Rainer H. Lange

Lipovitellin—phosvitin crystals with orthorhombic features: Thin-section electron microscopy, gel electrophoresis, and microanalysis in teleost and amphibian yolk platelets and a comparison with other vertebrates

Yolk-platelet crystals in the teleosts Pelvicachromis pulcher and Noemacheilus barbatulus and the amphibians Xenopus laevis, Rana temporaria, R. esculenta, and Triturus sp. have been studied by electron diffraction and imaging using a standardized processing (glutaraldehyde-osmium tetroxide fixation, glutaraldehyde-urea embedding, thin-section staining), by X-ray microanalysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of their constituents. The crystal lattice is orthorhombic having--following standardized processing--in three amphibians a = 9.0 nm, b = 17.6 nm, c = 19.2 nm, and in the two teleosts a = 8.9 nm, b = 17.6 nm, c = 20.0 nm (averages). These values are very close to X-ray data from wet crystals (Xenopus laevis). Crystal images in the three axial projections point to the presence of space group P212121 (or an approximation of it since the lipovitellin dimers cannot be fully equivalent in some cases), to differences between the phosvitins of the two teleosts, and to a highly unusual stain exclusion from large crystal constituents interpreted as representing lipovitellin dimers. Microanalysis in ultrathin cryosections and other preparations revealed K and Cl to be the prominent ions in the crystals (and in the superficial layer of the platelet). Gel electrophoresis (including data of cyclostomes) showed considerable molecular variations despite a closely similar crystal architecture, emphasizing a physiological significance of the architecture, which may have remained conserved for nearly 400 million years according to paleontologic views.

Yolk-platelet crystals in three ancient bony fishes: Polypterus bichir (polypteri), Amia calva L., and Lepisosteus osseus (L.) (Holostei)

SummaryYolk-platelet crystals in Amia calva L., Lepisosteus osseus (L.) and Polypterus bichir have orthorhombic features with unit-cell dimensions a= 8.3...8.8 nm, b= 16.4...16.9 nm and c= 18.6...19.8 nm as determined in electron-diffraction patterns of fixed, epoxy-resin embedded and thinsectioned material. Electron-diffraction patterns, crystal projections and the above unit-cell data make them extremely similar to the orthorhombic yolkplatelet crystals known for amphibians and teleosts. This observation fills a gap in yolk-platelet research and supports the view that the general architecture of yolk platelets has been conserved for nearly 400 million years. It follows that the peculiar platelet architecture itself has physiological significance.

Crystalline preparations of rhombohedral porcine insulin as studied by electron diffraction.

Porcine rhombohedral insulin crystals, mainly of the varieties containing 2.5 and 0.8% Zn, were studied by electron diffraction in the fixed, embedded, and thin-sectioned state. Stable and reproducible diffraction patterns (resolution limit 0.8 nm) were obtained only with 2.5% Zn-insulin crystals. Suitable fixatives were combinations of OsO 4 and glutaraldehyde; suitable embedding media were an epoxy resin and a glutaraldehyde-urea resin. The crystals, in the embedded state, were trigonal (rhombohedral cell) with a H = 7.4 nm, c = 3.1 nm as measured in the diffraction patterns. In discussing these values, a number of possible errors are considered, i.e., reciprocal lattice spikes, section compression, and mass loss from sections due to irradiation. For comparison wet unfixed 2.5% Zn-insulin crystals were also studied in X-ray powder patterns (Guinier, low angle), yielding a H = 8.2 nm, c = 3.3 nm (rhombohedral cell). These values indicate a close resemblance to porcine 2 Zn-insulin crystals (0.4% Zn), apparently shown here for the first time. Confidence intervals for a H and c were computed by use of their asymptotic distribution. The results are discussed in view of electron microscope studies of intracellular protein crystals, where, in conclusion, a reduction in length of approximately 8% is to be expected in reference to the hydrated state.

The lipoprotein crystals of cyclostome yolk platelets (Myxine glutinosa L., Lampetra planeri [Bloch], L. fluviatilis [L.])

The crystal-like main portion of the yolk platelets in a myxinoid ( Myxine glutinosa L.) and two petromyzonoids ( Lampetra planeri [Bloch], L. fluviatilis [L.]) is organized according to a monoclinic lattice (averaged unit cell: a = 20.0 nm, b = 9.0 nm, c = 9.2 nm, β = 107°, V = 1580 nm 3 ) as results from an electron diffraction study of material fixed with glutaraldehyde-OsO 4 , embedded in glutaraldehyde-urea resin, and double-stained in thin sections with uranyl acetate and lead citrate. The packing unit (there are two such per unit cell above) appears to be a thorn apple-like microchamber (approximately 10 × 10 × 10 nm 3 ) with an unestablished internal organization. The crystal can be interpreted as being a close-packed structure of such chambers forming layers in various spatial orientations; this interpretation results from the analysis of electron micrographs of crystals (11 indexed projections reproduced) and freeze-fracture replicas. This architecture of the yolk platelet must have been conserved in cyclostomes for 250 million years and its physiological relevance is seen in rendering the yolk platelet particularly suitable for electrolyte storage. Electron-probe microanalysis of frozen-dried cyclostome yolk platelets has, in fact, revealed the presence of Na, Mg, K, Ca, and Cl (!)—mostly absent following washing the platelets in distilled water—in addition to covalently bound P and S.

Crystalline islet B-granules in the grass snake [Natrix natrix (L.)]: Tilting experiments in the electron microscope

Thin-sectioned crystalline B-granules in the pancreas of the grass snake Natrix natrix (L.) have been studied by electron microscopy using a specimen tilting stage. On the basis of a rhombic dodecahedral model previously established, the following tilt angles were measured: between two 3-fold axes via 2-fold axis 75.0° ± 5.3° (n = 3; theoretical value 70.52°), between 3-fold and 4-fold axis via [211] projection 56.8° ± 4.9° (n = 9; theoretical value 54.74°), and between 4-fold and 2-fold axis 43.6° ± 4.6° (n = 9; theoretical value 45°). For reasons inherent in the techniques used, the measured angles are considered as supporting rather than contradicting the rhombic dodecahedral model.

The Vertebrate Yolk-Platelet Crystal: Comparative Analysis of an in Vivo Crystalline Aggregate

Publisher Summary This chapter provides a comparative analysis of an in vivo crystalline aggregate of the vertebrate yolk-platelet crystal. The vertebrate yolk-platelet crystal is a representative of the large number of naturally occurring, crystalline aggregates, many of which still await adequate analysis. An advantage of the presence of a crystalline aggregate is the structural redundancy, which allows structural information to be obtained from noisy images by an averaging process. Crystalline aggregates provide the most detailed macromolecular shape determinations available by electron microscopy. An aggregate developed by nature is likely to have some functional significance related to this structure. This aspect is absent with in vitro-grown crystals as used by crystallographers for molecular structure determination at the atomic level of resolution.

Lipoprotein crystals in the yolk platelet of a teleost, Pelvicachromis pulcher (Cichlidae)

SummaryYolk-platelet crystals in the oocytes of the teleost Pelvicachromis pulcher (Cichlidae) were shown, using electron diffraction and tilting of thinsectioned specimens, to possess an orthorhombic lattice with unit-cell sides a=8.3 nm, b=16.6 nm and c=18.0 nm. They thus closely resemble the crystals known for a newt (Triturus sp.) and a frog (Xenopus laevis).

Kristallographische Interpretation der Feinstruktur der B-Granula in den Langerhansschen Inseln der Ringelnatter, Natrix n. natrix (L.)

SummaryCrystalline B-granules of grass-snake islets of Langerhans have been shown to possess the shape of rhombic dodecahedra (a ≈ 11 nm, cubic body-centered lattice). Three-dimensional reconstructions from serial sections, optical diffractometry and comparison with crystal models were the techniques utilized. The significance of crystalline B-granules is discussed.ZusammenfassungKristalline B-Granula im Pankreas der Ringelnatter besitzen die Form von Rhombendodekaedern (a ≈ 11 nm, kubisch-raumzentriertes Gitter). Diese Diagnose wird abgeleitet von den Ergebnissen der dreidimensionalen Rekonstruktion von Serienschnitten, der optischen Diffraktometrie und aus dem Vergleich mit Kristallmodellen. Die Bedeutung kristalliner B-Granula wird erörtert.Kristalline B-Granula im Pankreas der Ringelnatter besitzen die Form von Rhombendodekaedern (a ≈ 11 nm, kubisch-raumzentriertes Gitter). Diese Diagnose wird abgeleitet von den Ergebnissen der dreidimensionalen Rekonstruktion von Serienschnitten, der optischen Diffraktometrie und aus dem Vergleich mit Kristallmodellen. Die Bedeutung kristalliner B-Granula wird erortert.

The vascular system of principal islets: semithin-section studies in teleosts fixed by perfusion.

The angioarchitecture of clearly zonated principal islets (central B-cell and peripheral A-cell region) from teleosts has been analyzed in five islets following perfusion fixation and serial sectioning. In contrast to the similar cell-type distribution in the species studied (Gadus morrhua , Myoxocephalus scorpius , Agonus cataphractus , Limanda limanda ) there were three different types of vasculature characterized by centripetal blood flow, centrifugal blood flow, or an intermediate type. The vascular pattern (and the development of the organ capsule) showed inter- and even intraspecies differences. It is suggested that differences in angioarchitecture reflect individually variable interactions of exocrine and endocrine tissue during organogenesis rather than mutual cell-type control via the vascular system.

Comparison between crystalline islet a granules in teleosts and crystals of the hormone glucagon

Abstract Pancreatic islet A cells in the cod, Gadus morrhua L., have been shown, by specimen tilting and accurate calibration of the electron microscope, to possess crystalline secretory granules of rhombic dodecahedral morphology (cubic system) and a unit cell length of a ≥ 4.3 nm in the embedded state. This value differs only by ≤ 9.1% from that of wet porcine glucagon crystals at neutral pH; a reduction in length of this order of size can be attributed to the preparation procedures for electron microscopy. Also, the crystal lattice of the intracellular granules shows features in the electron microscope not inconsistent with space group P 2 1 3 as found in porcine glucagon crystals.