Transmission electron microscopy of the bacterial nucleoid.

Water-containing biological material cannot withstand the vacuum of the transmission electron microscope. The classical solution to this problem has been to dehydrate chemically fixed biological samples and then embed them in resin. During such treatment, the bacterial nucleoid is especially prone to aggregation, which affects its global shape and fine structure. Initial attempts to deal with aggregation by optimizing chemical fixation yielded contradictory results. Two decades ago, the situation improved with the introduction of freeze-substitution. This method is based on dehydration of unfixed cryo-immobilized samples at low temperature, which substantially reduces aggregation. As a result, the global shape of the nucleoid can be fairly well defined. Overall, in actively growing bacteria, the nucleoids are dispersed and "coralline" but become more confined when growth ceases. However, it is usually impossible to determine the molecular arrangement of DNA in the nucleoids of freeze-substituted bacteria because crystallization and the subsequent removal of water during substitution result in unavoidable distortions at the ultrastructural level. Recently, cryo-electron microscopy of vitreous sections has enabled the fully hydrated bacterial nucleoid to be studied close to the native state. Such studies have revealed aspects of bacterial nucleoid organization that are not preserved by freeze-substitution, including locally parallel or twisted bundles of DNA filaments, which are more frequently observed once bacterial growth has stopped, whereas in actively growing bacteria, the DNA is seen to be in a mostly disordered pattern.

Geochemistry on manganese nodules from Timor

Fossil Mn nodules of Cretaceous age from western Timor exhibit chemical, structural and radioisotope compositions consistent with their being of deep-sea origin. These nodules show characteristics similar to nodules now found at depths of 3,500-5,000 m in the Pacific and Indian Oceans. Slight differences in the fine structure and chemistry of these nodules and modern deep-sea nodules are attributed to diagenetic alteration after uplift of enclosing sediments.

Os isotope ratios of sediments from the Eocene-Oligocene boundary

Osmium (Os) isotope analyses of bulk sediments from the South Atlantic, Equatorial Pacific, and the Italian Apennines yield a well-dated and coherent pattern of 187Os/188Os variation from the late Eocene to the early Oligocene. The resulting composite record demonstrates the global character of two prominent features of the low-resolution LL44-GPC3 Os isotope record (Pegram and Turekian, 1999, doi:10.1016/S0016-7037(99)00308-7). These are: (1) a pronounced minimum in 187Os/188Os (0.22-0.27) in the late Eocene, between 34 and 34.5 Ma, and (2) a subsequent rapid increase in 187Os/188Os, to approximately 0.6 by 32 Ma. An ultramafic weathering event and an increased influx of extraterrestrial particles to the Earth are discussed as alternative explanations for the late Eocene 187Os/188Os minimum. Comparison of the 187Os/188Os to benthic foraminiferal oxygen isotope records demonstrates that the nearly three-fold increase in 187Os/188Os from the late Eocene minimum coincides with the growth and decay of the first large ice sheet of the Oligocene (Oi1 (Miller et al., 1991, doi:10.1029/90JB02015)). The fine structure of the Os isotope record indicates that enhanced release of radiogenic Os, unrelated to the recovery from late Eocene minimum, lagged the initiation of the Oi1 event by roughly 0.5 Myr. This record, in conjunction with weathering studies in modern glacial soils (Blum, in: W.F. Ruddiman (Ed.), Tectonic Uplift and Climate Change, Plenum Press, New York, 1997, pp. 259-288; Peucker-Ehrenbrink and Blum, 1998, doi:10.1016/S0016-7037(98)00227-0), suggests that exposure of freshly eroded material during deglaciation following Oi1 enhanced chemical weathering rates, and may have contributed to ice sheet stabilization by drawing down atmospheric carbon dioxide. The improved temporal resolution and age control of the refined Eocene-Oligocene Os isotope record also makes it possible to illustrate the late Eocene Os isotope excursion as a tool for global correlation of marine sediments.

Sediment facies of the Indian-Pakistan continental margin, Indian Ocean Expedition

The studies described here base mainly on sedimentary material collected during the "Indian Ocean Expedition" of the German research vessel "Meteor" in the region of the Indian-Pakistan continental margin in February and March 1965. Moreover,samples from the mouth of the Indus-River were available, which were collected by the Pakistan fishing vessel "Machhera" in March 1965. Altogether, the following quantities of sedimentary material were collected: 59.73 m piston cores. 54.52 m gravity cores. 33 box grab samples. 68 bottom grab samples Component analyses of the coarse fraction were made of these samples and the sedimentary fabric was examined. Moreover, the CaCO3 and Corg contents were discussed. From these investigations the following history of sedimentation can be derived: Recent sedimentation on the shelf is mainly characterized by hydrodynamic processes and terrigenous supply of material. In the shallow water wave action and currents running parallel to the coast, imply a repeated reworking which induces a sorting of the grains and layering of the sediments as well as a lack of bioturbation. The sedimentation rate is very high here. From the coast-line down to appr. 50 m the sediment becomes progressively finer, the conditions of deposition become less turbulent. On the outer shelf the sediment is again considerably coarser. It contains many relicts of planktonic organisms and it shows traces of burrowing. Indications for redeposition are nearly missing, a considerable part of the fine fraction of the sediments is, however, whirled up and carried away. In wide areas of the outer shelf this stirring has gained such a degree that recent deposits are nearly completely missing. Here, coarse relict sands rich in ooids are exposed, which were formed in very shallow stirred water during the time when the sea reached its lowest level, i.e. at the turn of the Pleistocene to the Holocene. Below the relict sand white, very fine-grained aragonite mud was found at one location (core 228). This aragonite mud was obviously deposited in very calm water of some greater depth, possibly behind a reef barrier. Biochemic carbonate precipitation played an important part in the formation of relict sands and aragonite muds. In postglacial times the relict sands were exposed for long periods to violent wave action and to areal erosion. In the present days they are gradually covered by recent sediments proceeding from the sides. On the continental margin beyond the shelf edge the distribution of the sediments is to a considerable extent determined by the morphology of the sea bottom. The material originating from the continent and/or the shelf, is less transported by action of the water than by the force of gravity. Within the range of the uppermost part of the continental slope recent sedimentation reaches its maximum. Here the fine material is deposited which has been whirled up in the zone of the relict sands. A laminated fine-grained sediment is formed here due to the very high sedimentation rate as well as to the extremely low O2-content in the bottom water, which prevents life on the bottom of the sea and impedes thus also bioturbation. The lamination probaly reflects annual variation in deposition and can be attributed to the rhythm of the monsoon with its effects on the water and the weather conditions. In the lower part of the upper continental slope sediments are to be found which show in varying intensity, intercalations of fine material (silt) from the shelf, in large sections of the core. These fine intercalations of allochthonous material are closely related to the autochthonous normal sediment, so that a great number of small individual depositional processes can be inferred. In general the intercalations are missing in the uppermost part of the cores; in the lower part they can be met in different quantities, and they reach their maximum frequency in the upper part of the lower core section. The depositions described here were designated as turbid layer sediments, since they get their material from turbid layers, which transport components to the continental slope which have been whirled up from the shelf. Turbidites are missing in this zone. Since the whole upper continental slope shows a low oxygen-content of the bottom water the structure of the turbid layer sediments is more or less preserved. The lenticular-phacoidal fine structure does, however, not reflect annual rhythms, but sporadic individual events, as e.g. tsunamis. At the lower part of the continental slope and on the continental rise the majority of turbidites was deposited, which, during glacial times and particularly at the beginning of the post-glacial period, transported material from the zone of relict sands. The Laccadive Ridge represented a natural obstacle for the transport of suspended sediments into the deep sea. Core SIC-181 from the Arabian Basin shows some intercalations of turbidites; their material, however, does not originate from the Indian Shelf, but from the Laccadive Ridge. Within the range of the Indus Cone it is surprising that distinct turbidites are nearly completely missing; on the other hand, turbid layer sediments are to be found. The bottom of the sea is showing still a slight slope here, so that the turbidites funneled through the Canyon of the Swatch probably rush down to greater water depths. Due to the particularly large supply of suspended material by theIndus River the turbid layer sediments show farther extension than in other regions. In general the terrigenous components are concentrated on the Indus Cone. It is within the range of the lower continental slope that the only discovery of a sliding mass (core 186) has been located. It can be assumed that this was set in motion during the Holocene. During the period of time discussed here the following development of kind and intensity of the deposition of allochthonous material can be observed on the Indian-Pakistan continental margin: At the time of the lowest sea level the shelf was only very narrow, and the zone in which bottom currents were able to stir up material by oscillating motion, was considerably confined. The rivers flowed into the sea near to the edge of the shelf. For this reason the percentage of terrigenous material, quartz and mica is higher in the lower part of many cores (e.g. cores 210 and 219) than in the upper part. The transition from glacial to postglacial times caused a series of environmental changes. Among them the rise of the sea level (in the area of investigation appr. 150 m) had the most important influence on the sedimentation process. In connection with this event many river valleys became canyons, which sucked sedimentary material away from the shelf and transported it in form of turbidites into the deep sea. During the rise of the sea level a situation can be expected with a maximum area of the comparatively plane shelf being exposed to wave action. During this time the process of stirring up of sediments and formation of turbid layers will reach a maximum. Accordingly, the formation of turbidites and turbid layer sediments are most frequent at the same time. This happened in general in the older polstglacial period. The present day high water level results in a reduced supply of sediments into the canyons. The stirring up of sediments from the shelf by wave action is restricted to the finest material. The missing of shelf material in the uppermost core sections can thus be explained. The laminated muds reflect these calm sedimentation conditions as well. In the southwestern part of the area of investigation fine volcanic glass was blown in during the Pleistocene, probably from the southeast. It has thus become possible to correlate the cores 181, 182, 202. Eolian dust from the Indian subcontinent represents probably an important component of the deep sea sediments. The chemism of the bottom as well as of the pore water has a considerable influence on the development of the sediments. Of particular importance in this connection is a layer with a minimum content of oxygen in the sea water (200-1500 m), which today touches the upper part of the continental slope. Above and beyond this oxygen minimum layer somewhat higher O2-values are to be observed at the sea bottom. During the Pleistocene the oxygen minimum layer has obviously been locatedin greater depth as is indicated by the facies of laminated mud occuring in the lower part of core 219. The type of bioturbation is mainly determined by the chemism. Moreover, the chemism is responsible for a considerable selective dissolution, either complete or partial, of the sedimentary components. Within the range of the oxygen minimum layer an alkaline milieu is developed at the bottom. This causes a complete or partial dissolution of the siliceous organisms. Here, bioturbation is in general completely missing; sometimes small pyrite-filled burrowing racks are found. In the areas rich in O2 high pH-values result in a partial dissolution of the calcareous shells. Large, non-pyritized burrowing tracks characterize the type of bioturbation in this environment. A study of the "lebensspuren" in the cores supports the assumption that, particularly within the region of the Laccadive Basin, the oxygen content in the bottom sediments was lower than during the Holocene. This may be attributed to a high sedimentation rate and to a lower O2-content of the bottom water. The composition of the allochthonous sedimentary components, detritus and/or volcanic glass may locally change the chemism to a considerable extent for a certain time; under such special circumstances the type of bioturbation and the state of preservation of the components may be different from those of the normal sediment.

Concentrations of phosphates and silicon in near-bottom and interstitial waters of the Baltic Sea

Fine structure of vertical distributions of phosphorus and silicon in near-bottom layers and interstitial waters is studied in different regions of the Baltic Sea (Gulf of Finland, Bornholm area, Gotland trench). Data obtained are used to calculate fluxes of mineral forms of phosphorus and silicon in exchange processes between sediments and the near-bottom water layer. Depending on sediment types, values of nutrient fluxes vary from 9.8 to 632.6 µg-at/m**2/day for phosphorus and from 232.4 to 1881.1 µg-at/m**2/day for silicon. Fluxes calculated for different regions are compared.

Tailoring the Optical Properties of Silica Irradiated with Swift Heavy Ions

Irradiation with swift heavy ions (SHI), roughly defined as those having atomic masses larger than 15 and energies exceeding 1 MeV/amu, may lead to significant modification of the irradiated material in a nanometric region around the (straight) ion trajectory (i.e., latent tracks). In the case of amorphous silica it has been reported that SHI irradiation originates nano-tracks of either higher density than the virgin material (for low electronic stopping powers, Se < 7 keV/nm) [1] or having a low-density core and a dense shell (Se > 12 keV/nm) [2]. The intermediate region has not been studied in detail but we will show in this work that essentially no changes in density occur in this zone. An interesting effect of the compaction is that the refractive index is increased with respect to that of the surroundings. In the first Se region it is clear that track overlapping leads to continuous amorphous layers that present a significant contrast with respect to the pristine substrate and this has been used to produce optical waveguides. The optical effects of intermediate and high stopping powers, on the other hand, are largely unknown so far. In this work we have studied theoretically (molecular dynamics and optical simulations) and experimentally (irradiation with SHI and optical characterization) the dependence of the macroscopic optical properties (i.e., the refractive index of the effective medium, n_EMA) on the electronic stopping power of the incoming ions. Our results show that the refractive index of the irradiated silica is not increased in the intermediate region, as expected; however, the core-shell tracks of the high-Se region produce a quite effective enhancement of n_EMA that could prove attractive for the fabrication of optical waveguides at ultralow fluences (as low as 1E11 cm^-2). 1. J. Manzano, J. Olivares, F. Agulló-López, M. L. Crespillo, A. Moroño, and E. Hodgson, "Optical waveguides obtained by swift-ion irradiation on silica (a-SiO2)," Nucl. Instrum. Meth. B 268, 3147-3150 (2010). 2. P. Kluth, C. S. Schnohr, O. H. Pakarinen, F. Djurabekova, D. J. Sprouster, R. Giulian, M. C. Ridgway, A. P. Byrne, C. Trautmann, D. J. Cookson, K. Nordlund, and M. Toulemonde, "Fine structure in swift heavy ion tracks in amorphous SiO2," Phys. Rev. Lett. 101, 175503 (2008).

Phylogenetic assessment of length variation at a microsatellite locus

Sixty-six haplotypes at a locus containing a simple dinucleotide (CA)n microsatellite repeat were isolated by PCR–single-strand conformational polymorphism from populations of the horseshoe crab Limulus polyphemus. These haplotypes were sequenced to assess nucleotide variation directly. Thirty-four distinct sequences (alleles) were identified in a region 570 bp long that included the microsatellite motif. In the repeat region itself, CA-number varied in integer values from 5 to 11 across alleles, except that a (CA)8 class was not observed. Differences among alleles were due also to polymorphisms at 22 sites in regions immediately flanking the microsatellite repeats. Nucleotide substitutions in these regions were used to estimate phylogenetic relationships among alleles, and the gene phylogeny was used to trace the evolution of length variation and CA repeat numbers. A low correlation between size variation and genealogical relationships among alleles suggests that absolute fragment size (as normally scored in microsatellite assays) is an unreliable indicator of historical affinities among alleles. This finding on the molecular fine structure of microsatellite variation suggests the need for caution in the use of repeat counts at microsatellite loci as secure indicators of allelic relationships.

Birefringence Imaging Directly Reveals Architectural Dynamics of Filamentous Actin in Living Growth ConesV⃞

We have investigated the dynamic behavior of cytoskeletal fine structure in the lamellipodium of nerve growth cones using a new type of polarized light microscope (the Pol-Scope). Pol-Scope images display with exquisite resolution and definition birefringent fine structures, such as filaments and membranes, without having to treat the cell with exogenous dyes or fluorescent labels. Furthermore, the measured birefringence of protein fibers in the thin lamellipodial region can be interpreted in terms of the number of filaments in the bundles. We confirmed that birefringent fibers are actin-based using conventional fluorescence-labeling methods. By recording movies of time-lapsed Pol-Scope images, we analyzed the creation and dynamic composition of radial fibers, filopodia, and intrapodia in advancing growth cones. The strictly quantitative information available in time-lapsed Pol-Scope images confirms previously deduced behavior and provides new insight into the architectural dynamics of filamentous actin.

High-Voltage Electron Tomography of Spindle Pole Bodies and Early Mitotic Spindles in the Yeast Saccharomyces cerevisiaeV⃞

The spindle pole body (SPB) is the major microtubule-organizing center of budding yeast and is the functional equivalent of the centrosome in higher eukaryotic cells. We used fast-frozen, freeze-substituted cells in conjunction with high-voltage electron tomography to study the fine structure of the SPB and the events of early spindle formation. Individual structures were imaged at 5–10 nm resolution in three dimensions, significantly better than can be achieved by serial section electron microscopy. The SPB is organized in distinct but coupled layers, two of which show ordered two-dimensional packing. The SPB central plaque is anchored in the nuclear envelope with hook-like structures. The minus ends of nuclear microtubules (MTs) are capped and are tethered to the SPB inner plaque, whereas the majority of MT plus ends show a distinct flaring. Unbudded cells containing a single SPB retain 16 MTs, enough to attach to each of the expected 16 chromosomes. Their median length is ∼150 nm. MTs growing from duplicated but not separated SPBs have a median length of ∼130 nm and interdigitate over the bridge that connects the SPBs. As a bipolar spindle is formed, the median MT length increases to ∼300 nm and then decreases to ∼30 nm in late anaphase. Three-dimensional models confirm that there is no conventional metaphase and that anaphase A occurs. These studies complement and extend what is known about the three-dimensional structure of the yeast mitotic spindle and further our understanding of the organization of the SPB in intact cells.

Direct Visualization of a Protein Nuclear Architecture

Whether the cell nucleus is organized by an underlying architecture analagous to the cytoskeleton has been a highly contentious issue since the original isolation of a nuclease and salt-resistant nuclear matrix. Despite electron microscopy studies that show that a nuclear architecture can be visualized after fractionation, the necessity to elute chromatin to visualize this structure has hindered general acceptance of a karyoskeleton. Using an analytical electron microscopy method capable of quantitative elemental analysis, electron spectroscopic imaging, we show that the majority of the fine structure within interchromatin regions of the cell nucleus in fixed whole cells is not nucleoprotein. Rather, this fine structure is compositionally similar to known protein-based cellular structures of the cytoplasm. This study is the first demonstration of a protein network in unfractionated and uninfected cells and provides a method for the ultrastructural characterization of the interaction of this protein architecture with chromatin and ribonucleoprotein elements of the cell nucleus.

Stability of nonrelativistic quantum mechanical matter coupled to the (ultraviolet cutoff) radiation field

We announce a proof of H-stability for the quantized radiation field, with ultraviolet cutoff, coupled to arbitrarily many non-relativistic quantized electrons and static nuclei. Our result holds for arbitrary atomic numbers and fine structure constant. We also announce bounds for the energy of many electrons and nuclei in a classical vector potential and for the eigenvalue sum of a one-electron Pauli Hamiltonian with magnetic field.

Grass genomes

For the most part, studies of grass genome structure have been limited to the generation of whole-genome genetic maps or the fine structure and sequence analysis of single genes or gene clusters. We have investigated large contiguous segments of the genomes of maize, sorghum, and rice, primarily focusing on intergenic spaces. Our data indicate that much (>50%) of the maize genome is composed of interspersed repetitive DNAs, primarily nested retrotransposons that insert between genes. These retroelements are less abundant in smaller genome plants, including rice and sorghum. Although 5- to 200-kb blocks of methylated, presumably heterochromatic, retrotransposons flank most maize genes, rice and sorghum genes are often adjacent. Similar genes are commonly found in the same relative chromosomal locations and orientations in each of these three species, although there are numerous exceptions to this collinearity (i.e., rearrangements) that can be detected at the levels of both the recombinational map and cloned DNA. Evolutionarily conserved sequences are largely confined to genes and their regulatory elements. Our results indicate that a knowledge of grass genome structure will be a useful tool for gene discovery and isolation, but the general rules and biological significance of grass genome organization remain to be determined. Moreover, the nature and frequency of exceptions to the general patterns of grass genome structure and collinearity are still largely unknown and will require extensive further investigation.

Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica.

The marine slug Elysia chlorotica (Gould) forms an intracellular symbiosis with photosynthetically active chloroplasts from the chromophytic alga Vaucheria litorea (C. Agardh). This symbiotic association was characterized over a period of 8 months during which E. chlorotica was deprived of V. litorea but provided with light and CO2. The fine structure of the symbiotic chloroplasts remained intact in E. chlorotica even after 8 months of starvation as revealed by electron microscopy. Southern blot analysis of total DNA from E. chlorotica indicated that algal genes, i.e., rbcL, rbcS, psaB, psbA, and 16S rRNA are present in the animal. These genes are typically localized to the plastid genome in higher plants and algae except rbcS, which is nuclear-encoded in higher plants and green (chlorophyll a/b) algae. Our analysis suggests, however, that similar to the few other chromophytes (chlorophyll a/c) examined, rbcS is chloroplast encoded in V. litorea. Levels of psbA transcripts remained constant in E. chlorotica starved for 2 and 3 months and then gradually declined over the next 5 months corresponding with senescence of the animal in culture and in nature. The RNA synthesis inhibitor 6-methylpurine reduced the accumulation of psbA transcripts confirming active transcription. In contrast to psbA, levels of 16S rRNA transcripts remained constant throughout the starvation period. The levels of the photosystem II proteins, D1 and CP43, were high at 2 and 4 months of starvation and remained constant at a lower steady-state level after 6 months. In contrast, D2 protein levels, although high at 2 and 4 months, were very low at all other periods of starvation. At 8 months, de novo synthesis of several thylakoid membrane-enriched proteins, including D1, still occurred. To our knowledge, these results represent the first molecular evidence for active transcription and translation of algal chloroplast genes in an animal host and are discussed in relation to the endosymbiotic theory of eukaryote origins.