Distinct organization of adaptive immunity in the long-lived rodent Spalax galili.

A balanced immune response is a cornerstone of healthy aging. Here, we uncover distinctive features of the long-lived blind mole-rat (Spalax spp.) adaptive immune system, relative to humans and mice. The T-cell repertoire remains diverse throughout the Spalax lifespan, suggesting a paucity of large long-lived clones of effector-memory T cells. Expression of master transcription factors of T-cell differentiation, as well as checkpoint and cytotoxicity genes, remains low as Spalax ages. The thymus shrinks as in mice and humans, while interleukin-7 and interleukin-7 receptor expression remains high, potentially reflecting the sustained homeostasis of naive T cells. With aging, immunoglobulin hypermutation level does not increase and the immunoglobulin-M repertoire remains diverse, suggesting shorter B-cell memory and sustained homeostasis of innate-like B cells. The Spalax adaptive immune system thus appears biased towards sustained functional and receptor diversity over specialized, long-lived effector-memory clones-a unique organizational strategy that potentially underlies this animal's extraordinary longevity and healthy aging.

Cloning and characterization of serpin from red king crab Paralithodes camtschaticus.

Serpins are a family of serine protease inhibitors that are involved in numerous physiological processes and are known to regulate innate immunity pathways. To advance our understanding of their role in P. camtschaticus, a commercially significant species, we cloned and characterized a serpin from this species, designated serpin PC, that has anticoagulant and anticomplement effects on human blood. We found that serpin PC is a secreted protein with a typical serpin-like primary structure that is similar to other known crustacean serpins. Recombinant serpin PC was found to have inhibitory activity against R/K-specific bovine cationic trypsin. The reaction proceeds through the formation of a stable covalent complex of peptidase with P1 residue R383 of serpin PC. This interaction is characterized by a relatively high overall inhibition constant kass=(2.3 ±â€¯0.7) × 106 M-1s-1 and an SI of 4.7 ±â€¯0.8. Protein localization by western blotting showed that serpin PC is present in the muscles and, to a lesser extent, the heart, whereas it is transcribed predominantly in hemocytes and the heart. Through peptidase activity profiling of hemocytes and plasma, we found that serpin PC inhibits at least two R/K-specific activities and showed that it inhibits phenoloxidase (PO) activity induction in hemocytes.

[Application of the duplex-specific nuclease for fast analysis of single nucleotide polymorphisms and detection of target DNA in complex PCR products].

We have developed a simple method for fast analysis of single nucleotide polymorphisms and identification of target clones from cloned complex PCR products. The method utilizes Kamchatka crab duplex-specific nuclease and universal fluorescent probe and is alternative to laborious screening procedures using radioactive probes, restriction analysis followed by gel electrophoresis or expensive sequencing. The method efficacy was demonstrated in several model experiments.

[Preparation of prokaryotic cDNA for high-throughput transcriptome analysis].

High contents of non-coding RNA in total bacteria RNA complicates considerably transcriptome analysis using standard approaches like high-throughput sequencing, gene expression profiles, subtractive hybridization. We suggest a procedure of preparation of bacterial cDNA for transcriptomics that includes rRNA and tRNA depletion with preservation of relative abundance of coding sequences. The method is based on the second order hybridization kinetics and unique properties of Kanchatka crab duplex-specific nuclease. The method efficacy was demonstrated on a model experiments.

[Genetic screening among children with congenital and early childhood hearing loss].

Prevalence of mutations of genes of connexin 26, connexin 30 and mitochondrial DNA was studied among children with congenital and early childhood hearing loss. Screening with available methods of DNA diagnosis was conducted in some specialized schools. It was found that genetic disorders among children with congenital and early childhood hearing loss are rather frequent. Genetic consulting of parents with normal hearing carrying abnormal gene can help avoid birth of deaf children.

[A method for the preparation of normalized cDNA libraries enriched with full-length sequences].

We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, it effectively cleaves double-stranded DNA and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935-1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.

[DNA diagnosis in congenital and early childhood hypoacusis and deafness]. / DNK-diagnostika pri vrozhdennoi i rannei detskoi tugoukhosri i glukhote.

Congenital deafness affects 0.05-0.1% children. 90% of them were born from parents with normal hearing. There were no hearing defects in their families. In 70% of deafness cases deafness is the only symptom of the disease and is thought to be non-syndromal. Hypoacusis of unclear etiology may be congenital in 50% cases. Half of cases of severe autosomic-recessive non-syndromal hypoacusis (deafness) appear because of changes in only one gene--gene of connexine-26. Two thirds of the defects in this gene arise because of one mutation--35delG. Mean rate of this mutation carriage in Russia is over 2%. Identification of mutation 35delG in gene Cx26 is performed with the use of polymerase chain reaction. Genetic examination of 75 children with isolated hypoacusis (deafness) has detected deletion 35delG in both gene copies in 23 children (30%). 10 patients (13.4%) had mutation 35delG only in one copy of gene Cx26. Hearing defects in the latter may be related with the presence of another mutation in the same gene. A total of 33 patients (42%) carried deletion 35delG. According to our findings, the changed genotype is characterized primarily by bilateral neurosensory hypoacusis of the third-fourth degree. Weaker loss of hearing is rare. Thus, mutations in connexine gene26 present a problem for parents with normal hearing. Therefore, families with a deaf child should be referred for medicogenetic consultations.

[Sensorimotor neuropathy with X-linked dominant inheritance]. / Nasledstvennaia motorno-sensornaia nevropatiia s X-stseplennym dominantnym nasledovaniem.

Familial sensomotor neuropathy (FSMNX1) is reported to contribute significantly to FSMN spectrum. FSMNX1 is referred to FSMN type I but in respect to several signs is regarded as an intermediate one between type I and type II. A family including 26 patients in 5 generations has been described, 14 patients being examined in the study. Molecular genetic investigation showed that the disease was determined by mutation in connecsin gene 32 (Cx32). The mutation described is represented by a single nucleotide substitution 68T > C in codon 23 (GTA > GCA). A large proportion of presubclinical cases, preferentially in women, have been found in the family, that is in line with a type of inheritance. Substantial interfamilial disease polymorphism, especially by an age of onset, and symptoms rare for FSMN have been showed as well. Literature data on clinical genealogical, electrophysiological and molecular-genetic characteristics of FSMNX1 are analyzed comparing with own observations. Practical aspects of FSMNX1 diagnosis using DNA are discussed.