Bone substitute materials supplemented with prolyl hydroxylase inhibitors decrease osteoclastogenesis in vitro.

BACKGROUND AND OBJECTIVE Inhibition of prolyl hydroxylases stimulates bone regeneration. Consequently, bone substitute materials were developed that release prolyl hydroxylase inhibitors. However, the impact of prolyl hydroxylase inhibitors released from these carriers on osteoclastogenesis is not clear. We therefore assessed the effect of bone substitute materials that release prolyl hydroxylase inhibitors on osteoclastogenesis. MATERIAL AND METHODS Dimethyloxalylglycine, desferrioxamine, and l-mimosine were lyophilized onto bovine bone mineral and hydroxyapatite, and supernatants were generated. Osteoclastogenesis was induced in murine bone marrow cultures in the presence of the supernatants from bone substitute materials. The formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity were determined. To test for possible effects on osteoclast progenitor cells, we measured the effect of the supernatants on proliferation and viability. In addition, experiments were performed where prolyl hydroxylase inhibitors were directly added to the bone marrow cultures. RESULTS We found that prolyl hydroxylase inhibitors released within the first hours from bone substitute materials reduce the number and activity of TRAP-positive multinucleated cells. In line with this, addition of prolyl hydroxylase inhibitors directly to the bone marrow cultures dose-dependently reduced the number of TRAP-positive multinucleated cells and the overall resorption activity. Moreover, the released prolyl hydroxylase inhibitors decreased proliferation but not viability of osteoclast progenitor cells. CONCLUSION Our results show that prolyl hydroxylase inhibitors released from bone substitute materials decrease osteoclastogenesis in murine bone marrow cultures.

Clinical acid-base pathophysiology: disorders of plasma anion gap.

The plasma anion gap is a frequently used parameter in the clinical diagnosis of a variety of conditions. The commonest application of the anion gap is to classify cases of metabolic acidosis into those that do and those that do not leave unmeasured anions in the plasma. While this algorithm is useful in streamlining the diagnostic process, it should not be used solely in this fashion. The anion gap measures the difference between the unmeasured anions and unmeasured cations and thus conveys much more information to the clinician than just quantifying anions of strong acids. In this chapter, the significance of the anion gap is emphasized and several examples are given to illustrate a more analytic approach to using the clinical anion gap; these include disorders of low anion gap, respiratory alkalosis and pyroglutamic acidosis.

Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

Androgens are essential for sexual development and reproduction. However, androgen regulation in health and disease is poorly understood. We showed that human adrenocortical H295R cells grown under starvation conditions acquire a hyperandrogenic steroid profile with changes in steroid metabolizing enzymes HSD3B2 and CYP17A1 essential for androgen production. Here we studied the regulatory mechanisms underlying androgen production in starved H295R cells. Microarray expression profiling of normal versus starved H295R cells revealed fourteen differentially expressed genes; HSD3B2, HSD3B1, CYP21A2, RARB, ASS1, CFI, ASCL1 and ENC1 play a role in steroid and energy metabolism and ANGPTL1, PLK2, DUSP6, DUSP10 and FREM2 are involved in signal transduction. We discovered two new gene networks around RARB and ANGPTL1, and show how they regulate androgen biosynthesis. Transcription factor RARB stimulated the promoters of genes involved in androgen production (StAR, CYP17A1 and HSD3B2) and enhanced androstenedione production. For HSD3B2 regulation RARB worked in cooperation with Nur77. Secretory protein ANGPTL1 modulated CYP17A1 and DUSP6 expression by inducing ERK1/2 phosphorylation. By contrast, our studies revealed no evidence for hormones or cell cycle involvement in regulating androgen biosynthesis. In summary, these studies establish a firm role for RARB and ANGPTL1 in the regulation of androgen production in H295R cells.

Hypomagnesemia Induced by Long-Term Treatment with Proton-Pump Inhibitors.

In 2006, hypomagnesemia was first described as a complication of proton-pump inhibitors. To address this issue, we systematically reviewed the literature. Hypomagnesemia, mostly associated with hypocalcemic hypoparathyroidism and hypokalemia, was reported in 64 individuals on long-term proton-pump inhibitors. Hypomagnesemia recurred following replacement of one proton-pump inhibitor with another but not with a histamine type-2 receptor antagonist. The association between proton-pump inhibitors and magnesium metabolism was addressed in 14 case-control, cross-sectional studies. An association was found in 11 of them: 6 reports found that the use of proton-pump inhibitors is associated per se with a tendency towards hypomagnesemia, 2 found that this tendency is more pronounced in patients concurrently treated with diuretics, carboplatin, or cisplatin, and 2 found a relevant tendency to hypomagnesemia in patients with poor renal function. Finally, findings likely reflecting decreased intestinal magnesium uptake were observed on treatment with proton-pump inhibitors. Three studies did not disclose any relationship between magnesium metabolism and treatment with histamine type-2 receptor antagonists. In conclusion, proton-pump inhibitors may cause hypomagnesemia. In these cases, switching to a histamine type-2 receptor antagonist is advised.

Membrane Lipid Integrity Relies on a Threshold of ATP Production Rate in Potato Cell Cultures Submitted to Anoxia

In this paper we report on our study of the changes in biomass, lipid composition, and fermentation end products, as well as in the ATP level and synthesis rate in cultivated potato (Solanum tuberosum) cells submitted to anoxia stress. During the first phase of about 12 h, cells coped with the reduced energy supply brought about by fermentation and their membrane lipids remained intact. The second phase (12–24 h), during which the energy supply dropped down to 1% to 2% of its maximal theoretical normoxic value, was characterized by an extensive hydrolysis of membrane lipids to free fatty acids. This autolytic process was ascribed to the activation of a lipolytic acyl hydrolase. Cells were also treated under normoxia with inhibitors known to interfere with energy metabolism. Carbonyl-cyanide-4-trifluoromethoxyphenylhydrazone did not induce lipid hydrolysis, which was also the case when sodium azide or salicylhydroxamic acid were fed separately. However, the simultaneous use of sodium azide plus salicylhydroxamic acid or 2-deoxy-D-glucose plus iodoacetate with normoxic cells promoted a lipid hydrolysis pattern similar to that seen in anoxic cells. Therefore, a threshold exists in the rate of ATP synthesis (approximately 10 μmol g−1 fresh weight h−1), below which the integrity of the membranes in anoxic potato cells cannot be preserved.

THE EQUILIBRIUM CONSTANT FOR THE GLYCINE SYNTHASE REACTION (ONE-CARBON, METABOLISM, FOLATE)

The equilibrium constant (K(,c)) under physiological conditions (38(DEGREES)C, 0.25 M ionic strength (I), pH 7.0) for the glycine synthase (GS) reaction (E C 2.1.2.1.0) (Equation 1) has been determined. (UNFORMATTED TABLE FOLLOWS)^ 5,10-CH(,2)-H(,4)Folate NADH NH (,4)+ CO(,2) ^ K(,c) = Eq. 1^ H(,4)Folate NAD('+) GLY ^(TABLE ENDS)^ The enzymatic instability of the GS enzyme complex itself has made it necessary to determine the overall K(,c) from the product of constants for the partial reactions of GS determined separately under the same conditions. The partial reactions are the H(,4)Folate-formaldehyde (CH(,2)(OH)(,2)) condensation reaction (Reaction 1) the K(,c) for which has been reported by this laboratory (3.0 x 10('4)), the lipoate (LipS(,2)) dehydrogenase reaction (LipDH) (Reaction 2) and the Gly-Lip^ decarboxylase reaction (Reaction 3) forming reduced lipoate (Lip(SH)(,2)), NH(,4)('+), CO(,2) and CH(,2)(OH)(,2.) (UNFORMATTED TABLE FOLLOWS)(,)^ H(,4)Fote + CH(,2)(OH)(,2) 5,10-CH(,2)-H(,4)Folate (1)^ Lip(SH)(,2) + NAD('+) LipS(,2) + NADH + H('+) (2)^ H('+) + Gly + LipS(,2) Lip(SH)(,2) + NH(,4)('+) CO(,2) + CH(,2)(OH)(,2) (3)^(TABLE ENDS)^ In this work the K(,c) for Reactions 2 and 3 are reported.^ The K(,c)' for the LipDH reaction described by other authors was reported with unexplainable conclusions regarding the pH depend- ence for the reaction. These conclusions would imply otherwise unexpected acid dissociation constants for reduced and oxidized lipoate. The pK(,a)',s for these compounds have been determined to resolve discrepancy. The conclusions are as follows: (1) The K(,c) for the LipDH reaction is 2.08 x 10('-8); (2) The pK(,a)',s for Lip(SH)(,2) are 4.77(-COOH), 9.91(-SH), 11.59(-SH); for LipS(,2) the carboxyl pK(,a)' is 4.77; (3) Contrary to previous literature, the log K(,c)' for the LipDH reaction is a linear function of the pH, a conclusion supported by the values for the dissociation constants.^ The K(,c) for Reaction 3 is the product of constants for Reactions 4-7. (UNFORMATTED TABLE FOLLOWS)^ LipSHSCH(,2)OH + H(,2)O Lip(SH)(,2) + CH(,2)(OH)(,2) (4)^ H(,2)O + LipSHSCH(,2)NH(,3)('+) LipSHSCH(,2)OH + NH(,4)('+) (5)^ LipSHSCH(,2)NH(,2) + H('+) LipSHSCH(,2)NH(,3)('+) (6)^ Gly + LipS(,2) LipSHSCH(,2)NH(,2) + CO(,2) (7)^(TABLE ENDS)^ Reactions 4-6 are non-enzymatic reactions whose constants were determined spectrophotometrically. Reaction 7 was catalyzed by the partially purified P-protein of GS with equilibrium approached from both directions. The value for K(,c) for this reaction is 8.15 x 10('-3). The combined K(,c) for Reactions 4-7 or Reaction 3 is 2.4 M.^ The overall K(,c) for the GS reaction determined by combination of values for Reactions 1-3 is 1.56 x 10('-3). ^

CHARACTERIZATION OF HUMAN ACID ALPHA-GLUCOSIDASE

The goal of this study was to investigate the properties of human acid (alpha)-glucosidase with respect to: (i) the molecular heterogeneity of the enzyme and (ii) the synthesis, post-translational modification, and transport of acid (alpha)-glucosidase in human fibroblasts.^ The initial phase of these investigations involved the purification of acid (alpha)-glucosidase from the human liver. Human hepatic acid (alpha)-glucosidase was characterized by isoelectric focusing and native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four distinct charge forms of hepatic acid (alpha)-glucosidase were separated by chromatofocusing and characterized individually. Charge heterogeneity was demonstrated to result from differences in the polypeptide components of each charge form.^ The second aspect of this research focused on the biosynthesis and the intracellular processing and transport of acid (alpha)-glucosidase in human fibroblasts. These experiments were accomplished by immune precipitation of the biosynthetic intermediates of acid (alpha)-glucosidase from radioactively labeled fibroblasts with polyclonal and monoclonal antibodies raised against human hepatic acid (alpha)-glucosidase. The immune precipitated biosynthetic forms of acid (alpha)-glucosidase were analyzed by SDS-PAGE and autoradiography. The pulse-chase experiments demonstrated the existence of several transient, high molecular weight precursors of acid (alpha)-glucosidase. These precursors were demonstrated to be intermediates of acid (alpha)-glucosidase at different stages of transport and processing in the Golgi apparatus. Other experiments were performed to examine the role of co-translational glycosylation of acid (alpha)-glucosidase in the transport and processing of precursors of this enzyme.^ A specific immunological assay for detecting acid (alpha)-glucosidase was developed using the monoclonal antibodies described above. This method was modified to increase the sensitivity of the assay by utilization of the biotin-avidin amplification system. This method was demonstrated to be more sensitive for detecting human acid (alpha)-glucosidase than the currently used biochemical assay for acid (alpha)-glucosidase activity. It was also demonstrated that the biotin-avidin immunoassay could discriminate between normal and acid (alpha)-glucosidase deficient fibroblasts, thus providing an alternative approach to detecting this inborn error in metabolism. (Abstract shortened with permission of author.) ^

Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes.

Glutathione oxidants such as tertiary butyl hydroperoxide were shown previously to prevent microtubule assembly and cause breakdown of preassembled cytoplasmic microtubules in human polymorphonuclear leukocytes. The objectives of the present study were to determine the temporal relationship between the attachment and ingestion of phagocytic particles and the assembly of microtubules, and simultaneously to quantify the levels of reduced glutathione and products of its oxidation as potential physiological regulators of assembly. Polymorphonuclear leukocytes from human peripheral blood were induced to phagocytize opsonized zymosan at 30 degrees C. Microtubule assembly was assessed in the electron microscope by direct counts of microtubules in thin sections through centrioles. Acid extracts were assayed for reduced glutathione (GSH) and oxidized glutathione (GSSG), by the sensitive enzymatic procedure of Tietze. Washed protein pellets were assayed for free sulfhydryl groups and for mixed protein disulfides with glutathione (protein-SSG) after borohydride splitting of the disulfide bond. Resting cells have few assembled microtubules. Phagocytosis induces a cycle of rapid assembly followed by disassembly. Assembly is initiated by particle contact and is maximal by 3 min of phagocytosis. Disassembly after 5-9 min of phagocytosis is preceded by a slow rise in GSSG and coincides with a rapid rise in protein-SSG. Protein-SSG also increases under conditions in which butyl hydroperoxide inhibits the assembly of microtubules that normally follows binding of concanavalin A to leukocyte cell surface receptors. No evidence for direct involvement of GSH in the induction of assembly was obtained. The formation of protein-SSG, however, emerges as a possible regulatory mechanism for the inhibition of microtubule assembly and induction of their disassembly.

Acetate metabolism and the control of environmental pH in Candida albicans and Saccharomyces cerevisiae

Candida albicans is the most common opportunistic fungal pathogen of humans. The balance between commensal and pathogenic C. albicans is maintained largely by phagocytes of the innate immune system. Analysis of transcriptional changes after macrophage phagocytosis indicates the C. albicans response is broadly similar to starvation, including up-regulation of alternate carbon metabolism. Systems known and suspected to be part of acetate/acetyl-CoA metabolism were also up-regulated, importantly the ACH and ACS genes, which manage acetate/acetyl-CoA interconversion, and the nine-member ATO gene family, thought to participate in transmembrane acetate transport and also linked to the process of environmental alkalinization. ^ Studies into the roles of Ach, Acs1 and Acs2 function in alternate carbon metabolism revealed a substantial role for Acs2 and lesser, but distinct roles, for Ach and Acs1. Deletion mutants were made in C. albicans and were phenotypically evaluated both in vitro and in vivo. Loss of Ach function resulted in mild growth defects on ethanol and acetate and no significant attenuation in virulence in a disseminated mouse model of infection. While loss of Acs1 did not produce any significant phenotypes, loss of Acs2 greatly impaired growth on multiple carbon sources, including glucose, ethanol and acetate. We also concluded that ACS1 and ACS2 likely comprise an essential gene pair. Expression analyses indicated that ACS2 is the predominant form under most growth conditions. ^ ATO gene function had been linked to the process of environmental alkalinization, an ammonium-mediated phenomenon described here first in C. albicans. During growth in glucose-poor, amino acid-rich conditions C. albicans can rapidly change its extracellular pH. This process was glucose-repressible and was accompanied by hyphal formation and changes in colony morphology. We showed that introduction of the ATO1G53D point mutant to C. albicans blocked alkalinization, as did over-expression of C. albicans ATO2, the only C. albicans ATO gene to lack the conserved N-terminal domain. A screen for alkalinization-deficient mutants revealed that ACH1 is essential for alkalinization. However, addition of acetate to the media restored alkalinization to the ach1 mutant. We proposed a model of ATO function in which Atos regulated the cellular co-export of ammonium and acetate. ^

Alternative carbon metabolism: Virulence and regulation in Candida albicans

The interaction between C. albicans and innate immune cells is a key determinant to disease progression. Transcriptional profiling showed that C. albicans responds to macrophage phagocytosis by inducing pathways required for alternative carbon metabolism (beta-oxidation, the glyoxylate cycle, and gluconeogenesis), suggesting these pathways are important for virulence of C. albicans. ^ We have shown that deleting key genes (FOX2, FBP1) in these pathways results in virulence defects in an in vivo mouse model for systemic infection. Like icl1Δ/Δ mutants, fbp1Δ/Δ mutants are severely attenuated and fox2Δ/Δ mutants are mildly but significantly attenuated, indicating that carbon starvation is a relevant stress in vivo. ^ However, fox2Δ/Δ mutants also had unexpected phenotypes on certain carbon sources, unlike the case in Saccharomyces cerevisiae, suggesting these pathways are regulated differently in C. albicans. To test this, we identified the C. albicans regulators of these pathways based on those from S. cerevisiae and Aspergillus nidulans. ^ C. albicans has a partly conserved framework, but lacks two regulators (Oaf1p, Pip2p) controlling peroxisome biogenesis and beta-oxidation genes in yeast. Instead, C. albicans has a homolog, CTF1, of the A. nidulans fatty acid catabolism regulators FarA and FarB. We have shown that CTF1 is needed for growth on oleate (like FarA and FarB), expression of beta-oxidation and glyoxylate cycle genes, and full virulence. No function for CTF1 has previously been identified in C. albicans. Our data demonstrate a role for alternative carbon metabolism in the virulence of C. albicans and suggest that the regulation of these pathways is a mixture of the filamentous fungi and budding yeast systems. ^

BENZENE MYELOCLASTOGENICITY AND METABOLISM IN CD-1 MICE: A CORRELATION

Benzene was studied in its target organ of effect, the bone marrow, with the micronucleus test and metaphase chromosomal analysis. Groups of 5 or 10, male and female CD-1 mice were treated with one or two p.o. or i.p. doses of benzene (440 mg/kg) or toluene (430, 860 or 1720 mg/kg) or both, and sacrificed 30 or 54h after the first dose. Benzene-treated animals were pretreated with phenobarbital (PB), 3-methylcholanthrene (3MC), (beta)-naphthoflavone ((beta)NF), SKF-525A, or Aroclor 1254. Toluene showed no clastogenic activity and reduced the clastogenic effect of co-administered benzene. None of the pretreatments protected against benzene clastogenicity. 3MC and (beta)NF greatly promoted benzene myeloclastogenicity. Dose response curves for benzene myeloclastogenicity were much steeper with 3MC induction than without. Micronuclei (MN) were 4-6 times higher by p.o. than i.p. benzene administration. This was not due to bacterial flora since no difference was found between germ-free and conventional males gavaged with benzene. A sensitive high-pressure liquid chromatographic method was developed and used to explore the relation between metabolic profiles of benzene in urine and MN after various pretreatments. Phenol (PH), trans-trans-muconic acid (MA) and hydroquinone (HQ) in the 48h male mouse urine accounted, respectively, for 12.8-22.8, 1.8-4.7 and 1.5-3.7% of the single oral dose of benzene (880, 440 and 220 mg/kg). Catechol (CT) was seen in trace amounts. MA was identified by ultraviolet and infrared spectroscopy and elemental analysis. Urinary metabolites--especially MA, HQ, and phenol glucuronide--correlated well with MN and were dependent on both the dose and the metabolism of benzene. Benzene metabolism was most inducible by cytochrome P-448 enzyme inducers, by p.o. > i.p., in males > females, and inhibited by toluene. Ph, CT or HQ administered p.o., 250, 150 and 250 mg/kg, respectively, or at 150 mg/kg x 2 after 3MC pretreatment, failed to reproduce the potent myeloclastogenicity of benzene. In fact, only HQ was mildly clastogenic. ^

REGULATION OF ALTERNATIVE CARBON METABOLISM IN CANDIDA ALBICANS

Candida albicans is the most important fungal pathogen of humans. Transcript profiling studies show that upon phagocytosis by macrophages, C. albicans undergoes a massive metabolic reorganization activating genes involved in alternative carbon metabolism, including the glyoxylate cycle, β-oxidation and gluconeogenesis. Mutations in key enzymes such as ICL1 (glyoxylate cycle) and FOX2 (fatty acid β-oxidation) revealed that alternative carbon metabolic pathways are required for full virulence in C. albicans. These studies indicate C. albicans uses non-preferred carbon sources allowing its adaptation to microenvironments were nutrients are scarce. It has become apparent that the regulatory networks required for regulation of alternative carbon metabolism in C. albicans are considerably different from the Saccharomyces cerevisiae paradigm and appear more analogous to the Aspergillus nidulans systems. Well-characterized transcription factors in S. cerevisiae have no apparent phenotype or are missing in C. albicans. CTF1 was found to be a single functional homolog of the A. nidulans FarA/FarB proteins, which are transcription factors required for fatty acid utilization. Both FOX2 and ICL1 were found to be part of a large CTF1 regulon. To increase our understanding of how CTF1 regulates its target genes, including whether regulation is direct or indirect, the FOX2 and ICL1 promoter regions were analyzed using a combination of bioinformatics and promoter deletion analysis. To begin characterizing the FOX2 and ICL1 promoters, 5’ rapid amplification of cDNA ends (5’RACE) was used to identify two transcriptional initiation sites in FOX2 and one in ICL1. GFP reporter assays show FOX2 and ICL1 are rapidly expressed in the presence of alternative carbon sources. Both FOX2 and ICL1 harbor the CCTCGG sequence known to be bound by the Far proteins, hence rendering the motif as a putative CTF1 DNA binding element. In this study, the CCTCGG sequence was found to be essential for FOX2 regulation. However, this motif does not appear to be equally important for the regulation of ICL1. This study supports the notion that although C. albicans has diverged from the paradigms of model fungi, C. albicans has made specific adaptations to its transcription-based regulatory network that may contribute to its metabolic flexibility.

Characterization of the interaction between local cerebral metabolic rate for glucose and acid -base index in ischemic rat brain employing a double-isotope methodology

The association between increases in cerebral glucose metabolism and the development of acidosis is largely inferential, based on reports linking hyperglycemia with poor neurological outcome, lactate accumulation, and the severity of acidosis. We measured local cerebral metabolic rate for glucose (lCMRglc) and an index of brain pH--the acid-base index (ABI)--concurrently and characterized their interaction in a model of focal cerebral ischemia in rats in a double-label autoradiographic study, using ($\sp{14}$C) 2-deoxyglucose and ($\sp{14}$C) dimethyloxazolidinedione. Computer-assisted digitization and analysis permitted the simultaneous quantification of the two variables on a pixel-by-pixel basis in the same brain slices. Hemispheres ipsilateral to tamponade-induced middle cerebral occlusion showed areas of normal, depressed and elevated glucose metabolic rate (as defined by an interhemispheric asymmetry index) after two hours of ischemia. Regions of normal glucose metabolic rate showed normal ABI (pH $\pm$ SD = 6.97 $\pm$ 0.09), regions of depressed lCMRglc showed severe acidosis (6.69 $\pm$ 0.14), and regions of elevated lCMRglc showed moderate acidosis (6.88 $\pm$ 0.10), all significantly different at the.00125 level as shown by analysis of variance. Moderate acidosis in regions of increased lCMRglc suggests that anaerobic glycolysis causes excess protons to be generated by the uncoupling of ATP synthesis and hydrolysis. ^

Altered cholesterol metabolism in a hypercholesterolemia -resistant rabbit colony

A colony of rabbits has been developed at the University of Texas Medical School at Houston that is resistant to dietary-induced hypercholesterolemia. The liver of resistant rabbits had higher levels of ($\sp{125}$I) $\beta$-VLDL binding and 3-hydroxy-3-methylglutaryl (HMGCoA) reductase activity, but lower acyl coenzyme A:cholesterol acyltransferase (ACAT) activity than normal rabbits. Direct quantitation of intracellular cholesterol content of the liver revealed that the resistant rabbits had $<$10% of the intracellular free cholesterol present in normal rabbits. Fibroblasts isolated from normal and resistant rabbits exhibited differences in ($\sp{125}$I) LDL binding, HMGCoA reductase activity and ACAT activity that were similar to those found in the liver. No structural differences were found in the LDL receptor of normal and resistant fibroblasts that would account for the increased binding capacity of the resistant cells. The regulation of LDL receptor levels by exogenous oxygenated sterols was similar in normal and resistant fibroblasts. The regulation of LDL receptor binding capacity by LDL was attenuated in the resistant compared to normal fibroblasts, suggesting that the resistant fibroblasts have an alternate pathway for processing lipoprotein-derived cholesterol. Sterol-balance studies revealed that the cholesterol-fed resistant rabbits increased lithocholic acid excretion compared to the basal state, and had higher levels of deoxycholic acid excretion than cholesterol-fed normal rabbits. In addition, the specific activity and mRNA levels of cholesterol 7$\alpha$-hydroxylase (C7$\alpha$H) were higher in resistant rabbits than normal rabbits, suggesting that the increased bile acid excretion was due to an increase in bile acid synthesis. Increased clearance of cholesterol relieves the negative feedback inhibition cholesterol exerts on expression of the LDL receptor. The number of cell surface LDL receptors is then increased in resistant rabbits and allows rapid clearance of lipoproteins from the plasma compartment, thereby reducing plasma cholesterol levels. The low intracellular cholesterol level also relieves the negative feedback inhibition cholesterol exerts on HMGCoA reductase. Increased synthesis of cholesterol from acetate provides cells with cholesterol for bile acid synthesis and/or homeostasis. The activity of ACAT is then decreased due to the flux of cholesterol through the bile acid synthetic pathways. ^

Liposome -mediated delivery of all-{\it trans\/}-retinoic acid

Because of its antiproliferative and differentiation-inducing properties, all-trans-retinoic acid (ATRA) has been used as a chemopreventive and therapeutic agent, for treatment various cancers including squamous cell carcinomas (SCCs). Long-term treatment with ATRA is associated with toxic effects in patients leading to acute or chronic hypervitaminosis syndrome. Moreover, prolonged treatment with oral ATRA leads to acquired resistance to the differentiation-inducing effects of the drug. This resistance is attributed to the induction of cytochrome P-450-dependent catabolic enzymes that lead to accelerated ATRA metabolism and decline in circulating levels. Most of these problems could be circumvented by incorporating ATRA in liposomes (L-ATRA) which results in sustained drug release, decrease in drug-associated toxicity, and protection of the drug from metabolism in the host. Liposomes also function as a solubilization matrix enabling lipophilic drugs like ATRA to be aerosolized and delivered directly to target areas in the aerodigestive tract and lungs. Of the 14 formulations tested, the positively-charged liposome, DPPC:SA (9:1, w/w) was found to be most effective in interacting with SCC cell lines. This, L-ATRA formulation was stable in the presence of serum proteins and buffered the toxic effects of the drug against several normal and malignant cell lines. The positive charge attributed by the presence of SA was critical for increased uptake and retention of L-ATRA by SCC cell lines and tumor spheroids. L-ATRA was highly effective in mediating differentiation in normal and transformed epithelial cells. Moreover, liposomal incorporation significantly reduced the rate of ATRA metabolism by cells and isolated liver microsomes. In vivo studies revealed that aerosol delivery is an effective way of administering L-ATRA, in terms of its safety and retention by lung tissue. The drug so delivered, is biologically active and had no toxic effects in mice. From these results, we conclude that liposome-incorporation is an excellent way of delivering ATRA to target tissues. The results obtained may have important clinical implications in treating patients with SCCs of the aerodigestive tract. ^