[An infant with a swelling of the cheek].
A 2-weeks-old girl was presented with a swelling of the right parotid gland, due to acute suppurative parotitis caused by Staphylococcus aureus.
Acute Disease↗
Biomedical subjects
M de Graaf
Publications and source records attributed to M de Graaf.
Adenoviral vector-mediated expression of a gene encoding secreted, EpCAM-targeted carboxylesterase-2 sensitises colon cancer spheroids to CPT-11.
CPT-11 (irinotecan or 7-ethyl-10[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an anticancer agent in use for the treatment of colon cancer. In order to be fully active, CPT-11 needs to be converted into SN-38 (7-ethyl-10-hydroxycamptothecin) by the enzyme carboxylesterase (CE). In humans, only a minority of CPT-11 is converted to SN-38. To increase the antitumour effect of CPT-11 by gene-directed enzyme prodrug therapy, we constructed a replication-deficient adenoviral vector Ad.C28-sCE2 containing a fusion gene encoding a secreted form of human liver CE2 targeted to the surface antigen epithelial cell adhesion molecule (EpCAM) that is highly expressed on most colon carcinoma cells. By targeting CE2 to EpCAM, the enzyme should accumulate specifically in tumours and leakage into the circulation should be minimised. Ad.C28-sCE2-transduced colon carcinoma cells expressed and secreted active CE that bound specifically to EpCAM-expressing cells. In sections of three-dimensional colon carcinoma spheroids transduced with Ad.C28-sCE2, it was shown that C28-sCE2 was capable of binding untransduced cells. Most importantly, treatment of these spheroids with nontoxic concentrations of CPT-11 resulted in growth inhibition comparable to treatment with SN-38. Therefore, Ad.C28-sCE2 holds promise in gene therapy approaches for the treatment of colon carcinoma.
Adenoviridae↗
Secreted and tumour targeted human carboxylesterase for activation of irinotecan.
Irinotecan (CPT-11) is an anticancer agent for the treatment of colon cancer. CPT-11 can be considered as a prodrug, since it needs to be activated into the toxic drug SN-38 by the enzyme carboxylesterase. An approach to achieve tumour specific activation of CPT-11 is to transduce the cDNA encoding carboxylesterase into tumour cells. A secreted form of carboxylesterase may diffuse through a tumour mass and may activate CPT-11 extracellularly. This could enhance the antitumour efficacy by exerting a bystander effect on untransduced cells. In addition a secreted tumour-targeted form of carboxylesterase should prevent leakage of the enzyme from the site of the tumour into the circulation. We have constructed a secreted form of human liver carboxylesterase-2 by deletion of the cellular retention signal and by cloning the cDNA downstream of an Ig kappa leader sequence. The protein was secreted by transfected cells and showed both enzyme activity and efficient CPT-11 activation. To obtain a secreted, tumour-targeted form of carboxylesterase-2 the cDNA encoding the human scFv antibody C28 directed against the epithelial cell adhesion molecule EpCAM, was inserted between the leader sequence and carboxylesterase-2. This fusion protein showed CPT-11 activation and specific binding to EpCAM expressing cells. Importantly, in combination with CPT-11 both recombinant carboxylesterase proteins exerted strong antiproliferative effects on human colon cancer cells. They are, therefore, promising new tools for gene directed enzyme prodrug therapy approaches for the treatment of colon carcinoma with CPT-11.
Animals↗
A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.
Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug.
Animals↗
Cloning and characterization of human liver cytosolic beta-glycosidase.
Cytosolic beta-glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of beta-D-glycosides, including beta-D-glucoside and beta-D-galactoside. We therefore refer to this enzyme as cytosolic beta-glycosidase. We cloned the cDNA encoding the human cytosolic beta-glycosidase by performing PCR on cDNA prepared from total human liver RNA. Specific primers were based on human expressed sequence tags found in the expressed sequence tag database. The cloned cDNA contained 1407 nt with an open reading frame encoding 469 amino acid residues. Amino acid sequence analysis indicates that human cytosolic beta-glycosidase is most closely related to lactase phlorizin hydrolase and klotho protein. The enzyme was characterized by using cell lysates of COS-7 cells transfected with a eukaryotic expression vector containing the cDNA. The biochemical, kinetic and inhibition properties of the cloned enzyme were found to be identical with those reported for the enzyme purified from human liver.
Amino Acid Sequence↗
Hormone-inducible expression of secreted factors in zebrafish embryos.
The study of gene function at later stages of embryonic development by overexpression experiments is often complicated by genes exerting different functions at multiple stages of development, which renders analysis of stage-specific effects difficult. To address this problem an inducible expression system that supports timed expression of essentially any protein, including secreted proteins was designed. The system is based on a two step mechanism. A glucocorticoid inducible, Gal4-site binding chimeric transcription factor is expressed ubiquitously, whereas a gene of interest is placed under the control of a Gal4-site driven promoter. Treatment of zebrafish embryos injected with such constructs with the synthetic glucocorticoid dexamethasone results in readily detectable reporter activity within 3 h. The system was tested with induced expression of Xactivin(beta)B and X(wnt), which both were shown to induce morphological abnormalities, as well as alterations in the expression patterns of goosecoid and otx2, respectively. Coinjection of an inducible lacZ reporter vector served as an indicator for expressing cells in embryos. The present results demonstrate that this is a versatile inducible expression system for use in vertebrate embryos, that also supports expression of secreted proteins.
Activins↗
Development of visual function in hemihydranencephaly.
This study reports on the findings of longitudinal follow-up of visual function in a 12-year-old girl affected by congenital right hemihydranencephaly. This extremely rare unilateral brain malformation allowed the authors to gather new information on neuronal plasticity and functional compensations of the visual system across a period of 10 years. An extension of the preserved right visual hemifields above the middle line and strategical eye or head positions developed to increase visual functions are discussed. In addition, ophthalmological and orthoptical findings, as well as the development of monocular grating and linear acuity, are described.
Child↗
Autosomal recessive liver phosphorylase kinase deficiency caused by a novel splice-site mutation in the gene encoding the liver gamma subunit (PHKG2).
To facilitate mutation analysis of patients with an autosomal recessive form of liver phosphorylase kinase deficiency, the genomic structure of the gene encoding the testis/liver gamma subunit (PHKG2) was established. The gene consist of 10 exons. The translation start site is located in exon 2. Analysis of DNA from two female siblings, affected with liver phosphorylase kinase deficiency, by exon specific amplification followed by direct sequencing, revealed a single donor splice site mutation in the PHKG2 gene, IVS4 + 1(g --> a). The mutation leads to the skipping of exon 4, which results in a frameshift, starting at nucleotide 272, a premature stop codon after 32 additional amino acids, and subsequent loss of the catalytic site. It is concluded that deficiency of phosphorylase kinase in liver of the patients is caused by the IVS4 + 1(g --> a) mutation. In the patients described here, this genotype is associated with development of liver fibrosis.
Amino Acid Sequence↗
Q-fever in a patient with a ventriculo-peritoneal drain. Case report and short review of the literature.
Although Q-fever is still a relatively rare disease in the Netherlands, its incidence seems to be increasing. In this article we describe the case-history of a 65-year-old woman with a Pudenz-drain, who acquired Q-fever pneumonia while manuring her garden. The course of the disease was deviant, which most likely was caused by colonization of the ventriculo-peritoneal drain with Coxiella burnetii. Q-fever usually presents as a self-limiting illness. In the case of chronic Q-fever, complications such as endocarditis, hepatitis or meningo-encephalitis can be fatal and require long-term treatment. Patients with artificial drains or valves carry a greater risk of developing such complications. Therefore, especially in patients at risk, Q-fever should be included in the differential diagnosis when dealing with a patient with unexplained fever.
Aged↗
Detection of Chlamydia trachomatis in clinical specimens by the polymerase chain reaction.
Sequences derived from the endogenous plasmid of Chlamydia trachomatis and from the genes coding for ribosomal 16S RNA of Chlamydia psittaci were used as primers and oligonucleotide probes for detection of chlamydiae by the polymerase chain reaction. The endogenous plasmid primers generated specific amplified products of 517 bp with all known Chlamydia trachomatis serovars. No specific products of Chlamydia psittaci and Chlamydia pneumoniae could be detected using these primers. With the rRNA primers specific amplified products of 208 bp were generated with Chlamydia psittaci, Chlamydia trachomatis and Chlamydia pneumoniae. No specific amplified products were detected with DNA isolated from a variety of microorganisms from the urogenital and the respiratory tract. Of 156 clinical specimens used for evaluation of the polymerase chain reaction, 26 were found to be positive for Chlamydia trachomatis on culture. All 26 culture positive samples were also found to be positive for Chlamydia trachomatis DNA by the polymerase chain reaction with both primer sets. Two culture negative samples were also found to be positive by this technique. The polymerase chain reaction thus seems to be a sensitive and reliable method for detection of Chlamydia trachomatis.
Base Sequence↗
Detection and identification of FaeC as a minor component of K88 fibrillae of Escherichia coli.
A tribrid gene containing ompF, faeC, and lacZ sequences was constructed by subcloning a large central segment of the K88ab gene encoding the fibrillar subunit-like protein FaeC into the open reading frame expression vector pORF2. The resulting tribrid protein was isolated and used to raise antibodies against the FaeC protein. These antibodies were then used for the detection and subcellular localization of the FaeC protein in Escherichia coli harbouring the K88ab-encoding plasmid pFM205 or mutant derivatives. Immunoblotting of subcellular fractions and of purified fibrillae, and agglutination experiments using whole cells revealed that the FaeC protein is present in the periplasm and as a minor component in the K88ab fibrillae. FaeC was also detected in purified K88ac and K88ad fibrillae. Immunoelectron microscopy confirmed the presence of FaeC in K88ab fibrillae, particularly at the tips of the longer fibrillae.
Agglutination↗