Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The detection of human cytomegalovirus (HCMV) infected poly-morphonuclear leukocytes (PMNLs) for early finding of the pp65 antigen using automated image analysis has been improved. The routinely used immunoenzyme peroxidase (PO) labelling has been replaced by alkaline phosphatase (AP) using new fuchsin as substrate. The number of automatically detected false positive objects due to incomplete inactivation of endogenous peroxidase and strong variations in counterstain intensity could be reduced by 81% using this AP staining method. The number of detected truly positive cells with both staining methods was not significantly different. Furthermore, a new image analysis system providing processing of colour images was evaluated. Since plain differences in absorption wavelength are required for colour segmentation, the red immuno-staining was combined with a green counterstain using methyl green. Screening of AP- instead of PO-stained slides in combination with colour segmentation resulted in a further reduction of the number of falsely detected alarms from 61% to 11%. Consequently, the sensitivity of the automated detection was improved. For AP staining detection of cells in frequencies of approximately one to one million was demonstrated. Screening for CMV-positive, alkaline phosphatase labelled cells using an image analysis system with colour segmentation resulted in a reduced false alarm rate, a better visual interpretation of the images and subsequently an increase in the sensitivity of the automated screening process.
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PMID:Automated screening for cytomegalovirus infected cells using image analysis. Comparison of two immunoenzymatic staining methods with respect to colour segmentation. 753 31

Non-isotopic in situ hybridization with a novel phenytoin (PHE)-labeled probe was developed. The mixture of cloned cytomegalovirus (CMV) DNA fragments was labeled by random primer technique using PHE-11(spacer)-dUTP, instead of dTTP. The tissue sections were treated with 0.2 N HCl and with proteinase K (1 microgram/ml), and then heated at 70 degrees C in the presence of 50 or 75% formamide. The sections were hybridized with PHE-labeled probe at 37 degrees C overnight. The hybridization signal was visualized by alkaline phosphatase-5-bromo-4-chloro-3-indolyl phosphate (BCIP)/4-nitroblue tetazolium (NBT) system. Strong hybridization signals were detected in sections of the small intestine and the placenta, even when denatured in the presence of 50% formamide. In the case of small intestine, CMV DNA was also detected in the endothelial cells of the mucosa where apparent infected cell was not observed histologically. In the sections of the submaxillary gland, the lung, the adrenal gland and the ovary, hybridization signal was not detected when denatured in the presence of 50% formamide, but detected after denaturation with 75% formamide. Thus, in situ hybridization with the novel PHE-labeled probe is applicable to conventional formalin-fixed, paraffin-embedded tissue sections.
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PMID:Application of in situ hybridization with a novel phenytoin-labeled probe to conventional formalin-fixed, paraffin-embedded tissue sections. 760 5

Detection of human cytomegalovirus (CMV) DNA from peripheral blood and urine specimens taken from renal transplant and bone marrow transplant recipients was examined by using nested polymerase chain reaction (PCR). Results by the PCR were compared with those of healthy subjects. Oligonucleotide primers were used to amplify the immediate-early (IE) and the late antigen (V) genes of CMV. Amplified DNA products were identified by 2% agarose gel electrophoresis and by Southern hybridization with alkaline phosphatase-labeled probes. Twenty eight blood specimens from 118 healthy subjects were positive for the V gene fragment of CMV, whereas the IE gene fragment was more associated with a negative result (116 of 118 specimens). When amplified with mixed primer pairs, no blood and urine specimens simultaneously produced the PCR products of two separate CMV genes. In the case of transplant patients, however, 50 of 139 blood specimens and 34 of 117 urine specimens were positive with the V primers, and 23 blood and 17 urine specimens were positive with the IE primers. Using mixed primer pairs, amplification of two CMV genes was detected in 14 blood and 10 urine specimens. In three cases of bone marrow transplant recipients, amplification of two CMV genes was detectable prior to anti-CMV IgM development. From these results, we suggest that detection of CMV-DNA by nested PCR with mixed primer pairs may be a valuable tool for diagnosing CMV infections.
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PMID:[Detection of human cytomegalovirus DNA by nested polymerase chain reaction]. 788 68

We report three patients with intestinal microvillous dystrophy, two of whom were siblings. The relatively delayed clinical presentation and the lack of classical microvillous inclusions distinguish these cases from the previously described microvillous inclusion disease (MVID). There appears to be an underrecognized spectrum of microvillous disorders leading to fatal intractable secretory diarrhea in infants. In our three cases the diagnosis was suggested by periodic acid-Schiff (PAS) and alkaline phosphatase preparations of a jejunal biopsy specimen showing thinning or absence of brush border staining, which was confirmed by electron microscopy. The latter showed poorly developed and haphazardly arranged microvilli with intracytoplasmic vesicular bodies but no true inclusions. As in MVID, the prognosis of intestinal microvillous dystrophy is poor. The occurrence of the disease in two siblings of consanguinous parents suggests an autosomal recessive inheritance, and like MVID, genetic counselling of affected families is essential.
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PMID:Intestinal microvillous dystrophy: a variant of microvillous inclusion disease or a new entity? 767 89

In situ hybridization (ISH) provides a means for identifying viral genomes in the context of tissue pathology. We have developed a specific and sensitive ISH probe for the detection of cytomegalovirus (CMV) DNA in formalin-fixed, paraffin-embedded tissue sections. Digoxigenin-11-dUTP was incorporated into a 435-base pair fragment of the CMV Major Immediate Early (MIE) gene with use of the polymerase chain reaction (PCR). Hybridized probe was detected by reaction with antidigoxigenin antibody coupled to alkaline phosphatase and chromogenic substrates. This method has detected CMV infection in routine clinical specimens from a variety of tissue types, including colon, kidney, liver, and stomach. Infection in cells with and without characteristic inclusions is revealed with this probe. The background is so low that single infected cells are detected unambiguously. No cross-hybridization was observed with cells infected with other viruses of Herpesviridae. This approach may be useful for producing probes for the detection of other viral genomes in tissue sections.
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PMID:PCR production of a digoxigenin-labeled probe for the detection of human cytomegalovirus in tissue sections. 798 96

A 27-yr-old Jamaican male presented with a 2-month history of jaundice, pruritus, intermittent diarrhea, and right upper quadrant abdominal pain. Over the next month, his abdominal pain and diarrhea improved, but his jaundice and pruritus worsened. He was afebrile and profoundly jaundice, with a benign abdominal examination. Medical workup included a normal abdominal ultrasound, iron studies, ceruloplasm, and serum electrophoresis. Negative viral (Epstein-Barr virus, cytomegalovirus, mononucleosis, hepatitis A, B, C) studies, ANA, AMA, ASMA, RPR were noted. He denied any alcohol, drug, or toxin exposure. Liver tests revealed total bilirubin of 25.6 mg/dl, direct bilirubin of 13.9 mg/dl, alkaline phosphatase 278 IU/L, AST 45 IU/L, and ALT 71 IU/L. Liver biopsy demonstrated centrilobular zonal necrosis and cholestasis most consistent with a toxic reaction. The patient was again interviewed regarding potential toxins, and he admitted to the ingestion of ackee fruit, a native Jamaican fruit that is illegal in the United States. Shortly after he had ceased intake of the fruit, his symptoms resolved and his liver function tests returned to normal. We present a case of chronic ackee fruit ingestion that led to cholestatic jaundice, vomiting, and abdominal pain.
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PMID:Cholestatic jaundice due to ackee fruit poisoning. 807 44

The mode of formation and role of rheumatoid factors (RF) in transplantation were reviewed. RF might be produced through stimulation with altered transplantation antibody, which undergoes conformational changes resulting from the reaction with a graft. Some recipients might have such sources of infection as cytomegalovirus or human herpes virus 6 (HHV-6), because almost all patients were treated with immunosuppressive agents. Many bacteria and viruses have IgG Fc receptors on their surface. Herpes virus infection, such as herpes simplex virus, has been demonstrated to be capable of expressing Fc receptor on the infected cells. These Fc receptors could produce RF through the response of antibody production to anti-idiotype of Fc receptors. Also, IgG, which binds to Fc receptors, changes conformationally to produce RF. RF can also be produced as a result of graft versus host reaction. Although IgG RF reacting with homologous IgG is believed to have the most important role in pathogenic conditions, they have not been studied because of difficulties in their measurements. We developed an enzyme immunoassay for detection of IgG RF combining with homologous IgG. The wells of an assay plate were coated with Fc fragment of human IgG, and after incubation with tested serum, binding of IgG RF to the Fc was detected by means of goat anti-human IgG F (ab')2 antibodies followed by alkaline phosphatase conjugated rabbit anti-goat IgG and substrate. IgG RF were increased significantly more in transplantation sera of patients who rejected grafts than in those of patients who had satisfactory graft function. Some evidence for the role IgG RF in rejection, even if only circumstantial, has been provided.
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PMID:Rheumatoid factor: a review of the mode of formation and role in transplantation. 812 26

The biochemical properties of recombinant amphibian bone morphogenetic protein-4 (BMP-4), the cDNA of which has been cloned recently by screening of a Xenopus cDNA library, was characterized. The protein was expressed by the transfection of Chinese hamster ovary (CHO) cells with the cDNA cloned into expression vectors bearing a cytomegalovirus promoter or a simian virus 40 promoter. Northern-blot analysis showed that the latter vector was more efficient for Xenopus BMP-4 expression. Specific antiserum against Xenopus BMP-4 peptide demonstrated that the protein is synthesized as a large precursor, processed to the mature form and then secreted from the cells as a homodimer. Analysis of the biological activity in the conditioned medium revealed that Xenopus BMP-4 has a potent alkaline phosphatase-inducing activity on mouse osteoblastic cells.
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PMID:Biochemical properties of amphibian bone morphogenetic protein-4 expressed in CHO cells. 838 68

In a prospective study of HIV patients with suspected cytomegalovirus (CMV) disease (n = 144; 140 men, four women; aged 23-69 years, median 38 years; CD4 cells 0-400, median 20/microliters), 242 blood samples were examined for the presence of CMV-pp65 antigen in peripheral blood polymorphonuclear leucocytes by use of monoclonal antibodies and alkaline phosphatase-anti-alkaline phosphatase staining. All patients were thoroughly examined for existing CMV disease at first visit and during follow-up (at least 2 months or until death: 0-24 months, median 14 months). In 43/486 samples of patients with CMV disease, the antigen-test was positive and in 179/194 samples of patients without CMV disease the test was negative, resulting in a sensitivity of 90% and a specificity of 93% for the presence of CMV disease in HIV-infected patients.
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PMID:CMV-antigenemia in peripheral blood for the diagnosis of CMV disease in HIV-infected patients. 940 79

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.
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PMID:Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA. 864 53


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