Enzyme-Substrate Reporters for Evaluation of Substrate Specificity of HIF Prolyl Hydroxylase Isoforms.

An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD isoform preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of "paired" enzyme-substrate reporters to evaluate the potency of "branched tail" oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform.

[Diagnostics of sepsis in the patients presenting with pyo-inflammatory ENT pathology].

The objective of the present study was to develop the algorithm for early clinical, laboratory, and instrumental diagnostics of sepsis in the patients presenting with pyo-inflammatory ENT diseases. The study included 50 patients with pyo-inflammatory ENT diseases complicated by generalization of infection. The scheme (algorithm) developed for their examination allowed to diagnose sepsis at the early stages of hospitalization in the patients presenting with pyo-inflammatory ENT pathology. The diagnostic algorithm included the criteria for syndrome of systemic inflammatory reaction (SSIR), the Kalf-Kalif leukocytic intoxication index (LII), the procalcitonin test (PKT), the integral severity of illness scale (APACHE-II) and the organ failure assessment score (SOFA). Timely preoperative diagnostics of septic complications and sanation of the primary infection site in the combination with early intensive therapy under conditions of a resuscitation department made it possible to avoid further progress of generalization of the infection and thereby to reduce the number of secondary interventions, to improve prognosis of the disease, and lower the mortality rate down to 12% in the study group (compared with 44% in the control one).

Catalytic mechanism and substrate specificity of HIF prolyl hydroxylases.

This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely ß2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.

[Laboratory diagnosis of infected pancreonecrosis].

The results of treatment and examination were analyzed in 175 patients admitted to a clinic for sterile pancreonecrosis (PN). The significance of routinely available (clinical, biochemical, and microbiological) laboratory tests in the diagnosis of purulent complications was analyzed in patients with varying severity of pancreatic autolysis. Complex laboratory monitoring was found to be of importance in predicting and detecting infected PN with obligatory fine-needle punctures under ultrasound guidance and further microscopic studies of puncture specimens.

Structure-activity relationship for branched oxyquinoline HIF activators: Effect of modifications to phenylacetamide "tail".

HIF prolyl hydroxylase is a major regulator of HIF stability. Branched tail oxyquinolines have been identified as specific inhibitors of HIF prolyl hydroxylase and recently demonstrated clear benefits in various scenarios of neuronal failure. The structural optimization for branched tail oxyquinolines containing an acetamide bond has been performed in the present study using HIF1 ODD-luc reporter assay. The special attention has been paid to the length of a linker between acetamide group and phenyl ring, as well as substitutions in the phenyl ring in the other branch of the tail. The optimized version of branched tail oxyquinolines is 3-fold more potent than the original one identified before and shows a submicromolar EC50 in the reporter assay. The compounds have been studied in a "liver-on-a-chip" device to question their hepatotoxicity towards differentiated human HepaRG "hepatocytes": the absence of hepatotoxicity is observed up to 200 µM concentrations for all studied derivatives of branched tail oxyquinolines.

Microarray analysis of differentially expressed genes after exposure of normal human fibroblasts to ionizing radiation from an external source and from DNA-incorporated iodine-125 radionuclide.

Exposure of cells to ionizing radiation (IR) produces changes in the expression level of a large number of genes. However, less is known of gene-expression changes caused by local radiation exposure from radionuclides within cells. We studied changes in the genome-wide gene expression induced by decay of 125I incorporated into DNA as [125I]-iododeoxyuridine (125I-IUdR) in normal IMR-90 human lung fibroblasts and compared them with the changes produced by external gamma-radiation delivered at high (HDR) or low (LDR) dose rate. We found that more than 2000 genes were consistently up- or down-regulated following HDR and LDR gamma-radiation. The profiles of differentially expressed genes following HDR and LDR shared about 64% (up) and 74% (down) genes in common, with many genes identified as radiation-responsive for the first time. In contrast, in all only 206 genes changed their expression level in the 125I-IUdR-treated cells, even though the total number of DNA double-strand breaks (DSB) produced by 125I-IUdR exceeded that produced by the gamma-radiation. With few exceptions, the expression levels of 125I-IUdR-responsive genes were also altered following gamma-irradiation. Therefore, nuclear DNA-localized decays of 125I produce 10 times fewer differentially expressed genes than whole-cell exposure to gamma-radiation of comparable dose. These results suggest that the effect of IR on the changes in global gene expression depends on the distribution of energy depositions within the cell. In contrast to cell survival, DNA DSB may not be the major factor modulating changes in gene expression following irradiation.

[Mutations in the alpha-amylase gene of Bacillus amyloliquefaciens, leading to a decrease in the temperature of protein inactivation]. / Mutatsii v gene alpha-amilazy Bacillus amyloliquefaciens, privodiashchie k snizheniiu temperatury inaktivatsii belka.

Alpha-amylase genes of Bacillus amyloliquefaciens, coding proteins with reduced thermostability, had been obtained as a result of hydroxylamine mutagenesis. Temperature, pH and starch concentration dependences of two mutant alpha-amylases were investigated. The synthesis of the alpha-amylases by several B. subtilis strains with different levels of extracellular proteases was also studied. The mutation containing fragments were localized and the structures of the mutations were determined. It was found that the decrease of thermostability of mutant No 141 was due to Asp to Asn change at the position No 194 of the mature protein, and for mutant No 191--due to Glu to Lys change at the position No 185.

[Two-codon mutagenesis of alpha-amylase gene of Bacillus amyloliquefaciens]. / Dvukhkodonnyi mutagenez gena alpha-amilazy Bacillus amyloliquefaciens

The oligonucleotide encoding Bam HI recognition site having the structure pCGGGATC had been inserted into the recognition sites MspI of the B. amyloliquefaciens alpha-amylase gene, which was cloned in pTG29B plasmid. The alpha-amylase gene had no BamHI sites before mutagenesis. The set of pNSBamHI plasmids with BamHI site at four different positions was obtained. It was shown that all the mutant alpha-amylases possess different specific activities. One of the mutant proteins possesses reduced thermostability. The mutant alpha-amylases can be used for further experiments on protein-engineering of liquefying-type alpha-amylases.

[Experimental measles infection in rodents]. / Eksperimental'naia korevaia infektsiia u gryzunov.

The sensitivity of newborn hamsters to inoculation with the vaccine L-16 strain of measles virus and the Lec strain isolated from a patient with subacute sclerosing panencephalitis as well as the possibility of persistence of these viruses in the animals were studied. Intracerebral inoculation of the L-15 strain was shown to produce in hamsters acute meningoencephalitis leading to death in 85%-100% of cases. Over 30 days after inoculation, the infectious virus, the virus-specific antigen and virus genome were found in the brain. In the brains of the sick animals, all the structural proteins of measles virus with the exception of hemagglutinin were expressed. After inoculation with the Lec strain, the clinical signs of the disease were less manifest, and mortality was 40%. The infectious virus could be detected in the brain up to 20 days postinoculation, the genome, up to 31 days. All the structural proteins of measles virus were expressed in the brains of the inoculated animals. No persistence of L-16 and Lec strains of measles virus could be demonstrated at langer intervals after inoculation (90-180 days) in the brains of hamsters.

[Distribution of enzymes of nucleic acid biosynthesis and their precursors during fractionation of Escherichia coli MRE-600 extract]. / Raspredelenie fermentov biosinteza nukleinovykh kislot i ikh predshestvennikov pri fraktsionirovanii ékstrakta Escherichia coli MRE-600.

Distribution of the enzymes of template-dependent and template-independent polynucleotide syntheses (DNA-polymerase I, RNA-polymerase, polynucleotide phosphorylase) as well as those of the biosynthesis of nucleic acids precursors (nucleotide kinases, acetokinase and nucleoside deoxyribosyltransferases) during fractionation of Escherichia coli MRE-600 cell extract was studied. On the basis of the results obtained a technological scheme was developed that enabled to combine routine procedures of purification of the above mentioned enzymes.

[Dynamics of biochemical indices in a favorable course of sarcoidosis of the respiratory organs]. / Dinamika nekotorykh biokhimicheskikh pokazatelei pri blagopriiatnom techenii sarkoidoza organov dykhaniia.

Sarcoidosis ran a favourable course in 166 out of 179 patients. The active phase of sarcoidosis was characterized by dysproteinemia, high content of histamine, STH and beta-lipoproteins together with a decrease of HPI and total protein. More remarkable shifts were observed in patients afflicted with sarcoidosis of the intrathoracic lymph nodes and of the lungs. In the phase of regression, there was a unidirectional change in the majority of the parameters, which correlated with the process liability to the reverse development. It may be recommended that the content of histamine, HPI and beta-lipoproteins should be measured along with other parameters to estimate the process activity in patients with respiratory sarcoidosis.

[Zenker's diverticulum]. / Divertikul Tsenkera.

An experience with treatment of 14 women and 15 men aged from 41 to 83 years with Zenker's diverticulum was analyzed. Most liable to the disease were people at the age from 50 to 70 years. The average duration of the disease was 3-4 years. All the patients were subjected to improved surgical interventions: resection of the diverticulum (11 patients), operations under the control of the endoscopic technique (18 patients). Resections of the diverticulum were combined with cryopharyngeal myotomy. No complications were noted. There were no lethal outcomes. So, wider indications to operative treatment in patients with Zenker's diverticulum, endoscopic methods used at all stages of treatment as well as improved methods of performing the operation enabled the authors to obtain good immediate and long-term results.

[Use of oil preparations for hemostasis in gastroduodenal hemorrhage]. / Primenenie maslianykh preparatov s tsel'iu gemostaza pri gastroduodenal'nykh krovotecheniiakh.

The authors report on using oil preparations in endoscopic surgery for arrest of bleedings from vessels of the stomach and duodenum. Persistent hemostasis was obtained in 60 out of 64 patients.

[Clinico-endoscopic evaluation of the effectiveness of the treatment of uncomplicated gastroduodenal ulcers after local administration of solcoseryl and aevit]. / Kliniko-endoskopicheskaia otsenka éffektivnosti lecheniia neoslozhnennykh iazv zheludka i dvenadtsatiperstnoi kishki pri mestnom primenenii solkoserila i aevita.

The complex of conservative measures for noncomplicated peptic ulcers included curative endoscopy in 105 patients, in 61 of them Aevitum was used as an application with additional injections of Solcoseryl around the ulcers. In 44 patients Aevitum was used along with Solcoseryl which was injected into the submucous layer around the pathological focus of the stomach and duodenum. The results obtained gave a convincing evidence of advantages of interstitial administration of Aevitum allowing to considerably shorten time of treatment (up to 10-12 days) of noncomplicated ulcers of the stomach and duodenum.

[Open lavage of the abdominal cavity in diffuse suppurative peritonitis]. / Otkrytoe promivanie briushnoi polosti pri razlitykh gnoinykh peritonitakh.

Experimental investigations (240 rats) and clinical observations (30 patients) have shown repeated open lavages to be effective in the postoperative period for the treatment of purulent peritonitis.