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Biomedical subjects

E C Zwarthoff

Publications and source records attributed to E C Zwarthoff.

At least 37 records · Page 2Linked to original sources
PubMed1996

Regional localization of over 300 loci on human chromosome 22 using a somatic cell hybrid mapping panel.

A somatic cell hybrid panel, consisting of 25 cell lines, has been developed to localize loci subregionally on chromosome 22. Over 300 markers in the form of STSs or hybridization probes have been assigned to one of 24 unique regions or "bins" using this panel. This ordered collection of markers will aid in the assembly of physical maps and contigs of chromosome 22 and assist in positional cloning of disease loci mapped to chromosome 22.

Animals↗
PubMed1996

Neurofibromatosis and associated tumour suppressor genes.

Neurofibromatosis 1 and 2 (NF1 and NF2) are autosomal dominantly inherited disorders with close to 100% penetrance. NF1 is one of the most frequent human genetic diseases with an incidence of 1:3000. The incidence of NF2 is about 10 fold lower. NF1 is caused by mutations which inactivate the NF1 gene on chromosome 17q, while the NF2 gene is on chromsome 22. Both genes are tumour suppressor genes. The product of the NF1 gene, called neurofibromin, is a large protein of 2818 amino acids. The protein acts as a negative regulator in the ras signal transduction pathway and may also act downstream of ras. In the cell types that are affected in NF1 patients, the absence of neurofibromin leads to increased proliferation resulting in benign, and in some cases malignant tumours. The product of the NF2 gene is a protein of 595 amino acids. The protein displays in its N-terminal half considerable homology with proteins that are involved in contacts between the cytoskeleton and the cell membrane, and a similar function has been proposed for the NF2 protein. How the absence of the NF2 protein may lead to the development of Schwannomas and meningiomas, which are the major manifestations of NF2 in patients, is not clear at present.

Animals↗
PubMed1995

Characterization of four novel CAG repeat-containing cDNAs.

Stretches of CAG nucleotides coding for the amino acid glutamine are an important feature of many transcription factors and genes that are involved in neurodegenerative disorders. In an attempt to isolate CAG repeat-containing cDNAs expressed in nervous tissue, we screened a human fetal brain cDNA library with a probe containing a CAG repeat. Five different clones were characterized and found to contain CAG repeats. Sequence data revealed that four of these cDNAs were derived from novel genes. These cDNAs were designated CAG6, CAG12, CAG24, and CAG40 and were found to correspond to transcripts of 5.0, 7.5, 4.4, and 15 kb, respectively. The genes encoding CAG6, CAG12, CAG24, and CAG40 were assigned to chromosomes 12, 16, X, and 12, respectively. For the 5th gene, CAG26/pRHpA, a localization on two different chromosomes was established: 16 and X. None of the repeats showed any length polymorphisms in human DNA.

Base Sequence↗
PubMed1995

Modulation of intra-epithelial expansion of human T24 bladder-carcinoma cells in murine urothelium by growth factors and extracellular-matrix components.

The high recurrence rate of bladder cancer is probably due to an efficient repopulation of the bladder by residual transformed cells after resection of the tumour. However, the regenerating capacity of the normal urothelial cells is very high. To study the balance between regenerating normal urothelium and out-growth of transformed urothelial cells, we recently developed an in vitro co-cultivation model. With this model system we studied the effects of growth factors and extracellular-matrix components on the intra-epithelial expansion of human T24 bladder-carcinoma cells in primary mouse-bladder explants. Exposure of the cultures to acidic fibroblast growth factor (aFGF) and laminin led to a dramatic increase in the number of invasive T24 cells into the primary urothelium. Epidermal growth factor (EGF) and collagen types I and IV counteracted the infiltration of individual T24 cells. EGF, aFGF, laminin and collagen types I and IV did not directly affect the migration and proliferation of T24 cells. Apparently, the efficacy of invasion of transformed urothelial cells into primary urothelium is not only dependent on the intrinsic characteristics of the transformed cells, but can be influenced to a considerable extent by exogenous components exerting their influence on the normal urothelium. The clinical relevance of this observation needs to be studied further.

Animals↗
PubMed1995

The product of the NF2 tumour suppressor gene localizes near the plasma membrane and is highly expressed in muscle cells.

Neurofibromatosis type 2 (NF2) is a disease resulting in the formation of schwannomas of the eighth cranial nerve, and other central nervous system tumours. A tumour suppressor gene has been found to be responsible for this disorder. The 595 amino acid NF2 protein shows a great deal of homology to a superfamily of membrane organizing proteins. To generate antibodies against the NF2 protein four synthetic peptides (SP) were injected in rabbits. COS cells transfected with an NF2 cDNA construct in an expression vector were used for immunocytochemical staining experiments; lysates of transfected COS cells were used for Western blotting experiments, as were lysates of E. coli cultures transformed with an NF2 cDNA construct subcloned in a prokaryotic expression vector. In western blots all sera detected a band indicating the appropriate molecular weight in lysates of transfected COS cells and E. coli. Immunocytochemical staining experiments indicate that the NF2 protein localizes in or near the cell membrane. Immunohistochemical staining of human tissue sections demonstrated the presence of the NF2 protein in muscle-, and Schwann cells. These results support the hypothesis that the NF2 protein functions as a membrane organizing element.

Amino Acid Sequence↗
PubMed1995

Positional mapping of loci in the DiGeorge critical region at chromosome 22q11 using a new marker (D22S183).

The majority of patients with DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) and a minority of patients with non-syndromic conotruncal heart defects are hemizygous for a region of chromosome 22q11. The chromosomal region that is commonly deleted is larger than 2 Mb. It has not been possible to narrow the smallest region of overlap (SRO) of the deletions to less than ca 500 kb, which suggests that DGS/VCFS might be a contiguous gene syndrome. The saturation cloning of the SRO is being carried out, and one gene (TUPLE1) has been identified. By using a cosmid probe (M51) and fluorescence in situ hybridization, we show here that the anonymous DNA marker locus D22S183 is within the SRO, between TUPLE1 and D22S75 (probe N25). A second locus with weak homology to D22S183, recognized by cosmid M56, lies immediately outside the common SRO of the DGS and VCFS deletions, but inside the SRO of the DGS deletions. D22S183 sequences are strongly conserved in primates and weaker hybridizing signals are found in DNA of other mammalian species; no transcripts are however detected in polyA+ RNA from various adult human organs. Probe M51 allows fast reliable screening for 22q11 deletions using fluorescence in situ hybridization. A deletion was found in 11 out of 12 DGS patients and in 3 out of 7 VCFS patients. Two patients inherited the deletion from a parent with mild (atypical) symptoms.

Abnormalities, Multiple↗
PubMed1995

Cytogenetic, molecular genetic and pathological analyses in 126 meningiomas.

In a series of 126 meningiomas, tumor and patient characteristics were investigated and statistically analyzed. A combined cytogenetic and molecular genetic approach was used to study chromosomal abnormalities and loss of markers on chromosome 22q. This approach was successfully applied to 93 meningiomas. In 66 cases, complete or partial loss of chromosome 22 was observed and in at least 12 of them this chromosome was involved in structural aberrations. In addition to chromosome 22 changes, chromosomes 1, 6, 11, 13, 14, 18, 19, X, and Y were also frequently involved in structural and numerical aberrations. Statistical analysis revealed a significant association between the number of chromosomal abnormalities and tumor grade. Complex karyotypes predominated in the group of grade II/III meningiomas. Furthermore, other variables showed statistically (or marginally statistically) significant differences. Meningiomas from the convexity were more often grade II/III, displayed predominantly (partial) loss of chromosome 22 and had complex karyotypes more often. These features were frequently found in meningiomas from males. Base meningiomas, on the other hand, occurred more often in females; they were usually grade I, showed loss of (parts of) chromosome 22 less often and displayed fewer additional chromosomal abnormalities.

Adult↗
PubMed1995

IGF, type I IGF receptor and IGF-binding protein mRNA expression in the developing mouse lung.

The IGFs are important mitogens involved in lung growth and development. The regulation of IGF action depends not only on the expression of IGFs and IGF receptors, but also on the modulation of IGF activity by IGF-binding proteins (IGFBPs). In this study, we describe the mRNA expression of IGF-I, IGF-II, type I IGF receptor, IGFBP-2, IGFBP-4 and IGFBP-5 during mouse lung development as studied by in situ hybridization techniques. The IGF, type I IGF receptor and IGFBP-2, -4 and -5 genes were expressed in developing lung as early as embryonal day 12.5. Expression of IGFBPs-1, -3 and -6 was below detection. IGF and IGFBP-2 mRNAs were expressed both in mesenchymal and epithelial cells. Type I IGF receptor transcripts were also observed throughout the developing lung, with the exception of the epithelial cells of the bronchi after embryonal day 15. Furthermore, mRNA expression of IGFBPs-4 and -5 was noted in neighbouring cell types, and after embryonal day 15, co-expression of the type I IGF receptor and IGFBP-4 transcripts was detected. The observed expression patterns imply that the IGFBP-2, -4 and -5 genes are differentially regulated during embryonic development and suggest that each may have a discrete function. A possible role for IGFBPs-2, -4 and -5 is to participate in the regulation of cell-specific IGF responses during mouse lung development.

Animals↗
PubMed1995

Neurofibromatosis type 2 protein co-localizes with elements of the cytoskeleton.

The product of the neurofibromatosis type 2 (NF2) tumor suppressor gene is a 595-amino-acid protein bearing resemblance to a family of band-4.1-related proteins. These proteins, including ezrin, radixin, and moesin, probably function as molecular linking proteins, connecting the cytoskeleton to the cell membrane. On the grounds of the homology to the ezrin, radixin, and moesin proteins and on the basis of its predicted secondary structure, the NF2 protein is also thought to act as a cytoskeleton-cell membrane linking protein. Using monoclonal antibodies to amino- and carboxyl-terminal synthetic NF2 peptides we demonstrate the co-localization of the NF2 protein with elements of the cytoskeleton in a COS cell model system and in cultured human cells. Furthermore, the presence of the NF2 protein in tissue sections is shown. The monoclonal antibodies specifically stain smooth muscle cells and the stratum granulosum of the human epidermis. In cultured smooth muscle cells the NF2 protein co-localizes with actin stress fibers. Immunoelectron microscopy demonstrates the presence of the NF2 protein associated with keratohyalin granules and to a lesser extent with intermediate filaments in the human epidermis. We conclude that the NF2 protein is indeed associated with multiple elements of the cytoskeleton.

Actins↗
PubMed1994

E-cadherin expression determines the mode of replacement of normal urothelium by human bladder carcinoma cells.

The high recurrence rate of human bladder cancer can be attributed to intraepithelial expansion of tumor cells or shedding and subsequent implantation of tumor cells elsewhere in the bladder. E-Cadherin is a calcium-dependent cell-cell adhesion molecule, and loss of E-cadherin by tumor cells is associated with increased tumor aggressiveness. Here we demonstrate that E-cadherin is also an important determinant of the mechanisms which are involved in the recurrence rate of bladder cancer. In a recently developed in vitro cocultivation model, we studied the effect of E-cadherin expression on the intraepithelial expansion pattern of six different human bladder carcinoma cell lines into primary murine urothelium. Bladder carcinoma cells lacking E-cadherin infiltrate into the primary urothelium as individual cells (pagetoid pattern). In contrast, a sharp demarcation is observed between E-cadherin-positive bladder cancer cells and the primary urothelium (carcinoma in situ pattern). With the same model, we demonstrate that only E-cadherin-positive bladder carcinoma cell lines could attach to and colonize the intact primary urothelium. We hypothesize that it is the latter process that plays an important role in the high recurrence rate that is observed in some of the patients.

Animals↗
PubMed1994

Constitutional DNA-level aberrations in chromosome 22 in a patient with multiple meningiomas.

We describe a patient who developed multiple meningiomas but had no clear evidence of neurofibromatosis type 2. Four of the tumors, derived from three different sites, were analyzed cytogenetically and/or at the DNA level using chromosome 22 specific probes. All four tumors showed loss of the same copy of chromosome 22. On the chromosome that was retained in the tumors, we found two constitutional aberrations, a 1.5 kb deletion and a point mutation. The patient had inherited both alterations from her father. The father has not developed any meningiomas so far but he has been treated for a well-differentiated adenocarcinoma of the lung and a brain metastasis from this tumor. The mother and 75 unrelated individuals did not show any of the chromosome 22 alterations. The multiple tumors found in the patient suggest that she has a predisposing gene for the development of meningiomas. The finding that all investigated tumors lost the same, constitutionally normal copy of chromosome 22 could indicate that the predisposing gene resides on chromosome 22 and was affected by the constitutional mutations.

Adenocarcinoma↗
PubMed1994

An in vitro model of urothelial regeneration: effects of growth factors and extracellular matrix proteins.

Although the cellular turnover of resting urothelium is very low, its regenerative capacity is known to be outstanding. In organotypic mouse urothelial cultures closely mimicking the differentiation and multilayering of normal urothelium, we examined the cell biological mechanisms underlying urothelial regeneration and the specific role of growth factors and several extracellular matrix (ECM) components. Exposure to epidermal growth factor (EGF) and acidic fibroblast growth factor (aFGF) and culture on laminin resulted in enhanced expansion of the urothelium. Microscopy and assessment of proliferative activity revealed that enhanced urothelial expansion due to EGF could be attributed to increased proliferative activity and an increase in cell numbers, whereas aFGF-stimulated expansion must be considered the consequence of increased cellularity and migration. Laminin-enhanced urothelial expansion was shown to be the result of spreading of the entire urothelial organotypic culture. This was associated with a considerable decrease in the number of cell layers. A synergistic effect of growth factors and laminin was not found. This organotypic urothelial cell culture model seems to be very useful in studying strategies to improve urothelial regeneration.

Animals↗
PubMed1994

cDNA cloning and mRNA expression of the six mouse insulin-like growth factor binding proteins.

The insulin-like growth factor binding proteins (IGFBPs) comprise a family of six distinct proteins which modulate insulin-like growth factor action. We have isolated cDNAs encoding the six mouse IGFBPs (mIGFBPs). In addition, we studied the mRNA expression of the six mIGFBPs during development and in various adult tissues. Our results show that each of the six mIGFBPs is highly homologous to their human and rat counterparts, whereas only the N and C terminal ends are conserved between the six mIGFBPs. Northern blotting revealed that mIGFBP-2, -3, -4 and -5 genes are already expressed at gestational day 11.5, suggesting a role for these mIGFBPs in embryonal development. In liver, a peak of mIGFBP-1 mRNA expression was found around birth, suggesting a function for mIGFBP-1 in the newborn mouse. Finally, tissue-specific expression of the six mouse IGFBP genes was observed in adult tissues suggesting different roles or modes of actions in adult life.

Amino Acid Sequence↗
PubMed1993

Insulin-like growth factor binding protein-2, 28 kDa and 24 kDa insulin-like growth factor binding protein levels are decreased in fluid of dominant follicles, obtained from normal and polycystic ovaries.

In order to investigate potential changes in insulin-like growth factor binding proteins (IGFBPs) during human follicle maturation, we examined the IGFBP profiles in follicular fluid from follicles in different stages of maturation. Samples were obtained from ovaries of women with regular menstrual cycles and of subjects with cycle abnormalities and polycystic ovaries (diagnosed as polycystic ovary syndrome (PCOS)) and analyzed by Western ligand blotting. IGFBPs of 43 kDa, 37 kDa, 31 kDa, a doublet around 28 kDa and a minor band of 24 kDa were detected in follicle fluid of normal non-dominant (size < 10 mm) and atretic (androstenedione/estradiol ratio > 4) follicles of both regularly menstruating women and PCOS patients. The 43 and 37 kDa IGFBPs could be identified as IGFBP-3 and the 31 kDa IGFBP as IGFBP-2, whereas the 28 kDa IGFBP could not be identified as IGFBP-1, all by immunoblotting techniques. A dramatic decrease in IGFBP-2, the 28 kDa and 24 kDa IGFBPs was observed in follicular fluid of dominant follicles (size > 10 mm) of both regular menstruating individuals and one PCOS patient as compared with follicular fluid of normal non-dominant or atretic follicles. These observations indicate that the PCOS follicle may not be different from normal with respect to IGFBP profiles. Furthermore, these results suggest that at least one of these IGFBPs might be involved in human folliculogenesis.

Adult↗
PubMed1993

An in vitro model of intra-epithelial expansion of transformed urothelial cells.

Replacement of normal urothelium by pre-cancerous epithelium may explain the high recurrence rate of human bladder cancer. An in vitro model was designed in order to study the mechanisms of expansion of transformed urothelial cells at the expense of normal urothelium. For this purpose, mouse bladder explants were allowed to expand on a transparent porous membrane. Subsequently, cell sheets of the non-tumorigenic mouse urothelial cell line NUC-5, the tumorigenic mouse urothelial cell line NUC-5 Py and the human bladder-carcinoma cell line T24 were inoculated adjacent to the primary explant. Daily measurements of the outgrowth of the bladder explant were performed, and all cultures were terminated on day 24. At this time the post-confluent primary urothelial cell outgrowths still showed proliferative activity, as demonstrated by bromodeoxyuridine incorporation. In due course the non-tumorigenic NUC-5 cells were replaced by the bladder outgrowth. T24 and NUC-5 Py cells were able to inhibit the bladder outgrowth, or even infiltrate or replace the explant. This was confirmed by immunohistochemistry with (species-specific) anti-cytokeratin antibodies and by microscopic evaluation of cross-sections of the porous membrane. This co-cultivation model appears to be suitable for the in vitro study of the mechanisms of intra-epithelial expansion of transformed urothelial cell lines.

Animals↗
PubMed1993

Somatostatin receptor-positive primary breast tumors: genetic, patient and tumor characteristics.

In a series of 87 primary breast tumors, somatostatin receptor (SSR) expression was detected by in vitro autoradiography in 58 tumors. In 41 tumors the SSR expression was homogeneous and in 17 it was heterogeneous. Although the tumors were not selected by the investigators upon entry in the study, examination of the tumor and patient characteristics showed that a pre-selection had taken place for small tumors. Eighty percent of the tumors were classified as stage pT1 or pT2 tumors. This small tumor size and the large size of the tumor sections used for autoradiography can explain the high incidence of somatostatin expression in our series. Forty-three of these tumors, 30 SSR-positive and 13 SSR-negative, were tested for morphological and (immuno)histochemical markers of neuroendocrine differentiation. Three SSR-positive tumors were also positive for 2 or more other markers of neuroendocrine differentiation, suggesting that neuroendocrine breast tumors and SSR-positive breast tumors are overlapping, but independent, subgroups of tumors. To test whether specific genetic alterations are associated with SSR-positive or SSR-negative breast tumors, we examined in a selected series of 47 SSR-positive and 32 SSR-negative breast tumors a number of known genetic markers by Southern blotting. Deletions or rearrangements of the retinoblastoma (RB) tumor-suppressor gene were observed in 5 SSR-positive and 5 SSR-negative tumors. In 4 SSR-positive and also in 4 SSR-negative tumors an amplification of the neu oncogene was observed. Amplifications of the int-2 oncogene were found in 2 SSR-positive and 1 SSR-negative breast tumor. In one SSR-positive tumor an amplification of the c-myc oncogene was observed and in another SSR-positive tumor a rearrangement of the L-myc oncogene was found.

Autoradiography↗
PubMed1993

Isolation and characterization of 25 unique DNA markers for human chromosome 22.

Twenty-five single-copy anonymous DNA markers for human chromosome 22 were isolated. These markers were assigned to four different regions on the chromosome. Six markers recognize restriction fragment length polymorphisms. The relative positions of five of these polymorphic markers on the framework map of chromosome 22 were determined by linkage analysis. The sizes of the NotI fragments recognized by 22 markers were determined by pulsed-field gel analysis. The total length of the NotI fragments identified is at least 12 Mb, which represents about 20% of the entire chromosome.

Cell Line↗