From interaction of lipidic vehicles with intestinal epithelial cell membranes to the formation and secretion of chylomicrons.

Lipophilic drugs are carried by chylomicrons that are secreted by the small intestine and transported in lymph. This review discusses the digestion, uptake, and transport of dietary lipids and the impact that these processes have on the absorption of lipophilic drugs by the gastrointestinal tract. This chapter complements Dr. Chris Potter's chapter on the "pre-absorptive" events of drug processing and solubilization. This chapter reviews the digestion of lipids in the gastric and intestinal lumen and the role of bile salts in the solubilization of lipid digestion products for uptake by the gut. Both the passive and active uptake of lipid digestion products is discussed. How intestinal lipid transporters located at the brush border membrane may play a role in the uptake of lipids by the enterocytes is examined, as is the regulation of the absorption of cholesterol by the human ATP-binding cassette transporter-1 (ABC1). The intracellular trafficking and the resynthesis of complex lipids from lipid digestion products are explored, and the formation and secretion of chylomicrons are described.

Differentiation-dependent expression and localization of the class B type I scavenger receptor in intestine.

The current study used the human Caco-2 cell line and mouse intestine to explore the topology of expression of the class B type I scavenger receptor (SR-BI) in intestinal cells. Results showed that intestinal cells expressed only the SR-BI isoform with little or no expression of the SR-BII variant. The expression of SR-BI in Caco-2 cells is differentiation dependent, with little or no expression in preconfluent undifferentiated cells. Analysis of Caco-2 cells cultured in Transwell porous membranes revealed the presence of SR-BI on both the apical and basolateral cell surface. Immunoblot analysis of mouse intestinal cell extracts demonstrated a gradation of SR-BI expression along the gastrocolic axis of the intestine, with the highest level of expression in the proximal intestine and decreasing to minimal expression levels in the distal intestine. Immunofluorescence studies with SR-BI-specific antibodies also confirmed this expression pattern. Importantly, the immunofluorescence studies also revealed that SR-BI immunoreactivity was most intense in the apical membrane of the brush border in the duodenum. The crypt cells did not show any reactivity with SR-BI antibodies. The localization of SR-BI in the jejunum was found to be different from that observed in the duodenum. SR-BI was present on both apical and basolateral surfaces of the jejunum villus. Localization of SR-BI in the ileum was also different, with little SR-BI detectable on either apical or basolateral membranes. Taken together, these results suggest that SR-BI has the potential to serve several functions in the intestine. The localization of SR-BI on the apical surface of the proximal intestine is consistent with the hypothesis of its possible role in dietary cholesterol absorption, whereas SR-BI present on the basolateral surface of the distal intestine suggests its possible involvement in intestinal lipoprotein uptake.

Post-transcriptional regulation of phenylalanine ammonia-lyase expression in tobacco following recovery from gene silencing.

Introduction of a bean phenylalanine ammonia-lyase (PAL) transgene into tobacco plants results in epigenetic post-transcriptional gene silencing which is unstable, such that after self-pollination first generation progeny may become PAL over-expressors. The change from gene silencing to PAL over-expression is accompanied by a loss of cytosine methylation of the PAL transgene and reduced methylation of the endogenous tobacco PAL2 gene, but not the PAL1 gene. These changes are associated with the appearance of high levels of bean PAL and tobacco PAL2 transcripts in the total RNA fraction from PAL over-expressing plants. However, tobacco PAL2 transcripts are inefficiently recruited into polysomes, and tobacco PAL2 protein is not detected in leaves of PAL over-expressing or wild-type lines. Thus, in spite of the post-transcriptionally controlled increase in tobacco PAL2 transcripts in PAL over-expressors, the increased PAL activity is primarily the result of the increase in bean PAL transcripts and corresponding enzymatic activity. These results reveal a complex cross-talk between expression of the PAL transgene and the corresponding endogenous PAL genes at the levels of transcription, transcript stability and polysomal recruitment during sense transgene-mediated silencing and subsequent over-expresson of PAL in tobacco.

Carboxyl ester lipase activity in milk prevents fat-derived intestinal injury in neonatal mice.

Carboxyl ester lipase (bile salt-stimulated lipase) is a pancreatic enzyme capable of hydrolyzing esters of cholesterol and fat-soluble vitamins. It also efficiently digests triglycerides (TG) into free fatty acids and glycerol and is abundant in the milk of humans and several other species. We used the mouse as a model to test the hypothesis that milk-derived carboxyl ester lipase (CEL) digests milk TG and that without its activity milk lipids and their digestion intermediates can disrupt the intestinal epithelium of neonates. CEL protein and enzymatic activity were shown to be abundant in mouse milk. After 24-h administration of the CEL-specific inhibitor, WAY-121,751-5, the small intestines of treated and control neonates were analyzed histologically for signs of fat malabsorption and injury to their villus epithelium. In vehicle-fed controls, TG were digested and absorbed in the duodenum and jejunum, whereas, in inhibitor-fed littermates, large intracellular neutral lipid droplets accumulated in enterocytes of the ileum, resulting in damage to the villus epithelium. Similar results were observed in neonates nursed by CEL knockout females compared with heterozygous controls. The results suggest that lack of CEL activity causes incomplete digestion of milk fat and lipid accumulation by enterocytes in the ileum of neonatal mice.

Prospects for the metabolic engineering of bioactive flavonoids and related phenylpropanoid compounds.

The successful engineering of complex metabolic pathways will require, in addition to availability of cloned genes and promoters, knowledge of the regulatory mechanisms that control metabolic flux into the pathway including post-translational phenomena such as metabolite channeling. We are interested in modifying pathways for the synthesis of isoflavonoids and other bioactive phenylpropanoid compounds in transgenic plants. We describe studies on flux control utilizing transgenic tobacco plants that under- and over-express key biosynthetic enzymes, and outline experimental approaches for the molecular dissection of potential metabolic channels in the synthesis of antimicrobial flavonoid derivatives in alfalfa and other species.

Genetic variation in cholesterol absorption efficiency among inbred strains of mice.

The initial study utilized the outbred Black Swiss, the inbred 129/SvEv and their hybrid mice to test for possible genetic difference in cholesterol absorption efficiency. Female mice (10-12 wk old) were fed a lipid test meal containing [3H]cholesterol and beta-[14C]sitosterol by stomach tube. The amount of [3H]cholesterol excreted in the feces was determined as nonabsorbed cholesterol and was normalized based on the recovery of the nonabsorbable beta-[14C]sitosterol. The Black Swiss mice absorbed significantly less cholesterol than the 129/SvEv mice within a 24-h period. Cholesterol absorption efficiency of the hybrid mice varied widely and did not segregate with either parental group. Differences in cholesterol absorption efficiency were also observed among six different inbred strains of mice fed either a basal low fat diet or a high fat/high cholesterol diet for 3 wk. Cholesterol absorption efficiency did not differ among DBA/2, C57BL/6, C3H/He, BALB/c and AKR/J mice under basal dietary conditions. However, cholesterol absorption was significantly lower in the DBA/2 mice than in C57BL/6 and C3H/He mice after mice were fed a high fat/high cholesterol diet. Cholesterol absorption by the C57L/J mice did not differ from that of C57BL/6, C3H/He, BALB/c and AKR/J mice under basal diet conditions, but was significantly lower when mice were fed a high fat/high cholesterol diet. Cholesterol absorption efficiency differed between DBA/2 and C57L/J mice under both dietary conditions. These results suggest that cholesterol absorption is controlled by multiple genetic factors.

Reduced Lignin Content and Altered Lignin Composition in Transgenic Tobacco Down-Regulated in Expression of L-Phenylalanine Ammonia-Lyase or Cinnamate 4-Hydroxylase.

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry Similar reduction of lignin levels by down -regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of these results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors.

Overexpression of L-Phenylalanine Ammonia-Lyase in Transgenic Tobacco Plants Reveals Control Points for Flux into Phenylpropanoid Biosynthesis.

Transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the enzyme L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) were grown from seeds of a primary transformant containing the bean PAL2 gene, which had shown homology-dependent silencing of the endogenous tobacco PAL genes. Analysis of endogenous and transgene-encoded PAL transcripts and protein in the primary transformant (T0) and first-generation (T1) overexpressor plants indicated that the transgene-encoded PAL is the cause of the greater than wild-type levels of PAL activity (up to 5- and 2-fold greater in leaf and stem tissue, respectively) in the T1 plants. Leaves of PAL-overexpressing plants contained increased levels of the hydroxycinnamic acid ester chlorogenic acid but not of the flavonoid rutin, indicating that PAL is the key control point for flux into chlorogenic acid. In addition, levels of the glucoside of 4-coumaric acid increased in the overexpressing plants, suggesting that the 4-coumarate:coenzyme A ligase or coumarate hydroxylase reactions might have become limiting. These results help to define the regulatory architecture of the phenylpropanoid pathway and indicate the possibility of engineering-selective changes in this complex metabolic pathway by overexpression of a single early pathway gene.

Dietary free and esterified cholesterol absorption in cholesterol esterase (bile salt-stimulated lipase) gene-targeted mice.

The involvement of pancreatic cholesterol esterase (bile salt-stimulated lipase) in cholesterol absorption through the intestine has been controversial. We have addressed this issue by using homologous recombination in embryonic stem cells to produce mice lacking a functional cholesterol esterase gene. Cholesterol esterase knockout mice and their wild type counterparts were fed a bolus dose of [3H]cholesterol and a trace amount of [beta-14C]sitosterol by gavage. The ratio of the two radiolabels excreted in the feces over a 24-h period was found to be similar in the control and cholesterol esterase-null mice. Similar results were observed when the radiolabeled sterols were supplied in an emulsion with phospholipid and triolein or in lipid vesicles with phosphatidylcholine. Cholesterol absorption results were similar between the control and cholesterol esterase-null mice regardless of whether the animals were fed a low fat diet or a high fat/high cholesterol diet. The rate of [3H]cholesterol appearance in the serum of the gene-targeted mice paralleled that observed in control animals. In contrast to these results, when experiments were performed with [3H]cholesteryl oleate instead of [3H]cholesterol, a higher amount of the 3H radiolabel was found excreted in feces and dramatically less of the radiolabel was detected in the serum of the cholesterol esterase-null mice in comparison with that detected in control animals. Serum cholesterol levels were not significantly different between control and cholesterol esterase-null mice fed either control or an atherogenic diet. These results indicate that cholesterol esterase is responsible for mediating intestinal absorption of cholesteryl esters but does not play a primary role in free cholesterol absorption.

Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice.

Basic fibroblast growth factor (FGF-2) is a pleiotropic growth factor detected in many different cells and tissues. Normally synthesized at low levels, FGF-2 is elevated in various pathologies, most notably in cancer and injury repair. To investigate the effects of elevated FGF-2, the human full-length cDNA was expressed in transgenic mice under control of a phosphoglycerate kinase promoter. Overexpression of FGF-2 caused a variety of skeletal malformations including shortening and flattening of long bones and moderate macrocephaly. Comparison by Western blot of FGF-2 transgenic mice to nontransgenic littermates showed expression of human FGF-2 protein in all major organs and tissues examined including brain, heart, lung, liver, kidney, spleen, and skeletal muscle; however, different molar ratios of FGF-2 protein isoforms were observed between different organs and tissues. Some tissues preferentially synthesize larger isoforms of FGF-2 while other tissues produce predominantly smaller 18-kDa FGF-2. Translation of the high molecular weight isoforms initiates from unconventional CUG codons and translation of the 18-kDa isoform initiates from an AUG codon in the FGF-2 mRNA. Thus the Western blot data from the FGF-2 transgenic mice suggest that tissue-specific expression of FGF-2 isoforms is regulated translationally.

Transforming growth factor-beta 3 is required for secondary palate fusion.

Mice lacking TGF-beta 3 exhibit an incompletely penetrant failure of the palatal shelves to fuse leading to cleft palate. The defect appears to result from impaired adhesion of the apposing medial edge epithelia of the palatal shelves and subsequent elimination of the mid-line epithelial seam. No craniofacial abnormalities were observed. This result demonstrates that TGF-beta 3 affects palatal shelf fusion by an intrinsic, primary mechanism rather than by effects secondary to craniofacial defects.

In Trifolium subterraneum, chalcone synthase is encoded by a multigene family.

Chalcone synthase (CHS) catalyzes the first and key regulatory step in flavonoid biosynthesis. We report the existence and characterization of a CHS multigene family present in Trifolium subterraneum L. cultivar Karridale. The CHS family consists of at least four members, which are tightly clustered in a 15-kb region. The complete sequences of two of these genes (CHS1 and CHS2) are presented. The putative promoters of these genes have sequences which are homologous to those known, or implicated, in regulation of the expression of phenylpropanoid-encoding genes.

Characterization of a phenylalanine ammonia-lyase multigene family in Trifolium subterraneum.

The enzyme phenylalanine ammonia-lyase (PAL) was found to be encoded by a small gene family in the legume Trifolium subterraneum (subterranean clover). At least three of the family members are tightly clustered within approx. 20 kb of DNA. Sequencing of one of the genes established that it possesses two exons, the position of the single intron being identical to that found for PAL genes from other plants. The PAL protein consists of 725 amino acids, as deduced from the nucleotide sequence.

Developmental analysis of tropomyosin gene expression in embryonic stem cells and mouse embryos.

Tropomyosins (TMs) comprise a family of actin-binding proteins which play an important role in the regulation of contractility in muscle (cardiac, skeletal, and smooth) and nonmuscle cells. Although they are present in all cells, different isoforms are characteristic of specific cell types. In vertebrates, there are four different TM genes (alpha-TM, beta-TM, TM30, and TM4), three of which generate alternatively spliced isoforms. This study defines the expression patterns of these isoforms during murine embryogenesis, using both in vivo and in vitro conditions. The embryonic stem cell culture system, which has been shown to mimic different stages of mouse embryonic development, including the differentiation of primitive organ systems such as the myocardium, is used for our in vitro analysis. Our results demonstrate that several TM isoforms are expressed in specific developmental patterns, often correlated with the differentiation of particular tissues or organs. Surprisingly, other TMs, such as the striated muscle beta-TM and smooth muscle alpha-TM, are expressed constitutively. This study also demonstrates that there is an excellent correlation between the expression patterns of the TM isoforms observed in developing embryonic stem cells and mouse embryos. In addition, a quantitative molecular analysis of TM isoforms was conducted in embryonic, neonatal, and adult cardiac tissue. Our results show for the first time that the alpha- and beta-TM striated muscle transcripts are present in the earliest functional stages of the heart, and these TM isoforms are identical to those present throughout cardiac development.

DNA insertions distinguish the duplicated renin genes of DBA/2 and M. hortulanus mice.

In a survey of inbred and wild mouse DNAs for genetic variation at the duplicate renin loci, Ren-1 and Ren-2, a variant Not I hybridization pattern was observed in the wild mouse M. hortulanus. To determine the basis for this variation, the structure of the M. hortulanus renin loci has been examined in detail and compared to that of the inbred strain DBA/2. Overall, the gross features of structure in this chromosomal region are conserved in both Mus species. In particular, the sequence at the recombination site between the linked Ren-1 and Ren-2 loci was found to be identical in both DBA/2 and M. hortulanus, indicating that the renin gene duplication occurred prior to the divergence of ancestors of these mice. Renin flanking sequences in M. hortulanus, however, were found to lack four DNA insertions totaling approximately 10.5 kb which reside near the DBA/2 loci. The postduplication evolution of the mouse renin genes is thus characterized by a number of insertion and/or deletion events within nearby flanking sequences. Analysis of renin expression showed little or no difference between these mice in steady state renin RNA levels in most tissues examined, suggesting that these insertions do not influence expression at those sites. A notable exception is the adrenal gland, in which DBA/2 and M. hortulanus mice exhibit different patterns of developmentally regulated renin expression.

Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro.

In the mouse embryo, early organogenesis is characterized by the formation of a functional cardiac muscle, such that 9-day embryos exhibit beating, although not fully developed hearts. In light of this observation, we found it intriguing that mouse embryoid bodies (EB), which can develop in vitro from totipotential embryonic stem cells, undergo spontaneous contractile activity. To determine if these cells are capable of recapitulating aspects of cardiogenesis, a cDNA library was prepared from beating EB and screened with a chicken skeletal myosin heavy chain cDNA. We found that the predominant myosin transcripts in the library encode the alpha- and beta-cardiac isoforms. In addition, an embryonic skeletal myosin cDNA was isolated. The myosin heavy chain transcripts in both EB and 9-day embryonic hearts were found to be the same. Transcript-specific primers were prepared, and polymerase chain reaction analyses on single EB were carried out. The data show that a single EB is capable of expressing both the alpha- and beta-isoforms as well as very low amounts of the embryonic skeletal transcript. These data indicate that EB transcribe the appropriate tissue- and developmental stage-specific myosin heavy chain genes and therefore serve as a model system for studying early cardiogenic processes at the molecular level.

Expression of tissue-specific Ren-1 and Ren-2 genes of mice: comparative analysis of 5'-proximal flanking regions.

All inbred strains of mice carry the Ren-1 structural gene, which encodes the renin-1 isozyme, the classical renin activity found in kidneys. In addition, some strains carry a second renin structural gene, Ren-2, which encodes the predominantly expressed submaxillary gland renin isozyme, renin-2. Ren-1 and Ren-2 exhibit markedly different patterns of tissue-specific expression. In an effort to understand the molecular basis for this differential expression, detailed analysis of the genomic sequences corresponding to the Ren-1 and Ren-2 genes, and the transcripts originating from these loci, was undertaken. Sequence analysis of regions proximal to the structural genes indicated the presence of eucaryotic consensus sequences for transcription. These sequence motifs were strongly conserved between Ren-1 and Ren-2. Approximately 150 bases upstream from the major transcription initiation site, significant differences between these genes were apparent, including the presence of a repetitive DNA element in the Ren-2 copy as well as other breaks in homology and sequence curiosities. Strong homology between Ren-1 and Ren-2 resumed at a point ca. 200 bases further upstream on Ren-1. S1 analysis of submaxillary gland and kidney RNA populations indicated that the majority of transcripts initiate at homologous positions on Ren-1 and Ren-2. On a per cell basis, the accumulation of Ren-1 transcripts in the kidney and Ren-2 transcripts in the submaxillary gland are probably equivalent. These results suggest that it is tissue-specific utilization of the homologous start sites that is critical to their differential patterns of expression. Models which can account for this observation are presented. Interestingly, we found a minor fraction of transcripts initiating 5' to the major transcription start site. These transcripts encoded an open reading frame which may add an additional 23 amino acids to the N-terminus of the renin precursor.

Use of a cDNA recombinant for the gamma-subunit of mouse nerve growth factor to localize members of this multigene family near the TAM-1 locus on chromosome 7.

The gamma-subunit of mouse 7S nerve growth factor (gamma-NGF) is a member of a family of closely related serine proteases that includes kallikreins and tamases. We have isolated from a DBA/2J male submaxillary gland cDNA library a clone, pSM676, which codes for gamma-NGF. Sequence analysis of the clone shows that it codes for the C-terminal 138 amino acids of the protein plus 23 bases of the 3'-nontranslated portion of the message. The predicted amino acid sequence agrees with that determined by Thomas et al. (1) for the gamma-subunit of nerve growth factor from Swiss Webster mice except for the single, conservative substitution of glutamate for aspartate at amino acid 175. When used as a probe for Southern blot analysis, pSM676 hybridizes to at least twelve fragments of restricted mouse genomic DNA which correspond to several different serine protease genes. Using mouse-hamster hybrid cell lines and recombinant inbred strains of mice, we have demonstrated that all of the genes which show homology to pSM676 are located on mouse chromosome 7, clustered at or near the Tam-1 locus.