View PDF Version - RePub - Erasmus Universiteit Rotterdam

More documents

Recommendations

Info

Death occurs after a short period of physical retardation, characterized by progressive weight loss and cachexia. Pathology of deceased mice failed to reveal gross abnOlmalities and thus did not provide a clue to a cornmon cause of death, During their life TID mice behaved quite normally, although repeatedly tremors were noted, that were not observed in wt littermates, hinting at some neurological deficit. We did not detect apparent CNS myelination abnormalities (analysis of the nervus opticus and nervus ischiaticus). Table 1. cysteine content and protein composition of wt and TTD hair wt TTD %ofwt total cysteine content lOa,b (14t 7 (8) 70 (57) IFd fraction 27 54 200 CRP fraction 66 39 59 HGT fraction 6 6 100 " All values represent percentages of total hair protein. b Values for cysteine content represent the mean of three hair samples. C Typical values found for control and TTD human hair (Kleijer et al., 1994) are indicated between brackets. d IF, inter-mediate filament keratins; CRP, cysteine-rich matrix Skin and hair abnormalities of TTD mice The hallmark of TTD is the striking brittleness of hair and nails, due to a reduced content of cysteine-rich matrix proteins, which crosslink the keratin filaments. Newborn TID mice develop a normal coat (Figure 3a), but spontaneous hair loss was first noted on the head around day 19 (Figure. 3b). Thereafter, a dramatic progressive loss of hair continued until! at the age of four weeks, TTD mice had lost most of their hair except for the whiskers and a small band of fur at the feet and tail (Figure 3c). The naked condition lasted until the normal second cycle of hair growth initiated, starting at the head region until a complete new coat was grown (Figure 3d). A few days later the hair loss began again and this cycle of hairloss and regrowth was maintained throughout their life. However, since hair cycle synchrony deteriorates later in life, hairloss became more patchy and not complete. To further examine the hair defect, we performed scauning electron microscopy on skin of 4 week old TID mice, when most hairs were shed. Figure 3e shows, that broken TID hair fibers terminate in brush ends and are (virtually) devoid of the typical cuticular structure of nOlmal hair. Histological examination of the skin revealed follicular plugging and dilation (data not shown). The reduction in cysteine-rich matrix proteins (CRP, made up for up to 40% of cysteines) has a significant impact on the overall cysteine content of TID hair. As apparent from Table 1, the cysteine content 72 Chapter 4
of hair of TTD mice (of both first and second hair cycle) has dropped to a level found in hair of TID patients. To detennine whether this was due to the reduced synthesis of the class of CRPs, 2D gel electrophoresis of radiolabeled protein extracts of wt and TTD hair was perfOlmed. As shown in Figure 3f and the quantitation in Table I, TTD hair has a substantially lower content of CRPs and a corresponding relative increase of the fraction of intennediate filament (IF) keratins. We conclude that TID mice exhibit the hallmark of the human disorder: brittle hair due to a selective deficiency in the synthesis of the class of crosslinking CRPs. The scanning micrograph also revealed skin surface abnormalities in TTD mice (Figure 3e). Macroscopic examination of the skin of TID mice showed ichthyosis (especially in the neck region), and a flexible, oversized and wizened appearance later in life. Histologic analysis (Figure 3g) indicated thickening of the layer of nucleated cells, in particular the granular layer (acanthosis) and a more prominent cornified layer (hyperkeratosis), pointing to an epidermal differentiation defec!. DNA repair characteristics of TTD mice To examine the effect of the XPD R722W mutation on DNA repair, various NER parameters were analyzed in mouse embryonic fibroblasts (MEFs) derived from TTD and wt embryos. UV survival (Figure 4a) shows that the D37 value of 4 independent TID MEF lines was approximately 1.5 times lower than wt MEFs (3.6 Jim' versus 5.6 Jim', respectively), comparable to fibroblasts of patient TIDIBEL (unpublished results). Similarly, TID MEFs showed a slight but significant hypersensitivity to DMBA, a chemical carcinogen that fmms bulky DNA adducts which are substrates for NER (data not shown). Furthennore, the level of UVinduced unscheduled DNA synthesis (UDS) of mouse TTD cells is strongly reduced, reaching only 25 % of wt cells (Figure 4a), comparable to TIDIBEL fibroblasts. To verify whether the partial repair defect exhibited by the cultured TTD mouse cells is also reflected in vivo, acute sensitivity of the mice to genotoxic agents was tested. DMBA was applied to the shaven back of wt, TTD and totally NER-deficient XPA knockout mice (de Vries et a!., 1995). After one DMBA application) XPA-deficient mice showed extensive hairloss, crust fOlmation and epidennal hyperplasia on the application site (Figure 4b). Remarkably, these dramatic acute effects were absent in the skin of TID (as well as wt) mice, even after two applications of 10 flg DMBA. In contrast to DMBA, exposure of TTD mice to increasing UV doses indicated an enhanced erythernic response (unpublished data). In conclusion, the XPD R722W mutation induces a partial NER defect, very similar to that of patient TIDIBEL. Enhanced UV- and DMBAinduced skin cancer susceptibility was noted, as will be reported seperately (de Boer et ai., manuscript in prep.) A mouse model for TID 73
Page 1 and 2:
A MOUSE MODEL FOR TRICHOTHIODYSTROP
Page 3 and 4:
CONTENTS Aim of the thesis 5 Chapte
Page 5:
AIM OF THE THESIS Nucleotide excisi
Page 9 and 10:
NUCLEOTIDE EXCISION REPAIR AND HUMA
Page 11 and 12:
capacity to generate bulky base add
Page 14 and 15:
strong interaction between XPG and
Page 16 and 17:
XP is characterized by genetic hete
Page 18:
are diagnosed as a collodion baby (
Page 22 and 23: 23. Gerard, M., Fischer, L., Moncol
Page 24 and 25: associatcd with mutations in the DN
Page 27: CHAPTER 2 MOUSE MODELS TO STUDY THE
Page 30: NER DEFICIENCY AND GENOTOXIC SENSIT
Page 34 and 35: XPC-I-p53+1- mice were more aggress
Page 36 and 37: tumor development was absent in a g
Page 38 and 39: [8, 9] reported substantial embryon
Page 41 and 42: in male sterility. As suggested by
Page 43: A point of concern in the extrapola
Page 47: CHAPTER 3 DISRUPTION OF THE MOUSE X
Page 50: INTRODUCTION To counteract the dele
Page 57: RAD3 and RAD25 protein (the latter
Page 63: CHAPTER 4 A MOUSE MODEL FOR THE BAS
Page 77: Table 3. Comparison of the TTDIBEL
Page 81 and 82: model (UDS, UV survival, pathology,
Page 84 and 85: 33. Stefanini, M., Giliani, S., Nar
Page 88 and 89: INTRODUCTION Genomic instability is
Page 91: correlates very well with literatur
Page 96 and 97: mice, which are histologically noml
Page 98 and 99: proneness in mice cOlTesponds with
Page 101 and 102: 18, Lehmann, A. R., C. F, Arlclt, B
Page 103: CHAPTER 6 SYMPTOMS OF PREMATURE AGI
Page 106 and 107: INTRODUCTION The genome is under co
Page 108 and 109: osteosclerosis, carly depigmentatio
Page 110: oocyte that showed gemlinal vesicle
Page 114 and 115: DISCUSSION Human aging syndromes Ag
Page 116 and 117: lower levels of the anti-oxidants s
Page 118: 6. Nance, M.A and Ben·y, S.A. (199
Page 121 and 122:
SUMMARY AND CONCLUSIONS DNA damage
Page 123 and 124:
features are due to an impairment o
Page 125:
symptoms in normal aging. Oxidative
Page 130 and 131:
In Hoofdstuk 5 worden experimenten
Page 132 and 133:
LIST OF PUBLICATIONS Bitter, W., va
Page 134 and 135:
DANKWOORD Het schrijven van een dan
show all

View PDF Version - RePub - Erasmus Universiteit Rotterdam

Create successful ePaper yourself

Delete template?

Save as template?