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.     

Further similarities between astrocytes and perisinusoidal stellate cells of liver (Ito cells): colocalization of desmin and glial fibrillary acidic protein in astroglial primary cultures

In order to evaluate the effectiveness of an intensive care unit (ICU), the case-mix has to be considered. This was a cohort study. By using Acute Physiology and Chronic Health Evaluation scores (APACHE II score), we evaluated the case-mix and mortality rate of 282 patients who were treated in our postoperative ICU. The overall mortality rate was 10.6 per cent. Higher Acute physiology scores and emergency surgery in the presence of chronic health status were related to higher mortality, but age was not. However, the original APACHE II model could not precisely predict the mortality of Thai patients. We used stepwise logistic regression to determine the predictors of death and found the prediction model to be -7.24 + 0.37 (APACHE II score) + 1.46 (postemergency surgery). The actual mortality for patients with APACHE II score > 15 in our ICU was higher than that predicted by the original APACHE II model. The causes of this difference might be difference in methodology, characteristics of ICU and the quality of care.     

EPR Study of Cation Radicals of Short Chain Polyenes

Short chain polyene cation diene and triene radicals generated by X-ray irradiation of different polyenes diluted in a CFCl₃ matrix have been detected by EPR and the magnitudes of the g and isotropic hyperfine coupling constants have been obtained. The structure of the resulting cation radicals were deduced by comparing the experimental coupling constants to those derived from an INDO calculation. An estimate was obtained of the unpaired spin density as a function of chain length, suggesting that resolved hyperfine structure in the EPR spectra of long chain carotenoid cation radicals should not be observed.     

A single point mutation in the yeast TRP4 gene affects efficiency of mRNA 3' end processing and alters selection of the poly(A) site

The yeast TRP4 mRNA 3' end formation element is a bidirectional element which functions in both orien-tations in an artificial in vivo test system. In this study, the role of 3' end formation was analysed in the context of the entire TRP4 gene. The 3' untranslated region (3'UTR) of TRP4 was altered and changes were analysed for their influence on TRP4 gene expression. The 3'UTR in reverse orientation was fully functional and did not affect TRP4 gene expression. Exchanging the TRP4 3'UTR by the bidirectional ARO4 or the unidirectional GCN4 3' end formation element allowed efficient gene expression. Deletion of the entire TRP4 3'UTR resulted in 70% reduction of TRP4 mRNA and 50% reduced specific Trp4 enzyme activity in comparison to wild-type. A single point mutation within the TRP4 3'UTR caused the same effect on gene expression. This point mutation did not only affect the efficiency of 3' end formation, but also produced new poly(A) sites which were situated upstream of the wild-type poly(A) sites. Therefore this sequence motif in the TRP4 3'UTR acts simultaneously as both an efficiency and positioning element.