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.

[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.

[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.

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.

[A renoprotective effect of enalapril in chronic transplantation nephropathy]. / Renoprotektivnii éffekt énalaprila pri khronicheskoi transplantatsionnoi nephropatii.

AIM: To study safety and efficacy of ACE inhibitor enalapril in chronic transplantation nephropathy (CTN) as well as nephroprotective efficacy of this drug in various clinical variants of CTN. MATERIAL AND METHODS: A retrospective study covered 220 recipients with CRF. The patients were divided into the study group (n = 103) and the control group (n = 117). The study group was given ACE inhibitor enalapril the efficacy of which was assessed by arterial pressure (systolic, diastolic, mean) dynamics, 24 h proteinuria and the rate of CTN progression. This rate was suggested by probability of plasm creatinin doubling (Kaplan-Meier technique). RESULTS: Enalapril significantly inhibited CTN progression running with minimal or marked proteinuria, had a pronounced hypotensive effect, promoted stabilization of minimal proteinuria (in CTN with minimal proteinuria) or reduction of protein excretion (in a proteinuric variant of CTN). CONCLUSION: Use of enalapril in CTN in a daily dose 10 mg maximum is safe and can be used for inhibition of CTN progression.

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.

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.

Inactivation of Pasteurella (Mannheimia) haemolytica leukotoxin causes partial attenuation of virulence in a calf challenge model.

The leukotoxin of Pasteurella (Mannheimia) haemolytica is believed to play a significant role in pathogenesis, causing cell lysis and apoptosis that lead to the lung pathology characteristic of bovine shipping fever. Using a system for Cre-lox recombination, a nonpolar mutation within the lktC transacylase gene of the leukotoxin operon was created. The lktC locus was insertionally inactivated using a loxP-aph3-loxP cassette, and then the aph3 marker was excised from the chromosome by Cre recombinase expressed from a P. haemolytica plasmid. The resulting lktC strain (SH2099) secretes inactive leukotoxin and carries no known antibiotic resistance genes. Strain SH2099 was tested for virulence in a calf challenge model. We inoculated 3 x 10(8) or 3 x 10(9) CFU of wild-type or mutant bacteria into the lungs of healthy, colostrum-deprived calves via transthoracic injection. Animals were observed for clinical signs and for nasal colonization for 4 days, after which they were euthanized and necropsied. The lower inoculum (3 x 10(8) CFU) caused significantly fewer deaths and allowed lung pathology to be scored and compared, while the 3 x 10(9) CFU dose of either the wild-type or mutant was lethal to >/=50% of the calves. The estimated 50% lethal dose of SH2099 was four times higher than that of the wild-type strain. Lung lesion scores were reduced twofold in animals inoculated with the mutant, while clinical scores were nearly equivalent for both strains. The wild-type and mutant strains were equally capable of colonizing the upper respiratory tracts of the calves. In this study, the P. haemolytica lktC mutant was shown to be less virulent than the parent strain.

[Histomorphological tubulointerstitial changes in progression of chronic glomerulonephritis]. / Gistomorfologicheskie tubulointerstitsial'nye izmeneniia pri progressirovanii khronicheskogo glomerulonefrita

The role of histological changes associated with primary types of glomerulonephritides in progression of chronic glomerulonephritis (CGN) is analysed. It is shown that CGN progression acceleration occurs much more frequently if chronic renal failure arises within 7 years since the disease onset, in the presence of concomitant tubulointerstitial changes (TIC)in unfavorable morphological types--mesangiocapillary glomerulonephritis and focal-segmental hyalinosis/ sclerosis. It was found that more frequent occurrence of CGN accelerated progression in unfavorable morphological types and TIC depends on the presence of unfavorable clinical types--active nephritic and nephrotic-hypertensive types (classification of M. Ya. Ratner et al.). In TIC there were primarily unfavorable clinical types which contribute to accelerated progression of CGN. This relationship is explained by involvement of factors belonging to the above clinical types in the mechanism of the morphological changes. In these clinical types CGN accelerated progression takes place irrespectively of the presence of TIC and unfavorable morphological types.

[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.

[Implication of clinical and morphological types of chronic glomerulonephritis, tubulointerstitial changes in prognosis of disease progression]. / Znachenie klinicheskogo i morfologicheskogo tipov khronicheskogo glomerulonefrita i tubulointerstitsial'nykh izmenenii dlia prognoza progressirovaniia zabolevaniia.

A highly significant relationship has been established between rapid progression of chronic glomerulonephritis and belonging to unfavorable category of clinical types according to classification of M. Ya. Ratner et al. (chi-square = 84.3, p < 0.001), to unfavorable category of morphological types (chi-square = 13.2, p < 0.01) and the presence of tubulointerstitial changes (chi-square = 32, p < 0.0001).

[Prognostic factors of chronic glomerulonephritis and chronic non-inflammatory glomerulopathies rapid progression]. / Prognosticheskie faktory uskorennogo progressirovaniia khronicheskogo glomerulonefrita i khronicheskikh nevospalitel'nykh glomerulopatii.

AIM: To determine relationships between prognosis of rapid progression of chronic glomerulonephritis (CGN), chronic non-inflammatory glomerulopathies (CNG) with development of chronic renal failure and clinical, morphological types of the disease and tubulointerstitial component (TIC). MATERIALS AND METHODS: 137 CGN and CNG patients were followed up for 7 years. Favorable clinical types were found in 89, unfavorable ones in 48 patients. Favorable and unfavorable morphological types occurred in 87 and 50 patients, respectively. TIC was identified in 56 patients. RESULTS: Rapid progression of CGN and CNG appeared to associate with the unfavorable clinical type (active nephritic types of CGN and nephrotic-hypertensive type of CNG), the unfavorable morphological type (mesangiocapillary glomerulonephritis and focal segmentary sclerosis/hyalinosis) and TIC. CONCLUSION: Prognosis of rapid progression of CGN and CNG is most reliable in combination of the above three predictors or of the unfavorable clinical type with TIC.

[The dependence of the progression of chronic glomerulonephritis on the clinical and morphological types of glomerulonephritis and on the tubulointerstitial changes]. / Zavisimost' progressirovaniia khronicheskogo glomerulonefrita ot klinicheskogo i morfologicheskogo tipov glomerulonefrita i tubulointerstitsial'nykh izmenenii.

Progression of chronic glomerulonephritis (CGN) is strongly associated with morphologic type of the disease, tubulointerstitial changes, some clinical syndromes. The aim of the study was to trace relations between the onset of chronic renal failure within 7 years since the diagnosis (fast progression of CGN--FP CGN), CGN clinical variant according to M. Ia. Ratner et al. classification (1987) and histomorphological changes in the renal biopsy. Unfavorable clinical types (active nephritic types and nephrotic-hypertensive type) proved dominating predictor of FP CGN not only because of close relationship between these type and FP CGN but also due to FP CGN occurrence in morphologically unfavorable morphological types and tubulointerstitial changes in line with concomitant unfavorable clinical types.

[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.

Generation of targeted nonpolar gene insertions and operon fusions in Pasteurella haemolytica and creation of a strain that produces and secretes inactive leukotoxin.

An efficient method for targeted gene inactivation and generation of chromosomal gene fusions in Pasteurella haemolytica has been devised and used to create an lktC::cat operon fusion by allelic exchange at the leukotoxin gene cluster (lktCABD). A copy of the lktC gene was insertionally inactivated by using a nonpolar, promoterless cat cassette and then delivered into P. haemolytica on a shuttle vector. Plasmid incompatibility was used to detect clones where double recombination events had occurred at the chromosomal locus. The insertion in lktC did not affect expression of the downstream genes, and the mutant strain secreted an antigenic proleukotoxin that was neither leukotoxic nor hemolytic. Expression of the lktC gene in trans restored the wild-type phenotype, confirming that LktC is required for activation of the proleukotoxin to the mature leukotoxin. Construction of the lktC::cat operon fusion allowed us to quantitate leukotoxin promoter activity in P. haemolytica and to demonstrate that transcription was maximal during early logarithmic growth phase but was reduced following entry into late logarithmic phase. This allelic exchange system should be useful for future genetic studies in P. haemolytica and could potentially be applied to other members of Haemophilus-Actinobacillus-Pasteurella family, where genetic manipulation is limited.