[Nephron epithelial changes of the obstructive kidney in unilateral ureteral obstruction (experimental study)].

INTRODUCTION: The high prevalence of kidney diseases caused by urinary tract obstruction has led to the need for experimental studies of the dynamics of pathological processes in their lesions. Despite the fact that the general patterns of development of obstructive uropathy are known, the features of renal tissue damage, in particular structural and molecular biological changes in this pathology, remain insufficiently studied. OBJECTIVE: to study the dynamics of changes in the phenotype of epithelial cells of the nephron of an obstructive kidney with unilateral ureteral obstruction using an experimental model. MATERIALS AND METHODS: The experimental study was carried out on the basis of the Rostov State Medical University. The model of unilateral ureteral obstruction was reproduced in adult rabbits. The studies were carried out on the 7th, 14th and 21st days of complete obstruction of the left ureter. Immunophenotyping of obstructive kidney tissue samples was performed for markers of epithelial phenotype (cytokeratin 7, E-cadherin) and mesenchymal phenotype (vimentin, - smooth muscle actin). RESULTS: The sequence of changes in the phenotype of nephron epithelial cells during ureteral obstruction has been established. The first signs of an epithelial-mesenchymal transition (EMT) appear by day 7 in the form of a decrease in visualization of markers of the epithelial phenotype. On the 14th day, the expression of both epithelial and mesenchymal markers is noted. Significant changes in the phenotype of nephron epithelial cells: loss of epithelial markers (cytokeratin 7, E-cadherin) and the acquisition of mesenchymal markers (vimentin, - smooth muscle actin), are noted by the 21st day of the experiment. CONCLUSION: An experimental model of unilateral ureteral obstruction revealed the transformation of the nephron tubule cell phenotype from epithelial to mesenchymal.

Differential actinodin1 regulation in zebrafish and mouse appendages.

The fin-to-limb transition is an important evolutionary step in the colonization of land and diversification of all terrestrial vertebrates. We previously identified a gene family in zebrafish, termed actinodin, which codes for structural proteins crucial for the formation of actinotrichia, rigid fibrils of the teleost fin. Interestingly, this gene family is absent from all tetrapod genomes examined to date, suggesting that it was lost during limb evolution. To shed light on the disappearance of this gene family, and the consequences on fin-to-limb transition, we characterized actinodin regulatory elements. Using fluorescent reporters in transgenic zebrafish, we identified tissue-specific cis-acting regulatory elements responsible for actinodin1 (and1) expression in the ectodermal and mesenchymal cell populations of the fins, respectively. Mutagenesis of potential transcription factor binding sites led to the identification of one binding site crucial for and1 expression in ectodermal cells. We show that these regulatory elements are partially functional in mouse limb buds in a tissue-specific manner. Indeed, the zebrafish regulatory elements target expression to the dorsal and ventral ectoderm of mouse limb buds. Absence of expression in the apical ectodermal ridge is observed in both mouse and zebrafish. However, cells of the mouse limb bud mesoderm do not express the transgene, in contrast to zebrafish. Altogether these results hint for a change in regulation of and1 during evolution that led to the downregulation and eventual loss of this gene from tetrapod genomes.

[Connexin 43 expression in human brain glial tumors]. / Ékspressiia konneksina 43 v glial'nykh opukholiakh golovnogo mozga cheloveka.

AIM: Тo conduct an immunohistochemical (IHC) study of the expression of connexin 43 in the samples of glial tumors of various grades: gemistocytic astrocytomas (Grade 2), oligodendrogliomas (Grade 2) and glioblastomas (Grade 4). MATERIAL AND METHODS: The material investigated was fragments of human brain glial tumors (grade 2 gemistocytic astrocytomas (n=2), grade 2 oligodendrogliomas (n=2), and grade 4 glioblastomas (n=14) and those of tumor-surrounding tissue (n=4). The material was fixed in 10% buffered formalin, dehydrated, and embedded in paraffin according to the standard technique. IHC studies of the slices applied primary rabbit polyclonal antibodies against connexin 43 ('Spring Bioscience', USA) and the Dako EnVision + Peroxidase (DAB) visualization system ('Dako', Denmark). After the immunohistochemical reaction, the cell nuclei were stained with Mayer's hematoxylin. RESULTS: Immunohistochemistry showed the changing pattern of connexin 43 expression as compared with intact tissue in the glial tumors. Instead of the fine-granular expression in the thin cellular processes in the neuropil, the tumors mainly displayed a coarse-grained cytoplasmic and even nuclear reaction. The morphology and localization of positive structures depended on the variant of an examined tumor. In addition, the most malignant brain gliomas generally exhibited a reduction in the expression of connexin 43, i.e. its quantity is inversely proportional to the degree of malignancy of the tumor. CONCLUSION: The low connexin 43 expression levels may reflect both a reduction in astroglial functional gap junctions and semicanals and a decrease in the amount of the protein itself that has independently antioncogenic properties. The observed cytoplasmic and nuclear expression of connexin 43 is most likely to be associated with the aberrant activity of a number of kinases, such as proto-oncogene tyrosine-kinase Src or protein kinase C (PKC).

[Antimicrobial resistance of uropathogens in patients with acute obstructive pyelonephritis].

RELEVANCE: Acute pyelonephritis is known to be the most complicated and severe urinary tract infection occurring in all age groups and accounting for 14% of all kidney diseases. The generally recognized standard antibiotic therapy cannot completely prevent the progression of the disease to its chronic form after relief of its acute manifestations thus leading to a high incidence of relapses. The aim of our study was to investigate the spectrum of uropathogens and their antibiotic sensitivity in acute obstructive pyelonephritis. MATERIALS AND METHODS: The study comprised 72 patients who underwent semi-rigid ureteroscopy and ultrasonic lithotripsy for ureteral stones. In all patients, bladder urine samples collected by a transurethral catheter were tested bacteriologically using an extended set of culture media within 3 hours after hospital admission. Antibiotics used in antibiotic sensitivity testing for all uropathogens, were grouped into 4 classes (carbapenems, fluoroquinolones, cephalosporins, penicillins). Etiotropic treatment was started upon the availability of the spectrum of microbial patterns, the level of bacteriuria and antibioticogram of uropathogens, 5-6 days after administering initial empirical antibiotic therapy. RESULTS: The study patients had a high detection rate (83.3%) of canonical uropathogens in the bladder urine identified due to using an extended set of culture media, with a bacteriuria of more or equal 103 CFU/mL. Given the results of local antibiograms, a rational antimicrobial therapy should include carbapenems, namely ertapenem or meropenem as initial empirical antibiotics. Using fluoroquinolones as the first line treatment can lead to an inadequate effect in 15.0 to 67.0% of the cases. The findings of the antibiotic resistance testing of uropathogens to cephalosporins and semisynthetic penicillins showed that they should not be used as initial empirical antibiotic therapy for acute obstructive pyelonephritis in the given department of urology.

[PECULIARITIES OF THE STRUCTURAL ORGANIZATION OF VENTRAL POSTEROMEDIAL, VENTRAL POSTEROLATERAL, AND RETICULAR NUCLEI OF RAT THALAMUS (AN IMMUNOHISTOCHEMICAL STUDY)

The aim of this work was an immunohistochemical study of the expression of neuronal and glial proteins, and of gap junctions proteins (connexin 36, connexin 43) in ventral posteromedial (VPMN), ventral posterolateral (VPLN) and reticular (RТN) nuclei of the thalamus in rats. It was found that VPMN and VPLN of the thalamus were characterized by a homogeneous distribution of synaptophysin, grouped arrangement of astrocytes, horizontal orientation of somatostatincontaining myelinated and unmyelinated nerve fibers, forming the bundles, and running through the barreloid septum, expression of connexin 36 and 43 as well as of parvalbumin revealing barreloids in 4 µm-thick sections. In RTN the content of myelin basic protein, neurofilaments, parvalbumin, and somatostatin was increased, while the amount of glial fibrillary acidic protein and connexin 43 was moderate, and synaptophysin and connexin 36 were absent.

Regional expression of three homeobox transcripts in the inner ear of zebrafish embryos.

The inner ear of all jawed vertebrates arises from the epithelium of the otic vesicle and contains three semicircular canals, otoliths, and sets of sensory neurons, all positioned precisely within the cranium to detect head orientation and movement. The msh-C gene and two new homebox genes, msh-D and a gene related to distal-less, dlx-3, are each expressed in distinct regions of the otic vesicle during its early development in zebrafish embryos. Cells in the ectoderm express dlx-3 before induction of the otic vesicle, suggesting that dlx-3 has an early function in this process. Later, cells aligned with the future axes of the semicircular canals specifically express either dlx-3 or msh-D. Even later, sensory hair cells express msh-C and msh-D, while other cells of the epithelium express dlx-3. The early expression of these genes could specify the orientation and morphogenesis of the inner ear, whereas their later expression could specify the fates of particular cell types.

Position dependence of hemiray morphogenesis during tail fin regeneration in Danio rerio.

The fins of actinopterygian can regenerate following amputation. Classical papers have shown that the ray, a structural unit of these fins, might regenerate independent of this appendage. Each fin ray is formed by two apposed contralateral hemirays. A hemiray may autonomously regenerate and segmentate in a position-independent manner. This is observed when heterotopically grafted into an interray space, after amputation following extirpation of the contralateral hemiray or when simply ablated. During this process, a proliferating hemiblastema is formed, as shown by bromodeoxyuridine incorporation, from which the complete structure will regenerate. This hemiblastema shows a patterning of gene expression domain similar to half ray blastema. Interactions between contralateral hemiblastema have been studied by recombinant rays composed of hemirays from different origins on the proximo-distal or dorso-ventral axis of the caudal fin. Dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocianine perchlorate labeling of grafted tissues was used as tissular marker. Our results suggest both that there are contralateral interactions between hemiblastema of each ray, and that hemiblastema may vary its morphogenesis, always differentiating as their host region. These non-autonomous, position-dependent interactions control coordinated bifurcations, segment joints and ray length independently. A morphological study of the developing and regenerating fin of another long fin mutant zebrafish suggests that contralateral hemiblastema interactions are perturbed in this mutant.

Developmental effects of ectopic expression of the glucocorticoid receptor DNA binding domain are alleviated by an amino acid substitution that interferes with homeodomain binding.

Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor family through their association with heat shock proteins and immunophilins in the absence of ligands. Heat shock protein association represses steroid receptor DNA binding and protein-protein interactions with other transcription factors and facilitates hormone binding. In this study, we investigated the hormone-dependent interaction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domains of octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our results indicate that the GR DBD binds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several other homeodomain proteins. As these results suggest that the determinants for binding to the GR DBD are conserved within the homeodomain, we examined whether the ectopic expression of GR DBD peptides affected early embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severely affected their development, beginning with a delay in the epibolic movement during the blastula stage and followed by defects in convergence-extension movements during gastrulation, as revealed by the abnormal patterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GR peptide with a point mutation that disrupted homeodomain binding or with mRNA encoding the DBD of the closely related mineralocorticoid receptor, which does not bind octamer factors, developed normally. Moreover, coinjection of mRNA encoding the homeodomain of Oct-2 completely rescued embryos from the effects of the GR DBD. These results highlight the potential of DNA-independent effects of GR in a whole-animal model and suggest that at least some of these effects may result from direct interactions with homeodomain proteins.

Vladimir Mikhailovich Bekhterev.

V.M. Bekhterev (1857-1927) was an outstanding Russian neurologist, psychiatrist, psychologist, morphologist, physiologist, and public figure, who authored over 1000 scientific publications and speeches. At the beginning of the twentieth century he created a new multidimensional multidisciplinary scientific branch - psychoneurology, which included the objective knowledge of the anatomy and physiology of the nervous system, psychology, psychiatry, neurology, philosophy, sociology, pedagogy, and other disciplines. Psychoneurology in V.M. Bekhterev's understanding has furthered the introduction into the idea of a "biosocial" essence of man of a third - psychological - component, thus having created a "biopsychosocial" model in the interpretation of human diseases.

The history of neurology in St. Petersburg.

This article expounds the history of the formation and development of neurology in St. Petersburg and emphasizes the original character of St. Petersburg school of neurology. The authors state that many prominent neurologists of St. Petersburg dedicated their work to the development of neurological concepts and have made an important contribution to different areas of neurology, including vascular and demyelinating diseases, diseases of the peripheral nervous system, neuroinfections, epilepsy, etc.

Specific craniofacial cartilage dysmorphogenesis coincides with a loss of dlx gene expression in retinoic acid-treated zebrafish embryos.

Treatments of zebrafish embryos with retinoic acid (RA), a substance known to cause abnormal craniofacial cartilage development in other vertebrates, result in dose- and stage-dependent losses of dlx homeobox gene expression in several regions of the embryo. Dlx expression in neural crest cells migrating from the hindbrain and in the visceral arch primordia is particularly sensitive to RA treatment. The strongest effects are observed when RA is administered prior to or during crest cell migration but effects can also be observed if RA is applied when the cells have entered the primordia of the arches. Losses of dlx expression correlate either with the loss of cartilage elements originating from hindbrain neural crest cells or with abnormal morphology of these elements. Cartilage elements that originate from midbrain neural crest cells, which do not express dlx genes, are less affected. Taken together with the observation that the normal patterns of visceral arch dlx expression just prior to cartilage condensation resemble the morphology of the cartilage elements that are about to differentiate, our results suggest that dlx genes are an important part of a multi-step process in the development of a subset of craniofacial cartilage elements.

Gene expression analysis on sections of zebrafish regenerating fins reveals limitations in the whole-mount in situ hybridization method.

The caudal fin of adult zebrafish is used to study the molecular mechanisms that govern regeneration processes. Most reports of gene expression in regenerating caudal fins rely on in situ hybridization (ISH) on whole-mount samples followed by sectioning of the samples. In such reports, expression is mostly confined to cells other than those located between the dense collagenous structures that are the actinotrichia and lepidotrichia. Here, we re-examined the expression of genes by performing ISH directly on cryo-sections of regenerates. We detected expression of some of these genes in cell types that appeared to be non-expressing when ISH was performed on whole-mount samples. These results demonstrate that ISH reagents have a limited capacity to penetrate between the regenerating skeletal matrices and suggest that ISH performed directly on fin sections is a preferable method to study gene expression in fin regenerates.

Characterization of two new zebrafish members of the hedgehog family: atypical expression of a zebrafish indian hedgehog gene in skeletal elements of both endochondral and dermal origins.

We have characterized two new members of the Hedgehog (Hh) family in zebrafish, ihha and dhh, encoding for orthologues of the tetrapod Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh) genes, respectively. Comparison of ihha and Type X collagen (col10a1) expression during skeletal development show that ihha transcripts are located in hypertrophic chondrocytes of cartilaginous elements of the craniofacial and fin endoskeleton. Surprisingly, col10a1 expression was also detected in cells forming intramembranous bones of the head and in flat cells surrounding cartilaginous structures. The expression of col10a1 in both endochondral and intramembranous bones reflects an atypical composition of the extracellular matrix of the zebrafish craniofacial skeleton. In addition, during fin ray regeneration, both ihha and col10a1 are detected in scleroblasts, osteoblast-like cells secreting the matrix of the dermal bone fin ray. The presence of cartilage markers suggests that the dermal fin ray possesses an intermediate phenotype between cartilage and bone.

Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblasts differentiation and function.

The zebrafish caudal fin provides a simple model to study molecular mechanisms of dermal bone regeneration. We previously showed that misexpression of Bone morphogenetic protein 2b (Bmp2b) induces ectopic bone formation within the regenerate. Here we show that in addition to bmp2b and bmp4 another family member, bmp6, is involved in fin regeneration. We further investigated the function of BMP signaling by ectopically expressing the BMP signaling inhibitor Chordin which caused: (1) inhibition of regenerate outgrowth due to a decrease of blastema cell proliferation and downregulation of msxb and msxC expression and (2) reduced bone matrix deposition resulting from a defect in the maturation and function of bone-secreting cells. We then identified targets of BMP signaling involved in regeneration of the bone of the fin rays. runx2a/b and their target col10a1 were downregulated following BMP signaling inhibition. Unexpectedly, the sox9a/b transcription factors responsible for chondrocyte differentiation were detected in the non-cartilaginous fin rays, sox9a and sox9b were not only differentially expressed but also differentially regulated since sox9a, but not sox9b, was downregulated in the absence of BMP signaling. Finally, this analysis revealed the surprising finding of the expression, in the fin regenerate, of several factors which are normally the signatures of chondrogenic elements during endochondral bone formation although fin rays form through dermal ossification, without a cartilage intermediate.

Anterior duplication of the Sonic hedgehog expression pattern in the pectoral fin buds of zebrafish treated with retinoic acid.

The Sonic hedgehog gene has been identified as a candidate for the signal mediating the function of the zone of polarizing activity (ZPA) during limb development in tetrapods. To better understand the early steps of development of paired fin buds in fish, we have analyzed the regulation of the zebrafish Sonic hedgehog gene (shh/vhh-1) in response to retinoic acid. Systemic administration of retinoic acid (RA) to zebrafish embryos during the initial stages of pectoral fin bud development resulted in the induction of ectopic expression of shh/vhh-1 on the anterior margin of the bud under the apical ectodermal ridge and in abnormal pectoral fin bud morphology. RA treatment also resulted in ectopic shh/vhh-1 expression in floor plate cells at the caudal end of the neural keel. These results suggest that the control of ZPA function during the initial stages of development of paired appendages has been conserved between fish and tetrapods.

[Adaptive biocontrol in the system of treating epilepsy patients]. / Adaptivnoe bioupravlenie v sisteme lecheniia bol'nykh épilepsiei.

Adjuvant treatment of seizures by means of adaptive bioregulation was developed on the basis of material comprising 53 epileptic patients. The onset of auras or predictors of seizures was the signal to begin self-regulating activity. Method of suppression of paroxysmal epileptic activity in the moment of autogenic submersion of the patient is described. Efficacy of autogenic submersion was controlled by reduction of paroxysmal activity on EEG during EEG-examination in the hospital. The approach proved superior to chemotherapy alone.

Evolution of the immunoglobulin kappa light chain locus in the rabbit: evidence for differential gene conversion events.

The rabbit kappa light chain gene family is characterized by the presence of two constant region (C kappa) genes; the C kappa 1 gene encodes the constant region of the principal rabbit immunoglobulin light chain, the C kappa 2 gene being not or very poorly expressed in domestic rabbits. There exist four major K1 alleles (b4, b5, b6, and b9), which are unequally expressed in heterozygous rabbits at the K1 locus. Here, we compare the nucleotide sequences of the joining (J) clusters of the kappa light chain gene (J kappa) linked to the b4K2 locus and to the b4 and b9 alleles at the K1 locus. As for C kappa genes, there is evidence for intergenic conversion between the J kappa 1 and J kappa 2 clusters as well as maximum divergence in the expressed J segments. The b9 J kappa 1 cluster differs from its b4 counterpart in that two out of the five J kappa segments (J1 and J2) are expressed instead of only one. This implies that preferential expression of the b4 allele as compared to the b9 allele is not only correlated to the number of available J kappa pieces. The b9 J2 segment is functional in spite of the presence of a termination codon immediately upstream of its coding region. Two major structural differences were observed between the J-C intron sequences of the b9 and b4 alleles; namely a 160-base-pair deletion of an A + T-rich sequence in b9 (which also occurs in the K2 locus) and a 10-base-pair deletion plus some substitutions in the region corresponding to the mouse kappa intron activating element. These differences could underlie the lower transcriptional rate of the b9 allele.

Rearrangement of the immunoglobulin kappa light chain genes in a b4 rabbit and a Basilea rabbit.

The immunoglobulin chi light chain gene family of the rabbit is characterized by the presence of two constant region exons, C chi 1 and C chi 2 encoded at the chi 1 and chi 2 loci, and linked to their own cluster of joining pieces (J chi). The gene segments at the two loci are very unequally expressed. Thus, in domestic rabbits, the immunoglobulin light chains are essentially of the chi 1 type, even though the gene segments at the chi 2 locus are structurally functional. We have investigated the origin of the weak expression of the genes at the chi 2 locus by analysing the pattern of rearrangement of the chi 1 and chi 2 J chi segments in rabbit B-cell populations. Southern blot analysis of B cells isolated from a rabbit expressing chi 1 light chains suggests that the genes at the chi 2 locus underwent very few, if any, rearrangements. However, using more sensitive approaches, it was possible to detect transcripts originating from the rearranged chi 2 locus. In contrast, in B cells isolated from a Basilea rabbit, which cannot express chi 1 chains, Southern blots revealed the rearrangement of the chi 2 genes, whereas the chi 1 rearranged fragments were barely detectable. These results could be explained either by preferential rearrangement of genes at the chi 1 locus or by clonal amplification of only cells producing chi 1. Furthermore, results of Southern blot analysis provide evidence that V-J recombination may be accompanied by an inversion of the intervening DNA region.

Interallelic and intergenic conversion events could induce differential evolution of the two rabbit immunoglobulin kappa light chain genes.

Contrary to the situation in humans or mice, where the constant region (C) of the Immunoglobulin (Ig) kappa (kappa) light chain is encoded by a single gene, the rabbit possesses two C kappa genes: C kappa 1 and C kappa 2. However, in domestic rabbits, the vast majority of the immunoglobulins have a light chain of the kappa 1 isotype, which is expressed under four complex, highly divergent allelic forms: b4, b5, b6 and b9. In previous papers, we have shown that this high level of divergence was due, at least partly, to conversion events of the kappa 1 by the kappa 2 locus. Up to now, little was known about the evolution of the C kappa 2 gene. Here, we report sequences of the C kappa 2 genes in three different haplotypes, and show that, in contrast to the situation in the kappa 1 locus, the three analysed C kappa 2 alleles are identical (or only differing by one silent substitution). This suggests that intergenic conversion, which introduced most of the divergence in the kappa 1 locus, is not reciprocal and is unidirectional from kappa 2 towards kappa 1. To explain the small number of silent substitutions in the C kappa 2 gene and its remarkable conservation, we propose an extended model of multigenic family evolution, which postulates that gene conversion events occur between linked genes as well as between alleles.

Unique functional properties of a sensory neuronal P2X ATP-gated channel from zebrafish.

We report here the structural and functional characterization of an ionotropic P2X ATP receptor from the lower vertebrate zebrafish (Danio rerio). The full-length cDNA encodes a 410-amino acid-long channel subunit zP2X(3), which shares only 54% identity with closest mammalian P2X subunits. When expressed in Xenopus oocytes in homomeric form, ATP-gated zP2X(3) channels evoked a unique nonselective cationic current with faster rise time, faster kinetics of desensitization, and slower recovery than any other known P2X channel. Interestingly, the order of agonist potency for this P2X receptor was found similar to that of distantly related P2X(7) receptors, with benzoylbenzoyl ATP (EC(50) = 5 microM) >> ATP (EC(50) = 350 microM) = ADP > alpha,beta-methylene ATP (EC(50) = 480 microM). zP2X(3) receptors are highly sensitive to blockade by the antagonist trinitrophenyl ATP (IC(50) < 5 nM) but are weakly sensitive to the noncompetitive antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid. zP2X(3) subunit mRNA is exclusively expressed at high levels in trigeminal neurons and Rohon-Beard cells during embryonic development, suggesting that neuronal P2X receptors mediating fast ATP responses were selected early in the vertebrate phylogeny to play an important role in sensory pathways.