Isolation, ultrastructure, and partial characterization of collagen from the perineurium of the Florida lobster, Panulirus argus.

Highly concentrated extracellular filaments in the perineurium of the Florida spiny lobster, Panulirus argus, were isolated using ultracentrifugation and linear sucrose gradients. The pellet obtained was highly enriched for the filaments as observed by transmission electron microscopy. Fibril diameter and axial periodicity measurements were obtained from filaments positively and negatively stained with uranyl acetate. A period between 14.0 and 25.0 nm and an average fibril diameter of 15.0 nm were observed. The filaments proved resistant to solubilization by most conventional agents and by several collagenases. NaOH (0.1 M at 100 degrees C) safely dissolved the filaments for measurements of protein content by the Lowry method and carbohydrate content with anthrone reagent. These tests revealed a protein content of approximately 84% and a high carbohydrate content of approximately 15%. Polyacrylamide electrophoresis of an acid-pepsin filament extract revealed a highly concentrated band (approximately 100,000) corresponding to the alpha-1 and alpha-2 bands of vertebrate type I collagen. Wide angle X-ray diffraction yielded meridional reflections that confirmed the filaments as collagen when compared with mammalian collagen X-ray diffraction. The amino acid composition was determined with a computer-assisted Beckman amino acid analyzer, which showed a glycine content of 279 residues/1000. Hydroxylysine and hydroxyproline were present in lower concentrations than expected.

Investigation of osmotically stable spheroplasts from two strains of Escherichia coli ML-35, W7-M5.

Osmotically stable spheroplasts were produced from Escherichia coli ML-35 and W7-M5 using either 1 min exposure to polymyxin B or 10 min exposure to Tris/EDTA, followed by 1 to 3 h incubation with lysozyme. Spheroplast membrane permeability studies were conducted using paired radioactive probes with E. coli ML-35. Experiments with 14C-sucrose-16 kD 3H-dextran indicated that the outer membrane had lost its barrier to 16 kD dextran. Parallel experiments with 81 kD 3H-dextran indicated that the outer membrane was impermeable to the larger dextran. EDTA treated cells also showed outer membrane permeability to 16 kD dextran. Cytoplasmic membrane integrity was confirmed using 14C-sucrose and 3H2O before and after exposure to polymyxin B and EDTA. Scanning electron microscopy showed that a rough surface on polymyxin B produced spheroplasts while Tris/EDTA spheroplasts showed the same smooth surface as control cells.

Ultrastructure studies of the extracellular filaments in the ventral nerve of Panulirus argus.

The ventral nerve cord of the spiny lobster, Panulirus argus was examined by transmission and scanning electron microscopy. Tannic acid mordant stain was used to enhance extracellular filaments. The ventral nerve cord is surrounded by an unusual perineurial sheath composed primarily of interwoven extracellular filaments. Gap junctions were found associated with the glial cells making up the perineurium. The axo-glial wrappings also contained extracellular filaments associated in bundles rather than uniformly around the axons. The extracellular filaments of the perineurium and axo-glial wrappings appeared to be morphologically identical with diameters ranging from 10-15 nm.

Double membrane-bounded intestinal microvilli in Oncopeltus fasciatus.

A double plasma membrane (DPM) surrounding intestinal microvilli of the migratory milkweed bug, Oncopeltus fasciatus, is described. Mutant and wild types of the phytophagous insect have been studied by conventional SEM and TEM procedures with the use of membrane-enhancing staining methods. Longitudinal and transverse sections revealed a DPM surrounding microvilli and continuing over the apical portions of the intestinal cell. The outer membrane of the DPM contributes to an intestinal lining or peritrophic membrane (PTM), which apparently accumulates in layers. SEM studies reveal a rugose intestinal surface and complete PTM in both starved and fed insects. Only rarely are exposed microvilli seen by SEM. SEM examinations also enable the observation of numerous blebs on the luminal side of the PTM apparently held in position by a neck-like attachment and apparently derived from the outer membrane of the DPM. Preliminary TEM studies of microvilli revealed unique microvesicle-like structures, lying just inside the inner membrane of the DPM, which may be of membrane origin based on their typical trilaminar appearance after en bloc staining with uranyl acetate. Highly ordered microfilaments were observed to occupy the most central aspect of the microvilli.

Nuclear membrane changes in herpes simplex virus-infected BHK-21 cells as seen by freeze-fracture.

The freeze-fracture technique, which produced high-resolution replicas of large internal faces of membranes, was used for an ultrastructural study of the nuclei of herpes simplex virus-infected BHK-21 cells and mock-infected controls. Crystalline arrays of viral nucleocapsids were found in the nucleoplasm of infected cells, and numerous nuclear membrane "blebs" and protrusions were observed. The numerous areas of membrane distortions were not found to contain nuclear pores. In addition, specific areas of normal protein intramembranous particles are deleted from certain areas of the nuclear membrane as a result of herpes simplex virus, type 2, infection.

Inverted gap and other cell junctions in cockroach hemocyte capsules: a thin section and freeze-fracture study.

Freeze-fracture and thin-section studies were done on cockroach hemocytes that had encapsulated implanted pieces of Araldite. Desmosome-like junctions and 'B' type gap junction were described. Freeze-fractured gap junctions displayed fused and clustered, but not hexagonally arrayed intramembranous practicles (approximately 130 A) on the B face and pitted areas on the A face of the plasmalemma. Gap junctions were quite numerous and counts of gap and non-gap particles indicated at least a five-fold particle density increase (4000/mu2) compared with B face particle densities (approximately 800/mu2) from free circulating blood cells where gap junctions had not been formed.

The distribution of orthogonal assemblies and other intercalated particles in frog sartorius and rabbit sacrospinalis muscle.

Freeze-fracture replicas have been prepared from two fast-acting vertebrate muscles (frog sartorius and rabbit sacrospinalis) and are described with particular reference to the distribution of intercalated particles in the plasma membrane. T-tubule and SR cisternae. Orthogonal assemblies of small particles are present on the A face plasma membrane in each instance, and their distribution (in sartorius) is found to be random with respect to the underlying myofibrillar sarcomere repeat. Such assemblies are not present on A or B faces of T-tubule or SR cisternae. Asymmetric particle distribution is described for fracture faces of the T-tubules and SR: the profuse particle packing of the SR A face is uniform from terminal cisternae to medial fenestrated collar. Intercalated particles are present on A and B faces of T-tubule fractures; more commonly on the latter. These results are compared with studies on insect muscles, and a comparative approach to further studies on the correlation between membrane structure and function is discussed.

Vinblastine-induced disruption of microtubules in cockroach hemocytes.

Intrahemocoelic injections of vinblastine (10(-3), 10(-4)M) resulted in the disappearance of microtubule marginal bundles in the hemocytes of Periplaneta americana L. concomitant with a change in lenticular cell shape as determined by electron microscopy. In contrast to control cells, which in section were elliptical in shape with smooth edges, treated cells displayed large vacuoles apparently resulting from folds in the cytoplasm and an irregular shape. Vinblastine concentrations lethal to insects (25micro1/insect, 10(-3)M) resulted in marginal bundle loss, and the formation of cytoplasmic linear material with associated ribosomes, although microtubule crystals were not found.