Activated conformations of the ras-gene-encoded p21 protein. 1. An energy-refined structure for the normal p21 protein complexed with GDP.

A complete three-dimensional structure for the ras-gene-encoded p21 protein with Gly 12 and Gln 61, bound to GDP, has been constructed in four stages using the available alpha-carbon coordinates as deposited in the Brookhaven National Laboratories Protein Data Bank. No all-atom structure has been made available despite the fact that the first crystallographic structure for the p21 protein was reported almost four years ago. In the p21 protein, if amino acid substitutions are made at any one of a number of different positions in the amino acid sequence, the protein becomes permanently activated and causes malignant transformation of normal cells or, in some cell lines, differentiation and maturation. For example, all amino acids except Gly and Pro at position 12 result in an oncogenic protein; all amino acids except Gln, Glu and Pro at position 61 likewise cause malignant transformation of cells. We have constructed our all-atom structure of the non-oncogenic protein from the x-ray structure in order to determine how oncogenic amino acid substitutions affect the three-dimensional structure of this protein. In Stage 1 we generated a poly-alanine backbone (except at Gly and Pro residues) through the alpha-carbon structure, requiring the individual Ala, Pro or Gly residues to conform to standard amino acid geometry and to form trans-planar peptide bonds. Since no alpha-carbon coordinates for residues 60-65 have been determined, these residues were modeled by generating them in the extended conformation and then subjecting them to molecular dynamics using the computer application DISCOVER and energy minimization using DISCOVER and the ECEPP (Empirical Conformational Energies for Peptides Program). In Stage 2, the positions of residues that are homologous to corresponding residues of bacterial elongation factor Tu (EF-Tu) to which p21 bears an overall 40% sequence homology, were determined from their corresponding positions in a high-resolution structure of EF-Tu. Non-homologous loops were taken from the structure generated in Stage 1 and were placed between the appropriate homologous segments so as to connect them. In Stage 3, all bad contacts that occurred in this resulting structure were removed, and the coordinates of the alpha-carbon atoms were forced to superimpose as closely as possible on the corresponding atoms of the reference (x-ray) structure. Then the side chain positions of residues of the non-homologous loop regions were modeled using a combination of molecular dynamics and energy minimization using DISCOVER and ECEPP respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protection of DNA damage by dietary restriction.

Dietary restriction is known to retard the aging processes and delay the onset of age-related neoplastic diseases. The mechanisms underlying these remarkable actions of nutritional intervention are not known in spite of recently intensified research efforts. However, the last couple of years' research on dietary restriction produced strong evidence indicating that its effective antiaging actions might be related to its ability to modulate free radical damage. In the present study, DNA damage and attenuation of the damage by dietary restriction were assessed by measuring 8-hydroxydeoxyguanosine 8-OH dG) in both nuclear DNA (nuDNA) and mitochondrial DNA (mitDNA) fractions. The data show that substantially more damage (approximately 15 times) occurred in mitDNA compared to nuDNA. More interestingly, the DNA damage was significantly attenuated in dietarily restricted rats.     

A peptide derived from the Shaker B K+ channel produces short and long blocks of reconstituted Ca(2+)-dependent K+ channels.

A 20 amino acid synthetic peptide, corresponding to the amino-terminal region of the Shaker B (ShB) K+ channel and responsible for its fast inactivation, can block large conductance Ca(2+)-dependent K+ channels from rat brain and muscle. The ShB inactivation peptide produces two kinetically distinct blocking events in these channels. At lower concentrations, it produces short blocks, and at higher concentrations long-lived blocks also appear. The L7E mutant peptide produces only infrequent short blocks (no long-lived blocks) at a much higher concentration. Internal tetraethylammonium competes with the peptide for the short block, which is also relieved by K+ influx. These results suggest that the peptide induces the short block by binding within the pore of Ca(2+)-dependent K+ channels. The long block is not affected by increased K+ influx, indicating that the binding site mediating this block may be different from that involved in the short block. The short block of Ca(2+)-dependent K+ channels and the inactivation of Shaker exhibit similar characteristics with respect to blocking affinity and open pore blockade. This suggests a conserved binding region for the peptide in the pore regions of these very different classes of K+ channel.     

The relationship between ovarian steroids and uterine estrogen receptors during late pregnancy

Although a direct interdependence exists between the ovarian steroids, estrogen and progesterone, the exact role of these two hormones during pregnancy, especially late pregnancy, is not completely understood. Investigations have been conducted to determine whether the circulating levels of progesterone and estrogen or changes in the ratio of progesterone/estrogen in relation to the concentration of uterine estrogen receptors are associated with triggering parturition. Ninety-day old female rats were sacrificed at gestation days 14, 16, 18, 20 and two days post-partum. The plasma levels of estradiol and progesterone were measured by solid-phase radioimmunoassay. Uterine cytosol was subjected to a charcoal binding assay to determine the concentration of estrogen receptors. Our findings demonstrate that there is a significant (p less than 0.05) drop in both plasma progesterone and estradiol during late pregnancy. Also indicated is a significant (p less than 0.05) increase in uterine estrogen receptors throughout late pregnancy. Finally, during this period there is a direct correlation between the shift in the progesterone/estrogen ratio and the increase in the concentration of uterine estrogen receptors in late pregnancy.     

Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.     

The biological and clinical significance of nicks in human chorionic gonadotropin and its free beta-subunit.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits, alpha and beta. Nicks or missing peptide linkages have been found in the beta 44-52 region of the beta-subunit of hCG, whether from pregnancy or trophoblast disease. This article reviews recent reports about the location of nicks in hCG, their origin and occurrence, their effects on the steroidogenic and receptor-binding activities of hCG, and on the immunological activities of hCG and its free beta-subunit. Taken together, the reports show: (1) nicks occur primarily between beta 47 and beta 48, and to a lesser extent between beta 44 and beta 45; (2) the extent of nicking in hCG samples varies widely, from undetectable to 100 percent of molecules; (3) nicks greatly reduce the steroidogenic activity of hCG in vitro (nicked molecules have less than 20 percent of the activity of the intact hormone); (4) nicks may occur at the trophoblast-myometrial interface or in the circulation by the action of human leucocyte elastase or similar leucocytic protease; (5) hCG testing kits using dimer-specific antibodies may not detect nicked molecules and may give different results from those using other antibodies; (6) hCG international reference preparations and the CR series of hCG standards are variably nicked (10 percent to 20 percent), complicating the problem of discordant hCG results in nick-sensitive assays; (7) results from commonly used immunoassays for measurement of the hCG free beta-subunit vary by as much as tenfold because some of the antibodies employed do not detect nick free beta-subunit.     

Ultrastructure and microtubule organization of isolated generative cells ofAllemanda neriifolia at interphase and prophase

Summary The ultrastructure of isolated generative cells ofAllemanda neriifolia at interphase and prophase was studied. The microtubule organization of the isolated cells was also investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. After the generative cells had been isolated from the growing pollen tubes by osmotic shock, most of the cells were at prophase and only a few were at interphase. The interphase cell is spindle shaped and contains an ellipsoidal nucleus. In addition to the usual organelles, the cytoplasm of the interphase cell contains numerous vesicles (each measuring 40–50 nm in diameter) and two sets of longitudinally oriented microtubule bundles — one in the cortical region and the other near the nucleus. Most of the prophase cells are spherical in shape. Based on the ultrastructure and the pattern of microtubule cytoskeleton organization three types of prophase cells can be recognized. (1) Early prophase cell, which contains the usual organelles, numerous vesicles, and a spherical nucleus with condensed chromosomes. Longitudinally oriented microtubule bundles can no longer be seen present in the early prophase cell. A new type of structure resembling a microtubule aggregate appears in the cytoplasm. (2) Mid prophase cell, which has a spherical nucleus containing chromosomes that appear more condensed than those seen in the early prophase cell. In addition to containing the usual organelles, the cytoplasm of this cell contains numerous apparently randomly oriented microtubules. Few vesicles are seen and microtubule aggregates are no longer present. (3) Late prophase cell, typified by the lack of a nuclear envelope. Consequently, the chromosomes become randomly scattered in the cytoplasm. Microtubules are still present and some become closely associated with the chromosomes. The changes in the ultrastructure and in the pattern of microtubule organization in the interphase and prophase cells are discussed in relation to the method of isolation of the generative cells.     

Restoration of embryogenic competence in repeatedly subcultured carrot cell lines

Summary The production of somatic embryos from carrot suspension cultures invariably decreases through simple, repeated subculturing. Extracellular, concentrated compounds extracted from already established embryo culture not only recovered the embryogenic capability, but also accelerated the embryo production as much as two-fold (up to 1600 embryos/ml) compared with that of a control culture. Sugars, which were only a small portion of the total concentrate, were excluded as possible causative factors. It is likely that a protein fraction that is generated directly by competent, embryogenic cultures is important for the restoration of embryogenic potential.     

Randomised controlled trial of short term treatment to eradicate Helicobacter pylori in patients with duodenal ulcer.

OBJECTIVE--To determine whether one week''s drug treatment is sufficient to eradicate Helicobacter pylori in patients with duodenal ulcer. DESIGN--Single blind, randomised controlled trial. SETTING--Specialised ulcer clinic in a teaching hospital. PATIENTS--155 patients with H pylori and a duodenal ulcer verified endoscopically which had either bled within the previous 24 hours or was causing dyspepsia. INTERVENTIONS--Patients were allocated randomly to receive either omeprazole for four weeks plus bismuth 120 mg, tetracycline 500 mg, and metronidazole 400 mg (all four times a day) for the first week (n = 78), or omeprazole alone for four weeks (n = 77). Further endoscopy was performed four weeks after cessation of all drugs. MAIN OUTCOME MEASURES--Presence or absence of H pylori (by urease testing, microscopy, and culture of antral biopsy specimens), duodenal ulcer, and side effects. RESULTS--Eradication of H pylori occurred in 70 (95%) patients taking the four drugs (95% confidence interval 86% to 97%) compared with three (4%) patients taking omeprazole alone (1% to 11%). Duodenal ulcers were found in four (5%) patients taking the four drugs (2% to 12%) and in 16 (22%) patients taking omeprazole alone (14% to 32%). Mild dizziness was the only reported side effect (six patients in each group) and did not affect compliance. CONCLUSIONS--A one week regimen of bismuth, tetracycline, and metronidazole is safe and effective in eradicating H pylori and reduces the number of duodenal ulcers four weeks after completing treatment.