Integrative method for three-dimensional imaging of the entire Golgi apparatus by combining thiamine pyrophosphatase cytochemistry and array tomography using backscattered electron-mode scanning electron microscopy.

Thiamine pyrophosphatase (TPPase) cytochemistry is an established method for specific labeling of the trans-Golgi cisterns in tissue sections. Herein, we combined this enzyme cytochemical method with array tomography using scanning electron microscopy (SEM), a new imaging technique based on collection of backscattered electron (BSE) images of consecutive resin-embedded sections on glass slides, to detect the entire three-dimensional (3D) organization of the Golgi apparatus with sufficient spatial resolution. As the signal intensity of BSE depends on the atomic number of the materials, lead precipitates confined to the trans-Golgi cisterns after TPPase cytochemistry were clearly observed by BSE-mode SEM. The mild fixative used for TPPase cytochemistry also enabled accurate identification of target gonadotropes in the composite pituitary tissue by immunocytochemical staining. By 3D reconstruction of the entire trans-Golgi cisterns based on serial ultrathin section images of tissues after TPPase cytochemistry, we detected ultrastructural differences in the 3D configuration of the Golgi apparatus between cerebellar Purkinje cells and pituitary gonadotropes. The appropriate combination of enzyme cytochemistry and/or immunostaining with array tomography will further clarify the relationship between the organization and functional states of the Golgi apparatus.

Testicular effects of monensin, a golgi interfering agent in male rats.

OBJECTIVE: The present study was undertaken to explore the effects of monensin, a potent Golgi disturbing agent on male fertility. METHODS: Male Wistar rats were administered monensin at the dose levels of 2.5, 5, and 10 mg/kg b wt. Animals were sacrificed after 67 days of the treatment. The activities of lactate dehydrogenase (LDH), ATPase, acid phosphatase and thiamine pyrophosphatase (TPPase) were measured in the testis. Cytochemical assay of Golgi body marker enzyme, thiamine pyrophosphatase was also performed. Ultrastructural changes in testis were studied by Transmission electron microscopy. Sperm number and motility were also examined. RESULTS AND DISCUSSION: The alterations in the activities of above mentioned enzymes indicate the pronounced effect of the drug on the functioning of spermatogenic cells. The findings from electron microscopy such as membrane disruption, swelling and disintegration of Golgi apparatus strongly suggest the interference of monensin with the functioning of Golgi apparatus in the spermatogenic cells. Data from the sperm number and motility as well as the fertility studies and the resulted litter size further points towards the antifertility effects of monensin in male rats. CONCLUSION: The findings from the present study strongly indicated the effects of monensin on the testis, involving alterations in key enzyme activities and changes at the ultrastructural level.

Identification and characterization of oxalate oxidoreductase, a novel thiamine pyrophosphate-dependent 2-oxoacid oxidoreductase that enables anaerobic growth on oxalate.

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO(2) fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO(2) or bicarbonate. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe(4)S(4)] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a k(cat) of 0.09 s(-1) and a K(m) of 58 µM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood-Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism.

Determination of the genetic, molecular, and biochemical basis of the Arabidopsis thaliana thiamin auxotroph th1.

2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate kinase/thiamin monophosphate pyrophosphorylase (HMPPK/TMPPase) is a key enzyme involved in thiamin biosynthesis. A candidate HMPPK/TMPPase gene identified in the Arabidopsis genome complemented the thiamin auxotrophy of the th1 mutant, thus proving that the th1 locus corresponds to the structural gene for the HMPPK/TMPPase. Sequence comparisons between the wild-type HMPPK/TMPPase gene and the th1-201 mutant allele identified a single point mutation that caused the substitution of a phenylalanine for a conserved serine residue in the HMPPK domain. Functional analyses of the mutant HMPPK/TMPPase in Escherichia coli revealed that the amino acid substitution in the HMPPK domain of mutant enzyme resulted in a conformational change that severely compromised both activities of the bifunctional enzyme. Studies were also performed to identify the chloroplast as the specific subcellular locale of the Arabidopsis HMPPK/TMPPase.

Effect of monensin on the enzymes of oxidative stress, thiamine pyrophosphatase and DNA integrity in rat testicular cells in vitro.

Monensin, a sodium specific ionophore was evaluated for its in vitro effects on rat testis by studying changes at biochemical parameters as well as at the DNA level. It was observed that monensin produced marked alterations in the activities of various enzymes associated with the testicular functions. The significant inhibition of different enzymes of oxidative defense system points toward the generation of reactive oxygen species (ROS) by monensin treatment. The significant depletion of reduced glutathione and elevation in the level of lipid peroxidation further support the above findings. The significant inhibition of the activities of lactate dehydrogenase and adenosine triphosphatase shows the interference of monensin with the normal energy supply in spermatogenesis. Moreover, the significant increase in the activities of acid phosphatase and thiamine pyrophosphatase demonstrates the interference of monensin with the Golgi-lysosomal complex of the rat testis. Induced DNA fragmentation indicates towards the impact of monensin on the DNA integrity and apoptosis. Further studies are needed to understand the important molecular mechanisms responsible for these effects.

Cytochemical study of the involvement of cell organelles in formation and accumulation of fibrillar amyloid in the pancreas of NORbeta transgenic mice.

Phosphatase ultrastructural cytochemistry was used to evaluate the participation of cytoplasmic organelles in the accumulation of fibrillar amyloid beta (Abeta) in exocrine acinar cells and in macrophages of the pancreas of transgenic mice overexpressing a carboxy-terminal fragment of Abeta protein precursor (ABPP). Nucleoside diphosphatase (NDPase) and glucose-6-phosphatase (G6Pase) were used as cytochemical markers of the endoplasmic reticulum (ER), thiamine pyrophosphatase (TPPase) as a marker of the Golgi apparatus (GA), and acid phosphatase (AcPase) as a marker of lysosomes. Monoclonal antibody 4G8 raised against the 17-24 aa sequence of human Abeta protein was used for immunogold localization of fibrillar Abeta. The results of this study indicate that the formation of Abeta in acinar cells occurs directly in the vacuolar areas of the rough ER (RER) without evident participation of the elements of the GA, whereas an intimate structural relation with primary lysosomes suggests their role in modification or digestion of the deposited amyloid. In macrophages, fibrillar amyloid was present in numerous cytoplasmic vacuoles located frequently in close proximity to flattened saccules of the ER. This structural pattern revealed similarity to that observed previously in microglial cells producing fibrillar PrP amyloid in scrapie-infected mice and Abeta in brains of human elderly patients and in Alzheimer's type brain pathology.

Hormonal control of enzyme activity during the plasma membrane transformation of uterine epithelial cells.

Ultrastructural and light microscopic catalytic histochemical methods were used to study the distribution and changes in distribution of four phosphatase enzymes; alkaline phosphatase, 5'-nucleotidase, thiamine pyrophosphatase and adenosine triphosphatase in uterine epithelial cells in response to the ovarian hormones, oestrogen, progesterone or a combination of both used in different regimes on ovariectomised rats. Reaction product for all four enzymes was clearly localised in the epithelial cells, especially with oestrogen priming. However, the four enzymes showed markedly different patterns of organisation of reaction product in response to other hormonal treatments. Our findings clearly show that the expression of these enzymes is under ovarian hormonal control. However, while all of the enzymes are upregulated by oestrogen, the response to progesterone is variable, which can upregulate or downregulate different enzymes. The findings are particularly obvious at the electron microscopic level on the apical plasma membrane of the uterine epithelial cells, which was the main focus of our study.

Nuclear changes and acrosome formation during spermiogenesis in Euchistus heros (Hemiptera: Pentatomidae).

Ultrastructural and cytochemical studies were carried out on nuclear changes and acrosome formation during the spermiogenesis of the phytophagous bug Euchistus heros. The development of the nucleus involves changes in the shape and in degree of chromatin condensation: initially it is dispersed and with a low-electron density, then assumes a fibrillar arrangement and finally compacts in an electron-dense material. The acrosome is formed by the Golgi complex and presents unusual morphological features during its development. The reaction product of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase activities were detected during various stages of acrosome development. In contrast, residues of alpha-N-acetylgalactosamine and basic proteins were only reported in the intermediate and late stages of the differentiation process, respectively.

Cytoskeleton mediating transport between the ER system and the Golgi apparatus in the green alga Scenedesmus acutus.

In the green alga Scenedesmus acutus, Golgi bodies are located near the nucleus and supplied with transition vesicles that bud from the outer nuclear envelope membrane. Using this alga, we have shown previously that thiamine pyrophosphatase (TPPase), a marker enzyme of Golgi bodies, migrates in vesicles from the Golgi bodies to the ER via the nuclear envelope in the presence of BFA (Noguchi et al., Protoplasma 201, 202-212, 1998). In this study we demonstrate that both cytochalasin B and oryzalin (microtubule-disrupting agent) inhibit the BFA-induced migration of TPPase from Golgi bodies to the nuclear envelope. However, only actin filaments--not microtubules--can be detected between the nuclear envelope and the Golgi bodies in both BFA-treated and untreated cells. These observations suggest that actin filaments mediate the BFA-induced retrograde transport of vesicles. This mechanism differs from that found in mammalian cells, in which microtubules mediate BFA-induced retrograde transport by the elongation of membrane tubules from the Golgi cisternae. We also discuss the non-participation of the cytoskeleton in anterograde transport from the nuclear envelope to the Golgi bodies.

The role of the Golgi apparatus during terminal differentiation of mouse urothelial surface cells.

The development of the Golgi apparatus in the surface cells of mouse urinary bladder during embryonic development was investigated by electronmicroscopic cytochemistry. The distributions of NADPase and TPPase activities were studied in the urinary bladder during day 15 to day 18 of gestation. At the early embryonic stage, the products of the NADPase and TPPase reactions were visible exclusively in 1 to 2 medial and/or trans Golgi saccules. The strongest increment of NADPase and TPPase positive Golgi cisternae was detected at day 17 when the activity of the urothelial cells was very prominent. At this age, NADPase activity was detected also in lysosomes and on the apical surface of the urothelial cells. The highest distribution pattern of NADPase and TPPase activities observed at this stage rapidly decreases at day 18 of fetal life. The results suggest that the organization of the Golgi apparatus reflected the intensity of the processes occuring in the urothelial cells during gestation.

Ultrastructural and cytochemical studies of the spermatozoa of Acrosternum aseadum (Hemiptera: Pentatomidae) after copulation.

Beside the morphofunctional modifications undergone during spermiogenesis, the spermatozoon could undergo other modifications after copulation. Since no structural modification occurs in the spermatozoon of Acrosternum aseadum after copulation, we used cytochemical studies to show the enzymatic activities variations of acid phosphatase, thiamine pyrophosphatase, glucose-6-phosphatase and cytochrome C oxidase, when the spermatozoon passes through the spermatheca. The enzymatic activity, few hours after copulation, is strong and specifically located. However, 40 h after copulation, there is considerable loss of enzymatic activity, with the exception of thiamine pyrophosphatase, which shows the same activity. This result indicates that the spermatozoon of A. aseadum undergoes physiological modifications when passing through the spermatheca and that these modifications may be involved with survival in this organ as well as with the fertilization process.

Differential alterations in the distribution of three phosphatase enzymes during the plasma membrane transformation of uterine epithelial cells in the rat.

Ultrastructural and light microscopic cytochemical methods were used to study the distribution and changes in distribution of three phosphatase enzymes: 5'-nucleotidase (5N); thiamine pyrophosphatase (TPP); and adenosine triphosphatase (ATP) in the rat endometrium during early pregnancy up to the time of blastocyst attachment. The authors were particularly interested in changes in the apical plasma membrane and reaction product for all three enzymes was clearly localized along this membrane especially on day 1 of pregnancy. However, the three enzymes showed markedly different patterns of organization of reaction product at later times during early pregnancy. 5N, while showing a continuous lining along the microvilli on day 1 was virtually undetectable by day 6. TPP was also strongly present apically on day 1, but reaction product was not always found as a continuous lining. Again, by day 6, there was no presence of this enzyme along the apical surface. ATP differed from the other two in that it produced a strong, and relatively unchanged reaction product along the apical plasma membrane from day 1 through to day 6 of pregnancy. The changes in distribution of these enzymes was particularly obvious at the electron microscopic level and we consider their contribution to the process of 'plasma membrane transformation' of early pregnancy.

Characterization of thiamine pyrophosphatase positive phagocytic cells in the neural lobe of rat pituitary.

Here, using a histochemical staining for a microglia/phagocyte marker TPP-ase (Murabe, Sano 1981), and an electron microscopy we characterized the population of pituitary phagocytic cells activated by cerebral ischemia. An intense thiamine pyrophosphatase (TPP-ase) activity was demonstrated in glial cells and some cells of blood vessels of neural lobe, late period (12 months) after experimental ischemia. TPP-ase positive cells were ultrastructurally identified as pituicytes, microglia, pericytes and perivascular cells. The product characteristic for TPP-ase activity was seen on plasma membrane of these cells. Our electron-microscopic histochemical results provide strong support for a role of pituicytes, pericytes and perivascular cells as a phagocytic cells involved in mechanism of elimination of ischemically damaged axonal endings in neural lobe.

Chronic submandibular sialadenitis: ultrastructure and phosphatase histochemistry.

Eleven specimens of chronic submandibular sialadenitis were examined. A reduction in secretory material in acinar cells was seen with increasing atrophy until the acini resembled intercalary ducts. Myoepithelial cells and basement membrane were sometimes more conspicuous. Striated ducts showed a reduction of the folding of the plasma membranes in the basal part, and striated and excretory ducts showed a reduction in mitochondria. This possibly represents a functional atrophy secondary to reduced salivary flow. Very atrophic parenchyma largely consisted of simple cells. Phagosomes and apoptotic bodies were occasionally seen, and appear to be involved in the atrophy. Thiamine pyrophosphatase in the Golgi apparatus and acid phosphatase in the GERL were demonstrated in moderately atrophic parenchyma. This is similar to normal and indicates continuing synthetic activity. Acid phosphatase was demonstrated in lysosomes, which appear to be involved in the atrophy by their role in phagy. Alkaline phosphatase was occasionally demonstrated at luminal surfaces, and is likely to be involved in resorption of obstructed luminal contents. The changes are similar to those seen in experimentally obstructed glands and indicate that much of the parenchyma survives by adaptation to the altered environment which forms the basis for the successful results following conservative therapy.

Ultrastructural localization of thiamine pyrophosphatase in plasma membranes of brain phagocytes long time after cardiac arrest.

Brain phagocytes are members of a heterogeneous family of microglial cells. In this study we investigated the membrane activity of the enzyme, thiamine pyrophosphatase (TPP-ase), in brain phagocytes. Studies were performed on rats subjected to a transient ischemia because ischemic incident precipitates proliferation of microglial cells in the brain. Brain tissue was sampled from animals that survived 12 months after experimentally evoked cardiac arrest. The product characteristic for TPP-ase activity was present in the Golgi cisterns of neurons, basement membranes of endothelia and capillary pericytes. TPP-ase activity was present on plasma membranes of brain phagocytes. The phagocyte TPP-ase activity did not depend on the anatomical localization of the cell in the brain (cortex, hippocampus, hypothalamus). Thus, TPP-ase activity can be considered as a marker of brain phagocytes.

Endoplasmic reticulum and Golgi-like elements in Entamoeba.

The cytoplasm of Entamoeba is characterized by the presence of a large number of vesicles of different size and shape. Previous electron microscopic studies have not clearly revealed the presence of the endoplasmic reticulum and the Golgi complex. In the present study two approaches were used aimed at the identification of these two structures in trophozoites of Entamoeba moshkovskii and Entamoeba histolytica: (a) cytochemical techniques associated with transmission electron microscopy, such as osmium tetroxide-zinc iodide, localization of glucose-6-phosphatase and thiaminopyro-phosphatase, and (b) labeling of the structures with the fluorescent dyes DiOC6 and C6-NBD ceramide followed by visualization of the labeled cells by confocal laser scanning microscopy. Our observations suggest that some of the cytoplasmic vacuoles may correspond to components of the endoplasmic reticulum and the Golgi complex of Entamoeba.

Dose-dependent effects of chloroquine on secretory granule formation in the melanotroph.

Chloroquine diverts secretory peptides from the regulated to the constitutive secretory pathway. The exact site and mechanism of the effect are not known. We studied the effect of increasing doses of chloroquine on the morphology of cultured melanotrophs from the rat pituitary. 40 microM chloroquine for 2 h, which perturbs intracellular pH gradients in melanotrophs without affecting secretion, caused swelling of a subpopulation of immature secretory granules. 200 microM chloroquine for 2 h, which diverts secretory peptides from the regulated to the constitutive pathway in the AtT-20 cell line, caused pronounced swelling of immature secretory granules, vacuolization of the trans-Golgi region and the appearance of myeloid bodies and multivesicular bodies in the cytoplasm. Golgi stacks were retained and Golgi cisternae only slightly dilated at both chloroquine concentrations. Mature secretory granules were not affected. Cationized ferritin was internalized and transported to the trans-Golgi region in the presence of 40 microM chloroquine while 200 microM chloroquine arrested internalised ferritin in peripheral multivesicular bodies. The study shows a heterogeneous effect of lower doses of chloroquine on immature secretory granules, providing a tool for studies on the relationships between condensation, acidification and peptide processing during granule formation. Chloroquine of 200 microM caused morphological changes typical for chloroquine toxicity and arrest of endocytic traffic.

[Cytochemical study on the activity of enzymes in trophozoites of Entamoeba histolytica from the cecum of guinea pigs with invasive and non-invasive amebiasis]. / Cytochemiczne badania aktywnosci enzymów w trofozoitach Entamoeba histolytica pochodzacych z jelita slepego swinek morskich z inwazyjna i nieinwazyjna ameboza.

Experimental amoebiasis was inflicted on guinea pigs using the PS-2 strain of Entamoeba histolytica. Cytoenzymatic studies were conducted on the trophozoites sampled from the caecum of hosts with non-invasive and invasive amebiasis. The results show higher intensity of the processes of intracellular digestion, transportation, and anaerobic respiration in the amoebae originating from intestine of the host with infective amoebiasis.

A decrease in neural sialyltransferase activity in Alzheimer's disease.

The activities of both a particulate and soluble form of the sialyltransferase enzyme have been examined in post-mortem brain samples from Alzheimer's disease patients and age-matched controls. There was a considerable decrease in the activity of both the soluble and membrane-bound forms of the enzyme in the frontal and temporal cortical lobes, although no change was observed in the hippocampus. There was, however, no change in activity of the Golgi marker enzyme thiamine pyrophosphatase. Therefore, it is suggested that the decrease in sialyltransferase enzyme activity may be a specific biochemical event associated with the AD-like neurodegeneration.

Brefeldin A inhibits the transcytotic vesicular transport of horseradish peroxidase in intrahepatic bile ductules isolated from rat liver.

The fungal metabolite Brefeldin A (BFA) has become a valuable tool to address mechanisms of membrane transport in eukaryotic cells. The aim of the study was to investigate the action of BFA on the endocytic and transcytotic pathways in the biliary epithelium. Intrahepatic bile ductules were isolated from rat liver by collagenase digestion and mechanical separation of biliary tree from parenchymal tissue. Tissue remnants were first incubated in L-15 culture medium in absence or presence of BFA (10 or 20 mumol/L) or a BFA-inactive analog (B-36, 10 or 20 mumol/L) for 20 minutes at 37 degrees C. They were then exposed to horseradish peroxidase (HRP) (10 mg/mL) for 3 minutes at 37 degrees C and finally prepared for electron microscopy immediately (time 0) or after further 5, 10, 15, 20, 60, or 120 minutes' incubation in HRP-free medium with or without BFA. In control cells, HRP was predominantly found in regularly shaped, spherical vesicles. In the presence of BFA but not of its analog, HRP was retained in a prominent tubular juxtanuclear network. Part of this network was labeled for thiamine pyrophosphatase (TPP), a Golgi enzyme marker. A morphometric analysis of HRP-containing structures was performed to quantify the intracellular distribution of HRP. In presence of BFA, the volume density (VD = % area) of HRP-containing structures in the basolateral region was not significantly different with respect to control cells at 0 (1.08 +/- 0.11 vs. 1.32 +/- 0.11) or 5 minutes, respectively (1.33 +/- 0.19 vs. 1.40 +/- 0.13). On the contrary, VD or HRP-containing structures in the apical region at 15 minutes decreased from 1.95 +/- 0.19 in control cells to 1.12 +/- 0.20 (P < .02) in BFA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)