Search

Background: It is difficult to predict vancomycin trough concentrations in critically ill patients as their pharmacokinetics change with the progression of both organ failure and medical intervention. This study aims to develop a model to predict vancomycin trough concentration using machine learning (ML) and to compare its prediction accuracy with that of the population pharmacokinetic (PPK) model. Methods: A single-center retrospective observational study was conducted. Patients who had been admitted to the intensive care unit, received intravenous vancomycin, and had undergone therapeutic drug monitoring between 2013 and 2020,were included. Thereafter, ML models were developed with random forest, LightGBM, and ridge regression using 42 features. Mean absolute errors (MAE) were compared and important features were shown using LightGBM. Results: Among 335 patients, 225 were included as training data and 110 were used for test data. A significant difference was identified in the MAE by each ML model compared with PPK;4.13 ± 3.64 for random forest, 4.18 ± 3.37 for LightGBM, 4.29 ± 3.88 for ridge regression, and 6.17 ± 5.36 for PPK. The highest importance features were pH, lactate, and serum creatinine. Conclusion: This study concludes that ML may be able to more accurately predict vancomycin trough concentrations than the currently used PPK model in ICU patients.

The development of laser powder bed fusion (LPBF) has made it possible to produce complex three-dimensional components for high-temperature applications. The LPBF process needs to be refined to address several key factors, such as high-temperature elongation, microstructure heterogeneity, and mechanical anisotropy. Hastelloy-X Ni-based superalloy was used to illustrate these issues in this study. First, yttrium (Y) was added to Hastelloy-X alloy to prevent grain boundary embrittlement. The second step involves post-heat treatment at a higher temperature to eliminate the microstructural heterogeneity and creeps anisotropy. We used Hastelloy-X (HX) as reference material, along with 0.046 wt.% Y (HX-y). The aging was done at 850°C for 2 h (DA) for carbides and solution heat-treatment at 1240°C for 8 h upon aging at 850°C for 2 h (HTA) to eliminate microstructural heterogeneity. The creep properties were studied at 900°C/ 80 MPa. The HX-y specimen was strengthened by solid solution and Y-rich oxides and stabilizing oxygen-based contamination at the grain boundary. The DA and HTA HX-y specimens had better creep properties than HX specimens. The HX-y specimen showed superior creep properties to the HX specimen due to the presence of carbides M6C and Cr23C6 inside grains and at grain boundaries. However, carbides remained stable even at high temperatures within grains and at grain boundaries. Nevertheless, the HTA HX-y specimen exhibited superior isotropic creep properties. As a result of grain boundary pinning, serrated grain boundaries prevented grain boundary sliding. In contrast, HX specimens exhibited poor creep properties.

The photosensitizer talaporfin has been widely used in photodynamic therapy (PDT), but the mechanism of uptake and excretion has not been identified yet. We do research on the metabolism of talapor...

The effects of the addition of yttrium (Y) on the creep and tensile properties of Hastelloy-X built by a selective laser melting (SLM) process were studied. Two alloys were chosen: Hastelloy-X (Y-free) as a reference material and a specimen with 0.046 wt% yttrium (Y-added). Post-processing solution heat treatment (ST) was conducted at 1177 °C in air for 2 h, followed by air cooling to obtain appreciable mechanical properties. In the as-built condition, the Y-free specimen showed the formation of cracks; this was mainly due to the segregation of solute elements such as W, Si, and C at the grain boundaries and interdendritic regions. The Y-added specimen was free of cracks and showed longer creep life and rupture elongation than the Y-free specimen because the reduced Si content in the Y-added alloy eliminated cracking. In addition, the formation of Y2O3 oxides improved tensile elongation and creep properties by stabilizing solute oxygen contamination. After ST, both vertical and horizontal Y-added specimens presented better creep properties than ST Y-free specimens. This was mainly due to grain morphological change, carbides, and oxides formation at the grain boundary after ST heat treatment in the Y-added specimens.

The effect of solution heat treatment as well as primary and secondary aging treatment conditions on the microstructural evolution of a high-entropy superalloy was investigated. The as-cast sample shows coarsened &gamma, &prime, precipitates and other extra phases at interdendrite region due to microsegregation. This microsegregation makes &gamma, solvus unclear and decreases the solidus. After conducting the solution treatment determined in this study, primary aging conditions showing an aligned cubic &gamma, phase at 1050 &deg, C for 4 h and random spherical &gamma, precipitates at 950 &deg, C for 20 h in similar size were found. By using both samples, secondary aging conditions showing coarsened secondary &gamma, precipitates and &gamma, particles inside the &gamma, precipitates at 800 &deg, C for 20 h and fine secondary &gamma, precipitates at 850 and 870 &deg, C for 20 h were found.

Photodynamic therapy (PDT) using the photosensitizer talaporfin sodium (talaporfin) is a new mode of treatment for cancer. However, the metabolic mechanism of talaporfin has not been clarified. Thus, we investigated the uptake, transportation, and elimination mechanisms of talaporfin in carcinoma and sarcoma. The results showed that talaporfin co‐localized in early endosomes and lysosomes. Talaporfin uptake was via clathrin‐ and caveolae‐dependent endocytosis and a high amount of intracellular ATP was essential. Inhibition of lysosomal enzymes maintained intracellular talaporfin levels. Inhibition of K‐Ras signaling reduced talaporfin uptake in carcinoma and sarcoma cell lines. Talaporfin was taken up by clathrin‐ and caveolae‐dependent endocytosis, translocated from early endosomes to lysosomes, and finally degraded by lysosomes. We also demonstrated that ATP is essential for the uptake of talaporfin and that activation of K‐Ras is involved as a regulatory mechanism. These results provide new insights into the metabolism of talaporfin in cancer cells for the enhancement of PDT for carcinoma and sarcoma., Intracellular talaporfin was co‐localized with early endosomes and lysosomes but not with mitochondria in osteosarcoma cells.

Strength of Ni-base single-crystal superalloys under high temperature and low stress creep usually is enhanced by formation of γ/γ′ raft structure and larger aspect ratio of γ′ phase in the γ/γ′ raft structure. Elastic misfit between γ and γ′ phases is one of the most important factors to control the aspect ratio of the γ′ phase in the γ/γ′ raft structure formed under external stress. The aspect ratio of the γ′ phase is controlled by kinetics for the γ/γ′ raft structure formation, which is affected by a strain inhomogeneity caused by this elastic misfit between the γ and γ′ phases under external stress. To realize a new alloy design approach to control the aspect ratio of the γ′ phase in the γ/γ′ raft structure, this research aimed to obtain the regression equations which can predict elastic modulus of the individual γ and γ′ phases for multi-component Ni-base single-crystal superalloys based on measurements of elastic modulus of Ni-base single-crystal alloys. Elastic modulus of the individual γ and γ′ phases of various kinds of Ni-base single-crystal alloys was measured by using rectangular parallelepiped resonance (RPR) method. Using the analyzed and referenced elastic modulus, regression equations for predicting longitudinal elastic modulus of the individual γ and γ′ phases and its temperature and composition dependence were obtained. Detailed analysis of the elastic modulus and its composition dependence was executed to clarify the contribution of each element on the elastic modulus. At 900 °C, Re, Ta, Ti, Al, and Mo reduce the longitudinal elastic modulus in the γ phase. On the other hand, Ru, Re, Ta, Ti, Al, W, and Mo enlarge the elastic modulus in the γ′ phase.

Photodynamic diagnosis/therapy (PDD/PDT) are novel modalities for the diagnosis and treatment of cancer. The photosensitizer protoporphyrin IX is metabolized from 5-aminolevulinic acid (5-ALA) intracellularly, and PDD/PDT using 5-ALA have been approved in dermatologic malignancies and gliomas. However, the molecular mechanism that defines the efficacy of PDD/PDT is unknown. In this study, we analyzed the functions of ATP-binding cassette (ABC) transporters in PDD using 5-ALA. Most of the human gastrointestinal cancer line cells examined showed a homogenous staining pattern with 5-ALA, except for the pancreatic cancer line PANC-1, which showed heterogeneous staining. To analyze this heterogeneous staining pattern, single cell clones were established from PANC-1 cells and the expression of ABC transporters was assessed. Among the ABC transporter genes examined, ABCG2 showed an inverse correlation with the rate of 5-ALA-positive staining. PANC-1 clone #2 cells showed the highest level of ABCG2 expression and the lowest level of 5-ALA staining, with only a 0.6% positive rate. Knockdown of the ABCG2 gene by small interfering RNAs increased the positive rate of 5-ALA staining in PANC-1 wild-type and clone cells. Interestingly, PANC-1 clone #2 cells showed the high sphere-forming ability and tumor-formation ability, indicating that the cells contained high numbers of cancer stem cells (CSCs). Knockdown or inhibition of ABCG2 increased the rate of 5-ALA staining, but did not decrease sphere-forming ability. These results indicate that gastrointestinal cancer cell lines expressing high levels of ABCG2 are enriched with CSCs and show low rates of 5-ALA staining, but 5-ALA staining rates can be improved by inhibition of ABCG2.

A mechanism for phonon Hall effect (PHE) in non-magnetic insulators under an external magnetic field is theoretically studied. PHE is known in (para)magnetic compounds, where the magnetic moments and spin-orbit interaction play an essential role. In sharp contrast, we here show that a non-zero Berry curvature of acoustic phonons is induced by an external magnetic field due to the correction to the adiabatic Born-Oppenheimer approximation. This results in the finite thermal Hall conductivity $\kappa_H$ in nonmagnetic band insulators. Our estimate of $\kappa_H$ for a simple model gives $\kappa_H \sim 1.0\times 10^{-5} $[W/Km] at $ B=10 $[T] and $ T=150 $[K]., Comment: 5 pages, 2 figures

Time-frequency duality plays a pivotal role in modern optical science and engineering. Conventional duality, which is connected by one-dimensional Fourier transformation, is insufficient for characterizing quantum mechanical correlations in the time-frequency behavior of multiple photons; a higher-dimensional treatment is required. The authors directly measure the two-photon distributions of generated biphotons in both frequency and time domains, and show that they satisfy the Fourier-limited condition in two-dimensional time and frequency space, but not in conventional one-dimensional space. This study helps to pave the way to tomorrow's quantum optical technologies.

Atomic force microscopy (AFM) has been systematically used to image the surface of a femoral head made of alumina/zirconia composite (henceforth referred to as zirconia-toughened alumina, ZTA), in comparison with two types of commercially available femoral heads made of monolithic zirconia. AFM experiments were conducted before and after in vitro exposure in water moist environment. All materials contained zirconia partly stabilized with yttria. AFM observations were performed on wide areas of several heads of each type (i.e., in the order of several tens of μm2), in order to ensure statistical reliability in the topologic measurements. Tetragonal-to-monoclinic phase transformation, which was quantitatively characterized by confocal Raman spectroscopy, showed significant difference among the investigated samples. Such differences were similarly found with respect to both roughness and time needed for topologic changes to occur. Variation of topologic statistic parameters, such as skewness and kurtosis of surface height histograms, confirmed the main impact of zirconia grain size on the environmental stability of the head surface, the finer the grain size the higher the stability.

The permeability of higher molecular weight substances was investigated in mouse chorioallantoic labyrinthine hemotrichorial placenta, using horseradish peroxidase as a tracer. At the same time, ultrastructural localizations of some important enzymes, such as alkaline phosphatase (ALP), acid phosphatase (ACP), Ca++-ATPase and guanylate cyclase were elucidated in this organ by means of the enzyme-cytochemical technique. Peroxidase easily entered the space between layers I and II, and no penetration of this tracer beyond layer II was observed. The reaction products for ALP activity were found mainly on the maternal side of the plasma membrane of the layer II tro-phoblast. ACP activity was confined to the lysosomes of this layer II cell. In short, peroxidase stopped at the cell surface of the layer II trophoblast, and both ALP and ACP coexisted in this layer II cell. These observations strongly suggest that the layer II trophoblast, especially the surface plasma membrane of this cell, may have an important role in regulating the materno-fetal transfer of substances in mouse chorioallantoic placenta.

The aim of this study was to perform a surface morphology assessment with nanometer scale resolution on femoral heads made of an advanced zirconia toughened alumina (ZTA) composite. Femoral heads were characterized to a degree of statistical accuracy in the as-received state and after exposures up to 100 h in severe vapor-moist environment. Surface screening was made using an atomic force microscope (AFM). Scanning was systematically repeated on portions of surface as large as several tens of micrometers, randomly selected on the head surface, to achieve sufficient statistical reliability without lowering the nanometer-scale spatial resolution of the roughness measurement. No significant difference was found in the recorded values of surface roughness after environmental exposure (at 134°C, under 2 bar), which was always comparable to that of the as-received head. Surface roughness safely lay

We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett's brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells.

The effects of motion on the anterior cruciate ligament were examined using knee joints. When the knees of rats and rabbits subjected to motion in a horseradish peroxidase (HRP) solution were compared with those undisturbed in an identical solution, it was found that a greater quantity of HRP had infiltrated into the exercised knees. We concluded that motion facilitates nutrient infiltration into the anterior cruciate ligament.

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containing beta-galactosidase (beta-gal) gene in vitro, and the expression of beta-gal in CM was evaluated by X-gal staining and beta-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and the beta-gal expression increased to 31.1 +/- 4.6 ng/mg protein in a MOI-dependent manner (MOI: 10(4) to 10(6) particles/cell). The beta-gal expression was also increased in an incubation period-dependent manner (1-24 h). beta-gal expression was maximal at day 3 and then gradually decreased with time. However, beta-gal expression at day 14 was almost the same level as that at day 1 (45.5 +/- 5.9 v 55.2 +/- 6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZ ex vivo. When the beta-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.

Human scalp hair is important as a diagnostic clue to many diseases, in medical jurisprudential investigations, and also as a subject of cosmetic treatments. While many ultrastructural studies of the human hair root including the hair follicle have been reported, few studies have been done on the human hair shaft. We report here the ultrastructure of human scalp hair shafts prepared by a rapid-freezing technique followed by freeze-substitution fixation that allows the observation of fine cell structures. Healthy scalp hair shafts from Japanese females 12–13 years of age were rapid-frozen and then freeze-substituted in OsO4-acetone. In addition, this technique was applied to the study of some changes of the hair shafts (i.e., hair damaged by thioglycolic acid cold permanent waving and white hair). By this method, the hair shaft was rapid-frozen throughout without appreciable ice damage although the hair shaft was nearly 100 μm in diameter. The rapid-freezing technique resulted in excellent preservation of the ultrastructure of the hair shafts: lamellar structures in the cuticle and fine fibrous ultrastructures in the cortex were observed without chemical treatments. Thioglycolic acid treatment affected the ultrastructure of both the cuticle and the cortex. Except for the absence of melanin granules, no significant differences in the ultrastructure were observed between white hair and black hair. The rapid-freezing technique followed by freeze-substitution fixation appears to be the most reliable approach for the morphological evaluation of fully keratinized cells and tissues. Anat. Rec. 251:406–413, 1998. © 1998 Wiley-Liss, Inc.

Glucose-6-phosphatase is generally accepted as a functional component of rough endoplasmic reticulum and has been histochemically examined in many organs. The aim of this study is to know the ultracytochemical localization of glucose-6-phosphatase in each type of hormone-producing cell constituting the anterior pituitary gland in the rat. Pituitaries of male Sprague-Dawley rats were perfused with 1.5% glutaraldehyde from the left ventricles. After buffer washing 40 microns sections were incubated in the medium of Hugon et al. for 60 min at 37 degrees C. The sections were then postfixed with 1% osmium tetroxide, embedded in epoxy resin and observed under an electron microscope. The reaction product for glucose-6-phosphatase was observed in the lumen of rough endoplasmic reticulum and nuclear envelope of all anterior pituitary cells. The enzyme activities in thyroid-stimulating hormone-producing cells and luteinizing hormone/follicle-stimulating hormone-producing cells (LH/FSH cells) were stronger than those in growth hormone-producing cells and prolactin-producing cells; adrenocorticotropic hormone-producing cells and folliculo-stellate cells presented intermediate activity. In LH/FSH cells, the activity in dilated cisternae of endoplasmic reticulum had weaker density than that in flattened cisternae. In addition, substantial reaction product was also frequently observed in the cis saccules of the Golgi apparatus. These findings suggest that glucose-6-phosphatase may play different functional roles in hormone synthesis within different types of anterior pituitary cells.

The purpose of this study was to characterize the ultrastructural and enzyme-histochemical features of Hofbauer cells in the placental villi of second-trimester abortions. Hofbauer cells in the abortive placenta had more numerous cytoplasmic processes and more prominent phagosomes as compared to gestational age-matched normal control placenta. Acid phosphatase activity was demonstrated in both the lysosomes and on phagosomal membranes of these cells. Our results indicate that Hofbauer cells in second-trimester abortions are activated or stimulated in situ. These activated Hofbauer cells might play important roles in the pathophysiology of preterm parturition.

During the past decade, freeze-fracture cytochemistry, the combination of freezefracture electron microscopy with cytochemistry, has greatly contributed to the investigation of the macromolecular architecture of biological membranes. The application of enzyme cytochemical labeling technique to freeze-fracture cytochemistry (i. e., freeze-fracture enzyme cytochemistry) has not received considerable attention although by enzyme cytochemical labeling technique many enzyme molecules can be labeled with metal cations and enzyme cytochemistry has been of great utility in cell biology studies. In this study, we report freeze-fracture enzyme cytochemistry: “label-fracture method” and “fracture-label method”. Freeze-fracture enzyme cytochemistry was applied to the study of acid phosphatase, 5′-nucleotidase in the proximal tubular epithelium of the rat kidney, ectoadenosine triphosphatase in human neutrophils and alkaline phosphatase in rat neutrophils, and was shown to be a technique that visualized these enzyme activities on replicas. Cerium as the capture agent was a useful enzyme cytochemical probe in this technique. Lead was applicable for labeling of plasma membrane-associated enzyme molecules. This technique leads to new applications that may extend the usefulness of freeze-fracture cytochemistry for the analysis of biomembrane structure.

A cytochemical study of glucose-6-phosphatase (G-6-Pase) was made to see the changes in mucopolysaccharide metabolism in the synovial membrane during osteoarthritis, because the tissue is thought to play an important role in synovial fluid production by providing lubrication to the wear-and-tear surface of the synovial cartilage in osteoarthritis. In normal and osteoarthritic synovial membranes, a high G-6-Pase activity was clearly demonstrated in the cisternal spaces of the rough endoplasmic reticula, especially in the B cells of the thickened synovial membrane. The existence of an AB cells intermediate in character between A and B, was noted in having a high G-6-Pase activity in the accumulated rough endoplasmic reticula in the cytoplasm as B cells do located at the deeper part of the membrane, but also whose cell surface extends to the top of the membrane surface reaching into the synovial cavity, and protrude microvilli of a ruffled border indicating a high phagocytotic activity as A cells do. The existence of the AB cell is clearly one of intermediate structure and function, contributive to the compensatory and regenerative action of the synovial membrane.

Cyclic nucleotide metabolizing enzymes were studied cytochemically to estimate the changes in these enzymes in the synovial membrane, because one of the functions of this tissue is estimated to clean up the debris from the synovial fluid to facilitate lubrication in the knee joints by filtering up small fragments of cartilage which have been formed as the result of wear and tear from the articular cartilage in osteoarthritis. Since the cyclic nucleotides are assumed to regulate the biological activity of phagocytosis, it may be reasonable to expect changes in this activity during the disability of osteoarthritis. In normal and osteoarthritic synovial membranes, a high adenylate cyclase (ACase) activity was clearly observed in the A cells at the surface of the synovial membrane, positive on the plasma membranes of the microvilli and on the ruffled borders as well as in pinocytotic vesicles underneath. The activity of guanylate cyclase (GCase) was observed to be rather weak; however, a strong 5′-nucleotidase (5′-N) activity was evidenced, indicating the existence of a regulatory function with the cyclic nucleotides in the phagocytotic and pinocytotic activities during osteoarthritis.

We present a novel freeze-fracture cytochemistry method based upon enzyme cytochemistry. By this method, freeze-fractured membranes are labeled with cerium as an enzyme cytochemical marker on replicas. The cerium capture method was suitable for freeze-fracture enzyme cytochemistry because the cerium phosphate reaction product is stable after replica-cleaning. As a model system, acid phosphatase, which is a well-known lysosomal marker, was detected on freeze-fractured membranes of lysosomes in the proximal tubular epithelium of the rat kidney. This technique should be a useful addition for analyzing the ultrastructure of biological membranes.

The aim of this study was to perform a surface morphology assessment with nanometer scale resolution on femoral heads made of an advanced zirconia toughened alumina (ZTA) composite. Femoral heads were characterized to a degree of statistical accuracy in the as-received state and after exposures up to 100 h in severe vapor-moist environment. Surface screening was made using an atomic force microscope (AFM). Scanning was systematically repeated on portions of surface as large as several tens of micrometers, randomly selected on the head surface, to achieve sufficient statistical reliability without lowering the nanometer-scale spatial resolution of the roughness measurement. No significant difference was found in the recorded values of surface roughness after environmental exposure (at 134 degrees C, under 2 bar), which was always comparable to that of the as-received head. Surface roughness safely lay10 nm after environmental exposures up to 100 h, which corresponded to an exposure time in vivo of several human lifetimes (i.e., according to an experimentally derived thermal activation energy). In addition, the roughness results were significantly (about one order of magnitude) lower as compared to those recorded on femoral heads made of monolithic zirconia tested under the same conditions.

Our objective was to evaluate a method that incorporates a freezing and thawing process to detect cytochrome c oxidase in normal mitochondria in myogenic cell lines in monolayer culture, without the loss of enzyme activity. We used electron microscopy to detect and investigate cytochrome c oxidase activity. When cell preparations were subjected to a freezing and thawing process after being fixed, 91.4% of mitochondria were positively stained. No positive staining was obtained in cells treated by conventional procedures without the freezing process. This method facilitates the positive staining of mitochondria and should be useful in analyzing pathological conditions of mitochondria in cultured cells.

The precise establishment for the subcellular localization of guanylate cyclase would very much aid in clarifying a number of important involvements of cGMP metabolism on biological phenomena. Therefore, as reported, attempts were made to develop a cytochemical method for guanylate cyclase (GCase), using lead citrate as capture agent. Lead citrate acts as an inhibitor of GCase but 21.3% of its activity remains reserved in the tissue. The optimum pH examined on cytochemical sections was between 7.5 to 8.5, the manganese ion working as activator. In the rat retina given immersion fixation, the GCase activity was defined in the space of the disc membranes on the rod outer segment. However, after application of rapid freeze substitution fixation on the retina, the enzyme activity appeared at the cytoplasmic side of the disc membranes, showing good correlation with the biochemical estimations.

It is of increasing interest to observe the precise localization of cyclic 3′, 5′-nucleotide phosphodiesterase (PDEase) activity in rod outer segments at the ultrastructural level. The cytochemical method of PDEase localization at the ultrastructural level was developed by Florendo et al. However, there were two major difficulties in the use of snake venom as an exogenous 5′-Nucleotidase (5′-Nase): 1) the marked ultrastructural damage due to snake venom, and 2) the inadequate penetration of exogenous 5′-Nase into tissues. Therefore, two improvements for the demonstration of PDEase activity were carried out by the case of: 1) a purified 5′-Nase as exogenous 5′-Nase for a better preservation of cell morphology, and 2) 40μm sections made with the use of a freezing microtome for a better penetration of 5′-Nase into the tissues.PDEase activity using purified 5′-Nase was observed along the disc in the rod outer segment, and no retinal detachment was observed between the outer segments and pigment epithelium, the ultrastructure being excellently preserved, as compared with the use of snake venom. By applying this improved method to freeze-substitution technique, the catalytic site of PDEase was localized on the cytoplasmic surface of the disc membranes. This new technique would be useful in detecting the precise localization of PDEase activity.

Pattern recognition receptors, which include the toll-like receptors (TLRs), are considered to play an important role in the response against lipopolysaccharide (LPS). In this study, we performed a reverse transcriptase/polymerase chain reaction (RT-PCR) study, Western analysis, immunohistochemical staining, and RT-PCR-amplified in situ hybridization of TLR2 and TLR4 in the case of LPS-induced lung injury. The expression of TLR2 and TLR4 increased in the lung rapidly after LPS inhalation and peaked at 24 h, followed by a gradual decrease. TLR2 and TLR4 expression was observed on the bronchial epithelium and tissue macrophages. In the early hours after inhalation of fluorescein-isothiocyanate (FITC)-labeled LPS, LPS was detected mainly on the bronchial epithelium and on a few of tissue macrophages. One day after inhalation, the LPS signals disappeared in the lungs of the mice, except for a few alveolar macrophages. The expression of TLR2, TLR4, and CD14 was coincident with the signals of FITC-labeled LPS. Instillation of liposome-encapsulated dichloromethylene diphosphonate induced a significant decrease in alveolar macrophages. In the macrophage-depleted mice, however, expression of TLR2 and TLR4 mRNA or protein was slightly suppressed in the lung after LPS inhalation. These data suggest that the bronchial epithelium and macrophages play crucial roles in LPS-induced lung injury through TLR2 and TLR4.

We describe a new freeze-fracture cytochemical technique consisting of combined immunocytochemistry and enzyme cytochemistry. This technique reveals the relationship between molecules in biological membranes by double labeling with two different cytochemical markers (i.e., immunogold probes and cerium). In this method, antigens were detected with specific primary antibodies and appropriate secondary immunoprobes. Subsequently, alkaline phosphatase activity was detected with cerium as the capture agent on the same replicas. Octyl-glucoside (OG) digestion before the cytochemical reactions was crucial to the success of this combined method. OG is an efficient detergent and OG digestion can preserve both immunocytochemical antigenicity and enzyme activity on replicas. As an initial examination, we applied this technique to the study of glycosyl-phosphatidyl-inositol-anchored proteins and adhesion molecules in human neutrophils. The method described here should serve as a unique additional approach for the study of topology and dynamics of molecules in biomembranes.

Cyclic guanosine monophosphate (cGMP)-phosphodiesterase (PDE) in the outer segment of vertebrate retinal rod cells is one of key enzymes mediating phototransduction. We report here on light-induced PDE activity in intact rat retinal rod cells processed by rapid-freezing enzyme cytochemistry, a new morphological technique that is a combination of rapid-freezing, freeze-substitution fixation, and subsequent enzyme cytochemistry. This technique quickly immobilizes and preserves both enzyme activity and the cell ultrastructure in a state approximating living conditions; consequently, it has proved useful for cytochemical detection of light-induced PDE activity. Using this technique we observed that the catalytic site of PDE molecules in rapid-frozen outer segments was predominantly located in the extradiscal spaces; PDE activity was significantly greater in light than in darkness; and illumination elicited marked increases in PDE activity in dark-adapted cells. Light-induced PDE activity was first cytochemically detected after 3 s of illumination and reached a peak within 5 s, at which time it was in virtually the same as that seen in fully light adapted cells. In addition, cytochemical guanosine triphosphatase (GTPase) activity in dark-adapted cells, as well as corresponding PDE activity, increased in a time-dependent manner with illumination duration; this acceleration in GTPase activity closely paralleled the PDE activity. Thus, our results suggest, in part, the existence of reciprocal regulation of PDE and activated transducin alpha subunit, thereby modulating light adaptation in rod cells.

We show a new freeze-fracture enzyme cytochemistry technique, i.e. fracture-labelling by enzyme cytochemistry. Freeze-fracture replication is carried out first and subsequently enzyme molecules in the split-membrane halves of cellular membranes are labelled with enzyme cytochemical markers. A replica-digestion treatment before the cytochemical reactions is a key step in this method. Triton X-100, saponin, and ultrasonication provided adequate cleaning of the replicas with good preservation of enzyme activity. Enzyme cytochemical fracture-labelling was applied to the study of alkaline phosphatase (ALPase)-positive granules in unstimulated human neutrophils. ALPase activity was found primarily on the exoplasmic membrane halves of intracellular small granules. Using X-ray microanalysis, we confirmed that the electron-dense deposit on replicas was the reaction product demonstrating ALPase activity. The results obtained by this method should provide unique information for the understanding of structure and function of biological membranes.