Biomedical subjects

P Ohana

Publications and source records attributed to P Ohana.

12 recordsLinked to original sources

PubMed2005

H19 expression in hepatic metastases from a range of human carcinomas.

AIMS: To investigate the expression of the imprinted oncofetal H19 gene in hepatic metastases derived from a range of human carcinomas and assess its prognostic value with the view of developing a DNA based treatment for such metastases. METHODS: Non-radioactive in situ hybridisation for H19 RNA was performed on paraffin wax embedded sections of liver biopsies or partial hepatectomy specimens, taken from 80 patients with hepatic metastases derived from carcinomas from several medical centres in Israel. The degree of expression was graded qualitatively according to the number of cells expressing H19 and the intensity of staining. The medical files were searched for demographic data and survival times before and after diagnosis of hepatic metastases. RESULTS: H19 expression was found in the hepatic metastases of 64 of 80 patients. High expression (higher staining grades) of H19 in the metastases was found in 43 of 80 patients. However, H19 expression status in the hepatic metastases did not correlate with either the length of time to development of metastasis or overall survival. CONCLUSIONS: H19 is highly expressed in more than half of hepatic metastases derived from a range of carcinomas. Thus, these metastases may be suitable candidates for H19 DNA based treatment. Further studies are needed to determine whether H19 expression has prognostic value in metastatic liver disease using larger numbers of specific subtypes of primary carcinomas.

Adenocarcinoma↗

PubMed2003

Inhibition of tumor growth by DT-A expressed under the control of IGF2 P3 and P4 promoter sequences.

The human IGF2 P3 and P4 promoters are highly active in a variety of human cancers. We here present an approach for patient oriented therapy of TCC bladder carcinoma by driving the diphtheria toxin A-chain (DT-A) expression under the control of the IGF2 P3 and P4 promoter regulatory sequences. High levels of IGF2 mRNA expression from P3, P4 or both promoters were detected in 18 TCC samples (n = 29) by ISH or RT-PCR. Normal bladder samples (n = 4) showed no expression from either promoter. The activity and specificity of the IGF2 P3 and P4 regulatory sequences were established in human carcinoma cell lines by means of luciferase reporter gene assay. These sequences were used to design DT-A expressing, therapeutic vectors (P3-DT-A and P4-DT-A). The activity of both was determined in cell lines (in vitro) and the activity of P3-DT-A was determined in a heterotopic animal model (in vivo). The treated cell lines highly responded to the treatment in a dose-response manner, and the growth rate of the developed tumors in vivo was highly inhibited (70%) after intratumoraly injection with P3-DT-A compared to non-treated tumors (P < 0.0002) or tumors treated by luciferase gene expressing LucP3 vector (P < 0.002).

Carcinoma, Transitional Cell↗

PubMed2001

Imaging transgene expression in live animals.

Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assessing the effectiveness of systems of gene therapy delivery. We applied a new light-detection cooled charged-coupled device (CCCD) camera for continuous in vivo assessment of commonly used gene therapy delivery systems (such as ex vivo manipulated cells, viral vectors, and naked DNA), without the need to kill animals. We examined a variety of criteria related to real-time monitoring of luciferase (luc) gene expression in tissues including bone, muscle, salivary glands, dermis, liver, peritoneum, testis, teeth, prostate, and bladder in living mice and rats. These criteria included determination of the efficiency of infection/transfection of various viral and nonviral delivery systems, promoter specificity, and visualization of luciferase activity, and of the ability of luciferin to reach various organs. The exposure time for detection of luc activity by the CCCD camera is relatively short (approximately 2 minutes) compared with the intensified CCD camera photon-counting method (approximately 15 minutes). Here we transduce a variety of vectors (such as viruses, transfected cells, and naked DNA) by various delivery methods, including electroporation, systemic injection of viruses, and tail-vein, high-velocity-high-volume administration of DNA plasmids. The location, intensity, and duration of luc expression in different organs were determined. The distribution of luciferin is most probably not a barrier for the detection of in vivo luciferase activity. We showed that the CCCD photon detection system is a simple, reproducible, and applicable method that enables the continuous monitoring of a gene delivery system in living animals.

Adenoviridae↗

PubMed2000

Use of transcriptional regulatory sequences of telomerase (hTER and hTERT) for selective killing of cancer cells.

Telomerase (hTER and hTERT) plays a crucial role in cellular immortalization and carcinogenesis. Telomerase activity can be detected in about 85% of different malignant tumors, but is absent in most normal cells. In situ hybridization analysis showed that high levels of hTER and hTERT expression are present in bladder cancer, while no signal was detected in normal tissue. Therefore, in this work we propose to use hTER and hTERT transcriptional regulatory sequences to control the expression of a cytotoxic gene in bladder tumor cells, resulting in the selective destruction of this cell population. Expression vectors containing the diphtheria toxin A-chain (DT-A) gene were linked to hTER and hTERT transcriptional regulatory sequences, respectively. Inhibition of protein synthesis occurred in bladder and hepatocellular carcinoma cells transfected with the plasmids containing the DT-A gene under the control of the hTER or hTERT promoters in correlation with their activity. These studies support the feasibility of using hTER and hTERT transcriptional regulatory sequences for targeted patient-oriented gene therapy of human cancer.

Base Sequence↗

PubMed2000

Characterization of human and mouse H19 regulatory sequences.

H19 is expressed in a large percentage of bladder tumors, but not expressed in healthy bladder tissue. The aim of this study is to define H19 optimal transcriptional regulatory sequences in tumor cells, which can potentially be used to control expression of a toxin gene in constructs to be used in bladder cancer gene therapy trials in mice and human. Transient expression assays revealed that elements responsible for promoter activity are contained within the 85 bp upstream region. The transcriptional activity of this region was strongly inhibited by the methylation of the Hpa II sites. A modest cell specificity is conferred by the upstream sequences. The human and murine promoter activities were significantly increased by the human H19 4.1 kb enhancer sequence. The 85 bp H19 upstream region contains all the elements to interact with the enhancer. We showed that the human H19 promoter is highly active in a murine bladder carcinoma cell line, justifying its use to drive the expression of a cytotoxin gene in gene therapy trials in mice.

Animals↗

PubMed1999

The expression of the H19 gene and its function in human bladder carcinoma cell lines.

The human H19 gene is a paternally imprinted oncofetal gene, highly expressed in several fetal tissues, down-regulated in nearly all adult tissues but re-expressed in carcinomas of tissues which express the gene in fetal life. It has no known protein product and till today, no function could be designated to H19 RNA. Cells derived from bladder carcinomas and hepatocellular carcinomas were transfected with plasmids carrying a luciferase reporter gene under the control of a 800 nucleotides long promoter region of the H19 gene either alone or together with different parts of a 5 kb downstream region, previously shown to possess enhancer activity. Our results provide evidence that three regions of the 3' downstream sequence can independently stimulate the H19 promoter activity in a tissue and cell specific manner. The growth rate of two cell populations, both derived from the same bladder carcinoma cell line and which differ in their H19 RNA content, were compared. The cells with a high H19 RNA level stopped their proliferation after 48 h when cultivated in a low serum containing media while the cells lacking H19 RNA continued their proliferation for at least an additional 48 h period.

Gene Expression↗

PubMed1998

Identification of a novel triterpenoid saponin from Pisum sativum as a specific inhibitor of the diguanylate cyclase of Acetobacter xylinum.

A specific and highly potent inhibitor of diguanylate cyclase, the key regulatory enzyme of the cellulose synthesizing apparatus in the bacterium Acetobacter xylinum, was isolated from extracts of etiolated pea shoots (Pisum sativum). The inhibitor has been purified by a multistep procedure, and sufficient amounts of highly purified compound (3-8 mg) for spectral analysis were obtained. The structure of this compound was established as 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1--> 2)-beta-D-glucuronopyranosyl soyasapogenol B 22-O-alpha-D-glucopyranoside. The structure was elucidated on the basis of susceptibility to various enzymes, chemical and spectral methods, such as GC-MS, FAB-MS, and the following types of 2D-NMR: COSY, ROESY, TOCSEY, HMQC, HMBC analyses. An identical or a very similar compound with identical biological activity was also isolated from A. xylinum, strongly suggesting that at least certain aspects of cellulose synthesis in the bacteria and in higher plants may be regulated in a similar manner. The content of this saponin in etiolated plants was about 0.04 mumol (g fresh tissue)-1.

Carbohydrate Conformation↗

PubMed1998

Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes.

Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of beta-1,4-glucan (cellulose) synthase in Acetobacter xylinum. The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate, cdg1, cdg2, and cdg3. Within each cdg operon, a pdeA gene lies upstream of a dgc gene. cdg1 contains two additional flanking genes, cdg1a and cdg1d. cdg1a encodes a putative transcriptional activator, similar to AadR of Rhodopseudomonas palustris and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the cdg operons, such that cdg1 contributes 80% of cellular DGC and PDEA activities and cdg2 and cdg3 contribute 15 and 5%, respectively. Disruption of dgc genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process.

Amino Acid Sequence↗

PubMed1997

c-di-GMP-binding protein, a new factor regulating cellulose synthesis in Acetobacter xylinum.

A protein which specifically binds cyclic diguanylic acid (c-di-GMP), the reversible allosteric activator of the membrane-bound cellulose synthase system of Acetobacter xylinum, has been identified in membrane preparations of this organism. c-di-GMP binding is of high affinity (KD 20 nM), saturable and reversible. The equilibrium of the reaction is markedly and specifically shifted towards the binding direction by K+. The c-di-GMP binding protein, structurally associated with the cellulose synthase, appears to play a major role in modulating the intracellular concentration of free c-di-GMP and thus may constitute an essential factor in regulating cellulose synthesis in vivo.

Allosteric Regulation↗

PubMed1992

beta-Furfuryl-beta-Glucoside: An Endogenous Activator of Higher Plant UDP-Glucose:(1-3)-beta-Glucan Synthase : Biological Activity, Distribution, and in Vitro Synthesis.

In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of beta-furfuryl-beta-glucoside (FG), an endogenous activator of plant UDP-glucose:(1-->3)-beta-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized alpha-furfuryl-beta-glucoside also stimulates callose synthase, exhibiting a slightly higher K(a) of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using beta-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca(2+), is proposed.

Journal Article↗

PubMed1991

beta-Furfuryl-beta-glucoside. An endogenous activator of higher plant UDP-glucose: (1----3)-beta-glucan synthase.

We have recently established the existence of endogenous activators of higher plant UDP-glucose: (1----3)-beta-glucan synthase (Callaghan, T., Ross, P., Weinberger-Ohana, P., and Benziman, M. (1988) Plant Physiol. 86, 1099-1103). Here we report the purification and chemical analysis of the most abundant and specific compound, termed Activator I, isolated from Vigna radiata. This compound was extensively purified by a multistep procedure which yielded 0.1 mg of purified activator/g of fresh tissue. Enzyme digestion, neutral sugar analysis, GC/MS of permethylated derivatives, and NMR analysis of native Activator I indicated that the compound contains a single beta-linked glucosyl residue. High resolution FAB-MS indicated an elemental composition of C11H16O7 (Mr = 260), with a calculated Mr of 98 for the aglycone. 13C, DEPT, and COSY NMR spectra showed that the aglycone molecule is an oxygen heterocycle of 5 carbons, consistent with a structure of beta-furfuryl alcohol. Comparison of IR and GC/EI-MS spectra of authentic beta-furfuryl alcohol with native aglycone confirmed the conclusion that Activator I is beta-furfuryl-beta-glucoside. Chemically synthesized beta-furfuryl-beta-glucoside has identical chemical properties and biological activity when compared with the purified endogenous activator (Ka = 50 microM).

Enzyme Activation↗

PubMed1991

Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants.

To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either [32P]c-di-GMP or [alpha-32P]UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene. We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.

Affinity Labels↗