Folate receptor-specific cell-cell adhesion by using a folate-modified peptide-based anchor.

We report here a folate-modified membrane anchor for cell surface modification to induce cell adhesion to target cells. The membrane anchor region, which was consisted of cationic lysine residues and palmitoyl group-modified residues, was modified with folate through an oligoethlene glycol linker. The peptide anchor was modified on to the cell membrane by using ß-cyclodextrin as a solubilizer of the peptide anchor. After modification, the peptide anchor disappeared from the cell membrane via endocytotic uptake or dissociation from the cell membrane. However, the endocytosed peptide was represented on the cell surface via recycling endosome pathway. The obtained folate-modified cells successfully adhered on to target cells which expressed folate receptor α via ligand-receptor specific interaction and adhesion continued at least 4 hours.

Facilitating the presentation of antigen peptides on dendritic cells for cancer immunotherapy using a polymer-based synthetic receptor.

The introduction of proteins into dendritic cells (DCs) ex vivo is a critical step for the DC-based immunotherapy of cancer. Here, we developed a biotin-modified polymer with multiple hydrophobic membrane anchors for cells that functions as a synthetic receptor for an antigen protein, ovalbumin (OVA), to introduce it efficiently into DCs compared with the conventional pulsing method. Our method showed significant advantages, including the rapid incorporation of OVA and the activation of antigen-specific T cells in a MHC-restricted manner. When mice were immunized by DCs treated with our method, tumor growth was completely suppressed, indicating that our method can be used to prepare adjuvant DCs.

Optimum design of amphiphilic polymers bearing hydrophobic groups for both cell surface ligand presentation and intercellular cross-linking.

Amphiphilic polymers bearing hydrophobic alkyl groups are expected to be applicable for both ligand presentation on the cell surface and intercellular crosslinking. To explore the optimum design for each application, we synthesized eight different acyl-modified dextrans with varying molecular weight, alkyl length, and alkyl modification degree. We found that the behenate-modified polymers retained on the cell surface longer than the palmitate-modified ones. Since the polymers were also modified with biotin, streptavidin can be presented on the cell surface through biotin-streptavidin recognition. The duration of streptavidin on the cell surface is longer in the behenate-modified polymer than the palmitate-modified one. As for the intercellular crosslinking, the palmitate-modified polymers were more efficient than the behenate-modified polymers. The findings in this research will be helpful to design the acyl-modified polymers for the cell surface engineering.

Rapid and serum-insensitive endocytotic delivery of proteins using biotinylated polymers attached via multivalent hydrophobic anchors.

We have designed biotinylated polymers as synthetic receptors that have multiple alkyl groups for endocytotic delivery of target proteins. The polymers were stably attached to a cell surface via multivalent anchoring. The presented biotin was bound to streptavidin (SA) on the cell surface, and, via an endocytotic pathway, the cell rapidly internalized the biotinylated polymer/SA complex. The cell's uptake of the complex was not inhibited by the presence of 10% fetal bovine serum, and its efficacy for the uptake of SA was the highest when compared with commercial reagents and single-anchored-type synthetic receptors. The synthetic receptor-mediated endocytosis can be used generally for other kind of protein by using SA as an adaptor molecule between a target protein and the cell-surface presented biotin.

Selective inhibition of human type-5 17ß-hydroxysteroid dehydrogenase (AKR1C3) by baccharin, a component of Brazilian propolis.

The human aldo-keto reductase (AKR) 1C3, also known as type-5 17ß-hydroxysteroid dehydrogenase and prostaglandin F synthase, has been suggested as a therapeutic target in the treatment of prostate and breast cancers. In this study, AKR1C3 inhibition was examined by Brazilian propolis-derived cinnamic acid derivatives that show potential antitumor activity, and it was found that baccharin (1) is a potent competitive inhibitor (K(i) 56 nM) with high selectivity, showing no significant inhibition toward other AKR1C isoforms (AKR1C1, AKR1C2, and AKR1C4). Molecular docking and site-directed mutagenesis studies suggested that the nonconserved residues Ser118, Met120, and Phe311 in AKR1C3 are important for determining the inhibitory potency and selectivity of 1. The AKR1C3-mediated metabolism of 17-ketosteroid and farnesal in cancer cells was inhibited by 1, which was effective from 0.2 µM with an IC(50) value of about 30 µM. Additionally, 1 suppressed the proliferation of PC3 prostatic cancer cells stimulated by AKR1C3 overexpression. This study is the first demonstration that 1 is a highly selective inhibitor of AKR1C3.

Formation of interacting spins on flavosemiquinone and tyrosine radical in photoreaction of a blue light sensor BLUF protein TePixD.

Light-induced radicals were detected by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) in the BLUF-domain protein TePixD of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The illumination of TePixD at 5-200 K derived an EPR signal with a separation of 85 G between the main peaks around g = 2, showing a typical Pake's pattern of magnetic dipole-dipole interaction between two nearby radicals. Longer illumination induced an EPR signal at g = 2.0045, which was assigned as a neutral flavosemiquinone FADH(*). The FADH(*) formation occurred in parallel with a decrease in Pake's doublet. The Pake's doublet was not detected in a mutant TePixD protein in which a tyrosine residue was replaced with phenylalanine (Y8F protein). A pulsed ENDOR study suggested that the Pake's doublet had arisen from the interaction between a neutral flavosemiquinone radical and a neutral tyrosine radical, i.e., the FADH(*)-Y8(*) state. An EPR simulation of the Pake's doublet showed that the distance between FAD and Y8 is 2.2 A shorter than that calculated from the X-ray crystallography structure in the dark-adapted state, suggesting the modification of the protein conformation in the photoinduced FADH(*)-Y8(*) state. The Pake's doublet signal was detected by 10 K illumination in the sample which was immediately frozen after 273 K illumination, corresponding to the red-shifted state F(490). On the other hand, the signal was not detected in the sample which was incubated for 10 min at 273 K in the dark after 273 K illumination, corresponding to the dark-adapted state D(471). In the sample annealed at 160 K for 10 min after 160 K illumination, corresponding to the partially red-shifted state J(11), the Pake's doublet signal was detected by the 10 K illumination. On the basis of these observations, we concluded that the interaction with the FADH(*)-Y8(*) state occurred after the second photoexcitation of the photoinduced red-shifted states in the photocycle of TePixD.

[Successful delivery following subcutaneous heparin administration in a 7-weeks pregnant patient suffering from cerebral venous thrombosis due to secondary protein S deficiency].

A 25-year-old, 7-weeks pregnant woman was admitted to the Nagaoka Red Cross Hospital in a state of confusion, following fever, headache and vomiting. Brain CT and MRI showed swelling in the bilateral thalami, basal ganglia and splenium of corpus callosum, and thrombosis of the internal cerebral veins and straight sinus. Initial treatment by intravenous heparin and glycerol was successful, and she regained her consciousness, leaving antegrade amnesia and childish character change. Her free protein S antigen was 32% (normal 60-127) and subsequently rose to 70% after delivery. She was diagnosed as having secondary protein S deficiency associated with pregnancy. Because warfarin can be teratogenic, subcutaneous heparin injection was prescribed in order to prevent thrombosis and the patient subsequently had a successful delivery. This was the first case in Japanese of successful delivery after subcutaneous heparin treatment in a patient with cerebral venous thrombosis.

[A case of acute distigmine bromide intoxication in the therapeutic dosage for treatment of underactive neurogenic bladder].

Distigmine bromide (Ubretid) is a long-acting anti-cholinesterase, widely used for the treatment of underactive neurogenic bladder and myasthenia gravis. Our study concerns a 73-year-old man treated with a potentially life-threatening cholinergic state due to distigmine bromide. He had been administered distigmine bromide orally for over two years at a daily dosage of 10 mg as a treatment for underactive neurogenic bladder. He suddenly developed diarrhea and consciousness disturbance during treatment of his urinary tract infection. Bradycardia and miosis were noted. Blood examination revealed extremely low levels of the plasma cholinesterase activity. The condition was diagnosed as distigmine bromide intoxication. All cholinergic symptoms disappeared in several days after the administration of distigmine bromide was terminated. Cholinergic crisis due to overdosage with anticholinesterases is well known, and the myasthenic patients are usually supervised in the early stages of dosage regulation to guard against the possibility of cholinergic crisis. However the use of oral distigmine bromide, even in therapeutic doses for urinary retention, could result in cholinergic crisis. We therefore conclude that extreme caution must be used in administering distigmine bromide.

[Factors related to discrepancy in evaluation on functional capacity between reports by community-dwelling older people with cognitive decline and their family members].

Overestimation or underestimation of functional capacity in community-dwelling older people with cognitive impairment was evaluated between the responses of subjects and family members (proxies) by cognitive function level. Out of all the residents aged 65 years and over living in Yoita town, Niigata Prefecture in 2000 (n = 1,673), 1,544 voluntarily participated in the interview survey held at community halls or at home (92.3% response). They underwent the Mini-Mental State Examination (MMSE) for assessment of cognitive function and answered questionnaires comprising socio-demographic, psychological, physical and medical, and social activity items (2000/11). According to the age of the subject and MMSE score, we defined cognitive decline (MMSE scores < 1 SD below age-specific means, n = 371). 158 pairs among 371 subjects with cognitive decline and their proxies participated in a follow-up survey (2001/11). The subjects themselves underwent MMSE again. 136 subject-proxy pairs reported any complaints of memory-related problem and evaluated higher-level functional capacity (TMIG-IC, Tokyo Metropolitan Institute of Gerontology Index of Competence). We established criteria at follow-up survey as follows: control (n = 29), MMSE scores > 1 SD below age-specific means and CDR (Clinical Dementia Rating) = 0: mild cognitive decline (MCD) (n = 54), 21 < or = MMSE scores < 1 SD below age-specific means or CDR = 0.5); and severe cognitive decline (SCD) (n = 53), MMSE scores 20 < or = CDR > 0.5. SCD subjects significantly overestimated total and Instrumental Self-Maintenance scores in TMIG-IC more than control or SCD subjects. Multiple logistic regression analyses indicated that complaints of memory by the proxy, response by spouse, and higher levels of education were extracted as significantly independent variables affecting overestimation for functional capacity. On the other hand, aging affected underestimation.

[Toward constructing a system for detecting and coping with senile dementia in early stages among community-dwelling older people].

OBJECTIVES: To establish a community health care system for screening community-dwelling older people with mild cognitive decline (MCD) and early diagnosis by a medical specialist. METHODS: Out of all the elderly residents aged 65 years and over living in Yoita town in 2000 (n = 1673), 1544 participated in the interview survey held at community halls or at home (92.3% response). They underwent a Mini-Mental State Examination (MMSE) for assessment of cognitive function and answered questionnaires covering socio-demographic, psychological, physical and medical, and social activity items (2000/11). We defined cognitive decline as an MMSE < or = 1 SD below the age-specific mean (n = 371). Out of a total eligible population of 332, 158 pairs of subjects and their proxies participated in a follow-up survey (2001/11). The subjects themselves underwent MMSE again, and were asked for complaints of memory-related problems. Proxies answered about functional capacity and memory-related problems of subjects, for which we assessed the level of dementia with the Clinical Dementia Rating (CDR). We established criteria for encouragement to undergo detailed examination by a medical specialist as follows. 1) MMSE scores < or = 1 SD below age-specific means at both baseline and follow-up surveys, or 2) CDR > or = 0.5, and 3) not due to mental retardation. RESULTS: Non-participants had significant higher MMSE scores but were younger in the follow-up survey. Out of 96 subjects eligible for the detailed examination, 47 participants showed a tendency for older age with lower MMSE scores or younger age with higher MMSE scores than the average in the follow-up survey. The detailed examinations confirmed dementia of Alzheimer's type in 22 and vascular dementia in 13 on DSM-IV or magnetic resonance imaging. During our screening, 8 community care saloons were opened and the number of facilities for older people with cognitive decline is increasing. CONCLUSIONS: To establish a community health care system to screen and cope with community-dwelling older people with MCD in earlier stages, increased awareness of residents with slight MCD is especially important. Our attempt also suggested the importance of educational lectures, methods for testing cognitive function, and dementia care activity.

[MRI abnormalities of the oculomotor nerve in a case of ophthalmoplegic migraine].

A 15-year-old girl presented with headache in the left retro-orbital area associated with double vision. She had a history of migraine headaches since the age of 9, and her mother also had suffered from migraine. The patient had experienced a similar episode at the age of 13, which resolved without any treatment in a day. On examination, two days after the onset of diplopia the patient had left ptosis and paresis of inwards and downwards in the left eye. Her pupils were isocoric, and the light reflex was prompt. MRI of the brain showed thickening and enhancement of the left third nerve through the cistern portion, especially at the oculomotor midbrain exit. No cavernous sinus involvement was noted. Results of the hemogram, ESR, and CSF were normal. The oculomotor palsy resolved spontaneously within six days. A follow-up MRI two months later demonstrated a marked lessening of enhancement at the oculomotor nerve but did not completely disappeared. These serial MRI abnormalities are thought to be important and typical findings seen in the patients of ophthalmoplegic migraine.

[Successful use of mild hypothermia therapy in an adult patient of non-herpetic acute encephalitis with severe intracranial hypertension].

A 41-year-old man was admitted to our hospital suffering from generalized convulsion with a high fever and disturbed consciousness one week after exhibiting flu-like symptoms. We made a diagnosis of acute viral encephalitis, based on the clinical features and the evidence of pleocytosis with an increase in protein in the CSF. On admission, MRI was normal and CRP was negative. The levels of transaminase, ammonia, and blood sugar were normal, so that an adult Reye's syndrome could be ruled out. Herpes simplex encephalitis and influenza encephalopathy were also ruled out because of viral examinations, and specific agents could not be determined. Clinical symptoms subsided once after he was treated with dexamethasone, acyclovir, and anti-convulsants, until generalized convulsion accompanied by a high fever again occurred on the 9th day. On the 18th day, the patient showed anisocoria and ataxic respiration due to severe brain edema. Mild hypothermia therapy to rectal temperature 35 degrees C was induced under mechanical ventilation. Cranial CT taken 3 days after the therapy began to show the improvement of the brain edema. After 7 days of the therapy, his clinical symptoms began to recover dramatically. On the 46th day, he was discharged from hospital without showing almost any neurological symptoms. Mild hypothermia therapy should be considered for adult patients as well as non-adult patients suffering from acute encephalitis with severe intracranial hypertension.

[A Japanese case of Streptococcus suis meningitis associated with lumbar epidural abscess].

A 47-year-old man working at a meat processing factory was admitted to our hospital after suffering high fever and severe headache for a 2-day period. He was alert and showed no neurological symptoms apart from neck stiffness. The CSF was turbid, with 701/microliter of polymorphnuclear leukocytes, a protein concentration of 218 mg/ml, and a sugar concentration of 33 mg/dl. He was diagnosed as suffering from Streptococcus suis (S. suis) serotype 2 meningitis based on CSF culture. Severe headache and high fever subsided after the treatment with intravenous antibiotics and immunoglobin. He developed, however, severe lumbargo on the third day, which was proven to be caused by the lumbar epidural abscess. Also, on the 8th day he developed hearing loss. These symptoms were treated and ameliorated with the administration of dexamethasone. On the 73rd day, the patient was discharged in good clinical condition, although he still suffered from a decrease in hearing perception. S. suis, found as a commensal in the nasopharynx of swine, is responsible for meningitis in the people who are in close contact with swine or unprocessed pork. S. suis meningitis seems to be the result of a preventable occupational infection. Although most of the patients come from other countries in Asia, our patient is the first documented case occurring in Japan. Those who have an occupational exposure to swine and pork are strongly recommended to use protective gloves. Prevention through public health surveillance is important.

Detection and identification of Mycobacterium species isolates by DNA microarray.

Rapid identification of Mycobacterium species isolates is necessary for the effective management of tuberculosis. Recently, analysis of DNA gyrase B subunit (gyrB) genes has been identified as a suitable means for the identification of bacterial species. We describe a microarray assay based on gyrB gene sequences that can be used for the identification of Mycobacteria species. Primers specific for a gyrB gene region common to all mycobacteria were synthesized and used for PCR amplification of DNA purified from clinical samples. A set of oligonucleotide probes for specific gyrB gene regions was developed for the identification of 14 Mycobacterium species. Each probe was spotted onto a silylated glass slide with an arrayer and used for hybridization with fluorescently labeled RNA derived from amplified sample DNA to yield a pattern of positive spots. This microarray produced unique hybridization patterns for each species of mycobacteria and could differentiate closely related bacterial species. Moreover, the results corresponded well with those obtained by the conventional culture method for the detection of mycobacteria. We conclude that a gyrB-based microarray can rapidly detect and identify closely related mycobacterial species and may be useful in the diagnosis and effective management of tuberculosis.

Orthopedic treatment of metastatic bone fracture from hepatocellular carcinoma.

We report on a 73-year-old female patient with a small hepatocellular carcinoma, which was detected by ultrasonography in September 1993. She was treated by ethanol injection and repeated courses of transcatheter arterial embolization for local recurrence. Five years after the initial treatment of the primary hepatocellular carcinoma in 1998, a solitary metastatic tumor from hepatocellular carcinoma was detected in the middle of the left humerus by fluorine-18-fluoro-2-deoxy-D glucose positron emission tomography and technetium-99m methylene diphosphonate bone scintigraphy. About one year later, bone fracture occurred at the site of metastatic lesion in spite of local radiation therapy. Reconstruction by three enderpines relieved the fracture-associated pain and improved the quality of life. The patient continued to enjoy a good quality of life without pain until death by liver failure in September 2000.

[A case of Hashimoto's encephalopathy associated with Graves' disease].

A 23-year-old woman with Graves' disease was first admitted to a hospital because of generalized convulsion, consciousness disturbance, and tachycardia. Investigations showed biochemical hyperthyroidism and positive thyroid binding inhibitory immunoglobulin (TBII). She was treated with anticonvulsant and antithyroid therapy. One month later the patient developed the second episode of generalized convulsion, followed by cognitive deficits especially concerning short-term memory, when she was transferred to our hospital. Although thyroid function was normalized at that time, anti-thyroid peroxidase (TPO) antibodies level were elevated to 5,850 U/ml in the serum, and 4.9 IU/ml in CSF. CSF protein was elevated to 133 mg/dl. Brain SPECT showed global decrease of cerebral perfusion, and EEG demonstrated diffuse slowing of the background rhythm without any signs of epileptic activity, although brain MRI was normal. Based on a diagnosis of Hashimoto's encephalopathy associated with Graves' disease, therapy with steroids was started, which definitely improved her clinical symptoms. Anti-TPO antibodies disappeared in CSF and decreased in the serum after the treatment. Hashimoto's encephalopathy should always be kept in mind in patients with Hashimoto's disease and Graves' disease.