Scaling up nano-milling of poorly water soluble compounds using a rotation/revolution pulverizer.

We previously reported that a rotation/revolution pulverizer (NP-100) could mill a small amount of a drug (0.1 g) into nanoparticles in several minutes. In this investigation, scale up from the milligram to the kilogram scale of the nano-milling process by the rotation/revolution pulverizer was studied. Phenytoin was used as a model drug with low solubility in water. After confirming the improvement of the phenytoin bioavailability by milling to nanoparticles using NP-100, scaling parameters were evaluated using NP-100 and the middle scale model of NP-100 (ARV-3000T). A theoretical equation for the specific collisional energy was adapted for wet milling; this suggested that the relative centrifugal acceleration of revolution (revolution G) and the drug concentration in the suspension were the two most important parameters. The results obtained using NP-100 and ARV-3000T correlated well when these two parameters were identical. These results were applied to the large scale model of NP-100 (ARV-10KT), where 2 kg (1 kg x 2) of phenytoin nanoparticles were obtained in 60 min. The results from PXRD and DSC indicated that the milled phenytoin by ARV-3000T and ARV-10KT maintained its crystallinity. These results suggest nano-milling using a rotation/revolution pulverizer will be widely applicable to the development of nano-medicine.

Safety and efficacy of edoxaban, an oral factor xa inhibitor, for thromboprophylaxis after total hip arthroplasty in Japan and Taiwan.

Edoxaban, an oral direct factor Xa inhibitor, has proven antithrombotic efficacy. In a multicenter, phase II study, 264 total hip arthroplasty (THA) patients randomly received edoxaban 15 or 30 mg once daily or enoxaparin 2000IU (20-mg) twice daily for 11-14 days. Thromboembolic event incidences were 3.8% (3/78), 2.8% (2/72), and 4.1% (3/74) for edoxaban 15-mg, 30-mg, and enoxaparin, respectively (P=1.00). Edoxaban-induced prolongation of prothrombin time, international normalized ratio, and activated partial thromboplastin time were proportional to plasma edoxaban concentration. Major or clinically relevant non-major bleeding incidences were 2.2% (2/89), 1.2% (1/85), and 2.3% (2/87) for edoxaban 15-mg, 30-mg, and enoxaparin, respectively (P=1.00). Once-daily edoxaban showed similar efficacy and safety to enoxaparin for prevention of thromboembolic events in patients undergoing THA.

Pebbles and sand on asteroid (162173) Ryugu: In situ observation and particles returned to Earth.

The Hayabusa2 spacecraft investigated the C-type (carbonaceous) asteroid (162173) Ryugu. The mission performed two landing operations to collect samples of surface and subsurface material, the latter exposed by an artificial impact. We present images of the second touchdown site, finding that ejecta from the impact crater was present at the sample location. Surface pebbles at both landing sites show morphological variations ranging from rugged to smooth, similar to Ryugu's boulders, and shapes from quasi-spherical to flattened. The samples were returned to Earth on 6 December 2020. We describe the morphology of >5 grams of returned pebbles and sand. Their diverse color, shape, and structure are consistent with the observed materials of Ryugu; we conclude that they are a representative sample of the asteroid.

D-serine regulation: a possible therapeutic approach for central nervous diseases and chronic pain.

D-Serine, an endogenous modulator of NMDA receptors has been shown to play a vital role in many neuropsychiatric functions such as learning, memory, nociception and implicated in pathological conditions like schizophrenia and Alzheimer's disease. We propose possible therapeutic approaches for some CNS diseases and chronic pain, targeting the D-serine levels by manipulating its uptake, biosynthesis and metabolism.

Hayabusa2 arrives at the carbonaceous asteroid 162173 Ryugu-A spinning top-shaped rubble pile.

The Hayabusa2 spacecraft arrived at the near-Earth carbonaceous asteroid 162173 Ryugu in 2018. We present Hayabusa2 observations of Ryugu's shape, mass, and geomorphology. Ryugu has an oblate "spinning top" shape, with a prominent circular equatorial ridge. Its bulk density, 1.19 ± 0.02 grams per cubic centimeter, indicates a high-porosity (>50%) interior. Large surface boulders suggest a rubble-pile structure. Surface slope analysis shows Ryugu's shape may have been produced from having once spun at twice the current rate. Coupled with the observed global material homogeneity, this suggests that Ryugu was reshaped by centrifugally induced deformation during a period of rapid rotation. From these remote-sensing investigations, we identified a suitable sample collection site on the equatorial ridge.

Nocistatin attenuated the nociceptin induced c-Fos expression in the mouse hippocampus.

Nocistatin and nociceptin/orphaninFQ (N/OFQ) are the two new peptides which may have roles in nociception, memory, anxiety, and other biological functions. Nocistatin acts as a functional antagonist to N/OFQ in several functions, but their neuro-anatomical sites of interaction are unknown. We investigated the effect of combined intracerebroventricular (i.c.v.) injection of nocistatin with N/OFQ, on N/OFQ induced c-Fos expression in the mouse hippocampus, using c-Fos immunohistochemistry. We found that co-injection of nocistatin with N/OFQ significantly attenuated N/OFQ induced c-Fos expression in the hippocampus.

Non-destructive elemental analysis of a carbonaceous chondrite with direct current Muon beam at MuSIC.

Electron- or X-ray-induced characteristic X-ray analysis has been widely used to determine chemical compositions of materials in vast research fields. In recent years, analysis of characteristic X-rays from muonic atoms, in which a muon is captured, has attracted attention because both a muon beam and a muon-induced characteristic X-ray have high transmission abilities. Here we report the first non-destructive elemental analysis of a carbonaceous chondrite using one of the world-leading intense direct current muon beam source (MuSIC; MUon Science Innovative Channel). We successfully detected characteristic muonic X-rays of Mg, Si, Fe, O, S and C from Jbilet Winselwan CM chondrite, of which carbon content is about 2 wt%, and the obtained elemental abundance pattern was consistent with that of CM chondrites. Because of its high sensitivity to carbon, non-destructive elemental analysis with a muon beam can be a novel powerful tool to characterize future retuned samples from carbonaceous asteroids.

Lhermitte sign during yawning associated with congenital partial aplasia of the posterior arch of the atlas.

We describe the case of a 26-year-old man who presented with symptoms compatible with Lhermitte sign that occurred during yawning. It was associated with congenital partial aplasia of the posterior arch of the atlas. Cervical multisection-detector CT myelography during yawning showed compression of the upper cervical cord due to the inward mobility of the isolated posterior tubercle. The symptoms completely disappeared following removal of the isolated posterior tubercle.

Clinical significance of CD151 gene expression in non-small cell lung cancer.

Transmembrane 4 superfamily (TM4SF) is a recently described gene family, and TM4SF members are known to play roles in the signal transduction pathways and to regulate cell activation, development, proliferation, and motility. MRP-1/CD9, KAI1/CD82, and ME491/CD63, members of the TM4SF, have been reported to suppress tumor progression or metastasis. Previously, we showed that MRP-1/CD9 suppressed cell motility and metastatic potential to lungs. Moreover, reduction of MRP-1/CD9 and KAI1/CD82 gene expression was found to be a factor in a poor prognosis for patients with non-small cell lung cancer. However, among TM4SF, CD151 is identical to an existing gene, PETA-3, which may promote tumor metastasis of malignant cells, and its expression may be involved in the malignant progression of cancer. The function of CD151 is opposite that of the metastasis suppressor genes, MRP-1/CD9 and KAI1/CD82. On the basis of these results, we used reverse transcription-PCR and immunohistochemical techniques for a retrospective study of CD151 gene expression in tumor tissues from 145 lung cancer patients; 72 tumors were stage I, 29 stage II, 27 stage IIIA, and 17 stage IIIB. Whereas 86 patients had tumors positive for the CD151 gene, 59 had tumors that were negative for the CD151 gene. The overall survival rate of patients with CD151-positive tumors was much lower than that of CD151-negative patients (51.9% versus 73.1%; P = 0.013). Our findings suggest that high CD151 gene expression in lung cancer may be associated with a poor prognosis. Assessment of CD151 could be instrumental for improvements in lung cancer diagnosis and therapies.

An androgen receptor mutation causing androgen resistance in undervirilized male syndrome.

The molecular basis of androgen resistance was investigated in a patient with undervirilized male syndrome. Binding studies of the androgen receptors in the patient's genital skin fibroblasts revealed a normal binding capacity of 5 alpha-dihydrotestosterone, although the affinity to androgen was slightly lower than the normal control value. The androgen binding of the patient's receptor showed a moderate thermal instability when the assay temperature was raised from 30 to 41 C. Nucleotide sequencing analysis of the androgen receptor gene revealed a single nucleotide substitution in exon F, resulting in an amino acid alteration from leucine (CTC) to phenylalanine (TTC) at position 789 within the steroid-binding domain of androgen receptor. When expressed in COS-7 cells, the mutant androgen receptor harboring phenylalanine at position 789 showed thermolabile androgen-binding properties similar to those observed in the patient's genital skin fibroblasts. Cotransfection experiments with an androgen-inducible reporter gene demonstrated a decreased transactivational capability of the mutant receptor. These results indicate that this point mutation modified the receptor function and caused androgen resistance in this patient. This mutation caused the mildest form of all androgen insensitivity syndromes ever examined for mutations in the androgen receptor gene.

An unusual repetitive structure of caerulein mRNA from the skin of Xenopus laevis.

The nucleotide sequence of a 784-bp segment of cloned caerulein mRNA obtained from the skin of Xenopus laevis was determined. It codes for five heterogeneous procaerulein peptides interspersed with three 147-bp intercaerulein segments (ICS). The ICSs contain six inverted repeats and five eukaryotic enhancer-like sequences. Evidence for the presence of multiple forms of caerulein mRNA is presented.

Familial carpal tunnel syndrome due to amyloidogenic transthyretin His 114 variant.

We studied two patients from a Japanese family with carpal tunnel syndrome (CTS). The biopsy samples obtained during CTS surgical release revealed deposits of amyloid that stained with antihuman transthyretin (TTR) antiserum. Single-strand conformation polymorphism analysis and sequence analysis of polymerase chain reaction (PCR)-amplified exons of the proband's TTR gene revealed a point mutation resulting in a substitution of histidine for tyrosine at position 114. The mutation was confirmed by PCR-primer-induced restriction analysis. Our findings account for clinical heterogeneity of TTR-derived amyloidosis, and suggest the importance of substitution itself for deposits of amyloid in CTS.

A new direct solid-phase radioimmunoassay for carcinoembryonic antigen without pretreatment of serum samples.

We examined some fundamental conditions for establishing a new simplified radioimmunoassay system for CEA. The system consists of antibody-coated plastic beads and radiolabeled tracer antibody. The reactivity of this system with several purified CEA preparations can vary due to differences in the nature of antibody preparations used either for coating beads or for tracer antibody. Among purified CEA preparations, we also found some differences in reactivity with a given immobilized antibody preparation. The assay system that resulted, referred to as bead-direct-RIA, needs neither pretreatment of serum samples nor centrifugation in any of its steps, and has a wide assay range, measuring CEA concentrations of 1-300 ng/ml using only 50 microliters of a serum sample. Its accuracy, reproducibility, dilution effect, and analytical recovery proved the reliability of this method. The mean value +/- S.D. for 48 normal adults was 2.5 +/- 1.0 ng/ml. It was found by comparative studies that the Z-gel method of Hoffmann-La Roche generally gave the highest values, the antibody-disc method of Dainabot gave the lowest, and the values by bead-direct-RIA fell between those of the 2 other methods and correlated well with either value. Follow-up studies on patients with malignancy indicated that the values by bead-direct-RIA fluctuated in parallel with those by Roche and Dainabot kits.

Synthesis and structure-activity relationships of dynorphin A-(1-8) amide analogues.

In order to study the structure-activity relationships of dynorphin A-(1-8) amide [Dyn(1-8)-NH2], 20 analogues were synthesized by the solution method. Their biological activities were determined in the three bioassays [guinea pig ileum (GPI), mouse vas deferens (MVD), and rabbit vas deferens (RVD)] and in the mouse tail-pinch test after intravenous administration. Some analogues that showed interesting activity in the bioassays and/or in the analgesic tests were further characterized in mu-, delta-, and kappa-representative binding assays. The obtained data indicate that modification of the enkephalin segment to give metabolically stable analogues with high affinity and selectivity for the kappa receptor is strictly limited and that introduction of MeArg in position 7 protects the Arg6-Arg-7 bond from enzymatic degradation without potency drop and change of opioid receptor selectivity. [MeTyr1,MeArg7,D-Leu8]Dyn(1-8)-NHEt (18) [IC50 (nM) = 0.3 (GPI), 7.4 (MVD), and 2.6 (RVD); tail pinch ED50 (mg/kg) = 0.75] showed opioid activity similar to that of dynorphin A in the three bioassays and relatively high kappa-receptor selectivity in the binding assays and produced a 2.5-fold more potent analgesic effect than morphine. [D-Cys2-Cys5,MeArg7,D-Leu8]Dyn(1-8)-NHEt (20) showed a 40-60-fold more potent opioid activity than 18 in the three bioassays and produced a 3.4-fold more potent analgesic effect than 18. In the binding assays, however, 20 showed higher affinity for mu and delta receptors than for the kappa receptor.

Anti-nociceptive responses produced by human putative counterpart of nocistatin.

b-nocistatin is a heptadecapeptide produced from bovine prepronociceptin and blocks the induction of hyperalgesia and touch-evoked pain (allodynia) by intrathecal administration of nociceptin or prostaglandin E2 (PGE2). Human prepronociceptin may generate a 30-amino acid peptide different in length from b-nocistatin. Here, we examine whether the human putative counterpart of nocistatin (h-nocistatin) possessed the same biological activities as b-nocistatin. Simultaneous intrathecal injection of h-nocistatin in mice blocked the induction of allodynia by nociceptin and PGE2 in a dose-dependent manner with ID50 values of 329 pg kg(-1) and 16.6 ng kg(-1), respectively. h-nocistatin was about 10 times less potent than b-nocistatin. h-nocistatin also attenuated the nociceptin- and PGE2-induced hyperalgesia. These results demonstrate that h-nocistatin is biologically active and may be involved in the processing of pain at the spinal level in humans.

Enhancement of cell killing of HL-60 cells by ultrasound in the presence of the photosensitizing drug Photofrin II.

Enhancement of acute cell killing of HL-60 cells by low-level ultrasound in the presence of the photosensitizing drug Photofrin II was examined. HL-60 cells were exposed to ultrasound (270 kHz) at intensities of 150, 300 and 450 mW/cm2 for 60 s in the presence of Photofrin II (200 micrograms/ml). Cell survival after treatment was 49.6 +/- 5.1%, 34.5 +/- 3.1% and 27.4 +/- 3.9%, respectively. Ultrasound exposure alone at 150, 300 and 450 mW/cm2 resulted in a decrease in cell survival to 92.9 +/- 1.5%, 82.3 +/- 2.2% and 77.9 +/- 7.2%, respectively. The cell survival rate after the addition of Photofrin II alone showed no significant cell killing. These results indicate that Photofrin II was sensitized by low-level, non-thermal ultrasound to enhance the cell killing of HL-60 cells.

Liver tissue damage by ultrasound in combination with the photosensitizing drug, Photofrin II.

We have investigated whether ablation of normal rat liver by ultrasound can be enhanced after administration of a photosensitizing agent (Photofrin II). After laparotomy and administration of Photofrin II (30 mg/kg), ultrasound (210 kHz, 1.3 W/cm2) was directed directly to the surface of the liver for a total duration of 3 min by a plane type unfocused transducer (6 mm x 6 mm). The depth of tissue damage was histologically compared to rats exposed to ultrasound alone. The mean maximum lesion depth on rats applied with ultrasound and Photofrin II was 5.7 +/- 0.9 mm (mean +/- SD) whereas in rats treated with ultrasound alone this was 3.0 +/- 0.4 mm. There was a significant difference between the two groups (P < 0.05). The present study suggests the possible use of a photosensitizing agent as an enhancer for the treatment of liver tumors by ultrasound.

Identification and characterization of an endogenous ligand for opioid receptor homologue ROR-C: its involvement in allodynic response to innocuous stimulus.

We reported here purification and characterization of a novel heptadecapeptide in bovine brain as an endogenous ligand for ROR-C, an opioid receptor homologue cloned from rat cerebrum. The amino acid sequence of the peptide that we purified is identical to those recently identified as nociceptin in rat brain and orphanin FQ in porcine brain. The peptide inhibited the forskolin-induced cyclic AMP accumulation in ROR-C expressing Chinese hamster ovary cells. Studies on inhibitory activity of cyclic AMP accumulation and Northern blot analysis showed that the peptide and its precursor mRNA are present in a number of brain regions, less abundant in the spina cord, and negligible in the cerebellum. In situ hybridization analysis revealed that hybridization-positive neurons were distributed in the superficial layer (lamina I) of the dorsal horn and were also interspersed between the tract of Lissauer in the spinal cord. Intrathecal administration of the peptide into conscious mice induced allodynia, a pain response to innocuous tactile stimuli, in a beli-shaped manner. These results demonstrate that the peptide exists in the brain and spinal cord and plays an important role in pain transmission.

Doxorubicin-heparin complex: reduction of cardiotoxicity of doxorubicin.

We have compared the antitumor activity and cardiotoxicity of free doxorubicin (Dox) and doxorubicin-heparin complex in vivo and in vitro. Dox and Dox-heparin complex equally inhibited the DNA synthesis of leukemic cells and showed a similar anticancer activity against tumor-bearing mice. Acute toxicity of Dox at the dose of 20 mg/kg or 30 mg/kg was significantly more profound than that of the Dox-heparin complex, which was demonstrated by survival rate (P < 0.01). Chronic toxicities of Dox and the Dox-heparin complex were compared by giving the respective reagent (2 mg/kg) weekly for 20 weeks. The weight gains of the mice given Dox-heparin complex were greater than those of the mice given Dox alone (P < 0.01). The pathological damage to the cardiac tissue in mice treated with Dox-heparin complex was significantly less severe than that of mice treated with Dox. Thus, the present study indicates that complexing with heparin diminished the acute and chronic toxicity of Dox without reducing its antitumor activity in mice, and suggests a possible clinical application of Dox-heparin complex in humans.

Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed "cementoin".

Elafin was shown to be a new type of proteinase inhibitor which has an anchoring sequence. Human elafin, a potent inhibitor specific for elastase and proteinase 3, has a unique repeating sequence in its prosegment that is rich in Gln and Lys residues. The prosegment, termed "cementoin," exhibits high homology with the repetitive element of seminal vesicle clotting protein, which is known as a good substrate for prostate transglutaminase. The cross-linking of cementoin by tissue transglutaminase showed that the cementoin moiety is indeed a preferable substrate for transglutaminase. In addition, transglutaminase-mediated cross-linking between cementoin and laminin was observed in vitro, suggesting that cementoin has the ability to covalently attach to other extracellular matrix proteins. To determine whether or not this type of covalent gluing of elafin through the cementoin moiety occurs in vivo, we determined the molecular size of cementoin-elafin in the trachea mucous epithelium by Western blotting; the rationale of this approach is that (i) the trachea is the richest source of cementoin-elafin, as shown below, and (ii) if cementoin-elafin is covalently associated with other proteins, it should migrate as a higher M(r) species on SDS-polyacrylamide gel electrophoresis; cementoin-elafin immunoreactivity was indeed detected at a position corresponding to 50 kDa, a value much higher than that of its monomeric form. RNase protection analysis and immunohistochemical staining revealed that cementoin-elafin is densely distributed in the skin and trachea, and moderately in the stomach, duodenum and small intestine. These sites of localization are consistent with the locations where elastic fibers are abundant.(ABSTRACT TRUNCATED AT 250 WORDS)