Preparation, Biochemical Analysis, and Structure Determination of Methyllysine Readers.

In-depth in vitro characterization of methyllysine reader domains and their association with cognate methyllysine substrates is essential to better understand fundamental mechanisms of chromatin regulation and to design targeted therapeutics that disrupt these interactions. In this chapter, we summarize commonly used methods for preparation, biochemical characterization, and determination of structures of methyllysine reader domains. We provide a detailed protocol for the preparation of a GST-tagged methyllysine reader domain and for analysis of histone-binding activities using a combination of pull-down, tryptophan fluorescence, and NMR assays, and describe initial steps toward crystallization of the complexes.

High degree of genetic diversity of non-polio enteroviruses identified in Georgia by environmental and clinical surveillance, 2002-2005.

Enterovirus surveillance data are useful for establishing temporal and geographical patterns of circulation and for virus characterization to determine phylogenetic relationships between strains. Almost no information is available on circulating enteroviruses in Georgia and the surrounding region. To describe enterovirus circulation in Georgia, determine relationships with previously characterized strains and assess the role of environmental and clinical enterovirus surveillance, this study analysed a total of 112 non-polio enterovirus isolates identified during 2002-2005 from sewage and human stool samples. Viruses were isolated in cell culture using standard methods and typed by partial sequencing of the VP1 gene. A total of 20 different non-polio enterovirus serotypes were identified over the 4-year period. The most commonly detected enteroviruses included echovirus (E) 6 (21 isolates; 18.8 %), E20, E3 and E7 (11 isolates each; 9.8 %), E11, coxsackievirus (CV) B4 and CVB5 (seven isolates each; 6.3 %), and E13, E19 and E30 (six isolates each; 5.4 %). Phylogenetic analysis showed that many serotypes were represented by more than one genetic lineage. The present study showed a very high degree of enterovirus diversity in Georgia and demonstrated the added value of environmental enterovirus surveillance, particularly in settings with limited clinical surveillance. Several serotypes would not have been detected without having both clinical and environmental surveillance in place. Several serotypes detected in Georgia were among those rarely reported in the USA and Europe (e.g. E3, E20 and E19). As the emergence of new genetic lineages of enterovirus in a particular area is often associated with large-scale outbreaks, continued monitoring of enterovirus strains by both environmental and clinical surveillance and genetic characterization should be encouraged.

[Combined treatment for rectal cancer using different radiomodification means].

The paper presents an evaluation of the results of surgical and combined treatment received by 961 patients at the Center's Clinics within 28 years. The analysis was based on 3 protocols of prospective randomized study. It dealt with assessment of the end results of surgical and combined treatment for operable rectal cancer using one preoperative hyperfractonated STD of 5-25 Gy. That was combined with local microwave hyperthermia and two radio modifiers--local microwave hyperthermia+intrarectal administration of a preparation containing metronidazolum. Due to the latter factor, the rate of 3-year relapse-free survival rose considerably. Also, loco-regional and distant metastasis incidence was cut down due to superior ablasticity of surgery.

Histone H3K4me3 binding is required for the DNA repair and apoptotic activities of ING1 tumor suppressor.

Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair, and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with histone H3 trimethylated at Lys4 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 A-resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-pi contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild-type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage-induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I, and G221V mutations, found in human malignancies, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.

[Transfer of a child from children's home to family: medical aspects].

The project "Leaving of babies at children's homes and deinstitutionalization" was implemented at the children's home of Tbilisi. The purpose was to find out what influence was produced by deinstitutionalization on the physical and psychomotor development of deprived babies. The study lasted for 12 months. 3 groups of children were investigated. Group 1 comprised children selected for deinstitutionalization, age-matching healthy children who stayed at the children's home were in group 2, and healthy children randomly selected from 2 nurseries of Tbilisi were in group 3. The parameters of anthropometry and psychomotor development were evaluated according to the Denver screening test at the very beginning and in 12 months. The children from the children's home almost caught up, in 12 months after deinstitutionalization, with their control matches. Whereas, the children, who were brought up at the children's home, were behind the group-1 children and controls by all parameters. Projects promoting the upbringing of deprived children in their own or foster families are recommended for implementation.

Phosphatidylethanolamine has an essential role in Saccharomyces cerevisiae that is independent of its ability to form hexagonal phase structures.

Two yeast enzymes, Psd1p and Psd2p, catalyze the decarboxylation of phosphatidylserine to produce phosphatidylethanolamine (PtdEtn). Mitochondrial Psd1p provides approximately 90% of total cellular phosphatidylserine decarboxylase activity. When the PSD1 gene is deleted, the resultant strain (psd1Delta) grows normally at 30 degrees C in glucose and in the absence of exogenous choline or ethanolamine. However, at elevated temperature (37 degrees C) or on the nonfermentable carbon source lactate, the growth of psd1Delta strains is minimal without ethanolamine supplementation. The reduced growth and viability correlate with a PtdEtn content below 4% of total phospholipid. These results suggest that there is a critical level of PtdEtn required to support growth. This theory is supported by growth data revealing that a psd1Delta psd2Delta dpl1Delta strain can only grow in the presence of ethanolamine. In contrast, a psd1Delta psd2Delta strain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine. psd1Delta psd2Delta cells grown in 2 mm propanolamine accumulate a novel lipid, which was determined by mass spectrometry to be phosphatidylpropanolamine (PtdPrn). PtdPrn can comprise up to 40% of the total phospholipid content in supplemented cells at the expense of phosphatidylcholine and PtdEtn. The absolute level of PtdEtn required for growth when PtdPrn is present appears to be 1% of the total phospholipid content. The essential function of the PtdEtn in the presence of propanolamine does not appear to be the formation of hexagonal phase lipid, insofar as PtdPrn readily forms hexagonal phase structures detectable by (31)P NMR.

Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes.

Specific recognition of phosphoinositides is crucial for protein sorting and membrane trafficking. Protein transport to the yeast vacuole depends on the Vam7 t-SNARE and its phox homology (PX) domain. Here, we show that the PX domain of Vam7 targets to vacuoles in vivo in a manner dependent on phosphatidylinositol 3-phosphate generation. A novel phosphatidylinositol-3-phosphate-binding motif and an exposed loop that interacts with the lipid bilayer are identified by nuclear magnetic resonance spectroscopy. Conservation of key structural and binding site residues across the diverse PX family indicates a shared fold and phosphoinositide recognition function.

Structural mechanism of endosome docking by the FYVE domain.

The recruitment of trafficking and signaling proteins to membranes containing phosphatidylinositol 3-phosphate [PtdIns(3)P] is mediated by FYVE domains. Here, the solution structure of the FYVE domain of the early endosome antigen 1 protein (EEA1) in the free state was compared with the structures of the domain complexed with PtdIns(3)P and mixed micelles. The multistep binding mechanism involved nonspecific insertion of a hydrophobic loop into the lipid bilayer, positioning and activating the binding pocket. Ligation of PtdIns(3)P then induced a global structural change, drawing the protein termini over the bound phosphoinositide by extension of a hinge. Specific recognition of the 3-phosphate was determined indirectly and directly by two clusters of conserved arginines.

Signaling with phosphoinositides: better than binary.

When the outside of cell is stimulated,the inside generates a flurry of signals. Phosphates are sprinkled over lipids and proteins,where they are recognized within diverse signaling pathways. The kinases that congregate beneath the cell surface to provide the phosphate tags that mediate signaling have become major targets of new wave of drug design. Phosphoinositide signaling presents a particularly intriguing network whose many mysteries are now being unlocked. Research into protein domains that specifically recognize phosphoinositides have established the ENTH, FYVE,Phox,and pleckstrin homology domains s four cornerstones of phosphoinositide signaling.

A DNA architectural protein couples cellular physiology and DNA topology in Escherichia coli.

In Escherichia coli, the transcriptional activity of many promoters is strongly dependent on the negative superhelical density of chromosomal DNA. This, in turn, varies with the growth phase, and is correlated with the overall activity of DNA gyrase, the major topoisomerase involved in the elevation of negative superhelicity. The DNA architectural protein FIS is a regulator of the metabolic reorganization of the cell during early exponential growth phase. We have previously shown that FIS modulates the superhelical density of plasmid DNA in vivo, and on binding reshapes the supercoiled DNA in vitro. Here, we show that, in addition, FIS represses the gyrA and gyrB promoters and reduces DNA gyrase activity. Our results indicate that FIS determines DNA topology both by regulation of topoisomerase activity and, as previously inferred, by directly reshaping DNA. We propose that FIS is involved in coupling cellular physiology to the topology of the bacterial chromosome.

Phosphatidylinositol 3-phosphate recognition by the FYVE domain.

Recognition of phosphatidylinositol 3-phosphate (Ptdlns(3)P) is crucial for a broad range of cellular signaling and membrane trafficking events regulated by phosphoinositide (PI) 3-kinases. PtdIns(3)P binding by the FYVE domain of human early endosome autoantigen 1 (EEA1), a protein implicated in endosome fusion, involves two beta hairpins and an alpha helix. Specific amino acids, including those of the FYVE domain's conserved RRHHCRQCGNIF motif, contact soluble and micelle-embedded lipid and provide specificity for Ptdlns(3)P over Ptdlns(5)P and Ptdlns, as shown by heteronuclear magnetic resonance spectroscopy. Although the FYVE domain relies on a zinc-binding motif reminiscent of RING fingers, it is distinguished by ovel structural features and its ptdlns(3)P-binding site.

[Detection of poliomyelitis viral strains in natural isolates and identification of them by polymerase chain reaction]. / Obnaruzhenie shtammov virusa poliomielita v prirodnykh izoliatakh i ikh identifikatsiia s pomoshch'iu polimeraznoi tsepnoi reaktsii.

One hundred and fifty nucleotide-long VP1/2A junction regions were sequenced in the RNAs of 19 strains isolated in 1990-1991 from patients with paralytic poliomyelitis in different regions of the former USSR. On the basis of the alignments of these sequenced RNAs, four pairs of 19-25 base-long oligodeoxynucleotide PCR primers were designed capable of detecting polio RNAs in isolated strains and of discriminating between polio genotypes. PCR with 520 polio virus strains isolated from patients, normal subjects, and environmental objects showed 428 of these strains to be related to Sabin's vaccine strains, whereas the rest were referred to A (30), T (24), and G (1) genotypes of serotype 1 and to C-genotype (37) of serotype 3. The designed primers were highly specific and did not cross-react between themselves and with primers specific for Sabin's vaccine strains in PCR.

Geographical genotypes (geotypes) of poliovirus case isolates from the former Soviet Union: relatedness to other known poliovirus genotypes.

A 150 nucleotide long region corresponding to adjoining segments of the genes encoding polypeptides VP1 and 2A of 84 poliovirus strains recently isolated from patients with paralytic poliomyelitis over the territory of the former Soviet Union (FSU) were characterized by sequencing and/or PCR amplification using specially designed primers. Eighteen isolates were found to be very closely related to one or another of the three Sabin vaccine strains. Three distinct classes of geographical genotypes (geotypes) were discerned among 42 wild-type (non-Sabin) strains of serotype 1. One such geotype (called A) was widely circulating in 1990-91 in the Caucasian (Azerbaijan and Georgia) as well as Asian (Kyrgyzstan and Turkmenistan) Republics; this geotype exhibited only weak relatedness to known strains isolated outside the FSU. On the other hand, a subset of strains belonging to another geotype (T) of serotype 1, which circulated in 1991 in Tajikistan, demonstrated very close relatedness to contemporaneous strains isolated in Pakistan, India and Jordan. Strains that were somewhat different, but belonging to the same T-geotype, were found also in Moldova and Georgia. Strikingly, the primary structure of the VP1/2A junction of certain T-geotype isolates differed from the corresponding region of Sabin 1 only in 13-15% of positions, thereby not reaching the upper limit accepted for a geotype. This observation raises, though does not prove, the possibility that at least the relevant segment of the T-geotype RNA originated from the vaccine strain. The third geotype of serotype 1 was represented by a single, perhaps imported, isolate. Four distinct subsets of a common geotype (C) were discerned among 24 wild-type isolates belonging to serotype 3. These strains exhibited a broad geographical distribution being found, in particular, in Armenia, Azerbaijan, Georgia, Turkmenistan and Tajikistan; on the other hand, the C-geotype strains exhibited only a relatively distant relatedness to a strain isolated outside of the FSU (in Oman).

[The procedure for treating kidney cancer patients with metastases to the bones]. / Taktika lecheniia bol'nykh rakom pochki s metastazami v kosti.

Active surgical intervention in patients with kidney carcinoma and metastases in the bones helped in prolonging the life of previously doomed patients for several years. Of course, we are far from the idea that such tactics is justified in all patients. An individual approach is necessary. The fundamentals of successful treatment is thorough diagnosis. The extent of the neoplastic process cannot always be determined, errors are often made in establishing the diagnosis, which leads to faulty choice of the therapeutic tactics.

[Dynamics of metastasis of breast cancer to the bones]. / Dinamika metastazirovaniia raka molochnoi zhelezy v kostiakh.

A total of 120 cases with Stages I (n = 32) and IIa (n = 88) mammary carcinoma who later developed metastases to the bones are analyzed. The authors come to a conclusion that the time course of the metastases development directly depends on the tumor process stage. Postoperative thiophosphamide chemotherapy is the most effective method for the prevention of metastases to the bones; postoperative radiotherapy is conducive to a most rapid dissemination of the metastases in the bones.

Nucleoside 5'-triphosphates modified at sugar residues as substrates for calf thymus terminal deoxynucleotidyl transferase and for AMV reverse transcriptase.

Terminal deoxynucleotidyl transferase from calf thymus and RNA-directed DNA polymerase (reverse transcriptase) from the avian myeloblastosis virus catalyze the incorporation of 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates, as well as some of their 3'-derivatives, 3'-amino-3'-deoxyarabinonucleoside 5'-triphosphates and some other nucleoside 5'-triphosphates modified at sugar residues. After incorporation of the appropriate 5'-mononucleotide residue into the DNA, further chain elongation is blocked. This finding opens up a possibility for selective inhibition of DNA synthesis catalyzed by a certain enzyme.

3'-Hydroxymethyl 2'-deoxynucleoside 5'-triphosphates are inhibitors highly specific for reverse transcriptase.

dNTP(3'-OCH3), a 3'-O-methyl derivative of dNTP, is a chain terminator substrate for DNA synthesis catalyzed by AMV reverse transcriptase. The enzyme seems to be the only DNA polymerase susceptible to the inhibitor while all the other DNA polymerases tested are fully resistant to the nucleotide analog. The resistant polymerases are: E. coli DNA polymerase I, Klenow's fragment of DNA polymerase I, phage T4 DNA polymerase, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and calf thymus terminal deoxyribonucleotidyl transferase.

[Survival and prognosis in adjuvant chemotherapy for osteogenic sarcoma patients]. / Vyzhivaemost' i prognoz v usloviiakh ad"iuvantnoi khimioterapii u bol'nykh osteogennoi sarkomoi.

The effect of adjuvant chemotherapy on survival and prognosis in osteogenic sarcoma versus basic clinico-roentgenomorphological characteristics of tumor was studied in 108 cases. Prognosis was found to depend on patient's age, site and size of tumor, but mainly on the roentgeno-morphological pattern of the latter. Osteolytic form appeared to have a better course in cases of adjuvant chemotherapy. This was matched by osteoplastic form requiring surgical treatment only. The results suggest a strictly differentiated approach to selection of treatment modality, particularly in cases of post-operative chemotherapy.