Intestinal apolipoprotein B secretion is inhibited by the flavonoid quercetin: Potential role of microsomal triglyceride transfer protein and diacylglycerol acyltransferase

Recent studies have yielded evidence that plant flavonoids reduce hepatic lipid and apolipoprotein B (apoB) secretion. However, the possible role of flavonoids in regulating lipid and apoB secretion by the intestine has not been studied. The purpose of our study was to examine the effects of quercetin, a common dietary flavonoid, on TAG and apoB secretion in a human intestinal cell-line, CaCo-2. Differentiated postconfluent CaCo-2 cells grown on filters and pretreated with quercetin for 8 h were shown by ELISA to inhibit basolateral apoB secretion in a dose-dependent manner. At 15 μM, the secretion of both apoB-100 and apoB-48 were inhibited similarly. This effect was shown to be specific, as quercetin did not affect the incorporation of [³⁵S]methionine/cysteine into secreted TCA-precipitable proteins. To determine the mechanism underlying this inhibitory effect, we examined two regulatory points: IAG availability and lipid transfer to the lipoprotein particle. Quercetin inhibited TAG synthesis under both basal and lipid-rich conditions, indicating that lipid availability is a determining factor in the regulation of apoB secretion by quercetin. The reduction was due at least in part to a decrease in diacylglycerol acyltransferase activity. We next examined lipid transfer or lipidation of the lipoprotein particle by analyzing microsomal IAG transfer protein (MTP) activity. Quercetin decreased MTP activity moderately. In summary, the data demonstrated that pharmacological concentrations of quercetin are a potent inhibitor of intestinal apoB secretion and that reduced lipid availability and lipidation in the lipoprotein assembly step are the mechanism for the suppression of apoB-containing lipoprotein secretion by quercetin in CaCo-2 cells.     

A knowledge-based system for the recycling of non-hazardous industrial residuals in civil engineering applications

Abstract: It is estimated that 4.6 billion tons of non‐hazardous solid waste materials are produced annually in the USA. The potential reuse for a portion of the materials in the construction of highways and roads suggests that valuable benefits in terms of economic and environmental gains are possible. This paper describes the development of a prototype computer‐assisted tool or expert system to help manufacturers assess and analyze their industrial residuals as potential road construction material. This represents an expansion in the application of intelligent systems to domains where a few, hard‐to‐find technical reports have represented the main source of expertise available to practitioners for years. The system, developed through the use of an object‐oriented software shell, Level5 Object, was designed in a user‐friendly Windows environment which allows users with little or no computer training to effectively evaluate material residuals.     

Memory liabilities associated with hypnosis: does low hypnotizability confer immunity?

Retrospective analyses of data from the authors' program of research on hypnosis and memory are presented, with special emphasis on effects observed among low hypnotizable individuals. In Experiment 1, participants completed seven forced-recall trials in an attempt to remember a series of pictures that had been shown 1 week earlier. For half the participants, the middle five trials were carried out using hypnotic procedures; the remaining participants performed all recall attempts in a motivated waking condition. Hypnosis failed to enhance correct recall for either high or low hypnotizable participants beyond the hypermnesia and reminiscence effects associated with repeated retrieval attempts over time. However, whereas high hypnotizable participants produced substantial numbers of confident recall errors (i.e., intrusions) independent of the use of hypnosis, low hypnotizable participants exposed to hypnotic procedures reported significantly more intrusions than their counterparts in the waking condition. In Experiment 2, participants were asked to identify whether specific recollections, reported during two forced-interrogatory recall tests conducted 1 week earlier, had originated in the first or second of those tests. A general bias to misattribute previously reported recollections to the first of two recall occasions was observed; however, the effect was greatest among low hypnotizables who had undergone the second recall attempt in hypnosis. The findings imply that highly hypnotizable individuals are not unique in their vulnerability to distortions of memory induced by hypnotic techniques. Individuals of lesser hypnotic capacity also manifest memory alterations when exposed to such procedures.     

Three-dimensional structure of lipoamide dehydrogenase from Pseudomonas fluorescens at 2.8 A resolution. Analysis of redox and thermostability properties

The structure of Pseudomonas fluorescens lipoamide dehydrogenase, a dimeric flavoenzyme with a molecular mass of 106,000 daltons, was solved by the molecular replacement method and refined to an R-factor of 19.4% at 2.8 A resolution. The root-mean-square difference from ideal values for bonds and angles is 0.019 A and 3.8 degrees, respectively. The structure is closely related to that of the same flavoprotein from Azotobacter vinelandii. The root-mean-square difference for 932 C alpha atoms is 0.64 A, with 84% sequence identity. The residues in the active site are identical, while 89% of the interface residues are the same in the two enzymes. A few structural variations provide the basis for the differences in thermostability and redox properties between the two homologous proteins. Particularly, in the A. vinelandii molecule a threonine to alanine (T452A) mutation leaves a buried carbonyl oxygen, located at the subunit interface and in proximity of the flavin ring, unpaired to any H-bond donor, probably providing an explanation for the lower stability of the A. vinelandii enzyme with respect to the P. fluorescens enzyme. Six surface loops, which previously could not be accurately positioned in the A. vinelandii structure, are well defined in P. fluorescens lipoamide dehydrogenase. On the basis of the P. fluorescens structure, the six loops could be correctly defined also in the A. vinelandii enzyme. This is an unusual case where similar refinement methodologies applied to two crystal forms of closely related proteins led to electron density maps of substantially different quality. The correct definition of these surface residues is likely to be an essential step for revealing the structural basis of the interactions between lipoamide dehydrogenase and the other members of the pyruvate dehydrogenase multienzyme complex.     

The role of fine-needle aspiration biopsy in the initial diagnosis of pediatric bone and soft tissue tumors: an institutional experience

A 13-year-old girl developed atonic seizure at 2 years of age. At the age of 10 years, gelastic seizures were noted. Magnetic resonance imaging (MRI) revealed a hypothalamic mass protruding down into the basal cistern and up into the third ventricle. An interictal electroencephalogram (EEG) showed paroxysmal spike and wave complex discharges. Since the seizures failed to respond to medical therapy, it was decided to try to control them by removing the mass. The operation was carried out through an interhemispheric trans-lamina terminalis approach. The lesion was so similar to normal brain tissue that the resection had to be limited enough to avoid complications. Histological examination of the mass showed a hamartoma. Postoperative MRI showed residual mass, but no seizure has been noted since the operation. The EEG recorded one year after the operation showed no spike and wave complex discharge, although she was still on anticonvulsant drugs. The authors propose that surgical therapy should be considered as a treatment for intractable gelastic epilepsy with hypothalamic hamartoma and that the first operation should be conservative enough to avoid complications, because it can bring about good results even if it is only a partial resection.     

Laparoscopic management of ovarian tumors

BACKGROUND: Laparoscopy can be used with minimal operative morbidity to evaluate adnexal masses. We report our experience with the endoscopic approach to the diagnosis and treatment of ovarian tumors. In particular, we describe 11 patients who incidentally underwent laparoscopy and in whom the ovarian masses were found to be malignant. METHODS: Between September 1994 and September 1996, 292 patients with 316 ovarian tumors were treated laparoscopically in the Department of Obstetrics-Gynaecology, University of Ulm. We assessed vaginal ultrasonography, clinical assessment, the tumor marker CA 12-5, and the intraoperative low-power magnification for their value in predicting the final diagnosis in all laparoscopically treated ovarian tumors. RESULTS: From a total of 292 patients with ovarian tumors, 11 were diagnosed, intraoperatively or after final histologic examination, as having a malignant or borderline ovarian tumor. All applied pre- and intraoperative diagnostic procedures were by themselves too unreliable to exclude early stages of ovarian carcinoma exactly. CONCLUSIONS: On the basis of the present findings, we are tempted to conclude that laparoscopic surgery is justified in the management of ovarian tumors. Even with an accurate preoperative selection of suitable patients for laparoscopic surgery, the presence of an undetected ovarian carcinoma cannot be entirely excluded.     

An assessment of the osteoinductive potential of commercial demineralized freeze-dried bone in the murine thigh muscle implantation model

Early studies have demonstrated that implantation of laboratory preparations of demineralized freeze dried bone (DFDB) into the thigh muscle of mice induces ectopic osteoinduction. However, with the development of commercial preparations of DFDB for clinical use, concerns have been raised as to the osteoinductive properties of such preparations. The aim of this study was to investigate the osteoinductive potential of some commercial preparations of DFDB compared to a newly developed product which incorporates DFDB into a collagen sponge. Commercial preparations of DFDB or the DFDB/collagen sponge were inserted into the thigh muscles of 60 adult Swiss CD-1 mice. At the completion of each experimental period (7, 14, 30, 90 and 180 days), the animals were sacrificed, and the hindquarters of the mice were radiographed. The area where each graft had been placed was then excised, processed for light microscopy, and stained with hematoxylin and eosin or von Kossa's stain. Histological analysis of the DFDB/collagen sponges demonstrated significant remineralization which increased with time. Remineralization of the DFDB/collagen sponges was verified by radiographs which showed a significant increase in radiopacity over time. There was no radiographic evidence of mineralized tissue formation or remineralization in any of the commercial DFDB samples studied. At all time points studied, histological analyses failed to show evidence of bone formation for any of the preparations. The results suggest that commercially available DFDB is not osteoinductive in the murine model and question the use of such materials in clinical periodontics. The results found for the DFDB/collagen sponge indicate a different mechanism of activity from DFDB as evidenced by its rapid remineralization. The role this remineralization process has in osteoinduction is unknown and requires further study.     

Diabetic-like retinopathy: early and late intervention therapies in galactose-fed rats

PURPOSE: To determine whether the diabetic-like thickening of retinal capillary basement membrane (RCBM) that develops in the galactose-fed rat model of diabetic ocular complications could be halted or ameliorated after 4 or 8 months of galactosemia by treatment with ARI-509, a potent new aldose reductase inhibitor (ARI), or by withdrawal of the galactose diet. METHODS: Weanling female Sprague-Dawley rats were randomized into eight groups and fed laboratory chow plus 50% starch, control group (CON); 50% D-galactose, galactose-fed group (GAL); 50% D-galactose with ARI-509 at 25 mg/kg or 10 mg/kg body wt per day, high-dose prevention group (HDP) and low-dose prevention group (LDP), respectively; 50% D-galactose for 4 or 8 months and then intervention by addition of ARI-509 (25 mg/kg body wt per day), 4-month intervention group (4IN) and 8-month intervention group (8IN), respectively; or 50% D-galactose for 4 or 8 months and then intervention by withdrawing galactose and replacing it with the 50% starch diet, 4-month galactose withdrawal group (4GW) and 8-month galactose withdrawal group (8GW), respectively. After 4, 8, 16, and 24 months of the experimental diets, the levels of carbohydrates in tissues and the extent of RCBM thickening in capillaries of the outer plexiform layer were determined in all groups. RESULTS: Retinal polyol was reduced by 95% in all ARI-treated groups and by 100% in the 4GW and 8GW groups after withdrawal of the galactose. The mean RCBM thickness increased rapidly in GAL rats, becoming almost two times greater (189 +/- 9.4 nm) than in CON rats (103 +/- 3.4 nm) by 24 months. Treatment with ARI-509 in high and low doses (HDP, LDP) initiated with the introduction of the galactose diet significantly prevented RCBM thickening at all time points (P < 0.05). In contrast, intervention by withdrawing galactose from the diet or by adding the high dose of ARI-509 had no significant effect (P < 0.05) on RCBM thickening until the 24-month time point (4IN, 166 +/- 10.3 nm; 8IN, 161 +/- 8.2 nm; 4GW, 136 +/- 5.1 nm; 8GW, 163 +/- 9.6 nm). CONCLUSIONS: Both early and late interventions decreased RCBM thickening compared with that in untreated GAL rats. The decreased thickening, however, was not evident until 16 to 20 months after the intervention. Because RCBM thickening is one of the earliest changes in diabetic and galactosemic retinopathy, the findings suggest that RCBM thickening and possibly subsequent retinal lesions are caused by early biochemical alterations induced by the galactose diet that are not readily reversed. The delayed response to therapy is consistent with that observed in the Diabetes Control and Complications Trial. The cumulative evidence indicates that intervention should begin as early after onset of diabetes as possible, and long follow-up periods should be used to evaluate efficacy.     

In vitro disulfide-coupled folding of guanylyl cyclase-activating peptide and its precursor protein

Guanylyl cyclase-activating peptide II (GCAP-II), an endogenous ligand of particulate guanylyl cyclase C (GC-C), is processed from the precursor protein and circulates in human blood. GCAP-II consists of 24 amino acid residues and contains two disulfide bridges. The correct disulfide paring of GCAP-II is an absolute requirement for its biological activity. This study shows that the folding of the peptide from the reduced form yields a peptide with the native disulfide paring as a minor product and with non-native ones as major products, regardless of the presence or absence of reduced and oxidized glutathione. The results suggest that GCAP-II does not possess sufficient information to permit the adoption of the native conformation and to effectively form the correct disulfide pairing and, as a result, that GCAP-II is correctly folded by assistance of a factor(s) such as an intra- or intermolecular chaperone. We studied whether a peptide in the pro-leader sequence of the precursor protein (proGCAP-II) contains sufficient information to facilitate the folding of GCAP-II. For this purpose, we prepared proGCAP-II in Escherichia coli by a recombinant technique and examined the disulfide-coupled folding of proGCAP-II from the reduced form. proGCAP-II was quantitatively recovered with the correctly folded structure from the reduced form both in the presence and in the absence of reduced and oxidized glutathione. The protein contains only disulfide linkages at the same positions as the mature form of proGCAP-II, GCAP-II, and the biologically active isomer of GCAP-II in the molecule. These results provide evidence that the propeptide of proGCAP-II is a critical factor in the formation of the correct disulfide paring in the folding of the protein.