Larval skin of the flathead sole,Hippoglossoides elassodon

Summary The skin of newly-hatched larval flathead sole, Hippoglossoides elassodon, is described by light and electron microscopy. The epidermis is usually two cells thick and shows differentiation into both squamous and mucous cells. The squamous cells are characterized by numerous cytoplasmic filaments, typical desmosomes, and lack of keratinization; the mucous cells are distended with mucous droplets, which appear to originate in the Golgi apparatus. A basement membrane is present, although thinner and less dense than that of older fish, and the dermis contains loose formations of collagen and pigment cells.This work was supported in part by USPHS research grant CA-08158 from the National Cancer Institute.     

Effects of colchicine on ultrastructure of the lactating mammary cell: Membrane involvement and stress on the Golgi apparatus

Summary The effects of colchicine on ultrastructure of the lactating mammary cell in the rat and goat were studied by electron microscopy. Changes in tissue of the rat were examined over time (1, 2 and 4 h). The goat gland was evaluated by comparing ultrastructure of tissue at the time of maximum milk flow suppression induced by the drug with that of untreated tissue. Colchicine produced notable changes in the tissue of both species: 1) the secretion of lipid droplets and Golgi vesicle contents (exocytosis) was inhibited and the droplets and vesicles became randomly distributed throughout the cell, 2) the Golgi apparatus was significantly reduced in size, 3) casein and lipid continued to be synthesized as evidenced by greater numbers of secretory vesicles and increased sizes of casein micelles and lipid droplets, 4) secretory vesicles showed a propensity to cluster around lipid droplets, 5) isolated microtubules were found occasionally in the control tissue, ordinarily in the vicinity of the Golgi apparatus, but rarely in the colchicine-treated tissue. These observations indicate that colchicine has two effects leading to suppression of exocytosis in the mammary cell: one involves early interference with capacity of secretory vesicle membranes to fuse and a further effect, related to higher concentrations of colchicine, causes intracellular disorganization and loss of polarity. Microtubules were not seen as directly involved in the mechanisms of exocytosis. The secretion of milk fat globules is coupled to exocytosis and thereby is also inhibited by colchicine.Supported in part by grant HL 03622 of the U.S. Public Health Service     

High expression of the human hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP) gene in the mammary gland of lactating transgenic mice. Secretion into the milk and purification of the HIP/PAP lectin.

The human hepatocarcinoma-intestine-pancreas/pancreatic-associated protein (HIP/PAP) gene was previously identified because of its increased expression in primary liver cancers and during the acute phase of pancreatitis. In normal tissues, HIP/PAP is expressed both in endocrine and exocrine cells of the intestine and pancreas. HIP/PAP is a lactose binding C-type lectin which acts as an adhesion molecule for rat hepatocytes. The aim of the work was to study the HIP/PAP secretory pathway and to produce high levels of HIP/PAP in the milk of lactating transgenic mice. In view of its lactose C-type lectin properties, we have studied the consequences of the expression of HIP/PAP on mammary epithelial cells. In homozygous mice, production reached 11.2 mg.mL-1 of milk. High levels of soluble and pure HIP/PAP (18.6 mg) were purified from 29 mL of milk. The purified protein was sequenced and the N-terminal amino acid of the mature HIP/PAP was identified as Glu27, thus localizing the site of cleavage of the signal peptide. The HIP/PAP transgene was only expressed in the mammary gland of lactating transgenic mice. HIP/PAP was detected by immunofluorescence in the whole gland, but labelling was heterogeneous between alveolar clusters, with strongly positive sparse cells. Using immuno electron microscopy, HIP/PAP was observed in all the compartments of the secretory pathway within the mammary epithelial cells. We provide evidence that HIP/PAP is secreted through the Golgi pathway. However, the number of distended Golgi saccules was increased when compared to that found in wild-type mouse mammary cells. These modifications could be related to HIP/PAP C-type lectin specific properties.     

Use of nitrogen mustard N-oxide for the fixation of electron microscopic tissues

Summary Nitrogen mustard N-oxide was tried for the fixation of tissue for electron microscopy. A fixative consisting of 1% nitrogen mustard N-oxide, 1% glutaraldehyde and 1% paraformaldehyde buffered at pH 7.4 followed by 1% O_sO₄ buffered at pH 7.4 was found useful for the tissues examined: thyroid, anterior pituitary, adrenal gland and oviduct of mice.If the tissues are fixed and the sections are stained with uranyl acetate and lead acetate doubly, the follicle colloid, colloid droplets, and secretory granules containing thyroglobulin in the thyroid become higher in electron density. The cisterna of the maturing face of the Golgi apparatus, secretory granules, ribosomes, nucleolus and chromatin in the cells examined are extremely electron dense. Tubular elements of smooth endoplasmic reticulum in the adrenal cortical cell and microtubules in all the cells examined are also well preserved. The fixative containing nitrogen mustard N-oxide is useful also for cytochemistry. Using tissue fixed by this method and stained en bloc by uranyl acetate, the noradrenaline and adrenaline cells in the adrenal medulla are clearly distinguished by light microscopy.This study was supported by a grant from the Japan Educational Ministry     

Ultrastructural distribution of morphology of the mammary gland with observations on the size fat droplets in milk of the Weddell seal Leptonychotes weddelli (Pinnipedia)

Ultrastructural observations were made of the non-lactating and lactating mammary gland of the Weddell seal, and measurements of several cell components were compared with those in other species to determine whether there are any morphological modifications within the gland to explain the unusual milk composition and very rapid growth of sucking young in this species.
The mammary parenchyma in non-lactating and lactating Weddell seals is almost identical morphologically to that found in other mammals. Although the relative volumes of most organelles are similar to those in other eutherians, the relative volume of rough endoplasmic reticulum (RER) is reduced in the Weddell seal. In addition, the absolute and relative volumes of Golgi vacuoles are smaller in the Weddell seal, probably associated with the low lactose and water content of the milk. The synthesis and secretion of milkspecific proteins and fat droplets by mammary secretory cells in the Weddell seal appear to be identical to other eutherians. Relatively small numbers of fat droplets less than 1 μm diameter are present in epithelial cells, alveolar lumina and milk samples, and there is a far greater contribution by large fat droplets to the total fat volume of Weddell seal milk than milk from other mammals.     

The subcellular localization of apomucin and nonreducing terminal N-acetylgalactosamine in porcine submaxillary glands

Antibodies prepared against enzymatically deglycosylated porcine submaxillary gland mucin (apomucin), which were unreactive with native mucin and its partially deglycosylated derivatives, were used to immunolocalize apomucin in situ. Electron microscopy of sections of Lowicryl K4M-embedded tissue reacted successively with antibodies and protein A-gold complexes showed apomucin exclusively in mucous cells within the rough endoplasmic reticulum, transitional elements of the endoplasmic reticulum, and vesicles at the cis side of the Golgi apparatus. The Golgi apparatus, forming mucous droplets, and mucous droplets contained no apomucin. Although the rough endoplasmic reticulum contained most of the apomucin in mucous cells, some cisternae of the endoplasmic reticulum and the nuclear envelope were devoid of apomucin. Examination of tissue sections treated with the glycosidases used to prepare apomucin revealed immunolabel for apomucin throughout the secretory pathway. Colloidal gold coated with Helix pomatia lectin was used to detect nonreducing N-acetylgalactosamine residues. In mucin-producing cells lectin-gold was found in the mucous droplets, the forming mucous droplets, and throughout the Golgi apparatus but mostly in the cis portion of this organelle. In tissue sections reacted successively with lectin-gold and anti-apomucin/protein A-gold, both types of gold complex could be found in the cis side of the Golgi apparatus. These data indicate that the O-glycosylation of mucin is a posttranslational event that occurs in the Golgi apparatus and begins in the cis side of the Golgi apparatus.     

LIPID SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION : II. Electron Microscopic Radioautographic Study of the Mouse Lactating Mammary Gland

In the mammary glands of lactating albino mice injected intravenously with 9, 10-oleic acid-³H or 9, 10-palmitic acid-³H, it has been shown that the labeled fatty acids are incorporated into mammary gland glycerides. The labeled lipid in the mammary gland 1 min after injection was in esterified form (> 95%), and the radioautographic reaction was seen over the rough endoplasmic reticulum and over lipid droplets, both intracellular and intraluminal. At 10–60 min after injection, the silver grains were concentrated predominantly over lipid droplets. There was no concentration of radioactivity over the granules in the Golgi apparatus, at any time interval studied. These findings were interpreted to indicate that after esterification of the fatty acid into glycerides in the rough endoplasmic reticulum an in situ aggregation of lipid occurs, with acquisition of droplet form. The release of the lipid into the lumen proceeds directly and not through the Golgi apparatus, in contradistinction to the mode of secretion of casein in the mammary gland or of lipoprotein in the liver. The presence of strands of endoplasmic reticulum attached to intraluminal lipid droplets provides a structural counterpart to the milk microsomes described in ruminant milk.     

The Golgi apparatus is a primary site of intracellular damage after photosensitization with Rose Bengal acetate

The aim of the present investigation was to elucidate whether the Golgi apparatus undergoes photodamage following administration of the fluorogenic substrates Rose Bengal acetate (RBAc) and irradiation at the appropriate wavelength. Human HeLa cells were treated in culture and the changes in the organization of the Golgi apparatus were studied using fluorescence confocal microscopy and electron microscopy, after immunocytochemical labeling. To see whether the cytoskeletal components primarily involved in vesicle traffic (i.e., microtubules) might also be affected, experiments of tubulin immunolabeling were performed. After treatment with RBAc and irradiation, cells were allowed to grow in drug-free medium for different times. 24 hr after irradiation, the cisternae of the Golgi apparatus became packed, and after 48-72 hr they appeared more fragmented and scattered throughout the cytoplasm; these changes in the organization of the Golgi cisternae were confirmed at electron microscopy. Interestingly enough, apoptosis was found to occur especially 48-72 h after irradiation, and apoptotic cells exhibited a dramatic fragmentation of the Golgi membranes. The immunolabeling with anti-tubulin antibody showed that microtubules were also affected by irradiation in RBAc-treated cells.     

The steady-state distribution of glycosyltransferases between the Golgi apparatus and the endoplasmic reticulum is approximately 90:10

Several lines of evidence support a novel model for Golgi protein residency in which these proteins cycle between the Golgi apparatus and the endoplasmic reticulum (ER). However, to preserve the functional distinction between the two organelles, this pool of ER-resident Golgi enzymes must be small. We quantified the distribution for two Golgi glycosyltransferases in HeLa cells to test this prediction. We reasoned that best-practice, quantitative solutions would come from treating images as data arrays rather than pictures. Using deconvolution and computer calculated organellar boundaries, the Golgi fraction for both endogenous beta1,4-galactosyltransferase and UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase 2 fused with green fluorescent protein (GFP) was 91% by fluorescence microscopy. Immunogold labeling followed by electron microscopy and model analysis yielded a similar value. Values reflect steady-state conditions, as inclusion of a protein synthesis inhibitor had no effect. These data strongly suggest that the fluorescence of a GFP chimera with an organellar protein can be a valid indicator of protein distribution and more generally that fluorescent microscopy can provide a valid, rapid approach for protein quantification. In conclusion, we find the ER pool of cycling Golgi glycosyltransferases is small and approximately 1/100 the concentration found in the Golgi apparatus.     

Histo- and cytophysiology of the lactating mammary gland of the African elephant (Loxodonta africana)

The lactating mammary gland of the African elephant (Loxodonta africana) has been studied with a panel of morphological techniques focusing on (1) the functional changes during the secretory process, (2) proliferative process [by application of proliferating cell nuclear antigen (PCNA) immunohistochemistry] and apoptotic phenomena [by use of the TUNEL technique] in the individual lobules, and (3) components of milk and milk-fat-globule membrane. In the lactating gland, the lobules are variably differentiated; within a lobule, however, the alveoli are usually similarly differentiated. The morphology of their alveoli suggests a classification of the lobules into types 1–3. Lobules of type 1 are composed of immature tubular alveoli with mitotic figures and numerous PCNA-positive nuclei; advanced type 1 alveoli contain abundant glycogen and specific secretory granules. Lobules of type 2 are further subdivided. In type 2a lobules, the epithelial cells of the alveoli form tall apical protrusions, which in part are occupied by small lipid droplets and which are pinched off in an apocrine fashion. The number of lysosomes varies considerably. Type 2b is the most common type, with striking basal membrane foldings, abundant rough endoplasmic reticulum cisterns, large Golgi apparatus, numerous mitochondria, lipid droplets, and protein vesicles with 30- to 90-nm-wide casein micelles. The lipid droplets are pinched off with minimal amounts of cytoplasm. Type 2c is composed of alveoli with a cuboidal epithelium and few signs of secretory activity. Increasing expression of peanut-agglutinin-binding sites parallels the maturation and differentiation of the glandular cells. Type 3 lobules are marked by numerous TUNEL-positive nuclei and large lipid droplets and are apparently degenerating structures. Cytokeratin (CK) 14 is usually present in the myoepithelial cells; CK 19 and CK 7 mark ductal and immature alveolar epithelia. Milk protein content varies between 2.6% and 6.3%, and casein micelles range from 35 to 90 nm in diameter. The diameter of intra-alveolar milk fat globules ranges from 5 to 25 µm and the membranes bear a filamentous surface coat composed of membrane-anchored mucins; gel-electrophoretic analysis of these mucins from different individuals demonstrates the presence of mucin MUC 1, which is expressed with considerable genetic heterogeneity.     

Morphometric evaluation of lipid droplet associations with secretory vesicles,mitochondria and other components in the lactating cell

The size, cellular location, and identity of surface-associated components were determined for lipid droplets in lactating cells. Transmission electron-microscopic measurements were made involving 3801 droplets in approximately 211 cells from three rats and 1197 droplets in 66 cells from a mouse. For the purposes of droplet evaluation, cells were divided into seven locations ranging from basal to secreting positions. Droplets were also categorized with respect to contact with other droplets, basolateral plasma membrane, mitochondria, Golgi apparatus, secretory vesicles, and endoplasmic reticulum-cytoplasm (ERC). Data on droplet size showed that droplet growth occurs mainly in the secretory position, confirming previously published findings. Lipid droplets from mouse tissue, although somewhat smaller in size showed similar growth trends to those of the rat. Data on numbers of droplet contacts and percentages of droplet circumferences involved in associations with other cell components showed that the dominant interaction of lipid droplets was with the ERC. However, intimate association of droplets with mitochondria was noted in all cellular locations. In addition, nursed animals exhibited a greater proportion of droplet surface association with secretory vesicles and less in contact with mitochondria in comparison to those not nursed. The significance of these relationships to milk synthesis and secretion is discussed.     

Post-Golgi apparatus localization and regional expression of rat intestinal sialyltransferase detected by immunoelectron microscopy with polypeptide epitope-purified antibody

During studies on the Golgi apparatus immunolocalization of beta-galactoside alpha 2,6-sialyltransferase in intestinal cells, immunostaining of a number of post-Golgi apparatus structures including mucus droplets and plasma membrane were observed. In order to determine if this labeling was in fact due to sialyltransferase and not carbohydrate-specific antibodies in the polyclonal antiserum preparation, fusion protein to sialyltransferase was used to epitope purify polypeptide-specific antibodies. The affinity purification was performed on a column containing a beta-galactosidase-sialyltransferase fusion protein expressed in Escherichia coli. Using such antibodies we present evidence that in intestinal cells sialyltransferase is not only present in the Golgi apparatus cisternal stack but also its transtubular network and various post-Golgi apparatus structures. In absorptive enterocytes, post-Golgi apparatus vesicles, the brush border and basolateral plasma membrane, multivesicular bodies, and lysosome-like structures were labeled. In goblet cells the limiting membrane and lumen of forming and mature mucus droplets as well as the plasma membrane exhibited label for sialyltransferase. The results provide evidence for "ecto-sialyltransferase" in the plasma membranes of these cells, and suggest that most of the sialyltransferase is released from the Golgi membranes and becomes secreted with the goblet cell mucus. In addition, the polypeptide epitope-purified antibody was also used to examine regional expression of sialyltransferase in the rat intestinal epithelium. Immunolabel was restricted to the large intestine and not found in duodenum, jejunum, and ileum. Direct measurement of the enzyme activity was found to correlate with the immunoelectron microscopic data. This observation suggests that there is regional specific expression of the beta-galactoside alpha 2,6-sialyltransferase.     

Sexual differences in the Golgi apparatus of rat hepatocytes: three-dimensional analysis

Lipid metabolism takes place in the Golgi apparatus, but at a higher rate in female than in male rats. I therefore examined the Golgi apparatus by morphometric means for differences between the sexes at the light-and electron-microscopic level. The Golgi apparatus was stained in situ by a zinc-iodide-osmium method. The counts of the Golgi apparatus in cross-sections in female hepatocytes by light microscopy were approximately twice that in male hepatocytes. Upon ovariectomy, these counts were greatly reduced but were reestablished after estrogen supplement. To clarify this phenomenon, three-dimensional reconstructions of the Golgi apparatus were produced from electron-microscopic images of serially cut 160-nm sections. The Golgi apparatus of both male and ovariectomized females had the shape of a small ring, whereas it took the form of a large elongated cylinder in normal females and in castrated males after treatment with estrogen. The numerical difference in Golgi apparatus counts by light microscopy of in males and females is, therefore, apparently attributable to the size and shape of the Golgi apparatus, and is controlled by the estrogen level.     

Immunolocalization of blood group A gene specified alpha 1,3N-acetylgalactosaminyltransferase and blood group A substance in the trans-tubular network of the Golgi apparatus and mucus of intestinal goblet cells

The subcellular distribution of blood group A gene specified alpha 1,3N-acetylgalactosaminyltransferase and its product was studied in human intestinal goblet cells by immunoelectron microscopy. The O-glycosylation step yielding blood group A-active glycoconjugates occurred in the trans region of the Golgi apparatus as indicated by the presence of immunolabel for both antigens. In the Golgi apparatus, immunoreactive alpha 1,3N-acetylgalactosaminyltransferase was detectable in trans cisternae and in the trans-tubular network which was found to be continuous with the cisternal stack and exhibited acid phosphatase activity. This demonstrates that in intestinal goblet cells (i) the trans-tubular network does not constitute a compartment distinct from trans cisternae, and (ii) structures corresponding to GERL are structurally and functionally part of the Golgi apparatus. In addition to immunolabel for transferase at the inner surface of the cisternal membranes, luminally located immunolabel indicating the presence of free, not membrane-associated transferase became first detectable in the trans-tubular network and early forming mucus droplets contained therein. Further, the content of mature mucus droplets as well as the extracellular mucus layer were labeled. Absence of immunolabel in blood group 0 subjects lacking the blood group A gene specified transferase and the apparent non-reactivity of the antibodies with carbohydrate epitopes indicates that free alpha 1,3N-acetylgalactosaminyltransferase is present in mucus droplets and becomes secreted by intestinal goblet cells.     

X-Ray Fluorescence Microscopy Reveals Accumulation and Secretion of Discrete Intracellular Zinc Pools in the Lactating Mouse Mammary Gland

Background

The mammary gland is responsible for the transfer of a tremendous amount of zinc (∼1–3 mg zinc/day) from maternal circulation into milk during lactation to support the growth and development of the offspring. When this process is compromised, severe zinc deficiency compromises neuronal development and immune function and increases infant morbidity and/or mortality. It remains unclear as to how the lactating mammary gland dynamically integrates zinc import from maternal circulation with the enormous amount of zinc that is secreted into milk.

Methodology/Principal Findings

Herein we utilized X-ray fluorescence microscopy (XFM) which allowed for the visualization and quantification of the process of zinc transfer through the mammary gland of the lactating mouse. Our data illustrate that a large amount of zinc first accumulates in the mammary gland during lactation. Interestingly, this zinc is not cytosolic, but accumulated in large, discrete sub-cellular compartments. These zinc pools were then redistributed to small intracellular vesicles destined for secretion in a prolactin-responsive manner. Confocal microscopy identified mitochondria and the Golgi apparatus as the sub-cellular compartments which accumulate zinc; however, zinc pools in the Golgi apparatus, but not mitochondria are redistributed to vesicles destined for secretion during lactation.

Conclusions/Significance

Our data directly implicate the Golgi apparatus in providing a large, mobilizable zinc storage pool to assist in providing for the tremendous amount of zinc that is secreted into milk. Interestingly, our study also provides compelling evidence that mitochondrial zinc pools expand in the mammary gland during lactation which we speculate may play a role in regulating mammary gland function.     

Nuclear coactivator protein p100 is present in endoplasmic reticulum and lipid droplets of milk secreting cells

We have identified the p100 protein, previously known as a novel cellular coactivator, as a constituent of endoplasmic reticulum and cytosolic lipid droplets from milk secreting cells. Cytosolic lipid droplets of terminally differentiated mammary epithelial cells are secreted as milk lipid globules. However, milk lipid globules did not have detectable amounts of p100 protein. The p100 protein was found also in cytosol from lactating mammary gland, in storage lipid droplets from mouse adipocytes, and in endoplasmic reticulum from liver. Immunofluorescence microscopy of mammary epithelial cells confirmed the presence of p100 in non-nuclear regions of these cells. Partial sequence analysis of tryptic peptides from p100 from cow mammary gland showed extensive homology with the reported sequence of p100 determined from a human cDNA. Antibodies against a peptide synthesized to duplicate a sequence in human p100 recognized a protein of the size of p100 in cow, mouse and rat cell fractions.     

VEGFR1 receptor tyrosine kinase localization to the Golgi apparatus is calcium-dependent

Vascular endothelial growth factor receptor 1 (VEGFR1) is an essential receptor tyrosine kinase that regulates mammalian vascular development and embryogenesis but its function is not well understood. Herein, we present evidence whereby endothelial VEGFR1 is largely resident within the Golgi apparatus but translocates to the plasma membrane via a calcium-regulated process. Primary human endothelial cells reveal differing VEGFR1 and VEGFR2 intracellular distribution and dynamics. The major proportion of the full-length VEGFR1 membrane protein was resident within the Golgi apparatus in primary endothelial cells. Whereas VEGFR2 displayed down-regulation in response to VEGF-A, VEGFR1 was not significantly affected arguing for a significant intracellular pool that was inaccessible to extracellular VEGF-A. This intracellular VEGFR1 pool showed significant co-distribution with key Golgi residents. Brefeldin A caused VEGFR1 Golgi fragmentation consistent with redistribution to the endoplasmic reticulum. Metabolic labeling experiments and microscopy using domain-specific VEGFR1 antibodies indicated that the mature processed VEGFR1 species and an integral membrane protein was resident within Golgi apparatus. Cytosolic calcium ions play a key role in VEGFR1 trafficking as treatment with either VEGF-A, histamine, thrombin, thapsigargin or A23187 ionophore caused VEGFR1 redistribution from the Golgi apparatus to small punctate vesicles and plasma membrane. We thus propose a model whereby the balance of VEGFR1 and VEGFR2 plasma membrane levels dictate either negative or positive endothelial signaling to influence vascular physiology.     

Expression patterns of genes encoding endomembrane proteins support a reduced function of the Golgi in wheat endosperm during the onset of storage protein deposition.

Wheat storage proteins are deposited in the vacuole of maturing endosperm cells by a novel pathway that is the result of protein body formation by the endoplasmic reticulum followed by autophagy into the central vacuole, bypassing the Golgi apparatus. This model predicts a reduced role of the Golgi in storage protein accumulation, which has been supported by electron microscopy observations. To study this issue further, wheat cDNAs encoding three distinct proteins of the endomembrane system were cloned and characterized. The proteins encoded were homologues (i) of the ER translocon component Sec61 alpha, (ii) the vacuolar sorting receptor BP-80 which is located in the Golgi and clathrin-coated prevacuole vesicles (CCV), and (iii) the Golgi COPI coatomer component COP alpha. During endosperm development, the levels of all three mRNAs were highest in young stages, before the onset of storage protein synthesis, and declined with seed maturation. However, the relative mRNA levels of BP-80/Sec61 alpha and the COP alpha/Sec61 alpha were lower during the onset of storage protein synthesis than at earlier stages of endosperm development. These results support previous studies, suggesting a reduced function of the Golgi apparatus in wheat storage protein transport and deposition.     

The fine structure of the gall bladder epithelium of the sheep

Summary The electron microscopy of the gall bladder epithelium in the sheep shows that the cells are secretory. They have an extensive Golgi apparatus and sparse endoplasmic reticulum with secretory droplets localised in the apical region and have been referred to the group known as mucoid cells. The intercellular spaces which are elaborately developed in those species whose gall bladder is primarily absorptive are poorly developed. Pinocytosis is not a prominent feature. It is believed that the features noted are correlated with the reported absence of absorption in the ungulate gall bladder.