QSAR model to predict Kp,uu,brain with a small dataset, incorporating predicted values of related parameter.

The unbound brain-to-plasma concentration ratio (Kp,uu,brain) is a parameter that indicates the extent of central nervous system penetration. Pharmaceutical companies build prediction models because many experiments are required to obtain Kp,uu,brain. However, the lack of data hinders the design of an accurate prediction model. To construct a quantitative structure-activity relationship (QSAR) model with a small dataset of Kp,uu,brain, we investigated whether the prediction accuracy could be improved by incorporating software-predicted brain penetration-related parameters (BPrPs) as explanatory variables for pharmacokinetic parameter prediction. We collected 88 compounds with experimental Kp,uu,brain from various official publications. Random forest was used as the machine learning model. First, we developed prediction models using only structural descriptors. Second, we verified the predictive accuracy of each model with the predicted values of BPrPs incorporated in various combinations. Third, the Kp,uu,brain of the in-house compounds was predicted and compared with the experimental values. The prediction accuracy was improved using five-fold cross-validation (RMSE = 0.455, r2 = 0.726) by incorporating BPrPs. Additionally, this model was verified using an external in-house dataset. The result suggested that using BPrPs as explanatory variables improve the prediction accuracy of the Kp,uu,brain QSAR model when the available number of datasets is small.

Evaluation of sensitization to the crude extract of Dermatophagoides farinae and its derived allergens, Der f 2 and Zen 1, in dogs with atopic dermatitis in Southern Brazil.

BACKGROUND: Atopic dermatitis is associated with the production of IgE antibodies against environmental allergens and allergens of the house dust miteDermatophagoides farinae are frequently implicated in the disease. OBJECTIVES: We aimed to observe the allergen-specific IgE against crudeD. farinae, Der f 2 and Zen 1 in dogs with atopic dermatitis and report if these dogs are in contact with material that could shelter mite allergens. METHODS: 100 dogs with clinical diagnosis of atopic dermatitis were included after exclusion of other forms of pruritic skin disease and dogs that already received specific or non-specific immunotherapy. These dogs were of different breeds and ages and they were presented at a veterinary teaching hospital and a private service of veterinary dermatology, both located in Curitiba, Southern Brazil. At the time of anamnesis, some questions were applied to know the possibility of these dogs having had contact with furniture and textile material which could shelter house dust mites. Sera samples were obtained and further analyzed by ELISA assay to measure serum IgE levels against these allergens with an established cut-off of 0.200 IgE optical density. RESULTS: The allergen-specific IgE positivity against crudeD. farinae (92 %) and Zen 1 (77 %) was higher than Der f 2 (56 %). There was a correlation in sensitization to crude D. farinae and Zen 1 that was not observed between crude D. farinae and Der f 2 and Der f 2 and Zen 1. The sensitization to D. farinae and its allergens was associated with an unrestricted exposition to furniture and textile material. CONCLUSION & CLINICAL RELEVANCE: dogs with atopic dermatitis are frequently sensitized to D. farinae and its allergens, Der f 2 and Zen 1, may be considered major allergens in these dogs. Zen 1 may be the main allergen responsible for the sensitization to crude D. farinae.

Ocean (de)oxygenation from the Last Glacial Maximum to the twenty-first century: insights from Earth System models.

All Earth System models project a consistent decrease in the oxygen content of oceans for the coming decades because of ocean warming, reduced ventilation and increased stratification. But large uncertainties for these future projections of ocean deoxygenation remain for the subsurface tropical oceans where the major oxygen minimum zones are located. Here, we combine global warming projections, model-based estimates of natural short-term variability, as well as data and model estimates of the Last Glacial Maximum (LGM) ocean oxygenation to gain some insights into the major mechanisms of oxygenation changes across these different time scales. We show that the primary uncertainty on future ocean deoxygenation in the subsurface tropical oceans is in fact controlled by a robust compensation between decreasing oxygen saturation (O2sat) due to warming and decreasing apparent oxygen utilization (AOU) due to increased ventilation of the corresponding water masses. Modelled short-term natural variability in subsurface oxygen levels also reveals a compensation between O2sat and AOU, controlled by the latter. Finally, using a model simulation of the LGM, reproducing data-based reconstructions of past ocean (de)oxygenation, we show that the deoxygenation trend of the subsurface ocean during deglaciation was controlled by a combination of warming-induced decreasing O2sat and increasing AOU driven by a reduced ventilation of tropical subsurface waters.This article is part of the themed issue 'Ocean ventilation and deoxygenation in a warming world'.

Validity and reliability of the Family Empowerment Scale for caregivers of adults with mental health issues.

WHAT IS KNOWN ON THE SUBJECT?: Empowerment of family caregivers of adults with mental health issues has received increasing attention among mental health nurses in Japan and has been recognized as a new goal of family interventions. The Family Empowerment Scale (FES) was originally developed to measure the empowerment status of parents of children with emotional disorders. However, it was later applied to broader health issues. WHAT THIS PAPER ADDS TO EXISTING KNOWLEDGE?: We developed a Japanese version of the FES for family caregivers of adults with mental health issues (FES-AMJ) and examined the validity and reliability among parents. Results showed that the FES-AMJ had acceptable concurrent validity and reliability; however, insufficient construct validity was found, especially for the subscale regarding the service system. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Further studies need to modify the scale. Clarification of ideal family empowerment status in the service system through discussion with mental health nurses and family caregivers may be important. ABSTRACT: Introduction The Family Empowerment Scale (FES) was originally developed for parents of children with emotional disorders. In Japan, family empowerment is gaining increasing attention and may be one goal of nursing interventions. Aim To develop a Japanese version of the FES for family caregivers of adults with mental health issues and to study the validity and reliability of this scale among parents. Method We translated the FES into Japanese and administered this self-report questionnaire to 275 parents. Results The multitrait scaling analysis revealed acceptable convergent validity and insufficient discriminant validity among all subscales. In particular, all items of the Service system subscale had insufficient discriminant and/or convergent validity. Each subscale significantly correlated with the indicator of empowerment. The intraclass correlation coefficients of each subscale were .855-.917. Cronbach's alpha of each factor ranged from .867 to .895. Discussion The Service system subscale may not linearly reflect family empowerment, and instead may depend on unclear roles of family caregivers of adults, disorder severity or insufficient services. Implications for practice Further studies need to modify the scale. Clarification of ideal family empowerment status in the service system through discussion with mental health nurses and family caregivers may be important.

Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods.

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.

Assessment of residual tumor viability in thymic carcinoma by sequential thallium-201 SPECT: comparison with CT and biopsy findings.

We present a case of 201TI accumulating thymic carcinoma, in which sequential CT scans demonstrated a steady decrease in tumor volume. The presence of a residual mass on CT scans after the completion of therapy presented the clinical dilemma of whether or not a viable tumor remained. Sequential 201TI SPECT images demonstrated a marked decrease in tumor uptake. At 2 wk after therapy, no significant accumulation of 201TI in the region of the residual mass was observed, indicating a lack of viable tumor. A biopsy specimen revealed no tumor cells. Sequential histopathologic findings were correlated well with the findings of 201TI SPECT rather than those of CT. Thallium-201 SPECT is of great clinical value in assessing tumor viability in the course of therapy.

Multiple myeloma evaluated with 201Tl scintigraphy compared with bone scintigraphy.

UNLABELLED: The purpose of this study was to investigate the clinical usefulness of 201Tl chloride scintigraphy in the diagnostic evaluation of 20 patients with multiple myeloma (19/20 patients) or extramedullary plasmacytoma (1/20 patients) in comparison with bone scintigraphy. METHODS: Both 201Tl and bone scintigraphy were performed to obtain planar images on the same instrument. RESULTS: 201Tl scintigraphy showed increased uptake in 15 of 20 patients (75%) and was negative in 5 of 20 patients (25%). In addition, 201Tl scintigraphy of multiple myeloma was more useful in detecting the lesions in 11 of 17 patients and less useful in 6 of 17 patients than bone scintigraphy. CONCLUSION: The combination of 201Tl and bone scintigraphy, compared with bone scintigraphy alone, shows promise in more accurately diagnosing multiple myeloma.

Effects of semotiadil fumarate, a novel Ca2+ antagonist, on cytosolic Ca2+ level and force of contraction in porcine coronary arteries.

1. The mechanisms of action of semotiadil fumarate, a novel Ca2+ antagonist, were examined by measuring the cytosolic Ca2+ level ([Ca2+]i) and force of contraction in porcine coronary arteries, and by determining [3H]-pyrilamine binding to bovine cerebellar membranes. 2. Semotiadil or verapamil (0.1 and 1 microM) inhibited both the high KCl-induced increases in [Ca2+]i and force in a concentration-dependent manner. 3. Histamine (30 microM) produced transient increases followed by sustained increases in [Ca2+]i and force, which were inhibited by semotiadil and verapamil (1 and 10 microM). The agents were different in that semotiadil reduced the maximum [Ca2+]i and force responses to histamine, but not pD2 values, whereas verapamil did reduce the pD2 values for histamine, but not the maximum responses. 4. Verapamil (10 microM), but not semotiadil, inhibited histamine-induced increases in [Ca2+]i and force in Ca(2+)-free solution. Neither semotiadil nor verapamil affected the increases in [Ca2+]i and force induced by caffeine. Semotiadil even at the higher concentration (10 microM) did not displace specific binding of [3H]-pyrilamine to bovine cerebellar membranes. 5. These results suggest that semotiadil inhibits both KCl- and histamine-induced contractions mainly by blocking voltage-dependent L-type Ca2+ channels.

Up-regulation of glial fibrillary acidic protein in the retina of primate eyes with experimental glaucoma.

OBJECTIVE: To identify molecular mechanisms of retinal responses to intraocular pressure elevation in primate experimental glaucoma. METHODS: An experimental glaucoma model was created by repeated laser trabeculophotocoagulation. After the preparation of complementary DNAs from extracted total RNAs in the retinas, polymerase chain reaction (PCR) experiments were performed for the following screening target genes: beta-tubulin beta 2 and beta 5 and glial fibrillary acidic protein (GFAP). To investigate the amplified sequences derived from the PCR experiments, sequencing, subcloning, and Southern blot analysis of PCR products were performed. In addition, an immunohistochemical analysis was performed in an attempt to show the distribution of the target gene products in the retinas. RESULTS: A series of PCR experiments suggested up-regulation of gene expression for GFAP but not for beta-tubulins. Sequencing of the PCR products and results of the Southern blot analysis showed that the amplified sequences were derived mainly from the target gene, GFAP, and that increased expression of GFAP was found despite the severity of glaucoma. Immunohistochemical studies also demonstrated increased expression of GFAP proteins in Müller cells and astrocytes in the retinas of primate eyes with experimental glaucoma. CONCLUSIONS: Our study showed up-regulation of GFAP at gene and protein levels, which suggests that glial components in the retina may contribute to the pathologic processes induced by elevated intraocular pressure.

Effect of pyocin R1 on the glucose metabolism of sensitive cells of Pseudomonas aeruginosa.

The effect of pyocin R1 on the glucose metabolism of sensitive Pseudomonas cells was investigated. Upon treatment with pyocin R1, although the rate of O2 uptake of the sensitive cells for glucose or gluconate was not very much affected at first, the final level of O2 uptake was greatly reduced. When 2-oxogluconate was used as a substrate, O2 uptake was immediately halted by pyocin. By determining the amounts of glucose, gluconate, and 2-oxogluconate before and after the reaction and the amount of O2 consumed, it was concluded that glucose was exclusively metabolized via the following pathway with quantitative accumulation of 2-oxogluconate after pyocin treatment. (Formula: see text). The possible mechanism of this change is discussed.

The effect of defocusing on the electron microscopic image of the extended particle of pyocin R1.

The effect of defocusing on the electron microscopic image of the extended particle of pyocin R1 was examined with the aid of optical diffraction. The results show that the through-focus image of the cross-striated particle changed to a fishbone-like image with a little under-focusing, which had been considered to be a transitional state during the sheath contraction of phage G. In the optical diffraction pattern of the defocused image, a strong meridional reflection corresponding to the distance of cross-striations in the through-focus image almost disappeared, supporting the change to the fishbone-like image on defocusing.

Isolation of characterization of a major outer membrane protein of Pseudomonas aeruginosa. Evidence for the occurrence of a lipoprotein.

In the outer membrane of P. aeruginosa, a protein of apparent molecular weight 8,000 (protein I) is present as a major protein. Purification and chemical analysis of protein I were carried out. This protein was purified by essentially the same procedure as for the purification of the E. coli lipoprotein, which was developed by Inouye et al. (J. Bacteriol. (1976) 127, 555--563). The amino acid composition of protein I was determined. Protein I lacks proline, valine, isoleucine, phenylalanine, tryptophan, and half-cystine. Fatty acid analysis of the protein revealed that it contained 0.89 mol of fatty acids per mol of protein. Among the fatty acids hexadecanoic acid (C16:0) was predominant. In an in vivo labeling experiment, [2-3H]glycerol was incorporated into protein I. A protein with similar mobility to protein I on urea-SDS polyacrylamide gel electrophoresis was isolated from the purified peptidoglycan of P. aeruginosa by trypsin digestion. The amino acid composition of this protein was essentially the same as that of protein I. These results indicate that the outer membrane of P. aeruginosa contains a protein analogous to the E. coli lipoprotein, although considerable differences were observed in the amino acid composition and the fatty acid content.

Biochemical properties of a new flexuous bacteriocin, pyocin F1, produced by Pseudomonas aeruginosa.

Pseudomonas aeruginosa strain P15 produces not only pyocin R1 and phage PS10, but also a substance having a flexuous rod structure, the nature of which is so far unknown. A variant strain (P15--40) was obtained which produced these flexuous particles more effectively than the original strain, and the particles were purified to homogeneity and investigated. Several strains of P. aeruginosa were found to be killed by the particles. It was concluded that the flexuous rod-like particles are not related to pyocin R1 or phage PS10, but represent a new pyocin, which we have designated as pyocin F1. Pyocin F1 showed a different action spectrum and a different pattern on SDS-polyacrylamide gel electrophoresis from either pyocin R1 or phage PS10. The killing activity of pyocin F1 was of single-hit type. The activity was not affected by anti-R1, anti-R1-core or anti-PS10, or by DNase, RNase, pronase or trypsin, but was completely destroyed by treatment at 70 degrees C for 10 min. Some cofactor was required for the adsorption of this pyocin on sensitive bacteria. Another flexuous bacteriocin was also found and named pyocin F2.

Isolation and characterization of major outer membrane proteins of Pseudomonas aeruginosa strain PAO with special reference to peptidoglycan-associated protein.

The outer membrane of Pseudomonas aeruginosa PAO contains six major proteins (proteins D, E, F, G,H, and I). Two of them (protein F and protein H) were found to be retained by the peptidoglycan layer when cell envelopes were extracted with 2% sodium dodecyl sulfate (SDS) solution at 35 degrees C. At higher temperature (greater than 55 degrees C), no proteins were retained by peptidoglycan. By making use of this property, purification of protein F and protein H was achieved. Three other major outer membrane proteins, D, E, and I were also isolated and characterized. Their amino acids compositions were determined. Circular dichroism spectra of these isolated proteins were measured in SDS solution. Protein F was rich in beta-structure, while protein I was rich in alpha-helix. When isolated protein F was heated (100 degrees C-15 min) in SDS solution, the circular dichroism spectrum changed significantly. In parallel with the conformational change, the electrophoretic mobility of protein F on urea-SDS polyacrylamide gel also changed. These results indicate that protein F is a so-called heat-modifiable protein.

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.

Subunit arrangement in the extended sheath of pyocin R.

An optical diffraction study has been made of electron micrographs of extended sheaths of pyocin R. It has been demonstrated that the extended sheath consists of annuli of six subunits, which are arranged in a helix of 3.57 annuli per one turn. The annulus repeat in the helix direction is 35A. The dimensions and probably the helical parameters of pyocin R sheath are different from those of T4 phage tail, but a structural correlation appears to exist between the extended sheath of pyocin R and that of the phage tail.

Comparative study of F-type pyocins of Pseudomonas aeruginosa.

Pseudomonas aeruginosa strain PAF41 was found to produce a new F-type pyocin, pyocin F3, the action spectrum of which was different from those of previously reported pyocins F1 and F2. These three F-type pyocins were compared with respect to their structure and biological properties. These pyocins were almost the same with regard to the structure and the dimensions, and have similar amino acid compositions and S values. The particle weights of these pyocins were also suggested to be similar. Analyses of subunit proteins by SDS-polyacrylamide slab gel electrophoresis showed that these pyocins were composed of 5 major (bands 1, 2, 3, 4, and 6) and 2 minor (bands 5 and 7) subunit proteins and that no difference in the mobilities of these subunit proteins could be detected among the pyocins except that of the second major subunit protein (band 4), which did differ. Pyocins F1, F2, and F3 were immunologically cross-reactive, and carried common antigens as well as specific ones. It was shown that band 6 was a common antigen among the three pyocins and that band 4 was antigenically different in pyocins F1 and F3 by immunological reaction after protein blotting. Electron microscopic observation of pyocin particles treated with anti-sera revealed that the common antigens were located on the rod part and the specific ones were on the fiber part. Pyocin F3 was neutralized by both anti-F3 and anti-F1 sera showing apparent first order rate kinetics, whereas the neutralization for pyocin F1 by these sera did not show such kinetics, but a considerable increment of pyocin F1 activity was observed when small amounts of the sera were added. The increment seemed to be due to the antibodies common to pyocins F1, F2, and F3. A phage, which had a flexuous rod-like tail, was found to be immunologically cross-reactive with the three pyocins and was named KF1.

A fluorescent probe response to the interaction of pyocin R1 with sensitive cells.

Additon of pyocin R1, a bacteriocin of Pseudomonas aeruginosa, to sensitive cells caused a fluorescence increase of 8-anilino-1-naphthalenesulfonate (ANS) in the cell suspension. The reaction was rapid, starting with a short time lag after adsorption of pyocin onto the cells and finishing within several minutes. The fluorescence response was attributed to the interaction of the cell body and ANS, not to that of the medium outside the cells and ANS. The maximal amplitude of fluorescence after pyocin addition was dependent on temperature, and the relation appeared to be biphasic. Similarly, Arrhenius plots of the initial rate of fluorescence change were biphasic. The transition of slopes in both cases occurred in the temperature range between 18 and 19 degrees. These results suggest that ANS interacts with lipids in the cell envelope and that pyocin causes a structural change of the cell envelope leading to increased fluorescence of ANS.

Isolation and characterization of a new bacteriophage, KF1, immunologically cross-reactive with F-type pyocins.

Pseudomonas aeruginosa strain NIH S produced a bacteriophage, KF1, immunologically cross-reactive with F-type pyocins. Phage KF1 was neutralized by both anti-pyocin F1 and anti-pyocin F3 sera, although the efficiency was very low. About eleven polypeptides were detected by SDS-polyacrylamide gel electrophoresis of the phage. Most of the subunit proteins were different from those of F-type pyocins, but the molecular weights of minor subunit proteins P3 and P6 seemed to be the same as those of band 1 and band 5 of F-type pyocins, respectively. The head of the phage appeared to have an icosahedral structure, approximately 63 nm in diameter, with a long (190 nm, 11 nm wide and about 45 striations) flexuous tail connected to a fiber structure (about 53 nm in length). The density in CsCl and the sedimentation coefficient of the phage were 1.54 g/ml and 392S, respectively. Some other biochemical properties were described. The nucleic acid of the phage was linear, double stranded DNA of molecular weight 4 x 10(7). The density of the DNA in CsCl was 1.719 g/ml, the melting temperature was 95.4 degrees C. The guanine plus cytosine content was calculated to be 60 to 64%.