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Purpose: Mu-opioid receptors (MORs) are widely expressed in the central nervous system (CNS), peripheral organs, and immune system. This study measured the whole body distribution of MORs in rhesus macaques using the MOR selective radioligand [ 11 C]carfentanil ([ 11 C]CFN) on the PennPET Explorer. Both baseline and blocking studies were conducted using either naloxone or GSK1521498 to measure the effect of the antagonists on MOR binding in both CNS and peripheral organs., Methods: The PennPET Explorer was used for MOR total-body PET imaging in four rhesus macaques using [ 11 C]CFN under baseline, naloxone pretreatment, and naloxone or GSK1521498 displacement conditions. Logan distribution volume ratio (DVR) was calculated by using a reference model to quantitate brain regions, and the standard uptake value ratios (SUVRs) were calculated for peripheral organs. The percent receptor occupancy (%RO) was calculated to establish the blocking effect of 0.14 mg/kg naloxone or GSK1521498., Results: The %RO in MOR-abundant brain regions was 75-90% for naloxone and 72-84% for GSK1521498 in blocking studies. A higher than 90% of %RO were observed in cervical spinal cord for both naloxone and GSK1521498. It took approximately 4-6 min for naloxone or GSK1521498 to distribute to CNS and displace [ 11 C]CFN from the MOR. A smaller effect was observed in heart wall in the naloxone and GSK1521498 blocking studies., Conclusion: [ 11 C]CFN total-body PET scans could be a useful approach for studying mechanism of action of MOR drugs used in the treatment of acute and chronic opioid use disorder and their effect on the biodistribution of synthetic opioids such as CFN. GSK1521498 could be a potential naloxone alternative to reverse opioid overdose., (© 2024. The Author(s).)

Abnormal α-synuclein (α-syn) aggregation characterizes α-synucleinopathies, including Parkinson's disease (PD) and multiple system atrophy (MSA). However, no suitable positron emission tomography (PET) radiotracer for imaging α-syn in PD and MSA exists currently. Our structure-activity relationship studies identified 4-methoxy- N -(4-(3-(pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)benzamide ( 4i ) as a PET radiotracer candidate for imaging α-syn. In vitro assays revealed high binding of 4i to recombinant α-syn fibrils (inhibition constant ( K i ) = 6.1 nM) and low affinity for amyloid beta (Aβ) fibrils in Alzheimer's disease (AD) homogenates. However, [ 3 H]4i also exhibited high specific binding to AD, progressive supranuclear palsy, and corticobasal degeneration tissues as well as PD and MSA tissues, suggesting notable affinity to tau. Nevertheless, the specific binding to pathologic α-syn aggregates in MSA post-mortem brain tissues was significantly higher than in PD tissues. This finding demonstrated the potential use of [ 11 C]4i as a PET tracer for imaging α-syn in MSA patients. Nonhuman primate PET studies confirmed good brain uptake and rapid washout for [ 11 C]4i .

Concomitant with the increasing density of semiconductors, various attacks that threaten the integrity and security of dynamic random access memory (DRAM) have been devised. Among these, a side-channel attack called RAMBleed is a prolific one that utilizes a general user-level account without special rights to read secret information. Studies have reported that it can be used to obtain OpenSSH secret keys. However, a technique for deriving the Rivest-Shamir-Adleman (RSA) secret keys used in OpenSSL under realistic parameters and environments has not been reported. We propose a method that uses RAMBleed to obtain OpenSSL secret keys and demonstrate its efficacy using the example of an Apache server. The proposed method exploits the fact that, in the operation of an Apache server that uses OpenSSL, the RSA private keys are deployed on DRAM at a set time. Although the result of reading this secret information contains a few errors, error-free secret information is obtainable when it is used with RSA cryptanalysis techniques. We performed a series of attacks incorporating RAMBleed and eventually retrieved the OpenSSL RSA private key, indicating that secret information is obtainable with high probability. The proposed method can easily and externally be executed without administrator privileges on a server using DRAM that is vulnerable to RAMBleed, showing that RAMBleed is also a major threat to OpenSSL.

An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time from the Escherichia coli expression system using short Strep-tag II (ST II). The recHPL-ST II was solubilized using 8 M urea from E.coli lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses in the presence of glycerol and Ca2+ for 2 days followed by gel filtration, 1.8-6 mg of active recHPL-ST II was obtained from 1 L of culture. The recHPL was non-glycosylated, but showed almost equal specific activity, pH-dependency and time-dependent stability compared to those of native porcine pancreatic lipase (PPL) at 37°C. However, the recHPL lost its lipolytic activity above 50°C, showing a lower heat-stability than that of native PPL, which retained half its activity at this temperature.

Piperonal-catabolizing microorganisms were isolated from soil, the one (strain CT39-3) exhibiting the highest activity being identified as Burkholderia sp. The piperonal-converting enzyme involved in the initial step of piperonal metabolism was purified from strain CT39-3. Gene cloning of the enzyme and a homology search revealed that the enzyme belongs to the xanthine oxidase family, which comprises molybdoenzymes containing a molybdopterin cytosine dinucleotide cofactor. We found that the piperonal-converting enzyme acts on piperonal in the presence of O 2 , leading to formation of piperonylic acid and H 2 O 2 . The growth of strain CT39-3 was inhibited by higher concentrations of piperonal in the culture medium. Together with this finding, the broad substrate specificity of this enzyme for various aldehydes suggests that it would play an important role in the defense mechanism against antimicrobial compounds derived from plant species.

To develop patch testing (PT) that better reflects the skin irritation resulting from repeated applications, we methodically classified PT results and examined correlations between these results with results from repeated application tests. A total of 115 commercial topical drugs were used for patch testing. In two groups of 30 healthy individuals with normal back skin, 55 or 60 commercial topical drugs were applied to the back for 24 h. Skin reactions were scored for each group at 2 and 24 h after patch removal. Based on time-dependent changes in skin reaction at each evaluation time, PT results were classified into six different types. For repeated application testing, a total of 17 products with different types of PT results were selected. A total of 104 healthy individuals divided into three groups (n = 22-52 each) received application of 6-7 test materials, differing for each group, for 3 weeks on both arms. Incidences of positive symptoms induced by repeated drug application excluding transient erythema showed a greater correlation with reaction levels at 24 h after patch removal than 2 h after patch removal. Many test materials that triggered a high incidence of positive symptoms after repeated application also exhibited erythema or greater reaction up to 24 h after patch removal. PT offers a useful test system for predicting skin irritation that develops after repeated application of topical drug. Moreover, assessment at 24 h after patch removal is suggested as the optimal evaluation time point to reflect the results of repeated application tests., (© 2015 Japanese Dermatological Association.)

Brassinosteroids (BRs) are involved in many developmental processes and regulate many subsets of downstream genes throughout the plant kingdom. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice (Oryza sativa), we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1:OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. In addition, compared to the wild type, the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (Oryza sativa brassinosteroid insensitive1, d61) and a rice G protein alpha subunit (RGA1) mutant d1. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos. These results indicate that BU1 may participate in some other unknown processes modulated by BR in rice.

A lesion mimic mutant that we designated Spotted leaf 18 (Spl18) was isolated from 13,000 activation-tagging lines of rice produced by our modified activation-tagging vector and further characterized. Spl18 was dominant and its phenotype was linked to the T-DNA insertion. An ORF was located about 500 bp downstream of the inserted T-DNA, and the deduced protein, designated OsAT1, showed sequence similarity to an acyltransferase whose expression is induced by hypersensitive reaction in tobacco. The transcriptional level of OsAT1 was very low in the WT leaf blade but high in Spl18 leaf blade. In wild-type rice, OsAT1 was transcribed mainly in the young panicle, in the panicle just after heading, and in the leaf sheath. In addition, transcription of the genes for PR protein was upregulated in Spl18, accumulation of phytoalexins (both momilactone A and sakuranetin) was increased, and resistance to blast disease was improved. We then combined OsAT1 genomic DNA downstream of the modified 35S promoter and re-transformed it into rice. Lesion mimic and blast resistance phenotypes were detected in the transgenic lines produced, clearly indicating that overexpression of OsAT1 caused the Spl18 phenotypes. In addition, plants overexpressing OsAT1 showed resistance to bacterial blight.

We investigated the incidence of atopic dermatitis and related factors at infant physical examination at health centers. Subjects were 900 infants (290 four-month-old infants, 298 one-year and six month-old infants, 312 three-year-old infants) who participated in infant physical examinations in Kanazawa-ku, Yokohama City. Overall, we analyzed 696 infants whose mothers had cooperated with the survey by completing questionnaires during physical examinations, and who submitted to examination by consulting dermatologists. The incidence of atopic dermatitis was 11.6% in 4-month-old infants, 12.2% in 1.5-year-old infants, and 12.1% in 3-year-old infants. The following were found to be related to the atopic dermatitis of infants. 1. Family history of atopic dermatitis in their mothers and older siblings. 2. Mothers' limited diet during pregnancy (avoiding some food which are suspected allergens). 3. Past history of molluscum contagiosum. 4. The frequency of taking bath. While epidemiological surveys of atopic dermatitis have previously been performed, the criteria at each survey was not identical and results could not be compared precisely. In this survey, 1. Dermatologists specializing in atopic dermatitis performed examinations. 2. All diagnoses were made according to standardized criteria which are applied nationwide. 3. All subjects were from a specific region. Because of this approach, this survey provides important information that can form the basis of comparison for future epidemiological surveys of atopic dermatitis.

Studies were done on the effectiveness and safety of panipenem/betamipron, a new carbapenem antibiotic, in infections in the surgical domain and its safety when dissolved in infusions containing lactate. The obtained results are summarized as follows. The preparation, 0.5 g/0.5 g or 1.0 g/1.0 g, was administered by intravenous drip infusion 2 to 3 times a day to 31 cases of patients with infections in the surgical domain. A physiological saline solution was used as the solvent in 21 cases (group A) of them and Solita T3, an infusion containing lactate, was used as the solvent in the 10 remaining cases (group B). As for its clinical effects, results were rated as "excellent" in 20, "good" in 7, "fair" in 3 and "no response" in 1 out of the 31 cases, and the efficacy rate was 87.1%. Regarding its bacteriological effects, results were rated as "disappeared" in 22, "decreased" in 2, "unchanged" in 1 and "unknown" in 1 out of 26 cases from which bacteria were isolated, hence the bacteria-clearance rate was 88.0%. As for side effects, skin rash was seen in 1 case and slight increases of GOT and GPT were noted as abnormal changes in laboratory data in 2 cases. These side effects were all observed among the cases in group A but not at all in group B where a lactate containing infusion was used as the solvent.

In primary 2-day cultured bovine adrenocortical cells, adrenaline stimulated the steroidogenesis, while the effect of adrenaline did not appear in the freshly isolated cells. Thus the primary 2-day cultured cells were used to study the effect of adrenaline on steroidogenesis. Adrenaline showed the steroidogenesis-stimulating effect at concentrations higher than 10(-9) M, and the maximum effect was obtained between 10(-6) M and 10(-5) M in the primary 2-day cultured cells. The maximum effect of adrenaline was 50-70% of that of adrenocorticotropic hormone (ACTH). Noradrenaline, isoproterenol and phenylephrine also stimulated the steroidogenesis. However, the order of the potency was isoproterenol much greater than adrenaline = noradrenaline much much greater than phenylephrine. Propranolol and alprenolol inhibited the effect of adrenaline, but phenoxybenzamine and phentolamine did not inhibit the effect. Moreover, adrenaline stimulated the cyclic AMP production dose-dependently at concentrations higher than 10(-8) M. These results suggest that there are steroidogenesis-linked adrenergic receptors in primary 2-day cultured bovine adrenocortical cell membrane and that the steroidogenesis-stimulating effect of adrenaline occurs through the beta-adrenergic receptor.

The effect of acetylcholine (ACh) on corticoidogenesis in primary cultured bovine adrenocortical cells was examined. One hour exposure to 10(-3) M ACh resulted in a stimulative effect on corticoidogenesis in the freshly isolated cells, and the effect of ACh grew intense during primary culture and reached the maximum on day 2. ACh showed the effect at a higher concentration than 10(-6) M. Thus the primary 2-day cultured cells were used. The corticoidogenic effect of ACh was inhibited by atropine but not by hexamethonium. The effect of ACh was dose dependent, and the extracellular Ca++ was obligatory in inducing the effect. These results suggest that the corticoidogenic effect of ACh may be due to an increase in Ca++-influx via muscarinic receptor in adrenocortical cells.

In order to elucidate the relationship between cyclic AMP and the Ca2+-calmodulin system in the steroidogenic response to adrenocorticotropic hormone (ACTH), the effects of calmodulin inhibitors (trifluoperazine and W-7) on cortisol production and cellular cholesterol ester hydrolysis induced by ACTH or dibutyryl cyclic AMP in bovine adrenocortical cells were examined in the absence of extracellular Ca2+. These calmodulin inhibitors inhibited not only the cortisol production and the cholesterol ester hydrolysis induced by ACTH in the absence of extracellular Ca2+, but also inhibited the dibutyryl cyclic AMP-induced cortisol production and the cholesterol ester hydrolysis in the absence of extracellular Ca2+. These results suggested the possibility that cyclic AMP action was mediated by the Ca2+-calmodulin system in the activation process of cellular cholesterol ester hydrolysis in the steroidogenic response to ACTH.