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Conjugated linoleic acids are a group of geometric and positional isomers of linoleic acid that decrease body fat in growing animals by a poorly understood mechanism. The objective of this study was to investigate the isomer-specific effect of CLA on the proliferation and differentiation of pig preadipocytes in primary culture. The effect of CLA on preadipocyte proliferation was determined using cleavage of the tetrazolium salt, WST-1, as a marker for proliferation. Preadipocyte number was decreased in a dose-dependent fashion by trans-12,cis-10 CLA (P < 0.05). No other fatty acid affected preadipocyte number. Differentiation was monitored on d 10 after induction morphologically, enzymatically, and by measuring the mRNA abundance of key adipogenic transcription factors. Both a crude CLA preparation containing a mixture of CLA isomers (CLA-mix) and the pure trans-10,cis-12 CLA isomer inhibited glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent fashion, with trans-10,cis12 CLA being more potent (P < 0.01) than the CLA-mix. Cis-9,trans-11 CLA failed to decrease GPDH activity; however, increasing concentrations of cis-9,trans-11 CLA tended to blunt the inhibitory effect of trans-10,cis-12 CLA on GPDH activity (P < 0.09), suggesting that cis-9,trans-11 CLA may antagonize the action of trans10,cis-12 CLA in porcine adipocytes. Finally, the isomer-specific effect of CLA on adipogenic transcription factor gene expression was investigated. Trans-10,cis-12 CLA decreased expression of peroxisome proliferator-activated receptor [gamma] (PPAR[gamma], P < 0.01) and sterol regulatory element-binding protein-1c (SREBP-1c; P < 0.05) mRNA, while failing to alter the expression of CCAAT/enhancer binding protein [alpha] (C/EBP[alpha]) mRNA. Interestingly, both the CLA-mix and the trans-10,cis-12 CLA isomer increased the mRNA abundance of chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF; P < 0.002). No other fatty acid affected COUP-TF mRNA levels. Collectively these data support the concept that CLA decreases fat accretion in pigs, in part by inhibiting preadipocyte proliferation and differentiation, with trans-10,cis-12 CLA being an active isomer eliciting these effects. Furthermore, trans-10,cis-12 CLA inhibits porcine preadipocyte differentiation by a mechanism that involves the down-regulation of PPAR [gamma] and SREBP-1c mRNA. This mechanism is independent of changes in C/EBP [alpha] mRNA abundance and may involve COUP-TF. Key Words: Adipogenesis, Chicken Ovalbumin Upstream Promoter Transcription Factor 1, Conjugated Linoleic Acid, Primary Culture, Swine

All-trans retinoic acid (ATRA) potently inhibits the differentiation of porcine preadipocytes in primary culture; however, the mechanism by which ATRA exerts this effect in pigs is poorly understood. The objective of this study was to use retinoid receptorspecific ligands to investigate the mechanism underlying the antiadipogenic action of retinoids in cultured pig preadipocytes by identifying the retinoid receptor mediating this action and examining the effect of retinoids on the expression of key adipogenic transcription factors. Stromal-vascular cells were harvested from porcine adipose tissue and cultured in serum-free medium. Glycerol-3-phoshphate dehydrogenase (GPDH) activity, a late marker of preadipocyte differentiation, was decreased (P < 0.01) by the addition of 0 to 10 [micro]M of either ATRA, a nonspecific agonist for both the retinoic acid receptor (RAR) and the retinoid X receptor (RXR) or the selective RAR agonist, 4-(E-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-l-propenyl) benzoic acid (TTNPB). Addition of increasing amounts of Ro-61, a RAR-specific antagonist (0 to 10 [micro]M) prevented ATRA and TTNBP from decreasing GPDH activity. Addition of methoprene acid, an RXR specific agonist, increased (P < 0.01) GPDH activity. Preadipocytes were then continuously treated with 10 nM of TTNPB in the presence or absence of 1 [micro]M Ro61, and mRNA was isolated on d 2 and 8. Addition of TTNPB decreased (P < 0.001) the expression of peroxisome proliferator-activated receptor [gamma] (PPAR[gamma]), sterol regulatory element-binding protein-1c (SREBP-1c), retinoid X receptor st (RXR[alpha]), and adipocyte fatty acid binding protein (aP2) mRNA transcripts, whereas these effects were prevented by the presence of Ro-61. Interestingly, TTNBP increased (P < 0.001) the mRNA abundance of the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1), whereas Ro-61 prevented this increase. These changes were independent of alterations in the mRNA abundances of the retinoic acid receptor [alpha], and CCAAT/enhancer binding protein [alpha] and [beta] (C/EBP[beta]; C/EBP[alpha]) genes. These results indicate that retinoic acid inhibits porcine preadipocyte differentiation by a mechanism that involves activation of the RAR and downregulation of PPAR[gamma],RXR[alpha], and SREBP-1C mRNA. This mechanism is independent of changes in C/EBP[beta] and C/EBP[alpha] mRNA abundance and may involve COUP-TF. Key Words: Adipogenesis, Chicken Ovalbumin Upstream Promoter Transcription Factor 1, Primary Culture, Retinoic Acid, Swine

The highest occupied molecular orbital (HOMO) and next-highest occupied molecular orbital (NHOMO) valence orbital electron density distributions of 1,4-diazabicyclo[2.2.2]octane (DABCO) have been investigated by electron momentum spectroscopy, a technique that probes the orbital-like nature of valence (frontier) electron transfer out of a molecule. The experimental results are compared to a range of 6-311++G ** calculations to assess the relative merits of three different orbital models that have commonly been used in chemistry. The delocalized (correlated) canonical Kohn-Sham orbitals calculated using the B3LYP or B3PW91 functionals and density functional theory provide near quantitative agreement with the observed valence electron momentum density distributions, and the delocalized canonical molecular orbitals of Hartree-Fock (independent particle) theory are in semiquantitative agreement. In contrast, Pauling's widely used and taught valence bond (hybridization) model, which is equivalent to a localized molecular orbital description, does not correspond at all to the experimental measurements. It follows that, for considerations of electron transfer, the "lone pairs" of DABCO are not localized or hybridized, but rather exist as non-degenerate orbitals that are delocalized differently over the molecular framework. The existence of two different experimental valence orbital electron densities of DABCO provides direct confirmation of the frontier orbital HOMO-NHOMO energy splitting and reordering predicted many years ago by Hoffmann et al. using extended Huckel theory, and interpreted in terms of "through bond" and "through space" interactions. Key words: orbitals, lone pairs, orbital interaction, hybridization.

Niemann-Pick type C disease (NP-C) is an inherited neurovisceral lipid storage disorder characterized by progressive neurodegeneration. Most cases of NP-C result from inactivating mutations of NPC1, a recently identified member of a family of genes encoding membrane-bound proteins containing putative sterol sensing domains. By using a specific antipeptide antibody to human NPC1, we have here investigated the cellular and subcellular localization and regulation of NPC1. By light and electron microscopic immunocytochemistry of monkey brain, NPC1 was expressed predominantly in perisynaptic astrocytic glial processes. At a subcellular level, NPC1 localized to vesicles with the morphological characteristics of lysosomes and to sites near the plasma membrane. Analysis of the temporal and spatial pattern of neurodegeneration in the NP-C mouse, a spontaneous mutant model of human NP-C, by amino - cupric-silver staining, showed that the terminal fields of axons and dendrites are the earliest sites of degeneration that occur well before the appearance of a neurological phenotype. Western blots of cultured human fibroblasts and monkey brain homogenates revealed NPC1 as a 165-kDa protein. NPC1 levels in cultured fibroblasts were unchanged by incubation with low density lipoproteins or oxysterols but were increased 2- to 3-fold by the drugs progesterone and U-18666A, which block cholesterol transport out of lysosomes, and by the lysosomotropic agent N[H.sub.4]Cl. These studies show that NPC1 in brain is predominantly a glial protein present in astrocytic processes closely associated with nerve terminals, the earliest site of degeneration in NP-C. Given the vesicular localization of NPC1 and its proposed role in mediating retroendocytic trafficking of cholesterol and other lysosomal cargo, these results suggest that disruption of NPC1-mediated vesicular trafficking in astrocytes may be linked to neuronal degeneration in NP-C.

We studied the effect of retinoic acid on the differentiation of cultured porcine preadipocytes. Porcine preadipocytes were cultured in serum-free medium (DME/F12 medium containing 100 nM insulin, 10 [[micro]gram]/mL transferrin, and 50 ng/mL hydrocortisone). Addition of increasing amounts of retinoic acid (1 nM to 20 [[micro]molar]) to the medium reduced glycerol-3-phosphate dehydrogenase (GPDH) activity, a late marker of preadipocyte differentiation. At lower concentrations (0.1 to 10 nM), retinoic acid had no effect on the GPDH activity. Addition of retinoic acid (10 [[micro]molar]) for 24 h during the early stage of development (d 1) greatly inhibited the GPDH activity. After the cells were differentiated, however, retinoic acid no longer had any effect. Therefore, retinoic acid was most effective in undifferentiated cells. Following a 24-h exposure of porcine preadipocytes to retinoic acid at d 1, Northern blot analysis showed that there was a decrease in lipoprotein lipase and adipsin mRNA levels. The results suggest that retinoic acid is a potent inhibitor of porcine preadipocyte differentiation in primary culture. Key Words: Adipocyte, Cell Culture, Retinoids, Pigs

The objective of this study was to establish optimal conditions for the primary culture of pig preadipocytes. We cultured pig preadipocytes for 10 d and studied the effects of insulin, hydrocortisone, and triiodothyronine ([T.sub.3]) added to serum-free basal medium on differentiation and gene expression of lipoprotein lipase an early marker, and adipsin, a late marker of preadipocyte differentiation. Insulin alone and hydrocortisone alone stimulated a low level of cell differentiation, as indicated by an increase in glycerol-3-phosphate dehydrogenase activity. When added together, insulin and hydrocortisone had a synergistic effect on cell differentiation. When combined with insulin or hydrocortisone, [T.sub.3] had no effect on cell differentiation, indicating that [T.sub.3] is not required in porcine preadipocyte culture. Gene expression studies also showed that removal of insulin or hydrocortisone from complete serum-free medium reduced both early and late marker mRNA. As expected, removal of [T.sub.3] had no effect on the gene expression of early and late marker mRNA. We conclude that insulin and hydrocortisone, but not [T.sub.3], are required for the differentiation of pig preadipocytes in primary culture. Key Words: Adipose, Pigs, Hormones, Cell Culture, Development

Stromal-vascular (S-V) cells isolated from adipose tissue of newborn pigs (NBPC) and mature pigs (MPC) by collagenase digestion were used to evaluate differences in preadipocyte culture and development. Cells were seeded at a density of 3 x [10.sup.4] cells/[cm.sup.2] on six-well (35-mm) tissue culture plates in 3 mL of DMEM/HAM's F12 medium plus 10% fetal calf serum and cultured at 37 [degrees] C under a humidified atmosphere of 95% air:5% C[O.sub.2] for 24 h. Cells were then washed thoroughly in DMEM/HAM's F12 medium without fetal calf serum and maintained in serum free (SF) medium or SF medium supplemented with 2.5% newborn pig serum (NBPS) or mature pig serum (MPS) for 12 d. After 1 d, more NBPC adhered to the culture plates, as indicated by DNA values. After 12 d, protein per culture well was not significantly different, but DNA concentration per well remained higher (P [is less than] .05) in cultures of NBPC than in the MPC cultured in the same medium, indicating fewer MPC. Protein:DNA ratios were higher (P [is less than] .05) in cultures of MPC regardless of the medium, reflecting larger cell size. More cells containing fat deposits were seen with NBPC in all conditions in comparison with MPC, and more fat was deposited in NBPC in SF than in SF plus NBPS or MPS. The NBPC had higher (P [is less than] .05) sn-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) per protein than MPC regardless of the medium. For both cell types, GPDH activity in either serum was less than activity of cells grown in SF. This study demonstrated that adipose S-V cells from NBPC attach to the plate more efficiently, maintain a smaller cell size, and exhibit more differentiation when using morphological criteria and GPDH activity/milligram of protein. The results also indicated that the intrinsic activity of the S-V cells, rather than serum-borne factors, are responsible for successful adipose tissue culture and development.

The effect of porcine somatotropin (PST) on the lipid profiles of adipose tissue and muscle was investigated. Sixteen crossbred barrows were injected daily with either 3 mg of PST or a placebo. After slaughter, total lipid and fatty acid composition of raw subcutaneous (SC) adipose and intermuscular (IM) adipose tissue and longissimus muscle were determined. The SC adipose tissue from pST-treated pigs had a 7.5% decrease in total lipid content; specific fatty acids 16:0, 18:0, and 18:1(n-9)c decreased most. The IM fat from pST-treated pigs had lower levels of 16:0 and 20:0. There was no effect of PST treatment on the lipid profile of the longissimus muscle. The data suggest that PST treatment produces small but significant changes in the saturated fatty acid content of adipose tissue in pigs. Key Words: Pigs, Somatotropin, Fatty Acids, Lipids, The effects of porcine somatotropin on lipids and fatty acids in subcutaneous and intermuscular adipose tissue and longissimus muscles were studied in sixteen crossbred barrows given daily injections of either porcine somatotropin (pST) or placebo. Results show that pST decreases total lipid content of subcutaneous adipose tissue, reduces saturated fatty acids but does not affect the content of polyunsaturated fatty acids.

Autosomal dominant hypocalcified amelogenesis imperfecta (ADHCAI; OMIM #130900) is a genetic disorder exhibiting severe hardness defects and reduced fracture toughness of dental enamel. While the condition is nonsyndromic, it can be associated with other craniofacial anomalies, such as malocclusions and delayed or failed tooth eruption. Truncation mutations in FAM83H (OMIM *611927) are hitherto the sole cause of ADHCAI. With human genetic studies, Fam83h knockout and mutation–knock-in mouse models indicated that FAM83H does not serve a critical physiologic function during enamel formation and suggested a neomorphic mutation mechanism causing ADHCAI. The function of FAM83H remains obscure. FAM83H has been shown to interact with various isoforms of casein kinase 1 (CK1) and keratins and to mediate organization of keratin cytoskeletons and desmosomes. By considering FAM83H a scaffold protein to anchor CK1s, further molecular characterization of the protein could gain insight into its functions. In this study, we characterized 9 kindreds with ADHCAI and identified 3 novel FAM83H truncation mutations: p.His437*, p.Gln459*, and p.Glu610*. Some affected individuals exhibited hypoplastic phenotypes, in addition to the characteristic hypocalcification enamel defects, which have never been well documented. Failed eruption of canines or second molars in affected persons was observed in 4 of the families. The p.Glu610* mutation was located in a gap area (amino acids 470 to 625) within the zone of previously reported pathogenic variants (amino acids 287 to 694). In vitro pull-down studies with overexpressed FAM83H proteins in HEK293 cells demonstrated an interaction between FAM83H and SEC16A, a protein component of the COP II complex at endoplasmic reticulum exit sites. The interaction was mediated by the middle part (amino acids 287 to 657) of mouse FAM83H protein. Results of this study significantly extended the phenotypic and genotypic spectrums of FAM83H -associated ADHCAI and suggested a role for FAM83H in endoplasmic reticulum–to–Golgi vesicle trafficking and protein secretion (dbGaP phs001491.v1.p1). [ABSTRACT FROM AUTHOR]