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Biomedical subjects

A de Vries

Publications and source records attributed to A de Vries.

At least 55 records · Page 3Linked to original sources
PubMed1998

Detection of microorganisms in vessel wall specimens of the abdominal aorta: development of a PCR assay in the absence of a gold standard.

A procedure in which the "Invitrogen Easy-DNA" kit was followed by a silica-based method for the isolation of DNA was developed for extraction of PCR-inhibitor-free DNA from up to 300 mg of human vessel wall tissue. Optimally designed PCR assays were developed for the detection of at least one infected cell in this amount of tissue. Details of the procedure are given for the detection of DNA of Chlamydia pneumoniae, cytomegalovirus and herpes simplex virus type 1 and type 2 in human vessel walls. The procedure can serve as a reference method or as a gold standard when a high-performance method is needed.

Aorta, Abdominal↗
PubMed1997

Low incidence of p53 mutations in UVA (365-nm)-induced skin tumors in hairless mice.

Mutations with clear "UVB fingerprints" have been observed in the p53 gene of human nonmelanoma skin tumors and of experimentally UVB-induced murine skin tumors. Although UVA (315-400 nm) radiation is also a complete carcinogen, its contribution to sunlight-induced mutagenesis remains poorly characterized. There is experimental evidence that the production of reactive oxygen species plays a more dominant role with long-wave UVA than with UVB radiation. We have induced skin tumors (n = 42) in hairless SKH:HR1 mice (n = 14) by daily exposure to long-wave UVA (365-nm) radiation. The incidence of p53 alterations in these tumors is low compared to UVB-induced tumors; positive staining for the p53 protein was observed in only 50% of the tumors, and less than 15% of the tumors showed a mutation in one of the exons 5, 7, or 8 of the p53 gene. The pattern of p53 staining was more irregular and less dense compared to UVB, and the mutations (all C-->T) were mainly (six of seven) located at codon 267. Besides a general p53 hotspot, this codon is also the main hotspot for UVB-induced skin tumors in these mice. No mutations specific for UVA, ie., mutations specific for reactive oxygen species, could be detected.

Animals↗
PubMed1997

Effect of Brussels sprouts on oxidative DNA-damage in man.

The effect of consumption of Brussels sprouts on the excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) into human urine was investigated. Ten non-smoking volunteers (five males and five females) were randomly assigned to two groups. Five volunteers started on a diet of 300 g of glucosinolate-free vegetables whereas the other five consumed 300 g of Brussels sprouts per day. After 1 week dietary regimes were reversed. Levels of 8-oxodG in 24-h urine samples were measured by high-performance liquid chromatography. In four of five males a reduction in 8-oxodG was found, whereas in the fifth male the 8-oxodG excretion was high in the control period and was even much higher in the sprouts period. In females no effect of consumption of Brussels sprouts on excretion of 8-oxodG was found. Our previous and present findings support the results of epidemiologic studies that consumption of brassica vegetables may diminish cancer risk.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Relative susceptibilities of XPA knockout mice and their heterozygous and wild-type littermates to UVB-induced skin cancer.

Although xeroderma pigmentosum (XP) patients are rare, carriers of XP genes (heterozygotes) are much more common. Whether such carriers have an increased skin cancer risk is unknown. Recently developed mouse models for XP have opened up the possibility of determining the skin cancer risk of heterozygotes relative to wild types. Therefore, the XPA knockout trait has been crossed into hairless mice, and squamous cell carcinomas of the skin have been induced by low daily UVB exposures for 500 days in all three genotypes (-/-, +/-, and +/+). The carcinogenic response of the heterozygotes did not significantly differ from that of their wild-type littermates. Tumors in the XPA -/- animals appeared with a latency time that was decreased by a factor of 4.2. From this, we estimate that a functional XPA gene provides a "protection factor" of 60 (95% confidence interval, 15-250) against UV carcinogenesis, which is greater protection than that against acute UV effects, such as erythema and edema (protection factor between 7 and 16). Deficient nucleotide excision repair appears to have a more dramatic impact on skin cancer susceptibility than on sensitivity to acute UV effects.

Animals↗
PubMed1997

Metabotropic glutamate receptor mGluR5 in astrocytes: pharmacological properties and agonist regulation.

Metabotropic glutamate receptor (mGluR) agonists induce extensive phosphoinositide (PI) hydrolysis in astrocytes grown in a chemically defined medium with select growth factors. These astrocytes express mGluR5 transcripts, but none of the splice variants of mGluR1, thus permitting the characterization of mGluR5 in a native CNS cell without interference from mGluR1 activity. mGluR5 activation (1) was not associated with stimulation of cyclic AMP formation, (2) showed high sensitivity to the removal of extracellular versus intracellular Ca2+, (3) displayed high coupling efficiency relative to receptor density, and (4) induced PI hydrolysis that was suppressed by phorbol esters with low potency. The rank order of agonist potency was similar to that observed in mGluR1 and mGluR5 transfected cells. The phenylglycine antagonists tested were effective in blocking responses to 1-aminocyclopentane-1S,3R-dicarboxylic acid, but not to glutamate. Prolonged exposure to agonists induced a two-phase desensitization of mGluR5 function, an initial phase (completed by 1 h and plateaus for another 3 h) and a late phase (progressive decrease to approximately 30% of control levels by 24 h). Only the latter phase was associated with receptor down-regulation. Desensitization of mGluR5 function did not involve receptor internalization or phosphorylation mediated by protein kinase C or A; it was purely homologous, and reversible. Resensitization after short agonist treatment did not require prior receptor sequestration. Recovery after prolonged agonist exposure required new protein synthesis, but the restoration of function was more rapid than normalization of receptor protein levels, indicating that regulation also involves other components of the transduction system. The protracted desensitization of mGluR5 in astrocytes suggests that the functions mediated by this receptor are maintained under a variety of conditions ranging from repetitive stimulation to injury responses.

Animals↗
PubMed1997

Induction of DNA adducts and mutations in spleen, liver and lung of XPA-deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development.

We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild-type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA-deficient mice.

Animals↗
PubMed1997

Loss of Apc and the entire chromosome 18 but absence of mutations at the Ras and Tp53 genes in intestinal tumors from Apc1638N, a mouse model for Apc-driven carcinogenesis.

The Apc1638N mouse carries a targeted mutant allele at the endogenous adenomatous polyposis coli (Apc) gene and represents a unique in vivo model to study intestinal tumor formation and progression. Heterozygous Apc+/Apc1638N mice progressively develop 5-6 adenomas and adenocarcinomas of the small intestine within the first 6 months of life following a histologic sequence similar to that observed in human intestinal tumors. Here, we present the somatic mutation analysis of a total of 57 tumors. The results indicate that in > or = 75% of the lesions tested the wild type copy of the Apc gene is lost and that this LOH event extends to the entire mouse chromosome 18. Unexpectedly, mutations at the K-, N- and H-ras genes have not been found in these tumors. Immunohistochemical analysis of the Apc1638N tumors failed to detect accumulation of the Tp53 protein. Also, no mutations have been found in exons 7 and 8 of the Tp53 gene. These results indicate that, although the genetic inactivation of Apc is involved in the initiating event of the human as well as murine intestinal tumorigenesis, tumor growth and progression follow different mutational pathways in these two species.

Adenocarcinoma↗
PubMed1997

The role of UV-B light in skin carcinogenesis through the analysis of p53 mutations in squamous cell carcinomas of hairless mice.

Mutation spectra of the p53 gene from human skin carcinomas have been connected to solar UV radiation. For comparison we have characterized the mutation spectrum of the p53 gene in a very large sample of squamous cell carcinomas from hairless mice induced with UV of wavelength 280-320 nm (UV-B), which have substantiated the mutagenic effects of UV-B radiation in vivo. Tumors from hairless mice, random bred SKH:HR1 as well as inbred SKH:HRA strains, which are analyzed for mutations in the conserved domains of the p53 protein present a very specific mutation spectrum. The observed mutation frequency after chronic UV-B radiation in the p53 gene ranged from 54% (SKH-HRA) to 73% (SKH-HR1) among the 160 tumors analyzed. Over 95% of the mutations were found at dipyrimidine sites located in the non-transcribed strand, the majority were C-->T transitions and 5% were CC-->TT tandem double mutations. Four distinct UV-B mutation hot spots have been identified for the first time: two major ones at codons 267 (33%) and 272 (19%) and two minor ones at codons 146 (10%) and 173 (4%). The codon 267 hot spot consists of a CpG preceded by a pyrimidine, which confirms in vivo an important role for this UV-B mutable site in UV-B-induced skin tumors that is not found in other types of mouse tumors. Comparison with mutation spectra from human skin carcinomas fully validates the merits of the hairless mouse model for studying the molecular mechanisms of skin carcinogenesis. For example, the murine hot spot at codon 272 does have a full equivalent in human skin carcinomas. In contrast, the human equivalent of the murine codon 267 lacks the dipyrimidine site and therefore fails to be a pronounced hot spot in human skin carcinomas; however, this site is one of the major hot spots in human internal cancers (evidently not induced by UV radiation but probably by deamination of the 5 methyl cytosine).

Animals↗
PubMed1997

Use of E mu-PIM-1 transgenic mice short-term in vivo carcinogenicity testing: lymphoma induction by benzo[a]pyrene, but not by TPA.

E mu-pim-1 transgenic mice are predisposed to develop lymphomas. Due to their low spontaneous tumour incidence and their increased sensitivity towards the lymphomagen ethylnitrosourea these mice may present an interesting model for short-term carcinogenicity testing. Here, we report on the further exploration of this transgenic mouse model with two additional carcinogens known to have, among others, the lymphohaematopoietic system as target, i.e. benzo[a]pyrene (B[a]P) and 12-O-tetradecanoylphorbol-13-acetate (TPA). B[a]P, given three times a week (by gavage) for 13 weeks at 4.3, 13 or 39 mg/kg body weight, resulted in a dose-related increase in lymphomas up to a 90% incidence in E(mu)-pim-1 mice during the observation period of 40 weeks. B[a]P also induced tumours of the forestomach within this observation period, though at a lower incidence and apparently equally effective in wildtype and transgenic mice. TPA, on the other hand, was unable to induce lymphomas (or tumours in any other organ) in either transgenic or wildtype animals within the observation period of 44 weeks, when applied dermally at the maximum tolerated dose of 3 microg/mouse, twice a week for 35 weeks. Molecular analysis showed that B[a]P-induced lymphomas in transgenic mice were of T-cell origin, 80% of which had elevated levels of c-myc expression. None of the lymphomas had increased N-myc expression and mutation analysis of the ras-gene family revealed a K-ras mutation in only one out of eight tumours investigated. Also, none of the lymphomas showed aberrant expression of p53 as determined by immunohistochemistry. It is concluded that the E mu-pim-1 mouse model will not be very suitable for short-term carcinogenicity testing in general: only genotoxic chemicals that have the lymphohaematopoietic system as target for carcinogenesis in wild-type mice, appear to be efficiently identified.

Animals↗
PubMed1997

Species identification of Chlamydia isolates by analyzing restriction fragment length polymorphism of the 16S-23S rRNA spacer region.

The genetic diversity of the 16S-23S rRNA spacer region of 12 Chlamydia pneumoniae isolates, 7 Chlamydia trachomatis isolates (human biovars: the trachoma serovars B and C, the urogenital serovars D, E, and F, and the lymphogranuloma venereum serovar L2; and a mouse biovar), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and one Chlamydia pecorum isolate was studied. The 16S-23S rRNA spacer region was amplified by PCR and digested with the restriction enzymes MseI, PstI, AluI, BglII, NlaIV, and BclI. All 26 isolates could be amplified by using one genus-specific primer pair, yielding an amplicon with a size of 803 bp. The analytical sensitivity of the PCR assay was < or = 100 inclusion-forming units per reaction. Digestion with MseI or AluI made it possible to differentiate the four species from each other, the C. trachomatis human biovars from the mouse biovar, and the C. psittaci avian isolates from the feline isolate. The MseI profiles were more distinct than the AluI profiles. Phylogenetic analysis of the results for all enzymes combined supported the current classification of four Chlamydia species: C. pneumoniae, C. trachomatis, C. psittaci, and C. pecorum. Phylogenetic analysis also suggested subdivision of isolates of C. trachomatis and C. psittaci into subgroups that coincide with their host range. In conclusion, we have developed an easy and rapid method for species and subspecies identification of Chlamydia based on restriction fragment length polymorphism analysis of the 16S-23S rRNA spacer region.

Bacterial Typing Techniques↗
PubMed1997

Effect of the estrogen receptor locus on reproduction and production traits in four commercial pig lines.

We investigated the effect of the estrogen receptor (ESR) gene on growth and reproductive traits in four Large White-based commercial pig lines. A total of 9,015 litter records from 4,262 sows genotyped at the ESR locus were analyzed to determine whether ESR influenced total number born (TNB) or number born alive (NBA). Teat number (TN), test ADG, ADFI, feed:gain ratio (F/G), and ultrasonic backfat (BF) were also analyzed to determine effects of ESR. The TNB and NBA were increased per favorable allele of ESR (P < .01) with additive effects of .42 (.31) and .39 (.31) pigs/litter in the first parity (later parities), respectively. Dominance effects were near zero in parity one, but they were .16 and .14 pigs for TNB and NBA, respectively, in later parities (P < .05). A favorable additive pleiotropic effect was detected for BF (P < .001; -.11 mm per copy of the favorable litter size allele). There were no detectable effects on ADG or F/G (P > .10), although ADF was reduced 18 g/d per copy of the favorable litter size allele (P < .05). Average TN was 13.1 for pigs carrying the favorable litter size allele vs 13.2 for noncarriers (P < .05). Marker-assisted selection using ESR is warranted to increase litter size in the Large White-based lines considered here and will be of considerable economic value to pork producers.

Alleles↗
PubMed1997

Spontaneous liver tumors and benzo[a]pyrene-induced lymphomas in XPA-deficient mice.

Defects in the xeroderma pigmentosum complementation group A-correcting (XPA) gene, which encodes a component of the nucleotide excision repair (NER) pathway, are associated with the cancer-prone human disease xeroderma pigmentosum. We previously generated mice lacking the XPA gene, which develop normally but are highly sensitive to ultraviolet-B and 7,12-dimethylbenz[a] anthracene-induced skin tumors. Here we report that XPA-deficient mice spontaneously developed hepatocellular adenomas at a low frequency as they aged. Furthermore, oral treatment of XPA-deficient mice with the carcinogen benzo[a]pyrene (B[a]P) resulted in the induction of mainly lymphomas. These tumors appeared earlier and with a higher incidence than in B[a]P-treated wild-type and heterozygous mice. Our results show for the first time that XPA-deficient mice also displayed an increased sensitivity to developing tumors other than tumors of the skin.

Animals↗
PubMed1996

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Animals↗
PubMed1996

Xpa knockout mice.

The xeroderma pigmentosum group A correcting (XPA) gene encodes a DNA binding zinc-finger protein that recognizes DNA damage. As such the XPA protein participates in the initial step of the process of nucleotide excision repair. The multicomponent nucleotide excision repair pathway is one of the most thoroughly studied mechanisms that defends both eukaryotic and prokaryotic cells against the deleterious effects of UV-B and several chemical components. In the absence of nucleotide excision repair common cellular processes like transcription and replication are disturbed by persisting (unrepaired) DNA lesions (adducts), which may lead to the accumulation of gene mutations and ultimately to cancer. Xeroderma pigmentosum patients have a > 2000 fold increased risk to develop skin cancer at sun-exposed areas. Here we describe that XPA-deficient transgenic mice show features that mimic the phenotype found in humans. Furthermore, the possible use of Xpa- and other nucleotide excision repair deficient mice in cancer research will be outlined in more detail.

Animals↗
PubMed1996

Bimanual movement coordination in spastic hemiparesis.

A Fitts' task was used to examine whether the large movement asymmetry in subjects with spastic hemiparesis can be reduced or eliminated when both limbs are required to perform functionally equivalent tasks. Furthermore, it was determined whether any such benefit was expressed as mutual accommodation, or whether one hand "slaves" the other. Finally, the effect of increased task constraints on the magnitude of the asymmetry was considered. A group of ten students served as controls. Subjects had to grasp small balls and subsequently place them into holes. As expected, large total response time differences were present between the hands of the hemiparetic subjects in the unimanual conditions. However, 92% of the difference between hands was eliminated in the bimanual conditions. It is argued that the observed temporal invariance, or time locking, between hands in the bimanual conditions might be facilitated through the activity of bilateral controls exerted from each hemisphere and neural crosstalk at different levels of the central nervous system. Still, an asymmetric tendency remained in the bimanual conditions: a tendency existed for the impaired hand to reach the "target" later in time compared with the dominant hand. This tendency was enlarged as the asymmetry in task demands for the two limbs increased.

Adolescent↗
PubMed1996

Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma-infected Chlamydia sp. strains.

Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.

Base Sequence↗
PubMed1995

Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.

Xeroderma pigmentosum patients with a defect in the nucleotide-excision repair gene XPA are characterized by, for example, a > 1,000-fold higher risk of developing sunlight-induced skin cancer. Nucleotide-excision repair (NER) is involved in the removal of a wide spectrum of DNA lesions. The XPA protein functions in a pre-incision step, the recognition of DNA damage. To permit the functional analysis of the XPA gene in vivo, we have generated XPA-deficient mice by gene targeting in embryonic stem cells. The XPA-/-mice appear normal, at least until the age of 13 months. XPA-/-mice are highly susceptible to ultraviolet (UV)-B-induced skin and eye tumours and to 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumours. We conclude that the XPA-deficient mice strongly mimic the phenotype of humans with xeroderma pigmentosum.

9,10-Dimethyl-1,2-benzanthracene↗