutant fibroblasts. Chemical inhibition of purine nucleoside phosphorylase did not improve deoxyguanosine recycling by dGK in WT cells. We conclude that the activity of SAMHD1 contributes to the pathological phenotype of dGK deficiency. Our results prove the importance of SAMHD1 in the regulation of all dNTP pools and suggest that dGK inside mitochondria has the function of recycling the deoxyguanosine derived from endogenous dGTP degraded by SAMHD1 in the nucleus.     

Rapid chemical reaction techniques developed for use in investigations of membrane-bound proteins (neurotransmitter receptors)

New techniques for investigating chemical reactions on cell surfaces in the microsecond-to-millisecond time region are described. Reactions mediated by membrane-bound neurotransmitter receptors that control signal transmission between 10¹² cells of the nervous system are taken as an example. Cells with receptors on their plasma membrane are equilibrated with photolabile, biologically inactive precursors of the neurotransmitters. Photolysis of these compounds releases free neurotransmitter that interacts with the receptors, leading to the transient opening of transmembrane receptor-formed channels that are permeant to small inorganic ions. The current thus induced can be measured. The technique can be used to measure the elementary steps of the receptor-mediated reactions. To illustrate the approach it was shown that an understanding of the mechanism of inhibition of the nicotinic acetylcholine receptor by the drug cocaine was obtained and led to the first proof that compounds exist that alleviate the inhibition.     

Role of DNMT3B in the regulation of early neural and neural crest specifiers

The de novo DNA methyltransferase DNMT3B functions in establishing DNA methylation patterns during development. DNMT3B missense mutations cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. The restriction of Dnmt3b expression to neural progenitor cells, as well as the mild cognitive defects observed in ICF patients, suggests that DNMT3B may play an important role in early neurogenesis. We performed RNAi knockdown of DNMT3B in human embryonic stem cells (hESCs) in order to investigate the mechanistic contribution of DNMT3B to DNA methylation and early neuronal differentiation. While DNMT3B was not required for early neuroepithelium specification, DNMT3B deficient neuroepithelium exhibited accelerated maturation with earlier expression, relative to normal hESCs, of mature neuronal markers (such as NEUROD1) and of early neuronal regional specifiers (such as those for the neural crest). Genome-wide analyses of DNA methylation by MethylC-seq identified novel regions of hypomethylation in the DNMT3B knockdowns along the X chromosome as well as pericentromeric regions, rather than changes to promoters of specific dysregulated genes. We observed a loss of H3K27me3 and the polycomb complex protein EZH2 at the promoters of early neural and neural crest specifier genes during differentiation of DNMT3B knockdown but not normal hESCs. Our results indicate that DNMT3B mediates large-scale methylation patterns in hESCs and that DNMT3B deficiency in the cells alters the timing of their neuronal differentiation and maturation.     

Chromatography of human urinary erythropoietin and granulocyte colony-stimulating factor on insolubilized phytohaemagglutinin

Human urinary erythropoietin was adsorbed to phytohaemagglutinin coupled to agarose or porous glass and quantitatively eluted by a saturated solution of MgCl₂. This method provides a means of separating erythropoietin from several of its contaminants, presumably on the basis of its carbohydrate side chains. Erythropoietin which had been purified by chromatography on insolubilized phytohaemagglutinin was sufficiently free of toxicity to be assayable in tissue culture even when crude urine was used as a starting material.