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Crystalline structures in secretion from gastric fundus glands
Experimental
CRYSTALLINE
Cell Research 54 (1969) 150-l 56
STRUCTURES IN SECRETION FROM GASTRIC FUNDUS GLANDS H. F. HELANDER
Department
of Anatomy, University of Umed, Umed, Sweden
SUMMARY Crystalline material was observed in the secretion from the gastric mucosa in rats during the first 10 days after birth. This material was present in the lumen of the fundus glands as well as in the intracellular canaliculi of the parietal cells. Three basically different patterns were observed in sections of the crystals, viz. (1) Rows of - 50 A particles, disposed in two planes at right angles to each other. The spacing was about 75 A in both planes. (2) Parallel dense and less dense lines with a center-to-center distance of about 105 A. (3) Rows of -50 A narticles aligned in three planes at about 60” angle to each other. The center-to-center distance- between the particles in this hexagonal lattice was about 75 A. Transitional forms as well as irregular variations in these structures were common. Selected area diffraction was attempted on thin sections of the crystals but did not result in any specific pattern. Nor did polarization microscopy reveal any birefringence. It is suggested that the unit cell of the crystals is a face-centered 105 A cube. The material of the crystals probably originates from the parietal cells, and may contain mucus.
In the course of electron microscopic investigations of the gastric mucosa in rats of various ages [6, 71 crystalloid structures were sometimes observed in the lumen of the fundus glands and in the intracellular canaliculi of parietal cells. Since such structures have not been previously reported in the gastric mucosa, a more detailed study seemedjustified. MATERIAL
AND METHODS
Sprague-Dawley rats of the following ages were studied: &6 h (before sucking), 1, 3, 5, 10, 20 days, and adult. Furthermore, rat embryos of 16, 17, 18, 19, 20, and 21 days age were investigated. The latter were removed from their mothers under Pentobarbital anesthesia (0.03 mg/g body weight). From 1 to 10 days age the animals were fasted for about 16 h at 34°C before sacrifice [6]. The adult animals were fasted for about 48 h at room temperature, under conditions which restricted coprophagia. At least four animals in each group were studied. A few unfasted rats were also investigated. The animals were sacrificed by decapitation, and pieces of the gastric wall from the middle part of the greater curvature were fixed by immersion in an ice-cold, bloodisotonic 1 % 0~0, solution, buffered with Verona1 acetate to pH 8.5 [5]. Alternatively fixation was carried out in a 2 % solution of vacuum distilled [2] glutaraldehyde solution, buffered with 0.1 M Sorensen phosphate buffer Exptl
Cell Res 54
to pH 7.2. After rinsing in the same buffer these specimens were postfixed in a 1 % 0~0, solution in the same phosphate buffer. The tissue blocks were then rinsed, dehydrated with ethanol, and embedded in Vestopal. Most of the sections were stained with lead hydroxide and uranyl acetate. The sections were studied in a Siemens Elmiskop and/or a Philips EM 300 electron microscope, as a rule at 60 kV. For selected area diffraction the sections were mounted in a goniometer stage in the Philips microscope. In all several hundreds of crystals were studied in the electron microscope. Some of the electron micrograuhs were analvsed bv means of optical diffraction,-mainly according to a method described by Gall 131.Measurements were also performed on enlarged paper-prints of well-focused negatives, using a magnifier and a scale with 0.1 mm divisions. Thick sections (1 p) were examined in a polarizing light microscope in order to detect any birefringence.
RESULTS In all the examined animals between the 20th day of gestation and adult age, a substance of moderate to high density is observed in the lumen of some of the fundus glands (figs 1, 2) and in the intracellular canaliculi of some of the parietal cells (fig. 3). This substance is found more frequently in the young rats, from birth
0ystals
in gastric ~‘ecretion 1.51
Fig. 1. Parietal cell from a
lo-day-old rat. The lumen of the fundus gland (L) is partly filled with moderately dense material. The irregularly polygonal outline of this material appears to mak,eimpressionson the parietat cell surface.B, basalcell surface; M, mitochondrion; MC, mucous neck cells. x 18,000.
up to 10 days age, and is usually confined to only a part of the lumen or canaliculus. Sometimes the regions are irregularly outlined, in other cases polygons are formed, which only seldom display any symmetry. The size of the regions varies but rarely exceeds2 p. Although generally homogeneous the substance may exhibit a finely granular texture and the electron density may vary. In the fasted young rats between birth and IO days of age, more or less of the substance exhibits a crystalline pattern. This pattern generally appears in one of the following three forms: The first form (fig. 4) displays regularly spaced
dense particles, about 50 A in size, aligned in two sets of parallel rows at about right angle to each other. The center-to-center distance between the rows measures about ‘75A (range 68-85 ra>. The second form (fig. 5) exhibits alternating parallel denseand less denselines of abont equal thickness with a center-to-center distance of some 105 A (range 97-116 A). Sometimes short portions of the denselines can be seento consist of dense particles, about 50 A in size. In some sections the dense lines are resolved into two dense and an intermediate less dense line of about equal thickness.
152 H. F. Helander
Fig. 2. Fundus gland lumen from a lo-dav-old rat containing secretory material with a rhombic outline. 44, mitochondrion; N, nucleus; x 31,000. Fiz. 3. Parietal cell from a lo-day-old rat. Some of the intracellular canaliculi (ZC) are almost filled with squ&& of moderately dense material. B, basal &Al surface; L, lumen of fundus gland; M, mitochondrion ; MC, mucous neck cells. x 22,000. Exptl
Cell Res 54
Crystals in gastric secretion
153
Fig. 4. Secretorymaterial in fundus gland lumen of a lo-day-old rat. A cryslalline pattern is visible, consistent with the 100 plane of a facecentered cube (cf. model in inset in lower left corner). An optical diffractogram of this electron micrograph (rotated 45”) is demonstrated in the lower right corner. :i 300,000 (main figure).
In the third form (fig. 6) dense -50 A particles are regularly spaced in three planes at about 60” angle to each other. The center-tocenter distance between the particles in this hexagonal lattice measures about 75 A (range 70-86 pi).
Several transitional forms arc observed between these three main patterns. Irregular variations in the structure within a crystal are also frequently encountered. Also in the unfasted rats the luminal substance is present, but no crystalline pattern is observed. Selectedarea diffraction at 20,40,60, and 80 kV of thin sections of the crystals does not result in any specific pattern, which can be registered with the electron microscope. Thick sections (1 ,u) containing crystals subjected to polarization microscopy do not reveal any birefringence.
YISCUSSION For a detailed analysis of the packing arrangement in crystalloid structures diffraction methods are required. In the present investigation X-ray diffraction was not possible because of the small size of the crystals and interference from surrounding material, and attempts at electron diffraction in the sections did not result in any specific pattern, presumably because the crystalline material was too small in amount or too thin. Lacking these valuable methods other less precise ways of analysis must be employed. Polarization microscopy of thick sections should reveal birefringence typical of crystals other than cubes. The lack of birefringencc in the present investigation should be interpreted cautiously, however, one reason could be a cubic arrangement in the crystals.
154 H. F. Helander
Fin. 5. Crystalline material in- the lumen of a fundus gland from a lo-day-old rat. This lattice corresponds to the 110 mane of a facecentered cube, as shown in the model in the lower left corner. In some cases the dense lines are resolved into -50 A particles. However, these are visible only in small numbers at irregular intervals, or else they would have influenced on the diifractogram of this figure (inset in upper right corner, rotated 45”). x 180.000(main figure). ’ ’
Electron microscopy of thin sections remains at present the most important method for the analysis of the lattice structures. However, the thickness of the sections which by far exceeds the size of the unit cell in the crystals is a serious limitation of this method. The results obtained by electron microscopy suggest that the unit cell is a cube or more precisely a face-centered cube. The first, quadratic pattern with a spacing of about 75 A would correspond to the 100 plane. The second pattern, consisting of dense and less dense lines would fit to the 110 plane. The spacing between the lines in this plane is about 105 A, which is about equal to the theoretical value of 75 x VT= 106A derived from the dimensions in the 100 plane. The 111 plane finally, would agree with the third, hexagonal pattern. The electron micrographs do not conform with the packing of bodycentered cubes. A simple cubic packing, finally, can also be excluded on geometrical grounds. Exptl Cell Res 54
Moreover no known substance crystallizes in this form [9]. The distance in the electron micrographs between the lattice planes varies slightly, since the cutting plane is not always parallel to a crystal plane. For this reason systematic measurements of the distances between the planes are of limited value, and were performed only to obtain approximate mean values. When the sections are not quite parallel to the 110 plane the dense lines will appear somewhat thicker than when the cutting is absolutely parallel. In these cases the selective staining of the two surfaces of the sections with lead and uranium compounds could create the impression of split denselines (cf [l 11). In the 110 plane the denselines were resolved into dense particles only infrequently. It seems illogical to ascribe this lack of resolution to an over-layering of slightly displaced crystal planes in other layers of the section, since the particles
Crystals in gastric secretion
I55
Fig. 6. Crystal in lumen of fundus gland from a IO-dayold rat. This regular hexagonal patterncorresponds to the 111 plane of a face-centered cube (inset model in upper left corner). No other regular pattern is present in this crystal, as evidenced by the optical diffractogram of this electron micrograph (inset in upper right corner). x 160,000 (main figure).
the 100 and 111 planes could readily be demonstrated. However, Hamilton et al. evidently experienced a similar lack of resolution in salamander liver crystals (cf fig. 12 in their article I41). Crystalloid structures have been observed in electron micrographs of many tissues, but only in a few caseshas the interpretation of the findings been carried out so far that a unit cell could be suggested. Hamilton et al. [4] in their study of cytoplasmic liver crystals demonstrated that the unit cell evidently was 100 A face-centered cube. They concluded that these crystals, which are similar in appearance to those studied in the present investigation, presumably contained serum lipo-protein. In a series of articles the structure of the yolk platelet crystals has been analyzed, using X-ray diffraction, electron and polarizing microscopy (cf Honjin et al. [lo]). In this case a hexagonal packing has been revealed. Phosphoprotein and in
a lipoprotein appear to be the main components of the yolk platelet crystals 1141. Although the secretory substancesfrom several glands under pathological conditions may precipitate in the form of crystals (e.g. the urine and the bile), crystalline material has apparently not previously been demonstrated electron microscopically in the secretory ducts from any gland. It cannot be determined whether the observed crystals are present also in vivo, or if they appear as a result of an altered chemical and physical environment during the processing of the tissue. Since a back-flow of secretory substances into the intracellular canaliculi of the parietal cells seems less likely, it may be assumed that the material contained within the crystals is secreted by the parietal cells. In certain parietal cells cytoplasmic granules have been observed which react histochemi~ally asneutral, diastase-resistantmucosubstances[12]. ‘Thesecells apparently secretemucus in addition
156 H, F. Helander to HCI, and it is hence possible that mucus is a component of the crystalline material. The granule-containing parietal cells are common in young rats, but rather rare in adult ones [7], which could explain the apparent paucity of crystals in the adult animals. The intraluminal, sometimes crystalline substance should not be discussedisolated from the problems relating HCI secretion. It has been vigorously debated whether the HCI is secreted in a dissociated form, or if the acid is attached to some compound, which neutralizes it and thus protects the epithelial cells against corrosion (cf Babkin [l]). This hypothetical HCIbinding complex would then dissociate in the gastric cavity. The nature of this acid-binding compound as well as its possible relationship to the crystalline material remains to be investigated. This investigation was supported by grants from the Swedish Medical Research Council (no. B68-12X-2298Ol), Magnus Bergvalls stiftelse, and Stiftelsen Therese
Exptl Cell Res 54
and Johan Anderssons Minne. A brief report of the results was presented at the annual meeting of the Scandinavian Society for Electron Microscopy in Stockholm, Sweden, June 4, 1968 [8].
REFERENCES 1. Babkin, B P, Secretory mechanism of the digestive glands, 2nd edn. Hoeber, New York (1950). 2. Fahimi, H D & Drochmans, P, J micr 4 (1965) 725. 3. Gall, J G, J cell sci 2 (1967) 163. 4. Hamilton, D W, Fawcett, D W & Christensen, A K, Z Zellforsch 70 (1966) 347. 5. Helander, H F, J ultrastruct res, Suppl. 4 (1962). 6. - Gastroenterol. In press. 7. - Ibid. In press. - J ultrastruct res 25 (1968) 170. :: Holden, A & Singer, P,.Crystals and crystal growing. Doubleday, New York (1960). 10. Honjin, R,’ Nakamura,‘T & Shimasaki, S, J ultrastruct res 12 (1965) 404. 11. Maunsbach, A B, J ultrastruct res 14 (1966) 167. 12. Spicer, S S & Sun, D C H, Ann NY acad sci 140 (1967) 762. 13. Townsend, S F, Am j anat 109 (1961) 260. 14. Wallace, R A, Biochim biophys acta 74 (1963) 505. Received June 24, 1968
CRYSTALLINE
Cell Research 54 (1969) 150-l 56
STRUCTURES IN SECRETION FROM GASTRIC FUNDUS GLANDS H. F. HELANDER
Department
of Anatomy, University of Umed, Umed, Sweden
SUMMARY Crystalline material was observed in the secretion from the gastric mucosa in rats during the first 10 days after birth. This material was present in the lumen of the fundus glands as well as in the intracellular canaliculi of the parietal cells. Three basically different patterns were observed in sections of the crystals, viz. (1) Rows of - 50 A particles, disposed in two planes at right angles to each other. The spacing was about 75 A in both planes. (2) Parallel dense and less dense lines with a center-to-center distance of about 105 A. (3) Rows of -50 A narticles aligned in three planes at about 60” angle to each other. The center-to-center distance- between the particles in this hexagonal lattice was about 75 A. Transitional forms as well as irregular variations in these structures were common. Selected area diffraction was attempted on thin sections of the crystals but did not result in any specific pattern. Nor did polarization microscopy reveal any birefringence. It is suggested that the unit cell of the crystals is a face-centered 105 A cube. The material of the crystals probably originates from the parietal cells, and may contain mucus.
In the course of electron microscopic investigations of the gastric mucosa in rats of various ages [6, 71 crystalloid structures were sometimes observed in the lumen of the fundus glands and in the intracellular canaliculi of parietal cells. Since such structures have not been previously reported in the gastric mucosa, a more detailed study seemedjustified. MATERIAL
AND METHODS
Sprague-Dawley rats of the following ages were studied: &6 h (before sucking), 1, 3, 5, 10, 20 days, and adult. Furthermore, rat embryos of 16, 17, 18, 19, 20, and 21 days age were investigated. The latter were removed from their mothers under Pentobarbital anesthesia (0.03 mg/g body weight). From 1 to 10 days age the animals were fasted for about 16 h at 34°C before sacrifice [6]. The adult animals were fasted for about 48 h at room temperature, under conditions which restricted coprophagia. At least four animals in each group were studied. A few unfasted rats were also investigated. The animals were sacrificed by decapitation, and pieces of the gastric wall from the middle part of the greater curvature were fixed by immersion in an ice-cold, bloodisotonic 1 % 0~0, solution, buffered with Verona1 acetate to pH 8.5 [5]. Alternatively fixation was carried out in a 2 % solution of vacuum distilled [2] glutaraldehyde solution, buffered with 0.1 M Sorensen phosphate buffer Exptl
Cell Res 54
to pH 7.2. After rinsing in the same buffer these specimens were postfixed in a 1 % 0~0, solution in the same phosphate buffer. The tissue blocks were then rinsed, dehydrated with ethanol, and embedded in Vestopal. Most of the sections were stained with lead hydroxide and uranyl acetate. The sections were studied in a Siemens Elmiskop and/or a Philips EM 300 electron microscope, as a rule at 60 kV. For selected area diffraction the sections were mounted in a goniometer stage in the Philips microscope. In all several hundreds of crystals were studied in the electron microscope. Some of the electron micrograuhs were analvsed bv means of optical diffraction,-mainly according to a method described by Gall 131.Measurements were also performed on enlarged paper-prints of well-focused negatives, using a magnifier and a scale with 0.1 mm divisions. Thick sections (1 p) were examined in a polarizing light microscope in order to detect any birefringence.
RESULTS In all the examined animals between the 20th day of gestation and adult age, a substance of moderate to high density is observed in the lumen of some of the fundus glands (figs 1, 2) and in the intracellular canaliculi of some of the parietal cells (fig. 3). This substance is found more frequently in the young rats, from birth
0ystals
in gastric ~‘ecretion 1.51
Fig. 1. Parietal cell from a
lo-day-old rat. The lumen of the fundus gland (L) is partly filled with moderately dense material. The irregularly polygonal outline of this material appears to mak,eimpressionson the parietat cell surface.B, basalcell surface; M, mitochondrion; MC, mucous neck cells. x 18,000.
up to 10 days age, and is usually confined to only a part of the lumen or canaliculus. Sometimes the regions are irregularly outlined, in other cases polygons are formed, which only seldom display any symmetry. The size of the regions varies but rarely exceeds2 p. Although generally homogeneous the substance may exhibit a finely granular texture and the electron density may vary. In the fasted young rats between birth and IO days of age, more or less of the substance exhibits a crystalline pattern. This pattern generally appears in one of the following three forms: The first form (fig. 4) displays regularly spaced
dense particles, about 50 A in size, aligned in two sets of parallel rows at about right angle to each other. The center-to-center distance between the rows measures about ‘75A (range 68-85 ra>. The second form (fig. 5) exhibits alternating parallel denseand less denselines of abont equal thickness with a center-to-center distance of some 105 A (range 97-116 A). Sometimes short portions of the denselines can be seento consist of dense particles, about 50 A in size. In some sections the dense lines are resolved into two dense and an intermediate less dense line of about equal thickness.
152 H. F. Helander
Fig. 2. Fundus gland lumen from a lo-dav-old rat containing secretory material with a rhombic outline. 44, mitochondrion; N, nucleus; x 31,000. Fiz. 3. Parietal cell from a lo-day-old rat. Some of the intracellular canaliculi (ZC) are almost filled with squ&& of moderately dense material. B, basal &Al surface; L, lumen of fundus gland; M, mitochondrion ; MC, mucous neck cells. x 22,000. Exptl
Cell Res 54
Crystals in gastric secretion
153
Fig. 4. Secretorymaterial in fundus gland lumen of a lo-day-old rat. A cryslalline pattern is visible, consistent with the 100 plane of a facecentered cube (cf. model in inset in lower left corner). An optical diffractogram of this electron micrograph (rotated 45”) is demonstrated in the lower right corner. :i 300,000 (main figure).
In the third form (fig. 6) dense -50 A particles are regularly spaced in three planes at about 60” angle to each other. The center-tocenter distance between the particles in this hexagonal lattice measures about 75 A (range 70-86 pi).
Several transitional forms arc observed between these three main patterns. Irregular variations in the structure within a crystal are also frequently encountered. Also in the unfasted rats the luminal substance is present, but no crystalline pattern is observed. Selectedarea diffraction at 20,40,60, and 80 kV of thin sections of the crystals does not result in any specific pattern, which can be registered with the electron microscope. Thick sections (1 ,u) containing crystals subjected to polarization microscopy do not reveal any birefringence.
YISCUSSION For a detailed analysis of the packing arrangement in crystalloid structures diffraction methods are required. In the present investigation X-ray diffraction was not possible because of the small size of the crystals and interference from surrounding material, and attempts at electron diffraction in the sections did not result in any specific pattern, presumably because the crystalline material was too small in amount or too thin. Lacking these valuable methods other less precise ways of analysis must be employed. Polarization microscopy of thick sections should reveal birefringence typical of crystals other than cubes. The lack of birefringencc in the present investigation should be interpreted cautiously, however, one reason could be a cubic arrangement in the crystals.
154 H. F. Helander
Fin. 5. Crystalline material in- the lumen of a fundus gland from a lo-day-old rat. This lattice corresponds to the 110 mane of a facecentered cube, as shown in the model in the lower left corner. In some cases the dense lines are resolved into -50 A particles. However, these are visible only in small numbers at irregular intervals, or else they would have influenced on the diifractogram of this figure (inset in upper right corner, rotated 45”). x 180.000(main figure). ’ ’
Electron microscopy of thin sections remains at present the most important method for the analysis of the lattice structures. However, the thickness of the sections which by far exceeds the size of the unit cell in the crystals is a serious limitation of this method. The results obtained by electron microscopy suggest that the unit cell is a cube or more precisely a face-centered cube. The first, quadratic pattern with a spacing of about 75 A would correspond to the 100 plane. The second pattern, consisting of dense and less dense lines would fit to the 110 plane. The spacing between the lines in this plane is about 105 A, which is about equal to the theoretical value of 75 x VT= 106A derived from the dimensions in the 100 plane. The 111 plane finally, would agree with the third, hexagonal pattern. The electron micrographs do not conform with the packing of bodycentered cubes. A simple cubic packing, finally, can also be excluded on geometrical grounds. Exptl Cell Res 54
Moreover no known substance crystallizes in this form [9]. The distance in the electron micrographs between the lattice planes varies slightly, since the cutting plane is not always parallel to a crystal plane. For this reason systematic measurements of the distances between the planes are of limited value, and were performed only to obtain approximate mean values. When the sections are not quite parallel to the 110 plane the dense lines will appear somewhat thicker than when the cutting is absolutely parallel. In these cases the selective staining of the two surfaces of the sections with lead and uranium compounds could create the impression of split denselines (cf [l 11). In the 110 plane the denselines were resolved into dense particles only infrequently. It seems illogical to ascribe this lack of resolution to an over-layering of slightly displaced crystal planes in other layers of the section, since the particles
Crystals in gastric secretion
I55
Fig. 6. Crystal in lumen of fundus gland from a IO-dayold rat. This regular hexagonal patterncorresponds to the 111 plane of a face-centered cube (inset model in upper left corner). No other regular pattern is present in this crystal, as evidenced by the optical diffractogram of this electron micrograph (inset in upper right corner). x 160,000 (main figure).
the 100 and 111 planes could readily be demonstrated. However, Hamilton et al. evidently experienced a similar lack of resolution in salamander liver crystals (cf fig. 12 in their article I41). Crystalloid structures have been observed in electron micrographs of many tissues, but only in a few caseshas the interpretation of the findings been carried out so far that a unit cell could be suggested. Hamilton et al. [4] in their study of cytoplasmic liver crystals demonstrated that the unit cell evidently was 100 A face-centered cube. They concluded that these crystals, which are similar in appearance to those studied in the present investigation, presumably contained serum lipo-protein. In a series of articles the structure of the yolk platelet crystals has been analyzed, using X-ray diffraction, electron and polarizing microscopy (cf Honjin et al. [lo]). In this case a hexagonal packing has been revealed. Phosphoprotein and in
a lipoprotein appear to be the main components of the yolk platelet crystals 1141. Although the secretory substancesfrom several glands under pathological conditions may precipitate in the form of crystals (e.g. the urine and the bile), crystalline material has apparently not previously been demonstrated electron microscopically in the secretory ducts from any gland. It cannot be determined whether the observed crystals are present also in vivo, or if they appear as a result of an altered chemical and physical environment during the processing of the tissue. Since a back-flow of secretory substances into the intracellular canaliculi of the parietal cells seems less likely, it may be assumed that the material contained within the crystals is secreted by the parietal cells. In certain parietal cells cytoplasmic granules have been observed which react histochemi~ally asneutral, diastase-resistantmucosubstances[12]. ‘Thesecells apparently secretemucus in addition
156 H, F. Helander to HCI, and it is hence possible that mucus is a component of the crystalline material. The granule-containing parietal cells are common in young rats, but rather rare in adult ones [7], which could explain the apparent paucity of crystals in the adult animals. The intraluminal, sometimes crystalline substance should not be discussedisolated from the problems relating HCI secretion. It has been vigorously debated whether the HCI is secreted in a dissociated form, or if the acid is attached to some compound, which neutralizes it and thus protects the epithelial cells against corrosion (cf Babkin [l]). This hypothetical HCIbinding complex would then dissociate in the gastric cavity. The nature of this acid-binding compound as well as its possible relationship to the crystalline material remains to be investigated. This investigation was supported by grants from the Swedish Medical Research Council (no. B68-12X-2298Ol), Magnus Bergvalls stiftelse, and Stiftelsen Therese
Exptl Cell Res 54
and Johan Anderssons Minne. A brief report of the results was presented at the annual meeting of the Scandinavian Society for Electron Microscopy in Stockholm, Sweden, June 4, 1968 [8].
REFERENCES 1. Babkin, B P, Secretory mechanism of the digestive glands, 2nd edn. Hoeber, New York (1950). 2. Fahimi, H D & Drochmans, P, J micr 4 (1965) 725. 3. Gall, J G, J cell sci 2 (1967) 163. 4. Hamilton, D W, Fawcett, D W & Christensen, A K, Z Zellforsch 70 (1966) 347. 5. Helander, H F, J ultrastruct res, Suppl. 4 (1962). 6. - Gastroenterol. In press. 7. - Ibid. In press. - J ultrastruct res 25 (1968) 170. :: Holden, A & Singer, P,.Crystals and crystal growing. Doubleday, New York (1960). 10. Honjin, R,’ Nakamura,‘T & Shimasaki, S, J ultrastruct res 12 (1965) 404. 11. Maunsbach, A B, J ultrastruct res 14 (1966) 167. 12. Spicer, S S & Sun, D C H, Ann NY acad sci 140 (1967) 762. 13. Townsend, S F, Am j anat 109 (1961) 260. 14. Wallace, R A, Biochim biophys acta 74 (1963) 505. Received June 24, 1968