Benign familial hyperphosphatasaemia as a cause of unexplained increase in plasma alkaline phosphatase activity.

AIMS--To consider a possible genetic origin for the persistent unexplained increase in plasma alkaline phosphatase (ALP) in five non-related patients referred over an 18 month period. METHODS--Plasma ALP isoenzyme activities were measured in patients and their first degree relatives. RESULTS--In each patient there was a noticeable increase in intestinal plasma ALP, either alone or accompanied by an increase in bone or liver ALP. Family studies showed an unexpected increase in plasma ALP and similar isoenzyme changes in first degree relatives. The findings were consistent with autosomal dominant inheritance. CONCLUSION--Inherited raised plasma ALP activity is a reasonably common cause of persistent unexplained hyperphosphatasaemia which deserves wider recognition.     

Evidence for anion channels in secretory vesicles

Secretory vesicles from bovine neurohypophysis were reconstituted into lipid bilayers. Electrical measurements on the lipid bilayers under voltage clamp demonstrated the presence of channels that are permeable to chloride ions, are blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and are slightly voltage dependent. When several different membrane fractions were used for the reconstitution, the probability of finding channels correlated with the fraction of secretory vesicle membrane in the membrane fraction, indicating that the secretory vesicles are the source of the channels. The observed anion channel can provide a pathway for the anion transport that has previously been described for secretory vesicles. The secretory vesicle anion channel may play a role in calcium-induced secretion.     

Isoenzymes of alkaline phosphatase in epileptic patients receiving carbamazepine monotherapy.

The effects of carbamazepine monotherapy were investigated in 20 female and 21 male epileptic patients to determine whether treatment would induce an increase in serum alkaline phosphatase (ALP) activity, a known effect of many anticonvulsant drugs. Serum total ALP activity was increased in nine out of the 41 patients (22%), serum bone ALP activity was increased in 10 (24%), and serum non-bone ALP activity was increased in three (7%). There was no significant difference when the mean of the patients' serum total ALP was compared with that of the controls. Twenty per cent of the patients with increased serum bone ALP had normal serum total ALP, indicating that increased serum bone isoenzyme activity may precede an increase in the total enzyme activity. This should be considered when interpreting results of increased total ALP in epileptic patients.     

The amphicrine pancreatic cell line AR42J: a model system for combined studies on exocrine and endocrine secretion

Summary Secretory vesicles of both the exocrine and the endocrine pancreas have been isolated and characterized in molecular terms from pancreatic tissue and primary cell cultures. Studies on pancreatic secretory processes could be further facilitated by the use of permanent cell lines that respond to secretory stimuli with a regulated secretory response. We now present biochemical, morphological and secretory studies on the rat pancreatic acinar cell line AR42J. This cell line is characterized by the presence of digestive enzyme-containing dense core vesicles, which are released in response to cholecystokinin. In addition, we present evidence that these cells also contain small neuroendocrine-specific vesicles, as evidenced by the expression of the neuroendocrine-specific vesicle proteins synaptophysin and S.V.2. Corresponding to these mixed exocrine-neuroendocrine features, we also found considerable amounts of the neurotransmitters glycine, glutamine and gammaaminobutyric acid (GABA), as well as the rate-limiting enzyme in GABA synthesis, glutamic acid decarboxylase (GAD) (EC 4.1.1.15) expressed in these cells. We demonstrated a specific uptake mechanism for radioactively-labelled GABA by these cells. In addition, GABA was released from intracellular storage pools by nicotinic receptor stimulation or membrane depolarization. In summary, AR42J cells represent the first amphicrine pancreatic cell line with the combined expression of exocrine and neuroendocrine secretory organelles, both of which follow a regulated secretory pathway in response to various secretory stimuli.Abbreviations DCV dense core vesicles - GABA gammaaminobutyric acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Mechanisms responsible for SCN increase in resistance of in vitro frog gastric mucosa

Thiocyanate (SCN) inhibits H+ secretion and increases the resistance and potential difference (PD) of the gastric mucosa. These results support our separate-site electrogenic theory of HCl secretion. Recent work shows that an ATP-driven mechanism in the gastric mucosa can produce H+ by a neutral exchange of K+ for H+. The SCN increase in resistance and PD, if due to an inhibition of a high-conductance mechanism(s) in the secretory plasma membrane, is not easily compatible with the neutral mechanism. Therefore, the possibility was examined that SCN induces the increase in resistance by other mechanisms. The HCl and NaCl concentration profiles in the pit and tubular lumina were calculated. The effects of SCN were determined with isotonic, hypotonic, hypertonic, buffered, and high [H+] secretory solutions. The results indicate that SCN produces an increase in resistance of about 130 omega. cm2 of the plasma membranes of the tubular cells. A scheme is proposed that incorporates the neutral K+-H+ mechanism into an electrogenic system.     

Ultracytochemical localization of guanylate cyclase in the oviduct of estrogen-stimulated rat

Many reports have indicated that estrogens increase uterine guanosine 3′,5′-cyclic monophosphate (cGMP)levels via increasing the activity of guanylate cyclase. In the present study, guanylate cyclase activity was studied cytochemically in the oviduct of immature rats 24 hours after one or two doses (20 μg/kg, subcutaneously) of diethylstilbestrol (DES), one dose per day. The reaction product indicating guanylate cyclase was localized in the plasma membrane of epithelial cells, endothelial cells, and smooth muscle cells of all DES-treated animals, but was absent from those of the vehicle control immature rats. The Golgi saccules of secretory cells and the periciliary membrane of ciliated cells were stained for the enzyme 24 hours after the first and second dose of DES, but the activity seemed diminished 24 hours after the second dose. Likewise, reaction product indicating guanylate cyclase was more prominent in the plasma membrane of epithelial cells of animals treated with one dose of DES than those animals treated with two doses of DES. However, in the endothelial and smooth muscle cells, guanylate cyclase activity increased after two doses of the estrogen. Further, pinocytotic vesicles or caeolae in these cells were also strongly stained for the enzyme after one and two doses of DES. These findings confirm the suggestion that guanylate cyclase plays a significant role in the growth, differentiation, and function (secretion and transport) of the oviduct.     

Inactivation of human serum bactericidal activity by a trypsinlike protease isolated from Porphyromonas gingivalis.

A protease was isolated from an outer membrane vesicle preparation of Porphyromonas gingivalis ATCC 33277 and assessed for its ability to inactivate the bactericidal activity of normal human serum. The enzyme, which was activated by reducing agents, was found to be a trypsinlike protease with a molecular mass of approximately 80 kDa. Prior to being tested in the bactericidal assay, pooled human serum was preincubated with the partially purified enzyme. Under conditions in which the trypsinlike protease was activated, a strong reduction of the serum bactericidal activity against Capnocytophaga ochracea was noted. On the other hand, no reduction of the bactericidal action of serum was observed when the serum-protease mixture was preincubated in the presence of an inhibitor of the enzyme. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protease was shown to degrade immunoglobulins G and M as well as complement factor C3. This study confirms the previous hypothesis that the proteases of P. gingivalis can interfere with the protective action of human serum.     

Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons

Recent evidence shows that secretory phospholipase A2 (sPLA2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA2 on vesicle exocytosis. External application of group IIA sPLA2 (purified crotoxin subunit B or purified human synovial sPLA2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on calcium and sPLA2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.     

Dipeptidyl peptidase IV as a differentiation marker of the human endometrial glandular cells.

To investigate the involvement of membrane-bound peptidases in the human endometrial function, we examined the expression of dipeptidyl peptidase (DPP) IV and its enzyme activity. Immunohistological studies revealed that DPP IV was detected on human endometrial glandular cells and endometrial surface epithelium, but not on endometrial stromal cells or decidual cells in the first trimester of pregnancy. DPP IV expression on glandular cells and surface epithelium was weak in the proliferative phase, began to increase gradually in the early secretory phase, and was strong in mid-to late secretory phase and in the first trimester of pregnancy. DPP IV enzyme activity was detected histochemically in glandular cells and surface epithelium in the mid-secretory phase, and became stronger in the late secretory phase, but was rarely detected in the proliferative phase and early secretory phase. During the first trimester of pregnancy DPP IV enzyme activity in glandular cells and surface epithelium was slightly weaker than in the late secretory phase. Endometrial stromal cells and decidual cells, however, had no detectable DPP IV enzyme activity at any time throughout the menstrual cycle or during the first trimester of pregnancy. These findings indicate that DPP IV is a differentiation marker for glandular cells and surface epithelium and that active DPP IV is present in both areas during the peri-implantation period and thereafter.     

Fine structural studies of rat seminal vesicle in castrated and intact animals following estrogen treatment

The effect of estradiol and/or testosterone upon secretion by seminal vesicle in castrated and intact rats was assessed in young adult Sprague-Dawley rats, using light microscopy (LM), transmission (TEM) and scanning (SEM) electron microscopy. Hormones were injected daily for ten days beginning ten days after castrations were performed. The normal rat seminal vesicle, as revealed by SEM, was characterized by a large saccular lumen with highly folded walls. Cell surfaces were covered with microvilli, or occasionally displayed a protruding, ruffled surface, sparsely covered with short microvilli. Cytology was normal in testosterone-treated animals. Estradiol treatment of castrated animals stimulated secretion by seminal vesicle epithelial cells as evidenced by the presence of normal secretory bodies, the presence of RER, and moderately hypertrophied Golgi complexes. These glands were not heavier than were glands from castrated, untreated animals, although the epithelial cells were significantly taller. Secretion was maintained in intact animals treated with estradiol, although glands were smaller and epithelial height was reduced. Estradiol and testosterone treatment in combination did not appear to have an additive effect on secretion, weight of the gland, or epithelial height. The following results support the hypothesis that estrogen-induced prolactin synthesis and release may be involved in the mechanism by which estradiol effected stimulation of seminal vesicle epithelium. Prolactin-treated, castrated animals exhibited focal areas of stimulated epithelium. In hypophysectomized animals (untreated controls), the seminal vesicle epithelium retained some secretory bodies and secretory fluid in the glandular lumen; epithelial height was taller than that in castrated controls. Estrogen treatment reduced the epithelial height to that of castrated controls; there was no evidence of secretion. This suggests that in the absence of anterior pituitary hormones, including prolactin, the stimulatory effect of estradiol on seminal vesicle epithelium was nullified. In adrenalectomized/castrated animals, estradiol treatment stimulated secretion in seminal vesicle epithelium just as in non-adrenalectomized/castrated animals. This indicates that the adrenal gland plays a non-essential role in the action of estrogen on seminal vesicle epithelium.     

Soluble mediators of cytolytic activity in hemocytes of the Asian clam, Corbicula fluminea

Serum-free hemocytes from the clam, Corbicula fluminea, are cytolytic to mammalian erythrocytes (RBCs) as demonstrated in a hemoglobin release assay. The reaction is hemocyte dose- and temperature-dependent, and is mediated by the release of soluble hemolytic factors from clam cells. Initiation of hemocyte secretory activity does not appear to require contact with target RBCs. The chemical nature of the secreted lysin(s) has not been determined; however, PAGE analysis of hemocyte extracts (lysates) reveals the presence of at least five peaks of lytic activity. This activity is sensitive to heat and proteolytic enzyme treatment, and can be removed by absorption with fixed, homologous RBCs. Thus it is likely that the secreted hemocyte lysin is a heat-labile protein which exerts its hemolytic activity by binding directly to target RBC membrane constituents.     

Enrichment of osteogenic cell populations from rat bone marrow stroma

The presence of multiple cell types in bone marrow and their varying proportions from isolation to isolation may count for the considerable variation in the outcome of different experiments. The presence of these multiple subpopulations suggests a need for a method that can purify the osteogenic component, i.e. osteoprogenitors, from other components. The availabilities of monoclonal antibodies recognizing subpopulations of osteoblasts are providing means for antibody-based methods. The cell surface antigens STRO-1, ALP and HOP-26 were used for cell sorting experiments with fluorescence activated cell sorting (FACS). These cell populations were analyzed on differential gene expression, cell proliferation and differentiation into the osteoblastic lineage. The oligo-microarray results showed that only the ALP positive cell population expressed genes of the extracellular matrix; like different collagens, ECM-1 and matrix protease MMP-14. The real-time polymerase chain reaction (QPCR) results showed that STRO-1 and ALP positive cells had an upregulation in expression of lipoprotein lipase, osteocalcin, and collagen type I. Integrin beta-3 was only upregulated for ALP positive cells, while for these cells downregulation occurred for the genes myosine, alkaline phosphatase and integrin beta-1. HOP-26 positive cells showed an upregulation in collagen type I compared to control group. The DNA analysis revealed that the cells of the control group and the HOP-26 positive cells showed a 5 times higher cell growth compared to the STRO-1 and ALP positive cells. The alkaline phosphatase activity showed no activity for the control group. The STRO-1 and ALP positive cells had a higher activity compared to the HOP-26 positive. The calcium measurements revealed only for the control group calcium at day 24. Based on the results of our study, we conclude that the FACS method had no negative effect on the proliferating as well as differentiating response of the cells. Further, we conclude that by using an antibody-based cell selection method, different cell populations with different mRNA expression profiles and different osteogenic characteristics can be obtained.     

Latency of dopamine β-hydroxylase in purified noradrenergic vesicles

Purified large dense core noradrenergic vesicles isolated from bovine splenic nerve were investigated to determine the percentage of total dopamine β-hydroxylase which existed in a latent or masked form. The effects of the nonionic detergent Triton X-100 in concentrations between 0.0025 and 0.1% were studied. At 0.01%, Triton produced a maximum three-fold increase in accessible dopamine β-hydroxylase activity to reach 0.64μmol octopamine per mg protein per min at 37°C, but released only a third of the enzyme into the soluble phase. About 90% of the noradrenaline, 42% of the phospholipids and 17% of the vesicle protein were released. At 0.05%, Triton produced the same maximum enzyme activity with complete solubilization of the enzyme. All the noradrenaline, 90% of the phospholipids, and 48% of the vesicle protein were released into the soluble phase. Ultrastructural examination of the treated vesicles revealed only partial lysis with 0.01% Triton, while 0.05% produced essentially complete disruption of vesicles into membrane fragments and fine granular matrix contents. Equilibrium centrifugation of the control and treated vesicles on a second sucrose-D₂O density gradient showed that emptied and partially depleted vesicles and released matrix granules redistributed from the heavy vesicle peak to less dense regions of the gradient, including the zone of the light vesicle peak.It is proposed that the latency of dopamine β-hydroxylase activity in this noradrenergic vesicle fraction is best explained by the occurrence of the enzyme as an inaccessible complex in the vesicle matrix which may include phospholipid.     

Initial in vitro interaction of osteoblasts with nano-porous alumina

In the present study we have used a characterised primary human cell culture model to investigate cellular interactions with nano-porous alumina. This material, prepared by anodisation, is being developed as a coating on titanium alloy implants. The structure of the alumina, as determined by X-ray diffraction and transmission electron microscopy, was amorphous. When studying cell/material interactions we used both biochemical and morphological parameters. Cell viability, proliferation and phenotype were assessed by measurement of redox reactions in the cells, cellular DNA, tritiated thymidine ([3H]-TdR) incorporation and alkaline phosphatase (ALP) production. Results showed a normal osteoblastic growth pattern with increasing cell numbers during the first 2 weeks. A peak in cell proliferation was seen on day 3, after which cell growth decreased, followed by an increase in ALP production, thus indicating that the osteoblastic phenotype was retained on the alumina. Cell adhesion was observed, the osteoblast-like cells having a flattened morphology with filipodia attached to the pores of the material. SDS-PAGE and western blot measurements showed that the nano-porous alumina was able to adsorb fibronectin. Trace amounts of aluminium ions were measured in the surrounding medium, but no adverse effect on cell activity was observed.     

Matrix vesicle biogenesis in vitro by rachitic and normal rat chondrocytes.

Calcifying matrix vesicles (MVs) are released from chondrocytes and osteoblasts in monolayer culture. In the present studies, we tested the ability of rachitic versus normal rat growth plate chondrocytes in micromass or monolayer primary cultures to produce MVs. Unlike earlier reports of in vitro MV biogenesis by chicken chondrocytes in which most MVs were released into the medium, we found that most of the released rat matrix vesicles were entrapped in a newly formed cartilaginous matrix enveloping the cells. These matrix-associated MVs could be isolated by mild collagenase treatment and concentrated by differential centrifugation. Vesicle production slowed in the older 2- to 4-week-old cultures and, unlike vesicle release from cultured chicken chondrocytes, active vesicle production did not show a second burst of activity at 3 to 4 weeks. Alkaline phosphatase (ALP) activity diminished with time in culture in cells and matrix vesicles, suggesting a decrease in differentiative expression. Protein profiles on SDS polyacrylamide gels of native matrix vesicles and culture-derived MVs from rachitic and normal cells were quite similar and showed a typical simplified protein pattern as compared to chondrocyte plasma membrane proteins. There were distinctive proteins migrating at 130, 80 to 95, 66, 43, 20, and 14 kd. Culture-derived MVs showed vigorous in vitro calcifying activity that was ALP related. We conclude that 1) rachitic chondrocytes are essentially normal in their matrix vesicle production; 2) matrix entrapment of MVs is a characteristic of rat chondrocyte cultures; and 3) culture-produced MVs are similar to native MVs in protein profile and calcifiability, and thus can be studied as a model for normal MV composition and calcification.     

热休克对小白鼠肾中碱性磷酸酶的影响

本文用酶组织化学方法研究了热休克对不同发育时期小鼠肾组织中碱性磷酸酶（ALP）的影响。结果表明，热休克处理后ALP活性有明显增高．提示ALP活性的增强是提高细胞的热耐受力、维持细胞正常生理功能的重要途径。     

A Monoclonal Antibody to Intestinal Alkaline Phosphatase Made Against D98/AH-2 (HeLa) Cells

A monoclonal antibody (AAP1) to human intestinal alkaline phosphatase (ALP) was produced by immunizing a mouse with D98/AH-2 (HeLa) cells, which produce the enzyme ectopically. The antibody, which did not inhibit enzyme activity using p -nitrophenyl phosphate as the substrate, was of the IgG2A class and did not show complement-dependent cytotoxicity. In trace binding assays AAP1 bound only to cells that expressed an intestinal-like form of human ALP, including some human intraspecific (D98/AH-2 × human lymphocyte or fibroblast) hybrids. Immuno-precipitation of immune complexes from cell-free extracts of D98/AH-2 cells, using protein A containing 5. aureus and AAP1 antibody, resulted in precipitation of all the ALP activity. The precipitated material had a subunit molecular weight of 80,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. In non-denaturing conditions, AAP1 antibody prevented the migration of ALP activity into the gel when cell-free extracts were made from human adult or fetal intestine, or D98/AH-2 cells. Similarly, AAP1 could be used to precipitate ALP activity from these extracts but not from extracts of human liver, kidney or placenta.     

Stereospecific inhibition of alkaline phosphatase by L-tetramisole prevents in vitro cartilage calcification

To clarify the role of alkaline phosphatase (ALP) and ATPase in skeletal mineralization, we studied the effect of the anthelmintic drug, l-tetramisole (levamisole), a stereospecific inhibitor of ALP activity, and its inactive isomer, d-tetramisole (dexamisole), on in vitro calcification of rachitic rat cartilage. ALP activity in homogenized rachitic rat proximal tibiae was inhibited by l-tetramisole in a dose-dependent manner. Histochemical and electron microscopic cytochemical analysis of intact epiphyseal plate rachitic rat cartilage showed that 5 x 10(-2) M and greater concentrations of l-tetramisole (1) virtually abolished ALP activity, (2) moderately reduced ATPase activity, and (3) prevented in vitro cartilage calcification, while preserving the structural integrity of the matrix vesicles. Concentrations of d-tetramisole as high as 1 x 10(-1) M failed to inhibit ALP activity in tibial homogenates by more than 10 per cent and did not alter histochemical staining of enzyme activity or calcification in intact cartilage slices. Heating the cartilage slices destroyed ALP activity, prevented calcification, and disrupted matrix vesicle integrity. These data show that there is a close association between ALP activity and in vitro calcification of rachitic rat cartilage. In the absence of ALP activity, intact matrix vesicles do not promote calcification. Our data also suggest that some ATPase activity of rachitic rat cartilage may be distinct from ALP activity.     

Comparative enzymatic acetylation of carnitine and choline by human placenta syncytiotrophoblast membrane vesicles

Microvillous membrane vesicle preparations from the maternal surface of human placental syncytiotrophoblast were examined for the presence of carnitine and choline acetyltransferase activity. Radiometric assay for acetylcholine employed butyronitrile-tetraphenylboron extraction of the quaternary ions. Acetylcarnitine was assayed by anion exchange chromatography. The data reveal that carnitine is the primary substrate for the vesicle acetyltransferase enzyme(s), whereas choline appears to be a minor substrate. For acetylcarnitine synthesis, the Km is 0.749 mM carnitine and Vmax is 641 pmol X mg protein-1 X minute-1, respectively; for acetylcholine synthesis, the Km is 0.5 mM choline and Vmax is 53 pmol X mg protein-1 X minute-1, respectively. Approximately ten times more acetylated product was formed with carnitine than with choline. The carnitine-mediated reaction obeyed Michaelis-Menten kinetics, whereas the choline reaction exhibited anomalous behavior. Vesicle preparations were stable for 21 days at -80 degrees C. Preliminary studies on hypotonically lysed vesicles demonstrate that the acetyltransferase is particulate and is bound to the membrane of the vesicle. These findings demonstrate that carnitine acetyltransferase activity is in the plasmalemma membrane of the syncytiotrophoblast and suggest a role for this enzyme, analogous to the mitochondrial fatty acid shuttle system, in the maternofetal translocation of fatty acyl residues.