Efficient photodynamic therapy against drug-resistant prostate cancer using replication-deficient virus particles and talaporfin sodium.

To enhance the potency of photosensitizer, we developed a novel photosensitizer, Laserphyrin®-HVJ-E (L-HVJ-E), by incorporating talaporfin sodium (Laserphyrin®, Meiji Seika Pharma) into hemagglutinating virus of Japan envelope (HVJ-E). In this study, we examined the optimal Laserphyrin® concentration for preparation of Laserphyrin®-HVJ-E which had photocytotoxicity and maintained direct cytotoxicity derived from HVJ-E. Then, potency of Laserphyrin®-HVJ-E and Laserphyrin® were compared in vitro using castration-resistant prostate cancer cell line (PC-3). A laser diode (L660P120, Thorlabs, USA) with a wavelength of 664 nm was used for light activation of Laserphyrin®, which corresponds to an absorption peak of Laserphyrin® and provides a high therapeutic efficiency. The photocytotoxicity and direct cytotoxicity of Laserphyrin®-HVJ-E prepared using various Laserphyrin® concentrations were evaluated using PC-3 cell in vitro. We categorized the treatment groups as Group 1: 50 µL of D-MEM treatment group, Group 2: HVJ-E treatment group, Group 3: Laserphyrin®-HVJ-E treatment group, and Group 4: Laserphyrin® treatment group. Group 3 was subjected to different concentrations of Laserphyrin®-HVJ-E suspension, and all groups were subjected to different incubation periods (24, 48 h), (30 min, 1 h, or 3 h,) respectively, without and after PDT. Laserphyrin®-HVJ-E prepared using 15 mM Laserphyrin® had high photocytotoxicity and maintained HVJ-E's ability to induce direct cytotoxicity. Therapeutic effect of Laserphyrin®-HVJ-E was substantially equivalent to that of Laserphyrin® alone even at half Laserphyrin® concentration. By utilizing Laserphyrin®-HVJ-E, PDT could be performed with lower Laserphyrin® concentration. In addition, Laserphyrin®-HVJ-E showed higher potency than Laserphyrin® by combining cytotoxicities of HVJ-E and PDT.

Photodynamic therapy by lysosomal-targeted drug delivery using talaporfin sodium incorporated into inactivated virus particles.

BACKGROUND: Photodynamic therapy (PDT), a minimally invasive cancer treatment involving the activation of photosensitizer by a specific wavelength of light, is considered to be a promising treatment option for drug-resistant prostate cancer. Hemagglutinating virus of Japan envelope (HVJ-E) has the potential to serve as a highly effective cancer therapy through selective drug delivery and enhancement of the anti-tumor immune response. OBJECTIVES: To improve therapeutic efficacy and selective accumulation of photosensitizer into tumor cells, we developed a novel photosensitizer, Laserphyrin®-HVJ-E (L-HVJ-E), by incorporating talaporfin sodium (Laserphyrin®, Meiji Seika Pharma) into HVJ-E. MATERIALS AND METHODS: The therapeutic effect of PDT with Laserphyrin® or L-HVJ-E was evaluated in the human prostate cancer cell line PC-3 in vitro. The subcellular localizations of Laserphyrin® and L-HVJ-E were observed by confocal microscopy. Apoptosis or necrosis following PDT was detected by annexin V-fluorescein/propidium iodide double staining. RESULTS: The cytotoxic effect of Laserphyrin®- and L-HVJ-E-mediated PDT were determined by evaluating cell survival rate and production of reactive oxygen species. The cytotoxicity of L-HVJ-E-mediated PDT was dependent on drug concentration and light dose. Laserphyrin® and L-HVJ-E gradually entered cells as incubation time increased, and both agents tended to be distributed in lysosomes rather than mitochondria. Time and dose dependent increase in ROS production was observed, and induction of both apoptotic and necrotic cell death was confirmed. CONCLUSIONS: Laserphyrin® and L-HVJ-E were distributed mainly in lysosomes and induced cell death by both apoptosis and necrosis. Furthermore, L-HVJ-E-mediated PDT effectively killed cultured PC-3 cells and exerted higher photocytotoxicity than Laserphyrin®-mediated PDT.

A neonatal case of chronic granulomatous disease, initially presented with invasive pulmonary aspergillosis.

Chronic granulomatous disease (CGD) often presents with infectious illness, such as repeating bacterial and fungal infections, due to the inability to generate superoxide, which would destroy certain infectious pathogens, and is usually diagnosed in childhood. We describe a CGD case diagnosed in neonatal period, who initially presented with invasive aspergillosis. Neonatal invasive pulmonary aspergillosis is very rare and, to the best of our knowledge, this might be the youngest case in Japan.

Expression of mRNAs for the diacylglycerol kinase family in immune cells during an inflammatory reaction.

Phosphoinositide metabolism is intimately involved in cellular signal transduction. In response to extracellular stimuli, it generates diacylglycerol (DG), which serves as a lipid second messenger molecule to activate various proteins in various organs under pathophysiological conditions. Diacylglycerolkinase (DGK) constitutes an enzyme family that catalyzes conversion of DG to phosphatidic acid. It is therefore regarded as a regulator of the DG signal. Previous studies have revealed the critical role of α and ζ types of DGK in T cell functions. Nevertheless, little is known about the expression patterns of the DGK family in immune cells of various kinds. After examination of the expression profile of DGK isozymes in immune cells that are isolated from human blood, we investigated whether their mRNA expression levels would be changed during an inflammatory reaction. Results showed that DGK isozyme mRNAs are widely expressed in immune cells, except for DGKß and DGKι. During an inflammatory reaction, DGKε mRNA was increased transiently in the initial phase (20-40 min) of stimulation with both LPS and IL-2 in T cell-derived HUT-102 cells and macrophage-derived RAW264 cells. At the organismal level, an intraperitoneal injection of LPS also induced upregulation of DGKε mRNA in the spleen in a similar,but not identical, manner. These results suggest that DGKε is involved in inflammatory processes of the cellular immune system.

Development of icterus gravis in a preterm infant with G71R UGT1A1 polymorphism.

BACKGROUND: Uridine diphosphate-glucuronosyltransferase (UGT) gene family is involved in the detoxification of biomaterials and drugs in the liver. Among the UGT gene family members, only UGT1A1 is involved in bilirubin conjugation. As a result, deficient UGT1A1 activity causes jaundice. One disease that is characterized by reduced UGT1A1 activity is Gilbert's syndrome. Two prevalent UGT1A1 polymorphisms responsible for Gilbert's syndrome have been identified: G71R in exon 1 and A(TA)7TAA in the TATA box of the promoter region. Recently, the G71R polymorphism has been associated with breastfeeding jaundice and neonatal hyperbilirubinemia in term infants. However, its association with jaundice in very low birth weight infants (VLBWIs) has never been reported. CASE PRESENTATION: The patient was a female born at 28 weeks, 4 days gestation with a birth weight of 1172 g. On day 21, intense yellowing of the skin and eyes was noted, and the patient's total bilirubin level was 23.7 mg/dL (her direct bilirubin level was 2.1 mg/dL). Therefore, an exchange transfusion was conducted. She had neither blood type incompatibility nor a family history of constitutional jaundice. Metabolic screens for amino and organic acids were negative. No elevation of any of the examined antibody titers was noted, and no evidence of an inflammatory reaction was observed. In addition, no hematological abnormalities were detected. The direct/indirect Coombs test, irregular antibody test and red blood cell antibody dissociation test were all negative, and her thyroid function was normal. We performed sequence analysis of the UGT1A1 gene after the patient's parents provided written informed consent. Exon 1 of the UGT1 gene on chromosome 2 was analyzed by direct sequencing. A heterozygous substitution from G to A (211G→A: G71R) in base 211 was noted. CONCLUSION: We speculated that this preterm infant with carrying the G71R polymorphism reduced UGT1A1 activity and developed severe jaundice that was likely triggered by factors such as breast feeding and medications. The polymorphism appears at some frequency among VLBWIs, which would necessitate adequate care of severe jaundice even after the acute phase.

Postnatal development of tyrosine hydroxylase mRNA-expressing neurons in mouse neostriatum.

The striatum harbors a small number of tyrosine hydroxylase (TH) mRNA-containing GABAergic neurons that express TH immunoreactivity after dopamine depletion, some of which reportedly resembled striatal medium spiny projection neurons (MSNs). To clarify whether the TH mRNA-expressing neurons were a subset of MSNs, we characterized their postnatal development of electrophysiological and morphological properties using a transgenic mouse strain expressing enhanced green fluorescent protein (EGFP) under the control of the rat TH gene promoter. At postnatal day (P)1, EGFP-TH+ neurons were present as clusters in the striatum and, thereafter, gradually scattered ventromedially by P18 without regard to the striatal compartments. They were immunonegative for calbindin, but immunopositive for enkephalin (54.5%) and dynorphin (80.0%). Whole-cell patch-clamp recordings revealed at least two distinct neuronal types, termed EGFP-TH+ Type A and B. Whereas Type B neurons were aspiny and negative for the MSN marker dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32), Type A neurons constituted 75% of the EGFP+ cells, had dendritic spines (24.6%), contained DARPP-32 (73.6%) and a proportion acquired TH immunoreactivity after injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 3-nitropropionic acid. The membrane properties and N-methyl-d-aspartate : non-N-methyl-d-aspartate excitatory postsynaptic current ratio of Type A neurons were very similar to MSNs at P18. However, their resting membrane potentials and spike widths were statistically different from those of MSNs. In addition, the calbindin-like, DARPP-32-like and dynorphin B-like immunoreactivity of Type A neurons developed differently from that of MSNs in the matrix. Thus, Type A neurons closely resemble MSNs, but constitute a cell type distinct from classical MSNs.

Enzymatic activity and gene expression of diacylglycerol kinase isozymes in developing retina of rats.

Photoreceptors contain highly specialized structures for phototransduction, which is mediated by rhodopsins and heterotrimeric G-proteins. The signal is transmitted through the cGMP cascade, which controls cGMP-gated cation channels in mammals, while in flies it is operated by phosphoinositide (PI) cascade through a second messenger diacylglycerol (DG), which engenders the opening of Ca2+ channels. Recent studies suggest that PI-related signaling cascade is also involved in the phototransduction in mammalian retina. This study examined whether one PI-related enzyme, diacylglycerol kinase (DGK), which is regarded as a regulator of the DG signal through its metabolism, is expressed in mammalian retina. Enzymatic assay, Northern blot and RT-PCR analyses, and in situ hybridization histochemistry were performed to assess the expression profile of DGK isozymes and their cellular localization. In rat retina DGKε, DGKζ, and DGKι are the dominant species with distinct patterns of expression. At the cellular level, DGKε is the only one detected intensely in the photoreceptor layer, although DGKι and DGKζ are observed in bipolar and ganglion cell layers. These results suggest that each DGK isozyme plays a different role in the signal transduction in distinct cell types and that DGKε is a candidate involved in the photoreceptor PI signaling machinery.

Excitotoxicity by kainate-induced seizure causes diacylglycerol kinase ζ to shuttle from the nucleus to the cytoplasm in hippocampal neurons.

Diacylglycerol kinase (DGK), which consists of several isozymes, plays a pivotal role in lipid second-messenger diacylglycerol metabolism. A nuclear isozyme, DGKζ, which is translocated from the nucleus to the cytoplasm in hippocampal neurons under transient ischemic stress, is implicated in nuclear events of delayed neuronal death. Kainate (KA)-induced seizure is another model used to study excitotoxic stress. Therefore, we examined whether DGKζ is implicated in a different type of degenerative excitotoxicity in hippocampal neurons. We conducted immunohistochemical analysis of rat hippocampi after KA-induced seizures. DGKζ in hippocampal neurons shuttles from the nucleus to the cytoplasm. It never relocates to the nucleus during KA-induced seizures. Marked change in the immunoreactivity is first observed in CA1 pyramidal neurons 2h after injection during stage 3 seizures. Immunoreactivity for DGKι remains unchanged in the cytoplasm. That for NeuN remains mostly unchanged in the nucleus. Results show that nucleocytoplasmic translocation of DGKζ also occurs in a different model of excitotoxicity that results in apoptotic neuronal death. Cytoplasmic translocation of DGKζ might be involved in early events of the apoptotic cell death pathway in hippocampal neurons under stressed conditions.

Localization of fatty acid binding proteins (FABPs) in the cochlea of mice.

Various fatty acids (FAs) are involved in many different functions in the organism as a source of energy, as essential ingredients of membranous lipids as well as intracellular signaling molecules. Intracellular fatty acid binding proteins (FABPs) comprise a family of soluble lipid binding proteins with low molecular masses and which can make long chain FAs soluble to allow intracellular translocation in the aqueous cytosol. To clarify the possible involvement of FAs and FABPs in hearing function, the present study investigated the localization of FABPs in the cochlea of adult mice using immunohistochemical procedures. Among various FABP species, H (heart-type)-FABP was localized in inner and outer pillar cells and outer phalangeal cells, while B (brain-type)-FABP was localized in border cells and cells of Hensen, and fibrocytes in the spiral limbus and spiral prominence. In the spiral ganglion, moderate to low H-FABP immunoreactivity was observed in almost all neurons, while B-FABP immunoreactivity was found in satellite cells. The discrete localization of the two FABPs in different non-receptor cells in the Organ of Corti suggests that the FABP species and/or their ligands, FAs, play important roles in the regulation of the hearing function.

Cardiac sympathetic degeneration correlates with olfactory function in Parkinson's disease.

Autonomic and olfactory dysfunctions are considered markers for preclinical diagnosis in Parkinson's disease (PD), because pathological changes in these systems can start before motor symptoms develop. We investigated whether cardiac sympathetic function and olfactory function are associated in PD. Participants comprised 40 nondemented patients with idiopathic PD, and age-matched controls. Cardiac sympathetic function was evaluated by (123) I-metaiodobenzylguanidine (MIBG) uptake, in terms of the heart to mediastinum (H/M) ratio in both early and delayed images, and the washout rate (WR). Olfactory function was evaluated using the Odor Stick Identification Test for Japanese, which evaluates the detection of 12 odorants familiar to Japanese participants. Smell identification scores were significantly lower (P < 0.001) in patients with PD than in controls. Smell identification scores correlated positively with early (P < 0.05) and delayed H/M ratios (P < 0.01), and inversely with the WR (P < 0.005) especially in patients with early PD (below 5 years of the start of motor symptoms), whereas smell identification scores did not correlate with any parameters of MIBG in the advanced PD (above 5 years of the start of motor symptoms). There was no correlation between motor symptom scores and smell identification scores, H/M ratios, or WR. The results suggest that the cardiac sympathetic nervous system might degenerate in parallel with the olfactory system in patients with early PD, and that these two systems might degenerate at a different rate of speed in advanced PD.

Usefulness of curry odorant of odor stick identification test for Japanese in olfactory impairment screening.

CONCLUSION: The curry odorant of the odor stick identification test for Japanese (OSIT-J) is useful in screening for olfactory impairment in Japanese subjects. OBJECTIVE: The present study was designed to determine the most useful odorant of the OSIT-J in screening for olfactory impairment in Japanese subjects. SUBJECTS AND METHODS: We studied olfactory impairment screening with the OSIT-J in 83 participants (49 male, 34 female; average age 50 years) in an executive check-up at NTT West Kanazawa Hospital. Olfactory discrimination acuity was evaluated with three odorants of the OSIT-J (rose, curry, and sweaty-smelling clothes), each known to be significantly correlated with the assessment of the Japanese standard olfaction test (T&T olfactometer). Those participants who did not score full marks in tests with the three odors were assessed with another nine odorants of the OSIT-J. RESULTS: The positive predictive value was 100% in the screening with the curry odorant. In 38 participants who did not identify all three odors correctly, the identification of the curry odor was significantly correlated with the scores for all 12 odors (p<0.005). Identification of the curry odor was not significantly correlated with identification of the menthol odor of OSIT-J.

Localization of fatty acid binding protein of epidermal type common to dendritic cells and presumptive macrophages in Peyer's patches and epithelial M cells of mouse intestine.

Fatty acid binding protein of epidermal type (E-FABP) was expressed/localized in most, if not all, populations of the dendritic cells in the subepithelial domes, follicles and interfollicular regions of Peyer's patches and presumptive macrophages in their germinal centers, and all M cells in the follicle-associated epithelium of mouse intestine. The immunoreactivity in both of the cell populations makes it easy to recognize the accumulation of DCs in the subepithelial domes in close proximity to the base of M cells, which is essential for luminal antigens to be transported to Peyer's patches. E-FABP may play some important roles in the mucosal immune reaction through Peyer's patches and associated structures.

Discrete localization of various fatty-acid-binding proteins in various cell populations of mouse retina.

Various fatty acids (FAs) are involved as an energy source in many different functions in the organism. They are also essential ingredients of membranous lipids and act as intracellular signaling molecules. Intracellular fatty-acid-binding proteins (FABPs) comprise a family of soluble lipid-binding proteins with low molecular masses and solubilize long-chain FAs to allow intracellular translocation in the aqueous cytosol. To clarify the functions of FABPs in the retina, which is remarkably rich in polyunsaturated FAs, we have investigated the localization of B (brain type)-, H (heart type)-, E (epidermal type)-, and A (adipocyte type)-FABPs in adult mouse retinae by immunohistochemistry. In order to determine the possible involvement of FABPs in retinal degenerative diseases, we have also examined changes in FABP expression in light-induced photoreceptor cell degeneration (photic injury). The discrete localization of B-, H-, E-, and A-FABP species in various cell populations of the retina has been clarified: B-FABP is mainly localized in the cone photoreceptor cells, H-FABP in some populations of amacrine/bipolar/horizontal interneurons, and E-FABP in ganglion cells, with A-FABP-like immunoreactivity being located in resident microglia of normal retinae. E-FABP has further been localized in invasive macrophages in damaged retinae following photic injury, allowing discrete identification of the resident microglia and invasive macrophages by A- and E-FABP immunoreactivity, respectively.

Handedness: dependent asymmetrical location of the human primary gustatory area, area G.

In magnetoencephalogram studies, the primary gustatory area, area G, is not always seen in the same coronal plane in both hemispheres. We investigated possible asymmetry in right-handed and left-handed individuals by functional MRI. Group analyses revealed a significant difference in the antero-posterior coordinates of the area G between the right and left hemispheres in the right-handed group, but not in the left-handed group, indicating significant morphometric asymmetry in the former group and ambiguous morphometric asymmetry in the latter. However, in left-handed individuals with motor speech areas detected in the right hemisphere, area G was more posteriorly located in the right than in the left hemisphere. These findings suggest that the motor speech area contributes to the asymmetric location of area G.

Localization of EFA6A, a guanine nucleotide exchange factor for ARF6, in spermatogenic cells of testes of adult mice.

ADP ribosylation factors (ARFs) of small GTPase are molecular switches regulating various membrane dynamics. Among them, ARF6 has recently been highlighted because of its function to facilitate the interaction between the cytoskeleton and the plasma membrane. Each ARFs has its preferable or even specific guanine nucleotide exchange factors (GEFs) as its activators. According to our previous RT-PCR analysis, EFA6A, a guanine nucleotide exchange factor for ARF6, was restrictedly expressed in the brain, retina and testis. Different from previous studies on neurons, EFA6A, a guanine nucleotide exchange factor for ARF6, was first shown to be localized intensely in nuclei of spermatocytes of adult mouse testes in the present immunohistochemical study. This suggests a possible involvement of EFA6A-ARF6 signaling in the karyokinesis and cytokinesis.

Smell identification in Japanese Parkinson's disease patients: using the odor stick identification test for Japanese subjects.

OBJECTIVE: The purpose of the present study was to clarify the olfactory functions of Japanese patients with idiopathic Parkinson's disease (IPD) using the odor stick identification test for Japanese (OSIT-J). METHODS: Fifty-four non-demented IPD patients (33 men and 21 women), ranging in age from 43 to 81 years (69.7+/-8.1 years) and 50 age- and gender-matched healthy controls who reported having no olfactory complaints were enrolled. OSIT-J consisted of 12 odorants familiar to Japanese subjects. Each subject sniffed each odor that was applied to paraffin paper. Next the subject chose 1 of 6 answers: 4 pictures associated with the odors labeled with their names, one of which was correct, and 2 other ones ("unknown" and "not detected"). RESULTS: The number of correct answers was significantly lower in the IPD group (4.4+/-2.7) than in the normal group (8.3+/-2.2) (p<0.0001). Even in IPD patients who could smell normal strength odors in subjective symptom, the number of correct answers decreased. The number of correct answers was not correlated with motor function, disease duration, or medication. CONCLUSION: The present study demonstrated that the smell identification ability of Japanese IPD patients was impaired based on the OSIT-J.

First disclosure of lipid droplet substructure and myelin translucency in embedment-free section electron microscopy.

In conventional transmission electron microscopy (EM), thinly sectioned specimens embedded in epoxy resin are observed. However, because of a substantial level of electron density of epoxy resin, the possibility cannot be ruled out that bio-structures having electron density similar to that of epoxy resin are not clearly recognized and thus are neglected or misinterpreted in conventional EM. This was the reason to require for embedment-free EM. Embedment-free sections have already been made available reliably by transient embedding in polyethylene glycol (PEG) and subsequent de-embedding through immersion in water, and further by critical-point drying, and this embedment-free EM is thus termed PEG-EM. However, this PEG-EM has not been successful to attract reasonable attention from electron microscopists and instead been misunderstood as a non-reliable method. In this paper, the remarkably enhanced contrast and electron translucency of any observation targets in PEG-EM are clearly demonstrated by comparing with images in conventional EM of adipocytes and neural myelin as examples. These features of PEG-EM, together with faithful correspondence in EM images of any individual substructures between the two methods, confirm the reliability of PEG-EM. Furthermore, the much higher thickness of embedment-free sections together with these features makes the PEG-EM more advantageous than the conventional EM for three-dimensional appreciation of structural elements, which is made by stereo-viewing of sections or by EM tomography. Therefore, the PEG-EM is regarded as an important adjunct to the conventional EM for histological studies and wide application of this method may unravel a new level of histology.

Lipid messenger, diacylglycerol, and its regulator, diacylglycerol kinase, in cells, organs, and animals: history and perspective.

Diacylglycerol kinase (DGK) metabolizes diacylglycerol (DG), a glycerolipid containing two acyl chains, to convert phosphatidic acid. DG is produced through phosphoinositide turnover within the membrane and is well known to act as a second messenger that modulates the activity of protein kinase C in the cellular signal transduction. Recent studies have revealed that DG also activates several proteins, including Ras guanine-nucleotide releasing protein and ion channels such as transient receptor potential proteins. Therefore, DGK is thought to participate in a number of signaling cascades by modulating levels of DG. Previous studies have disclosed that DGK is composed of a family of the isozymes, which differ in the structure, enzymological property, gene expression and localization, subcellular localization, and binding molecules. The present review focuses on the stories of phosphoinositide turnover and DG, including historical views, structural features, metabolism, and relevant cellular phenomena, together with the characteristics of DGK isozymes and the pathophysiological findings on animal studies using knockout mice and models for human diseases. Now it is being revealed that the structural and functional diversity and heterogeneity of and around DGK support the proper arrangement of the complex signal transduction machinery.

Visualization of spatiotemporal differentiation of dopaminergic interneurons in adult mouse olfactory bulb using transgenic mice.

Olfactory bulb (OB) interneurons, predominantly periglomerular (PG) and granule (GR) cells, are derived from neural stem cells in the subventricular zone (SVZ) throughout life and migrate to the OB in the rostral migratory stream (RMS). In the adult superficial GR layer transgene expression was found, either enhanced green fluorescent protein or LacZ reporter driven by a 9 kb tyrosine hydroxylase (TH) promoter, that marked the dopamine (DA) phenotype. To demonstrate that the reporters and endogenous TH were similarly regulated expression of both parameters was shown to decline in the OB PG cells ipsilateral to odor deprivation produced by adult unilateral naris closure. The present findings suggested that DAergic differentiation might begin prior to progenitors reaching a PG position despite evidence that TH protein expression occurred only after PG cells received olfactory afferent stimulation. Of many genes previously hypothesized to regulate OB DA expression, regulated expression of the orphan receptor, Nurr1, but not the homeobox-containing genes, Dlx-1 and -2, was consistent with a role in regulation of the DA phenotype in adult mice OB. The studies show that transgenic lines are useful for analyzing spatiotemporal regulation by both intrinsic (programmed) and environmental factors in the neurogenesis of adult mouse OB DAergic interneurons.