Structures of SAS-6 suggest its organization in centrioles

Centrioles are cylindrical, ninefold symmetrical structures with peripheral triplet microtubules strictly required to template cilia and flagella. The highly conserved protein SAS-6 constitutes the center of the cartwheel assembly that scaffolds centrioles early in their biogenesis. We determined the x-ray structure of the amino-terminal domain of SAS-6 from zebrafish, and we show that recombinant SAS-6 self-associates in vitro into assemblies that resemble cartwheel centers. Point mutations are consistent with the notion that centriole formation in vivo depends on the interactions that define the self-assemblies observed here. Thus, these interactions are probably essential to the structural organization of cartwheel centers.

Primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease with an incidence estimated between 1:2,000 and 1:40,000. Ciliated epithelia line the airways, nasal and sinus cavities, Eustachian tube and fallopian tubes. Congenital abnormalities of ciliary structure and function impair mucociliary clearance. As a consequence, patients present with chronic sinopulmonary infections, recurrent glue ear and female subfertility. Similarities in the ultrastructure of respiratory cilia, nodal cilia and sperm result in patients with PCD also presenting with male infertility, abnormalities of left-right asymmetry (most commonly situs inversus totalis) and congenital heart disease. Early diagnosis is essential to ensure specialist management of the respiratory and otological complications of PCD. Diagnostic tests focus on analysis of ciliary function and electron microscopy structure. Analysis is technically difficult and labour intensive. It requires expertise for interpretation, restricting diagnosis to specialist centres. Management is currently based on the consensus of experts, and there is a pressing need for randomised clinical trials to inform treatment.

Morphofunctional adaptations of the olfactory mucosa in postnatally developing rabbits

Rabbits are born blind and deaf and receive unusually limited maternal care. Consequently, their suckling young heavily rely on the olfactory cue for nipple attachment. However, the postnatal morphofunctional adaptations of olfactory mucosa (OM) are not fully elucidated. To clarify on the extent and the pattern of refinement of the OM following birth in the rabbit, morphologic and morphometric analysis of the mucosa were done at neonatal (0-1 days), suckling (2 weeks), weanling (4 weeks), and adult (6-8 months) stages of postnatal development. In all the age groups, the basic components of the OM were present. However, proliferative activity of cells of the mucosal epithelium decreased with increasing age as revealed by Ki-67 immunostaining. Diameters of axon bundles, packing densities of olfactory cells, and cilia numbers per olfactory cell knob increased progressively with age being 5.5, 2.1, and 2.6 times, respectively, in the adult as compared with the neonate. Volume fraction values for the bundles increased by 5.3% from birth to suckling age and by 7.4% from weaning to adulthood and the bundle cores were infiltrated with blood capillaries in all ages except in the adult where such vessels were lacking. The pattern of cilia projection from olfactory cell knobs also showed age-related variations, that is, arose as a tuft from the tips of the knobs in neonates and sucklings and in a radial pattern from the knob bases in weanlings and adults. These morphological changes may be attributed to the high olfactory functional demand associated with postnatal development in the rabbit.

Anticancer drug vinblastine sulphate induces transient morphological changes on the olfactory mucosa of the rabbit

Vinblastine sulphate (VBS) is an anticancer drug that acts by disrupting microtubule dynamics of highly mitotic tissue cells. The consequences of VBS on the olfactory mucosa (OM), a tissue with high mitotic numbers, are not clearly understood. We used qualitative and quantitative methods to determine the structural changes that may be produced on the rabbit OM by VBS. Following a single dose (0.31 mg/kg) of this drug, the structure of the mucosa was greatly altered on the first 3-5 days. The alteration was characterized by disarrangement of the normal layering of nuclei of the epithelia, degeneration of axonal bundles, occurrence of blood vessels within the bundles, localized death of cells of Bowman's glands and glandular degeneration. Surprisingly on or after day 7 and progressively to day 15 post-exposure, the OM was observed to regenerate and acquire normal morphology, and the vessels disappeared from the bundles. Relative to control values, bundle diameters, olfactory cell densities and cilia numbers decreased to as low as 53.1, 75.2 and 71.4%, respectively, on day 5. Volume density for the bundles, which was 28.6% in controls, decreased to a lowest value of 16.8% on day 5. In contrast, the volume density for the blood vessels was significantly lower in controls (19.9%) than in treated animals at day 2 (25.8%), day 3 (34.3%) and day 5 (31.5%). These findings suggest that the changes induced on the rabbit OM by VBS are transient and that regenerative recovery leads to the restoration of the normal structure of the mucosa.

Histology of periapical lesions obtained during apical surgery

The aim of this was to evaluate the histology of periapical lesions in teeth treated with periapical surgery. After root-end resection, the root tip was removed together with the periapical pathological tissue. Histologic sectioning was performed on calcified specimens embedded in methylmethacrylate (MMA) and on demineralized specimens embedded in LR White (Fluka, Buchs, Switzerland). The samples were evaluated with light and transmission electron microscopy (TEM). The histologic findings were classified into periapical abscesses, granulomas, or cystic lesions (true or pocket cysts). The final material comprised 70% granulomas, 23% cysts and 5% abscesses, 1% scar tissues, and 1% keratocysts. Six of 125 samples could not be used. The cystic lesions could not be subdivided into pocket or true cysts. All cysts had an epithelium-lined cavity, two of them with cilia-lined epithelium. These results show the high incidence of periapical granulomas among periapical lesions obtained during apical surgery. Periapical abscesses were a rare occasion. The histologic findings from samples obtained during apical surgery may differ from findings obtained by teeth extractions. A determination between pocket and true apical cysts is hardly possible when collecting samples by apical surgery.

Occurrence of spontaneous tumours of the renal proximal tubules in oscars Astronotus ocellatus

This report describes the occurrence of renal papillary cystic adenomas and adenocarcinomas in oscars Astronotus ocellatus Cuvier, 1829. Samples from 5 oscars with abdominal swelling were collected between 1996 and 2004 and compared to a published case from the USA. Macroscopically, all cases revealed a large, well-demarcated, greyish-brown nodular mass in a retroperitoneal position within the body cavity, and originating from the posterior kidney. Histologically, these neoplasms were composed of epithelial cells, which were arranged in papillary cystic tubular structures and partly covered by cilia. In this study, microscopic and ultrastructural examination confirmed that the origin of the neoplasm was the proximal tubules of the kidney.

Lectin binding patterns in two cultured endothelial cell types derived from bovine corpus luteum

Epithelial cells of different phenotypes derived from bovine corpus luteum have been studied intensively in our laboratory. In this study, specific lectin binding was examined for cells of type 1 and 3, which were defined as endothelial cells. In order to confirm differences in their glycocalyx at the light microscopic level, five biotinylated lectins were applied to postconfluent cultures which had been fixed with buffered paraformaldehyde or glutaraldehyde. Cells were not permeabilized with any detergent. Lectin binding was localized with a streptavidin-peroxidase complex which was visualized with two different techniques. The DAB technique detected peroxidase histochemically, while the immunogold technique used an anti-peroxidase gold complex together with silver amplification. Neither cell type 1 nor cell type 3 bound a particular lectin selectively, yet each cell type expressed a particular lectin binding pattern. With the DAB technique, diverse lectin binding patterns were seen, probably indicating either "outside" binding, i.e., a diffuse pattern, a lateral-cell-side pattern and a microvillus-like pattern, or "inside" binding, i.e., a diffuse pattern, and a granule-like pattern. With the immunogold technique, only "outside" binding was observed. In addition, the patterns of single cilia or of single circles were detected, the latter roughly representing 3-micron-sized binding sites for concanavalin A. When localizing them at the ultrastructural level, single circles corresponded with micron-sized discontinuities of the plasma membrane. Shedding vesicles were detected whose outer membrane was labelled with concanavalin A. Our results confirm the diversity of the two cell types under study. The "inside" lectin binding may be caused by way of transient plasma membrane openings and related to shedding of right-side out vesicles ("ectocytosis").

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments.

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.

Sensory regulation of network components underlying ciliary locomotion in Hermissenda.

Ciliary locomotion in the nudibranch mollusk Hermissenda is modulated by the visual and graviceptive systems. Components of the neural network mediating ciliary locomotion have been identified including aggregates of polysensory interneurons that receive monosynaptic input from identified photoreceptors and efferent neurons that activate cilia. Illumination produces an inhibition of type I(i) (off-cell) spike activity, excitation of type I(e) (on-cell) spike activity, decreased spike activity in type III(i) inhibitory interneurons, and increased spike activity of ciliary efferent neurons. Here we show that pairs of type I(i) interneurons and pairs of type I(e) interneurons are electrically coupled. Neither electrical coupling or synaptic connections were observed between I(e) and I(i) interneurons. Coupling is effective in synchronizing dark-adapted spontaneous firing between pairs of I(e) and pairs of I(i) interneurons. Out-of-phase burst activity, occasionally observed in dark-adapted and light-adapted pairs of I(e) and I(i) interneurons, suggests that they receive synaptic input from a common presynaptic source or sources. Rhythmic activity is typically not a characteristic of dark-adapted, light-adapted, or light-evoked firing of type I interneurons. However, burst activity in I(e) and I(i) interneurons may be elicited by electrical stimulation of pedal nerves or generated at the offset of light. Our results indicate that type I interneurons can support the generation of both rhythmic activity and changes in tonic firing depending on sensory input. This suggests that the neural network supporting ciliary locomotion may be multifunctional. However, consistent with the nonmuscular and nonrhythmic characteristics of visually modulated ciliary locomotion, type I interneurons exhibit changes in tonic activity evoked by illumination.

The equine DNAH3 gene: SNP discovery and exclusion of an involvement in recurrent airway obstruction (RAO) in European Warmblood horses

Recurrent airway obstruction is one of the most common airway diseases affecting mature horses. Increased bronchoalveolar mucus, neutrophil accumulation in airways, and airway obstruction are the main features of this disease. Mucociliary clearance is a key component of pulmonary defense mechanisms. Cilia are the motile part of this system and a complex linear array of dynein motors is responsible for their motility by moving along the microtubules in the axonemes of cilia and flagella. We previously detected a QTL for RAO on ECA 13 in a half-sib family of European Warmblood horses. The gene encoding DNAH3 is located in the peak of the detected QTL and encodes a dynein subunit. Therefore, we analysed this gene as a positional and functional candidate gene for RAO. In a mutation analysis of all 62 exons we detected 53 new polymorphisms including 7 non-synonymous variants. We performed an association study using 38 polymorphisms in a cohort of 422 animals. However, after correction for multiple testing we did not detect a significant association of any of these polymorphisms with RAO (P>0.05). Therefore, it seems unlikely that variants at the DNAH3 gene are responsible for the RAO QTL in European Warmblood horses.

Control of ventricular ciliary beating by the melanin concentrating hormone-expressing neurons of the lateral hypothalamus: a functional imaging survey

The cyclic peptide Melanin Concentrating Hormone (MCH) is known to control a large number of brain functions in mammals such as food intake and metabolism, stress response, anxiety, sleep/wake cycle, memory, and reward. Based on neuro-anatomical and electrophysiological studies these functions were attributed to neuronal circuits expressing MCHR1, the single MCH receptor in rodents. In complement to our recently published work (1) we provided here new data regarding the action of MCH on ependymocytes in the mouse brain. First, we establish that MCHR1 mRNA is expressed in the ependymal cells of the third ventricle epithelium. Second, we demonstrated a tonic control of MCH-expressing neurons on ependymal cilia beat frequency using in vitro optogenics. Finally, we performed in vivo measurements of CSF flow using fluorescent micro-beads in wild-type and MCHR1-knockout mice. Collectively, our results demonstrated that MCH-expressing neurons modulate ciliary beating of ependymal cells at the third ventricle and could contribute to maintain cerebro-spinal fluid homeostasis.

Effects of anticancer drug docetaxel on the structure and function of the rabbit olfactory mucosa

Docetaxel (DCT) is an anticancer drug which acts by disrupting microtubule dynamics in the highly mitotic cancer cells. Thus, this drug has a potential to affect function and organization of tissues exhibiting high cellular turnover. We investigated, in the rabbit, the effects of a single human equivalent dose (6.26mg/kg, i.v.) of DCT on the olfactory mucosa (OM) through light and electron microscopy, morphometry, Ki-67 immunostaining, TUNEL assay and the buried food test for olfactory sensitivity. On post-exposure days (PED) 5 and 10, there was disarrangement of the normal cell layering in the olfactory epithelium (OE), apoptotic death of cells of the OE, Bowman's glands and axon bundles, and the presence (including on PED 3) of blood vessels in the bundle cores. A decrease in bundle diameters, olfactory cell densities and cilia numbers, which was most significant on PED 10 (49.3%, 63.4% and 50%, respectively), was also evident. Surprisingly by PED 15, the OM regained normal morphology. Furthermore, olfactory sensitivity decreased progressively until PED 10 when olfaction was markedly impaired, and with recovery from the impairment by PED 15. These observations show that DCT transiently alters the structure and function of the OM suggesting a high regenerative potential for this tissue.

Towards an Earlier Diagnosis of Primary Ciliary Dyskinesia: Which Patients Should Undergo Detailed Diagnostic Testing?

Primary ciliary dyskinesia is a rare heterogeneous recessive genetic disorder of motile cilia, leading to chronic upper and lower respiratory symptoms. Prevalence is estimated at around 1:10,000, but many patients remain undiagnosed, while others receive the label incorrectly. Proper diagnosis is complicated by the fact that the key symptoms such as wet cough, chronic rhinitis and recurrent upper and lower respiratory infection, are common and nonspecific. There is no single gold standard test to diagnose PCD. Presently, the diagnosis is made by augmenting the medical history and physical examination with in patients with a compatible medical history following a demanding combination of tests including nasal nitric oxide, high- speed video microscopy, transmission electron microscopy, genetics, and ciliary culture. These tests are costly and need sophisticated equipment and experienced staff, restricting use to highly specialised centers. Therefore, it would be desirable to have a screening test for identifying those patients who should undergo detailed diagnostic testing. Three recent studies focused on potential screening tools: one paper assessed the validity of nasal nitric oxide for screening, and two studies developed new symptom-based screening tools. These simple tools are welcome, and hopefully remind physicians whom to refer for definitive testing. However, they have been developed in tertiary care settings, where 10 to 50% of tested patients have PCD. Sensitivity and specificity of the tools are reasonable, but positive and negative predictive values may be poor in primary or secondary care settings. While these studies take an important step forward towards an earlier diagnosis of PCD, more remains to be done before we have tools tailored to different health care settings.

Functional analysis of the sea urchin {\it orthodenticle\/} -related gene, {\it SPOTX\/}, in aboral ectoderm-specific gene activation and aboral ectoderm cell fate specification

An important question in developmental biology is how embryonic cell types are derived from a fertilized egg. To address this question, this thesis investigates the mechanisms by which the aboral ectoderm-specific Spec2a gene is spatially and temporally regulated during sea urchin embryogenesis. The Spec2a gene of the sea urchin Strongylocentratus purpuratus has served as a valuable maker to understand the basis of lineage-specific gene activation and the role of transcription factors in cell fate specification. The hypothesis is that transcription factors responsible for cell type-specific gene activation are key components in the initial cell specification step. The Spec2a gene, which encodes a small cytosolic calcium-binding protein, is expressed exclusively in aboral ectoderm cell lineages. The 1516-bp control region of the Spec2a gene contains a 188-bp enhancer element required for temporal activation and aboral ectoderm/mesenchyme cell expression, while an unidentified element upstream of the enhancer represses expression in mesenchyme cells. Using an enhancer activation assay, combined with site-directed mutagenesis, I showed that three TAATCC/T sites within the enhancer are responsible for enhancer activity. Mutagenizing these sites and a fourth one just upstream abolished all activity from the Spec2a control region. A 77-bp DNA fragment from the Spec2a enhancer containing two of the TAATCC/T sites is sufficient for aboral ectoderm/mesenchyme cell expression. A cDNA encoding SpOtx, an orthodenticle-related protein, was cloned from S. purpuratus and shown to bind with high affinity to the TAATCC/T sequences within the Spec2a control region. SpOtx transcripts were found initially in all cells of the cleaving embryo, but they gradually became restricted to oral ectoderm and endoderm cells, suggesting that SpOtx might play a role in the initial temporal activation of the Spec2a gene and most likely has additional functions in the developing embryo. To reveal the broader biological functions of SpOtx, I injected SpOtx mRNA into living sea urchin eggs to determine what effects overexpressing the SpOtx protein might have on embryo development. SpOtx mRNA-injected embryos displayed dramatic alterations in development. Instead of developing into pluteus larvae with 15 different cell types, uniform epithelia balls were formed. These balls consisted of a thin layer of squamous cells with short cilia highly reminiscent of aboral ectoderm. Immunohistochemical staining and RT-PCR demonstrated that the SpOtx-injected embryoids expressed aboral ectoderm markers uniformly, but showed very weak or no expression of markers for non-aboral ectoderm cell types. These data strongly suggested that overexpression of SpOtx redirected the normal fate of non-aboral ectoderm cells to that of aboral ectoderm. These results show that SpOtx is involved in aboral ectoderm differentiation by activating aboral ectoderm-specific genes and that modulating its expression can lead to changes in cell fate. ^

Energetic considerations of ciliary beating and the advantage of metachronal coordination

The internal mechanism of cilia is among the most ancient biological motors on an evolutionary scale. It produces beat patterns that consist of two phases: during the effective stroke, the cilium moves approximately as a straight rod, and during the recovery stroke, it rolls close to the surface in a tangential motion. It is commonly agreed that these two phases are designed for efficient functioning: the effective stroke encounters strong viscous resistance and generates thrust, whereas the recovery stroke returns the cilium to starting position while avoiding viscous resistance. Metachronal coordination between cilia, which occurs when many of them beat close to each other, is believed to be an autonomous result of the hydrodynamical interactions in the system. Qualitatively, metachronism is perceived as a way for reducing the energy expenditure required for beating. This paper presents a quantitative study of the energy expenditure of beating cilia, and of the energetic significance of metachronism. We develop a method for computing the work done by model cilia that beat in a viscous fluid. We demonstrate that for a single cilium, beating in water, the mechanical work done during the effective stroke is approximately five times the amount of work done during the recovery stroke. Investigation of multicilia configurations shows that having neighboring cilia beat metachronally is energetically advantageous and perhaps even crucial for multiciliary functioning. Finally, the model is used to approximate the number of dynein arm attachments that are likely to occur during the effective and recovery strokes of a beat cycle, predicting that almost all of the available dynein arms should participate in generating the motion.