Alkaline Phosphatase Reaction Research Articles

Cytological and enzyme-histochemical investigations on the digestive organs of Nautilus pompilius (Cephalopoda, Tetrabranchiata).

The foregut, stomach, caecum, midgut, and rectum of the digestive tract of Nautilus pompilius L.were investigated with ultrastructural and enzyme-cytological methods. Three different cell types were identified within the lamina epithelialis mucosae: main cells, goblet cells, and cells with secretory granules. The main cell type is the epithelial cell with microvilli, a basal nucleus surrounded by dictyosomes, rough endoplasmic reticulum, mitochondria, and electron-dense granules identified as lysosomes in the apical part of the cell. In the caecum this cell type contains endosymbiotic bacteria. The presence of endocytotic vesicles and the storage of lipids in the caecum indicate that this organ is involved in the process of absorption. In the caecum and the longitudinal groove of the rectum the main cells are, in addition, ciliated, facilitating the transport of food particles and faeces. Two types of goblet cells are found in all organs except in the stomach, forming a gliding path for food particles and protecting the epithelium. In the foregut and rectum, cells with electron-dense granules were recognized as the third type. The conspicuous secretory cells of the rectum represent a delimited rectal gland; its possible biological function is discussed. The tunica muscularis in all organs of the digestive tract consists of obliquely striated muscle cells innervated by axons containing transparent, osmiophilic and dense-cored vesicles. Positive reactions for acid and alkaline phosphatase, monoamine oxidase, beta-glucuronidase, and trypsin- and chymotrypsin-like enzymes are localized in the lamina epithelialis mucosae.

Comparative histology of pineal calcification.

The pineal organ (pineal gland, epiphysis cerebri) contains several calcified concretions called "brain sand" or acervuli (corpora arenacea). These concretions are conspicuous with imaging techniques and provide a useful landmark for orientation in the diagnosis of intracranial diseases. Predominantly composed of calcium and magnesium salts, corpora arenacea are numerous in old patients. In smaller number they can be present in children as well. The degree of calcification was associated to various diseases. However, the presence of calcified concretions seems not to reflect a specific pathological state. Corpora arenacea occur not only in the actual pineal tissue but also in the leptomeninges, in the habenular commissure and in the choroid plexus. Studies with the potassium pyroantimonate (PPA) method on the ultrastructural localization of free calcium ions in the human pineal, revealed the presence of calcium alongside the cell membranes, a finding that underlines the importance of membrane functions in the production of calcium deposits. Intrapineal corpora arenacea are characterized by a surface with globular structures. Meningeal acervuli that are present in the arachnoid cover of the organ, differ in structure from intrapineal ones and show a prominent concentric lamination of alternating dark and light lines. The electron-lucent lines contain more calcium than the dark ones. There is a correlation between the age of the subject and the number of layers in the largest acervuli. This suggests that the formation of these layers is connected to circannual changes in the calcium level of the organ. The histological organization of the human pineal is basically the same as that of mammalian experimental animals. Pineal concretions present in mammalian animal species are mainly of the meningeal type. Meningeal cells around acervuli contain active cytoplasmic organelles and exhibit alkaline phosphatase reaction in the rat and mink, an indication of a presumable osteoblast-like activity. Using Kossa's method for the staining of calcium deposits, a higher calcium concentration was detected in the rat pineal than in the surrounding brain tissue. Since in parathyroidectomised rats calcified deposits are larger and more numerous than in controls, the regulation of the production of acervuli by the parathyroid gland has also been postulated. In most of submammalian species, the pineal organs (pineal-, parapineal organ, frontal organ, parietal eye) are photoreceptive and organized similarly to the retina. Acervuli were found in the pineal of some birds. The pineal organs of lower vertebrates (fish, amphibians, reptiles) exhibit a high calcium content by ultrastructural calcium histochemistry (PPA-method). However, concrements are not formed. The accumulation of Ca2+ seems to depend on the receptor function of the organ. Comparing pineal and retinal photoreceptors in the frog, the photoreceptor outer segments of pinealocytes as well as retinal cones and rods show a large amount of Capyroantimonate deposits. In dark adapted animals calcium ions are present in both sides of the photoreceptor membranes of the outer segment, whereas calcium is shifted extra-cellularly following light adaptation. Overviewing the data available about the pineal calcification, we can conclude that a multifactorial mechanism may be responsible for the calcification. The pineal of higher vertebrates is not just a simple endocrine gland, rather, its histological organization resembles a folded retina having both hormonal and neural efferentation. Mammalian pinealocytes preserve several characteristics of submammalian receptor cells and accumulate free Ca2+ on their membranes (1). In the thin walled retina and in the similarly organized pineal of submammalian species, the diffusion of extracellular calcium is probably easy and there is a lesser tendency to form concrements. (ABSTRACT TRUNCATED)

Identification and temporospatial distribution of bovine primordial germ cells prior to gonadal sexual differentiation.

The temporospatial distribution of bovine primordial germ cells was studied in 34 embryos (18 to 39 days). For a reliable identification of bovine primordial germ cells in varying localizations and at different developmental stages the alkaline phosphatase reaction combined with the use of selected lectins was applied. The first potential primordial germ cells were identified in an 18-day-old trilaminar embryo in the caudal wall of the proximal yolk sac at a distance of less than 100 microm from the germ disc. These cells are alkaline phosphatase-positive. but do not yet react with lectins. From 18 through 23 days, morphogenetic folding converts the flat trilaminar disc into a cylindrical embryonic body. During this folding process primordial germ cells located in the proximal yolk sac area are incorporated into the embryo when this portion of the yolk sac becomes the hind- and mid-gut. Consequently, in 23- to 25-day-old embryos putative primordial germ cells (alkaline phosphatase- and lectin-positive) are situated predominantly in the axial body region at the level of the mesonephros. When the gonadal ridge develops in this region (about day 27) it contains a certain number of primordial germ cells present from the very beginning. Thus, the assumptions of a long-range chemoattraction of primordial germ cells by the gonadal ridge, of active immigration from an extraembryonic site. or of a passive transportation via the blood stream are not necessary to explain the initial settlement of bovine primordial germ cells in the gonadal ridge. Within the gonadal ridge (days 27-31) and later in the still sexually indifferent gonadal fold (32-39 days) the primordial germ cells are unevenly distributed. Extragonadal potential primordial germ cells (alkaline phosphatase-positive, but with reduced or no lectin staining) are regularly present in large numbers in bovine embryos with indifferent gonads. Such cells occur predominantly in the paraaortal tissue and in the liver, but also in the branchial arches. The different locations of extragonadal primordial germ cells are discussed in the light of recent evidence that germ cells and haematopoietic cells share common ancestors.

Extraskeletal osteosarcomas: a clinicopathologic study of 25 cases.

A follow-up investigation of 25 cases of extraskeletal osteosarcomas diagnosed at the Center for Bone and Soft Tissue Tumors, Aarhus University Hospital, Denmark, in the period from 1970-1995 was undertaken. The immunohistochemical profile of these tumors was evaluated using a panel of 10 antibodies, and the value of alkaline phosphatase staining in differential diagnostic situations also was considered. The study revealed that this tumor is high-grade malignant and affects adults (median age, 67 years; range, 35-82 years) at diagnosis. The thigh (52%) was the most common tumor location. Seven tumors were superficial, whereas the remaining 18 were intramuscular. Two patients with superficial tumors previously received radiation to the area. Local recurrences developed in 9 (36%) patients and distant metastases developed in the lungs in 15 (60%) patients as the most common site. Median survival time was 24 months, and the cause-specific survival rate at 5 years was less than 25%. Thirteen (52%) intramuscularly located extraskeletal osteosarcomas were of the fibroblastic subtype, often with sparse amounts of osteoid. They could be separated from malignant fibrous histiocytoma on the basis of a strongly positive alkaline phosphatase reaction. Immunohistochemistry did not reveal characteristic features because positivity for vimentin, occasional positivity for desmin, actin, S-100, epithelial membrane antigen, cytokeratin, and p-53 may be observed in many other pleomorphic sarcomas. Various histopathologic factors, such as tumor size, tumor depth, histopathologic subtype, malignancy grade (IIIA versus IIIB), MIB-1, and p53 reactivity were analyzed in relation to clinical course. Only MIB proliferation was correlated to prognosis, with significantly longer survival in patients with tumors with MIB-1 values less than 24%. Our study has shown extraskeletal osteosarcoma to behave in a highly aggressive fashion. Alkaline phosphatase staining compared with immunohistochemistry proved to be superior in the differentiation from other pleomorphic sarcomas.

Analysis of Molecular Heterogeneity of Dahl/Iwai Salt-Sensitive Rats and Salt-Resistant Rats

Molecular evidence, using DNA fingerprint analyses, of extensive genetic heterogeneity between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and even within some of the WKY colonies has been reported. Thus we investigated the genetic relations between Dahl S and R rats newly inbred by Dr. Iwai. Genomic DNA was isolated from the liver of four Dahl S and four Dahl R rats, digested with the restriction enzyme HinfI or AluI, and separated in 1.2% agarose gel by electrophoresis. Then, DNA fingerprinting was performed by Southern blot analysis using the human myoglobin 33.6 minisatellite probe. Bands were detected in an alkaline phosphatase reaction system. Within the same strains, there was no heterogeneity of these fingerprinting patterns. The S and R rats shared 82% of the bands in the HinfI-digested DNA and 93% of those in the AluI-digested DNA. These shared values were much greater than the reported value (54%) between SHR and WKY from Charles River Laboratories. These newly inbred Dahl S and R rats may be appropriate, although still limited, experimental animals for investigating the pathophysiology of salt-sensitive hypertension.

Multi-colour brightfield in situ hybridisation on tissue sections.

We describe the brightfield microscopical detection of multiple DNA target sequences in cell and tissue preparations. For this purpose, chromosome-specific DNA probes labelled with biotin, digoxigenin or fluorescein were simultaneously hybridised and detected by enzyme cytochemistry using two horseradish peroxidase (PO) reactions and one alkaline phosphatase (APase) reaction. For triple-colour detection on single cell preparations, the combination of the enzyme precipitates PO/diaminobenzidine (DAB, brown colour), APase/fast red (FR, red colour) and PO/tetramethylbenzidine (TMB, green colour) resulted in an accurate detection of DNA targets. Embedding of the preparations in a thin cross-linked protein layer further stabilised the enzyme reaction products. For in situ hybridisation on tissue sections, however, this detection procedure showed some limitations with respect to both the stability of the APase/FR and PO/TMB precipitates, and the sequence of immunochemical layers in multiple-target procedures. For this reason, the APase/FR reaction was replaced by the APase/new fuchsin (NF, red colour) reaction and the washing steps after the PO/TMB reaction were restricted to the use of phosphate buffer pH 6.0. Furthermore, to improve the efficiency of the ISH reaction, APase/NF was applied in an avidin-biotin complex detection system and, to avoid target shielding in the triple-target ISH, the third primary antibody was applied prior to the second enzyme cytochemical reaction. These adaptations resulted in stable, well contrasting brown, red and green coloured precipitates. After quick haematoxylin counterstaining, the tissue preparations were directly mounted in phosphate buffer and, optionally, embedded in the cross-linked protein layer.

Intrachoroidal Microvascular Abnormality: A Previously Unrecognized form of Choroidal Neovascularization

To evaluate the histopathologic and histochemical characteristics of intrachoroidal microvascular abnormality. Forty eyes obtained at autopsy from human donors ranging in age from 20 to 91 years (25 diabetics, 15 nondiabetics) were analyzed. Choroids were processed for alkaline phosphatase flat-embedding. Vascular patterns were examined and analyzed before embedding and serial sectioning. Intrachoroidal microvascular abnormality had the most prominent alkaline phosphatase reaction product of choroidal vessels. These formations appeared as ameboid or cobweb-like vascular networks (area, 0.05 to 4.6 mm2) in the choroidal stroma external to the choriocapillaris. They appeared as both single or groups of formations in the posterior pole and equatorial regions in all subjects. Intrachoroidal microvascular abnormality was found in five subjects with diabetes: four with type I diabetes mellitus, and one with type II diabetes mellitus. One subject had proliferative diabetic retinopathy, three had background retinopathy, and one had no retinopathy. Intrachoroidal microvascular abnormality was connected with all levels of choroidal vasculature. Microaneurysms were observed within intrachoroidal microvascular abnormality formations in most subjects but not in other choroidal vessels. Intrachoroidal microvascular abnormality is a form of intrachoroidal neovascularization. This neovascularization has features similar to intraretinal microvascular abnormalities found in diabetic subjects but seems to form independently of the status of retinopathy. The presence of microaneurysms in intrachoroidal microvascular abnormalities and not other choroidal vessels supports the view that aneurysms may be aborted attempts at neovascularization.

Analysis of molecular heterogeneity of Dahl/Iwai salt-sensitive rats and salt-resistant rats

Molecular evidence, using DNA fingerprint analyses, of extensive genetic heterogeneity between spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and even within some of the WKY colonies has been reported. Thus we investigated the genetic relations between Dahl S and R rats newly inbred by Dr. Iwai. Genomic DNA was isolated from the liver of four Dahl S and four Dahl R rats, digested with the restriction enzyme HinfI or AluI, and separated in 1.2% agarose gel by electrophoresis. Then, DNA fingerprinting was performed by Southern blot analysis using the human myoglobin 33.6 minisatellite probe. Bands were detected in an alkaline phosphatase reaction system. Within the same strains, there was no heterogeneity of these fingerprinting patterns. The S and R rats shared 82% of the bands in the HinfI-digested DNA and 93% of those in the AluI-digested DNA. These shared values were much greater than the reported value (54%) between SHR and WKY from Charles River Laboratories. These newly inbred Dahl S and R rats may be appropriate, although still limited, experimental animals for investigating the pathophysiology of salt-sensitive hypertension. Am J Hypertens 1997;10:98S–101S © 1997 American Journal of Hypertension, Ltd.

Sex-dependent expression of placental (P)-cadherin during mouse gonadogenesis.

Placental (P)-cadherin is one of a family of cell adhesion molecules that participate in embryonic sorting and organogenesis. In previous work, P-cadherin was localized to Sertoli cells in the mouse testis as early as postnatal day 1. This early postnatal localization raised questions about when P-cadherin first appeared in the embryonic testis and whether P-cadherin was expressed differentially in the embryonic testis and ovary. The localization of P-cadherin, epithelial (E)-cadherin, and Müllerian inhibiting substance was determined in frozen sections of mouse gonads between embryonic days 10.5 and 18 using indirect immunohistochemistry. Alkaline phosphatase reactivity was used to identify germ cells. The expression of P-cadherin was traced back to the indifferent stage of gonadogenesis where uniform distribution was observed in the indifferent gonad of both sexes. However, after sexual differentiation, the expression of P-cadherin in the testis was localized to Sertoli cells in the testicular cords, while its expression in the ovary fell below detectable levels. The localization of P-cadherin in the male and female indifferent gonad is similar and cannot be used to distinguish the future testis and ovary. The localization of P-cadherin in the testis after sexual differentiation suggests a role for P-cadherin in testicular cord formation. The common temporal pattern of P-cadherin and Müllerian inhibiting substance expression in Sertoli cells is consistent with a shared regulatory mechanism.

Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study

The chemical phenotype of proneurotensin messenger RNA-expressing cells was determined in the acute haloperidol-treated rat striatum using a combination of [35S]-labelled and alkaline phosphatase-labelled oligonucleotides. Cellular sites of proneurotensin messenger RNA expression were visualized simultaneously on tissue sections processed to reveal cellular sites of preproenkephalin A messenger RNA or the dopamine and adenylate cyclase phosphoprotein-32, messenger RNA. The cellular co-expression of preproenkepahlin A and preprotachykinin messenger RNA was also examined within forebrain structures. Cellular sites of preproenkephalin A and dopamine and adenylate cyclase phosphoprotein-32 messenger RNAs were visualized using alkaline phosphatase-labelled oligonucleotides whilst sites of preprotachykinin and proneurotensin messenger RNA expression were detected using [35S]-labelled oligos. Cellular sites of enkephalin and dopamine and adenylate cyclase phosphoprotein-32 gene expression were identified microscopically by the concentration of purple alkaline phosphatase reaction product within the cell cytoplasm, whereas sites of substance P and proneurotensin gene expression were identified by the dense clustering of silver grains overlying cells.An intense hybridization signal was detected for all three neuropeptide messenger RNAs in the striatum, the nucleus accumbens and septum. Dopamine and adenylate cyclase phosphoprotein-32 messenger RNA was detected within the neostriatum but not within the septum. In all forebrain regions examined, with the exception of the islands of Cajella, the cellular expression of enkephalin messenger RNA and substance P messenger RNA was discordant; the two neuropeptide messenger RNAs were detected essentially in different cells, although in the striatum and nucleus accumbens occasional isolated cells were detected which contained both hybridization signals; dense clusters of silver grains overlay alkaline phosphatase-positive cells, demonstrating clearly that these dual-labelled cells expressed both messenger RNAs. By contrast, the hybridization signals for proneurotensin and enkephalin, and proneurotensin and dopamine and adenylate cyclase phosphoprotein-32 were generally coincident, at least within the neostriatum; most proneurotensin messenger RNA-positive cells expressed enkephalin messenger RNA and were also positive for dopamine and adenylate cyclase phosphoprotein-32 messenger RNA. However, occasional proneurotensin messenger RNA-positive striatal cells were identified that were single-labelled and did not express enkephalin messenger RNA. Within the septal nucleus, enkephalin messenger RNA and substance P messenger RNA were expressed essentially within segregated cell populations.These studies illustrate further the utility of co-expression techniques for investigating the chemical phenotype of cells within the CNS and demonstrate that the distribution of neuropeptide co-expressing cells is different within different brain regions. That several populations of proneurotensin messenger RNA-positive striatal cells may exist, of which one population is sensitive to haloperidol, co-expresses enkephalin messenger RNA and is positive for dopamine and adenylate cyclase phosphoprotein-32 messenger RNA may be of some significance in neuropsychiatric/neurological disorders given that the translated peptide, neurotensin, is known to influence and interact closely with the dopamine systems.

Relaxin and decidualization in mice: a reappraisal.

The nature of the physiological stimulus inducing decidualization in the endometrium is unknown. In this study we attempted to verify a recent report that relaxin can induce decidualization in intact mice primed with a high dose of estradiol valerate (5 micrograms) and a low dose (10 micrograms) of medroxyprogesterone acetate. In our study, neither s.c. nor intrauterine relaxin, nor intraluminal arachis oil, (an established deciduogenic stimulus) were able to induce decidualization. In addition, while oil was able to induce decidualization (increased uterine weight, and positive Pontamine Sky Blue and stromal alkaline phosphatase reactions) in ovariectomized mice treated with a regimen of estradiol and medroxyprogesterone acetate designed to produce optimum uterine sensitivity, no decidualization occurred in response to either s.c. or intraluminal relaxin. This study fails to provide any support for a role for relaxin as a deciduogenic stimulus.

Effect of single-dose, early luteal phase administration of mifepristone (RU486) on implantation stage endometrium in the rhesus monkey.

The characteristics of implantation stage endometrium following a single-dose, early luteal phase application of mifepristone (RU486) in proven conception cycles has been examined in the rhesus monkey in an attempt to understand the physiological basis of the anti-implantation activity of the drug. Endometrial samples were collected from monkeys subjected to vehicle (group 1, n = 14) and RU486 (2 mg/kg body weight; group 2, n = 12) on day 2 after the presumed day of ovulation of successfully mated cycles. The average diameter of glands (P < 0.05), number of vacuolated cells (P < 0.01), number of supranuclear vacuolated cells (P < 0.05) in glandular epithelium and amount of glandular secretion (P < 0.05) were significantly lower in RU486-treated endometrium compared with control tissue samples. Additionally, 18% of glandular epithelial cells showed apoptotic and degenerative features in RU486-treated tissue samples. These data, together with the observed significant decreases in precipitate area (P < 0.02) and in the optical absorbance of alkaline phosphatase reaction end-product (P < 0.05), confirm that retardation in glandular differentiation in the upper functionalis is a likely target of antiprogestin action in implantation stage endometrium. An increased frequency of mitosis in stromal cells (P < 0.05) and a greater degree of extravasation (P < 0.05) were also observed after RU486 exposure. Despite an apparent indication of constriction and regression in few RU486-exposed endometria compared with controls, morphometric analyses did not show any changes in capillary structure. Whether endometrial vasculature in progesterone-exposed uterus is a target of antiprogestin action during the peri-implantation stage remains to be determined. Further studies are required to explain the observed increase (P < 0.02) in the area of precipitate of von Willebrand (vW) factor with no change in vW factor-positive vessels, and the apparent increase in collagen IV immunostain in subepithelial and perivascular basement membrane in implantation stage endometrium after early luteal phase RU486 treatment in monkeys.

Histologic Studies of Brain Microemboli in Humans and Dogs After Cardiopulmonary Bypass.

Emboli become lodged in the brains of patients undergoing cardiopulmonary bypass (CPB) and can cause death, stroke, or more subtle neurological and neuropsychological deficits. Using a specialized vascular stain (for alkaline phosphatase) in autopsy tissues from the brains of patients who underwent CPB shortly before death, we found large numbers of microemboli. All of these microemboli contain lipid, some contain small birefringent particles, and some may contain aluminum or silicon. Within a few weeks after CPB, most of the microemboli had disappeared from the brain tissues, but some persisted for at least 6 months. After several days, the endothelial cells of some of the vessels containing microemboli showed subtle damage in the form of loss of alkaline phosphatase reactivity, and some vessels appeared to be degenerating. Sometimes the surrounding neuropil also showed degeneration. Since skin and muscle biopsies can be readily obtained before, during, and after CPB, we investigated their suitability as surrogate tissues for brain; however, they were unsuitable because they had so few microemboli. By injecting microspheres into dogs at various times during CPB, we have investigated the timing of the production of microemboli. We are also exploring the use of rat brains to trap microemboli from the injected blood of patients undergoing CPB. (ECHOCARDIOGRAPHY, Volume 13, September 1996)

A combined staining method for argyrophilic nucleolar organizer regions and for glial fibrillary acidic protein in astrocytes of human brain.

Different protocols are described for the combined staining method by which argyrophilic nucleolar organizer region sites can be evaluated in human astrocytes that are immunoreactive for glial fibrillary acidic protein. Among the four protocols studied, the following method was superior to others in terms of unambiguous visualization of the regions in glial fibrillary acidic protein-positive astrocytes; the first step was immunostaining for the protein with a blue colour reaction of alkaline phosphatase, followed by sequential colloidal silver staining for the regions. By this double staining method, we have demonstrated that the reactive astrocytes found in white matter around the metastatic lesion of carcinoma and the infarction, contain more argyrophilic nucleolar organizer regions in terms of the count as well as the area than glial fibrillary acidic protein-positive astrocytes present in the white matter of the normal brain. In conclusion, the double staining may provide valuable information on the cellular activity of astroglia when performed on routine formalin-fixed paraffin sections of the human brain.

Spectrophotometric Assay Using Streptavidin–Alkaline Phosphatase Conjugate for Studies on the Proteolysis of Polymer Bead-Bound Peptides

A simple method for indirectly measuring the concentration of biologically available peptides on the surface of polymer beads synthesized for the peptide or nonpeptide library is developed. To identify very small differences in the amount of cleaved peptides after α-chymotrypsin treatment, the so-called “shaving” process, a system of biotin and streptavidin–alkaline phosphatase conjugate was used. To this conjugate, 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (NBT) are coupled consecutively, to carry out alkaline phosphatase reaction and color reaction, respectively. The proteolytic action of α-chymotrypsin on surface peptides located on polymer beads was detected and cleaved peptides were quantified spectrophotometrically by analyzing unreacted NBT. Using this method, the enzymatic “shaving” process of surface peptides on polymer beads can be optimized by examining the effects of the morphology of polymer beads and the length of spacers on how easily enzymes such as α-chymotrypsin can access the surface of polymer beads. Quantitative understanding of molecular interactions on the bead surface will give us very useful information in designing polymer bead and bead-bound oligopeptide structures for the peptide or nonpeptide library.

A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation.

A non-radioactive 96-well microtitre plate reverse transcriptase (RT) assay, based on the use of covalently bound riboadenosine homopolymer in the wells and 5-bromodeoxyuridined 5'-triphosphate (BrdUTP) as dNTP, is described. The whole assay is performed in a single well, including the quantitative detection of incorporated BrdU, which is performed immunologically using alkaline phosphatase-conjugated anti-BrdU antibody and colorometric reading. The system also allows the use of variable amounts of primer. The kinetics and characteristics of the assay using BrdUTP is similar to the use of [3H]dTTP. The sensitivity of the assay can be varied either by altering the duration of RT assay time and/or by prolonging the alkaline phosphatase reaction. Thus the assay can detect < 0.02 pg of recombinant human-immunodeficiency-virus (HIV) type I RT, < 0.005 m unit of avian-myeloblastosis-virus RT or < 0.02 m unit of recombinant Moloney-murine-leukaemia-virus RT. The assay was found to be useful with various types of cell-culture material, and a comparative study of 16 HIV-infected lymphocyte cultures, using 10 microliters of supernatant medium for RT assay and 22.5 microliters for p24 antigen assay showed that the new RT assay was at least 25-fold more sensitive than the p24 antigen assay. The results also show a good correlation between the RT activities found and the p24-antigen level detected, with exception for HIV2 isolates, as they only became positive in the RT assay. The technical performance and the capacity of the test compared with other available RT kits is discussed, as well as its use for other applications.

Alkaline phosphatase reactivity in rabbit airway epithelium: a potentially useful marker for airway basal cells.

The purpose of this study was to investigate alkaline phosphatase (ALPase) reactivity in rabbit airway epithelial cells. Acetone-fixed, methyl benzoate and xylene-cleared (AMeX-treated) paraffin sections of trachea, bronchus, and lung tissue were stained by an azo dye coupling method for ALPase and examined by light microscopy. Electron histochemical staining was also performed in order to study the sensitivity and specificity of reactivity in each cell type. ALPase reactivity at the light microscopic level was observed exclusively in tracheo-bronchial basal cells, and not in bronchiolar basal cells. By electron microscopy, ALPase reactivity was noted in 97.9% of basal cells in the trachea, 97.0% of basal cells in the bronchus, and 94.5% of basal cells and 15.4% of Clara cells in the bronchiole. This was also true for dispersed tracheal epithelial cells. Reactivity was rarely observed in ciliated cells, non-goblet-type secretory cells, and undetermined cells. The reactivity was heat-labile, levamisole-sensitive, and of a non-specific type. These findings indicate that basal cells of rabbit trachea and bonchus have fairly high specificity for ALPase of a non-specific isozyme (92.2% and 95.6%, respectively). Therefore, ALPase is considered to be a useful marker for these cells.

ATP synthase subunit c storage in the polymorphonucleocytes of late infantile and juvenile batten patients

In late infantile and juvenile forms of neuronal ceroid lipofuscinosis, commonly known as Batten disease (BD), ATP synthase subunit c accumulates in the lysosomes of neural cells. By using polyclonal antibodies, raised against bovine liver subunit c and an image analysis system for the quantification of antibody-linked alkaline phosphatase reaction, we have demonstrated that polymorphonucleocytes (PMN) from a late infantile and a juvenile BD patient stored several-fold more subunit c as compared to normal PMN.

Growth of calcium phosphate on ion-exchange resins pre-saturated with calcium or hydrogenphosphate ions: an SEM/EDX and XPS study

Calcium phosphate formed on the surfaces of ion-exchange resins pre-saturated with either Ca2+ or HPO4 2- ions has been studied using a combination of scanning electron microscopy (SEM)/energy dispersive X-ray (EDX) analysis and X-ray photoelectron spectroscopy (XPS). Calcium phosphate was formed at a temperature of 36.5°C via two methods. On Ca2+ or HPO4 2--saturated resins, 1.5xSBF (simulated body fluid) solution was used while on Ca2+-saturated resins only, a novel biomimetic growth medium using the alkaline phosphatase-catalysed hydrolysis reaction of disodium p-nitrophenylphosphate as a source of inorganic phosphate was employed. SEM micrographs showed that the use of 1.5xSBF growth medium solution led to extensive coverage of the resins with calcium phosphate. In contrast, calcium phosphate coatings formed via the alkaline phosphatase-catalysed reaction were of a more variable quality whose morphology could be influenced by adding albumin and collagen to the growth medium. Average Ca:P ratios determined by EDX for coatings formed from the 1.5xSBF growth medium were in the range 1.62–1.74 suggesting that hydroxyapatite had formed. In contrast, Ca:P ratios for the calcium phosphate compounds formed on resins from the alkaline phosphatase reaction were lower at 1.50 suggesting that calcium-deficient hydroxyapatite had formed which was confirmed by inductively coupled plasma (ICP) analysis and X-ray diffraction of isolated amorphous and crystallized powder samples, respectively. Evidence from X-ray photoelectron studies supports a mechanism of formation of the coatings which involves diffusion of the ion out of the interior of the resin to create a high local concentration at the surface thus stimulating precipitation of the coating material on the resin beads.

Qualitative and quantitative detection of alkaline phosphatase coupled to an oligonucleotide probe for somatostatin mRNA after in situ hybridization using unfixed rat brain tissue

In situ hybridization (ISH) of somatostatin (SOM) mRNA was carried out on sections of rat brain using an alkaline phosphatase (AP) coupled oligonucleotide probe. Different hybridization and AP development conditions were tested for qualitative and quantitative detection of target mRNA on sections of unfixed tissue. Hybridization signal intensities after 24 h of hybridization were high. Comparison with adjacent formaldehyde-fixed tissue sections and hybridization for various lengths of time (2-42 h) indicated that in unfixed tissue retention of SOM mRNA was at least as high as after fixation, and that the mRNA was not degraded during hybridization. The use of tetranitroblue instead of nitroblue tetrazolium chloride in the AP detection medium provided a superior signal-to-noise ratio, and medium stability was improved for quantitative studies on unfixed sections by adding 10% polyvinyl alcohol at pH 8.5. Microphotometric measurements of mean optical densities (MOD) of the formazan reaction product in a defined area within individual neurons of the lateral central amygdaloid nucleus showed a linear increase over the first 23 h of AP reaction time. The mean MOD values per neuron were comparably high in various equally thick sections of the nucleus and increased with section thickness in a linear manner. The findings indicate that the ISH and detection reagents penetrate the entire section and that there is a linear relationship between the amount of AP reaction product measured and the amount of mRNA present in the measured area. Thus, ISH using an AP-coupled oligonucleotide on sections of unfixed tissue appears suitable for quantitative mRNA detection.