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Aims/Introduction Diabetic polyneuropathy (DPN) develops in the early stage of diabetes. However, no common diagnostic protocol has yet been established. Here, to verify that the flicker electroretinogram using a hand‐held device can detect the early dysfunction of the peripheral nervous system in patients with diabetes, we investigated the correlation between the progression of DPN and neuroretinal dysfunction. Materials and Methods In total, 184 participants with type 1 or 2 diabetes underwent a flicker electroretinogram (ERG) using a hand‐held device RETeval™ and nerve conduction study. Participants were also evaluated for intima‐media thickness, ankle‐brachial index, toe brachial index and brachial‐ankle pulse wave velocity. Parameters of the nerve conduction study were used to diagnose the severity according to Baba’s classification. A multiple regression analysis was used to examine the associations of ERG parameters with the severity of DPN categorized by Baba’s classification. Diagnostic properties of the device in DPN were evaluated using a receiver operating characteristic curve. Results A multiple regression model to predict the severity of DPN was generated using ERG. In the model, moderate‐to‐severe DPN was effectively diagnosed (area under the receiver operating characteristic curve 0.692, sensitivity 56.5%, specificity 78.3%, positive predictive value 70.6%, negative predictive value 66.1%, positive likelihood ratio 2.60, negative likelihood ratio 0.56). In the patients without diabetic retinopathy, the implicit time and amplitude in ERG significantly correlated with the parameters of the nerve conduction study, brachial‐ankle pulse wave velocity and intima‐media thickness. Conclusions Electroretinogram parameters obtained by the hand‐held device successfully predict the severity of DPN. The device might be useful to evaluate DPN., The progression of diabetic retinopathy and the dysfunction of neuroretina evaluated using the mydriasis‐free flicker electroretinogram showed a significant correlation. In patients without apparent diabetic retinopathy, the electroretinogram data correlated with parameters indicating vascular dysfunction, and with parameters indicating diabetic polyneuropathy, such as data of a nerve conduction study. Therefore, the electroretinogram data might reflect the neural and vascular impairments of the retina in patients with diabetes. The electroretinogram data were able to be used to predict the severity of diabetic polyneuropathy.

Aims/Introduction Diabetic polyneuropathy (DPN) and diabetic retinopathy (DR) are traditionally regarded as microvascular complications. However, these complications may share similar neurodegenerative pathologies. Here we evaluate the correlations in the severity of DPN and changes in the thickness of neuroretinal layers to elucidate whether these complications exist at similar stages of progression. Materials and Methods A total of 43 patients with type 2 diabetes underwent a nerve conduction study (NCS), a macular optical coherence tomography, and a carotid artery ultrasound scan. Diabetic polyneuropathy was classified according to Baba’s classification using NCS. The retina was automatically segmented into four layers; ganglion cell complex (GCC), inner nuclear layer/outer plexiform layer (INL/OPL), outer nuclear layer/photoreceptor inner and outer segments, and retinal pigment epithelium (RPE). The thickness of each retinal layer was separately analyzed for the fovea and the parafovea. Results Fourteen patients were classified as having moderate to severe diabetic polyneuropathy. The thicknesses of the foveal and parafoveal INL/OPL increased in patients with diabetic polyneuropathy compared with patients without. The thickness of the parafoveal retinal pigment epithelium decreased in patients with diabetic polyneuropathy. The thinning of parafoveal ganglion cell complex and foveal and parafoveal retinal pigment epithelium were positively correlated with deterioration of nerve functions in the nerve conduction study, but the thickening of INL/OPL was positively correlated with the nerve function deterioration. The thinning of parafoveal ganglion cell complex and foveal retinal pigment epithelium were positively correlated with the thickening of the carotid intima‐media. Conclusions Depending on the progression of diabetic polyneuropathy, the ganglion cell complex and retinal pigment epithelium became thinner and the INL/OPL became thicker. These retinal changes might be noteworthy for pathological investigations and for the assessment of diabetic polyneuropathy and diabetic retinopathy., (1) The thickness of the foveal and parafoveal inner nuclear layer/outer plexiform layer increased in patients with clinical diabetic polyneuropathy compared with patients without clinical diabetic polyneuropathy. (2) A decrease in the thickness of the parafoveal ganglion cell complex correlated with deterioration of nerve functions in the nerve conduction study. (3) A decrease in the thickness of the parafoveal ganglion cell complex was positively correlated with an increase in the parameters of atherosclerosis and cardiovascular risk factors.

The structure of [Arg8]vasopressin methylenedithioether ([AVP]CH2) has been determined in dimethylsulfoxide-d6. Two-dimensional DQF-COSY and NOESY spectra were measured and used to derive angle and distance constraints for restrained molecular dynamics (MD) calculations. In the MD trajectory, two types of beta-turn structure were found in the region from Tyr2 to Asn5, suggesting an equilibrium between type-I and type-II' beta-turn structures. When Halpha chemical shifts were used as an additional constraint, the type-I turn was favoured. To validate this result, an independent energy minimization procedure was used, using differences between calculated and observed chemical shifts. The two approaches gave essentially identical results. It is therefore concluded that the type-I turn predominates in solution. Analysis of calculated chemical shift contributions suggests that the beta-turn structure found in AVP is well preserved in [AVP]CH2, although the pressin ring size is expanded.

We used the combination of an atomic force microscope and a light microscope equipped with epifluorescence to serially image Plasmodium falciparum-infected erythrocytes. This procedure allowed us to determine unambiguously the presence and developmental stage of the malaria parasite as well as the number and size of knobs in singly, doubly, and triply infected erythrocytes. Knobs are not present during the ring stage of a malaria infection but a lesion resulting from invasion by a merozoite is clearly visible on the erythrocyte surface. This lesion is visible into the late trophozoite stage of infection. Knobs begin to form during the early trophozoite stage of infection and have a single-unit structure. Our data suggest the possibility that a two-unit structure of knobs, which was reported by Aikawa et al. (1996, Exp. Parasitol. 84, 339-343) using atomic force microscopy, appears to be a double-tipped image. The number of knobs per unit of host cell surface area is directly proportional to parasite number in both early and late trophozoite stages. These results indicate that knob formation by one parasite does not influence knob formation by other parasites in a multiply infected erythrocyte. In addition, knob volume is not influenced by either parasite stage or number at the late trophozoite stage, indicating that the number of component molecules per knob is constant throughout the parasite maturation process.

The first sign of neurogenesis in the embryo of grasshopper, Chortophaga viridifasciata (Orthoptera: Acrididae), is signaled by a partition of the ectodermal cells into non-neural ectodermal cells and neural eetodermal cells. The neuroblasts are differentiated from neural ectodermal cells. In the present study, we examined the pattern of mitotic activity in the developing embryo by tracing the incorporation of BrdU in S phase nuclei. The results indicate that the ectodermal cells in 6-day old embryos do not show any signs of differentiation. In 7-day old embryos, in which ectodermal cells become partitioned into 2 types, almost no neural ectodermal cells are incorporated with BrdU, whereas a constant incorporation is revealed in non-neural ectodermal cells. Among the mitotically quiescent neural ectodermal cells, which are arrested at the GI stage of the cell cycle, in 8-day old embryos, the neuroblasts are the first to resume their mitotic activity, while the other cells are then released from the mitotic quiescence. It seems that the mitotic quiescence may be an essential process to acquire a neural fate.

1. 1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches. 2. 2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation. 3. 3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition. 4. 4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size. 5. 5. The native molecular weight determined by light scattering method was 560 kDa. 6. 6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively. 7. 7. VG is a lipoprotein and comprises 92% protein and 8% lipid. 8. 8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

The mechanism of long-distance flight in insects was investigated by comparing lipid mobilization and transport in gregarious- and solitary-phase locusts and in the American cockroach. Unlike the gregarious-phase locust, both the American cockroach and the solitary locust were unable to form low-density lipophorin (loaded with increased amount of diacylglycerol) even when injected with adipokinetic hormone (AKH). The cockroach fat body responded to AKH. However, not only does the American cockroach lack apolipophorin-III (apoLp-III) in the haemolymph, but the fat body contains only an extremely small amount of diacylglycerol and a relatively large triacylglycerol pool. By contrast, the solitary-phase locust had apoLp-III in the haemolymph, but the fat body was only one-seventh or less in weight of the fat body of the gregarious locust. Furthermore, the fat body of the solitary locust contains a very small amount of triacylglycerol (1/20 or less of that of the gregarious locust) with only a trace of diacylglycerol. It was concluded that in the American cockroach and the solitary locust, the stores of fuel in the fat body are insufficient to maintain prolonged flight.

Using a combination of atomic force, scanning and transmission electron microscopy, we found that avian erythrocytes infected with the avian malaria parasite Plasmodium gallinaceum develop approximately 60 nm wide and approximately 430 nm long furrow-like structures on the surface. Furrows begin to appear during the early trophozoite stage of the parasite's development. They remain constant in size and density during the course of parasite maturation and are uniformly distributed in random orientations over the erythrocyte surface. In addition, the density of furrows is directly proportional to the number of parasites contained within the erythrocyte. These findings suggest that parasite-induced intraerythrocytic processes are involved in modifying the surface of host erythrocytes. These processes may be analogous to those of the human malaria parasite P. falciparum, which induces knob-like protrusions that mediate the pathogenic adherence of parasitized erythrocytes to microvessels. Although P. gallinaceum-infected erythrocytes do not seem to adhere to microvessels in the host chicken, the furrows might be involved in the pathogenesis of P. gallinaceum infections by some other mechanism involving host-pathogen interactions.

Detergent resistant membranes (DRMs) have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. Flotillins-1 and -2 have recently been shown to be large components of erythrocyte DRMs. In this study, we show that a Plasmodium falciparum infection disrupts the association of flotillins with erythrocyte DRMs. Flotillins are probably released from erythrocyte DRMs through the reduction of cholesterol and sphingomyelin levels during the course of a P. falciparum-infection. Although it is well known that a P. falciparum infection can modify the host erythrocyte membrane, this is the first report that P. falciparum can alter the DRM components of erythrocyte membranes.

In the medaka, Oryzias latipes, sex is determined chromosomally. The sex chromosomes differ from those of mammals in that the X and Y chromosomes are highly homologous. Using backcross panels for linkage analysis, we mapped 21 sequence tagged site (STS) markers on the sex chromosomes (linkage group 1). The genetic map of the sex chromosome was established using male and female meioses. The genetic length of the sex chromosome was shorter in male than in female meioses. The region where male recombination is suppressed is the region close to the sex-determining gene y, while female recombination was suppressed in both the telomeric regions. The restriction in recombination does not occur uniformly on the sex chromosome, as the genetic map distances of the markers are not proportional in male and female recombination. Thus, this observation seems to support the hypothesis that the heterogeneous sex chromosomes were derived from suppression of recombination between autosomal chromosomes. In two of the markers, Yc-2 and Casp6, which were expressed sequence-tagged (EST) sites, polymorphisms of both X and Y chromosomes were detected. The alleles of the X and Y chromosomes were also detected in O. curvinotus, a species related to the medaka. These markers could be used for genotyping the sex chromosomes in the medaka and other species, and could be used in other studies on sex chromosomes.

Both living and fixed specimens of the medically-important parasitic protozoa, Trypanosoma cruzi, Toxoplasma gondii, Giardia lamblia, Entamoeba histolytica, and Acanthamoeba spp. were studied by atomic force microscopy (AFM). The preparation of fixed specimens was similar to methods used for either scanning or transmission electron microscopy. AFM scanning was performed using both contact and tapping modes. A classical fixation procedure utilizing glutaraldehyde followed by ethanol dehydration was not suitable for all parasite species. AFM images could not be obtained from fixed samples of T. cruzi, T. gondii or E. histolytica. However, excellent topographic images could be obtained from specimens of G. lamblia and Acanthamoeba under identical conditions. Critical point drying permitted AFM imaging of both trypomastigote and epimastigote stages of T. cruzi. Phase imaging of T. cruzi elucidated unique surface details at a level of resolution not visible using any other imaging modalities. AFM elasticity map imaging of T. cruzi-infected and T. gondii-infected cells demonstrated that both parasites were markedly firmer than the surrounding host cell cytoplasm. The parasitophorous vacuole surrounding replicating T. gondii tachyzoites was also visualized by elasticity map imaging. These data suggest that although much remains to be learned about preparing parasitic protozoa for AFM imaging, the technique has the potential of providing unique and important insights into these disease causing organisms.

The carbohydrate compositions of lipophorin from Locusta migratoria were studied. The asparagine-linked oligosaccharides were liberated from the lipophorin by almond glycopeptidase ( N- oligosaccharide glycopeptidase, EC 3.5.1.52) digestion. This gave six molecular species [two major (E,F) and four minor (A,B,C,D)] oligosaccharides. All six oligosaccharides are classified as high-mannose type. Based on data obtained from thin-layer chromatography, gas-liquid chromatography (GLC) and colorimetric determination, the following compositions are proposed; Man5GlcNAc2 for A, Man6GlcNAc2 for B, Man7GlcNAc2 for C, Man8GlcNAc2 for D, Man9GlcNAc2 for E and Glc1Man9GlcNAc2 for F, respectively. It was also demonstrated that all six oligosaccharide types were associated with the heavy apoprotein or apo-I (mol. wt 250,000) but only three types (D,E,F) were associated with the light apoprotein or apo-II (mol. wt 85,000).

Atomic force microscope-based phase imaging in air is capable of elucidating variations in material properties such as adhesion, friction, and viscoelasticity. However, the interpretation of phase images of specimens in a fluid environment requires clarification. In this report, we systematically analyzed atomic force microscope-derived phase images of mica, glass, and collagen under the same conditions as used for living cells at various tapping forces; the resulting data provide critical information for the interpretation of phase images of living cells. The peripheral regions of COS-1 cells consistently show a more negative phase shift than the glass substrate in phase images at set-point amplitude: free amplitude (A sp /A 0 )=0.6–0.8. In addition, at all A sp /A 0 values suitable for phase imaging, tapping frequency appears to be high enough to ensure that phase shifts are governed primarily by stiffness. Consequently, phase imaging is capable of high resolution studies of the cellular surface by detecting localized variations in stiffness. We demonstrate that phase imaging of a bifurcating fiber in COS-1 cell cytoplasm is readily capable of a lateral resolution of ∼30nm.

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.

We describe the first cell biological application of carbon nanotube (CN) probes for atomic force microscopy studies. Topographic and phase images were collected from Plasmodium falciparum malaria-infected erythrocytes using both TappingMode Etched Silicon Probes (TESP probe) and CN probes. We estimate that the lateral resolution of a CN probe-generated topographic image is at least four-fold higher than that of the TESP probe. Carbon nanotube probe-generated phase images of P. falciparum-induced knobs on the surface of erythrocytes also show a markedly higher lateral resolution than comparable TESP probe-generate phase images of the same area. We conclude that CN probes are useful for cell biological atomic force microscopy studies and should play an increasingly important role in the future of this evolving discipline.