Characterization of mouse profilaggrin: Evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N-terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal anti-body raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

The ultrastructure of Macropodinium moiri and revised diagnosis of the Macropodiniidae (Litostomatea : Trichostomatia)

The ultrastructural features of Macropodinium moiri were investigated. The somatic cortex is composed of two lateral non-ciliated zones covered with trapezoidal plates and separated by a trough-like dorsoventral groove (DVG) which divides the cell into left and right halves. The somatic kineties occupy the margins of the DVG and are composed of monokinetids whose infraciliature shows a typical litostome pattern. The pellicular plates are lamellate, and separated by V-shaped grooves which are lined by thick-walled vacuoles. The DVG cortex is composed of electron-opaque U-shaped ribs which alternate with electron-lucent saccular structures. The DVG surface is composed of small regular pellicular sacs built up to form the ridges of the dorsal DVG. The vestibulum forms a laterally compressed cone with left/right differentiation. The basal section of its non-ciliated right side is internally lined (outer to innermost) by longitudinal fibres, nematodesmata and transverse microtubular ribbons. The left side bears the vestibular kineties and in its basal section is lined (outer to innermost) by small nematodesmata and transverse tubules. Cytoplasmic organelles include endoplasmic reticulum, starch granules and a single contactile vacuole surrounded by patches of nephridioplasm. Hydrogenosomes are absent and coccoid Gram-positive bacteria lie under the ciliated portions of the cell. This set of characteristics differs significantly from those of the all other trichostomes; Macropodiniidae is therefore designated Trichostomatia incertae sedis. A revised familial diagnosis of the Macropodiniidae is proposed.

GABA(A) receptor sites in the developing human foetus

GABA(A) receptor sites were characterised in cerebral cortex tissue samples from deceased neurologically normal infants who had come to autopsy during the third trimester of pregnancy. Pharmacological parameters were obtained from homogenate binding studies which utilised the 'central-type' benzodiazepine ligands [H-3]diazepam and [H-3]flunitrazepam, and from the GABA activation of [H-3]diazepam binding. It was found that the two radioligands behaved differently during development. The affinity of [H-3]flunitrazepam for its binding site did not vary significantly between preparations, whereas the [H-3]diazepam K-D showed marked regional and developmental variations: infant tissues showed a distinctly lower affinity than adults for this ligand. The density of [H-3]flunitrazepam binding sites increased similar to35% during the third trimester to reach adult levels by term, whereas [H-3]diazepam binding capacity declined slightly but steadily throughout development. The GABA activation of [H-3]diazepam binding was less efficient early in the trimester, in that the affinity of the agonist was significantly lower, though it rose to adult levels by term. The strength of the enhancement response increased to adult levels over the same time-frame. The results strongly suggest that the subunit composition of cortical GABA(A) sites changes significantly during this important developmental stage. (C) 2002 Elsevier Science B.V. All rights reserved.

Co-crystallization of sucrose at high concentration in the presence of glucose and fructose

Co-crystallization of sucrose from a highly concentrated sucrose syrup (less than or equal to 7% moisture, w/w) at 131 degreesC with 0, 5, 10, 15, and 20% of fructose, glucose, or a mixture of fructose and glucose was investigated. The crystallization of sucrose was delayed in presence of these lower molecular weight sugars. The DSC melting endotherm of cocrystallized samples exhibited a decrease in crystalline sucrose in the sample as a function of increased level of glucose and fructose. The mechanical strength of co-crystallized granules was found to be related to the moisture content and the amount of glucose or fructose content in the sample. The samples containing 10, 15, and 20% glucose in co-crystallized product demonstrated crystallization of glucose in its monohydrate form during 1 mo of storage.

A role for the dominant thalamus in language? A linguistic comparison of two cases subsequent to unilateral thalamotomy procedures in the dominant and non-dominant hemispheres

Background: Thalamotomy has been reported to be successful in ameliorating the motor symptoms of tremor and/or rigidity in people with Parkinson's disease (PD), emphasising the bona fide contribution of this subcortical nucleus to the neural circuitry subserving motor function. Despite evidence of parallel yet segregated associative and motor cortico-subcortical-cortical circuits, comparatively few studies have investigated the effects of this procedure on cognitive functions. In particular, research pertaining to the impact of thalamotomy on linguistic processes is fundamentally lacking. Aims: The purpose of this research was to investigate the effects of thalamotomy in the language dominant and non-dominant hemispheres on linguistic functioning, relative to operative theoretical models of subcortical participation in language. This paper compares the linguistic profiles of two males with PD, aged 75 years (10 years of formal education) and 62 years (22 years of formal education), subsequent to unilateral thalamotomy procedures within the language dominant and non-dominant hemispheres, respectively. Methods & Procedures: Comprehensive linguistic profiles comprising general and high-level linguistic abilities in addition to on-line semantic processing skills were compiled up to 1 month prior to surgery and 3 months post-operatively, within perceived on'' periods (i.e., when optimally medicated). Pre- and post-operative language performances were compared within-subjects to a group of 16 non-surgical Parkinson's controls (NSPD) and a group of 16 non-neurologically impaired adults (NC). Outcomes & Results: The findings of this research suggest a laterality effect with regard to the contribution of the thalamus to high-level linguistic abilities and, potentially, the temporal processing of semantic information. This outcome supports the application of high-level linguistic assessments and measures of semantic processing proficiency to the clinical management of individuals with dominant thalamic lesions. Conclusions: The results reported lend support to contemporary theories of dominant thalamic participation in language, serving to further elucidate our current understanding of the role of subcortical structures in mediating linguistic processes, relevant to cortical hemispheric dominance.

Population balance modelling of granulation with a physically based coalescence kernel

It was previously published by the authors that granules can either coalesce through Type I (when granules coalesce by viscous dissipation in the surface liquid layer before their surfaces touch) or Type II (when granules are slowed to a halt during rebound, after their surfaces have made contact) (AIChE J. 46 (3) (2000) 529). Based on this coalescence mechanism, a new coalescence kernel for population balance modelling of granule growth is presented. The kernel is constant such that only collisions satisfying the conditions for one of the two coalescence types are successful. One constant rate is assigned to each type of coalescence and zero is for the case of rebound. As the conditions for Types I and II coalescence are dependent on granule and binder properties, the coalescence kernel is thus physically based. Simulation results of a variety of binder and granule materials show good agreement with experimental data. (C) 2002 Elsevier Science Ltd. All rights reserved.

Axon navigation in the mammalian primary olfactory pathway: Where to next?

The process of establishing long-range neuronal connections can be divided into at least three discrete steps. First, axons need to be stimulated to grow and this growth must be towards appropriate targets. Second, after arriving at their target, axons need to be directed to their topographically appropriate position and in some cases, such as in cortical structures, they must grow radially to reach the correct laminar layer Third, axons then arborize and form synaptic connections with only a defined subpopulation of potential post-synaptic partners. Attempts to understand these mechanisms in the visual system have been ongoing since pioneer studies in the 1940s highlighted the specificity of neuronal connections in the retino-tectal pathway. These classical systems-based approaches culminated in the 1990s with the discovery that Eph-ephrin repulsive interactions were involved in topographical mapping. In marked contrast, it was the cloning of the odorant receptor family that quickly led to a better understanding of axon targeting in the olfactory system. The last 10 years have seen the olfactory pathway rise in prominence as a model system for axon guidance. Once considered to be experimentally intractable, it is now providing a wealth of information on all aspects of axon guidance and targeting with implications not only for our understanding of these mechanisms in the olfactory system but also in other regions of the nervous system.

Color constancy and the functional significance of McCollough effects

A central problem in visual perception concerns how humans perceive stable and uniform object colors despite variable lighting conditions (i.e. color constancy). One solution is to 'discount' variations in lighting across object surfaces by encoding color contrasts, and utilize this information to 'fill in' properties of the entire object surface. Implicit in this solution is the caveat that the color contrasts defining object boundaries must be distinguished from the spurious color fringes that occur naturally along luminance-defined edges in the retinal image (i.e. optical chromatic aberration). In the present paper, we propose that the neural machinery underlying color constancy is complemented by an 'error-correction' procedure which compensates for chromatic aberration, and suggest that error-correction may be linked functionally to the experimentally induced illusory colored aftereffects known as McCollough effects (MEs). To test these proposals, we develop a neural network model which incorporates many of the receptive-field (RF) profiles of neurons in primate color vision. The model is composed of two parallel processing streams which encode complementary sets of stimulus features: one stream encodes color contrasts to facilitate filling-in and color constancy; the other stream selectively encodes (spurious) color fringes at luminance boundaries, and learns to inhibit the filling-in of these colors within the first stream. Computer simulations of the model illustrate how complementary color-spatial interactions between error-correction and filling-in operations (a) facilitate color constancy, (b) reveal functional links between color constancy and the ME, and (c) reconcile previously reported anomalies in the local (edge) and global (spreading) properties of the ME. We discuss the broader implications of these findings by considering the complementary functional roles performed by RFs mediating color-spatial interactions in the primate visual system. (C) 2002 Elsevier Science Ltd. All rights reserved.

Age does not influence the bone response to treadmill exercise in female rats

Purpose: Because it is believed that bone may respond to exercise differently at different ages, we compared bone responses in immature and mature rats after 12 wk of treadmill running. Methods: Twenty-two immature (5-wk-old) and 21 mature (17-wk-old) female Sprague Dawley rats were randomized into a running (trained, N = 10 immature, 9 mature) or a control group (controls, N 12 immature, 12 mature) before sacrifice 12 wk later. Rats ran on a treadmill five times per week for 60-70 min at speeds up to 26 m.min(-1). Both at baseline and after intervention, we measured total body, lumbar spine, and proximal femoral bone mineral, as well as total body soft tissue composition using dual-energy x-ray absorptiometry (DXA) in vivo. After sacrificing the animals, we measured dynamic and static histomorphometry and three-point bending strength of the tibia. Results: Running training was associated with greater differences in tibial subperiosteal area, cortical cross-sectional area, peak load, stiffness, and moment of inertia in immature and mature rats (P < 0.05). The trained rats had greater periosteal bone formation rates (P < 0.01) than controls, but there was no difference in tibial trabecular bone histomorphometry. Similar running-related gains were seen in DXA lumbar spine area (P = 0.04) and bone mineral content (BMC; P = 0.03) at both ages. For total body bone area and BMC, the immature trained group increased significantly compared with controls (P < 0.05), whereas the mature trained group gained less than did controls (P < 0.01). Conclusion: In this in vivo model, where a similar physical training program was performed by immature and mature female rats, we demonstrated that both age groups were sensitive to loading and that bone strength gains appeared to result more from changes in bone geometry than from improved material properties.

Neonatal ethanol exposure reduces AMPA but not NMDA receptor levels in the rat neocortex

Fetal alcohol syndrome (FAS) is the leading cause of mental retardation in western society. We investigated possible changes in glutamate receptor levels in neonatal animals following ethanol exposure using radioligand binding and western blot analysis. We used a vapor chamber to administer ethanol to neonatal Wistar rats 3 h a day from postnatal day (PND) 4-9. A separation control group was separated from their mothers for the same time and duration as the vapor treatment, while a normal control group was left to develop normally. Daily ethanol administrations resulted in decreased brain weight and body weight, as well as microencephaly (decreased brain:body weight ratio). Neither the affinity nor maximum binding of [H-3]MK-801 (dizoclipine maleate) in the cortex of PND10 rats differed between treatment groups. Western blot analysis also failed to reveal any changes in NMDAR1, NMDAR2A, or NMDAR2B receptor levels. In contrast, the AMPA receptor subunit GluR1 was greatly reduced in vapor-treated pups compared with control pups, as revealed by western blot analysis. A similar reduction was found in westerns with an antibody recognizing the GluR2 and 4 subunits. These results indicate that ethanol reduces AMPA rather than NMDA receptors in the developing neocortex, possibly by blocking NMDA receptors during development. (C) 2002 Elsevier Science B.V. All rights reserved.

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.

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.

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.

The pyramidal cell of the sensorimotor cortex of the macaque monkey: Phenotypic variation

Recent studies have revealed striking differences in pyramidal cell structure among cortical regions involved in the processing of different functional modalities. For example, cells involved in visual processing show systematic variation, increasing in morphological complexity with rostral progression from V1 through extrastriate areas. Differences have also been identified between pyramidal cells in somatosensory, motor and prefrontal cortex, but the extent to which the pyramidal cell phenotype may vary between these functionally related cortical regions remains unknown. In the present study we investigated the structure of layer III pyramidal cells in somatosensory and motor areas 3b, 4, 5, 6 and 7b of the macaque monkey. Cells were intracellularly injected in fixed, flat-mounted cortical slices and analysed for morphometric parameters. The size of the basal dendritic arbours, the number of their branches and their spine density were found to vary systematically between areas. Namely, we found a trend for increasing complexity in dendritic arbour structure through areas 3b, 5 and 7b. A similar trend occurred through areas 4 and 6. The differences in arbour structure may determine the number of inputs received by neurons and may thus be an important factor in determining function at the cellular and systems level.

Mast cells and oral inflammation

Mast cells are mobile granule-containing secretory cells that are distributed preferentially about the microvascular endothelium in oral mucosa and dental pulp. The enzyme profile of mast cells in oral tissues resembles that of skin, with most mast cells expressing the serine proteases tryptase and chymase. Mast cells in oral tissues contain the pro-inflammatory cytokine tumour necrosis factor-alpha in their granules, and release of this promotes leukocyte infiltration during evolving inflammation in several conditions, including lichen planus, gingivitis, pulpitis, and periapical inflammation, through induction of endothelial-leukocyte adhesion molecules. Mast cell synthesis and release of other mediators exerts potent immunoregulatory effects on other cell types, while several T-lymphocyte-derived cytokines influence mast cell migration and mediator release. Mast cell proteases may contribute to alterations in basement membranes in inflammation in the oral cavity, such as the disruptions that allow cytotoxic lymphocytes to enter the epithelium in oral lichen planus. A close relationship exists among mast cells, neural elements, and laminin, and this explains the preferential distribution of mast cells in tissues. Mast cells are responsive to neuropeptides and, through their interaction with neural elements, form a neural immune network with Langerhans cells in mucosal tissues. This facilitates mast cell degranulation in response to a range of immunological and non-immunological stimuli. Because mast cells play a pivotal role in inflammation, therapies that target mast cell functions could have value in the treatment of chronic inflammatory disorders in the oral cavity.