Intraoperative ultrasound assessment in management of complex pancreatic pseudocysts.

Preoperative imaging studies and operative inspection may provide insufficient information to appropriately manage certain complex pancreatic pseudocysts. Intraoperative ultrasound accurately identifies and localizes peripancreatic fluid collections, cyst wall thickness, parenchymal and ductal anatomy, and relationships to adjacent visceral and vascular structures. Adjunctive use of intraoperative ultrasonography altered the surgical management in the clinical case described herein and is advocated for assessment of problematic pancreatic pseudocysts.

Prevalence and implications of perinatal substance use in Missouri.

A study was conducted to determine the prevalence of perinatal substance use in Missouri. The population sample studied was selected according to a multi-stage probability-proportional-to-size sampling. The weighted prevalence for perinatal exposure to alcohol, tobacco or illicit substances was 31.9%, 10.8% for illicit substances, 21.9% for tobacco use, and 7.9% for self-reported alcohol use. In 1993, an estimated 23,925 perinatal exposures to licit and illicit substances occurred.

Generation of the mitochondrial permeability transition does not involve inhibition of lysophospholipid acylation. Acyl-coenzyme A:1-acyllysophospholipid acyltransferase activity is not found in rat liver mitochondria.

The possible involvement of acyl-coenzyme A:1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles in regulating the mitochondrial permeability transition have been examined by direct methods. 1-Acyllysophospholipid acyltransferase activity found in mitochondrial preparations obtained by differential centrifugation is inhibited by several transition-inducing agents and by glutathione disulfide. However, marker enzyme analysis employing mitochondria prepared by Percoll density gradient centrifugation or fractionated by a shear force-dependent method indicate that this activity is associated with contaminating microsomes and not with mitochondria. The absence of phospholipid acylation-deacylation cycles in isolated mitochondria is demonstrated by the absence of 18O incorporation from H2(18)O into phospholipid acylester carbonyl groups, confirming conclusions arrived at from marker enzyme data by a definitive independent approach. Mitochondria prepared by differential centrifugation and Percoll density gradient centrifugation are shown to be equivalent in requirements for induction of the permeability transition and the apparent rate of this process. It is concluded that 1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles are not factors regulating the transition in isolated mitochondria. However, mitochondrial phospholipase A2 activity remains as a potential regulating factor, whereas the action of transition-inducing agents on microsomal 1-acyllysophospholipid acyltransferase may be important in mechanisms of cell injury.

Cyclosporin A is a potent inhibitor of the inner membrane permeability transition in liver mitochondria.

The immunosuppressive peptide cyclosporin A is a powerful inhibitor of the Ca2+-dependent permeability transition in rat liver mitochondria. When swelling is used to monitor the transition, the inhibitor is effective regardless of whether N-ethylmaleimide, Hg2+, WY-14643, t-butyl hydroperoxide, oxalacetate, rhein, phosphate, phosphoenolpyruvate, or ruthenium red plus uncoupler is used as the inducing agent. Twenty-five to fifty pmol/mg protein of cyclosporin A reduces the swelling response by 50% with complete inhibition obtained at about 150 pmol/mg protein. The compound, which does not inhibit Ca2+ uptake or mitochondrial phospholipase A2, is effective when added before or after the transition promoting agent. These findings, together with the shape of the inhibition dose-response curve, suggest that cyclosporin A essentially titrates a mitochondrial component which is present at 80-90 pmol/mg protein. It is proposed that this component is a solute unselective, regulated pore or a factor involved in controlling such a structure.

Hormonal triggering of the diurnal variation of sterol carrier protein.

Rat liver sterol carrier protein (SCP) is a major intracellular protein regulating lipid metabolism and transport. During a dark-light cycle, SCP undergoes a dramatic diurnal variation in synthesis and level, reflecting translational events. Several hormones participate in the control of SCP synthesis. Insulin was implicated when the circadian rhythm of SCP was lost in both diabetes and fasting, states where insulin is low. After a 12-h fast the amplitude of the diurnal rhythm is diminished; after a 48-h fast it disappears, although SCP synthesis and level remain high. When endogenous insulin secretion is increased in fasted rats by glucose administration, SCP increases 2-fold in less than 30 min. When food intake is manipulated, but the dark-light cycle is unchanged, the circadian rhythm of SCP corresponds to feeding patterns and not light cycling. During feeding, increases in SCP are triggered following the expected increase in serum insulin. However, SCP is rapidly and significantly elevated in response to insulin only when glucocorticoids are normally high or increased by injection of the synthetic glucocorticoid, dexamethasone. Hepatocyte SCP levels are also induced by a combination of insulin and dexamethasone (2.3-fold) or insulin alone (1.3-fold). Dexamethasone alone causes a striking depression of SCP (2.4-fold). Thus, insulin is a major regulator of the diurnal variation of SCP synthesis. Glucocorticoids and other hormones (e.g. triiodothyronine) are also essential for maximum induction of SCP but play permissive roles.

Corticotropin specifically stimulates the uptake and intracellular movements of sterol carrier protein in adrenals.

Sterol carrier protein (SCP), also known as liver fatty acid binding protein, is a major adrenal protein localized in the cytosol and inner mitochondrial membrane. SCP is synthesized in liver and intestine and then rapidly secreted into blood, where it associates primarily with the high density lipoprotein fraction. Studies using steroidogenesis inhibitors showed that corticotropin has a specific effect on the uptake of SCP and its movement with cholesterol to the inner mitochondrial membrane. Thus, SCP appears to be intimately involved in the mechanism(s) of adrenal steroidogenesis from cholesterol.

Role of sterol carrier protein in cholesterol metabolism.

This report summarizes our recent studies on the protein known as sterol carrier protein (SCP) or fatty acid binding protein (FABP). SCP is a highly abundant, ubiquitous protein with multifunctional roles in the regulation of lipid metabolism and transport. SCP in vitro activates membrane-bound enzymes catalyzing cholesterol synthesis and metabolism, as well as those catalyzing long chain fatty acid metabolism. SCP also binds cholesterol and fatty acids with high affinity and rapidly penetrates cholesterol containing model membranes. Studies in vivo showed SCP undergoes a remarkable diurnal cycle in level and synthesis, induced by hormones and regulated in liver by translational events. SCP rapidly responds in vivo to physiological events and manipulations affecting lipid metabolism by changes in level. Thus SCP appears to be an important regulator of lipid metabolism. Preliminary evidence is presented that SCP is secreted by liver and intestine into blood and then taken up by tissues requiring SCP but incapable of adequate SCP synthesis.

Fluorescence of delta 5,7,9(11),22-ergostatetraen-3 beta-ol in micelles, sterol carrier protein complexes, and plasma membranes.

The fluorescent sterol analogue delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was synthesized and purified by reverse-phase high-performance liquid chromatography. Dehydroergosterol in aqueous solution had a critical micelle concentration of 25 nM and a maximum solubility of 1.3 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of dehydroergosterol with purified rat liver squalene and sterol carrier protein (SCP). SCP increased the maximal solubility of dehydroergosterol in aqueous buffer. The fluorescence emission spectrum of dehydroergosterol was blue shifted upon addition of SCP. The fluorescence lifetime of dehydroergosterol in aqueous buffer was 2.3 ns; addition of SCP resulted in the appearance of a second lifetime component near 12.4 ns. The SCP increased the fluorescence polarization of monomeric dehydroergosterol in aqueous buffer from 0.033 to 0.086. Scatchard analysis of the binding data indicated that dehydroergosterol interacted with purified rat liver SCP with an apparent KD = 0.88 microM and Bmax = 4.8 microM. At maximal binding, 1.0 mol of dehydroergosterol was specifically bound per mole of SCP. The close molecular interaction of dehydroergosterol with SCP was also demonstrated by energy-transfer experiments. The intermolecular distance between SCP and bound dehydroergosterol was evaluated by fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene series of double bonds in dehydroergosterol. The transfer efficiency was 36%, and R, the apparent distance between the tyrosine energy donor and the dehydroergosterol energy acceptor, was 19 A. The significance of these data obtained in vitro for dehydroergosterol interaction with SCP was also tested in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Translational control of the circadian rhythm of liver sterol carrier protein. Analysis of mRNA sequences with a specific cDNA probe.

The striking changes in amount of rat liver SCP (sterol carrier protein) during a 24-h dark-light cycle are due to alterations in the relative synthetic rate of SCP. However, functional SCP mRNA, measured by a cell-free translational assay, does not fluctuate in the dark-light cycle. Since cell-free translational assays do not always reflect the actual abundance of an mRNA molecule, a specific cDNA hybridization assay was used to directly quantitate SCP mRNA sequences. The cDNA probe was selected from a rat liver library by hybridization to a mixture of synthetic oligonucleotides containing a portion of the sequence of SCP mRNA. The relative amount and size distribution of the SCP mRNA species (approximately 700-800 nucleotides) does not change during the diurnal cycle. To explore possible mechanisms of this translational control, the polysomal distribution of SCP mRNA was compared at the maximum and minimum points of SCP synthesis. No significant amounts of SCP mRNA were present in nonpolysomal ribonucleoprotein particles. Furthermore, no alteration in the relative level of SCP mRNA associated with polysomes or in polysome size occurs at the maximum and minimum points of SCP synthesis. Thus, changes in total SCP mRNA levels or its polysomal distribution cannot account for the diurnal variation in SCP synthesis.

Sterol and squalene carrier protein interactions with fluorescent delta 5,7,9(11)-cholestatrien-3 beta-ol.

The fluorescent sterol delta 5,7,9(11)-cholestatrien-3 beta-ol (cholestatrienol) was used as an analogue of cholesterol to determine the properties of the sterol in aqueous buffer and the interaction of cholesterol with sterol and squalene carrier protein (SCP). Cholestatrienol was synthesized and purified to a stable product by reverse phase high performance liquid chromatography. The critical micelle concentration of cholestatrienol in aqueous buffer was 1 nM while its maximum solubility was 1.15 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of cholestatrienol with purified rat liver SCP. The fluorescence emission spectrum of monomeric cholestatrienol in aqueous buffer was blue shifted upon addition of SCP. The fluorescence lifetime of monomeric cholestatrienol in aqueous buffer was increased by SCP from 5 to 12 ns. The SCP increased the fluorescence polarization of monomeric cholestatrienol from 0.002 to 0.38 in aqueous buffer. The close molecular interaction of cholestatrienol with SCP was also demonstrated by energy transfer experiments. Fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene fluorophore in cholestatrienol had a transfer efficiency of 59%. R, the apparent distance between the tyrosine energy donor and the cholestatrienol energy acceptor, was 16.3 A. Binding analysis indicated that cholestatrienol interacted with SCP with an apparent KD = 0.5 microM and a Bmax = 3.54 microM. One mol of cholestatrienol was bound per mol of SCP. These results demonstrate the utility of cholestatrienol not only as a membrane sterol probe molecule but also as a probe for sterol-protein interactions.

Correlation of tumor metastasis with sterol carrier protein and plasma membrane sterol levels.

Squalene and sterol carrier protein (SCP) levels and sterol/phospholipid molar ratios of whole cells and plasma membranes were measured in cultured primary tumor and metastatic cell lines. SCP is abundant in all cell lines. However, metastatic lines have significantly lower SCP levels and plasma membrane sterol/phospholipid ratios than do primary lines. The results indicate that extremely malignant, metastatic cells are unable to produce or maintain adequate levels of both SCP and plasma membrane sterols when grown in lipoprotein deficient media. This defect, in vivo, probably causes excess uptake of SCP and lipid.

Translational control of the circadian rhythm of liver sterol carrier protein.

Rat liver sterol carrier protein (SCP), a major regulator of lipid metabolism and transport, undergoes a rapid turnover and dramatic circadian variation in amount. The level of SCP was quantitated by a specific immunochemical assay using an antibody to homogeneous liver SCP. During a 12-h dark, 12-h light cycle, liver exhibits a biphasic pattern in SCP level. A 7-fold increase in SCP (i.e. from 1 to 7 mg/g of liver) occurs in the dark period, peaking at the midpoint and returning to basal levels by the beginning of the light period. A similar but smaller pattern of variation in SCP amount occurs in the light cycle. To elucidate the basic mechanism responsible for these changes in SCP level, the relative synthetic rate of SCP and mRNA functional activity for SCP were measured during the dark-light cycle. Alterations in the rate of SCP synthesis can account for the variations in SCP concentration. Although large changes occur in relative synthetic rate, no significant changes were found in the level of mRNA for SCP. Therefore, the circadian rhythm in SCP synthesis and amount does not reflect variations in the concentration of mRNA for SCP, but instead is caused by some mechanism controlling the efficiency of translation of SCP mRNA.

Intramitochondrial movement of adrenal sterol carrier protein with cholesterol in response to corticotropin.

Sterol carrier protein (SCP) is a highly abundant, ubiquitous, low molecular weight protein that has a rapid turnover and multifunctional roles in lipid metabolism and transport. It is also known as the fatty acid-binding protein. These and other characteristics led to studies on the possible role of SCP in mediation of steroidogenic responses of the adrenal gland to corticotropin. To quantitate the level of SCP in adrenal tissue and subcellular fractions, a specific immunochemical assay was developed using an antibody to homogeneous liver SCP. SCP is a major protein in adrenal cells (greater than 100 micrograms/mg of total protein). The bulk of it is present in the soluble (60%) and mitochondrial (35%) fractions. Nearly all mitochondrial SCP is present in the inner membrane. Adrenal SCP undergoes a dramatic diurnal variation, varying from 2% to 12% of total protein. When corticotropin was administered in vivo in the presence of aminoglutethimide, an inhibitor of steroidogenesis, there was a parallel movement of SCP with cholesterol to the inner mitochondrial membrane. Other work showed mitochondrial SCP levels increase only in situations where there is an increase in cholesterol levels. These findings strongly suggest that one of the functions of adrenal SCP is to participate in teh corticotropin-stimulated movement of cholesterol to the inner mitochondrial membrane for steroidogenesis.

Regulation of lipid metabolism by a lipid-carrying protein.

The overall conclusion to be made from the information presented here is that for many reasons SCP is a highly unusual protein. Some of these reasons are, first, SCP serves as cofactor for a number of different membrane-bound enzymes catalyzing specific steps in lipid metabolism. Second, SCP is involved in intracellular transport or movement of both cholesterol and fatty acids. Third, SCP is remarkably abundant and ubiquitous; its structure is conserved throughout nature. Fourth, SCP is exported to the blood stream from its site of synthesis by some, perhaps unique, mechanism and then rapidly taken up by specific tissues, e.g., the adrenal. Fifth, SCP is free in the cytosol and can also move to the inner mitochondrial membrane, where it is tightly bound. Sixth, SCP undergoes a dramatic diurnal variation in amount, reflecting changes in synthetic rate. Its half-life is less than an hour. Seventh, the diurnal variation in amount is triggered by feeding and influenced by several hormones. The diurnal variation is lost but a high level of SCP is maintained in the face of debilitating conditions, i.e., starvation, diabetes. Eighth, malignant cells exhibit defects in the uptake, synthesis, or turnover of SCP. Ninth, the synthesis of SCP is regulated by the efficiency of translation of its ever abundant mRNA. Tenth, there is much more to be learned about the functions and regulation of SCP.

Cholesterol accumulation in heterotransplanted renal cell cancer.

To date there have been no in vivo studies of the known propensity of human renal cell cancer (RCC) to accumulate large amounts of cholesterol. Caki-1, a human RCC line, was grown in the cheek pouches of immunosuppressed golden Syrian hamsters. Analysis of cholesterol levels revealed 2 to 4 times as much cholesterol in the heterotransplants as in any of the host tissues. After intraperitoneal injection of tumor-bearing hamsters with 4-14C-cholesterol, the tumors accumulated less radioactivity than did hamster liver and no more radioactivity than did hamster kidney or cheek pouch. It appears that accumulation of cholesterol by human RCC is not dependent on extensive uptake of extracellular cholesterol. Instead, an intrinsic defect in the cellular regulation of cholesterol synthesis and efflux is more likely to be responsible.

Synthesis of delta 5,22-cholestadien-3 beta-ol from delta 5,7,22-cholestatrien-3 beta-ol by a liver enzyme.

The rat liver enzyme system, which catalyzes reduction of delta 5,7,24-cholestatrien-3 beta-ol to cholesterol (delta 5-cholesten-3 beta-ol), converted radiolabeled delta 5,7,22-cholestatrien-3 beta-ol to delta 5,22-cholestadien-3 beta-ol, but not to cholesterol. This enzyme system thus contains membrane-bound delta 7- and delta 24-reductase and no delta 22-reductase. Kinetic and competition studies showed that the enzyme system contains a single delta 5,7-sterol delta 7-reductase, which is not influenced by unsaturation at the delta 22-position of the sterol side chain. The identity of delta 5,22-cholestadienol was established by chromatographic, spectral and chemical analyses. Use of the enzyme system and readily available delta 5,7,22-cholestatrienol provides a facile procedure for specific production of delta 5,22-cholestadien-3 beta-ol in quantity.