Structural studies of proteinase entrapment by human alpha 2-macroglobulin using 3-D electron microscopy

Human a2 -macroglobulin ( a2 M; homotetramer, Mr 720 kDa) is an essential scavenger of proteinases in the serum. Each of its four subunits has a ‘bait region’, with cleavage sequences for almost all endo-proteinases, an unusual thiol ester moiety and a receptor-binding domain (RBD). Bait region cleavage in native a2 M ( a2 M-N) by a proteinase results in rapid thiol ester breakage, with a large-scale structural transformation, in which a2 M uniquely entraps the proteinase in a cage-like structure and exposes receptor-binding domains for rapid endocytosis. Transformed a2 M ( a2 M-TR) contains up to two proteinases, which remain active to small substrates. 3-D electron microscopy is optimally suited to study this unusual structural change at resolutions near (1/30) Å−1. ^ The structural importance of the thiol esters was demonstrated by a genetically-engineered a2 M, with the cysteines involved in thiol ester formation mutated to serines, which appeared structurally homologous to a2 M-TR. This demonstrates that the four highly labile thiol esters alone maintain the a2 M-N structure, while the ‘closed trap’ formed by a2 M-TR is a more stable structural form. ^ Half-transformed a2 M ( a2 M-HT), with cleaved bait regions and thiol esters in only two of its four subunits, provides an important structural link between a2 M-N and a2 M-TR. A comparison with a2 M-N showed the two proteinase-entrapping domains were above and below the plane bisecting the long axis. Both a2 M-N and a2 M-TR consist of two dense, oppositely twisted strands with significant interconnections, indicating that the structural change involves a rotation of these strands. In a2 M-HT these strands were partially untwisted with large central openings, revealing the manner in which the proteinase enters the internal cavity of a2 M. ^ In reconstructions of a2 M-N, a2 M-HT and a2 M-TR labeled with a monoclonal Fab, the Fabs were located on distal ends of each constitutive strand, demonstrating an anti-parallel arrangement of the subunits. Separation between the top and bottom pairs of Fabs was nearly the same on all structures, but the pairs were rotated about the long axis. Taken together, these results indicate that upon proteinase cleavage the two strands in a2 M-N separate. The proteinase enters the structure, while the strands re-twist to encage it. In a2 M-TR, which displays receptor-binding arms, more than two subunits are transformed as strands in the transformed half of a2 M-HT were not separated. ^

Silicoflagellates and ebridians from ODP Leg 138 holes

The biostratigraphic distribution and abundance of middle Miocene to Pleistocene silicoflagellates is documented from Ocean Drilling Program (ODP) Leg 138 Holes 844B, 847B, 848B, 849B, 850B, 85 IB, 852B, and 854B from the eastern Equatorial Pacific Ocean. The silicoflagellates were generally abundant and well preserved and frequently exhibited an unusually large range of variation. The upper Miocene of near-equatorial sites includes an assemblage of Bachmannocena diodon nodosa, which includes a bridge across the width of the basal ring. Stratigraphically below this, at sites within 5° of the equator is a lengthy interval of specimens of Distephanus speculum tenuis, which have a fragile apical structure. Both the intervals of Bachmannocena diodon nodosa plexus and Distephanus speculum tenuis are biostratigraphically useful within 5° of the equator, but are less useful beyond that. An unusual range of variation also is observed for Dictyocha in the Pliocene sediments at about the point where D. perlaevis and D. messanensis appear in the geologic record. This variation may be explained by hybridization between diverging species.

Sedimentology of ODP Leg 126 deep-water volcaniclastics

Three sites were drilled in the Izu-Bonin forearc basin during Ocean Drilling Program (ODP) Leg 126. High-quality formation microscanner (FMS) data from two of the sites provide images of part of a thick, volcaniclastic, middle to upper Oligocene, basin-plain turbidite succession. The FMS images were used to construct bed-by-bed sedimentary sections for the depth intervals 2232-2441 m below rig floor (mbrf) in Hole 792E, and 4023-4330 mbrf in Hole 793B. Beds vary in thickness from those that are near or below the resolution of the FMS tool (2.5 cm) to those that are 10-15 m thick. The bed thicknesses are distributed according to a power law with an exponent of about 1.0. There are no obvious upward thickening or thinning sequences in the bed-by-bed sections. Spaced packets of thick and very thick beds may be a response to (1) low stands of global sea level, particularly at 30 Ma, (2) periods of increased tectonic uplift, or (3) periods of more intense volcanism. Graded sandstones, most pebbly sandstones, and graded to graded-stratified conglomerates were deposited by turbidity currents. The very thick, mainly structureless beds of sandstone, pebbly sandstone, and pebble conglomerate are interpreted as sandy debris-flow deposits. Many of the sediment gravity flows may have been triggered by earthquakes. Long recurrence intervals of 0.3-1 m.y. for the very thickest beds are consistent with triggering by large-magnitude earthquakes (M = 9) with epicenters approximately 10-50 km away from large, unstable accumulations of volcaniclastic sand and ash on the flanks of arc volcanoes. Paleocurrents were obtained from the grain fabric of six thicker sandstone beds, and ripple migration directions in about 40 thinner beds; orientations were constrained by the FMS images. The data from ripples are very scattered and cannot be used to specify source positions. They do, however, indicate that the paleoenvironment was a basin plain where weaker currents were free to follow a broad range of flow paths. The data from sandstone fabric are more reliable and indicate that turbidity currents flowed toward 150? during the time period from 28.9 to 27.3 Ma. This direction is essentially along the axis of the forearc basin, from north to south, with a small component of flow away from the western margin of the basin.

Ostracoda fauna of surface sediments from the Persian Gulf

During the "Meteor"-Expedition to the Persian Gulf in March-May 1965, approximately 300 samples were collected. Most of them have been already studied by various authors in sedimentological as well as micropaleontological respects. 49 samples were selected for ostracode studies. These samples are arranged to form a long-axis section ("Laengsprofil"), and 4 shorter cross-profiles, perpendicular to the long-axis profile in the Persian Gulf and Gulf of Oman. 52 species of ostracodes in this area were specifically determined; 39 of them are described under open nomenclature. 13 species are already known from surrounding sea areas: 2 species from the Red Sea; 2 species from the east coast of Africa; 1 species from the Mediterranean Sea; and others from the Indian and Pacific Oceans. 12 species show close relationships to species from the Indopacific Ocean. The ostracode species found in the area can be grouped after the method of BRAUN-BLANQUT into 2 bioassociations. Association 1 with the following 4 characteristic species : Cytherella cf. pulchra, Loxoconcha sp. A, Neomonoceratina sp. A, Alocopocythere reticulata. Association 2 with 1 characteristic species: Ruggieria (Ruggieria) sp. B. The association 1 is widespread in the entire studied area of the Persian Gulf, where it is considered to characterize the shallow water region down to 200 m. The association 2 is restricted to the deeper water below 200 m of the inner part of the Oman Gulf. Only a few species known from the shallow water association of the Persian Gulf are present. Within the two ostracode associations mentioned above 4 zones from the total studied area could be related to the water depth. The zones A-D are characterized more or less readily by the relative abundance of certain species: Zone A : 7-30 m depth, on substrates of poorly coarse-grained clayey marl; Zone B: 30-94 m depth, on substrates of richly coarse-grained calcareous marl; Zone C: 94-1961208 m depth, on substrates of richly coarse-grained calcareous marl; Zone D: 196/208-500 m depth, on substrates of calcareous clay, poor in benthos. The regional and bathymetric distribution of the ostracode fauna in the area studied was compared in relation to 10 environmental factors: water depth, temperature, salinity, water density, O2-concentration, phosphate-silica contents, pH-values, stratification of the water body, water currents and type of sediments. The major environmental factors which appear to control the ostracode distribution are water depth (as a complex factor), O2-concentration and the type of sediment. At 3 stations (GIK01058, GIK01074 and GIK01204) species of the shallow water association were found together with a few bathyal species. These stations are situated at the outer Biaban shelf, in an area where the bottom water of the Persian Gulf flows down the slope towards the Oman Gulf. Several samples of the Zone B in the major part of the Persian Gulf show also a high species diversity containing a high percentage of subfossil ostracode carapaces. It is probable that the recent biocoenosis has been mixed with a late quarternary thanatocoenosis.

Geologic-hydrologic setting and surface sedimentology in the Persian Gulf

1. Morphology and sedimentation The deepest parts of the Persian Gulf lie off the Iranian coast. Several swells separate the Persian Gulf into the Western Basin, the Central Basin and the Strait of Hormuz, which leads without noticeable morphological interruption onto the Biaban Shelf; the latter gradually drops off towards the continental slope, which itself has a strongly subdivided morphology. The sediment distribution in the Western Basin runs parallel to the basin's axis to a depth of 50 -60 m. This is caused by the shallow and uniform slope of the Iranian coast into the Western Basin, by clear exposure of the area to the Shamal-Winds and by tidal currents parallel to the basin's axis. Most other parameters also show isolines parallel to the coast line. Data from the sediment analyses show a net transport which extends out along the Central Swell: coarse fraction > 63 µ, total carbonate content, carbonate in fine fractions < 2 µ, 2-6 µ and 20-63 µ, calcite-aragonite ratios in the fine fractions 2-6 µ and 20-63 µ and quartz-dolomite ratios in fine fraction 2-6 µ. At least the uppermost 10-40 m of this sediment is late Holocene. This implies sedimentation rates of several meters per 1000 years. The slope from the Iranian coast into the Central Basin (max. depth 100 m) is generally steeper, with interspersed islands and flats. Both facts tend to disturb a sediment dustribition parallel to the basin's axis over extensive areas and may preclude any such trend from being detected by the methods and sample net used. The spatial distribution of the coarse fraction, however, seems to indicate sediment transport at greater water depths perpendicular to the basin's long axis and along the steepest gradients well into the Central Basin. The flats of the Central Basin have a sediment cover distinctly different from those of the deeper basin areas. Characteristic parameters are the extremely high percentages of coarse grained sediments, total content of carbonate CO2 over 40, low total organic carbon content, (however values are high if calculated on the basis of the < 63 µ fraction), low total N-content, and low C/N ratios. These characteristics probably result from the absence of any terrigenous material being brought in as well as from exposure to wave action. Finest terrigenous material is deposited in the innermost protected part of the Hormuz Bay. In the deep channel cut into the Biaban Shelf which carries the Persian Gulf out-flow water to the Indian Ocean, no fine grained sediment is deposited as shown by grain size data. 2. Geographic settings and sedimentation Flat lands border the Arabian coast of the Persian Gulf except for the Oman region. The high and steep Zagros Mountains form the Iranian coastline. Flat topography in combination with generally low precipitation precludes fluviatile sediment being added from the South. Inorganic and biogenic carbonates accumulating under low sedimentation rates are dominant on the shallow Arabic Shelf and the slopes into the Western and Central Basins. The fluviatile sediment brought in from the Iranian side, however decisively determine the composition of the Holocene sediment cover in the Persian Gulf and on the Biaban Shelf. Holocene sediments extend 20-30 km seaward into the Western Basin and about 25 km on to the Biaban Shelf. As mentioned before, sedimentation rates are of several meters/1000 years. The rocks exposed in the hinterland influence the sediments. According to our data the Redbeds of the Zagros Mountains determine the colour of the very fine grained sediments near the Iranian Coast of the Persian Gulf. To the West of Hormuz, addition of carbonate minerals is particularly high. Dolomite and protodolomite, deposited only in this area, as well as palygorskite, have proven to be excellent trace minerals. To the East of Hormuz, the supply of terrigenous carbonates is considerably lower. Clay minerals appear to bring in inorganically bound nitrogen thus lowering the C/N ratio in these sediments especially off river mouths. 3. Climate and sedimentation The Persian Gulf is located in a climatically arid region. This directly affects sedimentation through increased wind action and the infrequent but heavy rainfalls which cause flash floods. Such flash floods could be responsible for transporting sedheats into the Central Basin in a direction perpendicular to the Gulf's axis. Eolian influx is difficult to asses from our data; however, it probably is of minor importance from the Iranian side and may add, at the most, a few centimeters of fine sediment per 1000 years. 4. Hydrology and sedimentation High water temperatures favor inorganic carbonate precipitation in southern margin of the Gulf, and probably on the flats, as well as biogenic carbonate production in general. High evaporation plus low water inflow through rivers and precipitation cause a circulation pattern that is typical for epicontinental seas within the arid climate region. Surface water flows in from the adjoining ocean, in this case the Indian Ocean and sinks to the bottom of the Persian Gulf mainly in the northern part of the Western Basin, on the "Mesopotamischer Flachschelf" ard probably in the area of the "Arabischer Flachschelf". This sinking water continually rejuvenates the bottom out-flow water. The inflowing surface water from the Indian Ocean brings organic matter into the Persian Gulf, additional nutrients are added by the "fresh" upwelling waters of the Gulf of Oman. Both nutrients and organic matter diminish very rapidly as the water moves into the Persian Gulf. This depletion of nutrients and organic matter is the reasonfor generally low organic carbon contents of the Persian Gulf sediments. The Central Swell represents a distinct boundary, to the west of which the organic carbon content are lower than to the east when sediment samples of similar grain size distribution are compared. The outflow carries well oxygenated water over the bottom of the Persian Gulf and the resulting oxidation further decreases the content of organic matter. In the Masandam-Channel and in the Biaban-Shelf channel, the outflowing water prevents deposition of fine material and transports sediment particles well beyond the shelf margin. The outflowing water remains at a depth of 200-300 m depending on its density and releases ist suspending sediment load to the ocean floor, irrespectative of the bottom morphology. This is reflected in several parameters in which the sediments from beneath the outflow differ from nearby sediments not affected by the outflowing water. High carbonate content of total samples and of the individual size fraction as well as high aragonite and dolomite contents of individual size fractions characterize the sediment beneath the outflowing water. The tidal currents, which avt more or less parallel to the Gulf's axis, favor mixing of the water masses, they rework sediments at velocities reported here. This fact enlarges to a certain degree the extent of our interfaces which are based on only a few sample points (Persian Gulf and Biaban Shelf one sample per 620 km**2, continental slope one sample per 1000 km**2). The water on the continental slope shows and oxygen minimum at 200-1200 m which favors preservation of organically-bound carbon in the sediment. The low pH-values may even permit dissolution of carbonate minerals.

In situ atomic force microscopy study of Alzheimer’s β-amyloid peptide on different substrates: New insights into mechanism of β-sheet formation

We have applied in situ atomic force microscopy to directly observe the aggregation of Alzheimer’s β-amyloid peptide (Aβ) in contact with two model solid surfaces: hydrophilic mica and hydrophobic graphite. The time course of aggregation was followed by continuous imaging of surfaces remaining in contact with 10–500 μM solutions of Aβ in PBS (pH 7.4). Visualization of fragile nanoscale aggregates of Aβ was made possible by the application of a tapping mode of imaging, which minimizes the lateral forces between the probe tip and the sample. The size and the shape of Aβ aggregates, as well as the kinetics of their formation, exhibited pronounced dependence on the physicochemical nature of the surface. On hydrophilic mica, Aβ formed particulate, pseudomicellar aggregates, which at higher Aβ concentration had the tendency to form linear assemblies, reminiscent of protofibrillar species described recently in the literature. In contrast, on hydrophobic graphite Aβ formed uniform, elongated sheets. The dimensions of those sheets were consistent with the dimensions of β-sheets with extended peptide chains perpendicular to the long axis of the aggregate. The sheets of Aβ were oriented along three directions at 120° to each other, resembling the crystallographic symmetry of a graphite surface. Such substrate-templated self-assembly may be the distinguishing feature of β-sheets in comparison with α-helices. These studies show that in situ atomic force microscopy enables direct assessment of amyloid aggregation in physiological fluids and suggest that Aβ fibril formation may be driven by interactions at the interface of aqueous solutions and hydrophobic substrates, as occurs in membranes and lipoprotein particles in vivo.

Dynamics of contacts between lamellae of fibroblasts: Essential role of the actin cytoskeleton

We investigated actin cytoskeletal and adhesion molecule dynamics during collisions of leading lamellae of nontransformed and oncogene-transformed fibroblasts. By using real-time video microscopy, it was found that during lamellar collision there was considerable overlapping of leading lamellae followed by subsequent retraction. Overlapping of nontransformed fibroblasts was accompanied by formation of β-catenin-positive contact structures organized into strands oriented parallel to the long axis of the cell that were associated with bundles of actin filaments. Maintenance of such cell–cell contact structures critically depended on the contractility of actin cytoskeleton, as inhibition of contractility with serum-free medium or 2,3-butanedione 2-monoxime (BDM) resulted in loss of strand formation. Strand formation was recovered when cells in serum-free medium were incubated with the microtubule inhibitor nocodazole, which is known to increase contractility. Oncogene-transformed fibroblasts reacted to collisions with responses similar to nontransformed fibroblasts but did not develop well-organized cell–cell contacts. A model is presented to describe how differences in the organization of the actin cytoskeleton could account for the structurally distinct responses to cell–cell contact by polarized fibroblastic cells versus nonpolarized epithelial cells.

Propagating structure of Alzheimer’s β-amyloid(10–35) is parallel β-sheet with residues in exact register

The pathognomonic plaques of Alzheimer’s disease are composed primarily of the 39- to 43-aa β-amyloid (Aβ) peptide. Crosslinking of Aβ peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Aβ. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Aβ peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Aβ. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 Å. Analysis of these data establish that this central core of Aβ consists of a parallel β-sheet structure in which identical residues on adjacent chains are aligned directly, i.e., in register. Our data, in conjunction with existing structural data, establish that the Aβ fibril is a hydrogen-bonded, parallel β-sheet defining the long axis of the Aβ fibril propagation.

Time-Lapse Video Microscopy of Gliding Motility in Toxoplasma gondii Reveals a Novel, Biphasic Mechanism of Cell LocomotionV⃞

Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of ∼1.5 μm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180o of the turn, the parasite moves forward one body length at a rate of ∼1–3 μm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.

Dissipative metabolic patterns respond during neutrophil transmembrane signaling

Self-organization is a common theme in biology. One mechanism of self-organization is the creation of chemical patterns by the diffusion of chemical reactants and their nonlinear interactions. We have recently observed sustained unidirectional traveling chemical redox [NAD(P)H − NAD(P)+] waves within living polarized neutrophils. The present study shows that an intracellular metabolic wave responds to formyl peptide receptor agonists, but not antagonists, by splitting into two waves traveling in opposite directions along a cell's long axis. Similar effects were noted with other neutrophil-activating substances. Moreover, when cells were exposed to an N-formyl-methionyl-leucyl-phenylalanine (FMLP) gradient whose source was perpendicular to the cell's long axis, cell metabolism was locally perturbed with reorientation of the pattern in a direction perpendicular to the initial cellular axis. Thus, extracellular activating signals and the signals' spatial cues are translated into distinct intracellular dissipative structures.

Corneal collagen fibril structure in three dimensions: Structural insights into fibril assembly, mechanical properties, and tissue organization

The ability of the cornea to transmit light while being mechanically resilient is directly attributable to the formation of an extracellular matrix containing orthogonal sheets of collagen fibrils. The detailed structure of the fibrils and how this structure underpins the mechanical properties and organization of the cornea is understood poorly. In this study, we used automated electron tomography to study the three-dimensional organization of molecules in corneal collagen fibrils. The reconstructions show that the collagen molecules in the 36-nm diameter collagen fibrils are organized into microfibrils (≈4-nm diameter) that are tilted by ≈15° to the fibril long axis in a right-handed helix. An unexpected finding was that the microfibrils exhibit a constant-tilt angle independent of radial position within the fibril. This feature suggests that microfibrils in concentric layers are not always parallel to each other and cannot retain the same neighbors between layers. Analysis of the lateral structure shows that the microfibrils exhibit regions of order and disorder within the 67-nm axial repeat of collagen fibrils. Furthermore, the microfibrils are ordered at three specific regions of the axial repeat of collagen fibrils that correspond to the N- and C-telopeptides and the d-band of the gap zone. The reconstructions also show macromolecules binding to the fibril surface at sites that correspond precisely to where the microfibrils are most orderly.

Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals1

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.

Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.

Wild-type actin and a mutant actin were isolated from yeast (Saccharomyces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin. While similar to the properties of skeletal muscle actin, there are several important differences between the wild-type yeast and muscle actins. First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result from a difference in the steps involving formation of the nucleating species. Second, as measured with pyrene-labeled yeast actin, but not with intrinsic fluorescence, there is an overshoot in the fluorescence that has not been observed with skeletal muscle actin under the same conditions. Third, in order to simulate the polymerization process of wild-type yeast actin it is necessary to assume some fragmentation of the filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutant actin (R177A/D179A) has also been isolated and studied. The mutations are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elongation rates of the wild type and the mutant differ by only about 3-fold, and the slower polymerization of the mutant appears to result primarily from poorer nucleation.

Genetic engineering of vein grafts resistant to atherosclerosis.

Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.

Observations of spermiogenesis and epididymal sperm maturation in the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia)

Acrosomal development in the early spermatid of the rufous hare wallaby shows evidence of formation of an acrosomal granule, similar to that found in eutherian mammals, the Phascolarctidae and Vombatidae. Unlike the other members of the Macropodidae so far examined, the acrosome of this species appears to be fully compacted at spermiation and extends evenly over 90% of the dorsal aspect of the nucleus. During spermiogenesis, the nucleus of the rufous hare wallaby spermatid showed evidence of uneven condensation of chromatin; this may also be related to the appearance of unusual nucleoplasm evaginations from the surface of the fully condensed spermatid. This study was unable to find evidence of the presence of Sertoli cell spurs or nuclear rotation during spermiogenesis in the rufous hare wallaby. The majority of spermatozoa immediately before spermiation had a nucleus that was essentially perpendicular to the long axis of the sperm tail. Nuclei of spermatozoa found in the process of being released or isolated in the lumen of the seminiferous tubule were rotated almost parallel to the long axis of the flagellum; complete parallel alignment occurred during epididymal maturation. At spermiation spermatozoa have characteristically small cytoplasmic remnants compared to those of other macropods. Unlike the majority of macropodid spermatozoa so far described, the spermatozoa of the rufous hare wallaby showed little evidence of morphological change during epididymal transit. There was no formation of a fibre network around the midpiece or of plasma membrane specializations in this region; the only notable change was a distinctive flattening of midpiece mitochondria and scalloping of the anterior mitochondrial sheath to accommodate the sperm head. Preliminary evidence from spermiogenesis and epididymal sperm maturation supports the classification of the rufous hare wallaby as a separate genus but also indicates that its higher taxonomic position may need to be re-evaluated.