Dmitry V. Semenov

DNA-hydrolyzing activity of the light chain of IgG antibodies from milk of healthy human mothers

Various catalytically active antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Normal humans are generally considered to have no abzymes, since no obvious immunizing factors are present. Recently we have shown that IgG (its Fab and F(ab)2 fragments) from the milk of normal humans possesses DNase activity. Here we demonstrate for the first time that the light chain of IgG catalyzes the reaction of DNA hydrolysis. These findings speak in favor of the generation of abzymes in the tissue of healthy mothers, and since a mothers breast milk protects her infant from infections until the immune system is developed, they raise the possibility that these abzymes may contribute to this protective role.

Secretory immunoglobulin A from healthy human mothers' milk catalyzes nucleic acid hydrolysis.

The human milk secretory immune system is known to be the first line of protection for the newborn infant against various pathogens. Secretory IgA (sIgA), the typical immunoglobulin found in secretions, can fight infections through many mechanisms. Using different methods, we have shown that sIgA from the milk of healthy women possesses DNAse and RNAse activities. The catalytic center is localized in the light chain of catalytic sIgA, while the DNA-binding center is predominantly formed by its heavy chain. The enzymic properties and substrate specificity of catalytic sIgA distinguish it from other known DNases and RNases. It is reasonable to assume, that the milk DNA- and RNA-hydrolyzing antibodies are capable not only of neutralizing viral and bacterial nucleic acids by binding these antigens as well as by hydrolyzing them. The DNA-hydrolyzing activity of Abs raises the possibility that these catalytic Abs may provide protective functions for the newborn through the hydrolysis of viral and bacterial nucleic acids.

Catalytic DNA-and RNA-hydrolyzing antibodies from milk of healthy human mothers

Various catalytically active antibodies (Abs), or abzymes, have been detected recently in the sera of patients with autoimmune pathologies, in whom their presence is probably associated with autoimmunization. Normal humans are generally not considered to have abzymes, since no obvious immunizing factors are present. Here is shown by different methods that IgG from the milk of normal females possesses both DNase and RNase activities. The activities were also present in the IgG F(ab′)2 and Fab fragments.Affinity modification of IgG by the chemically reactive derivative of an oligonucleotide led to preferential modification of the L chain of IgG. After separation of the subunits by sodium dodecyl sulfate electrophoresis in a gel containing DNA, an in-gel assay showed DNase activity in the L chain. The L chain separated by affinity chromatography on DNA-cellulose was catalytically active. These findings speak in favor of the generation of cat alytic Abs by the immune system of healthy mothers. It is known that the treatment of adults with DNases and RNases offers protection from viral and bacterial diseases. Since breast milk protects the infants from infec tions until the immune system is developed, this raises the possibility that catalytic Abs like nucleases, may possess a protective role.

Human milk lactoferrin binds two DNA molecules with different affinities

Evidence is presented that lactoferrin (LF), an Fe3+‐binding glycoprotein, possesses two DNA‐binding sites with different affinities for specific oligonucleotides (ODNs) (K d1=8 nM; K d2∽0.1 mM). The high affinity site became labeled after incubation with affinity probes for DNA‐binding sites; like the antibacterial and polyanion‐binding sites, this site was shown to be located in the N‐terminal domain of LF. Interaction of heparin with the polyanion‐binding site inhibits the binding of ODNs to both sites. These data suggest that the DNA‐binding sites of LF coincide or overlap with the known polyanion and antimicrobial domains of the protein.

Regulatory Role of Small Nucleolar RNAs in Human Diseases

Small nucleolar RNAs (snoRNAs) are appreciable players in gene expression regulation in human cells. The canonical function of box C/D and box H/ACA snoRNAs is posttranscriptional modification of ribosomal RNAs (rRNAs), namely, 2′-O-methylation and pseudouridylation, respectively. A series of independent studies demonstrated that snoRNAs, as well as other noncoding RNAs, serve as the source of various short regulatory RNAs. Some snoRNAs and their fragments can also participate in the regulation of alternative splicing and posttranscriptional modification of mRNA. Alterations in snoRNA expression in human cells can affect numerous vital cellular processes. SnoRNA level in human cells, blood serum, and plasma presents a promising target for diagnostics and treatment of human pathologies. Here we discuss the relation between snoRNAs and oncological, neurodegenerative, and viral diseases and also describe changes in snoRNA level in response to artificial stress and some drugs.

Secretory immunoglobulin A from human milk catalyzes milk protein phosphorylation.

This article presents evidence that protein kinase activity is an intrin sic property of secretory immunoglobulin A (sIgA) from milk of healthy human mothers. Polyclonal sIgA was purified by sequential chromatog raphy on protein A-Sepharose, DEAE-cellulose, and gel filtration on Toyopearl HW-55 and Sepharose 4B columns. Its purity was established by one-and two-dimensional SDS-PAGE. The protein kinase activity was inhibited by specific antibodies (Abs) against sIgA, and was stable to acidic and alkaline conditions. Catalytic sIgA showed optimal reaction conditions (pH and MgCl2 concentration) and substrate specificity dif ferent from those of known protein kinases; i.e., sIgA phosphorylated the serine residues of various milk proteins in the presence of different γ-[32P]nucleoside-and deoxynucleoside-5’-triphosphates. The homoge neous Fab fragment of sIgA also showed kinase activity. An ATP-binding activity of fractions of sIgA was demonstrated by affinity chromatog raphy on ATP-Sepharose and by covalent binding of an affinity analog of ATP; this activity was mediated by the L chain of sIgA. The authors believe these observations are the first example of the catalytic activity of IgA Abs and of natural catalytic Abs with synthetic activity. In addition, the findings suggest the likelihood that catalytic Abs are generated by the immune system of healthy mothers.

Phosphorylation of different human milk proteins by human catalytic secretory immunoglobulin A

The ability of secretory immunoglobulin A (sIgA) from milk of healthy mothers to phosphorylate various milk proteins in the presence of γ‐[32P]‐ATP was shown to be a property of the antibodies. The polyclonal sIgA was purified by sequential chromatography on Protein‐A Sepharose and DEAE‐cellulose, and then separated by chromatography on the ATP‐Sepharose. The preparations containing all milk proteins except for protein kinases [integrated milk proteins (IMP)] were obtained by extraction of the kinase activities from the milk using their affinity to the insoluble crosslinked staphylococcus. Addition of sIgA fractions (having a different affinity to ATP) to the IMP led to phosphorylation of casein and several other milk proteins with different efficiencies.

Radionuclide transfer to reptiles

Reptiles are an important, and often protected, component of many ecosystems but have rarely been fully considered within ecological risk assessments (ERA) due to a paucity of data on contaminant uptake and effects. This paper presents a meta-analysis of literature-derived environmental media (soil and water) to whole-body concentration ratios (CRs) for predicting the transfer of 35 elements (Am, As, B, Ba, Ca, Cd, Ce, Cm, Co, Cr, Cs, Cu, Fe, Hg, K, La, Mg, Mn, Mo, Na, Ni, Pb, Po, Pu, Ra, Rb, Sb, Se, Sr, Th, U, V, Y, Zn, Zr) to reptiles in freshwater ecosystems and 15 elements (Am, C, Cs, Cu, K, Mn, Ni, Pb, Po, Pu, Sr, Tc, Th, U, Zn) to reptiles in terrestrial ecosystems. These reptile CRs are compared with CRs for other vertebrate groups. Tissue distribution data are also presented along with data on the fractional mass of bone, kidney, liver and muscle in reptiles. Although the data were originally collected for use in radiation dose assessments, many of the CR data presented in this paper will also be useful for chemical ERA and for the assessments of dietary transfer in humans for whom reptiles constitute an important component of the diet, such as in Australian aboriginal communities.

Unbiased approach to profile the variety of small non-coding RNA of human blood plasma with massively parallel sequencing technology.

Objective: Understanding structures of circulating RNA expands fundamental knowledge of cell communications and signaling pathways as well as allows developing new molecular diagnostic approaches. The aim of this study was to deploy a new approach to sequencing cDNA library construction which expands the capabilities of high-throughput sequencing analysis of small non-coding RNAs. With the approach, we performed massively parallel sequencing of human blood plasma RNA to document profile of common and peculiar RNA species normally circulating in blood of healthy individuals. Methods: Total RNA was extracted from blood plasma samples of eight apparently healthy individuals. To obtain comprehensive cDNA libraries RNA was dephosphorylated and then 5′-phosphorylated. 5′-Phosphorylated total plasma RNA was ligated with adapters, reverse transcribed and eight personalized cDNA libraries were constructed. Libraries were sequenced with SOLiD™ technology. Results/conclusion: Fragments of rRNA, mitochondrial transcripts, microRNAs, fragments of scRNAs, snRNA and snoRNA, fragments of several mRNAs as well as the set of newly discovered transcripts were found to be permanent representatives of human blood plasma RNAs. Advanced mapping allowed to identify circulating herpes virus and enterobacterial transcripts. Documented profile of circulating RNA of healthy individuals provides basis for development of new approaches in research and diagnosis of human pathology.

Characterisation of circulating DNA by parallel tagged sequencing on the 454 platform

BACKGROUND Fragments of genomic DNA that can be isolated from the blood and body fluids of vertebrates are also known as circulating DNA. This DNA has widely been investigated as a biomarker for cancer and other diseases but the origin and significance of circulating DNA have not been elucidated to date. METHODS We used a parallel tagged sequencing method to sequence circulating DNA obtained from control individuals as well as cancer patients on the GSFLX sequencer (454 life sciences). RESULTS Circulating DNA sequenced on one 16th of a picotiter plate produced approximately 3600 unique circulating DNA sequences which were distributed over the human genome and a higher frequency of mutations was observed in cancer patients compared to healthy controls. CONCLUSION Circulating DNA represents genomic DNA in the blood of an individual, some sequence related differences might be evident between circulating DNA from cancer individuals and controls but distribution over the genome is similar.