Morphologic evaluation of the antitumor activity of photodynamic therapy (PDT) using mono-L-aspartyl chlorin e6 (NPe6) against uterine cervical carcinoma cell lines.

In order to elucidate the antitumor effect and mechanism of action of photodynamic therapy (PDT) using the photosensitizing agent mono-L-aspartyl chlorin e6 (NPe6) and a semiconductor laser, we conducted a morphologic study on uterine cervical cancer cell lines. First, tumor shrinkage was confirmed in a tumor growth inhibition test. Next, morphologic changes after PDT were examined, and since the major change appeared to be tumor necrosis secondary to obstruction of the blood vessels around the tumor, an NPe6 cell uptake experiment was performed. The results confirmed that a significantly greater amount of NPe6 was incorporated by human umbilical vein endothelial cells (HUV-EC1) and the cervical cancer cell lines than by human umbilical cord-derived fibroblasts. Based on these findings it was concluded that NPe6 possesses tumor affinity, and necrosis secondary to vascular obstruction was postulated to be the principal mechanism of the antitumor effect of PDT using NPe6.

Asymmetric colocalization of Flamingo, a seven-pass transmembrane cadherin, and Dishevelled in planar cell polarization.

The Drosophila wing provides an appropriate model system for studying genetic programming of planar cell polarity (PCP) [1-4]. Each wing cell respects the proximodistal (PD) axis; i.e., it localizes an assembly of actin bundles to its distalmost vertex and produces a single prehair. This PD polarization requires the redistribution of Flamingo (Fmi), a seven-pass transmembrane cadherin, to proximal/distal cell boundaries; otherwise, the cell mislocalizes the prehair [5]. Achievement of the biased Fmi pattern depends on two upstream components in the PCP signaling pathway: Frizzled (Fz), a receptor for a hypothetical polarity signal, and an intracellular protein, Dishevelled (Dsh) [6-8]. Here, we visualized endogenous Dsh in the developing wing. A portion of Dsh colocalized with Fmi, and the distributions of both proteins were interdependent. Furthermore, Fz controlled the association of Dsh with cell boundaries, which association was correlated with the presence of hyperphosphorylated forms of Dsh. Our results, together with a recent study on Fz distribution [9], support the possibility that Fz, Dsh, and Fmi constitute a signaling complex and that its restricted localization directs cytoskeletal reorganization only at the distal cell edge.

Studies on the psychosomatic functioning of ill-health according to Eastern and Western medicine 6. Psychosomatic characteristics of anxiety and depression.

In the accompanying paper "Psychosomatic Characteristics of Anxiety and the Anxiety-Affinitive Constitution (Provisional Term) in Medical Students," we suggest that an assessment of the relationship between anxiety and the state of thoracic-abdominal balance of breathing, and the creation of an anxiety-affinitive constitution index (AACI) derived from that assessment, will be indispensable for furthering research in the future. Using the tenets of Oriental psychosomatic medicine, we examined in this research medical students (N=104) from whom we had received fully-informed consent for the research in writing, and patients who met the DSM-IV diagnostic criteria for anxiety disorders (N=18) and major depressive episodes (N=20). We then identified their anxiety and depressive psychosomatic traits in accordance with an assessment of the relationship between anxiety and the state of their thoracic-abdominal balance of breathing, and the AACI derived from that assessment. We performed a multiple regression analysis with the STAI trait anxiety as the dependent variable, and the parameters of the somatic tests as the independent variables. We obtained the formula that AACI=-62.9 + 72.9 x the thoracic/abdominal respiratory movement ratio (the fractal dimension value for the thoracic respiratory curve/the fractal dimension value for the abdominal respiratory curve) + 22.5 x the horizontal eye movement (the fractal dimension value) + 2.4 x the dental indentation of the tongue (existence, 1; none 0). We then obtained data regarding a symptomatological, constitutional, and nosological, diagnosis of anxiety and depression based on the AACI values of the Student's t test calculated for the medical students and the anxiety disorder patients and patients with major depressive episodes, and a statistical analysis using ANOVA. We believe the AACI we created in this research will be very important and significant for the preventive treatment of lifestyle illnesses and stress-related diseases.

Targeted disruption of cadherin-11 leads to a reduction in bone density in calvaria and long bone metaphyses.

The migration and adhesion of osteoblasts requires several classical cadherins. Cadherin-11, one of the classical cadherins, was expressed in mouse osteoblasts in skull bone and femur, revealed by immunohistochemistry. To elucidate the function of cadherin-11 in osteoblastogenesis, cadherin-11 null mutant mice were investigated. Although apparently normal at birth, Alizarin red staining of null mutant mice showed a reduced calcified area at the frontal suture that caused a round-shaped calvaria with increasing animal age to 3 months. Consequently, there was a reduction in bone density at the femoral metaphyses and the diploë of calvaria in null mutant mice. In the in vitro culture of newborn calvarial cells, the calcified area of mutant cells was smaller than those derived from wild-type littermates. These results show that absence of cadherin-11 leads to reduced bone density in some parts of skeletons including calvaria and long bone metaphyses, and thus suggest that cadherin-11 plays roles in the regulation of osteoblast differentiation and in the mineralization of the osteoid matrix.

Studies on the psychosomatic functioning of ill-health according to Eastern and Western medicine 5. Psychosomatic characteristics of anxiety and anxiety-affinitive constitution.

In successive studies of the psychosomatic functioning of ill-health according to Oriental and Western medicine in medical students, we established the existence of the psychosomatic characteristics we have provisionally termed the anxiety-affinitve constitution at the core of ill-health. Therefore, we conducted this research because our previous investigation showed this constitution included a high complexity of respiratory movement and eye movement with a significant correlation to the State Trait Anxiety Inventory (STAI). We examined the correlation between the STAI and somatic function of 88 medical students to identify the psychosomatic characteristics of anxiety and the anxiety-affinitive constitution. These tests included STAI, fractal (EEG, EOG, plethysmogram, respiratory curves, and EMG) and non-fractal (accelerated plethysmogram) dimension analyses, and malocclusion (based on Angle's classification). In particular, EOG, plethysmogram, and respiratory curves are known to have close association with trait anxiety. We were able to discover the correlation between (1) trait anxiety and thoracic and abdominal respiratory movements, and malocclusion (Class III), and (2) the correlation of state anxiety with thoracic respiratory movement, horizontal eye movement, a plethysmogram and an EEG-Pz (in males only). In subsequent study the relation between thoracic dominance and state-trait anxiety and between abdominal dominance and state-trait anxiety should be assessed to develop this research regarding the psychosomatic characteristics of anxiety and the anxiety-affinitive constitution. Further, it is essential to create an anxiety-affinitve constitution index based on multi-regression analysis.

Developmental roles and molecular characterization of a Drosophila homologue of Arabidopsis Argonaute1, the founder of a novel gene superfamily.

BACKGROUND: Arabidopsis Argonaute1 (AGO1) is the founder of a novel gene superfamily that is conserved from fission yeasts to humans. AGO1, and several other members of this superfamily are necessary for stem cell renewal or RNA interference. However, little has been reported about their roles in animal development or about the molecular activities of any of the members. RESULTS: We have isolated a Drosophila homologue of AGO1, dAGO1, in our attempt to search genetically for regulators of Wingless (Wg) signal transduction. dAGO1 is broadly expressed in the embryo and the imaginal disc. dAGO1 over-expression at wing margins suggested that it behaves as a positive regulator in the genetic background employed. Loss-of-function mutations of dAGO1, unexpectedly, did not give typical segment polarity phenotypes of the wg class; instead, dAGO1 maternal and zygotic mutant embryos showed developmental defects, with malformation of the nervous system being the most prominent. The mutant decreased in the numbers of several types of neurones and glia examined. The dAGO1 protein was distributed in the cytoplasm and co-sedimented with poly(U)- or poly(A)-conjugated beads. CONCLUSION: Our results suggest that the dAGO1 protein exerts its developmental functions by binding to RNA either directly or indirectly.

Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development.

In ventricular cells of the mouse telencephalon, differential expression of cadherin cell adhesion molecules defines neighbouring regions; R-cadherin delineates the future cerebral cortex, while cadherin-6 delineates the lateral ganglionic eminence. By using cell labelling analyses in the whole embryo culture system, we demonstrated that the interface between R-cadherin and cadherin-6 expression is a boundary for cell lineage restriction at embryonic day 10.5. Interestingly, when a group of cells with exogenous cadherin-6 were generated to straddle the cortico-straital boundary by electroporation at embryonic day 11.0, ectopic cadherin-6-expressing cortical cells were sorted into the striatal compartment, and the reverse was the trend for ectopic R-cadherin-expressing striatal cells. Although cadherin-6 gene knockout mice engineered in this study showed no obvious phenotype in telencephalic compartmentalisation, the preferential sorting of ectopic cadherin-6-expressing cells was abolished in this mutant background. Thus, the differential expression pattern of cadherins in the embryonic telencephalon is responsible for maintaining the cortico-striatal compartment boundary.

Studies on the psychosomatic functioning of ill-health according to eastern and western medicine 7. Psychoimmuno-endocrinological changes induced by Kampo medication and relaxation training.

Using the anxiety-affinitive constitution index (AACI) that we created, we measured the degree of the anxiety-affinitive constitution as an equivalent of ill-health for the study of psychoimmuno-endrocrinological changes induced by Kampo medication and relaxation training (RT). With 20 sixth-year Saga Medical School students (average age: 24.8 +/- 1.4) as the subjects, we obtained three results: (1) relaxation training produced lowered STAI trait and state anxiety scores and tended to transform the respiratory pattern from thoracic to abdominal; (2) Kampo medication generated lowered GHQ-30 total scores and lowered the depression factor scores for its symptom components; and (3) Kampo medication for overcoming blood stagnation induced lowered NK cell activity, whereas Kampo medication for providing a tonic effect on vital energy and enriching blood brought about elevated NK cell activity and blood cortisol concentration. These results strongly suggest that while Kampo medication alleviated psychological distress as demonstrated by lowered GHQ-30 scores, RT improved the respiratory pattern, as demonstrated by increased abdominal dominance. The utmost attention must be paid to the prolonged administration of drugs for overcoming blood stagnation because of their possible immunosuppressive action.

Hoxa3 regulates integration of glossopharyngeal nerve precursor cells.

In vertebrates, certain Hox genes are known to control cellular identities along the anterior-posterior (A-P) axis in the developing hindbrain. In mouse Hoxa3 mutants, truncation of the glossopharyngeal (IXth) nerve or the fusion of the IXth and vagus (Xth) nerves was reported, although its underlying mechanism is largely unknown. To elucidate the mechanism of the IXth nerve defects, we reexamined the phenotype of Hoxa3 mutant embryos. In Hoxa3 mutants, we observed an abnormal caudal stream of the migrating Hoxa3-expressing neural crest cells at the prospective IXth nerve-forming area. Dorsomedial migration of the placode-derived neuronal precursor cells of the IXth nerve was also affected. Motor neurons at rhombomere 6 (r6), where those of the IXth nerve were positioned, often projected axons to the Xth nerve. In summary, the Hoxa3 gene has crucial roles in ensuring the correct axon projection pattern of all three components of the IXth nerve, i.e., motor neurons and sensory neurons of the proximal and distal ganglia.

Requirement of the juxtamembrane domain of the cadherin cytoplasmic tail for morphogenetic cell rearrangement during myotome development.

During development, the activity of cadherin cell adhesion molecules is assumed to be regulated to allow for cell rearrangement or translocation. Previous studies suggest that the juxtamembrane (JM) domain of the cadherin cytoplasmic tail, which contains the site for binding to p120ctn, has a regulatory function in this adhesion system. To study the possible role of JM domain-dependent cadherin regulation in embryonic cell rearrangement, we ectopically expressed a series of N-cadherin mutants in developing somites of chicken embryos. When a JM domain-deficient N-cadherin was expressed, the morphogenetic expansion of the myotome was strongly suppressed. However, a triple alanine substitution in the JM domain, which specifically inhibited the p120ctn binding, had no effect on myotome development. Furthermore, a dominant negative N-cadherin, which had a deletion at the extracellular domain but maintained the normal cytoplasmic tail, did not affect myotome expansion; although it disrupted intersomite boundaries. Overexpression of p120ctn also did not affect myotome expansion, but it did perturb myofiber orientation. These and other observations suggest that the JM domain of N-cadherin has a regulatory role in myotome cell rearrangement in which molecules other than p120ctn are involved. The p120ctn molecule itself seems to play a critical role in the arrangement of myofibers.

Cadherin-dependent cell-cell adhesion.

Differential treatment of cells with trypsin can be used to distinguish Ca(2+)-dependent adhesion (CDS) from Ca(2+)-independent adhesion (CIDS). Cadherins appear to be a unique family of molecules whose structure and function as adhesion molecules are protected from trypsin in the presence of Ca(2+). This unit provides protocols for preparation and analysis of cells for cadherin-dependent adhesion in short-term and long-term aggregation assays. The functions of different cadherins can be assessed in mixed aggregate assays. Fluorescence antibody-based assays are used to identify specific cadherins and their associated catenins, and transformation of cells with specific constructs can be used to assay adhesion in cells with loss of cadherin activity.

Patterning of cell assemblies regulated by adhesion receptors of the cadherin superfamily.

During morphogenesis, cell-cell association patterns are dynamically altered. We are interested in how cell adhesion molecules can regulate the patterning of cellular assemblies. Cadherins, a group of cell-cell adhesion receptors, are crucial for the organized assembly of many cell types, but they also regulate dynamic aspects of cell association. For example, during neural crest emigration from the neural tube, the cadherin subtypes expressed by crest cells are switched from one subtype to another. Artificial perturbation of this switch results in blocking of their escape from the neural tube. Intracellular modulations of cadherin activity also seem to play a role in regulation of cell adhesion. We identified p120ctn as a regulator of cadherin function in carcinoma cells. With such regulators, cells may make a choice as to whether they should maintain stable cell contacts or disrupt their association. Finally, we found another type of cadherin-mediated cell patterning: Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity in Drosophila imaginal discs. Thus, the cadherin superfamily receptors control the patterning of cell assemblies through a variety of mechanisms.

Characterization of a mutant E-cadherin protein encoded by a mutant gene frequently seen in diffuse-type human gastric carcinoma.

The cell-cell adhesion molecule E-cadherin plays an essential role in the maintenance and function of epithelial tissues. Altered expression of E-cadherin has been implicated in tumor invasion. Analysis of mutations of the human E-cadherin gene in gastric carcinoma of the diffuse type has revealed that deletion of exon 8 or 9 in its cDNA appears to be predominant. In this study, we carried out structural and functional analyses of a mutant form of E-cadherin in a cell line, HSC45-M2, established from a human signet ring-cell carcinoma. Although immunohistochemical analysis showed that the mutant cadherin was localized at cell-cell contact sites as usually seen with the wild type, these cells did not form compact colonies. HSC45-M2 cells expressed aberrant E-cadherin with an m.w. larger than that of the wild type. In these cells, we found deletion of the exon 9-intron 9 boundary including the splicing donor site in E-cadherin genomic DNA. RT-PCR indicated 2 transcripts, which appeared to be caused by the splicing defect. Northern blotting, however, showed that the transcript lacking exon 9 was predominantly detected in these cells. The electrophoretic mobilities on SDS-PAGE of the mutant E-cadherin protein in HSC45-M2 cells and the protein expressed from cDNA lacking exon 9 appeared identical. Analysis of the amino-terminal region of the mutant E-cadherin protein revealed that the cadherin was capable of becoming a mature form by removal of its amino-terminal peptide. However, the mutant E-cadherin was susceptible to trypsinization in the presence of Ca(2+), which is not the case for wild-type E-cadherin, suggesting that the mutant E-cadherin frequently found in diffuse-type gastric carcinoma may have lost its Ca(2+)-binding ability, leading to disruption of the tight cell-cell association.

Abnormal illness behavior and psychiatric disorders: a study in an outpatient clinic in Japan.

Abnormal illness behavior, such as hypochondriacal attitude and inappropriate treatment-seeking, has been associated with various psychiatric disorders in which patients tend to abuse medical services and seek inappropriate treatment in general practice clinics rather than psychiatric clinics. However, the relationship between illness behavior and psychiatric disorders in Japan is yet to be elucidated. We examined the abnormal illness behavior of 243 patients who visited the outpatient department of psychiatry at Saga Medical School Hospital, Saga, Japan, using a Japanese version of the Illness Behavior Questionnaire (IBQ). Multivariate analysis indicated significant association between some of the IBQ scale scores and age, sex and employment status. Patients with anxiety disorder scored higher on five of the seven IBQ scales compared with patients with another major disorder (mood disorder, schizophrenia or somatoform disorder). When compared with the IBQ scale scores reported in Australian patients in a psychiatric hospital, most of the IBQ scale scores differed significantly in our patients; a higher score among Japanese patients on the general hypochondriasis scale was most prominent. A similar trend in IBQ scale scores was also noted among Japanese patients visiting the hospital's general medicine clinic in comparison with Australian patients visiting a general practice clinic. Japanese patients with anxiety disorder may display the most salient abnormal illness behaviors among patients with psychiatric disorders. Sociocultural background may contribute to the characteristic abnormal illness behaviors of Japanese patients.

Loss of cadherin-11 adhesion receptor enhances plastic changes in hippocampal synapses and modifies behavioral responses.

Cadherins organize symmetrical junctions between the pre- and postsynaptic membranes in central synapses. One of them, cadherin-11 (cad11), is expressed in the limbic system of the brain, most strongly in the hippocampus. Immunohistochemical studies of the hippocampus showed that cad11 proteins were densely distributed in its synaptic neuropil zones; in cultured hippocampal neurons, their distribution often overlapped with that of synaptophysin, and also occasionally with that of GluR1 at spines. To assess the role of cad11 in synaptic formation and/or function, we analyzed brains of cad11-deficient mice. In these mice, long-term potentiation (LTP) in the CA1 region of the hippocampus was, unexpectedly, enhanced; and the level of LTP saturation was increased. In behavioral tests, the mutant mice showed reduced fear- or anxiety-related responses. These results suggest that the cad11-mediated junctions may modulate synaptic efficacy, confining its dynamic changes to a limited range, or these junctions are required for normal development of synaptic organization in the hippocampus.

Morphologic fate of diencephalic prosomeres and their subdivisions revealed by mapping cadherin expression.

The expression of four cadherins (cadherin-6B, cadherin-7, R-cadherin, and N-cadherin) was mapped in the diencephalon of chicken embryos at 11 days and 15 days of incubation and was compared with Nissl stains and radial glial topology. Results showed that each cadherin is expressed in a restricted manner by a different set of embryonic divisions, brain nuclei, and their subregions. An analysis of the segmental organization based on the prosomeric model indicated that, in the mature diencephalon, each prosomere persists and forms a coherent domain of gray matter extending across the entire transverse dimension of the neural tube, from the ventricular surface to the pial surface. Moreover, the results suggest the presence of a novel set of secondary subdivisions for the dorsal thalamus (dorsal, intermediate, and ventral tiers and anteroventral subregion). They also confirm the presence of secondary subdivisions in the pretectum (commissural, juxtacommissural, and precommissural). At most of the borders between the prosomeres and their secondary subdivisions, changes in radial glial fiber density were observed. The diencephalic brain nuclei that derive from each of the subdivisions were determined. In addition, a number of previously less well-characterized gray matter regions of the diencephalon were defined in more detail based on the mapping of cadherin expression. The results demonstrate in detail how the divisions of the early embryonic diencephalon persist and transform into mature gray matter architecture during brain morphogenesis, and they support the hypothesis that cadherins play a role in this process by providing a framework of potentially adhesive specificities.

Studies on the psychosomatic functioning of ill-health according to Eastern and Western medicine. 4. The verification of possible links between ill-health, lifestyle illness and stress-related disease.

In our previous reports on the same general topic, we exploited research in Oriental psychosomatic medicine to obtain diagnoses through visual observation of the sublingual vein (useful for the early detection of vital energy stagnation and blood stasis) from a computerized color analysis of the tongue proper, its coating, and the sublingual vein. This led us to develop the concept of the anxiety-affinitive constitution, based on unbalanced qi, blood, and body fluid in ill-health as a causative factor for stress-related diseases and lifestyle illnesses. As a development of this research, the present report examines the verification of possible links of ill-health, lifestyle illness, and stress-related disease through the diagnosis and treatment of functional subclinical psychosomatic disorders detected as a clinical expression of the anxiety-affinitive constitution. First, a diagnosis of functional subclinical psychosomatic disorders (ill-health) in 197 medical school students, including 156 first-year students at medical and nursing schools made it clear that the trait anxiety forming the core of the anxiety-affinitive constitution is linked to Dr. Lester Breslow's seven good health habits, and the manner of respiration. Second, it was revealed during the treatment of functional subclinical psychosomatic disorders in eight medical students that kampo medication and relaxation training (RT) produce lowered scores for STAI trait anxiety, and transform respiration from thoracic pattern to a balanced thoracic and abdominal respiratory pattern. This was particularly true for RT. Finally, we concluded that a high score for trait anxiety correlates to the formation of inappropriate health habits, and the habituation of inadequate respiration (thoracic pattern). Therefore, lifestyle illness or stress-related diseases will develop unless an anxiety-affinitive constitution is improved with kampo medication and/or RT.

Blockade of cadherin-6B activity perturbs the distribution of PSD-95 family proteins in retinal neurones.

BACKGROUND: Synaptic junctions have cadherin-catenin complexes, but their functions are poorly understood. Using retinal neurones, we investigated the role of this adhesion machinery in synaptic organization. RESULTS: In cultures of chicken retinal cells, cadherin-6B (cad6B) and cadherin-7 (cad7) are expressed by distinct neurones, each being distributed in a punctate pattern along their neurites as well as in the soma. Double-immunostaining for cad6B and PSD-95/SAP90 or other PSD-95 family members, known to localize in the postsynaptic density, showed that their distributions overlapped each other. To assess the role for cad6B, we incubated retinal cells with antibodies that could specifically block cad6B-mediated adhesion. In the antibody-treated neurones, the localization pattern of PSD-95 family proteins was altered, that is, their staining signals tended to be reduced or disarranged. We then examined whether cadherins interacted molecularly with PSD-95: Cadherin immunoprecipitates from brain lysates did not contain PSD-95; nevertheless, this protein was co-precipitated with alphaN- and beta-catenins. When PSD-95 proteins were ectopically expressed in epithelial cells, some of these molecules were concentrated in cell-cell junctions, co-localizing with E-cadherin, and this junctional localization of PSD-95 was abolished by blocking of E-cadherin activity. CONCLUSION: These results suggest that cadherins play a role in the subcellular organization of postsynaptic density components through some, perhaps indirect, molecular interactions.

Differential expression of cadherin adhesion receptors in neural retina of the postnatal mouse.

PURPOSE: To determine the expression pattern of multiple subtypes of cadherin adhesion receptor in postnatal mouse neural retina. METHODS: The expression of N-cadherin, R-cadherin, cadherin-6, cadherin-8, and cadherin-11 in retinas at postnatal days 0 to 42 was analyzed by in situ hybridization of mRNA as well as by immunohistochemistry. RESULTS: Each cadherin was expressed by different cell populations of the retina, and the following expression patterns were established by postnatal day 14: in the ganglion cell layer, all these molecules were expressed, but each occurred only in a subset of the cells. Likewise, in the inner nuclear layer, R-cadherin and cadherin-6 and -8 were expressed by a restricted population of amacrine cells, and cadherin-8 also by a subpopulation of bipolar cells. All horizontal cells expressed R-cadherin, and Muller cells expressed N-cadherin and cadherin-11. Proteins of R-cadherin and cadherin-6 were concentrated in neuropil layers. CONCLUSIONS: The pattern of differential expression of the five cadherins supports the idea that these molecules may play a role in selective cell interactions within the heterogeneous cell pool of the neural retina.