TPPase Activity Research Articles

GhTPPA_2 enhancement of tobacco sugar accumulation and drought tolerance

Trehalose metabolism plays an important role in plant growth and response to abiotic stress. Trehalose-6-phosphate (Tre6P) can help regulate sugar homeostasis and act as an indication signal for intracellular sugar levels. Crop productivity can be greatly increased by altering the metabolic level of endogenous trehalose in plants, which can optimize the source-sink connection. In this study, the upland cotton GhTPP protein family was first homologously compared and 24 GhTPP genes were found. Transcriptome analysis revealed that GhTPP members had obvious tissue expression specificity. Among them, GhTPPA_2 (Gh_A12G223300.1) was predominantly expressed in leaves and bolls. The results of subcellular localization showed that GhTPPA_2 is localized in the chloroplast. Via PlantCare, we analyzed the promoters and found that the expression of GhTPPA_2 may be induced by light, abiotic stress, and hormones such as abscisic acid, ethylene, salicylic acid and jasmonic acid. In addition, GhTPPA_2 was overexpressed (TPPAoe) in tobacco, and we found that the TPPase activity of TPPAoe tobacco increased by 66 %. Soluble sugar content increased by 39 % and starch content increased by 27 %. Whereas, the transgenic tobacco had obvious growth advantages under 100 mM mannitol stress. Transcriptome sequencing results showed that the differential genes between TPPAoe and control were considerably enriched in functions related to photosynthesis, phosphate group metabolism, and carbohydrate metabolism. This study shows that GhTPPA_2 is involved in regulating sugar metabolism, improving soluble sugar accumulation and drought stress tolerance of tobacco, which provides theoretical basis for research on high yield and drought tolerance of crops.

Tripolyphosphate hydrolysis by bovine fast and slow myosin subfragment 1 isoforms

Polyphosphates are used in the meat industry to increase the water holding capacity of meat products. Tripolyphosphate (TPP) is a commonly used polyphosphate and it is metabolized into pyrophosphate and monophosphate in meat. The enzymes responsible for its metabolism have not been fully characterized. The motor domain of myosin (subfragment 1 or S1) is a likely candidate. The objectives of this study were to determine if bovine S1 hydrolyzes TPP, to characterize the TPPase activity of the fast (cutaneous trunci) and slow (masseter) isoforms, and to determine the influence of pH on S1 TPPase activity. S1 hydrolyzed TPP and in comparison with ATP as substrate, it hydrolyzed TPP 16-32% more slowly. Fast S1 hydrolyzed both substrates faster compared to slow S1 and the difference between the isoforms was greater with TPP as the substrate. The V(max) was 0.94 and 5.0 nmol Pi/mg S1 protein/min while the K(m) was 0.38 and 0.90 mM TPP for slow and fast S1, respectively. Pyrophosphate was a strong inhibitor of TPPase activity with a K(i) of 88 and 8.3 microM PPi for fast and slow S1 isoforms, respectively. Both ATPase and TPPase activities were influenced by pH with the activity being higher at low pH for both fast and slow S1 isoforms. The activity at pH 5.4 was 1.5 to 4-fold higher than that at pH 7.6 for the different isoforms and substrates. These data show that myosin S1 readily hydrolyzes TPP and suggest that it is a major TPPase in meat.

Study on the tripolyphosphatase (TPPase) property of bighead carp (Aristichthys nobilis) myosin subfragment-1

Myosin subfragment-1 was prepared from the myofibrils of bighead carp (Aristichthys nobilis). The myosin subfragment-1 was proved to have the activity of tripolyphosphatase (TPPase) responding to the hydrolysis of sodium tripolyphosphate (STPP). The optimum temperature and pH for the TPPase of myosin subfragment-1 were 30°C and pH 5.0, and at pH 8.0 the TPPase also showed a high activity. Mg2+ was necessary to TPPase. The TPPase activity of myosin subfragment-1 was activated by Mg2+ under low concentrations, but was inhibited when the concentration was over 17 mmolL−1. The TPPase activity was also affected by KCl. The optimum concentration of KCl for TPPase was 0.3 molL−1 under the condition of 17 mmolL−1 Mg2+. The TPPase activity was significantly inhibited by EDTA-Na2. Reagents such as KBr, KI and KIO3 could inhibit the TPPase effectively. K2Cr2O7 as well as KMnO7 and KNO3 exhibited weak inhibiting effects. The TPPase converted STPP to pyrophosphate (PP) and orthophosphate (Pi) stoichiometrically with a K M of 3.2mmolL−1.

Three-dimensional Dynamics of the Golgi Apparatus of Parotid Acinar Cells as Analyzed by Computer Graphics of Cytochemically-marked Serial Sections.

To correlate the structure and function of the Golgi apparatus, we employed computergenerated three-dimensional reconstruction from electron micrographs of serial sections treated for thiamine pyrophosphatase (TPPase) cytochemistry. Three-dimensional analyses were conducted for rat parotid acinar cells in a resting state, in the course of mitotic division, and during postnatal development.In resting acinar cells, the TPPase-positive trans elements are the same size as the TPPase-negative middle/cis elements horizontally, and they mutually overlap from side to side. During the mitotic stage, TPPase activity disappears from the disorganized Golgi apparatus and no secretory granule formation is observed. Recovery of the stacked structure and TPPase activity take place in daughter cells at the telophase, which are followed by secretory granule formation. During the differentiation of Golgi apparatus in postnatally developing acinar cells, TPPase activity is expressed following formation of the stacked structure, and then granule formation is initiated. These results clearly demonstrate the close relationship among the stacked structure, TPPase activity and the exocrine function of the Golgi apparatus.

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.

Selective translocation of different markers in the ante- and retrograde pathways between the Golgi apparatus and the rough endoplasmic reticulum in a hybridoma cell line

We examined the effects of brefeldin A (BFA, 10 micrograms/ml), an inhibitor of protein transport, on the redistribution of different markers of the Golgi apparatus (GA) in hybridoma H35 cells to examine selective transport of marker molecules between the rough endoplasmic reticulum (RER) and the GA. In H35 cells, the GAs had several cisternae with cis and trans faces as deduced by morphology such as relationship with RER and secretory granules. Thiamin pyrophosphatase (TPPase) was distributed in the trans elements, mannosidase II (man II) was in the cis-medial elements, and deposits of Zinc-Iodide-Osmium (ZIO) staining were localized in the cis/intermediate compartment. Upon BFA treatment for 5 min, man II and TPPase were redistributed in all cisternae. After 10 min of BFA treatment, TPPase activity was observed only in the RER, while the cis/intermediate compartment as evidenced by ZIO staining and man II remained. Upon clearance of BFA from the medium, cisternal structures with man II and ZIO staining reappeared at 30 min. TPPase activity was detected in the GA only after 120 min. Thus, in the retrograde pathway, the trans marker, TPPase moves earlier than the cismedial markers, man II and ZIO staining, whereas in the antegrade pathway, the cis-medial markers move earlier than the trans marker. These results suggest that BFA first alters the characteristic enzyme localization before the GA vanishes into the RER, and that selective transport mechanisms may exist for components of different stacks of the GA.

Three-dimensional Dynamics of the Golgi Apparatus in Mitotic Parotid Acinar Cells: Computer-aided Reconstruction from Cytochemically-marked Ultrathin Serial Sections.

For clarification of the structural and cytochemical dynamics of the Golgi apparatus of the parotid acinar cell during mitotic division in vivo induced by repeated injections of isoproterenol, computer-generated three-dimensional reconstruction was conducted of serial section electron micrographs in conjunction with Golgispecific enzyme cytochemistry. Outlines of the cells, nuclei, thiamine pyrophosphatase (TPPase) -positive Golgi elements (trans Golgi) and TPPase-negative ones (middle/cis Golgi) could be clearly seen through the use of a commercially available computer software program.In interphase acinar cells, the Golgi apparatus could be seen to spread out in the supranuclear region of the cytoplasm as a single continuous reticular structure with many branches and anastomosis. TPPase-positive trans elements were associated with the entire surface of the interphase Golgi stack. With the start of the mitotic phase, gradual disorganization of the stack into a loose structure followed by dispersion throughout the cytoplasm and marked disappearance of TPPase activity became evident. Up to the anaphase, the Golgi apparatus was completely disorganized as small TPPase-negative clusters comprised of tubulo-vesicular membranes were distributed throughout the cytoplasm. Recovery of the stack, single reticular continuity, supranuclear location and TPPase-reactivity of Golgi apparatus was apparent in daughter cells during the telophase. Distributional change of the Golgi apparatus of parotid cell during mitotic division was clearly confirmed by the present study. Dispersion and reassembly may be significant factors in the equal partitioning of the Golgi apparatus into daughter cells. TPPase activity would appear quite essential to the expression of the normal cellular secretory functions.

Thiamine pyrophosphatase cytochemistry in rat endometrium during the oestrous cycle.

The functional morphology of the Golgi apparatus was studied in various types of cells in the rat endometrium during the oestrous cycle. A cerium-based enzyme-cytochemical method was used for the ultrastructural visualization of the activity of thiamine pyrophosphatase (TPPase). The cerium-based method was evidently superior to the classical lead technique, which was used for comparison. TPPase activity in luminal and glandular epithelial cells displayed cyclical modulation and redistribution. It was restricted to only one or two narrow trans lamellae during dioestrus but extended during proestrus and oestrus into nearly all trans-to-cis lamellae of the well-compartmentated Golgi apparatus. A homogeneous staining reaction, which was particularly intense during the latter two phases and only partly due to unspecific alkaline phosphatase, was confined to the apical and basolateral plasma membranes of luminal epithelial cells. In the stromal fibroblasts, only one short Golgi saccule was positive at dioestrus, whereas three or more trans Golgi lamellae were filled with reaction product during oestradiol-dominated oestrus. TPPase activity was furthermore observed in the lysosomes in epithelial cells, stromal fibroblasts, capillary endothelial cells and pericytes. The present findings of cyclic changes in TPPase activity in epithelial cells and stromal fibroblasts provide the first evidence of cyclic modulation and redistribution of this enzyme in the endometrium.

Ultracytochemical Characteristics of Two Cell Types in the Rat Knee Synovial Membrane.

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.

Thiamine pyrophosphatase activity in secretory cells of the lateral prostate and seminal vesicle of normal and castrated guinea pigs and castrates treated with oestradiol.

Ultrastructural localization of thiamine pyrophosphatase (TPPase) activity was studied in secretory cells of the lateral prostate and seminal vesicle of normal and castrated guinea pigs and castrates treated with 17 beta-oestradiol benzoate. The present study has demonstrated that TPPase reaction product is consistently localized in the three to four trans cisternae of Golgi complexes in both the lateral prostate and the seminal vesicle. The reaction was intense and the reaction product often filled the cisternae completely. After castration there was a decrease in TPPase activity in both glands as revealed by the reduction in the amount of the reaction product which was found mainly in one to two trans cisternae of the regressed Golgi complex. The reaction product changed from a dense to a more particulate or granular pattern or to discrete deposits of high electron-density. Administration of 17 beta-oestradiol benzoate to the castrates caused changes in the localization and patterns of distribution of TPPase. In the lateral prostate there was an apparent increase in TPPase activity. The reaction product was found in two to four trans cisternae and occasionally in the trans-most cisternae of the dilated Golgi complex. The reaction product appeared as discrete, dense coarse precipitates. In the seminal vesicle TPPase reaction product was consistently found in one to two trans cisternae in cells with larger Golgi complexes. However, almost all cisternae of the smaller Golgi complexes were TPPase-positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes of cytochemical properties in the Golgi apparatus during in vivo differentiation of the ameloblast in developing rat molar tooth germs.

The cytochemical changes of the Golgi stacks occurring concomitantly with cell differentiation were examined in ameloblasts of developing rat molar tooth germs using osmium impregnation and cytochemistry with nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and acid phosphatase (Acpase). NADPase, TPPase, and Acpase activities were already present in the Golgi stacks of the inner enamel epithelial cells, the undifferentiated form of the ameloblast: NADPase activity existed in the medial Golgi cisternae, TPPase activity in the trans Golgi cisternae, and Acpase activity in almost all cisternae and strongly in the trans-most cisterna of the Golgi stack. At this stage, however, osmium deposits after impregnation were not observed in the cisterna of Golgi stacks but were present in some small vesicles. These vesicles were located throughout the cytoplasm. Osmiophilic cisternae in the Golgi stacks were apparent for the first time at the stage when the Golgi apparatus developed and migrated to the region distal to the nucleus with the progression of cell differentiation. These findings indicate that the cis subcompartment of the Golgi apparatus was incomplete in the inner enamel epithelial cells with regard to appearance of its cytochemical property, as compared with the medial and trans subcompartments. It is suggested that the cis compartment of the Golgi stack may be completed only in the last stage of the compartmentalized Golgi organization during differentiation of the ameloblast.

Ultrastructural and cytochemical studies of the effects of prolactin on the lateral prostate and the seminal vesicle of the castrated guinea pig.

Administration of ovine prolactin to castrated guinea pigs for 2 weeks induced hypertrophy of secretory cells in the lateral prostate when compared with the castrated controls. This was accompanied by an apparent increase in the number of profiles of granular endoplasmic reticulum and well developed Golgi complexes with dilated cisternae. An increase in the number of low-contrast electron-dense secretory granules was observed 4 weeks after prolactin treatment. In the seminal vesicle, dilatation and degranulation of granular endoplasmic reticulum and an apparent decrease in the number of secretory granules were observed 4 weeks after prolactin administration. Following castration and 2 weeks after prolactin treatment, thiamine pyrophosphatase (TPPase)-reaction product was mainly confined to 1-2 trans cisternae of the Golgi complexes in secretory cells of the lateral prostate and the seminal vesicle. In both glands, a reduction of TPPase activity was observed 2 weeks following prolactin administration, and the reaction product was totally absent after prolonged treatment for 4 weeks. The present study has provided morphological evidence that prolactin is capable of stimulating the secretory function of the lateral prostate while exerting some inhibitory effects on the seminal vesicle of the castrated guinea pig. In both glands, TPPase activity, and hence the process of glycosylation was inhibited after prolactin administration. The results from radioimmunoassay indicated that the action of prolactin on these glands could be a direct effect and not mediated through testosterone.

Thiamin mono- and pyrophosphatase activities from brain homogenate of Guamanian amyotrophic lateral sclerosis and parkinsonism-dementia patients

Thiamin-pyrophosphatase (TPPase) and thiamin-monophosphatase (TMPase) were determined using a spectrophotometric method at various pH values (5.5, 7.5, and 9.0) in brain tissue obtained at autopsy from amyotrophic lateral sclerosis (ALS) and parkiinsonism-dementia (PD) patients from Guamaand from Guamanian patients who died from other diseases (controls). TPPase separation by thin-layer polyacrylamide gel isoelectric focusing (IEF) was also performed using both gray and white matter. TPPase content, chemically determined at pH 9.0, was found to be significantly reduced in the frontal cortex of ALS and PD patients compared to controls TMPase content, on the contrary, was unchanged. IEF analysis showed 9 clear-cut bands with TPPase activity in the pH range 5.4–7.2 and a broad band at pH 4.7–5.2. The enzymatic activity was higher in gray than in white matter. In one patient the pattern was clearly different, with two additional bands observed at pH 7.1 and 6.7, and thought to be due to genetic microheterogeneity.

Structural and histochemical studies of Golgi complex differentiation in salivary gland cells during Drosophila development

Morphological alterations in the Golgi complex (GC) and changes in the distribution of acid phosphatase (AcPase), thiamine pyrophosphatase (TPPase), complex carbohydrates and reduced osmium tetroxide compounds in this organelle were studied in the salivary gland cells of Drosophila during larval and prepupal development. The morphology and the AcPase, TPPase and complex carbohydrates cytochemical patterns of the Golgi complex varied characteristically during cell differentiation. At the early 3rd instar period the Golgi complex consisted mainly of vesiculated cisternae, and AcPase activity was observed in all cisternae but not in the secretory granules. As development proceeded to the late 3rd instar the Golgi complex displayed its typical appearance, consisting of four to six cisternae, and only the two to three cisternae towards the trans-face as well as the trans-Golgi network and some of the immature secretory granules exhibited AcPase reactivity. In the course of a 'wave' of production of the 'glue' secretory granules proceeding proximally through the gland, the number of AcPase positive cisternae changed correspondingly. After secretion of the 'glue' secretory granules, the size of the Golgi complex decreased and almost all cisternae displayed AcPase reactivity. The detection of TPPase activity presented some specificity problems, since staining was observed not only in the GC cisternae but in the endoplasmic reticulum (ER) and microvilli. The reaction products were seen in a few GC vesicles during the early 3rd instar and in the trans side of the organelle at the end of the 3rd instar. During production of the secretory granules, every GC cisterna was intensely stained. These results agree with previous findings suggesting that AcPase and TPPase in secretory cells may be primarily involved in the processing of exportable proteins. The vicinal (vic)-glycol groups of the complex carbohydrates were detected using the periodic acid/thiocarbohydrazide/silver proteinate (PA-TCH-SP) technique. During synthesis of the 'glue' secretory granules, the reaction products were observed over the GC cisternae and the trans-Golgi network, with increasing intensity from the cis to the trans side of the organelle. No PA-TCH-SP staining was observed over the GC cisternae during the early 3rd instar. Following discharge of the 'glue' secretory granules, all GC cisternae displayed uniform PA-TCH-SP staining. After OsO4 impregnation, the reaction products were observed mainly in ER and mitochondria and rarely in the GC. In numerous cells, only the mitochondria were stained, while in many cases the ER of neighboring cells exhibited differential staining.(ABSTRACT TRUNCATED AT 400 WORDS)

Thiamine Pyrophosphatase Activity in the Golgi Apparatus of Calcitonin-treated Osteoclasts

The thiamine pyrophosphatase (TPPase) activity described by Novikoff and Goldfisher was examined in osteoclasts affected by calcitonin in order to elucidate whether the morphological and functional changes of the osteoclasts have an influence over the secretion function of their Golgi apparatus. The Golgi apparatus of osteoclasts of which the ruffled border had disappeared and bone resorption discontinued as the result of treatment with calcitonin showed a slight TPPase activity. The reaction products of the enzyme in these inactive osteoclasts were distinctly fewer than that of control osteoclasts, which were not affected by calcitonin. From these results, it is suggested that there may be a connection between the morphological and functional changes of osteoclasts and the secretion function of the Golgi apparatus.

Golgi apparatus of epithelial principal cells of the epididymal initial segment of the rat: Structure, relationship with endoplasmic reticulum, and role in the formation of secretory vesicles

The initial segment of the epididymis of rats, fixed with glutaraldehyde, was postfixed with reduced osmium, a technique that clearly delineates the membranes of cisternae of the endoplasmic reticulum (ER) and the various elements of the Golgi apparatus, or with tannic acid to enhance the coats of vesicles and ribosomes on ER cisternae. The material was also treated to demonstrate various phosphatase activities (NADPase, TPPase, CMPase, G-6-Pase) or impregnated with osmium tetroxide. In osmium-impregnated material, the Golgi apparatus of the epithelial principal cells of the initial segment appeared in the light microscope as a branching, anastomosing ribbon forming a large network in the supranuclear region. In the electron microscope, ER were of two types: the heavily granulated, flattened, rough ER seen in the infranuclear and juxtanuclear regions and the distended, tubular, sparsely granulated ER, showing only few ribosomes, seen interlaced with the Golgi ribbon in the supranuclear region and at the apical pole of the cell. Of particular interest in this cell was the fact that the sparsely granulated ER approximated the Golgi stack on both its cis- and trans-faces. On the cis-face of the Golgi stack, the sparsely granulated ER cisternae showed the usual finger- or bud-like protrusions directed toward the cis element of the Golgi stack and around which numerous small 80 nm vesicles or membranous tubules were clustered. The Golgi stack consisted of the following elements in a cis-trans axis: the cis osmiophilic element, a first saccule slightly dilated, saccules two to four (S2-S4), which were NADPase-positive, and saccules five to seven and the eight Golgi element, which were TPPase-positive. On the trans-aspect of the Golgi stacks, several (up to four) CMPase-positive trans-Golgi networks were observed often in close apposition to the sparsely granulated ER cisternae. One of the trans-Golgi networks showed a "peeling-off" configuration, i.e. part of it was closely apposed to the overlying Golgi element of the stack, whereas the remaining part was separated from the stack by a space occupied by a cisterna of sparsely granulated ER. The other trans-Golgi networks were completely separated from the stack and were often seen sandwiched between sparsely granulated ER cisternae. Thus, ER cisternae showed extensive areas of close apposition but no continuity with the trans-Golgi networks. Although the saccules of the Golgi stacks showed NADPase and/or TPPase activity, the trans-Golgi networks displayed CMPase activity, thus facilitating their identification from the closely associated unreactive sparsely granulated ER cisternae.(ABSTRACT TRUNCATED AT 400 WORDS)

Thiamine pyrophosphatase in brain, partial purification, regional distribution and ontogeny

Thiamine pyrophosphatase (TPPase) from sheep brain was purified to 1043-fold. During the course of purification a concomitant increase in phosphatase activity against phosvitin was noted to follow the increase in specific activity to TPPase. Final preparation with a characteristic high salt (0.5 M NaCl) requirement for solubilization, was found to have a specific activity of 928 and 87.5 mU respectively for thiamine pyrophosphate (TPP) and phosvitin. While no phosphatase activity was observed against casein and phosphoaminoacids, the enzyme was found to have high affinity towards adenosine diphosphate (ADP) (Km 0.73 mM). Both TPP and ADP were found to inhibit the phosphatase activity at higher substrate concentration with a optimum pH of 6.0. Regional distribution of enzyme activity in rabbit and human brain indicated a different pattern with a distinct species difference with reference to thalamus, striatum and hippocampus. Studies on ontogeny in rat brain indicated a gradual increase in TPPase activity during the prenatal extending over to postnatal period with the maximum activity attained on 4th day after birth to be followed by a gradual decrease in enzyme activity up to 30th day of age.

Proliferation of microglia in the injured cerebral cortex of mice as studied by thiamine pyrophosphatase histochemistry combined with 3H-thymidine autoradiography.

Proliferation of microglia in the mouse cerebral cortex around a stab wound was studied, using thiamine pyrophosphatase (TPPase) histochemistry combined simultaneously with 3H-thymidine (3H-TdR) autoradiography. Many cells with cell membrane TPPase activity (TPPase-positive cells) were scattered in the cortical parenchyma apart from the stab wound. Light and electron microscopically, TPPasepositive cells were identified as microglia but not as astrocytes, oligodendrocytes, or neurons. TPPasepositive cells were immunohistochemically negative for astrocytic markers, i. e., S-100 protein and glial fibrillary acidic protein. Autoradiography showed that approximately 92% of all cells labeled with 3H-TdR within 2 days after stab-wounding were TPPase-positive cells. Approximately 60% of all labeled cells 3 and 4 days after stab-wounding were also TPPase-positive cells. These results suggest that microglia, but not astrocyte, begins active proliferation immediately after injury and is the major cell type having high proliferative activity in the injured cerebral cortex.

Ultrastructural localization of phosphatases with cerium in a ciliated protozoan.

Acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) have been detected with cerium as a capture agent for the first time in ciliated protozoa. In the trophozoites of the parasitic ciliate Ichthyophthirius multifiliis, AcPase activity was previously reported in food vacuoles, primary lysosomes, golgian-like cisternae and in some alveolar sacs. These former results obtained by lead methods, have now been confirmed and improved by a cerium-based technique. TPPase activity was also observed in golgian-like cisternae and alveolar sacs. As the cell membrane of this ciliate proved to be highly impermeable to cerium ions, several hr of preincubation in buffer with cerium chloride and Triton X-100 were needed to detect phosphatases with cerium in intact ciliates. However, in sectioned ciliates the preincubation time could be greatly reduced. Since TPPase became inhibited after a long preincubation, it could only be detected with cerium in sectioned ciliates.

High-voltage electron microscopic observation of golgi complex in the rat small ganglion cell. Thiamine metabolism.

Thiamine mono- and pyrophosphatase (TMPase, TPPase) activities were demonstrated in the rat small ganglion cell under high-voltage electron microscopy. From a series of micrographs of the same field tilted in increments, a trans tubular network (TTN) consisting of looping and anastomosing tubules was found; this network showed relatively higher intense TMPase activity, and exhibited connections with TMPase-positive vesicle-like bodies. TPPase activity was localized in the trans Golgi saccules, and the TPPase-positive transmost saccule had a structural resemblance to the TTN. These results indicate that the TTN plays an important role in thiamine metabolism.