Bouveret Syndrome: A Rare Form of Gallstone Ileus.

Bouveret's syndrome is a rare variant of gallstone ileus characterized by a gastric outlet obstruction due to the impaction of a gallstone lodged in the duodenum, resulting from a cholecystoduodenal fistula. It accounts for only one to three percent of cases of gallstone ileus. We examine a case of Bouveret syndrome in an elderly Japanese female who presented with vomiting and decreased oral intake. Subsequent imaging found a gallstone ileus due to a bilioduodenal fistula. She underwent exploratory laparotomy enterolithotomy which found a large black gallstone located in the small bowel and confirmed the presence of the fistula. Despite its relative rarity, Bouveret syndrome carries a high risk of morbidity and mortality.

The combined impact of pH and activated carbon on the elemental composition of a liquid conifer embryogenic tissue initiation medium.

This study considered the elemental composition of plant tissue culture media in response to pH and two different types of activated C (AC; tissue culture and non acid-washed grades) in liquid media. When tissue culture medium is supplemented with AC the method of AC addition and pH adjustment can greatly impact the final medium pH, in turn, altering mineral availability. Over the pH range of 4-7, Cu and Zn adsorbed (95% and 50%) onto the two physically different ACs to the same extent. As the pH exceeded 5.8, precipitation became pronounced, resulting in 50% reductions in Mn and Fe and smaller reductions in Ca (20%), and P (15%), independent of AC. Non acid-washed AC released significant levels of Mg (65% increase) and Ca (10% increase) at pH 5.8 compared to the no-AC control. No adsorption was indicated for inorganic anions. Low levels for Cu and Zn are a concern when using AC, and low levels of Fe and Mn are a concern when the pH of the medium exceeds 5.8. Due to its impurity content and difficulty associated with its neutralization, non-acid-washed AC may be a poor choice for use in tissue culture medium.

The impact of Gelrite and activated carbon on the elemental composition of two conifer embryogenic tissue initiation media.

The elemental composition of plant tissue culture media was studied in response to (1) different levels of Gelrite and activated carbon (AC) in semisolid media and (2) different levels and types of AC in liquid media. Doubling the level of Gelrite from 2 g/l to 4 g/l reduced available magnesium (20%), calcium (16%), zinc (17%) and manganese (24%) and increased potassium (6%). AC adsorbed copper (90-95%) and zinc (35-51%) from both liquid and semisolid media. Two significantly different ACs gave minor differences in adsorption. No adsorption was indicated for inorganic anions. Nonacid-washed AC released significant levels of magnesium (44% increase), calcium (16% increase) and silica (a 75% increase to 1.8 mg/l). The elemental composition of media may need to be adjusted when increasing the Gelrite level or adding AC.

The cytotoxicity and mode of action of 2,3,4-trisubstituted pyrroles and related derivatives in human Tmolt4 leukemia cells.

4-Carbethoxy-1-methyl-2-phenacyl-3-phenylpyrrole (9), 4-carbethoxy-2-(4-methoxybenzoyl)-3-(4-methoxyphenyl)pyrrole (10) and 2-(4-methoxybenzoyl)-3,4-bis-(4-methoxyphenyl)pyrrole (11) proved to be potent cytotoxic agents against the growth of murine and human leukemias and lymphomas. Selective toxicity was demonstrated against the growth of solid tumors, e.g., human adenocarcinoma of the colon SW480 and ileum HCT-8, glioma U-87-MG, and rat UMR-106 osteosarcoma. A mode of action study in Tmolt4 leukemia cells demonstrated that the agents inhibited de novo purine synthesis at the regulatory sites PRPP-amido transferase, IMP dehydrogenase as well as dihydrofolate reductase resulting in significant inhibition of DNA synthesis in 60 min. Other biochemical sites which were affected significantly were thymidylate synthetase, DNA polymerase alpha, RNA polymerases, nucleoside kinase and ribonucleoside reductase.

Water and formula fluoride concentrations: significance for infants fed formula.

The independent contributions of formula and water to the total fluoride (F) intake from the diet of formula-fed infants is not fully documented. Although the precise timing and mechanism by which dental fluorosis occurs has not been fully defined, water F levels can be an important consideration in the risk of dental fluorosis for formula-fed infants. An assessment of 1,308 participants younger than 2 years old revealed that: 81% of homes received public water; 19% received well water; 26% of participants used bottled water; and 11% used some kind of filtration system. In this study, virtually all formulas consumed by the birth cohort and water sources used in the reconstitution of these formulas were assayed for F using a F ion specific electrode and direct read method, except for soy-based formulas, which were analyzed by microdiffusion (modified Taves). Among 78 commercially available bottled waters in Iowa, F levels ranged from 0.02 to 1.36 ppm (mean 0.18 ppm), 83% from 0.02 to 0.16 ppm, 7% from 0.34 to 0.56 ppm, 1% had a F level of 0.88, and 9% had F levels > 1.0 ppm. Among 47 casein (milk)-based formulas, 16 ready-to-feed (RTF) formulas had levels of 0.04-0.55 ppm F (mean 0.17 ppm), 14 liquid concentrates (LC) reconstituted with distilled water had levels of 0.04-0.19 ppm F (mean 0.12 ppm), and 17 powdered concentrates (PC) reconstituted with distilled water had levels of 0.05-0.28 ppm F (mean 0.14 ppm). The 17 soy-based formulas had a range of 0.04-0.47 ppm F (mean 0.26 ppm).(ABSTRACT TRUNCATED AT 250 WORDS)

The interface between an alternating CG motif and a random sequence motif displays altered nuclease activity.

Previously we described the B-Z junctions produced in oligomers containing (5meCG)4 segments in the presence of 5.0 M NaCl or 50 uM Co(NH3)6+3 [Sheardy, R.D. & Winkle, S.A., Biochemistry 28, 720-725 (1989); Winkle, S.A., Aloyo, M.C., Morales, N., Zambrano, T.Y. & Sheardy, R.D., Biochemistry 30, 10601-10606 (1991)]. The circular dichroism spectra of an analogous unmethylated oligomer containing (CG)4, termed BZ-IV, in 5.0 M NaCl and in 50 uM Co(NH3)+3 suggest, however, that this oligomer does not form a B-Z hybrid. BZ-IV possesses Hha I sites (CGCG) in the (CG)4 segment and an Mbo I site (GATC) at the terminus of the (CG)4 segment. BZ-IV is equally digestible in the presence and absence of cobalt hexamine by Hha I, further indicating that the structure of BZ-IV is fully B-like under these conditions. The Mbo I cleavage site at the juncture between the (CG)4 segment and the adjacent random segment displays enhanced cleavage by both Mbo I and its isoschizomer Sau3AI in the presence of cobalt hexamine. In addition, exonuclease III digestion of BZ-IV is inhibited at this juncture. Actinomycin inhibits Mbo I activity in the presence of cobalt hexamine but not in the absence. Together, these results suggest that enzymes recognize the interfaces of (CG)n and adjacent random sequences as altered substrates even in the absence of a B-Z junction formation.

Using lambda exonuclease inhibition assays to map carcinogen binding sites.

Previous restriction mapping studies (M.A. Mallamaci, D.P. Reed and S.A. Winkle, J. Biomolecular Structure and Dynamics, in press (1992)) have indicated that a small number of locations on the plasmid pBR322 may be high affinity binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF). PBR322 was reacted with acetoxyAAF to produce DNA with one, three or seven acetoxyAAF moieties per DNA molecule. Thus only the higher affinity binding sites are affected. Subsequent digestion with the restriction enzyme Hinf I produced fragments containing previously indicated locations of potential acetoxyAAF binding sites. Fragments thought not to contain binding sites were also examined as controls. The isolated fragments, singly 32P end-labeled, were digested with lambda exonuclease. The three fragments suspected of containing acetoxyAAF binding sites possess new lambda exonuclease inhibition sites when the fragments are obtained from acetoxyAAF reacted DNA. No such inhibition sites are found with the two fragments suggested previously not to contain acetoxyAAF binding sites. These carcinogen produced inhibition sites occur in sequences which are similar, suggesting that acetoxyAAF preferentially may target a small number of sequences.

Restriction enzyme mapping of carcinogen binding regions on pBR322.

Previous equilibrium binding experiments (S.A. Winkle and T.R. Krugh, Nucleic Acids Res. 9, 3175-3186 (1981)) suggested that the carcinogen N-hydroxy-N-acetyl-2-aminofluorene might exhibit preferential binding to a small number of sites on phiX174 DNA. To examine whether the covalently binding analogue N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) also possesses high affinity sites, the plasmid pBR322 was reacted with 3H labeled acetoxyAAF to give one to sixteen adducts per DNA molecule. Thus only higher affinity sites would be affected. The DNA was subsequently cleaved with either Alu I, Hae III, Hha I, Hinf I or Hpa II restriction endonuclease and the restriction fragments isolated by gel electrophoresis. Examination of the distribution of 3H acetoxyAAF among the fragments was not random but, rather, with each enzyme, the acetoxyAAF was found predominantly in a few fragments. The locations of the bands containing the acetoxyAAF for each enzyme overlap--suggesting that there are regions on pBR 322 which contain high affinity sites for acetoxyAAF binding.

Locating binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene using restriction enzyme inhibition assays.

Restriction enzyme inhibition studies have been employed to map the locations of high affinity binding sites of the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) on pBR322, phiX174 and SV40 DNAs. Bound carcinogen levels were kept low (less than 20 bound AAF moieties per DNA molecule) in order to observe only the binding to the high affinity sites. Inhibition of certain restriction enzymes was observed in a limited number of locations on these DNAs. Inhibition increased as bound AAF increased and the particular restriction enzymes inhibited varied with location. On all three DNAs, activities of these enzymes was not affected in other locations. Comparison of the sequences at the sites of inhibition on the three DNAs indicates that all sites have common sequence elements: the presence of either the sequence T(C/G)TT(G/C) or the sequence T(G/C)CTT(G/C).

Reversibility of the effects of dietary fish oil on the fatty acid composition of the brain and retina of growing chicks.

Dietary fish oil increases levels of (n-3) fatty acids in the brain and retina of younger animals but has less effect in adults. The duration of the effects of fish oil in young animals, as well as the extent of reversibility of the effects, are unknown. Laying hens were fed either a fish oil diet or a soybean oil-based control diet. Resulting chicks were assigned to three diet groups: chicks from fish oil and soybean oil hens were continued on fish oil and soybean oil diets, respectively, for 0, 3, 6, or 9 weeks, and additional chicks from the fish oil hens were fed the fish oil diet for 0, 3, or 6 weeks and then reversed to the soybean oil diet for a period of 3 weeks. The fatty acid composition of the brain, retina, liver, and serum of the reversal chicks was compared with chicks fed the fish oil diet only or the soybean oil diet only. Brain levels of docosahexaenoic acid (22:6(n-3)) decreased substantially when reversal from the fish oil diet to the control diet was begun at hatching, but did not decrease when reversal was begun at later times. Other (n-3) fatty acids in the brain, docosapentaenoic acid (22:5(n-3)) and eicosapentaenoic acid (20:5(n-3)), decreased substantially at all ages, and to a greater extent than 22:6(n-3). Brain arachidonic acid (20:4(n-6)), which was low in fish oil chicks, rose to control after reversal at hatching, but recovered only partially when reversal was begun at later times. A similar patterns was observed in the retina. Serum and liver (n-3) fatty acids fell to control in all reversal chicks, and (n-6) fatty acids increased to control, except in chicks reversed at 6 weeks. This study demonstrates that by 3 weeks of age the chick brain strongly resists diet-induced lowering of high levels of 22:6(n-3).

Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI.

We have been investigating the structure, dynamics, and ligand-binding properties of the interface that exists between a right-handed conformation and a left-handed conformation (i.e., a B-Z junction) in synthetic DNA oligomers. Since exo- and endonuclease activity is known to be sensitive to the conformation of the template DNA, we have designed and synthesized a DNA oligonucleotide of 20 base pairs (designated as BZ-III) with an MboI recognition site (GATC) at the location of a potential B-Z junction. The activity of the MboI enzyme toward this molecule and DNA oligomers that contain multiple MboI sites located at B-Z junctions was monitored in the absence and presence of the Z-conformation-inducing reagent cobalt hexaammine. In all cases, the activity of the enzyme was enhanced in the presence of cobalt hexaammine. The activity of MboI toward BZ-III, in the presence and absence of cobalt hexaammine, was also examined when the DNA oligomer is also in the presence of the DNA binding drugs actinomycin D, ametantrone, or ethidium bromide. In all cases, the activity of the enzyme was inhibited in the presence of drug. The results suggest that B-Z junctions are structurally unique and that this uniqueness may alter nuclease activity at sites in or near the junction.

Anomalous gel migration of DNA oligomers containing multiple conformational junctions.

We have previously shown that a short 16 base pair DNA oligomer can accommodate a B-Z conformational junction [Sheardy, R. D., & Winkle, S. A. (1989) Biochemistry 28, 720-725]. Results from 1H NMR studies indicated that only three base pairs were involved in the junction and that one of these base pairs was highly distorted. Being interested in the nature of this distortion, we constructed DNA oligomers which have the potential to contain multiple B-Z junctions for polyacrylamide electrophoretic studies. We report that the mobilities displayed by these molecules through acrylamide gels in the absence and presence of cobalt suggest that these molecules run shorter than they actually are. This anomalous migration may be due to structural/dynamic properties of the DNA helix manifested by the periodic distortions of the potential B-Z junctions.

Synthesis and characterization of deoxyguanosine-benzoquinone adducts.

Benzene expresses its carcinogenic potential in humans largely in the form of acute leukemia. Because an understanding of the formation of DNA adducts by benzene metabolites may help to explain the etiological role they play in benzene-induced bone marrow disease, we have synthesized, isolated and characterized adducts formed by the reaction of deoxyguanosine with hydroquinone and p-benzoquinone, two toxic metabolites of benzene. [3H]Deoxyguanosine and [14C]hydroquinone reacted in neutral aqueous buffer containing iron to form two dual-labeled products, which were separated using HPLC. When p-benzoquinone was substituted for hydroquinone, the same adducts were formed in the absence of added iron. The ultraviolet and fluorescence spectra of the less polar adduct, called Adduct 2, were distinctly different from the spectra of the starting materials. NMR and mass spectrometry suggested a compound with a mass of 357 with the p-benzoquinone moiety bound to the N-1 and N2 positions of deoxyguanosine. Based on these data it is proposed that Adduct 2 is (3'OH)benzetheno(1,N2)deoxyguanosine. The more polar product, Adduct 1, was found to have a unique ultraviolet spectrum but did not appear to be fluorescent. Both adducts were observed after calf thymus DNA was incubated with hydroquinone and digested to its constituent nucleosides.

Temperature-dependent CD and NMR studies on a synthetic oligonucleotide containing a B-Z junction at high salt.

It is now accepted that two or more conformations may exist within the same DNA molecule, thereby generating conformational junctions. The presence of B-Z junctions between right- and left-handed DNA conformations has been detected in plasmids by a number of techniques. Preliminary characterization of the first example of a B-Z junction is a short DNA oligonucleotide has recently been reported [Sheardy, R. D. (1988) Nucleic Acids Res. 16, 1153-1167]. We report additional CD and NMR data that support the existence of the junction in this model oligomer. These studies indicate that only three base pairs are involved in the junction and only one of these is dramatically distorted. Furthermore, the NMR saturation-transfer experiments suggest the junction's internal motion is temperature dependent.

Equilibrium binding of spermine and histamine to salmon sperm DNA and poly(dGdC).

Studies using the solute-enhanced phase partition technique demonstrate that the endogenous amines histamine and spermine bind to both salmon sperm DNA and poly(dGdC) in a markedly cooperative fashion. Both compounds exhibited DNA sequence-dependence effects in their mode of binding. The linear aliphatic spermine molecule binds more strongly than histamine to both DNA types.

Modulation of diethylnitrosamine-induced hepatocarcinogenesis and O6-ethylguanine formation in rainbow trout by indole-3-carbinol, beta-naphthoflavone, and Aroclor 1254.

Rainbow trout were fed a diet containing indole-3-carbinol (2000 ppm), beta-naphthoflavone (500 ppm), or Aroclor 1254 (100 ppm) for 6 weeks before a single 24-hr exposure to an aqueous solution of 250 ppm diethylnitrosamine (DEN). The fish were killed 42 weeks later to determine the carcinogenic response. DEN exposure produced an 80.2% incidence of liver tumors and an average of 3.47 tumors per tumor-bearing fish, whereas no tumors were detected in the sham-treated control fish. Tumor induction was inhibited by indole-3-carbinol (27.5% incidence, 1.89 tumors per tumor-bearing fish) but enhanced by beta-naphthoflavone (91.8% incidence, 3.60 tumors per tumor-bearing fish). Aroclor 1254 had no effect on DEN-induced hepatocarcinogenesis (80.0% incidence, 3.03 tumors per tumor-bearing fish). The effects of these modulators on O6-ethylguanine and 7-ethylguanine formation (measured by HPLC and fluorescence spectrophotometry) were examined. Liver DNA ethylguanine levels were reduced in indole-3-carbinol-pretreated fish and increased in beta-naphthoflavone-pretreated fish compared to untreated controls after DEN exposure. Aroclor 1254 pretreatment had no significant effect on DNA ethylguanine formation. Similar O6-ethylguanine to 7-ethylguanine ratios were found among the control and treated groups. The results of this study indicate that modulation of DEN hepatocarcinogenesis by indole-3-carbinol and beta-naphthoflavone may be mediated by their effects on O6-ethylguanine formation and, therefore, on the initiation phase of carcinogenesis.

Formation and persistence of ethylguanines in liver DNA of rainbow trout (Salmo gairdneri) treated with diethylnitrosamine by water exposure.

Diethylnitrosamine exposure via the water resulted in the formation of 7-ethylguanine and O6-ethylguanine in rainbow trout liver DNA. The modified bases were quantitated by high-pressure liquid chromatography and fluorescence spectrophotometry. In vivo 7-ethylguanine and O6-ethylguanine levels were directly proportional to DEN concentrations between 62.5 and 250 ppm. 7-Ethylguanine and O6-ethylguanine levels were approximately directly proportional to duration of exposure to DEN between 0 and 6 hr under the conditions used, with less than proportionate increases thereafter. Removal of ethylguanines from liver DNA following a 24-hr exposure to 250 ppm DEN (a known carcinogenic regimen) was biphasic; 24% of the O6-ethylguanine and 32% of the 7-ethylguanine found immediately after exposure were removed in 12 hr but no significant decline was found over the period from 12 to 96 hr after exposure. Alkyl acceptor protein activity in trout liver was examined to assess the role of enzymatic repair in the observed loss of O6-ethylguanine in vivo. Although an O6-alkylguanine repair system similar to the alkyltransferase system reported in rodents was found in trout liver, only 4% of the O6-ethylguanine lost from DNA after exposure to 250 ppm DEN can be accounted for by activity of the alkyl acceptor protein. The high incidence of liver tumours observed in DEN-treated rainbow trout may be related to the rapid formation and substantial persistence of the promutagenic O6-ethylguanine in liver DNA.

Enzyme immunoassay using a novel recombinant polypeptide to detect human immunodeficiency virus env antibody.

A unique antigen, CBre3, has been synthesized from a genetically engineered clone to detect human immunodeficiency virus (HIV) env antibodies with high sensitivity and specificity. The antigen contains sequences derived from both envelope proteins of HIV, i.e., gp120 and gp41, and was purified free of Escherichia coli proteins detectable by Coomassie stain or immunoblotting with E. coli antiserum. The purified recombinant polypeptides were used as antigen in an enzyme immunoassay (EIA) to screen serum samples from healthy and HIV-infected individuals. The same samples were also tested by radioimmunoprecipitation (RIP) for gp120 and gp160 HIV antibodies. All samples containing gp120 and gp160 antibodies by RIP had CBre3 EIA values greater than 0.35 (n, 122; range, 0.37 to 2.1+; median, 1.65). All RIP HIV antibody-negative samples had CBre3 EIA values less than 0.25 (n, 140; mean, 0.052; standard deviation, 0.045; range, 0.00 to 0.22). The endpoint titer of a standard positive control serum was 1:10,000 by RIP and by CBre3 EIA. The assay was 100% accurate in three proficiency panels. It easily detected six samples from individuals whose infections were confirmed by culture; these samples were reactive only with p24 by Western blot. The samples also were positive for gp120 and gp160 antibodies by RIP. These data suggest that the CBre3 EIA can detect env antibodies as sensitively and specifically as RIP and with more sensitivity than Western blot.

Mixed-mode hydrophobic ion exchange for the separation of oligonucleotides and DNA fragments using HPLC.

Hydrophobic anion exchangers were formed by cobonding both ionic and hydrophobic ligands to silica gel. These phases were used to separate single-stranded oligonucleotides and double-stranded DNA restriction fragments. By varying the ratio of n-octyldimethylsilane and either 3-chloropropyldimethylsilane or 4-chlorobutyldimethylsilane added during silanization a series of mixed-ligand or mixed-mode stationary phases was created. Concentration and ratio of bonded ligands were determined using a new gas chromatography fluorination method. Total ligand coverage was found to approach 2.1 ligands nm-2 for n-octyldimethylsilane. Bonding reproducibility for mixed-mode phases was good. Nucleic acid separations were achieved under gentle mobile phase conditions by using the stationary phase as an easily modifiable variable.

13C-NMR of ribosyl ApApA, ApApG and ApUpG.

The chemical shifts as well as the 13C-31P coupling constants of the carbon-13 nuclei in single-stranded ApApA, ApApG, and ApUpG are sensitive to sequence and temperature. ApApA and ApApG have similar properties with large shielding (up to 1.7 ppm) of many of the base carbons upon decreasing the temperature from 70 degrees C to 11 degrees C; the base carbons have smaller shielding changes in ApUpG. Large shielding and deshielding effects are observed for the 1', 3', 4' and 5'-carbons over this temperature range. Analysis of the 13C-31P couplings measured at the 4' ribose carbons show that the population of the anti rotamer about O5'-C5' varies from 98 to 75%, and is higher in ApApA and ApApG than in ApUpG. The CCOP coupling data at 2' and 4' is consistent with a blend of the -antiperiplanar/-synclinal nonclassical rotamers about the C3'-O3' bond, varying from 89/11% in ApApG to 55/45% in ApUpG. The coupling and chemical shift data support the thesis that ApUpG is stacked much less than the other two molecules. The stacked forms of all three trinucleotides is most easily interpreted by a standard A-RNA model. It is not necessary to invoke the "bulged base" hypothesis [Lee, C.-H. and Tinoco, Jr., I. (1981) Biophysical Chemistry 1, 283-294; Lankhorst, P.P., Wille, G., van Boom., J.H., Altona, C., and Haasnoot, C.A.G. (1983) Nucleic Acids Research 11, 2839-2856] to explain the contrast in 13C spectroscopic properties of ApUpG in comparison to ApApG and ApApA.