Alterations in the chromatin packaging, driven by transcriptional activity, revealed by AFM.

BACKGROUND: The gene expression differs in the nuclei of normal and malignant mammalian cells, and transcription is a critical initial step, which defines the difference. The mechanical properties of transcriptionally active chromatin are still poorly understood. Recently we have probed transcriptionally active chromatin of the nuclei subjected to mechanical stress, by Atomic Force Microscopy (AFM) [1]. Nonetheless, a systematic study of the phenomenon is needed. METHODS: Nuclei were deformed and studied by AFM. Non-deformed nuclei were studied by fluorescence confocal microscopy. Their transcriptional activity was studied by RNA electrophoresis. RESULTS: The malignant nuclei under the study were stable to deformation and assembled of 100-300 nm beads-like units, while normal cell nuclei were prone to deformation. The difference in stability to deformation of the nuclei correlated with DNA supercoiling, and transcription-depended units were responsive to supercoils breakage. The inhibitors of the topoisomerases I and II disrupted supercoiling and made the malignant nucleus prone to deformation. Cell nuclei treatment with histone deacetylase inhibitors (HDACIs) preserved the mechanical stability of deformed malignant nuclei and, at the same time, made it possible to observe chromatin decondensation up to 20-60 nm units. The AFM results were supplemented with confocal microscopy and RNA electrophoresis data. CONCLUSIONS: Self-assembly of transcriptionally active chromatin and its decondensation, driven by DNA supercoiling-dependent rigidity, was visualized by AFM in the mechanically deformed nuclei. GENERAL SIGNIFICANCE: We demonstrated that supercoiled DNA defines the transcription mechanics, and hypothesized the nuclear mechanics in vivo should depend on the chromatin architecture.

AFM imaging of the transcriptionally active chromatin in mammalian cells' nuclei.

BACKGROUND: Nuclear rigidity is traditionally associated with lamina and densely packed heterochromatin. Actively transcribed DNA is thought to be less densely packed. Currently, approaches for direct measurements of the transcriptionally active chromatin rigidity are quite limited. METHODS: Isolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM). RESULTS: Nuclei of the normal fibroblast cells were completely flattened under mechanical stress, whereas nuclei of the cancerous HeLa were extremely resistant. In the deformed HeLa nuclei, AFM revealed a highly-branched landscape assembled of ~400 nm closed-packed globules and their structure was changing in response to external influence. Normal and cancerous cells' isolated nuclei were strikingly different by DNA resistance to applied mechanical stress. Paradoxically, more transcriptionally active and less optically dense chromatin of the nuclei of the cancerous cells demonstrated higher physical rigidity. A high concentration of the transcription inhibitor actinomycin D led to complete flattening of HeLa nuclei, that might be related to the relaxation of supercoiled DNA tending to deformation. At a low concentration of actinomycin D, we observed the intermediary formation of stochastically distributed nanoloops and nanofilaments with different shapes but constant width ~ 180 nm. We related this phenomenon with partial DNA relaxation, while non-relaxed DNA still remained rigid. CONCLUSIONS: The resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM. GENERAL SIGNIFICANCE: A new outlook to studying internal nuclei structure is proposed.

Bifractal structure of chromatin in rat lymphocyte nuclei.

The small-angle neutron scattering (SANS) on the rat lymphocyte nuclei demonstrates the bifractal nature of the chromatin structural organization. The scattering intensity from rat lymphocyte nuclei is described by power law Q^{-D} with fractal dimension approximately 2.3 on smaller scales and 3 on larger scales. The crossover between two fractal structures is detected at momentum transfer near 10^{-1}nm^{-1}. The use of contrast variation (D_{2}O-H_{2}O) in SANS measurements reveals clear similarity in the structural organizations of nucleic acids (NA) and proteins. Both chromatin components show bifractal behavior with logarithmic fractal structure on the large scale and volume fractal with slightly smaller than 2.5 structure on the small scale. Scattering intensities from chromatin, protein component, and NA component demonstrate an extremely extensive range of logarithmic fractal behavior (from 10^{-3} to approximately 10^{-1}nm^{-1}). We compare the fractal arrangement of rat lymphocyte nuclei with that of chicken erythrocytes and the immortal HeLa cell line. We conclude that the bifractal nature of the chromatin arrangement is inherent in the nuclei of all these cells. The details of the fractal arrangement-its range and correlation/interaction between nuclear acids and proteins are specific for different cells and is related to their functionality.

[Left ventricular myocardial hypertrophy after transplantation of the kidney: risk factors and possible regress].

AIM: To study structural-functional changes in left ventricular (LV) myocardium in recipients of renal allograft (RA) after different postoperative period and to specify factors promoting persistence, progression or regression of LV hypertrophy (LVH). MATERIAL AND METHODS: The study included 240 recipients of primary RA (38% females and 62% males, age 16-69 years, mean age 42 +/- 11 years). A prospective study covered 143 patients. RESULTS: LVH was diagnosed in 52% patients. LVH incidence after renal transplantation (RT) had a wave-like dynamics: during 9 months after RT LVH presents in more than 50% patients; after 9-24 months after the operation it fell to 30% and after 3-7 years after the operation it affected at least 2/3 patients. After RT LVH risk factors were age, duration of chronic renal failure (CRF) and pretransplantation dialysis, reduced mass of the operating nephrons, arterial hypertension, anemia, functioning of arterio-venous fistula (AVF) and chronic inflammation syndrome. LVH was also associated with factors specific for RT: RA rejection crises, infections complicating massive immunosuppressive therapy. LVH is also associated with proteinuria which may indicate RA damage and can be considered as a marker of generalized endothelial dysfunction. 2-year and longer follow-up after RT confirmed complete LVH regression in 1/3 of the recipients. LVH regression was observed in normal RA function, normal blood pressure, the absence of proteinuria, hypoalbuminemia, anemia, AVF, infectious complications. CONCLUSION: LVH after RT is multifactorial and can completely regress in a favourable posttransplantation course.

Sodium ion cycle in bacterial pathogens: evidence from cross-genome comparisons.

Analysis of the bacterial genome sequences shows that many human and animal pathogens encode primary membrane Na+ pumps, Na+-transporting dicarboxylate decarboxylases or Na+ translocating NADH:ubiquinone oxidoreductase, and a number of Na+ -dependent permeases. This indicates that these bacteria can utilize Na+ as a coupling ion instead of or in addition to the H+ cycle. This capability to use a Na+ cycle might be an important virulence factor for such pathogens as Vibrio cholerae, Neisseria meningitidis, Salmonella enterica serovar Typhi, and Yersinia pestis. In Treponema pallidum, Chlamydia trachomatis, and Chlamydia pneumoniae, the Na+ gradient may well be the only energy source for secondary transport. A survey of preliminary genome sequences of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Treponema denticola indicates that these oral pathogens also rely on the Na+ cycle for at least part of their energy metabolism. The possible roles of the Na+ cycling in the energy metabolism and pathogenicity of these organisms are reviewed. The recent discovery of an effective natural antibiotic, korormicin, targeted against the Na+ -translocating NADH:ubiquinone oxidoreductase, suggests a potential use of Na+ pumps as drug targets and/or vaccine candidates. The antimicrobial potential of other inhibitors of the Na+ cycle, such as monensin, Li+ and Ag+ ions, and amiloride derivatives, is discussed.

[Rapidly progressive glomerulonephritis in ANCA-associated vasculitis: a course, treatment efficacy, prognosis].

AIM: To study efficacy of ANCA-RPGN treatment with corticosteroids and cyclophosphamide or mycophenolic acid drugs. MATERIAL AND METHODS: We treated 28 patients (17 males and 11 females aged 19-71 years) with morphologically verified ANCA-associated crescentic RPGN (crescentic median 79 (63:88)%. The patients received corticosteroids and cytostatics. RESULTS: The response to the treatment was registered in 22 (78%) patients in 8-16 weeks: a complete remission was achieved in 8 patients, a partial one--in 14 patients. In partial remission renal functions recovered incompletely (median Pcr 200 (180;255) mcmol/l) in persistence of moderate proteinuria (median 0.7 (0.6;1.3)g/day) and absence of microhematuria. Probability of the treatment success depended on severity of glomerulosclerosis and weakly depended on activity of extracapillary reaction. Severe renal failure was not an absolute predictor of treatment failure. CONCLUSION: In the absence of advanced nephrosclerosis early treatment with corticosteroid in combination with cytostatics can produce a positive effect in 70-80% patients with ANCA associated RPGN.

[Replacement renal therapy in complex treatment of severe acute pancreatitis].

The purpose of this study was to retrospectively analyze the efficiency of replacement renal therapy (RRT) in multimodality treatment for severe acute pancreatitis (SAP) concurrent with a systemic inflammatory response and multiple organ failure/dysfunction. The authors analyzed the results of treating 55 patients (14 women and 41 men) aged 22 to 72 years (mean 43.5 +/- 16.4 years) treated at the intensive care units of Moscow City Clinical Hospital Fifty-Two in January 1, 2000, to December 31, 2006. All the patients had multiple organ dysfunctions with the involvement of 2 to 5 organs (median 4 (3; 4) and were on RRT. RRT may be successfully used in multimodality treatment for SAP provided that the dose of dialysis is at least 35 ml/kg/hour. The severe condition rated by the APACHE HII and SAPS II scales and the dialysis dose of less than 35 ml/kg/hour are independent risk factors of death in SAP patients.

What can comparative genomics tell us about species concepts in the genus Aspergillus?

Understanding the nature of species" boundaries is a fundamental question in evolutionary biology. The availability of genomes from several species of the genus Aspergillus allows us for the first time to examine the demarcation of fungal species at the whole-genome level. Here, we examine four case studies, two of which involve intraspecific comparisons, whereas the other two deal with interspecific genomic comparisons between closely related species. These four comparisons reveal significant variation in the nature of species boundaries across Aspergillus. For example, comparisons between A. fumigatus and Neosartorya fischeri (the teleomorph of A. fischerianus) and between A. oryzae and A. flavus suggest that measures of sequence similarity and species-specific genes are significantly higher for the A. fumigatus - N. fischeri pair. Importantly, the values obtained from the comparison between A. oryzae and A. flavus are remarkably similar to those obtained from an intra-specific comparison of A. fumigatus strains, giving support to the proposal that A. oryzae represents a distinct ecotype of A. flavus and not a distinct species. We argue that genomic data can aid Aspergillus taxonomy by serving as a source of novel and unprecedented amounts of comparative data, as a resource for the development of additional diagnostic tools, and finally as a knowledge database about the biological differences between strains and species.

[Risk factors and pathogenetic mechanisms of left ventricular hypertrophy in progressive chronic kidney disease and after transplantation of the kidney].

AIM: To specify the trend in the incidence of left ventricular hypertrophy (LVH) at a predialysis stage of chronic kidney disease (CKD) in the course of its progression from stage III to stage V and after transplantation of the kidney (TK); to study correlations between homeostatic disorders caused by CKD progression and myocardial remodeling; to define the role of some hemodynamic and nonhemodynamic factors in formation of LVH. MATERIAL AND METHODS: The study enrolled 128 patients (58 males and 70 females, age 18-55 years, mean age 42 +/- 11 years) at a predialysis stage of CKD (group 1) and 225 recipients of renal allotransplant--RRA (group 2, 140 males and 85 females, age 18-69 years, mean age 43 +/- 12 years). General clinical examination, biochemical and immunological blood tests, echocardiography were made. RESULTS: At a predialysis stage of CKD, LVH was diagnosed in 56% patients. Incidence of LVH was directly related with age of the patients (p = 0.001), blood pressure (p < 0.001), duration of arterial hypertension (p = 0.004), severity of anemia (p = 0.017), the level of C-reactive protein (p = 0.003), blood phosphorus concentration and inversely correlated with glomerular filtration rate--GFR (p = < 0.001), albumin level (p = 0.023) and blood Ca (p < 0.001). LVH was followed up for 12 months in 35 patients with predialysis CKD. Factors of LVH progression and factors hindering its regression were systolic blood pressure, Hb and Ca in the blood. In group 2 of RRA incidence of LVH was 53%. Significant factors of LVH risk after transplantation were age (p = 0.002), hypertension (p = 0.005) and anemia (p = 0.04). Moreover, LVH closely correlated with proteinuria (p < 0.03), transplant dysfunction (p = 0.002) and posttransplantation ischemic heart disease (p < 0.037). Changes in LVH were analysed in 30 RRA. Frequency of LVH decreased for 2 years after transplantation (from 56 to 32%) but 36-60 and more months after transplantation it increased (46 and 64%, respectively). Transplant dysfunction was the leading factor hindering LVH regression after transplantation. CONCLUSION: The same mechanisms are involved in LVH pathogenesis after transplantation and at a predialysis stage of CKD. The significance of initial renal lesion signs--minimal proteinuria and hypercreatininemia--was higher after renal transplantation than in patients with CKD.

The COG database: new developments in phylogenetic classification of proteins from complete genomes.

The database of Clusters of Orthologous Groups of proteins (COGs), which represents an attempt on a phylogenetic classification of the proteins encoded in complete genomes, currently consists of 2791 COGs including 45 350 proteins from 30 genomes of bacteria, archaea and the yeast Saccharomyces cerevisiae (http://www.ncbi.nlm.nih. gov/COG). In addition, a supplement to the COGs is available, in which proteins encoded in the genomes of two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and shared with bacteria and/or archaea were included. The new features added to the COG database include information pages with structural and functional details on each COG and literature references, improvements of the COGNITOR program that is used to fit new proteins into the COGs, and classification of genomes and COGs constructed by using principal component analysis.

Plasmids for heterologous expression in Pasteurella haemolytica.

New cloning and expression vectors that replicate both in Pasteurella haemolytica and in Escherichia coli were constructed based on a native sulfonamide (SuR) and streptomycin (SmR) resistant plasmid of P. haemolytica called pYFC1. Each shuttle vector includes an MCS and a selectable antibiotic resistance marker that is expressed in both organisms. Plasmid pNF2176 carries the P. haemolytica ROB-1 beta-lactamase gene (blaP, ApR) and pNF2214 carries the Tn903 aph3 kanamycin resistance (KmR) element. The expression vector, pNF2176, was created by placing the MCS downstream of the sulfonamide gene promoter (PsulII) on pYFC1; this was used to clone and express the promoterless Tn9 chloramphenicol resistance gene (cat, CmR) in P. haemolytica (pNF2200). A promoter-probe vector (pNF2283) was constructed from pNF2200 by deleting PsulII.

Escherichia coli strain for thermoinducible T7 RNA polymerase-driven expression.

An Escherichia coli strain for thermoinducible T7Pol-driven transcription has been constructed. The strain was obtained by site-specific integration of the T7 gene 1 coding for T7Pol into the attB site of phage lambda in the E. coli chromosome. The expression of the inserted gene is regulated by cIts857 and major early promoter-operator regions of phage lambda.

[Cloning of the gene for extracellular Bacillus circulans RNAase]. / Klonirovanie gena vnekletochnoi RNKazy Bacillus circulans.

The gene for extracellular low molecular weight ribonuclease of Bacillus circulans BCF 247 was cloned. The strain was isolated from permafrost deposits of the Kolyma lowland. The gene for the ribonuclease from Bacillus intermedius (binase) was used as a specific probe. The cloning succeeded only in the E. coli strain producing the inhibitor of ribonuclease form Bacillus amyloliquefaciens. Selected clones secreted the active ribonuclease into the growth media. Deletion derivatives of the parental recombinant plasmid were constructed. The smallest DNA fragment which enclosed a functional ribonuclease gene in E. coli was determined to be 0.6 kb in length.

[Cloning the asd and lysC genes from Cornyebacterium glutamicum]. / Klonirovanie genov asd i LysC Corynebacterium glutamicum.

Plasmids carrying an asd gene from a mutant. S-(2-aminoaethyl)-L-cysteine resistant strain of Corynebacterium glutamicum were selected from a clonoteque constructed on a plasmid cloning vector pSL5 by complementation of asd mutation in Escherichia coli. Evidence has been obtained that the cloned chromosomal DNA fragment contains also a complete sequence for feed-back-resistant aspartokinase lysC gene.

[Cosmid libraries containing DNA from human chromosome 13]. / Issledovanie kosmidnykh bibliotek, soderzhashchikh DNK khromosomy 13 cheloveka.

We characterized two cosmid libraries constructed from flow-sorted chromosome 13 at the Imperial Cancer Research Fund (ICRF), UK (13,000 clones) and Los Alamos National Laboratory (LANL), USA (17,000 clones). After storage for two years, clones showed high viability (95%) and structural stability. EcoR I and Hind III restriction patterns were studied in more than 500 ICRF and 200 LANL cosmids. The average size of inserts was shown to be 35-37 kb in both the libraries. Most cosmids (83% and 93% of ICRF and LANL libraries, respectively) exceed the lower size limit of DNA fragments that can be packaged and represent a good source for physical mapping of chromosome 13. Total length of inserts is four and five genome equivalents in the ICRF and LANL libraries, respectively. ICRF cosmids showed hybridization to 22 of 24 unique probes tested, which corresponds to a 90% probability of having any DNA fragment represented in the library. More than 1 Mb of chromosome 13 is overlapped by 90 cosmids of 22 groups revealed. A chromosomal region of more than 150 kb, containing the ATP1AL1 gene for alpha-1 peptide of Na+, K(+)-ATPase, is covered by 12 cosmids forming a contig. The results of restriction and hybridization analyses are stored in a CLONE database. These data and all the cosmids described are publicly available.

[The importance of a new classification of chronic glomerulonephritis for determining the prognosis of the disease]. / Znachenie novoi klassifikatsii khronicheskogo glomerulonefrita dlia opredeleniia prognoza zabolevaniia.

Dependence of chronic glomerulonephritis upon its clinical type was studied on the basis of observation in 165 patients of chronic renal failure incidence during 7 years after the beginning of the disease.

[Disordered permeability of the glomerular filter for middle- and high-molecular serum proteins in various morphologic types and stages of chronic glomerulonephritis]. / Rasstroistvo pronitsaemosti klubochkovogo fil'tra dlia sredne- i vysokomolekuliarnykh syvorotochnykh belkov pri raznykh morfologicheskikh tipakh i stadiiakh khronicheskogo glomerulonefrita.

Investigation of glomerular filter permeability for serum immunoglobulins and albumins in 83 patients with chronic glomerulonephritis (CGN) with nephrotic proteinuria has shown that in mesangioproliferative glomerulonephritis permeability for IgM is rare, for IgA and IgG moderately raised; in a marked but not maximum degree of permeability for albumins, it is characterized by considerable variability. In maximum permeability for albumins there is a tendency to the reduction of the ratio of IgA and IgM clearances to albumin clearance, and variability of these indices. In membranous CGN the structure of nephron permeability for serum proteins is identical but it remains unchanged with a sharp rise of albumin fractional clearance. In mesangiocapillary CGN permeability for IgM is more frequent and enhanced for IgG. in focal-segmental glomerular hyalinosis/sclerosis and diffuse-fibroplastic GN permeability for IgG is considerably raised unrelated to a degree of permeability for albumins. Sclerotic changes are accompanied by elevated permeability for IgG.

[The prognostic significance of the morphological type of chronic glomerulonephritis and of the tubulointerstitial changes depending on the clinical type of the disease]. / Prognosticheskoe znachenie morfologicheskogo tipa khronicheskogo glomerulonefrita i tubulointerstitsial'nykh izmenenii v zavisimosti ot klinicheskogo tipa zabolevaniia.

The review of 200 cases of chronic glomerulonephritis (CG) led the authors to the conclusion that there is a statistically significant relationship between rapid progression (RP) of the disease (onset of chronic renal failure within 7 years since the diagnosis) and its morphological type (chi 2 = 37), tubulointerstitial changes (chi 2 = 34; p < 0.0000), clinical disease types according to M. Ia. Ratner, V. V. Serov et al. (chi 2 = 115; p < 0.0000). In both prognostically favourable and unfavorable morphological types RP occurred more frequently in concurrent unfavorable clinical types (chi 2 = 19; p < 0.0001). In prognostically unfavorable morphological types there were, as a rule, unfavorable clinical types, whereas in favorable ones-favorable clinical types. In the presence of tubulointerstitial changes RP occurred primarily in unfavorable clinical types which are encountered in these morphological changes significantly more frequently than in their absence (chi 2 = 48; p < 0.01). RP of CG in prognostically unfavorable morphological types and tubulointerstitial changes depends mainly on accompanying clinical types of CG.

[Prediction of accelerated progression of chronic glomerulonephritis basing on clinical and histomorphological data]. / Prognozirovanie uskorennogo progressirovaniia khronicheskogo glomerulonefrita na osnovanii klinicheskikh i gistomorfologicheskikh dannykh.

AIM: To find out predictive value of three factors in progression of chronic glomerulonephritis (CGN): unfavorable clinical course, unfavorable morphological type and tubulointerstitial changes. MATERIALS AND METHODS: 150 CGN patients entered the trial. Frequency of onset of chronic renal failure (CRF) within 7 years after the diagnosis was chosen as a criterium of accelerated progression of CGN (AP CGN). Chi-square criterium was used for testing relationships between AP CGN and the parameters under study. RESULTS: The findings support previously published data on statistically more frequent occurrence of AP CGN in unfavorable clinical types (active nephritic and nephrotically-hypertensive), in unfavorable morphological types (mesangiocapillary CGN and focal-segmental hyalinosis/sclerosis and tubulointerstitial lesions). In unfavorable clinical types there was a significantly more frequent occurrence of AP CGN irrespective of unfavorable morphological changes. In contrast, both in unfavorable and favorable clinical types, frequency of AP CGN in unfavorable morphological types of CGN and tubulointerstitial changes was the same. CONCLUSION: Clinical type of CGN is a valuable prognostic criterium for AP CGN.

[Diagnostic value of determining neutral alpha-glucosidase and N-acetyl-beta-D-hexosaminidase activity in the urine in kidney pathology]. / Diagnosticheskoe znachenie opredeleniia aktivnosti neitral'noi alpha-gliukozidazy i N-atsetil-beta-D-geksozaminidazy v moche pri patologii pochek.

As shown in studies of 123 patients with various nephropathies, activities of alpha-glucosidase and N-acetyl-beta-D-hexosaminidase in urine of the patients correlated with severity and stages of these diseases; at the same time, simultaneous estimation of both enzymatic activities in urine was shown to be more informative in diagnostic of kidney impairments. Low values of correlation coefficient (r = 0.35 +/- 0.09) between daily excretion of protein and the activity of neutral alpha-glucosidase in urine showed that the enzyme activity did not depend on proteinuria and was independent test for kidney impairment. Estimation of alpha-glucosidase activity could be used for control of the therapy; the enzymatic activity in urine correlated distinctly with the clinico-laboratory patterns of the patients studied.