Function of the p53-pRb-dependent path after exposure to DNA damaging agents in rats embryonal fibroblast cells, transformed by E1A and Ha-Ras oncogenes

The packing of the G-actin monomers within crystalline actin tubes was investigated at atomic detail. To achieve this, we have chosen an integrated structural approach which combines intermediate resolution electron microscopy based 3-D reconstruction and surface metal shadowing of crystalline actin tubes with atomic resolution X-ray data of the G-actin monomer. Distinct from the parallel, half-staggered packing of the actin subunits within F-actin filaments, the arrangement of actin monomers within the crystalline tubes involves antiparallel packing into dimers with p2 symmetry. Within the crystalline tubes, the actin monomers are oriented so that the filament axis runs parallel with the sheet plane and the intersubunit contacts in this direction are similar to those existing along the two long-pitch helical strands of the F-actin filament. The other intersubunit contacts within the crystalline tubes are not found in the actin filament. The ability of actin to form a variety of polymorphic oligomers is still not fully understood, and the functional implications of this variability have yet to be deciphered. Regularly packed actin assemblies such as sheets, tubes or ribbons may ultimately yield structural relationships to in vivo relevant actin oligomers such as, for example, the "lower dimer".     

Temperature-sensitive mutants of the EcoRI endonuclease

The EcoRI endonuclease is an important recombinant DNA tool and a paradigm of sequence-specific DNA-protein interactions. We have isolated temperature-sensitive (TS) EcoRI endonuclease mutants (R56Q, G78D, P90S, V97I, R105K, M157I, C218Y, A235E, M255I, T261I and L263F) and characterized activity in vivo and in vitro. Although the majority were TS for function in vivo, all of the mutant enzymes were stably expressed and largely soluble at both 30 degrees C and 42 degrees C in vivo and none of the mutants was found to be TS in vitro. These findings suggest that these mutations may affect folding of the enzyme at elevated temperature in vivo. Both non-conservative and conservative substitutions occurred but were not correlated with severity of the mutation. Of the 12 residues identified, 11 are conserved between EcoRI and the isoschizomer RsrI (which shares 50% identity), a further indication that these residues are critical for EcoRI structure and function. Inspection of the 2.8 A resolution X-ray crystal structure of the wild-type EcoRI endonuclease-DNA complex revealed that: (1) the TS mutations cluster in one half of the globular enzyme; (2) several of the substituted residues interact with each other; (3) most mutations would be predicted to disrupt local structures; (4) two mutations may affect the dimer interface (G78D and A235E); (5) one mutation (P90S) occurred in a residue that is part of, or immediately adjacent to, the EcoRI active site and which is conserved in the distantly related EcoRV endonuclease. Finally, one class of mutants restricted phage in vivo and was active in vitro, whereas a second class did not restrict and was inactive in vitro. The two classes of mutants may differ in kinetic properties or cleavage mechanism. In summary, these mutations provide insights into EcoRI structure and function, and complement previous genetic, biochemical, and structural analyses.     

Geographic patterns of low birth weight in Hawaii

This study examines areal variations in low birth weight, using the census tract as the unit of analysis. Reports from the 1980 U.S. census were used to develop summary indicators of environmental and socio-economic conditions, including poverty, employment, education and crowding, for 155 census tracts in the state of Hawaii. Maternal socio-demographic, prenatal care utilization, and medical risk indicators and low birth weight percentages for resident, single live births were extracted from the Hawaii 1979-1987 vital record live birth files and aggregated by census tract. Multiple regression analysis was used to develop a model that predicted 61% of the variation among census tracts in the percentage of low birth weight. Patterns of low birth weight were primarily associated with ethnic patterns of maternal residence and single marital status. There was no association between inadequate prenatal care and low birth weight at the census tract level.     

Differential diagnosis of hepatocellular nodular lesions

While certain neuroactive volatile organic compounds (VOCs) have been reported to have an uneven distribution in various anatomically distinctive brain regions, this has not yet been reported for the short-chain aliphatic halogenated hydrocarbons. Therefore, the uptake and regional brain distribution of 1, 1, 1-trichloroethane (TRI) in mice and rats following inhalation exposure were examined. Male Sprague-Dawley rats and CD-1 mice were exposed to TRI at either 3500 or 5000 ppm for 10, 30, 60, or 120 min. Seven brain regions from rats and three from mice were sampled, and TRI concentrations in the blood and brain tissues were determined by headspace gas chromatography. In both species, the medulla oblongata was found to have the highest TRI concentrations, while cortex (in both species) and hippocampus (only sampled in rats) contained the lowest TRI concentrations. Substantial differences were also observed between the two species, as the mice exhibited higher capacity to accumulate TRI in the blood as well as in the brain regions. It appears that lipid content is a main factor influencing the differential disposition of TRI among the brains regions. Physiological differences in the respiratory systems of the two species and the physiochemical properties of the chemical favoring diffusion toward lipid-rich compartments could also have been expected to account for the patterns of regional distribution and species differences.     

Total protein and acid phosphatase concentrations in prostatic fluid from patients with BPH compared to carcinoma

Efficacy of reperfusion therapy in acute myocardial infarction is strictly time dependent. As is evidenced by several studies, most benefit in terms of myocardial salvage and short- and long-term mortality is achieved with initiation of therapy within the first 60-90 minutes after onset of symptoms. Nearly exclusively, prehospital initiation of thrombolysis makes it possible to take advantage of this early time window. Moreover a time gain of more than 30 minutes, up to 130 minutes, is possible by prehospital initiation of thrombolysis, depending on local circumstances. Randomized studies yielded a better outcome when a time gain of > or = 90 minutes was achieved. Since it has been shown that prehospital diagnosis of an acute myocardial infarction is reliable and out-of-hospital initiation of therapy has no additional specific risk, patients seen within the first 60-90 minutes after onset of symptoms or for whom a relevant time gain of > or = 90 minutes can be expected are ideal candidates for, and therefore should receive, prehospital thrombolysis.     

Cysteine proteinase inhibitors block early steps in hemoglobin degradation by cultured malaria parasites

Erythrocytic malaria parasites degrade hemoglobin as a source of amino acids for parasite protein synthesis. Cysteine proteinase inhibitors have been shown to block the hydrolysis of globin by cultured parasites, indicating that a malarial cysteine proteinase is required for this process. In the present study, we have evaluated the role of parasite proteinases in earlier steps of hemoglobin degradation, namely the disassociation of the hemoglobin tetramer and the separation of heme from globin. Hemoglobin did not spontaneously denature or release heme under the pH and reducing conditions of the malarial food vacuole, suggesting that parasite enzymatic activity is necessary for early steps in hemoglobin degradation. The incubation of cultured parasites with cysteine proteinase inhibitors inhibited the denaturation of hemoglobin and the release of heme from globin. These results suggest that, in addition to its role in globin hydrolysis, a malarial cysteine proteinase participates in the dissociation of the hemoglobin tetramer and the release of heme from globin. Thus, the malarial cysteine proteinase is a promising target for antimalarial chemotherapy.     

Effects of carbon dioxide pneumoperitoneum, air pneumoperitoneum, and gasless laparoscopy on body weight and tumor growth

BACKGROUND: The oncologic consequences of intraperitoneal carbon dioxide (CO2) insufflation during the laparoscopic resection of cancer are under debate. The effect of other insufflating gases or gasless laparoscopy on cancer requires study. OBJECTIVE: To study body weight and tumor growth in rats after CO2 pneumoperitoneum, air pneumoperitoneum, and gasless laparoscopy. METHODS: On day 1, an 8-mg bolus of ROS-1 tumor was placed under the renal capsule of both kidneys in rats. In experiment A, rats had either CO2 insufflation (n=10) or a gasless laparoscopic bowel resection (n=10) on day 3 and were humanely killed after 7 days. In experiment B, rats had either a laparoscopic bowel resection with CO2 insufflation (n=11) or insufflation with air (n=11) on day 3 and were killed after 7 days. In both experiments, postoperative weight loss and tumor growth were measured, and the differences were tested with an analysis of covariance. RESULTS: Renal subcapsular tumor growth in the group having gasless laparoscopy was less than that in the group having CO2 pneumoperitoneum (P=.04). Postoperative weight loss in these groups showed no differences (P=.55). No differences in tumor growth or weight loss were found between rats having insufflation with CO2 and those having insufflation with air (P=.61 and P=.68, respectively). CONCLUSIONS: The restoration of body weight after a laparoscopic surgical procedure was similar with CO2, air, or gasless laparoscopy. Gasless laparoscopy was associated with less renal subcapsular tumor growth than was insufflation with CO2. Therefore, the application of gasless techniques in laparoscopic oncologic surgical treatment demands further study.     

Reaction paths of iron oxidation and hydrolysis in horse spleen and recombinant human ferritins

UV-visible spectroscopy, electrode oximetry, and pH stat were used to study Fe(II) oxidation and hydrolysis in horse spleen ferritin (HoSF) and recombinant human H-chain and L-chain ferritins (HuHF and HuLF). Appropriate test reactions and electrode responses were measured, establishing the reliability of oxygen electrode/pH stat for kinetics studies of iron uptake by ferritin. Stoichiometric ratios, Fe(II)/O2 and H+/Fe(II), and rates of oxygen uptake and proton production were simultaneously measured as a function of iron loading of the protein. The data show a clear distinction between the diiron ferroxidase site and mineral surface catalyzed oxidation of Fe(II). The oxidation/hydrolysis reaction attributed to the ferroxidase site has been determined for the first time and is given by 2Fe2+ + O2 + 3H2O --> [Fe2O(OH)2]2+ + H2O2 + 2H+ where [Fe2O(OH)2]2+ represents the hydrolyzed dinuclear iron(III) center postulated to be a mu-oxo-bridged species from UV spectrometric titration data and absorption band maxima. The transfer of iron from the ferroxidase site to the mineral core has been now established to be [Fe2O(OH)2]2+ + H2O --> 2FeOOH(core) + 2H+. Regeneration of protein ferroxidase activity with time is observed for both HoSF and HuHF, consistent with their having enzymatic properties, and is facilitated by higher pH (7.0) and temperature (37 degreesC) and by the presence of L-subunit and is complete within 10 min. In accord with previous studies, the mineral surface reaction is given by 4Fe2+ + O2 + 6H2O --> 4FeOOH(core) + 8H+. As the protein progressively acquires iron, oxidation/hydrolysis increasingly shifts from a ferroxidase site to a mineral surface based mechanism, decreasing the production of H2O2.