Regulation and mechanisms of gene amplification

Amplification in rodent cells usually involves bridge-breakage-fusion (BBF) cycles initiated either by end-to-end fusion of sister chromatids, or by chromosome breakage. In contrast, in human cells, resistance to the antimetabolite N-(phosphonacetyl)-L-aspartate (PALA) can be mediated by several different mechanisms that lead to overexpression of the target enzyme carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase (CAD). Mechanisms involving BBF cycles account for only a minority of CAD amplification events in the human fibrosarcoma cell line HT 1080. Here, formation of a 2p isochromosome and overexpression of CAD by other types of amplification events (and even without amplification) are much more prevalent. Broken DNA is recognized by mammalian cells with intact damage-recognition pathways, as a signal to arrest or to die. Loss of these pathways by, for example, loss of p53 or pRb tumour suppressor function, or by increased expression of ras and myc oncogenes, causes non-permissive rat and human cells to become permissive both for amplification and for other manifestations of DNA damage. In cells that are already permissive, amplification can be stimulated by overexpressing oncogenes such as c-myc or ras, or by damaging DNA in a variety of ways. To supplement genetic analysis of amplification in mammalian cells, an amplification selection has been established in Schizosaccharomyces pombe. Selection with LiCl yields cells with amplified sod2 genes in structures related to those observed in mammalian cells. The effect on amplification in S. pombe can now be tested for any mutation in a gene involved in repair of damaged DNA or in normal cellular responses to DNA damage.     

Membrane topology of Borrelia burgdorferi and Treponema pallidum lipoproteins

A critical issue regarding the molecular architectures of Treponema pallidum and Borrelia burgdorferi, the agents of venereal syphilis and Lyme disease, respectively, concerns the membrane topologies of their major lipoprotein immunogens. A related question is whether these lipid-modified membrane proteins form intramembranous particles during freeze fracture electron microscopy. To address these issues, native borrelial and treponemal lipoproteins were reconstituted into liposomes of diverse composition. The importance of the covalently associated lipids for membrane association of lipoproteins was revealed by the observation that nonlipidated recombinant forms of both B. burgdorferi OspA and the T. pallidum 47-kDa immunogen (Tpp47) showed very weak or no binding to model bilayer vesicles. In contrast to control liposomes reconstituted with bacteriorhodopsin or bovine rhodopsin, two well-characterized transmembrane proteins, none of the lipoprotein-liposomes contained particles when examined by freeze fracture electron microscopy. To extend these findings to prokaryotic lipoproteins with relatively amphiphilic polypeptides, similar experiments were conducted with a recombinant nonlipidated form of Escherichia coli TraT, a lipoprotein which has putative transmembrane domains. The nonlipidated TraT oligomers bound vesicles derived from E. coli lipids but, surprisingly, did not form particles in the freeze-fractured liposomes. These findings support (i) a proposed topology of spirochetal lipoproteins in which the polypeptide is extrinsic to the membrane surface and (ii) the contention that particles visualized in freeze-fractured spirochetal membranes represent poorly characterized transmembrane proteins.     

Studies of the coronary circulation in Chagas' heart disease

Pathogenesis of chronic Chagas' heart disease may include various disturbances in the coronary circulation, that could be responsible for the myocardial lesions seen in human hearts and in experimental models of the disease. In this paper we critically reviewed the anatomical and functional abnormalities described in chronic chagasic patients, pertaining to the so-called vascular pathogenetic theory of Chagas' disease. The epicardial coronary arteries are usually free of significant obstructive disease in nonselected groups of chagasic patients examined at autopsy or by coronary angiography. However, chagasic patients who were studied after an episode of acute myocardial infarction, show the same patterns of atherosclerotic coronary artery disease seen in the general nonchagasic population. Studies of chagasic patients with angiographically normal coronary arteries, by several scintigraphy methods, revealed myocardial perfusion abnormalities which may be caused by the microcirculatory derangements described in animals experimentally infected with the T. cruzi. Since hypoperfusion has been detected in regions with normal or mildly impaired wall motion, it is likely that the microvascular disturbances precede and may be causative mechanism for the subsequent myocardial damage. We speculate that hibernating ventricular areas may occur in chagasic patients, on the basis of the evidence gathered from these studies. Recent investigations of chronic patients with Chagas' disease and chest pain showed attenuation of the vasomotor responses to physiological and pharmacological stimuli, in the epicardial coronary arteries.     

Acadesine extends the window of protection afforded by ischaemic preconditioning in conscious rabbits

OBJECTIVE: Ischaemic preconditioning protects myocardium from infarction if the reperfusion interval between the brief and prolonged ischaemic intervals is less than 1 h. In anaesthetised rabbits acadesine (5-amino-4-imidazolecarboxamide riboside, AICAR), an adenosine enhancer which increases tissue adenosine during ischaemia, prolongs the window of protection to 2 h. The aim of this study was to try to determine the maximum extension of this window of protection, using chronically instrumented, unsedated rabbits. METHODS: Rabbits were instrumented with a balloon occluder around a major branch of the left coronary artery for reversible coronary occlusion. Five to seven days after surgery all animals underwent a 30 min coronary occlusion. Animals were randomised to one of seven groups: (1) No additional treatment (control); (2) Ischaemic preconditioning with 5 min regional ischaemia followed by 10 min reperfusion before the 30 min coronary occlusion; (3) and (4) Ischaemic preconditioning followed by 2 or 4 h of reperfusion before the 30 min occlusion, respectively; (5) Treatment with acadesine (2.5 mg.kg-1.min-1 intravenously for 5 min and then 0.5 mg.kg-1.min-1 beginning 45 min before and continuing until 30 min after release of the 30 min occlusion) without ischaemic preconditioning; (6) and (7) Treatment with the higher dose of acadesine for 5 min beginning 35 min before the 5 min ischaemic period, and then the lower dose continuing until 30 min after release of the 30 min coronary occlusion in rabbits with 4 or 6 h reperfusion intervals, respectively. RESULTS: Rabbits with ischaemic preconditioning with 10 min reperfusion preceding the 30 min coronary occlusion (group 2) had only 5.6(SEM 1.1)% infarction of the ischaemic zone. Ischaemic preconditioning followed by 2 h reperfusion (group 3) offered continued protection [18.2(2.2)% infarction] as compared to control animals [37.7(2.6)% infarction]. However, protection waned if ischaemic preconditioning was followed by 4 h reperfusion (group 4) [36.7(3.0)% infarction]. Additionally, treatment with acadesine alone did not modify infarct size (group 7) [39.5(4.0)%], but acadesine largely restored the protection of ischaemic preconditioning despite a 4 h reperfusion interval (group 5) [20.4(3.0)% infarction, P < 0.01 v control]. However, when reperfusion was extended to 6 h (group 6) acadesine could no longer restore protection [36.2(0.9)% infarction]. CONCLUSIONS: The protection afforded by a 5 min ischaemic preconditioning period lasts from 2 to 4 h in the awake, unsedated rabbit, and acadesine can extend the duration of this window of protection to at least 4 h but not to 6 h.     

A quantitative evaluation of the glycogen content of the hepatocytes from different lobular ares of the normal human liver and in chronic hepatitides of different etiologies

Investigation of glycogen function in hepatocytes of different liver lobule zones is particularly important in understanding glycogen metabolism in humans and animals in norm and pathology. The present study was done to investigate glycogen contents in hepatocytes of different lobule zones of human liver in norm, and in patients with chronic hepatitis of viral or alcohol etiology. Quantitative analysis of glycogen content in hepatocytes of portal and central lobule zones was conducted on slices of human liver (the material of series live punctional biopsies) stained using a quantitative variant of PAS-reaction (Kudryavtseva et al., 1970, 1974). The measurements were done by image analyzer , which allows to make jointly cytophotometric analysis of substance in cells and definition of cell localization in tissue. The results showed clear differences of glycogen contents in different lobule zones in normal liver and in liver during chronic viral and alcohol hepatitis. Glycogen contents in hepatocytes of portal lobule zone were significantly higher than in the central lobule zone in patients with chronic viral hepatitis. Opposite data were obtained in patients with chronic alcohol hepatitis. Significantly higher glycogen contents were found in hepatocytes of the central liver lobule zone. Possible mechanisms of such a phenomenon are discussed . Thus, if glycogen contents in hepatocytes may be taken as an indicator of liver chronic damage degree (as has been shown elsewhere: Kudryavtseva, 1987; Kudryavtseva et al., 1988) the pattern of distribution of hepatocytes with different glycogen content in the liver lobule can be used as an indicator of etiology of chronic hepatitis. The obtained data seem to be important and actual, particularly for diagnostic of subclinical and symptomless forms of these diseases. Further investigation is required to find out reasons and mechanisms of this phenomenon.     

Recurrent meningitis in a case of congenital anterior sacral meningocele and agenesis of sacral and coccygeal vertebrae

A rare case of recurrent meningitis due to congenital anterior sacral meningocele and agenesis of the sacral and coccygeal vertebrae is described. An autosomal dominant inheritance is demonstrated for lower cord malformation, and environmental factors (chromic acid or fumes) are discussed.