DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1.

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G(1) to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication.

[Revealing of allelic polymorphism in the populations of parthenogenetic lizards Darevskia dahli (Lacertidae) using locus-specific PCR].

Locus-specific PCR was used to study the genetic polymorphism in three populations of parthenogenetic lizard species Darevskia dahli. The analysis was carried at the two (GATA)n-containing loci (Du215 and Du281) using the sample of 26 individuals. A total of eight Du215 and three Du281 allelic variants were detected. It was demonstrated that all the lizards examined were heterozygous at these loci. In 12 animals, unusual Du215 allelic variant was revealed, the origin of which was thought to be associated with different types of genomic rearrangements, or segmental duplication. The populations studied were substantially different relative to the levels of allelic polymorphism, which could be explained by different habitation conditions, leading to accumulation of mutations in noncoding genome regions.

Regulation of the Arf/p53 tumor surveillance network by E2F.

Deregulation of the cell cycle machinery plays a critical role in tumorigenesis. In particular, functional inactivation of the retinoblastoma protein (pRB) is a key event. pRB's tumor suppressive activity is at least partially dependent on its ability to regulate the activity of the E2F transcription factors. E2F controls the expression of genes that encode the cellular proliferation machinery. E2F can also trigger apoptosis when it is inappropriately expressed. Here we present evidence that E2F acts to directly regulate the Arf/p53 tumor surveillance network. In normal cells, a single member of the E2F family, E2F3, participates in the transcriptional silencing of Arf. In response to oncogenic stress, the activating E2Fs, E2F1, 2, and E2F3A, all associate with Arf and promote its transcription. These findings raise the possibility that E2F acts as a sensor of inappropriate versus normal proliferative signals and determines whether or not the Arf/p53 tumor surveillance network is engaged.

[Variation of mini- and microsatellite DNA markers in populations of parthenogenetic rock lizard Darevskia rostombekovi]. / Izmenchivost' mini- i mikrosatellitnykh markerov DNK v populiatsiiakh partenogeneticheskoi skal'noi iashcheritsy Darevskia rostombekovi.

Variation and clonal diversity in populations of the parthenogenetic rock lizard Darevskia rostombekovi was examined by means of multilocus DNA fingerprinting using mini- and microsatellite DNA markers M13, (GATA)4, and (TCC)50). The animals examined were shown to exhibit a clonally inherited, species-specific pattern of DNA markers (fingerprint profile) that is different from the species-specific patterns of parthenogenetic species D. dahli, D. armeniaca, and D. unisexualis. The mean intraspecific similarity index S was 0.950 (0.003) for a sample of 19 animals from three isolated populations of North Armenia. This significantly differed from the estimate of this parameter for a sample of 21 animals including two individuals from mountainous, relict population from the vicinity of the Sevan Lake, which was equal to 0.875 (0.001). A comparison of DNA fingerprints showed differences between 21 individuals attaining 79 DNA fragments of 1801 mini- and microsatellite markers included in the analysis. The results obtained show that intraspecific variation in D. rostombekovi is higher than that in the previously studied parthenogenetic species D. dahli (S = 0.962) and D. unisexualis (S = 0.950) (P < 0.001). Taking into account that D. rostombekovi is considered monoclonal on the basis of allozyme data, the problem of clonal variability is discussed with regard to the evidence on nuclear DNA markers. It is suggested that the hybrid karyotype of D. rostombekovi, which is more unstable than that of D. dahli and D. unisexualis, generates a series of chromosomal rearrangements (mutations). This may lead to the appearance of a geographically isolated chromosomal race (clone) in the population inhabiting the southeastern coast of the Sevan Lake.

Multiple adaptive mechanisms affect asparagine synthetase substrate availability in asparaginase-resistant MOLT-4 human leukaemia cells.

Childhood acute lymphoblastic leukaemia is treated by combination chemotherapy with a number of drugs, almost always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance remain a problem. In vitro studies have demonstrated an adaptive increase in asparagine synthetase (AS) expression in ASNase-resistant cells, which is believed to permit ASNase-resistant human leukaemia cells to survive in vivo. The present results, obtained with ASNase-sensitive and -resistant human MOLT-4 leukaemia cell lines, illustrate that several other adaptive processes occur to provide sufficient amounts of the AS substrates, aspartate and glutamine, required to support this increased enzymic activity. In both cell populations, aspartate is derived almost exclusively from intracellular sources, whereas the necessary glutamine arises from both intracellular and extracellular sources. Transport of glutamine into ASNase-resistant cells is significantly enhanced compared with the parental cells, whereas amino acid efflux (e.g. asparagine) is reduced. Most of the adaptive change for the amino acid transporters, Systems A, ASC and L, is rapidly (12 h) reversed following ASNase removal. The enzymic activity of glutamine synthetase is also enhanced in ASNase-resistant cells by a post-transcriptional mechanism. The results demonstrate that there are several sites of metabolic adaptation in ASNase-treated leukaemia cells that serve to promote the replenishment of both glutamine and asparagine.

Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells.

Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12 h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate.

Highly conserved RNA sequences that are sensors of environmental stress.

The putative function of highly conserved regions (HCRs) within 3' untranslated regions (3'UTRs) as regulatory RNA sequences was efficiently and quantitatively assessed by using modular retroviral vectors. This strategy led to the identification of HCRs that alter gene expression in response to oxidative or mitogenic stress. Databases were screened for UTR sequences of >100 nucleotides that had retained 70% identity over more than 300 million years of evolution. The effects of 10 such HCRs on a standard reporter mRNA or protein were studied. To this end, we developed a modular retroviral vector that can allow for a direct comparison of the effects of different HCRs on gene expression independent of their gene-intrinsic 5'UTR, promoter, protein coding region, or poly(A) sequence. Five of the HCRs tested decreased mRNA steady-state levels 2- to 10-fold relative to controls, presumably by altering mRNA stability. One HCR increased translation, and one decreased translation. Elevated mitogen levels caused four HCRs to increase protein levels twofold. One HCR increased protein levels fourfold in response to hypoxia. Although nonconserved UTR sequences may also have a role, these results provide evidence that sequences that are highly conserved during evolution are good candidates for RNA motifs with posttranscriptional regulatory functions in gene expression.

An expression system for mammalian amino acid transporters using a stably maintained episomal vector.

Despite its versatility and effectiveness in numerous studies, the vaccinia/HeLa cell expression model may not be optimal for the study of all transport proteins. To evaluate an alternative expression model for amino acid transport Systems ASC and X-AG, the mRNA content and transport activity encoded by human hippocampal ASCT1 cDNA and rat hippocampal EAAC1 cDNA, respectively, were measured in pDR2-cDNA-transfected human embryonic kidney 293 cells made competent by stable transfection with the Epstein-Barr neutral antigen-1 (EBNA-1) cDNA (293c18 cells) to evaluate the EBNA-1/293c18 expression system. The results show that (i) the EBNA-1/293c18 expression system results in a larger increase over background of Systems ASCT1 (6.4x) and EAAC1 (39x) transport activity than does the vaccinia/HeLa expression system (2.6x and 22x, respectively); (ii) transfection and hygromycin B selection for the pDR2 vector do not affect the endogenous transport velocities of Systems ASC, X-AG, or A; and (iii) the endogenous transport velocities of Systems ASC and X-AG in 293c18 cells were not affected by the expression of exogenous EAAC1 or ASCT1. We conclude that the EBNA-1/293c18 cell expression model represents a useful transient expression regimen to characterize mammalian amino acid transport proteins, especially for transporters that may exhibit relatively low activity in transient expression systems lacking a selection mechanism.

Differential regulation of GRP78 and GLUT1 expression in 3T3-L1 adipocytes.

We tested the hypothesis that the constitutive glucose transporter (GLUT1) in 3T3-L1 adipocytes belongs to the family of glucose-regulated proteins which are transcriptionally regulated by glucose deprivation. Using cDNA probes for both GRP78 (BiP) and GLUT1, we show that the level of GRP78 mRNA increased by 15-fold within 24 h of glucose deprivation with little change in GLUT1 mRNA. The elevated GRP78 mRNA in turn led to a time-dependent increase in GRP78 protein. While glucose deprivation did not alter the expression of the normal glycoform of GLUT1, a lower molecular weight glycoform accumulated with extended deprivation. Mannose and fructose, but not galactose, prevented the induction of GRP78 and accumulation of the abnormal GLUT1. Because GRP78 acts as a chaperone in other cell systems, we also sought evidence to support this activity in 3T3-L1 adipocytes. Using the technique of co-immunoprecipitation, we demonstrate that GRP78 bound several proteins unique to the glucose-deprived state. No deprivation-specific proteins could be detected in association with GLUT1. These data lead us to conclude that GLUT1 does not display characteristics of the glucose-regulated proteins, at least in 3T3-L1 adipocytes, a widely used model for differentiation, hormone action, and nutrient control. However, the mechanisms for activating traditional members of this family appear intact.

[Surgical treatment of open and closed injuries of the duodenum]. / Khirurgicheskoe lechenie otkrytykh i zakrytykh povrezhdenii dvenadtsatiperstnoi kishki.

The article discusses a complex of methods for the diagnosis of injuries to the duodenum in 18 patients. Laboratory and X-ray findings and laparocentesis with introduction of a "feeling" catheter into the abdominal cavity with lavage of the cavity facilitated the discovery of blood and intestinal contents and allowed the correct diagnosis to be established before the operation. In inspection of the duodenum mobilization after Kocher is insufficient, in rupture of the posterior wall in the region of the inferior horizontal part the duodenum must be mobilized for its whole length. It is advisable that the defect in the duodenum is closed with a double-row suture applied with an atraumatic needle (the first inner row of interrupted sutures). The methods for applying hemostatic sutures suggested by the authors by means of created devices provide reliable hemostasis in combined injuries to the parenchymatous organs. The operation was completed by leaving glove-tube drains. The postoperative mortality was 16.6%.

[Changes in immunologic reactivity in patients with gastrointestinal bleeding]. / Izmenenie immunologicheskoi reaktivnosti u bol'nykh s zheludochno- kishechnym krovotecheniem.

The disorders in immunologic reactivity in acute gastrointestinal bleeding were studied in dynamics in 160 patients. Pronouncement of disorders in the T-system of immunity, activation of the reactions of specific sensibilization and autoallergic phenomena depend on severity of the blood loss. In ulcer location in the duodenum, the performance of an organ-preserving operation with vagotomy and elimination of the ulcer which is a source of antigen stimulation is preferable.

[Disorders and correction of lung functions in acute destructive cholecystitis]. / Narusheniia i korrektsiia funktsii legkogo pri ostrom destruktivnom kholetsistite.

On the day of operation for acute destructive cholecystitis functions of external ventilation prove to be disturbed due to deterioration of pulmonary blood flow (2-6 times) and ventilation (1.3-3 times). The degree of these disturbances are in direct dependence on the degree of the inflammatory process in gallbladder and time of preoperative observation. At the same terms after emergency cholecystectomy the lung functions were reestablished considerably quicker and was more valuable than after operations on urgent indications. Inclusion of adrenoblocking agents into conservative treatment of acute cholecystitis before and after operation facilitates more effective and quicker recovery of lung functions.

[Use of adrenergic blockaders for correction of pulmonary functions in the treatment of acute cholecystitis]. / Ispo'lzovanie adrenoblokatorov dlia korrektsii funktsii legkikh pri lechenii ostrogo kholetsistita.

The assessment of external respiration during a 2-day management of acute pain attack produced by cholecystitis disclosed a 1.2-1.5-fold decrease in the parameters of the function resultant from poor ventilation of the pulmonary zones and loss of coordination between the ventilation and relevant blood flow. On day 3 of the attack treatment of external respiration returned to normal functioning though in patients over 60 this return took a week, as they had a 1.2-2-fold drop in the blood flow and pulmonary ventilation. The attempts of administration of adrenoblockers in combined treatment of acute cholecystitis succeeded in restoration of pulmonary function during 3 days and in more rapid attenuation of attacks in acute cholecystitis.

[The role of a sympathetic pharmacologic blockade in restoring blood flow to the organ in acute cholecystitis]. / Rol' simpaticheskoi farmakologicheskoi blokady v vosstanovlenii organnogo krovotoka pri ostrom kholetsistite.

By means of a method of rheography, in 74 patients with acute cholecystitis before and after the operation, and in 26 healthy subjects as well, the blood flow of the brain, lungs, liver and lower extremities under conditions of the use of benzohexamethonium, obsidan, aminazine and neostigmine methylsulfate was studied. Resulting from the use of sympathetic pharmacologic blockade, already by day 2, the indices of cerebral, pulmonary and hepatic blood flow normalized or exceeded the normal level.

[Surgical treatment of intestinal injuries]. / Khirurgicheskoe lechenie povrezhdenii kishechnika.

The results of treatment of 235 sufferers with open (142) and closed (93) intestinal injuries are analysed. One hundred and twenty one patients had isolated, 114-combined intestinal injuries. Suturing of the small, large intestine and other hollow organs was performed in 192 sufferers, resection of the small intestine-in 28, resection of the large intestine-in 5, resection of the large intestine with unloading cecostomy-in 4, right hemicolectomy-in 3, colostomy-in 3. Twenty eight patients developed complications after the operation, 14 (6.4%) died.