Photoreceptor spectral tuning by colorful, multilayered facet lenses in long-legged fly eyes (Dolichopodidae).

The facet lenses of the compound eyes of long-legged flies (Dolichopodidae) feature a striking, interlaced coloration pattern, existing of alternating rows of green-yellow and orange-red reflecting facets, due to dielectric multilayers located distally in the facet lenses (Bernard and Miller. Invest Ophthalmol 7:416-434 (1968). We investigated this phenomenon in the dolichopodid Dolichopus nitidus by applying microspectrophotometry, electron microscopy and optical modeling. The measured narrow-band reflectance spectra, peaking at ~540 and ~590 nm with bandwidth ~105 nm, are well explained by a refractive index oscillating sinusoidally in six periods around a mean value of about 1.44 with amplitude 0.6. The facet lens reflectance spectra are associated with a spectrally restricted, reduced transmittance, which causes modified spectral sensitivities of the underlying photoreceptors. Based on the modeling and electroretinography of the dolichopodid Condylostylus japonicus we conjecture that the green and orange facets narrow the spectral bandwidths of blue and green central photoreceptors, respectively, thus possibly improving color and/or polarization vision.

Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction.

OBJECTIVE: In this study, we investigated the role of phenytoin (PHT) in death receptor-induced apoptosis of gingival fibroblasts to clarify the mechanism of PHT-induced gingival overgrowth. METHODS: Human gingival fibroblasts were cultured to semiconfluence and treated with PHT (0.025, 0.1, 0.25, and 1.0 µM) for 48 h, and then, the apoptotic cell numbers were relatively determined by absorptiometry. After 24 h of 0.25 µM PHT treatment, caspase activity was measured by absorptiometry, apoptotic and cell cycle phase distribution was analyzed by flow cytometry, expression levels of apoptotic genes were quantified by real-time qPCR, and expression of apoptotic proteins was detected by Western blot analysis. After 48 h of 0.25 µM PHT treatment, appearance of apoptotic cells was detected by TUNEL assay. RESULTS: PHT treatment decreased the proportion of apoptotic cells in gingival fibroblasts compared to a serum-free control culture in response to the protein changes as follows: PHT upregulated c-FLIP and, in turn, downregulated FADD, caspase-8, and caspase-3; PHT upregulated c-IAP2 and downregulated TRAF2; PHT downregulated caspase-9 and caspase-3 via decreased RIPK1 activity and increased Bcl-2 activity. CONCLUSION: PHT-induced gingival overgrowth may result from the above-mentioned mechanisms involving apoptosis inhibition in gingival fibroblasts.

Biochemical characterisation of urease from urease-positive thermophilic campylobacter (UPTC).

This study aims to characterise biochemically urease from an atypical Campylobacter lari, namely urease-positive thermophilic Campylobacter (UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, G and H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF8912 showed enzyme activity over a broad pH range (pH 6-10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20-60 degrees C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.

Oral toxicity to high level sodium fluoride causes impairment of autophagy.

Autophagy is a highly conserved intracellular digestion process that degrades damaged proteins and organelles but the biological roles of autophagy in pathological aspects of oral tissues remain largely unknown. We sought to elucidate the function of autophagy, especially its interplay with apoptosis and oxidative stress, in the oral toxicity induced by exposure to 5 mM sodium fluoride (NaF). Human cementoblasts (HCEM-2) in culture were exposed to 5 mM NaF for 5 min, after which cell viability and cell apoptosis were assessed using the MTS assay and an Annexin V-FITC/PI apoptosis detection kit, respectively. Quantitative RT-PCR and Western blotting were performed to characterize the expression levels of markers for autophagy, apoptosis, and oxidative stress. Senescence-resistant (SAMR1) mice were exposed to 5 mM NaF in their drinking water from 12 to 58 weeks. Micro-computed tomography was used to measure changes in their alveolar bone while immunohistochemistry and immunofluorescence staining was used to evaluate protein expression levels. HCEM-2 cells exposed to 5 mM NaF had decreased levels of autophagy, as shown by reduced expression levels of ATG5, Beclin-1 and LC3-II, elicited apoptosis, which in turn induced oxidative stress and inflammation, as manifested by elevated levels of Bax, cleaved caspase-3, SOD1 and phospho NF-κB. Treatment of mice with 5 mM NaF resulted in histological abnormalities in periodontal tissues, induced excessive oxidative stress and apoptosis, and reduced autophagy. Micro-computed tomography analysis demonstrated that 5 mM NaF caused a decrease in bone areas of mice compared with controls. Exposure to 5 mM NaF induced RANKL (receptor activator of nuclear factor κB ligand) and cathepsin K expression in periodontal tissues, while ATG5 and Beclin-1 expression was abrogated by 5 mM NaF. Taken together, our findings suggest that 5 mM NaF elicits oral toxicity that contributes to excessive apoptosis, oxidative stress, and defective autophagy, which aggravates periodontal tissue damage.

Identification of actin filaments in the rhabdomeral microvilli of Drosophila photoreceptors.

The phototransductive microvilli of arthropod photoreceptors each contain an axial cytoskeleton. The present study shows that actin filaments are a component of this cytoskeleton in Drosophila. Firstly, actin was detected in the rhabdomeral microvilli and in the subrhabdomeral cytoplasm by immunogold labeling with antiactin. Secondly, the rhabdomeres were labeled with phalloidin, indicating the presence of filamentous actin. Finally, the actin filaments were decorated with myosin subfragment-1. The characteristic arrowhead complex formed by subfragment-1 decoration points towards the base of the microvilli, so that the fast growing end of each filament is at the distal end of the microvillus, where it is embedded in a detergent-resistant cap. Each microvillus contains more than one actin filament. Decorated filaments extend the entire length of each microvillus and project into the subrhabdomeral cytoplasm. This organization is comparable to that of the actin filaments in intestinal brush border microvilli. Similar observations were made with the photoreceptor microvilli of the crayfish, Procambarus. Our results provide an indication as to how any myosin that is associated with the rhabdomeres might function.

Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration.

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.

Multiplex real-time PCR for exhaustive detection of diarrhoeagenic Escherichia coli.

AIMS: The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC. METHODS AND RESULTS: Primers and TaqMan probes were designed to amplify and quantify one gene (eae, stx1, stx2, elt, est, virB, aggR, astA, and afaB) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0.99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7.1 x 10(2) to 1.1 x 10(4) CFU ml(-1), depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g(-1)) were found to be positive by the method. CONCLUSIONS: The present system allows for the efficient and simultaneous determination of various DEC pathotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.

Light on the moth-eye corneal nipple array of butterflies.

The outer surface of the facet lenses in the compound eyes of moths consists of an array of excessive cuticular protuberances, termed corneal nipples. We have investigated the moth-eye corneal nipple array of the facet lenses of 19 diurnal butterfly species by scanning electron microscopy, transmission electron microscopy and atomic force microscope, as well as by optical modelling. The nipples appeared to be arranged in domains with almost crystalline, hexagonal packing. The nipple distances were found to vary only slightly, ranging from about 180 to 240 nm, but the nipple heights varied between 0 (papilionids) and 230 nm (a nymphalid), in good agreement with previous work. The nipples create an interface with a gradient refractive index between that of air and the facet lens material, because their distance is distinctly smaller than the wavelength of light. The gradient in the refractive index was deduced from effective medium theory. By dividing the height of the nipple layer into 100 thin slices, an optical multilayer model could be applied to calculate the reflectance of the facet lenses as a function of height, polarization and angle of incidence. The reflectance progressively diminished with increased nipple height. Nipples with a paraboloid shape and height 250 nm, touching each other at the base, virtually completely reduced the reflectance for normally incident light. The calculated dependence of the reflectance on polarization and angle of incidence agreed well with experimental data, underscoring the validity of the modelling. The corneal nipples presumably mainly function to reduce the eye glare of moths that are inactive during the day, so to make them less visible for predators. Moths are probably ancestral to the diurnal butterflies, suggesting that the reduced size of the nipples of most butterfly species indicates a vanishing trait. This effect is extreme in papilionids, which have virtually absent nipples, in line with their highly developed status. A similar evolutionary development can be noticed for the tapetum of the ommatidia of lepidopteran eyes. It is most elaborate in moth-eyes, but strongly reduced in most diurnal butterflies and absent in papilionids.

Possible pharmacotherapy for nifedipine-induced gingival overgrowth: 18α-glycyrrhetinic acid inhibits human gingival fibroblast growth.

BACKGROUND AND PURPOSE: This investigation aimed to establish the basis of a pharmacotherapy for nifedipine-induced gingival overgrowth. Gingival overgrowth has been attributed to the enhanced growth of gingival fibroblasts. In this study, we investigated the effects of 18-α-glycyrrhetinic acid (18α-GA) on growth, the cell cycle, and apoptosis and on the regulators of these processes in gingival fibroblasts isolated from patients who presented with nifedipine-induced gingival overgrowth. EXPERIMENTAL APPROACH: Gingival fibroblasts were cultured in medium containing 1% FBS with/without 10 µM 18α-GA for 24 or 48 h, and the cell number, cell cycle phase distribution, relative DNA content, apoptotic cell number and morphological characteristics of the cells undergoing apoptosis were measured together with the levels of proteins that regulate these processes and the level of caspase activity. KEY RESULTS: 18α-GA significantly decreased cell numbers and significantly increased the percentage of cells in the sub-G1 and G0 /G1 phases of the cell cycle and the number of apoptotic cells. Nuclear condensation and fragmentation of cells into small apoptotic bodies appeared in the fibroblasts treated with 18α-GA. In addition, 18α-GA significantly decreased the protein levels of cyclins A and D1, CDKs 2 and 6, phosphorylated Rb (ser(780) and ser(807/811)), Bcl-xL and Bcl-2 and increased the protein levels of p27, cytosolic cytochrome c, pro-caspase-3, and cleaved caspase-3 and the activities of caspases 3 and 9. CONCLUSIONS AND IMPLICATIONS: 18α-GA inhibited gingival fibroblast growth by suppressing the G1 /S phase transition and inducing apoptosis. In conclusion, 18α-GA may be used as a pharmacotherapy for nifedipine-induced gingival overgrowth.

Blocking type antithyrotropin receptor antibody in patients with nongoitrous hypothyroidism: its incidence and characteristics of action.

The incidence, characteristics of action, and pathogenetic importance of blocking type anti-TSH receptor antibody were examined in patients with autoimmune thyroiditis. Serum immunoglobulin G (IgG) from 8 of 20 patients with nongoitrous hypothyroidism contained substantial amounts of TSH binding inhibitor immunoglobulin (TBII) activity. Newborn infants of a patient with the greatest TBII activity had neonatal transient hypothyroidism. In sera of patients with goitrous hypothyroidism and euthryoid chronic thyroiditis, only weakly positive or negative TBII activity was found. IgGs of these patients and those of nongoitrous hypothyroid patients without strongly positive TBII activity did not inhibit TSH stimulation of thyroid adenylate cyclase activity. Seven of 8 IgGs which had strongly positive TBII activity significantly inhibited cAMP generation induced by 9.1 mU/ml TSH, and the eighth IgG inhibited stimulation with 0.5 mU/ml TSH. Although the modes of TSH binding inhibition were variable, markedly close correlation was found between TSH binding- and TSH stimulation-inhibiting activities of these 8 IgGs (r = 0.90; P less than 0.01). These IgGs may exert their inhibitory effects on adenylate cyclase activity by inhibiting TSH binding to its receptor.

Sequential serum measurements of thyrotropin binding inhibitor immunoglobulin G in transient familial neonatal hypothyroidism.

Infants with transient neonatal hypothyroidism, in whom TSH binding inhibitor immunoglobulin G (IgG) (TBII) were sequentially measured, are described. Their mother had been taking thyroid replacement for hypothyroidism due to nongoitrous autoimmune thyroiditis. IgGs inhibiting TSH binding were detected in maternal sera by radioreceptor assay. These IgGs also inhibited the adenylate cyclase response to TSH in human thyroid membranes. Three infants had frank hypothyroidism immediately after birth, and TBII were detected in two of them. In the two surviving infants, hypothyroidism was transient and improved when TBII disappeared from their sera. The profile of TBII in one patient corresponded to the IgG disappearance curve. These findings suggest that the transient neonatal hypothyroidism reported was caused by transplacental transfer of TBII.

Ultrastructure of the extraocular photoreceptor in the genitalia of a butterfly, Papilio xuthus.

This paper describes the ultrastructure of a sensory neuron found in the extraocular photoreceptive site on the butterfly genitalia. Our previous studies have shown that there are two pairs of the photoreceptive sites in a butterfly (four per individual). Each photoreceptive site is recognizable by a transparent area in the pigmented cuticle of the genitalia. From the nerve that extends from the transparent cuticle to the last abdominal ganglion, a sustained train of single unit spikes can be recorded in response to a light flash. The single unit spikes disappear when the transparent cuticle is covered, thus indicating that a single photoreceptor exists close to it. Here, we examined complete serial semithin sections of plastic-embedded photoreceptive sites of both male and female, and observed an ovoid structure close to the transparent cuticle. The structure contained the cell body of a sensory neuron whose axon was in the nerve branch from which the photoresponse had been recorded. Further electron microscopy revealed that the cell body extended a few distal processes that protrude tubular membrane from the tip, forming a structure resembling the phaosome (from Greek; phaos = light, some = body) which was first described in the earthworm dermal photoreceptors. The sensory neuron was also found in a surgically isolated nerve-photoreceptor preparation that responded to the light. We therefore propose that the phaosome-containing sensory neuron is the butterfly genital photoreceptor.

Morphogenesis of the photoreceptive site and development of the electrical responses in the butterfly genital photoreceptors during the pupal period.

This paper describes the process of morphogenesis of the photoreceptive site of the butterfly genital photoreceptors. Associated development of the electrical responses is also described. The photoreceptor is a sensory neuron whose cell body is located in the genitalia and has a photoreceptive site of the phaosome-type. This consists of the distal processes and the tubular membranes, which protrude from the tip of distal processes. Phaosome morphogenesis was studied using electron microscopy. The results indicate that morphogenesis occurs in the latter half of the pupal period and that the process is divided into five phases. First, the tubular membranes appear as small membrane protrusions (phase I). The short tubular membranes emerge from several portions of the cell body forming several membrane clusters (phase II). The clusters then collect to form a small phaosome. Short distal processes become evident (phase III). The phaosome volume increases, mainly due to the extensive elongation and bifurcation of both tubular membranes and distal processes (phase IV). Phase V achieves final adult morphology. The photoreceptors of phase II are already able to produce spikes in response to light stimulation, although the sensitivity was about one tenth of the adult. The sensitivity increase occurred in parallel with the increase in the phaosome volume.

Organization of actin filaments and immunocolocalization of alpha-actinin in the connecting cilium of rat photoreceptors.

A small discrete concentration of actin filaments in the connecting cilium of vertebrate photoreceptors appears to have a role in the morphogenesis of the phototransductive disk membranes (Williams et al., '88). We have visualized these actin filaments in rat rod photoreceptors by decorating them with myosin subfragment-1. At the site of disk morphogenesis, we observed a cluster of short filaments, with various orientations and their faster growing (barbed) ends at the ciliary plasma membrane. Their association with the liplike structure of an early nascent disk is consistent with their apparent involvement in the initiation of disk morphogenesis. A few longer decorated filaments extended along the core the connecting cilium, away from the site of disk morphogenesis, implying that they might have some function other than the shaping of a new disk. Most of the antiactin label was found in the region of the short filaments. The alpha-actinin immunolabel coincided with that of actin, suggesting that the filaments may be crosslinked by alpha-actinin.

Cytoskeletal components of the adherens junctions between the photoreceptors and the supportive Müller cells.

The outer limiting "membrane" (OLM) of the vertebrate retina comprises a series of heterotypic adherens junctions between the photoreceptors and the supportive Müller cells. These junctions appear to support the photoreceptors, which in teleosts, anurans, and birds are motile, and thus help them maintain their orientation with respect to incoming light. In an unusual role for this type of junction, they also provide a semipermeable barrier, preventing the diffusion of some proteins out of the extracellular space that surrounds the inner and outer segments of the photoreceptors. Using immunoelectron microscopy, we examined the association of actin, myosin, alpha-actinin, and vinculin with the OLM junctions of the adult chicken retina. Vinculin was detected close to the plasma membrane in the cytoplasmic plaques of the junctions, as it was in the adherens junctions of the retinal epithelium. Labelling of actin, myosin, and alpha-actinin was spread more throughout the plaques and was distributed unevenly about the junctions; labelling was much more extensive in the Müller cells than in the photoreceptors. Thus, the junctions of the OLM show similarity to the other adherens junctions in that their cytoplasmic plaques contain actin, myosin, alpha-actinin, and vinculin. But the large aggregation of actin, myosin, and alpha-actinin in the Müller cells, and their resulting asymmetrical distribution about the junctions, is unusual, and possibly an adaptation for the special function of the OLM junctions, in providing both structural support for the motile photoreceptors and a semipermeable barrier.

Acetylated alpha-tubulin in the connecting cilium of developing rat photoreceptors.

PURPOSE: To examine the distribution of acetylated alpha-tubulin in the connecting cilium of rat rod photoreceptors during different stages of photoreceptor development. METHODS: An antibody found to be specific for the acetylated form of alpha-tubulin was used in immunoelectron microscopy of retinas from animals of different ages. RESULTS: All the microtubules of the connecting cilium, including those of the basal bodies, were found to contain acetylated alpha-tubulin at the earliest stage of outer segment development, before the cilium has begun to grow out from the cell, and at all subsequent stages. CONCLUSIONS: These results indicate that the alpha-tubulin of the connecting cilium is acetylated either before, or at least very soon after, its assembly into microtubules. Given that acetylation of alpha-tubulin is correlated with stable microtubules, the results suggest that stable microtubules might be important in creating the foundation for the formation of the outer segment, as well as in helping maintain the polarity of the mature photoreceptor.

Butterfly wing colours: scale beads make white pierid wings brighter.

The wing-scale morphologies of the pierid butterflies Pieris rapae (small white) and Delias nigrina (common jezabel), and the heliconine Heliconius melpomene are compared and related to the wing-reflectance spectra. Light scattering at the wing scales determines the wing reflectance, but when the scales contain an absorbing pigment, reflectance is suppressed in the absorption wavelength range of the pigment. The reflectance of the white wing areas of P. rapae, where the scales are studded with beads, is considerably higher than that of the white wing areas of H. melpomene, which has scales lacking beads. The beads presumably cause the distinct matt-white colour of the wings of pierids and function to increase the reflectance amplitude. This will improve the visual discrimination between conspecific males and females.

Combination of physiological and anatomical methods for studying extraocular photoreceptors on the genitalia of the butterfly, Papilio xuthus.

This paper describes a combination of electrophysiological and anatomical techniques useful for characterizing sensory neurons in insects in our studies on the extraocular photoreceptors on the genitalia of the butterfly, Papilio xuthus. Genital photoreceptors were first electrophysiologically identified by recording photoreceptor spikes in response to light stimulation of the genitalia. The precise location and ultrastructure of these photoreceptors were then studied by light and electron microscopy. Both electrophysiological and anatomical techniques employed here were rather classical, but, as shown in this paper, they appear to be particularly useful for systems where intracellular penetration is difficult.

Cyanosis in atrial septal defect due to persistent eustachian valve.

Two cyanotic patients had venoarterial shunting from the inferior vena cava to the left atrium in an uncomplicated atrial septal defect with normal right ventricular pressures. Cyanosis was due to a large, anomalous inferior vena caval valve, the eustachian valve. The mechanism of cyanosis and technical problems for surgical repair are discussed.

Flow velocity of cardiac lymph and contractility of the heart: an experimental study.

The flow velocity of cardiac lymph in various abnormal conditions of the heart and in control situations was studied experimentally in dogs. The time needed for the cardiac lymph node to become stained after injection of contrast medium into the muscle layer of the left ventricular apex was measured as an indicator in determining flow velocity of cardiac lymph. Left ventricular contractility was studied simultaneously. The hypoxic dogs had a short staining time with a vigorous cardiac beat. (Short staining time means accelerated lymph flow through the heart). The hearts in which the coronary sinus was ligated revealed the shortest staining time with an insignificant contractile change. The exsanguinating dogs had a long staining time with reduced contractility. The dogs with ventricular fibrillation had the longest staining time. When the heart rate was fixed by pacing, the staining time reflected contractile change. The contractile force of the heart plays an important role in the flow velocity of cardiac lymph.