Inducible polymerization and two-dimensional assembly of the repeats-in-toxin (RTX) domain from the Pseudomonas aeruginosa alkaline protease.

Self-assembling proteins represent potential scaffolds for the organization of enzymatic activities. The alkaline protease repeats-in-toxin (RTX) domain from Pseudomonas aeruginosa undergoes multiple structural transitions in the presence and absence of calcium, a native structural cofactor. In the absence of calcium, this domain is capable of spontaneous, ordered polymerization, producing amyloid-like fibrils and large two-dimensional protein sheets. This polymerization occurs under near-physiological conditions, is rapid, and can be controlled by regulating calcium in solution. Fusion of the RTX domain to a soluble protein results in the incorporation of engineered protein function into these macromolecular assemblies. Applications of this protein sequence in bacterial adherence and colonization and the generation of biomaterials are discussed.

The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells.

The effects of different culture conditions, suspension and microcarrier culture and temperature reduction on the structures of N-linked glycans attached to secreted human placental alkaline phosphatase (SEAP) were investigated for CHO cells grown in a controlled bioreactor. Both mass spectrometry and anion-exchange chromatography were used to probe the N-linked glycan structures and distribution. Complex-type glycans were the dominant structures with small amounts of high mannose glycans observed in suspension and reduced temperature cultures. Biantennary glycans were the most common structures detected by mass spectrometry, but triantennary and tetraantennary forms were also detected. The amount of sialic acid present was relatively low, approximately 0.4 mol sialic acid/mol SEAP for suspension cultures. Microcarrier cultures exhibited a decrease in productivity compared with suspension culture due to a decrease in both maximum viable cell density (15-20%) and specific productivity (30-50%). In contrast, a biphasic suspension culture in which the temperature was reduced at the beginning of the stationary phase from 37 to 33 degrees C, showed a 7% increase in maximum viable cell density, a 62% increase in integrated viable cell density, and a 133% increase in specific productivity, leading to greater than threefold increase in total productivity. Both microcarrier and reduced temperature cultures showed increased sialylation and decreased fucosylation when compared to suspension culture. Our results highlight the importance of glycoform analysis after process modification as even subtle changes (e.g., changing from one microcarrier to another) may affect glycan distributions.

Phosphatase actions at the site of appositional mineralization in bisphosphonate-affected bones of the rat.

Tissue-nonspecific alkaline phosphatase (TNSALP) and Ca-ATPase are known to play roles in bone mineralization, but how these enzymes contribute to appositional mineralization has been illusive. Here we examined the active sites of these enzymes in appositional mineralization using the bones of young rats being administered with 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) for 5 days. The doses of HEBP totally abolished mineralization of newly formed bone matrix except in matrix vesicles (MVs), and hence allowed precise localization of MVs and phosphatase reactions within non-mineralized extracellular matrix. Intense TNSALP and ATPase reactions were confirmed along the limited portions of osteoblast membranes where intimate cell-cell contacts were maintained. Diffuse reactions of these enzymes were throughout the osteoid implicating efflux of TNSALP and ATPase molecules into extracellular matrix from the osteoblast membranes. Phosphatase reactions associated with MVs varied both in intensity and location among the individual vesicles; newly formed MVs were almost free of reactions but appeared to gain those activities later in the osteoid. These data suggest that TNSALP and ATPase are released from the osteoblast membrane and later integrated into MVs within the osteoid. The osteoblasts may thus regulate appositional mineralization of bone from a distance at least in part by providing phosphatases via MVs.

Areal and subcellular localization of the ubiquitous alkaline phosphatase in the primate cerebral cortex: evidence for a role in neurotransmission.

The ubiquitous enzyme TNAP (tissue non-specific alkaline phosphatase) is found in numerous tissues such as liver, kidney and bone, but little attention has been paid to its expression and role in the brain. Observations in TNAP-KO mice, which analyzed the role of this enzyme in osteogenesis, had suggested that TNAP might be involved in GABA neurotransmission. Apart from its presence in endothelial cells, here we show a specific and strong alkaline phosphatase (AP) activity in the neuropile, matching the pattern of thalamo-cortical innervation in layer 4 of the primate sensory cortices (visual, auditory and somatosensory). Such a pattern is also evident in rodents and carnivores, making AP a powerful marker of primary sensory areas. Remarkably, AP activity is regulated by sensory experience as demonstrated by monocular deprivation paradigms in monkeys. The areal and laminar distribution of AP activity matches that of the GAD(65), the GABA synthesizing enzyme found in presynatic terminals. As our electron microscopic investigations indicate that AP is found at the neuronal membranes and in synaptic contacts, it is proposed that the neuronal AP isoform (NAP), may be a key enzyme in regulating neurotransmission and could therefore play an important role in developmental plasticity and activity-dependent cortical functions.

Insertion of a glycosylphosphatidylinositol-anchored enzyme into liposomes.

Incorporation of alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein, into liposomes containing detergent, followed by detergent removal with hydrophobic resin was performed. Incorporation media were collected during different steps of detergent removal and were analyzed by flotation in sucrose gradient. The presence of protein was checked by measuring enzymatic activity, while the presence of (3)H-radio-labelled liposomes was followed by determination of the radioactivity. The incorporation yield of the protein into liposomes increased with incubation time in presence of hydrophobic resin. Protein was also incorporated at different protein/lipid ratios. At the highest protein lipid ratio, our data showed that 260 molecules of GPI-linked AP (AP-GPI) could be associated with one liposome, corresponding to 65% vesicle coverage. Finally, observations by electron cryomicroscopy indicated (i) that the protein seemed exclusively associated with the lipid bilayer via the GPI-anchor, as shown by the distance-about 2.5 nm-between the protein core and the liposome membrane; (ii) that the AP-GPI distribution was heterogeneous on the liposome surface, forming clusters of protein.

Enhanced ultrastructural localization of acid phosphatase using microwave irradiation during enzyme incubation / Localización ultraestructural intensificada de la fosfatasa alcalina utilizando irradiación de microondas durante la incubación enzimática

Microwave irradiation in enzyme cytochemistry is not only advantageous for shortening procedures, but will also improve the sensitivity of histochemical detection since short periods and less fixative are required to obtain good preservation. Moreover, shorter incubation procedures will reduce the diffusion and loss of enzyme activity. Here we compared conventional incubations in a shaking waterbath at 37°C, with incubations carried out in a microwave oven. Lysosomal and extralysosomal acid phosphatase were identified using microwave irradiation. Microwave irradiation elicited enhanced reactivity of the chemicals present in the tissue, leading to the detection of minuscule concentrations of enzyme activity, which was most pronounced in the detection of extralysosomal enzyme activity. A major advantage of the microwave is the accelerated formation of reaction product. Furthermore, enzymatic preincubation or chemical treatment of the tissues is no longer necessary. By shortening the incubation time, the danger of artificial precipitations and diffusion of the final reaction product are reduced. The sensitivity and reproducibility of the enzymatic reaction are increased. Consequently, microwave irradiation during enzyme incubation leads to a highly specific localization of acid phosphatase (AU)

En la histoquímica enzimática la irradiación con microondas no sólo ofrece ventajas para acortar los procedimientos sino también es capaz de mejorar la sensibilidad de la detección histoquímica puesto que se precisan de periodos cortos y menos fijador para obtener una buena preservación. Además, procedimientos de incubación más cortos reducirán la difusión y pérdida de actividad enzimática. En este trabajo, comparamos las incubaciones convencionales, llevadas a cabo en un baño de agua con agitación a 37º C con las llevadas a cabo en un horno de microondas. Se identificaron la fosfatasa lisosomial y extralisosomial utilizando la irradiación con microondas. La irradiación con microondas suscitó una reactividad intensificada de los reactivos presentes en los tejidos; esto fue muy patente en la detección de concentraciones muy bajas de actividad enzimática. Una ventaja principal de las microondas es la formación acelerada del producto de la reacción. Adicionalmente, la pre-incubación enzimática o el tratamiento químico de los tejidos ya es necesario. Al acortar el tiempo de incubación, se reducen tanto el riesgo de las precipitaciones artificiales como la difusión del producto final de la reacción. La sensibilidad y la reproducibilidad de la reacción enzimática son aumentadas. En consecuencia, la irradiación durante la incubación enzimática conduce a una localización altamente específica de la fosfatasa alcalina (AU)

Fine structural and cytochemical observations of dental epithelial cells during the enameloid formation stages in red stingrays Dasyatis akajei.

The fine structure and the localization of nonspecific acid phosphatase (ACPase), nonspecific alkaline phosphatase (ALPase), and calcium-dependent adenosine triphosphatase (Ca-ATPase) activities in the dental epithelial cells in tooth germs of Dasyatis akajei in the later stages of enameloid formation were investigated. Numerous invaginations of the distal cell membrane of the inner dental epithelial (IDE) cells were observed at the early stage of enameloid maturation. The invaginations contain many fine granular and filamentous substances; the lamina densa, which was thicker during the former stages, is obscure. Granules exhibiting defined ACPase activity were usually found in the IDE cells during the stages of enameloid mineralization and maturation. IDE cells are putatively involved in the removal of degenerated enameloid matrix during these stages. Marked ALPase activity was detected at the proximal and the lateral cell membranes of the IDE cells from the late stage of enameloid matrix formation to the early stage of enameloid maturation. Strong activity of Ca-ATPase was localized at the proximal and the lateral cell membranes of the IDE cells during the stages of enameloid mineralization and maturation. ALPase and Ca-ATPase activity is probably related to crystal formation in the enameloid and the removal of degenerated enameloid matrix from the enameloid.

Ultrastructure and function of the fractalkine mucin domain in CX(3)C chemokine domain presentation.

Fractalkine (FKN), a CX(3)C chemokine/mucin hybrid molecule on endothelium, functions as an adhesion molecule to capture and induce firm adhesion of a subset of leukocytes in a selectin- and integrin-independent manner. We hypothesized that the FKN mucin domain may be important for its function in adhesion, and tested the ability of secreted alkaline phosphatase (SEAP) fusion proteins containing the entire extracellular region (FKN-SEAP), the chemokine domain (CX3C-SEAP), or the mucin domain (mucin-SEAP) to support firm adhesion under flow. CX3C-SEAP induced suboptimal firm adhesion of resting peripheral blood mononuclear cells, compared with FKN-SEAP, and mucin-SEAP induced no firm adhesion. CX3C-SEAP and FKN-SEAP bound to CX(3)CR1 with similar affinities. By electron microscopy, fractalkine was 29 nm in length with a long stalk (mucin domain), and a globular head (CX(3)C). To test the function of the mucin domain, a chimeric protein replacing the mucin domain with a rod-like segment of E-selectin was constructed. This chimeric protein gave the same adhesion of peripheral blood mononuclear cells as intact FKN, both when immobilized on glass and when expressed on the cell surface. This implies that the function of the mucin domain is to provide a stalk, extending the chemokine domain away from the endothelial cell surface to present it to flowing leukocytes.

Ultracytochemical studies of the effects of aluminum on the blood-brain barrier of mice.

We studied the effect of chronic exposure (6 weeks and 6 months) of mice to drinking (tap) water containing 1.76% (0.06 M) aluminum lactate on some cytochemical properties of the blood-brain barrier (BBB). The plasmalemma-bound enzymatic activities of alkaline phosphatase (AP) and Ca(2+)-activated adenosine triphosphatase (Ca(2+)-ATPase) were studied at the ultrastructural level. Anionic sites were localized with cationized ferritin in a pre-embedding procedure and with cationic colloidal gold in a post-embedding procedure applied to brain samples embedded in Lowicryl K4M. Intravenously injected Evans blue and horseradish peroxidase (HRP) were used for evaluation of the functional state of the BBB. The results indicate that chronic exposure to aluminum does not noticeably affect barrier function of the endothelium of cerebral cortex blood microvessels. Focal leakage of larger than capillary microvessels (presumably arterioles and venules) was observed only in a few areas, such as the basal ganglia and amygdaloid nuclei. The localization of both enzymatic activities (AP and Ca(2+)-ATPase) in microvessels remained essentially unchanged. The localization of anionic sites was also unchanged except on the luminal surface of the endothelium of a few blood microvessels located in areas of the brain where leakage of the injected HRP was noted. In these vessels the injected HRP was often attached to the luminal surface of the endothelial cells, suggesting its increased stickiness. These data, compared with our previous observations on brain microvascular endothelial cells growing in vitro, indicate that cytotoxicity of aluminum is evidently less pronounced in the living organism, presumably due to action of detoxicating and regulatory mechanisms.

Changes of activity and ultrastructural localization of alkaline phosphatase in cerebral cortical microvessels of rat after single intraperitoneal administration of mercuric chloride.

Inorganic mercury salts administered systemically at low mg/ml doses produce neurotoxic effects without penetrating the cerebral microvascular endothelial cells which form the blood-brain barrier (BBB). This phenomenon promoted investigations testing a hypothesis relating inorganic mercury-induced brain dysfunction to its interference with the BBB transport. In the present study, we tested the effect of a single i.p. administration of mercuric chloride (MC) (6 mg/kg body weight) on the activity and ultrastructural localization of cerebral alkaline phosphatase (AP), a cerebromicrovascular marker enzyme primarily located on luminal plasmalemma of endothelial cells. At 1h after MC administration, light microscopy revealed a virtual absence of AP in cerebral cortical layers II and III, and its dramatic reduction in the remaining layers. Electron microscopy confirmed the disappearance of the AP reaction product from luminal endothelial cell membranes, and luminal phasmalemma revealed pinocytic vesicles and invaginations likely to manifest changes in BBB transport. At 18h post-treatment, a moderate enzyme activity appeared on abluminal endothelial plasmalemma and on basement membrane, but remained absent from luminal plasmalemma. A similar picture persisted through day 5 post-treatment. The inhibition and subsequent translocation of AP activity from luminal to abluminal site and the accompanying ultrastructural changes are typical of the formation of "leaky" microvessels, previously reported for a variety of neuropathological conditions associated with BBB damage.

Combined immunocytochemistry and enzyme cytochemistry on ultra-thin cryosections: a new method.

We combined immunocytochemistry and enzyme cytochemistry to localize two different proteins on the same ultrathin cryosection. In this method the immunocytochemical localization is visualized with colloidal gold probes and the enzyme cytochemical detection is achieved with cerium as the capture agent. The immunocytochemistry is conducted first so that any potential adverse effects of the enzyme cytochemical procedure will not alter the antibody binding properties of the cryosections.

Ultrastructural localization of alkaline and acid phosphatase activities in dental plaque.

Ultrastructural cytohistochemical techniques showed presence of acid and alkaline phosphatases in dental plaque. Both phosphatases had intra- and extramicrobial localization. In the extracellular matrix, phosphatases were associated with small vesicles of bacterial origin, or were freely scattered in the matrix without apparent connection with microbial structures. Intracellularly, alkaline (AlkP) and acid (AcP) phosphatases were observed in Gram-negative and Gram-positive bacteria, showing a different localization. The AlkP was mainly located in the periplasmic space, while AcP had a double preferential localization: along the outer surface of the cell wall and in the periplasmic space. Less frequently an intracellular phosphatase reaction was seen in the cytoplasm.

Alkaline phosphatase activity in the osteoclasts induced by experimental tooth movement.

The localization of alkaline phosphatase (Alpase) activity in the osteoclasts was examined cytochemically. Alpase activity was located in the basolateral membrane in mature osteoclasts having ruffled borders and clear zones, and also in the basolateral membrane in the osteoclasts lacking a ruffled border or a clear zone on the bone surface. But in the preosteoclasts situated away from the bone surface the enzyme activity was noted in the whole plasma membrane. These results suggest that the localization of Alpase activity may be altered in relation to the changes in morphology associated with the functional activity of the osteoclasts.