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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 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.

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.

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 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

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.

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.

The synovial lining layer consists of two cell types ultrastructurally. It is said that the function of type A cell is phagocytosis and one of type B cell is secretion of synovial fluid, but the details are still obscure. For a better understanding of these mechanisms, the localizations of five phosphatase activities, i.e. Mg2+-activated adenosine triphosphatase (Mg2+-ATPase), Ca2+ -activated adenosine triphosphatase (Ca2+ -ATPase), ouabain-sensitive, potassium-dependent adenosine triphosphatase (Na+, K+ -ATPase), acid phosphatase (ACPase) and thiamine pyrophosphatase (TPPase) activities, were studied ultracytochemically on the rat knee joints. Type A cell had Mg2+ -ATPase and Ca2+ -ATPase activities on the plasma membrane, especially on the filopodia. Type B cell had these activities at the pinocytotic vesicles. Na+, K+ -ATPase, ACPase, TPPase activities were found at the Golgi apparatus of type B cell. ATPase on the plasma membrane is the enzyme which supplies the energy needed at active transport or cytosis. So the localizations of these enzyme activities may raise the possibility that both type A and B cells might have mechanisms governing the intraarticular homeostasis of the synovial fluid. And on type B cell, basic data were given about the function of the Golgi apparatus. Furthermore in the synovial lining layer, type A and B cells will be distinguishable not only structurally but also cytochemically.

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.

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.

Objectives: Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial β-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo. Methods: VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0×105 to 1.0×107. Expression of β-galactosidase (β-gal) in VSMC was evaluated by X-gal staining and a β-gal ELISA method. Excised rat aortas were incubated with medium containing AAV-lacZ. Expression of β-gal in the aortic segments was evaluated by X-gal staining. Results: With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the β-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transduced endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC. Conclusions: AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.

We identified five cDNA clones of the Six gene family which are expressed in retina. They are Six2, Six3 alpha and Six3 beta (which are derived from alternative splicing forms), Six5, and AREC3/Six4. All of these Six family genes possess extensive sequence similarity among each other in the so-homologous region (Six domain and homeodomain) but differ greatly in structure in some other regions. The amino acid sequence similarity of the so-homologous region to the previously identified AREC3/Six4 is 70.1% for Six2, 57.3% for Six3 alpha and Six3 beta, and 70.3% for Six5. The expression of these genes was observed in inner and outer nuclear layer, ganglion cell layer, and pigment epithelium of mouse retina by in situ hybridization. The so-homologous region of each Six family protein has specific DNA binding activity. Six5 and Six2 bind to the same sequence as does AREC3/Six4, while Six3 does not. These observations suggest that some of the Six family genes can regulate the same target genes.

Cholinesterase (ChE) activity was ultracytochemically demonstrated in mouse digital (Meissner-like) corpuscles by Karnovsky's or Koelle-Friedenwald's method. Intense enzyme activity was found in the caveolae of lamellar cell processes, in the interlamellar spaces and in the narrow perineural spaces between the nerve endings and the apposing lamellar processes. Prominent enzyme activity was also found in the cistern of the rough endoplasmic reticulum as well as in the nuclear envelope of the lamellar cell body. These findings suggest that the enzyme is synthesized in the lamellar cells and released into the interlamellar and perineural spaces in the corpuscle. The enzyme activity was inhibited by eserine 10-4M, DFP 10-5M, or iso-OMPA 10-4M, but not by BW284C51 at a concentration lower than 10-4M. This result indicates that the enzyme activity studied is not that of acetylcholinesterase (AChE) but of nonspecific cholinesterase.

Dissociated cerebella of newborn rats were cultured in vitro as a monolayer for a long period, and ependymal cell differentiation was investigated by scanning and transmission electron microscopy. Cells with actively beating cilia were recognized on the 3rd day, and gradually increased in number thereafter. At nine months of culturing, a number of areas appeared, some of which consisted of several hundreds of ciliated cells. Cultured ependymal cells showed a striking resemblance in their fine structure to the intact ependyma. They were always found to grow upon the feltwork of astrocytic processes which served as a subependymal structure, as observed in the intact ependyma. It is possible that astrocytes may play some role in ependymal differentiation. Cultured ependymal cells are helpful in understanding the control mechanism for the metachronal cilial movement.

Acetylcholinesterase (AChE) activity in the developing chick retina was histochemically studied by electron microscopy. The enzyme activity in the 1-day-old chick retina was confined within the ganglion, amacrine and horizontal cells as well as in the inner plexiform layer. In the inner plexiform layer reaction product was found in the intercellular spaces between nerve processes. Synapses in this layer did not always display reaction product. In the developing chick retina, AChE activity was found in the innermost layer of 6-day-old retina in ovo. The stage of the appearance of histochemically demonstrable AChE activity in the developing ganglion and amacrine cells mostly coincided with that of the beginning of morphological maturity of these cells. These findings indicate that the ganglion, amacrine and horizontal cells are functionally different from other types of neurons, and that synthesis of the enzyme starts at almost the same time as those cells begin to manifest the morphological characteristics of each cell type.

Ultrastructural as well as ultracytochemical observations were made On rat hepatic parenchymal cells after glucagon perfusion (200-600μg/50ml of Krebs-Ringer's bicarbonate solution) via portal vein. The irregularly shaped lysosomes, such as rod-shaped, dumbbell-shaped or the ones with tails, were observed even 5 min after glucagon perfusion. The acid phosphatase ctivity was clearly positive in the matrix of these unusual structures.These irregularly shaped lysosomes were considered to be the sheet-like extensions from the original body of the pre-existing lysosomes. In connection with the presence of the irregularly shaped lysosomes an autophagic process was evident and the double-membraned (inner and outer) autophagolysosomes were frequently observed. Results obtained in the present investigation indicate that the autophagolysosomes are formed by the wrapping mechanism of the irregularly shaped lysosomes in the hepatic parenchymal cells of the rat following glucagon administration.

Since acrolein can penetrate more quickly and deeply into tissue blocks than glutaraldehyde, the possibility of the use of this aldehyde as a prefixative in enzyme cytochemistry was reinvestigated. At low concentrations, acrolein preserves the activities of the enzymes investigated, including those of glucose-6-phosphatase, which is known as one of the most vulnerable to aldehyde fixation; thus, acrolein is usable in enzyme ultracytochemistry. Enzyme activities are also preserved in tissues fixed with acrolein and glutaraldehyde combined. The rapid penetration of acrolein enables fixation in larger tissue blocks and provides greater freedom in specimen selection, especially important advantages when encountering heterogeneous materials as in pathology.

This study was carried out to elucidate the mechanism of an autophagy of peroxisomes seen in hepatic parenchymal cells in the CPIB-treated rat liver followed by partial hepatectomy or by glucagon administration. The catalase activity demonstrated cytochemically by the diaminobenzidine reaction was used as a marker for peroxisomes and the acid phosphatase activity demonstrated by the lead method for lysosomes.The large size and number of peroxisomes induced by the CPIB administration in the rat hepatic parenchymal cell showed a gradual decrease in number after partial hepatectomy with concomitant increase in the number of lysosomes of various sizes including autophagolysosomes. Autophagoly-sosome formation involving peroxisomes was also evident in the hepatic parenchymal cells in the CPIB-treated and glucagon-administered rat liver. Furthermore, participation of the lysosomal wrapping mechanism in autophagocytosis of peroxisomes was frequently observed.It was concluded in the present investigation that the lysosome participates in the catabolism of peroxisomes observed in the CPIB-treated rat liver followed by partial hepatectomy or glucagon administration.

The ultracytochemical localizations of adenosine nucleotidase activities were investigated in the human term placenta. The enzymes studied were as follows: 5′-nucleotidase (5′-N), Ca++-activated adenosine triphosphatase (Ca++ ATPase), Mg++-activated adenosine triphosphatase (Mg++ ATPase) and nucleotide diphosphatase (NDPase). 5′-N activity was demonstrated using not only the lead nitrate method of Wachstein and Meisel but also the recently developed cerium and neodymium method as well.The reaction products for 5′-N activity were found on the external surface of the microvillous plasma membrane of the syncytiotrophoblast. Alpha-beta-methylene adenosine diphosphate (AOPCP), at a concentration of 2.0mM, effectively inhibited 5′-N activity. Ca++ATPase and Mg++ATPase activity were observed strongly on the microvillous membrane of the syncytiotrophoblast and weakly on the basal plasma membrane of the syncytiotrophoblast. NDPase, using ADP as a substrate, was localized on the microvillous membrane.These observations suggest that the syncytiotrophoblast is active in the nucleotide metabolism and that microvillous surface of the syncytium may play an important role in regulating the feto-placental-maternal microcirculation in the human term placenta.