Paramecium, a Model to Study Ciliary Beating and Ciliogenesis: Insights From Cutting-Edge Approaches.

Cilia are ubiquitous and highly conserved extensions that endow the cell with motility and sensory functions. They were present in the first eukaryotes and conserved throughout evolution (Carvalho-Santos et al., 2011). Paramecium has around 4,000 motile cilia on its surface arranged in longitudinal rows, beating in waves to ensure movement and feeding. As with cilia in other model organisms, direction and speed of Paramecium ciliary beating is under bioelectric control of ciliary ion channels. In multiciliated cells of metazoans as well as paramecia, the cilia become physically entrained to beat in metachronal waves. This ciliated organism, Paramecium, is an attractive model for multidisciplinary approaches to dissect the location, structure and function of ciliary ion channels and other proteins involved in ciliary beating. Swimming behavior also can be a read-out of the role of cilia in sensory signal transduction. A cilium emanates from a BB, structurally equivalent to the centriole anchored at the cell surface, and elongates an axoneme composed of microtubule doublets enclosed in a ciliary membrane contiguous with the plasma membrane. The connection between the BB and the axoneme constitutes the transition zone, which serves as a diffusion barrier between the intracellular space and the cilium, defining the ciliary compartment. Human pathologies affecting cilia structure or function, are called ciliopathies, which are caused by gene mutations. For that reason, the molecular mechanisms and structural aspects of cilia assembly and function are actively studied using a variety of model systems, ranging from unicellular organisms to metazoa. In this review, we will highlight the use of Paramecium as a model to decipher ciliary beating mechanisms as well as high resolution insights into BB structure and anchoring. We will show that study of cilia in Paramecium promotes our understanding of cilia formation and function. In addition, we demonstrate that Paramecium could be a useful tool to validate candidate genes for ciliopathies.

Towards characterization of photo-excited electron transfer and catalysis in natural and artificial systems using XFELs.

The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ray emission spectra, using an energy dispersive spectrometer, at ambient conditions, and used this approach to study the room temperature structure and intermediate states of the photosynthetic water oxidizing metallo-protein, photosystem II. Moreover, we have also used this setup to simultaneously collect the X-ray emission spectra from multiple metals to follow the ultrafast dynamics of light-induced charge transfer between multiple metal sites. A Mn-Ti containing system was studied at an XFEL to demonstrate the efficacy and potential of this method.

Effect of visual biofeedback of posterior tongue movement on articulation rehabilitation in dysarthria patients.

Articulation is driven by various combinations of movements of the lip, tongue, soft palate, pharynx and larynx, where the tongue plays an especially important role. In patients with cerebrovascular disorder, lingual motor function is often affected, causing dysarthria. We aimed to evaluate the effect of visual biofeedback of posterior tongue movement on articulation rehabilitation in dysarthria patients with cerebrovascular disorder. Fifteen dysarthria patients (10 men and 5 women; mean age, 70.7 ± 10.3 years) agreed to participate in this study. A device for measuring the movement of the posterior part of the tongue was used for the visual biofeedback. Subjects were instructed to produce repetitive articulation of [ka] as fast and steadily as possible between a lungful with/without visual biofeedback. For both the unaffected and affected sides, the range of ascending and descending movement of the posterior tongue with visual biofeedback was significantly larger than that without visual biofeedback. The coefficient of variation for these movements with visual biofeedback was significantly smaller than that without visual biofeedback. With visual biofeedback, the range of ascent exhibited a significant and strong correlation with that of descent for both the unaffected and affected sides. The results of this study revealed that the use of visual biofeedback leads to prompt and preferable change in the movement of the posterior part of the tongue. From the standpoint of pursuing necessary rehabilitation for patients with attention and memory disorders, visualization of tongue movement would be of marked clinical benefit.

Differences in biomechanical features of tongue pressure production between articulation and swallow.

Summary The purpose of this study was to compare the tongue pressure against the hard palate during the articulation of a monosyllable with that during swallowing. The participants were 20 healthy adults without swallowing or articulation disorder (10 men and 10 women, mean age ± standard deviation: 22·5 ± 0·9 years). Tongue pressure during articulation of [ki] (articulatory pressure) and during dry swallowing (swallowing pressure) was recorded by a 0·1-mm-thick sensor sheet with five measuring points attached to the hard palate. Biomechanical parameters such as maximal magnitude, duration, integrated value and slope gradient were compared between articulatory pressure and swallowing pressure at each measuring point. Although swallowing pressure was produced at each measuring point, articulatory pressure was found only in the posterior circumferential parts of the hard palate and was smaller in magnitude (14·9-16·7% of swallowing pressure) and integrated value, which meant the amount of work by tongue pressing (7·0-7·9%), shorter in duration (26·6-31·8%) and shallower in slope gradient, which meant the speed of tongue pressing (26·9-27·4%). Maximal magnitude was closely correlated with duration (R(2) = 0·386) and slope gradient (R(2) = 0·843) for articulatory pressure. These results first show the biomechanical differences between articulation and swallowing in terms of tongue contact with the hard palate. The findings suggest that tongue pressure measurement might be a useful investigation for patients with tongue motor disorder.

Annexin-A1 identified as the oral epithelial cell anti-Candida effector moiety.

Innate and adaptive immunity are considered critical to protection against mucosal candidal infections. Among innate anti-Candida mechanisms, oral and vaginal epithelial cells have antifungal activity. The mechanism is fungistatic, acid-labile and includes a requirement for cell contact by intact, but not necessarily live, epithelial cells. The purpose of this study was to use the acid-labile property to further characterize the effector moiety. Surface material extracted from phosphate-buffered saline (PBS) -treated, but not acid-treated, epithelial cells significantly inhibited the growth of Candida blastoconidia in a dose-dependent manner which was abrogated by prior heat and protease treatment. Proteins extracted from PBS-treated cells bound blastoconidia and hyphae more intensely than those from acid-treated cells. Proteins from PBS-treated cells eluted from Candida revealed two unique bands of approximately 33 and 45 kDa compared with acid-treated cells. Mass spectrometry identified these proteins as Annexin-A1 and actin, respectively. Oral epithelial cells stained positive for Annexin-A1, but not actin. Western blots showed reduced Annexin-A1 in proteins from acid-treated epithelial cells compared with those from PBS-treated epithelial cells. Lastly, it was demonstrated that immunoprecipitation of Annexin-A1 from proteins extracted from PBS-treated oral epithelial cells resulted in abrogation of inhibitory activity. Taken together, these results indicate that Annexin-A1 is a strong candidate for the epithelial cell anti-Candida effector protein.

Factors associated with acute otitis media in children due to penicillin intermediately resistant Streptococcus pneumoniae.

OBJECTIVES: This study was conducted to analyze the association between recent antimicrobial use and acute otitis media (AOM) due to Streptococcus pneumoniae intermediately resistant to penicillin (PISP). The influence of drug resistance on the clinical course of AOM was also assessed. METHODS: A retrospective cohort study was conducted in infants at Japanese Red Cross Medical Center, Tokyo. Children included in the study were under 24 months of age and diagnosed with AOM due to infection with S. pneumoniae between April 2002 and March 2007. Crude risk ratios (cRR) of PISP infection in cases with recent antibiotic use and other factors were obtained. The Mantel-Haenszel estimate was applied for the adjustment of cRR. RESULTS: Of 35 children, 13 had AOM due to penicillin-susceptible S. pneumoniae (PSSP) and 22 had AOM due to PISP. The adjusted risk ratio (aRR) of penicillin antibiotic use within 1 month was 1.99 (95% confidence interval (CI): 1.36-2.92), and the aRR of penicillin antibiotic use within 1 week was 1.93 (95% CI: 1.33-2.67). Recent use of penicillin antibiotics was an associated factor for AOM due to PISP. The clinical course was not clearly different between cases infected with PSSP and those with PISP. CONCLUSION: Recent use of penicillin antibiotics might be a selective pressure for PISP.

Acute otitis media due to Haemophilus influenzae with antibiotic resistance: experience of a tertiary hospital in Tokyo city.

OBJECTIVE: The objective of the study was to clarify the associated factors of pediatric acute otitis media (AOM) due to Haemophilus influenzae with antibiotic resistance. METHODS: A retrospective chart review was conducted for all AOM cases due to H.influenzae in children younger than 2 years of age that were presented at the Department of Otolaryngology, Japanese Red Cross Medical Hospital (JRCMC), Tokyo, from April 2005 to March 2007. RESULTS: Seventy-six children were enrolled in this study. Multivariate analysis showed that residing at the JRCMC infant home (OR, 3.7; [95% CI, 1.1-12.8]; P=0.039) and a history of consuming macrolide in the past month (OR, 4.7; [95% CI, 1.1-21.5]; P=0.041) were the associated factors for AOM due to beta-lactamase positive, ampicillin-resistant H. influenzae. CONCLUSIONS: A history of consuming macrolide in the past month should be noted on antibiotic prescriptions for children with AOM who are younger than 2 years of age.

Oral and vaginal epithelial cell anti-Candida activity is acid labile and does not require live epithelial cells.

BACKGROUND: Candida albicans is the causative agent of oral and vaginal candidiasis. Innate host defenses against C. albicans are important against each infection. Among these are oral and vaginal epithelial cells that have anti-Candida activity. The mechanism of action includes a requirement for cell contact with no role for soluble factors, and a putative role for carbohydrates based on the sensitivity of the activity to periodic acid. METHODS: Periodic acid treatment of epithelial cells as well as the property of partial resistance of antifungal activity to fixation was used to further dissect the mechanism of action. RESULTS: The results herein effectively now challenge a role for carbohydrates alone. Firstly, the putative carbohydrate(s) released into supernatants of periodic acid-treated epithelial cells could not compete with fresh epithelial cells for activity, and equivalent abrogation of activity was observed by periodic acid-treated cells irrespective of the amount of carbohydrate released. Instead, the similar abrogation of activity following treatment with other acids or when cocultured under acidic conditions suggests that the activity is acid-labile. Finally, while activity requires intact epithelial cells, it does not require live cells; activity was minimally affected by fixing epithelial cells prior to coculture where the majority of cells remained impermeable to Trypan blue but were defined as non-viable by positive nuclear staining with propidium iodide. CONCLUSION: These results suggest that antifungal activity is dependent on contact by intact, but not necessarily live, epithelial cells through an acid-labile mechanism.

Efficacy of ozone on survival and permeability of oral microorganisms.

In the present study, we examined the effect of ozonated water on oral microorganisms and dental plaque. Almost no microorganisms were detected after being treated with ozonated water (4 mg/l) for 10 s. To estimate the ozonated water-treated Streptococcus mutans, bacterial cells were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Fluorescence microscopic analysis revealed that S. mutans cells were killed instantaneously in ozonated water. Some breakage of ozonated water-treated S. mutans was found by electron microscopy. When the experimental dental plaque was exposed to ozonated water, the number of viable S. mutans remarkably decreased. Ozonated water strongly inhibited the accumulation of experimental dental plaque in vitro. After the dental plaque samples from human subjects were exposed to ozonated water in vitro, almost no viable bacterial cells were detected. These results suggest that ozonated water should be useful in reducing the infections caused by oral microorganisms in dental plaque.

Toll-like receptor-4 is expressed by macrophages in murine and human lipid-rich atherosclerotic plaques and upregulated by oxidized LDL.

BACKGROUND: Inflammation is implicated in atherogenesis and plaque disruption. Toll-like receptor 2 (TLR-2) and TLR-4, a human homologue of drosophila Toll, play an important role in the innate and inflammatory signaling responses to microbial agents. To investigate a potential role of these receptors in atherosclerosis, we assessed the expression of TLR-2 and TLR-4 in murine and human atherosclerotic plaques. METHODS AND RESULTS: Aortic root lesions of high-fat diet-fed apoE-deficient mice (n=5) and human coronary atherosclerotic plaques (n=9) obtained at autopsy were examined for TLR-4 and TLR-2 expression by immunohistochemistry. Aortic atherosclerotic lesions in all apoE-deficient mice expressed TLR-4, whereas aortic tissue obtained from control C57BL/6J mice showed no TLR-4 expression. All 5 lipid-rich human plaques expressed TRL-4, whereas the 4 fibrous plaques and 4 normal human arteries showed no or minimal expression. Serial sections and double immunostaining showed TLR-4 colocalizing with macrophages both in murine atherosclerotic lesions and at the shoulder region of human coronary artery plaques. In contrast to TLR-4, none of the plaques expressed TLR-2. Furthermore, basal TLR-4 mRNA expression by human monocyte-derived macrophages was upregulated by ox-LDL in vitro. CONCLUSIONS: Our study demonstrates that TLR-4 is preferentially expressed by macrophages in murine and human lipid-rich atherosclerotic lesions, where it may play a role to enhance and sustain the innate immune and inflammatory responses. Moreover, upregulation of TLR-4 in macrophages by oxidized LDL suggests that TLR-4 may provide a potential pathophysiological link between lipids and infection/inflammation and atherosclerosis.

Molecular monitoring of bleomycin-induced pulmonary fibrosis by cDNA microarray-based gene expression profiling.

Pulmonary fibrosis is a progressive disorder whose molecular pathology is poorly understood. Here we developed an in-house cDNA microarray ("lung chip") originating from a lung-normalized cDNA library. By using this lung chip, we analyzed global gene expression in a murine model of bleomycin-induced fibrosis and selected 82 genes that differed by more than twofold intensity in at least one pairwise comparison with controls. Cluster analysis of these selected genes showed that the expression of genes associated with inflammation reached maximum levels at 5 days after bleomycin administration, while genes involved in the development of fibrosis increased gradually up to 14 days after bleomycin treatment. These changes in gene expression signature were well correlated with observed histopathological changes. The results show that microarray analysis of animal disease models is a powerful approach to understanding the gene expression programs that underlie these disorders.

High-dose recombinant apolipoprotein A-I(milano) mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage content in apolipoprotein e-deficient mice. Potential implications for acute plaque stabilization.

BACKGROUND: Repeated doses of recombinant apolipoprotein A-I(Milano) phospholipid complex (apoA-I(m)) reduce atherosclerosis and favorably change plaque composition in rabbits and mice. In this study, we tested whether a single high dose of recombinant apoA-I(m) could rapidly mobilize tissue cholesterol and reduce plaque lipid and macrophage content in apoE-deficient mice. METHODS AND RESULTS: High cholesterol-fed, 26-week-old apoE-deficient mice received a single intravenous injection of saline (n=16), 1080 mg/kg dipalmitoylphosphatidylcholine (DPPC; n=14), or 400 mg/kg of recombinant apoA-I(m) complexed with DPPC (1:2.7 weight ratio; n=18). Blood was sampled before and 1 and 48 hours after injection, and aortic root plaques were evaluated for lipid content and macrophage content after oil-red O and immunostaining, respectively. One hour after injection, the plasma cholesterol efflux-promoting capacity was nearly 2-fold higher in recombinant apoA-I(m)-treated mice compared with saline and DPPC-treated mice (P<0.01). Compared with baseline values, serum free cholesterol, an index of tissue cholesterol mobilization, increased 1.6-fold by 1 hour after recombinant apoA-I(m) injection, and it remained significantly elevated at 48 hours (P<0.01). Mice receiving recombinant apoA-I(m) had 40% to 50% lower lipid content (P<0.01) and 29% to 36% lower macrophage content (P<0.05) in their plaques compared with the saline- and DPPC-treated mice, respectively. CONCLUSIONS: A single high dose of recombinant apoA-I(m) rapidly mobilizes tissue cholesterol and reduces plaque lipid and macrophage content in apoE-deficient mice. These findings suggest that this strategy could rapidly change plaque composition toward a more stable phenotype.

[Clinical laboratory data and reference intervals standardized in Fukuoka].

In Fukuoka whose population is approximately five million inhabitants, surveys on the accuracy of laboratory data have been performed by the Fukuoka Prefecture Medical Association for the last 30 years. We have been attempting to evaluate the data for routine use since 1988, and it has become possible to share laboratory data between all institutions in Fukuoka prefectures. As a result, reference intervals for 23 clinical chemistry analytes were established in 1995, to which were added in 1996 five serum protein constituents that have been utilized for clinical examinations. Methods for documentations and monitorings the data obtained in the prefecture were also established, standardization of the above analytes extended to 97% of the institutions in the prefecture. Results for 14 of the 23 clinical chemistry analytes have become highly reliable and clinically useful as differences between institutions in terms of results have narrowed. Standardization of other analytes is now in progress.

Standardization of laboratory data and establishment of reference intervals in the Fukuoka Prefecture: a Japanese perspective.

Standardization of 22 clinical chemistry analytes and five serum protein constituents has been performed in the Fukuoka Prefecture, which has a population of approximately five million. The standardization project was established to determine reference intervals for these analytes by educating physicians, medical technologists and staff of medical institutions, and by daily or monthly monitoring the use of common control samples through e-mail. Standardization extended to 97% of the institutions in the prefecture. Results for 14 of the 22 clinical chemistry analytes have become highly reliable and differences between institutions decreased. Standardization of other analytes is now in progress. Regional collaboration based on international guidelines led to a significant improvement in interlaboratory comparability. Areas where further improvements are needed have been identified.

Atomic resolution structure analysis of beta' polymorph crystal of a triacylglycerol: 1,2-dipalmitoyl-3-myristoyl-sn-glycerol.

The crystal structure of the beta'-2 form of a mixed chain triacylglycerol (TAG), 1,2-dipalmitoyl-3-myristoyl-sn-glycerol (PPM), was determined to a final reliability factor of 0.074. This work is the first to resolve the atomic-level structure of the beta' polymorph, which is of the highest functionality among multiple polymorphs in asymmetric TAG. In particular, fat crystals present in food emulsions are in beta', whose transformation into beta causes deterioration in their physical properties. beta'-2, one of the two beta' forms of PPM, forms a monoclinic unit cell with a space group of C2; Z = 8, a = 16.534(5) A, b = 7.537(2) A, c = 81.626(9) A; beta = 90.28(2) degrees, V = 10171(3) A(3), density = 1.018 g/cm(3), and mu = 4.96 cm(-1). The following characteristics were obtained: 1) two asymmetric units, named A and B, form a hybrid-type orthorhombic perpendicular subcell; 2) the two asymmetric units reveal different glycerol conformations: trans for sn-1 palmitic acid and sn-2 palmitic acid, but gauche for sn-3 myristic acid in A; and trans for sn-2 palmitic acid and sn-3 myristic acid, but gauche for sn-1 palmitic acid in B; 3) a unit lamellae reveals a four-chain-length structure consisting of two double-layer leaflets; 4) the two double-layer leaflets are combined end-by-end in a unit lamellae; and 5) the chain axes are alternatively inclined against the lamellar interface. -- Sato, K., M. Goto, J. Yano, K. Honda, D. R. Kodali, and D. M. Small. Atomic resolution structure analysis of beta' polymorph crystal of a triacylglycerol: 1,2-dipalmitoyl-3-myristoyl-sn-glycerol. J. Lipid Res. 2001. 42: 338--345.

Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain.

The giant myofibrillar protein titin contains within its C-terminal region a serine-threonine kinase of unknown function. We have identified a novel muscle specific RING finger protein, referred to as MURF-1, that binds in vitro to the titin repeats A168/A169 adjacent to the titin kinase domain. In myofibrils, MURF-1 is present within the periphery of the M-line lattice in close proximity to titin's catalytic kinase domain, within the Z-line lattice, and also in soluble form within the cytoplasm. Yeast two-hybrid screens with MURF-1 as a bait identified two other highly homologous MURF proteins, MURF-2 and MURF-3. MURF-1,2,3 proteins are encoded by distinct genes, share highly conserved N-terminal RING domains and in vitro form dimers/heterodimers by shared coiled-coil motifs. Of the MURF family, only MURF-1 interacts with titin repeats A168/A169, whereas MURF-3 has been reported to affect microtubule stability. Association of MURF-1 with M-line titin may potentially modulate titin's kinase activity similar to other known kinase-associated proteins, whereas differential expression and heterodimerization of MURF1, 2 and 3 may link together titin kinase and microtubule-dependent signal pathways in striated muscles.

Pravastatin treatment increases collagen content and decreases lipid content, inflammation, metalloproteinases, and cell death in human carotid plaques: implications for plaque stabilization.

BACKGROUND: The clinical benefits of lipid lowering with statins are attributed to changes in plaque composition leading to lesion stability, but supporting clinical data from human studies are lacking. Therefore, we investigated the effect of 3 months of pravastatin treatment on composition of human carotid plaques removed during carotid endarterectomy. METHODS AND RESULTS: Consecutive patients with symptomatic carotid artery stenosis received 40 mg/d pravastatin (n=11) or no lipid-lowering therapy (n=13; control subjects) for 3 months before scheduled carotid endarterectomy. Carotid plaque composition was assessed with special stains and immunocytochemistry with quantitative image analysis. Plaques from the pravastatin group had less lipid by oil red O staining (8.2+/-8.4% versus 23.9+/-21.1% of the plaque area, P<0.05), less oxidized LDL immunoreactivity (13.3+/-3.6% versus 22.0+/-6.5%, P<0.001), fewer macrophages (15.0+/-10.2% versus 25.3+/-12.5%, P<0.05), fewer T cells (11.2+/-9.3% versus 24.3+/-13.4%, P<0.05), less matrix metalloproteinase 2 (MMP-2) immunoreactivity (3.6+/-3.9% versus 8.4+/-5.3%, P<0.05), greater tissue inhibitor of metalloproteinase 1 (TIMP-1) immunoreactivity (9.0+/-6.2% versus 3.1+/-3.9%, P<0.05), and a higher collagen content by Sirius red staining (12.4+/-3.1% versus 7.5+/-3.5%, P<0.005). Cell death by TUNEL staining was reduced in the pravastatin group (17.7+/-7.8% versus 32.0+/-12.6%, P<0.05). CONCLUSIONS: -Pravastatin decreased lipids, lipid oxidation, inflammation, MMP-2, and cell death and increased TIMP-1 and collagen content in human carotid plaques, confirming its plaque-stabilizing effect in humans.

Genomic analysis of a mouse model of immunoglobulin A nephropathy reveals an enhanced PDGF-EDG5 cascade.

The molecular mechanism of immunoglobulin A nephropathy (IgAN), the most common primary renal glomerular disease worldwide, is unknown. HIGA (high serum IgA) mouse is a valid model of IgAN showing almost all of the pathological features, including mesangial cell proliferation. Here we elucidate a pattern of gene expression associated with IgAN by analyzing the diseased kidneys on cDNA microarrays. In particular, we showed an enhanced expression of several genes regulating the cell cycle and proliferation, including growth factors and their receptors, as well as endothelial differentiation gene-5 (EDG5), a receptor for sphingosine 1-phosphate (SPP). One of the growth factors, platelet-derived growth factor (PDGF) induces a marked upregulation of EDG5 in proliferative mesangial cells, and promotes cell proliferation synergistically with SPP. The genomic approach allows us to identify families of genes involved in a process, and can indicate that enhanced PDGF-EDG5 signaling plays an important role in the progression of IgAN.