[Eyeball trauma caused by confetti particles]. / Bulbustrauma durch Konfettipartikel.

The two presented case examples demonstrate that scleral perforation through highly accelerated, seemingly harmless confetti particles is possible. In addition to penetrating injuries that should be treated directly, contusions with serious consequential complications can also occur. A careful assessment and examination are essential to promptly provide the patient with the right treatment and to minimize complications.

Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection.

The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.

Glomerular basement membrane polyanion distribution and nitric oxide in spontaneous hypertensive rats: effects of salt loading and antihypertensive therapy with propranolol.

Cationic colloidal gold (CCG), a polycationic histochemical probe, was used to analyze the distribution of glomerular basement membrane (GBM) polyanions, mainly heparan sulfate proteoglycan in spontaneous hypertensive rats (SHR) with or without salt loading and antihypertensive treatment with propranolol. The changes of mean GBM width and anionic sites distribution were assessed by electron microscopy. Plasma and urinary nitrates (NO(x)) were measured by nitrite (NO2) + nitrate (NO3), stable metabolites of NO. SHR had decreased NO production and increased GBM width (27%) compared with the control Wistar-Kyoto (WKY) rats. The chronic high dietary salt intake resulted in a significant increase in blood pressure, proteinuria, and renal function in the SHR rats. The chronic high salt dietary intake resulted in a decrease in NO in the WKY and a further reduction in NO production in the SHR. The GBM anionic sites count was similar in the SHR and WKY nonsalt-loaded groups, 13.5 +/- 0.5 and 12.8 +/- 0.4 CCG counts/microm GBM, respectively, but significantly lower in both salt-loaded SHR and WKY, 9.9 +/- 0.55 (P < .01) and 9.6 +/- 0.55 (P < .01) CCG counts/microm GBM, respectively. Antihypertensive treatment with propranolol in the salt-loaded SHR group resulted in lower blood pressure, a further decrease in NO production, but no significant changes in GBM width and anionic sites count. It is concluded that chronic high salt intake may be deleterious to the permselectivity of the GBM. A low NO production state that results from chronic salt loading in already hypertensive rats will result in more severe organ (renal) damage, most probably by the addition of the loss of GBM permselectivity to the existing pathomorphologic changes.

DNA protection by stress-induced biocrystallization.

The crystalline state is considered to be incompatible with life. However, in living systems exposed to severe environmental assaults, the sequestration of vital macromolecules in intracellular crystalline assemblies may provide an efficient means for protection. Here we report a generic defence strategy found in Escherichia coli, involving co-crystallization of its DNA with the stress-induced protein Dps. We show that when purified Dps and DNA interact, extremely stable crystals form almost instantaneously, within which DNA is sequestered and effectively protected against varied assaults. Crystalline structures with similar lattice spacings are formed in E. coli in which Dps is slightly over expressed, as well as in starved wild-type bacteria. Hence, DNA-Dps co-crystallization is proposed to represent a binding mode that provides wide-range protection of DNA by sequestration. The rapid induction and large-scale production of Dps in response to stress, as well as the presence of Dps homologues in many distantly related bacteria, indicate that DNA protection by biocrystallization may be crucial and widespread in prokaryotes.

Peritubular dentin formation: crystal organization and the macromolecular constituents in human teeth.

Peritubular dentin (PTD) is a relatively dense mineralized tissue that surrounds the tubules of coronal tooth dentin. It is composed mainly of crystals of carbonated apatite together with a small amount of collagen. Its mode of formation has been investigated by studying the relatively dense particles isolated from a powdered preparation. Electron microscopic examination of the PTD particles, including 3-dimensional image reconstruction and electron diffraction, shows that the organization of the crystals of PTD is very similar to that of the adjacent intertubular dentin (ITD). The latter contains relatively large amounts of collagen and the carbonated apatite crystals are closely associated with the collagen matrix. The proteins present in the PTD particles are soluble after decalcification and stain with Stains All. The principal protein has higher molecular weight and a quite different amino acid composition than the phosphophoryns of the intertubular dentin. The interface between the PTD and the ITD shows structural continuity. These data show how two distinct carbonated apatite-based mineralized tissues can be organized and formed contiguously within the same organ by utilizing different sets of matrix proteins.

Rostrum of a toothed whale: ultrastructural study of a very dense bone.

The rostral bones of the toothed whale, Mesoplodon densirostris, consist mainly of hypermineralized secondary osteons and have yielded among the highest values for density (2.6 g/cm3) and mineral content (86.7%) yet reported for any bone. Scanning and transmission electron microscopy show parallel rods of mineral oriented along the length of the rostrum. These consist of platey crystals of carbonated hydroxyapatite, which, judging from electron diffraction, are extremely well and coherently aligned. The collagen component of the rostral bone consists largely of very thin fibrils aligned in longitudinal register to form tubular networks. The collagen fibrils are also aligned with the lengths of the mineral rods, which are apparently accommodated in the tubular spaces of the collagenous network. This peculiar ultrastructure clearly differs from the densely packed mineralized fibrils commonly observed in vertebrate lamellar osseous tissues, although histological examination has indicated some vestiges of "normal" primary bone surrounding the secondary osteons. Thus, the bone tissue in the rostrum is characterized by a remarkably sparse collagenous component. This ultrastructure can explain the high density, stiffness, and brittleness of the rostrum that have been observed. It also raises interesting questions about possible modes of crystal growth during ongoing mineralization in normal bone, and may have some relevance in the mechanical behavior of dense bones in pathological conditions.

Rotated plywood structure of primary lamellar bone in the rat: orientations of the collagen fibril arrays.

A basic structural motif of lamellar bone is the arrays of parallel collagen fibrils, with successive arrays having different orientations to form a plywood-like structure. Measurements of the angles between adjacent arrays from cryomicrotomed and vitrified thin sections of demineralized rat bone, cut approximately parallel to the lamellar boundary plane, show that most angles are around 30 degrees, although a subset are around 70 degrees. A structural model for collagen organization based on these measurements is proposed in which an individual lamellar unit (thick and thin lamellae together with transition zones) is composed of five arrays of parallel collagen fibrils, each offset by 30 degrees.

Transitional structures in lamellar bone.

Scanning electron micrographs of fractured surfaces of mineralized bone show a lamellar structure with alternating smooth and rough regions. These have been interpreted as corresponding to two distinct collagen fibril and mineral crystal orientations in a rotated plywood structure. However, in various bones, there are clear indications of transition zones between lamellae in which the fibrils, as well as the plate-like crystals, have intermediate orientations. Strong evidence for intermediate collagen fibril orientations comes from vitrified cryo-sections of demineralized bone. These show zones of fibril segments graded in length between more homogenous regions of fibrils roughly parallel to the specimen section. Evidence for intermediate crystal orientations comes from transmission electron micrographs and electron diffraction patterns of crushed bone fragments. A tentative scheme is presented for an interlamellar transition zone, involving rotation about the collagen fibril axis as well as tilting of this axis parallel to the plane of the interlamellar boundary. Although it may be convenient to think of the structure of lamellar bone as being composed of alternating thick and thin lamellae, it is probably more correct and biologically more relevant to consider one pair of lamellae as the product of a single depositional cycle of varyingly oriented collagen fibrils that subsequently mineralize.

Supercoiling-regulated liquid-crystalline packaging of topologically-constrained, nucleosome-free DNA molecules.

Electron microscopy and circular dichroism studies of cholesteric aggregates derived from topologically-constrained DNA molecules indicate that the overall morphology and structural properties of these aggregates are fundamentally different from those characterizing condensed structures of nonconstrained DNA species. Specifically, the cholesteric pitch and twist of all hitherto characterized lyotropic mesophases of biopolymers--including those obtained from linear DNA--depend predominantly upon environmental parameters such as the dielectric constant of the solvent. In contrast, the properties of aggregates derived from closed circular supercoiled DNA are found to be solely and directly dictated by the superhelical density and handedness. On the basis of these results, as well as on the demonstrated ubiquity of liquid-crystalline DNA organizations in vivo, we suggest that supercoiling-regulated liquid crystallinity represents an effective packaging mode of nucleosome-free, topologically-constrained DNA molecules in living systems.

Ultrastructural studies of bones from patients with osteogenesis imperfecta.

Bone samples from patients suffering from osteogenesis imperfecta (OI) types I, II, III and IV, as well as normal controls, were studied by scanning (SEM) and transmission electron microscopy (TEM). SEM views of normal bone at low magnification show coherent structure, with regular striations due to a lamellar plywood-like arrangement of the mineralized collagen fibrils. Compact lamellar bone was also found in various OI specimens, but in limited disconnected regions separated by open spaces. Furthermore, some OI, but not normal, bones have regions of loose unconnected fibers and others of apparently abnormally dense mineral deposition. High resolution TEM studies of OI bone fragments have served to elucidate the structures of these different textures. There appears to be a substantial, though reduced, proportion of normal lamellar bone even in quite severe OI. However, the regions of loose fibers are largely unmineralized and probably contain abnormal collagen. Other regions are overmineralized, with generally small unorganized apatite crystals deposited onto fibril surfaces or in separate clusters. These structural abnormalities, together with the paucity of normal bone, may explain the fragility of OI bones.

Electron cryomicroscopy of Bacillus stearothermophilus 50 S ribosomal subunits crystallized on phospholipid monolayers.

50 S ribosomal subunits from Bacillus stearothermophilus have been crystallized as 2-dimensional periodic arrays on phospholipid monolayer films at the water-air interface. These crystals were preserved in vitreous ice and imaged with 100 keV electrons under low dose and low temperature conditions. The unit cell parameters of the crystals are a = 371.3(+/- 3.8) A, b = 152.3(+/- 1.6) A, gamma = 96.3(+/- 1.0) degrees. Some of the image arrays of these crystals have twofold rotational symmetry with a phase residual of less than 25 degrees. The mean figure of merit of the merged structure factors from these image arrays out to 20 A resolution is higher than 0.87. The 2-dimensional projection map shows a level of detail not seen in previous structural studies of the 50 S ribosome subunit. Some of these features may be related to the current 3-dimensional model of the subunit. This analysis illustrates the potential of using the electron crystallographic approach for determining the 3-dimensional structure of the 50 S ribosomal subunit crystallized on a monolayer surface. In addition, the structural information retrieved by electron crystallography might be useful for phasing X-ray data towards an atomic resolution model of the ribosome.

Electron diffraction and imaging of uncompressed monolayers of amphiphilic molecules on vitreous and hexagonal ice.

A new approach is described for probing domains of ordered self-assemblies of amphiphilic monolayers at the aqueous solution interface. The method has potential importance for the study of membrane structure, Langmuir-Blodgett films, and nucleation processes of two-and three-dimensional crystals. Electron diffraction (ED) patterns indicative of two-dimensional crystalline self-assembly were obtained from samples, which were examined by cryo-electron microscopy, of monolayers of water-insoluble amphiphiles on vitrified aqueour substrates. The apparent hexagonal symmetry of an ED pattern from a C(16)H(33)OH monolayer was interpreted in terms of multiple twinning. Monolayers of the CL(31)H(63)OH and cadmium salt of C(19)H(39)CO(2)H that were studied by dark-field techniques displayed faceted two-dimensional crystallites with a maximal size of 1 to 2 micrometers. Epitaxial nucleation of hexagonal ice by the C(31)H(63)OH monolayer has also been demonstrated by ED.

Origin of mineral crystal growth in collagen fibrils.

Collagen fibrils from young turkey-leg tendons, just beginning to mineralize, were stained with uranyl acetate and examined by electron microscopy. Small needle-like mineral crystals were observed and located, in relation to the collagen banding pattern, as originating at the e band in the gap region and near the surface of the fibrils. These are evidently the sites of crystal nucleation. They lie near binding locations on collagen fibrils of two glycosylated proteins believed to be implicated in the mineralization process, as well as the sites of early crystals in embryonic fowl bones.

DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system.

Evidence for a conceptually novel DNA packaging process is presented. X-ray scattering, electron microscopy, and circular dichroism measurements indicate that in the presence of positively charged micellar aggregates and flexible anionic polymers, such as negatively charged polypeptides or single-stranded RNA species, a complex is formed in which DNA molecules are partially embedded within a micellar scaffold and partially condensed into highly packed chiral structures. Based on studies of micelle-DNA and micelle-flexible anionic polymer systems, as well as on the known effects of a high charge density upon the micellar organization, a DNA packaging model is proposed. According to this model, the DNA induces the elongation of the micelles into rodlike aggregates, forming a closely packed matrix in which the DNA molecules are immobilized. In contrast, the flexible anionic polymers stabilize clusters of spherical micelles which are proposed to effect a capping of the rodlike micelles, thus arresting their elongation and creating surfactant-free segments of the DNA that are able to converge and collapse. Thus, unlike other in vitro DNA packaging systems, in which condensation follows encounters between charge-neutralized DNA molecules, a prepackaging phase where the DNA is immobilized within a matrix is proposed in this case. Cellular and nuclear membranes have been implicated in DNA packaging processes in vivo, and negatively charged polyelectrolytes were shown to be involved in the processes. These observations, combined with the basic tenets of the DNA condensation system described here, allow for the progression to the study of more elaborate model systems and thus might lead to insights into the nature and roles of the intricate in vivo DNA-membrane complexes.

Dentin phosphophoryn binding to collagen fibrils.

The interaction of rat incisor phosphophoryn with native turkey tendon collagen fibers has been examined by electron microscopy. The binding of phosphophoryn to the tendon fibril surfaces is quite selective. The phosphophoryn is seen as positively or negatively stained globular particles predominantly at the "e" band in the collagen gap region in transmission electron micrographs of the phosphophoryn-reacted fibrils. The selectivity of binding to the fibrils was obtained in the presence of calcium ions, which bind avidly to phosphophoryn. The specific association of phosphophoryn at the "e" band suggests a possible regulation of mineral deposition within the gap regions of the collagen fibrils.

Activity of sarafotoxin/endothelin peptides in the heart and brain of lower vertebrates.

The effects of sarafotoxin-b (SRTX-b) and endothelin-1 (ET-1) were tested in the fish tilapia (Ore niloticus x O. aureus hybrids) and torpedo (Torpedo ocellata), the toad (Bufo viridis), the agama lizard (Agama stellio) and water snake (Natrix tessellata). In isolated heart preparations of the fish and agama, peptide doses of 0.05-0.5 micrograms/ml induced positive inotropic effects, reduction of the contraction rate and arrhythmia, leading to cardiac arrest. In the toad, a negative inotropic effect and a reduction of the contraction rate were observed, whereas the water snake was hardly affected by either SRTX-b or ET-1. In the agama, an i.v. injection of 15 micrograms of SRTX-b caused changes in the ECG, culminating in A-V block that led to cardiac arrest, while in the toad an injection of 45 micrograms induced only transient disturbances in the ECG. Binding studies with 125I-SRTX-b revealed specific binding sites for SRTX-b and ET-1 in the heart and brain preparations of fish (tilapia and torpedo) and agama, whereas no specific binding could be demonstrated in the toad or in the snake. These results suggest that most vertebrates tested are sensitive to SRTX/ET, while the snake may possess receptors that are of a different structure.

Growth of mineral crystals in turkey tendon collagen fibers.

Bone and several other vertebrate mineralized tissues are formed by the organized growth of crystals of carbonated apatite within a matrix of type 1 collagen fibers. The development of this process in isolated fibrils of young turkey leg tendons has been studied by transmission electron microscopy. Collagen banding, presumably due to ion concentration, precedes the appearance of any crystals. The smallest crystals observed are short needles in bands near the surface of the fibrils. Longer needles, up to the length of the collagen gap regions, were also seen, and, evidently at a later stage, single crystal belts extending partly or wholly through the fibrils. Finally, in mature tendon crystal platelets, seemingly derived from the cracking of belts, extend partly into the collagen overlap zone. In the least mineralized tendon, extrafibrillar mineral-containing vesicles have occasionally been observed adjacent to regions of radiating needle crystal growth in the fibrils, and, more commonly, smaller particles adjacent to bands of very small needles.

Study of vitrified, unstained frozen tissue sections by cryoimmunoelectron microscopy.

We describe the development and application of a novel approach to high-resolution ultrastructural analysis of cells and tissues. It is based on the preparation of ultrathin frozen sections of fixed tissues, rinsing of the sections, followed by their embedding on the grid in a layer of vitrified ice, and direct observation with a cryoelectron microscope. Examination of smooth muscle, kidney and heart tissues showed that although no heavy metal staining was used, high-contrast images are obtained. Fine details of cytoplasmic filaments and organelles, membranes and membrane-associated structures, as well as connective-tissue elements are all visible. The new method is suitable for immunolabeling, including high resolution localization of specific molecules within the cytoplasm.

Three-dimensional reconstruction of innermost chorion layer of Drosophila grimshawi and Drosophila melanogaster eggshell mutant fs(1)384.

A low-resolution three-dimensional structure of the crystalline innermost chorionic layer (ICL) of the Hawaiian species Drosophila grimshawi and the Drosophila melanogaster eggshell mutant fs(1)384 has been calculated from electron microscope images of tilted negatively stained specimens. The isolated ICL of Drosophila grimshawi is a three-layer structure, about 36 nm thick, whereas the ICL of Drosophila melanogaster eggshell mutant fs(1)384 is a single layer, about 12 nm thick. Each unit in both crystalline structures includes octamers made up of four heterodimers. Crosslinks between the structural elements, both within and between unit cells form an interconnecting network, apparently important in maintaining the integrity of the layer. A model which may account for the ICL self-assembly formation in vivo and the ICL observed lattice polymorphism is proposed, combining data from the three-dimensional reconstruction work and secondary structure features of the ICL component proteins s36 and s38.