Interaction of non-histone chromosomal proteins HMG1 and HMG2 with DNA

Interaction between non-histone protein HMG1 or HMG2 and DNA has been studied by using thermal denaturation and circular dichroism (CD) spectroscopy. We have made the following observations. 1. The binding of each of these two proteins to DNA stabilizes the latter, as shown by an increase in melting temperature of 20 degrees C (from 45 degrees C to about 65 degrees C). 2. There are 6.0 amino acids/nucleotide in HMG1-bound DNA and 5.0 in HMGI-bound DNA which suggests that each HMB1 moleculae would cover about 20 base pairs of DNA and each HMG2 molecule would cover about 25 base pairs. 3. The alpha-helical content of these two non-histone proteins in the complexes, estimated from the CD value at 220 nm, is about one third to one half that of total proteins in calf thymus chromatin. 4. DNA conformation is distorted only slightly by the binding of protein HMG1 or HMG2. 5. Neither the melting nor the CD properties of HMG1-DNA or HMG2-DNA complexes differ substantially whether they are prepared by NaCl-gradient dialysis in urea or by direct mixing of protein and DNA at 0.15 M NaCl, followed by dialysis against the same buffer i.e. 0.25 mM EDTA (pH 8.0).     

Effects of 2,2'-dipyridyl and related compounds on platelet prostaglandin synthesis and platelet function

2,2'-dipyridyl, a chelator of ferrous iron and inhibitor of platelet aggregation, was studied together with several similar compounds to determine the mechanism of their effects on platelets. All of these compounds were more potent inhibitors of arachidonic-acid-mediated aggregation (IC50, 0.17-1.8 mM) than of ADP-mediated aggregation (IC50, 7.6-19.7 mM). At low concentrations required to inhibit arachidonic-acid-mediated aggregation, 2,2'-dipyridyl, 4,4'-dipyridyl and 2-chloropyridine specifically inhibited the platelet cyclo-oxygenase. The mechanism of inhibition of ADP-induced aggregation was investigated, but was not explained. At concentrations needed to inhibit ADP-induced aggregation, 2,2'-dipyridyl did not alter cell ultrastructure, serotonin or nucleotide content or interfere with release of [14C]arachidonic acid or calcium movements. Therefore, our results indicate that 2,2'-dipyridyl and related compounds have two effects on platelets, both due to the unprotonated form. The inhibition of cyclo-oxygenase by low concentrations of these compounds is not due to bidentate iron chelation, since 4,4'-dipyridyl was almost as effective as 2,2'-dipyridyl, but is compatible with binding of these inhibitors to the iron in the heme of the cyclo-oxygenase.     

Measurement of wavelength variation of mode field radius using far-field pattern method

The far-field pattern measuring technique is used to measure the wavelength dependence of the mode field radius in standard single-mode fibres. Chromatic dispersion calculated from these results is shown to be accurate using as few as three lasers. Both theoretical and experimental results are presented.     

Orally active, hydrolytically stable, semisynthetic, antimalarial trioxanes in the artemisinin family

In only three chemical operations, natural trioxane lactone artemisinin (1) was converted into a series of C-10 carbon-substituted 10-deoxoartemisinin compounds 4-9. The three steps involved lactone reduction, replacement of the anomeric lactol OH by F using diethylaminosulfur trifluoride, and finally boron trifluoride-promoted substitution of F by aryl, heteroaryl, and acetylide nucleophiles. All of these C-10 nonacetal, chemically robust, enantiomerically pure compounds 4-9 have high antimalarial potencies in vitro against Plasmodium falciparum malaria parasites, and furans 5a and 5b and pyrrole 7a are antimalarially potent also in vivo even when administered to rodents orally.     

Use of cyclodextrins for manipulating cellular cholesterol content

Previous studies from this laboratory have demonstrated that exposure of tissue culture cells to cyclodextrins results in rapid cholesterol depletion. In the present study, we have developed experimental systems for using solutions of cyclodextrins, either 2-hydroxypropyl beta-cyclodextrin or methylated beta-cyclodextrin, complexed with varying amounts of free cholesterol to manipulate cell cholesterol content. Cholesterol delivered via the cyclodextrin has been found to be metabolically active, as measured by the acyl-coenzyme A:cholesterol acyltransferase (ACAT)-mediated esterification of [3H]cholesterol in Fu5AH rat hepatoma cells and Chinese hamster ovary cells. The methylated beta-cyclodextrin was found to be a more efficient donor in all cell types studied, with an average cholesterol uptake of at least 100 microg cholesterol/mg protein within 6 h. By modifying the cyclodextrin:cholesterol molar ratio, it is possible to manipulate the cellular cholesterol content of cells, producing conditions ranging from net cholesterol enrichment to depletion. The use of cyclodextrins provides a convenient, precise and reproducible method for modulating the cholesterol content of tissue culture cells.     

Retinal dystrophy in long chain 3-hydroxy-acyl-coA dehydrogenase deficiency

We report a case of Bellini duct carcinoma of the left kidney with invasive growth pattern. A 39-year-old man was admitted to our hospital with the chief complaint of gross hematuria. Ultrasonography showed left renal swelling but normal reniform configuration of the kidney was maintained. Computed tomography demonstrated a low density tumor infiltrating into the renal cortex and with tumor extension into the renal vein. Renal angiography revealed a hypovascular tumor. We suspected a left renal cell carcinoma with tumor extension into the left renal vein, and performed radical nephrectomy. Macroscopically, the resected kidney had a normal outer contour. The tumor with infiltrative growth pattern existed in renal medulla. Histopathologic examination revealed a papillary adenocarcinoma originated in Bellini duct (pT3bN2M0). The patient underwent systemic chemotherapy (M-VAC). This case showed invasive growth pattern, which were different from the usual renal cell carcinoma and Bellini duct carcinoma.     

Regioselectivity of nitroglycerin denitration by flavoprotein nitroester reductases purified from two Pseudomonas species

Two species of Pseudomonas capable of utilizing nitroglycerin (NG) as a sole nitrogen source were isolated from NG-contaminated soil and identified as Pseudomonas putida II-B and P. fluorescens I-C. While 9 of 13 laboratory bacterial strains that presumably had no previous exposure to NG could degrade low concentrations of NG (0.44 mM), the natural isolates tolerated concentrations of NG that were toxic to the lab strains (1.76 mM and higher). Whole-cell studies revealed that the two natural isolates produced different mixtures of the isomers of dinitroglycerol (DNG) and mononitroglycerol (MNG). A monomeric, flavin mononucleotide-containing NG reductase was purified from each natural isolate. These enzymes catalyzed the NADPH-dependent denitration of NG, yielding nitrite. Apparent kinetic constants were determined for both reductases. The P. putida enzyme had a Km for NG of 52 +/- 4 microM, a Km for NADPH of 28 +/- 2 microM, and a Vmax of 124 +/- 6 microM x min(-1), while the P. fluorescens enzyme had a Km for NG of 110 +/- 10 microM, a Km for NADPH of 5 +/- 1 microM, and a Vmax of 110 +/- 11 microM x min(-1). Anaerobic titration experiments confirmed the stoichiometry of NADPH consumption, changes in flavin oxidation state, and multiple steps of nitrite removal from NG. The products formed during time-dependent denitration reactions were consistent with a single enzyme being responsible for the in vivo product distributions. Simulation of the product formation kinetics by numerical integration showed that the P. putida enzyme produced an approximately 2-fold molar excess of 1,2-DNG relative to 1,3-DNG. This result could be fortuitous or could possibly be consistent with a random removal of the first nitro group from either the terminal (C-1 and C-3) positions or middle (C-2) position. However, during the denitration of 1,2-DNG, a 1.3-fold selectivity for the C-1 nitro group was determined. Comparable simulations of the product distributions from the P. fluorescens enzyme showed that NG was denitrated with a 4.6-fold selectivity for the C-2 position. Furthermore, a 2.4-fold selectivity for removal of the nitro group from the C-2 position of 1,2-DNG was also determined. The MNG isomers were not effectively denitrated by either purified enzyme, which suggests a reason why NG could not be used as a sole carbon source by the isolated organisms.     

Sequence analysis of equine adenovirus 2 hexon and 23K proteinase genes indicates a phylogenetic origin distinct from equine adenovirus 1

We report the first nucleotide sequence data on equine adenovirus 2 (EAdV2) which corroborate on the molecular level that EAdV2 is distinct from equine adenovirus 1 (EAdV1). Based on sequence homology with Eadv1 the hexon gene of Eadv2 was identified. HindIII restriction fragments containing the hexon and eight other viral genes were cloned into the plasmid pUC19 and the nucleotide sequence of the hexon and the 23K proteinase genes completely determined. Amino acid (aa) comparison of sequence fragments with published adenovirus (AdV) proteins identified the genes for the E1B/19K, IVa2, DNA polymerase, terminal protein, pVI, DNA binding and 100K proteins, usually with highest similarities to human AdV. The nine EAdV2 genes appeared to be in the same relative order as homologous genes of other AdV. The EAdV2 hexon was encoded between the minor capsid precursor protein pVI upstream and the 23K proteinase gene downstream and comprised 2712 nucleotides which translated into 903 aa residues. It was more closely related to the human AdV48 hexon with 71.6% identical and 82.7% functionally similar aa than to the EAdV1 hexon gene with 69.3% aa identity and 80.7% functional similarity. The deduced aa sequence of the EAdV2 23K proteinase gene was 201 residues; it shared 59.7% identical and 75% similar aa residues with the bovine AdV3 23K proteinase as the closest relative. Phylogenetic analysis of the hexon and 23K proteinase genes indicated that EAdV2 does not share an immediate common ancestor with EAdV1 and other AdV.