Is Cadinella ornatissima a chomodorid? Sperm ultra structure in an enigmatic nudibranch (Opisthobranchia Mollusca)

Sperm ultrastructure is described for the nudibranch gastropod Cadlinella ornatissima, type species of the genus Cadlinella (Thiele). Although C. ornatissima exhibits most of the sperm features characteristic of other Opisthobranchia and the Pulmonata (a small, rounded acrosomal vesicle, a complex, helical, mitochondrial derivative - partially paracrystalline, coarse fibres associated with the axoneme), it also possesses a number of previously undescribed and possibly unique features (a longitudinally inrolled acrosomal pedestal, an axial structure within the cavity of the acrosomal pedestal, an electron-dense collar at the anterior region of the acrosomal pedestal, the presence of crystalloid bodies within the glycogen helices of the mitochondrial derivative). To our knowledge this is the first report of crystalloid bodies in mature sperm of any mollusc. Collectively this evidence raises questions concerning the affinities and systematic position of Cadlinella within the Nudibranchia. The peculiar nature of the sperm differences, in comparison with other investigated nudibranchs, suggest that Cadlinella is not easily linked to either the Cadlinidae or Chromodorididae, and should be considered incertae sedis.

Comparative sperm ultrastructure of the Baikalian endemic prosobranch gastropods

Mature euspermatozoan ultrastructure is described for seven species of the rissooidean family Baicaliidae (endemic to Lake Baikal, Russia)-Liobaicalia stiedae, Teratobaikalia ciliata, T. macrostoma, Baicalia carinata, Pseudobaikalia pulla, Maackia bythiniopsis, M. variesculpta, and M. herderiana. For comparison with these species and previously investigated Rissooidea, two species of the Lake Baikal endemic genus Benedictia (B. cf. fragilis and B. baicalensis; Hydrobiidae: Benedictiinae of some authors, Benedictiidae of other authors) in addition to Lithoglyphus naticoides (Hydrobiidae: Lithoglyphinae) and Bythinella austriaca (Hydrobiidae: Bythinellinae) were also investigated. Paraspermatozoa were not observed in any of the species examined, supporting the view that these cells are probably absent in the Rissooidea. In general, the euspermatozoa of all species examined resemble those of many other caenogastropods (basally invaginated acrosomal vesicle, mid-piece with 7-13 helical mitochondria, an annulus, glycogen piece with nine peri-axonemal tracts of granules). However, the presence of a completely flattened acrosomal vesicle and a specialized peri-axonemal membranous sheath (a scroll-like arrangement of 4-6 double membranes) at the termination of the mid-piece, clearly indicates a close relationship between the Baicaliidae and other rissooidean families possessing these features (Bithyniidae, Hydrobiidae, Pyrgulidae, and Stenothyridae). Euspermatozoa of Benedictia, Lithoglyphus, Bythinella, and Pyrgula all have a solid nucleus, which exhibits a short, posterior invagination (housing the centriolar complex and proximal portion of the axoneme). Among the Rissooidea, this form of nucleus is known to occur in the Bithyniidae, Hydrobiidae, Truncatellidae, Pyrgulidae, Iravadiidae, Pomatiopsidae, and Stenothyridae. In contrast, the euspermatozoa of the Baicaliidae all have a long, tubular nucleus, housing not only the centriolar derivative, but also a substantial portion of the axoneme. Among the Rissooidea, a tubular nuclear morphology has previously been seen in the Rissoidae, which could support the view, based on anatomical grounds, that the Baicaliidae may have arisen from a different ancestral source than the Hydrobiidae. However, the two styles of nuclear morphology (short, solid versus long, tubular) occur widely within the Caenogastropoda, and sometimes both within a single family, thereby reducing the phylogenetic importance of nuclear differences within the Rissooidea. More significantly, the occurrence of the highly unusual membranous sheath within the mid-piece region in the Baicaliidae appears to tie this family firmly to the Bithyniidae + Hydrobiidae + Stenothyridae + Pyrgulidae assemblage. Eusperm features of Benedictia spp. strongly resemble those of hydrobiids and bithyniids, and neither support recognition of a distinct family Benedictiidae (at best this is a subfamily of Hydrobiidae) nor any close connection with the hydrobiid subfamily Lithoglyphinae.

Treatment of non-resectable hepatocellular carcinoma with autologous tumor-pulsed dendritic cells

Background: The response of hepatocellular carcinoma (HCC) to therapy is often disappointing and new modalities of treatment are clearly needed. Active immunotherapy based on the injection of autologous dendritic cells (DC) co-cultured ex vivo with tumor antigens has been used in pilot studies in various malignancies such as melanoma and lymphoma with encouraging results. Methods: In the present paper, the preparation and exposure of patient DC to autologous HCC antigens and re-injection in an attempt to elicit antitumor immune responses are described. Results: Therapy was given to two patients, one with hepatitis C and one with hepatitis B, who had large, multiple HCC and for whom no other therapy was available. No significant side-effects were observed. The clinical course was unchanged in one patient, who died a few months later. The other patient, whose initial prognosis was considered poor, is still alive and well more than 3 years later with evidence of slowing of tumor growth based on organ imaging. Conclusions: It is concluded that HCC may be a malignancy worthy of DC trials and sufficient details in the present paper are given for the protocol to be copied or modified. (C) 2002 Blackwell Publishing Asia Pty Ltd.

Neural control of the heart: developmental changes in ionic conductances in mammalian intrinsic cardiac neurons

The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, I-h, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, I-K(I), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+. Using the perforated-patch recording configuration, an ATP-sensitive K+ (K-ATP) conductance was identified in greater than or equal to 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs. glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion. (C) 2002 Elsevier Science B.V All rights reserved.

Stress correlation function evolution in lattice solid elasto-dynamic models of shear and fracture zones and earthquake prediction

It has been argued that power-law time-to-failure fits for cumulative Benioff strain and an evolution in size-frequency statistics in the lead-up to large earthquakes are evidence that the crust behaves as a Critical Point (CP) system. If so, intermediate-term earthquake prediction is possible. However, this hypothesis has not been proven. If the crust does behave as a CP system, stress correlation lengths should grow in the lead-up to large events through the action of small to moderate ruptures and drop sharply once a large event occurs. However this evolution in stress correlation lengths cannot be observed directly. Here we show, using the lattice solid model to describe discontinuous elasto-dynamic systems subjected to shear and compression, that it is for possible correlation lengths to exhibit CP-type evolution. In the case of a granular system subjected to shear, this evolution occurs in the lead-up to the largest event and is accompanied by an increasing rate of moderate-sized events and power-law acceleration of Benioff strain release. In the case of an intact sample system subjected to compression, the evolution occurs only after a mature fracture system has developed. The results support the existence of a physical mechanism for intermediate-term earthquake forecasting and suggest this mechanism is fault-system dependent. This offers an explanation of why accelerating Benioff strain release is not observed prior to all large earthquakes. The results prove the existence of an underlying evolution in discontinuous elasto-dynamic, systems which is capable of providing a basis for forecasting catastrophic failure and earthquakes.

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton [1-4]. In the context of stable adhesion [1], cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis [2]. Here actin polymerization has been proposed to drive cell surfaces together [5], although F-actin reorganization also occurs as cell contacts mature [6]. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere [7]. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol Chem 277, 25867-25869) described novel caveolin- and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed caves, using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.

Genes induced by growth hormone in a model of adipogenic differentiation

A substantial number of GH regulated genes have been reported in mature hepatocytes. but genes involved in GH-initiated cell differentiation have not yet been identified. Here we have studied a, ell-characterised model of GH-dependent differentiation, adipogenesis of 3T3-F442A preadipocytes, to identify genes rapidly induced by GH. Using the suppression subtractive hybridisation technique, we have identified eight genes induced within 60 min of GH treatment, and verified these by northern analysis. Six were identifiable as Stat 2. Stat 3, thrombospondin-1. oncostatin M receptor beta chain. a DEAD box RNA helicase. and muscleblind. a developmental transcription factor. Bioinformatic approaches assigned one of the two remaining unknown genes as a novel 436 residue serine,threonine kinase. As each of the identified genes hake important developmental roles. they may be important in initiating GH-induced adipogenesis. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Cauda epididymal spermatozoa of the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia) imaged by electron and confocal microscopy

The ultrastructure of mature Lagorchestes hirsutus spermatozoa is described for the first time, revealing unusual aspects of sperm structure in macropodid species. The sperm head is ovoid rather than cuneiform, lacks a ventral nuclear groove and has an acrosomal distribution over approximately 85-90% of its dorsal surface. Immediately adjacent to the nuclear membrane the peripheral nucleoplasm in most spermatozoa form an irregular series of distinctive evaginations previously not described in the spermatozoa of any other marsupial. The midpiece is extremely thickened and short, containing no helical network or peripheral plasma membrane specializations. Axonemal structure is unspecialized with no connecting lamellae; dense outer fibres are closely adherent to axonemal doublets. The sperm morphology of this species is highly aberrant in comparison to other macropod taxa and supports the retention of Lagorchestes as a distinctive genus. In light of this new information, skeletal and serological data should be re-evaluated to determine the true taxonomic and phylogenetic position of this species.

Differences in benthic fauna and sediment among mangrove (Avicennia marina var. australasica) stands of different ages in New Zealand

Management of coastal environments requires understanding of ecological relationships among different habitats and their biotas. Changes in abundance and distribution of mangroves, like those of other coastal habitats, have generally been interpreted in terms of changes in biodiversity or fisheries resources within individual stands. In several parts of their range, anthropogenically increased inputs of sediment to estuaries have led to the spread of mangroves. There is, however, little information on the relative ecological properties, or conservational values, of stands of different ages. The faunal, floral and sedimentological properties of mangrove (Avicennia marina var. australasica) stands of two different ages in New Zealand has been compared. Older (>60 years) and younger (3-12 years) stands showed clear separation on the basis of environmental characteristics and benthic macrofauna. Numbers of faunal taxa were generally larger at younger sites, and numbers of individuals of several taxa were also larger at these sites. The total number of individuals was not different between the two age-classes, largely due to the presence of large numbers of the surface-living gastropod Potamopyrgus antipodarum at the older sites. It is hypothesized that as mangrove stands mature, the focus of faunal diversity may shift from the benthos to animals living on the mangrove plants themselves, such as insects and spiders, though these were not included in the present study. Differences in the faunas were coincident with differences in the nature of the sediment. Sediments in older stands were more compacted and contained more organic matter and leaf litter. Measurement of leaf chemistry suggested that mangrove plants in the younger stands were able to take up more N and P than those in the older stands. (C) 2003 Elsevier Science B.V. All rights reserved.

Identification of intracellular calcium oxalate crystals in Chamelaucium uncinatum (Myrtaceae)

Intracellular inclusions in the pedicel and calyx-tube tissues of Chamelaucium uncinatum Schauer ( Myrtaceae) flowers are irregular in shape. They were shown, by polarised light and scanning electron microscopy, to be birefringent 8.9-29.5 mum druse (i.e. aggregate) crystals. Energy-dispersive X-ray spectroscopy showed that these crystals were predominantly composed of calcium. Histochemical and acid-solubility tests indicated that the crystals were calcium oxalate. Raman microprobe spectroscopy was used to confirm this chemical identity. The calcium oxalate crystals were located in xylem-vessel lumens and also in parenchyma cells adjacent to vascular tissues. Thus, the crystals may function to regulate soluble calcium concentrations in C. uncinatum tissues near sites where calcium is unloaded from the xylem.

Influence of planting date, method of handling and seed size on supersweet sweet corn seed quality

A supersweet sweet corn hybrid, Pacific H5, was planted at weekly intervals (P-1 to P-5) in spring in South-Eastern Queensland. All plantings were harvested at the same time resulting in immature seed for the last planting (P-5). The seed was handled by three methods: manual harvest and processing (M-1), manual harvest and mechanical processing (M-2) and mechanical harvest and processing (M-3), and later graded into three sizes (small, medium and large). After eight months storage at 12-14degreesC, seed was maintained at 30degreesC with bimonthly monitoring of germination for fourteen months and seed damage at the end of this period. Seed quality was greatest for M-1 and was reduced by mechanical processing but not by mechanical harvesting. Large and medium seed had higher germination due to greater storage reserves but also more seed damage during mechanical processing. Immature seed from premature harvest (P-5) had poor quality especially when processed mechanically and reinforced the need for harvested seed to be physiologically mature.

Ultrastructure of developing tooth plates in the Australian lungfish, Neoceratodus forsteri (Osteichthyes : Dipnoi)

While the lungfish dentition is partially understood as far as morphology and light microscopic structure is concerned, the ultrastructure is not. Each tooth plate is associated with a dental lamina that develops from the inner layer of endodermal cells that form the oral epithelium. Dentines, bone and cartilage of the jaws differentiate from mesenchyme cells aggregating beneath the oral endothelium. Enamel, in the developing and in the mature form, has similarities to that of other early vertebrates, but unusual characters appear as development proceeds. Ameloblasts are capable of secreting enamel, and, with mononuclear osteoclasts, of remodelling the bone below the tooth plate. The forms of dentine, all based largely on an extracellular matrix of collagen and mineralised with biological apatite, differ from each other and from the underlying bone in the ultrastructure of associated cells and in the mineralised extracellular matrices produced. Cell processes emerging from the odontoblasts and from the osteoblasts vary in length, degree of branching and of anastomoses between the processes, although all of the cell types have large amounts of rough endoplasmic reticulum. Mineralisation of the extracellular matrices varies among the enamel, dentines and bone in the tooth plate. In addition, the development of the hard tissues of the tooth plates indicates that many of the similarities in fine structure of the dentition in lungfish, to tissues in other fish and amphibia, apparent early in development, disappear as the dentition matures. (C) 2003 Elsevier Ltd. All rights reserved.

CRIM1 Regulates the Rate of Processing and Delivery of Bone Morphogenetic Proteins to the Cell Surface

The Crim1 gene is predicted to encode a transmembrane protein containing six von Willebrand-like cysteine-rich repeats (CRRs) similar to those in the BMP-binding antagonist Chordin (Chrd). In this study, we verify that CRIM1 is a glycosylated, Type I transmembrane protein and demonstrate that the extracellular CRR-containing domain can also be secreted, presumably via processing at the membrane. We have previously demonstrated Crim1 expression at sites consistent with an interaction with bone morphogenetic proteins (BMPs). Here we show that CRIM1 can interact with both BMP4 and BMP7 via the CRR-containing portion of the protein and in so doing acts as an antagonist in three ways. CRIM1 binding of BMP4 and -7 occurs when these proteins are co-expressed within the Golgi compartment of the cell and leads to (i) a reduction in the production and processing of preprotein to mature BMP, (ii) tethering of pre-BMP to the cell surface, and (iii) an effective reduction in the secretion of mature BMP. Functional antagonism was verified by examining the effect of coexpression of CRIM1 and BMP4 on metanephric explant culture. The presence of CRIM1 reduced the effective BMP4 concentration of the media, thereby acting as a BMP4 antagonist. Hence, CRIM1 modulates BMP activity by affecting its processing and delivery to the cell surface