Use of a novel human immunodeficiency virus type 1 reporter virus expressing human placental alkaline phosphatase to detect an alternative viral receptor.

We report here on the construction and use of a novel human immunodeficiency virus (HIV) type 1 reporter vector, HIV-AP, that encodes human placental alkaline phosphatase. Upon staining with chromogenic alkaline phosphatase substrates 24 to 36 h postinfection, cells infected with HIV-AP develop an intense purple color and can then be counted under a dissecting microscope. Alternatively, HIV-AP infectivity can be quantitated and infected cells can be sorted by a fluorescence-activated cell sorter after staining with a fluorescent alkaline phosphatase substrate. The assay is rapid and accurate, has very low background in a variety of cell lines and primary cells, and is not restricted to use in human cells. Infectious HIV-AP can be pseudotyped by various HIV or murine leukemia virus envelope glycoproteins. Using this virus, we have addressed the long-standing question of CD4-independent infection of cells by HIV. Our results confirm the presence on a human osteosarcoma cell line of an alternative receptor for HIV infection that functions with an efficiency approximately 1/20 that of CD4.     

Candida glabrata shuttle vectors suitable for translational fusions to lacZ and use of beta-galactosidase as a reporter of gene expression

The functionality of beta-galactosidase encoded by the E. coli lacZ gene as a reporter of gene expression in C. glabrata was investigated. C. glabrata/E. coli shuttle vectors were constructed, containing both a C. glabrata CEN-ARS cassette, to allow regular segregation and episomal replication of the plasmids, and the lacZ coding sequence of E. coli. The functionality of beta-galactosidase in C. glabrata was verified by inserting the promoter and the 5' coding region of the HIS3 gene from C. glabrata directionally upstream of the lacZ gene. By fusing the promoter of the copper-controlled MTII gene to the lacZ reporter, we showed that beta-galactosidase activity can be differentially induced in C. glabrata. beta-galactosidase reporter activities were detected qualitatively by an indirect filter assay and quantitatively from permeabilized cells.     

Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice

The human placental alkaline phosphatase (PLAP) gene was analysed for its utility as a histochemically detectable reporter gene in transgenic mice. A reporter gene was made by linking the PLAP structural gene to an enhancerpromoter element from the human -actin gene. This gene was inserted into the mouse genome by transfection of embryonic stem cells, and by microinjection of fertilized eggs. Histochemical staining showed that the transgene was uniformly expressed in four of four stable ES cell lines, and in all ten tissues examined from adult animals from five lines of transgenic mice. Non-transgenic cells did not stain. These results suggest that the human PLAP gene will be of utility in studies requiring phenotypic marking of cells in tissues of mice.     

Regional mapping of the human placental alkaline phosphatase gene (ALPP) to 2q37 by in situ hybridization

In order to determine the subchromosomal location of the gene for human placental alkaline phosphatase (ALPP; EC 3.1.3.1.), a cDNA probe encompassing most of the ALPP translated sequences was hybridized in situ to metaphase chromosomes. Our results confirm previous assignment of the gene to chromosome 2 and allow its regional mapping to band q37.     

Structural studies of human placental alkaline phosphatase in complex with functional ligands

The activity of human placental alkaline phosphatase (PLAP) is downregulated by a number of effectors such as l-phenylalanine, an uncompetitive inhibitor, 5'-AMP, an antagonist of the effects of PLAP on fibroblast proliferation and by p-nitrophenyl-phosphonate (PNPPate), a non-hydrolysable substrate analogue. For the first two, such regulation may be linked to its biological function that requires a reduced and better-regulated hydrolytic rate. To understand how such disparate ligands are able to inhibit the enzyme, we solved the structure of the complexes at 1.6A, 1.9A and 1.9A resolution, respectively. These crystal structures are the first of an alkaline phosphatase in complex with organic inhibitors. Of the three inhibitors, only l-Phe and PNPPate bind at the active site hydrophobic pocket, providing structural data on the uncompetitive inhibition process. In contrast, all three ligands interact at a remote peripheral site located 28A from the active site. In order to extend these observations to the other members of the human alkaline phosphatase family, we have modelled the structures of the other human isozymes and compared them to PLAP. This comparison highlights the crucial role played by position 429 at the active site in the modulation of the catalytic process, and suggests that the peripheral binding site may be involved in the functional specialization of the PLAP isozyme.     

The developmental regulation of the human zeta-globin gene in transgenic mice employing beta-galactosidase as a reporter gene.

We have investigated the developmental and tissue specific expression of the human embryonic zeta-globin gene in transgenic mice. A construct containing 550 bp of zeta-globin 5' flanking region, fused to a beta-galactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like alpha positive regulatory element (alpha PRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire zeta-globin gene linked to the alpha PRE or the beta LCR. Data showed that 12% of mice generated from eggs injected with zeta-promoter/lacZ/alpha PRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire zeta-globin gene linked to the alpha PRE or the beta LCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, possibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that zeta-globin promoter activity was most pronounced at 8.5-9.5 days of development and was restricted to erythroid cells. By 15 days of development, no zeta-globin promoter activity was detected. These results suggest that the alpha PRE can direct high level expression from the zeta-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human zeta-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the zeta-globin gene or 3' of the cap site do not appear to be necessary for correct zeta-globin developmental regulation.     

Prediction and molecular modeling of T-cell epitopes derived from placental alkaline phosphatase for use in cancer immunotherapy

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYFPEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.     

Delivery of secreted placental alkaline phosphatase (SEAP) gene in vitro and in vivo as a component of recombinant avian adenovirus (CELO)

Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.     

He partial sequencing of human nonspecific (bone/liver/kidney) alkaline phosphatase gene

• 1.1. A charon 4A human fetal liver genomic library was screened for human nonspecific alkaline phosphatase gene using the cloned human bone cDNA as a hybridization probe.
• 2.2. A clone 2.2 Kb DNA was sequenced and found to contain a piece of sequences encoding the 4–44th amino acids of NH; terminus.
• 3.3. The other cloned 1.6Kb DNA contains two segments of sequences each corresponding to two separate regions of the cDNA for alkaline phosphatase. The first segment of the DNA codes for the 83–141st amino acids whereas the second for 141–199th.

     

Utility of human placental alkaline phosphatase as a genetic marker for cell tracking in bone and cartilage

It was the aim of the current study to evaluate the utility of human placental alkaline phosphatase (hPLAP) as a genetic marker for cell tracking in bone and cartilage, using transgenic Fischer 344 rats expressing hPLAP under the control of the ubiquitous R26 promoter [F344-Tg(R26-hPLAP)]. hPLAP enzyme activity was retained during paraffin and methylmethacrylate (MMA) embedding, and was best preserved using 40% ethanol as fixative. Endogenous alkaline phosphatase activity could be completely blocked by heat inactivation in paraffin and MMA sections, allowing histochemical detection of hPLAP in the complete absence of background staining. In addition, sensitive detection of hPLAP was also possible using immunohistochemistry. F344-Tg(R26-hPLAP) rats demonstrated ubiquitous expression of hPLAP in hematopoietic bone marrow cells and stromal cells such as osteoblasts, osteocytes, and chondrocytes. Osteoclasts only weakly expressed hPLAP. In conclusion, hPLAP provides superb detection quality in paraffin and plastic sections, and constitutes an excellent genetic marker for cell tracking in hard and soft tissues.     

A comparative study of alkaline phosphatases among human placenta, bovine milk, hepatopancreases of shrimp Penaeus monodon (Crustacea: Decapoda) and clam Meretrix lusoria (Bivalvia: Veneidae): to obtain an alkaline phosphatase with improved characteristics as a reporter

1. Alkaline phosphatases were purified from human placenta, bovine milk, shrimp and clam with a final spec. act. of 67,000, 32,000, 22,000 and 15,000 U/mg of protein respectively. 2. The alkaline phosphatase from Meretrix lusoria is unique with its thermostability at 65 degrees C for 30 min; whereas the remaining enzymes studied, including the human placental alkaline phosphatase, are inactivated and have negligible activities. 3. The alkaline phosphatase from Penaeus monodon can be differentiated by its pH optimum at 9.0; the remaining enzymes studied have their optimal pH at 10.0. 4. The alkaline phosphatases from shrimp and clam are proposed to be applied as "reporters" in the study of mammalian cells.     

Sex ratio in man: an analysis of the relationship with ABO blood groups and placental alkaline phosphatase phenotype.

Secondary sex ratio (SR) in man is influenced by various genetic and environmental factors. It has been observed that SR in subjects of blood group B compatible with their mothers is higher than in other subjects. The analysis of 676 newborns of the Rome population and 1,684 newborns of the New Haven (Connecticut) population have confirmed a higher SR in B group subjects compatible with their mothers. The data also indicate that placental alkaline phosphatase is another genetic factor influencing SR in man and that there is a strong interaction among ABO phenotype, fetomaternal ABO compatible status and PAP phenotype concerning their effects on SR.     

Severe hypophosphatasia due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene

Hypophosphatasia is a rare autosomal recessive inborn error of metabolism characterized by a defective bone mineralisation and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase activity. We report the characterisation of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutation in a patient affected by infantile hypophosphatasia. This boy was the first child of non affected, non related parents. At 1 month of age he presented with palsy of the left upper limb with hypotonia. Length was - 2SD. The anterior fontanel was large. There was a markedly decreased ossification of all bones. All limbs were shortened. Ultrasonographic examination of the kidneys showed nephrocalcinosis. Level of alkaline phosphatases was decreased in the child as well as in the parents. Bone density was decreased. At 2 years of age development was delayed. Weight was - 3,5 SD and OFC - 3SD. The child had craniosynostosis. Molecular studies showed 2 missense mutations, both in exon 6 of the TNSALP gene.