Preparation of thin, fine-grained, tantalum metal replicas for freeze-fracture electron microscopy.

Two critical factors in the preparation of metal films on biological specimens are the type of metal used and the potentially damaging effects of radiant energy from the hot metal source. The excessive heating of surfaces is a major limitation to the replication of heat-sensitive aqueous specimens with refractory metals such as tungsten and tantalum, although these metals are known to form smaller grains and thinner films than the more commonly used platinum/carbon deposited under similar conditions. We describe here an electron gun designed for the evaporation of pure tantalum; surface heating is reduced through intermittent deposition controlled by varying the open/closed intervals of a fast shutter that operates in ultra-high vacuum.

Purified lac permease and cytochrome o oxidase are functional as monomers.

Purified lac permease, the 46.5-kDa product of the lac Y gene that catalyzes lactose/H+ symport, or purified cytochrome o, a terminal oxidase of the Escherichia coli respiratory chain composed of four subunits with a composite molecular mass of 140 kDa, was reconstituted into proteoliposomes individually or in combination. The preparations were then examined by freeze-fracture electron microscopy employing conventional platinum/carbon replicas or by means of a new technique using thin tantalum replicas.

The osmotic response of large unilamellar vesicles studied by quasielastic light scattering.

Large unilamellar vesicles of two phosphatidylcholines, one saturated (DMPC) and the other unsaturated (DOPC), prepared by the reverse-phase evaporation method were studied using the quasielastic light scattering technique. The accurate sizing obtained by this technique showed an osmotic response for the two kinds of vesicles when the salinity of the external medium was diluted. The elastic moduli of lipid vesicles bilayers in the liquid phase were then estimated according to the elasticity theory of spherical shells taking into account salt leakage data known from the literature.

Membrane fusion events in the Ca2+/ionophore-induced acrosome reaction of ram spermatozoa.

An acrosome reaction was induced in ejaculated ram spermatozoa by treatment with calcium and the ionophore A23187. Samples were fixed at different times after initiation of induction, and the morphological changes within the head membranes that took place as exocytosis occurred were studied in freeze-fracture replicas. Reacted acrosomes appeared in individual spermatozoa within the calcium/ionophore-treated population at different times after the start of treatment; the first cells had reacted by 10 min, whereas some took more than 40 min to react.

Shape and fine structure of nucleoids observed on sections of ultrarapidly frozen and cryosubstituted bacteria.

Very rapidly frozen cells of Escherichia coli and Bacillus subtilis were substituted at low temperature into acetone with 1% OsO4 and embedded in Epon. They showed ribosome-free spaces filled with globular and fibrillar material of up to 15 nm. The sizes of structures seen do not exclude DNA superstructures such as supercoils, aggregates, and nucleosomes.

Bovine enteric coronavirus structure as studied by a freeze-drying technique.

A strain of bovine coronavirus (F15) was studied by electron microscopy using a freeze-drying technique. Purified coronavirus preparations show three different categories of image: (i) 'blackberry-like' virions, (ii) virions with a smooth depression at their surface, and (iii) apparently broken particles showing very clearly the areas of spike insertion in the virus membrane. Virus projections resemble 'mushrooms' with the 'stalk' inserted at the virus membrane.

A new pentraxin (serum amyloid P-component) in the rat: evidence for two quaternary structures and effect of ligands on self-association.

A new rat serum protein has been isolated by affinity chromatography using ethanolamine- or phosphoethanolamine-substituted agarose gels. This protein has the morphological and functional characteristics of serum amyloid P-component and C-reactive protein. It comprises C5 cyclic symmetry structure with non covalently cross-linked subunits which have calcium-dependent binding sites.

Chromatin freeze fracture electron microscopy: a comparative study of core particles, chromatin, metaphase chromosomes, and nuclei.

Chromatin gels, metaphase chromosomes, and intact nuclei were studied by freeze fracturing followed by electron microscopy. The results complement and extend those obtained by classical electron microscopy techniques as they are obtained without fixation or dehydration. The freeze fracturing technique permits a determination of the hydrated diameters of nucleosomes in chromatin and in nuclei to be 13 nm by comparing to simultaneously studied test objects.

The tight junctions of rabbit choroid plexus and ciliary body epithelia. A comparative study using the double replica freeze-fracture technique.

The spatial arrangement of tight junctions in choroid plexus and ciliary body rabbit epithelia has been determined by studying freeze-fracture complementary replicas. In the choroid plexus epithelium, the interruptions of the junctional P-face fibrils were measured to be 14% in their total length. In the ciliary body epithelium, where the fibrils were found to be more fragmented than in the choroid plexus, the P-face fibril interruption accounted for 12% of the total length of the zonulae occludentes sealing the non-pigmented cells and 30% in the focal linear tight junctions connecting the non-pigmented cells at their apices.

Jimpy mouse myelin revisited with freeze-fracture.

Corpus callosum, cerebellum, and spinal cord from Jimpy mice, and control littermates, 15 and 21 days old, were prepared for freeze-fracture in a "cryofract" apparatus. The few myelinated axons in the Jimpy exhibited a striking paucity of particles in myelin P faces, though tight junctions were present. In addition, small maculae of particles were found on these P faces.

[Rapid freezing of biologic tissue. Measurement of temperature and rate of freezing by thin-layer thermocouple].

An apparatus for rapid freezing of biological tissues by contact with a copper block cooled by liquid helium has been devised to reduce the contamination of copper block surface. It prevents the precooling of the specimen while going through the layers of cold helium gas surrounding the copper block and reduces the quantity of helium necessary for freezing. It also enables one to obtain easily reproducible results from freezing by immersion in liquid coolants.

[Cryofractures of biological material performed at very low temperatures in ultravacuum].

A new instrument has been designed for freeze-fracturing of biological material in ultra high vacuum. Complementary replicas of the freeze-fracture faces can be done at a temperature reaching to --260 degrees C in a short time, without any contamination of fracture faces being observed.