Multiple DNA damage recognition factors involved in mammalian nucleotide excision repair.

The nucleotide excision repair (NER) subpathway operating throughout the mammalian genome is a versatile DNA repair system that can remove a wide variety of helix-distorting base lesions. This system contributes to prevention of blockage of DNA replication by the lesions, thereby suppressing mutagenesis and carcinogenesis. Therefore, it is of fundamental significance to understand how the huge genome can be surveyed for occurrence of a small number of lesions. Recent studies have revealed that this difficult task seems to be accomplished through sequential actions of multiple DNA damage recognition factors, including UV-DDB, XPC, and TFIIH. Notably, these factors adopt completely different strategies to recognize DNA damage. XPC detects disruption and/or destabilization of the base pairing, which ensures a broad spectrum of substrate specificity for global genome NER. In contrast, UV-DDB directly recognizes particular types of lesions, such as UV-induced photoproducts, thereby vitally recruiting XPC as well as further extending the substrate specificity. After DNA binding by XPC, moreover, the helicase activity associated with TFIIH scans a DNA strand to make a final search for the presence of aberrant chemical modifications of DNA. The combination of these different strategies makes a crucial contribution to simultaneously achieving efficiency, accuracy, and versatility of the entire repair system.

HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro.

A protein complex which specifically complements defects of XP-C cell extracts in vitro was previously purified to near homogeneity from HeLa cells. The complex consists of two tightly associated proteins: the XPC gene product and HHR23B, one of two human homologs of the Saccharomyces cerevisiae repair gene product Rad23 (Masutani et al., EMBO J. 13:1831-1843, 1994). To elucidate the roles of these proteins in "genome-overall" repair, we expressed the XPC protein in a baculovirus system and purified it to near homogeneity. The recombinant human XPC (rhXPC) protein exhibited a high level of affinity for single-stranded DNA and corrected the repair defect in XP-C whole-cell extracts without extra addition of recombinant HHR23B (rHHR23B) protein. However, Western blot (immunoblot) experiments revealed that XP-C cell extracts contained excess endogenous HHR23B protein, which might be able to form a complex upon addition of the rhXPC protein. To investigate the role of HHR23B, we fractionated the XP-C cell extracts and constructed a reconstituted system in which neither endogenous XPC nor HHR23B proteins were present. In this assay system, rhXPC alone weakly corrected the repair defect, while significant enhancement of the correcting activity was observed upon coaddition of rHHR23B protein. Stimulation of XPC by HHR23B was found with simian virus 40 minichromosomes as well as with naked plasmid DNA and with UV- as well as N-acetoxy-2- acetylfluorene-induced DNA lesions, indicating a general role of HHR23B in XPC functioning in the genome-overall nucleotide excision repair subpathway.

Identification and characterization of XPC-binding domain of hHR23B.

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.

Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.

XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.

Retinoblastoma susceptibility protein, Rb, possesses multiple BRCT-Ws, BRCA1 carboxyl-terminus-related W regions with DNA break-binding activity.

The BRCT region, the carboxyl-terminus of BRCA1 (the breast cancer susceptibility gene 1 product), is ubiquitous in several proteins that participate in cell cycle checkpoints and DNA repair. We have previously shown that the BRCT regions of TopBP1 (DNA topoisomerase II binding protein 1) and BRCA1 bound DNA breaks. A BRCT-related region, BRCT-W1, in the retinoblastoma susceptibility gene product (Rb) also could bind DNA fragments, independently of DNA sequences. Five BRCT-W regions were found in the Rb family. All BRCT-Ws of Rb bound DNA fragments. Electron microscopy and treatment with an exonuclease showed that BRCT-Ws bound double-strand DNA breaks. Since some BRCTs are exceptional common relating elements in tumor suppression, our findings reveal novel aspects of the tumor suppression mechanism.

Abrogation of p53-mediated transactivation by SV40 large T antigen.

p53 is known to bind specifically to the 44-bp human DNA sequence in an immunoprecipitation assay. We show here that the transcription of the reporter CAT gene linked with the herpesvirus thymidine kinase (tk) promoter containing the 44-base sequence is enhanced by mouse wild-type but not mutant-type p53 in F9 and p53-null Saos-2 cells. The p53-mediated transactivation was dramatically abrogated by introduction of SV40 large T antigen (SVLT) in Saos-2 cells in which p53 was clearly associated with SVLT. Furthermore, the p53-SVLT complex did not bind to the 44-base sequence at all. Thus, SVLT sequesters the transactivation function of the wild-type p53 by inhibiting the binding of p53 to the 44-base sequence. This is good evidence to show 'loss of functions' in the product of a tumor-suppressor oncogene by a dominant oncogene product at a molecular level.

Risk factors for neurological complications after acoustic neurinoma radiosurgery: refinement from further experiences.

PURPOSE: Further actuarial analyses of neurological complications were performed on a larger population treated by stereotactic radiosurgery at our institution, to establish the optimal treatment parameters. METHODS AND MATERIALS: Between June 1990 and September 1998, 138 patients with acoustic neurinomas underwent stereotactic radiosurgery at Tokyo University Hospital. Of these, 125 patients who received medical follow-up for 6 months or more entered the present study. Patient ages ranged from 13 to 77 years (median, 53 years). Average tumor diameter ranged from 6.7 to 25.4 mm (mean, 13. 9 mm). Maximum tumor doses ranged from 20 to 40 Gy (mean, 29.8 Gy) and peripheral doses from 12 to 25 Gy (mean, 15.4 Gy). One to 12 isocenters were used (median, 4). Follow-up period ranged from 6 to 104 months (median, 37 months). The potential risk factors for neurological complications were analyzed by two univariate and one multivariate actuarial analyses. Neurological complications examined include hearing loss, facial palsy, and trigeminal nerve dysfunction. Variables included in the analyses were four demographic variables, two variables concerning tumor dimensions, and four variables concerning treatment parameters. A variable with significant p values (p < 0.05) on all three actuarial analyses was considered a risk factor. RESULTS: The variables that had significant correlation to increasing the risk for each neurological complication were: Neurofibromatosis Type 2 (NF2) for both total hearing loss and pure tone threshold (PTA) elevation; history of prior surgical resection, tumor size, and the peripheral tumor dose for facial palsy; and the peripheral tumor dose and gender (being female) for trigeminal neuropathy. In facial palsies caused by radiosurgery, discrepancy between the course of palsy and electrophysiological responses was noted. CONCLUSION: Risk factors for neurological complications seem to have been almost established, without large differences between institutions treating a large number of patients by radiosurgery. Radiosurgical doses and tumor dimensions were considered the two important risk factors for the 7th and 5th nerve injuries. Neurofibromatosis Type 2 was an important factor for hearing loss.

Analyses of neuro-otological complications after radiosurgery for acoustic neurinomas.

PURPOSE: To find out the optimum treatment parameters and the proper indications for treatment of acoustic neurinomas, univariate and multivariate actuarial analyses of neuro-otological complications after stereotactic radiosurgery for acoustic neurinomas were performed. METHODS AND MATERIALS: The subjects were 46 patients with acoustic neurinomas who underwent unilateral radiosurgery between June 1990 and June 1994 and were followed up at the University of Tokyo. Age ranged from 13 to 77 years (median, 54 years). Tumor diameter ranged from 0 to 25 mm (mean, 12 mm) at the cerebellopontine angle and from 2 to 15 mm (mean, 8.3 mm) in the internal auditory meatus. Maximum tumor doses ranged from 20 to 40 Gy (mean, 31.4 Gy), and peripheral doses from 12 to 25 Gy (mean, 16.8 Gy). One to eight isocenters were used (mean, 3.2). Median follow-up was 39 months. Eight events concerning neuro-otological complications were chosen, and the potential risk factors for them were analyzed by the actuarial analyses (univariate and multivariate). The events examined include hearing loss, vestibular function loss, facial palsy, and trigeminal nerve dysfunction. In order to point out potential risk factors for neuro-otological complications, univariate analyses were performed using both the Wilcoxon test and the log rank test, and multivariate analyses were performed with the Cox proportional hazards model. Variables nominated as potential risk factors were 1) demographic variables such as patient age and sex, 2) tumor dimensions, 3) treatment variables such as tumor doses and number of isocenters, and 4) pretreatment hearing levels. A variable with significant p-values (p < 0.05) in two or more of the three actuarial analyses (two univariate and one multivariate) was considered a possible risk factor. RESULTS: The possible variables that increase the risk for each event analyzed were: neurofibromatosis type II (NF2) and the number of isocenters for total hearing loss; experience of prior operation, the tumor diameter in the internal auditory meatus, and NF2 for hearing threshold elevation; peripheral tumor dose for vestibular function loss; patient age or midporus transverse tumor diameter (the two variables were correlated), and the number of isocenters for facial palsy; and the number of isocenters for trigeminal neuropathy. CONCLUSION: NF2 and the tumor diameter were the common risk factors for hearing loss in previous studies and ours. For the 5th/7th nerve dysfunction, the tumor diameter was the common risk factor. The risk of using more isocenters remains controversial. The difference in risk factors for hearing impairment and vestibular function loss suggests different mechanisms for the two. Further studies with larger populations and longer follow-up periods are required in order to draw conclusions on the risk factors in radiosurgery.

Acidification of phagosomes and degradation of rod outer segments in rat retinal pigment epithelium.

PURPOSE: The authors investigated the phagocytic processes of the rod outer segments (ROS) in rat retinal pigment epithelium (RPE) cells, and the appearance of lysosomal enzymes, acidification, and degradation of the contents in the phagolysosomes. In particular, they examined the effect of bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, on the degradation of ROS in the RPE cells in vivo. METHODS: A lysosomal enzyme (cathepsin D), a lysosomal membrane protein (LGP107), and opsin were localized in the RPE cells by the immunogold electron microscopic technique. Bafilomycin A1 was injected into the vitreous, and acidification of the phagosomes was measured in vivo by injecting 3-[2,4-dinitroanilino]3'amino-N-methyldipropylamine (DAMP) in the vitreous and detecting the accumulation of DAMP in the phagolysosomes using anti-dinitrophenol antibody. RESULTS: Opsin was abundantly detected in phagosomes that did not contain cathepsin D, but the immunolabeling of opsin rapidly disappeared soon after the appearance of cathepsin D. By double staining with cathepsin D and DAMP, it was shown that the pH of the phagosomes dramatically decreased after fusion with lysosomes. When bafilomycin A1 was injected into the vitreous, many large phagolysosomes containing cathepsin D appeared in the RPE cells, in which the immunoreactivity of opsin was well preserved. CONCLUSIONS: Degradation of opsin and acidification proceeded almost parallel with the appearance of cathepsin D in the phagolysosomes. Bafilomycin A1 did not inhibit the fusion of phagosomes with lysosomes, but it increased intraphagosomal pH and markedly inhibited the degradation of ROS in the phagolysosomes. This result indicates that vacuolar-type H(+)-ATPase is essential for acidifying the lumen of phagolysosomes and subsequent protein degradation of ROS in the RPE cells.

Characterization of the 100,000 dalton material produced by chromatin cross-linking reaction with dithio-bis(succinimidyl propionate).

A chemical cross-linking reagent, dithio-bis(succinimidyl propionate), is known to be capable of cross-linking histones in nucleosomes so as to give one major product with the molecular weight of about 100,000. Because the product has been supposed to represent the cross-linked histone octamer, the reaction has been used for studying the movements of core histones in nucleosomes. However, the precise protein composition of the product has not been determined thus far, so that the use of the reaction was limited. We report here that the 100 kilodalton product is composed of the core histones, and does not contain significant amounts of any other proteins. Moreover, quantitative analysis of the content of each core histone confirmed that the four types of core histones participate in the product with an equal molar ratio. As one can specifically observe the behaviors of histone octamers with this reaction, it should be useful for research in various fields related to the dynamics and functions of the nucleosome.

Changes in electrovestibular brainstem responses after aminoglycoside intoxication in guinea pigs.

OBJECTIVE: To verify the usefulness of short-latency vestibular responses evoked by a combination of round window electrical stimulation and sinusoidal rotation (electrovestibular brainstem responses; EVBRs) as a new monitoring tool of the vestibular function in animal experiments. METHODS: EVBRs were obtained before, during, and after treatment with aminoglycosides, along with compound action potential (CAP) audiograms. The changes in EVBRs were compared with morphological changes observed by scanning electron microscopy. RESULTS: EVBR amplitudes did not change in the group of guinea pigs treated with amikacin, but markedly decreased in the streptomycin and gentamicin- treated groups. CAP audiograms indicated a significant threshold elevation in the amikacin group, a moderate elevation in the gentamicin group, and no change in the streptomycin group. Under scanning electron microscopy, the loss of the sensory hair cells observed in the cristae ampullares was slight to moderate in the amikacin group, moderate to severe in the streptomycin group, and severe in the gentamicin group. CONCLUSION: EVBRs reflect overall pathological changes undergone by vestibular hair cells, and support the vestibular specificity of EVBRs.

Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro.

The XPC-HR23B complex, a mammalian factor specifically involved in global genomic nucleotide excision repair (NER) has been shown to bind various forms of damaged DNA and initiate DNA repair in cell-free reactions. To characterize the binding specificity of this factor in more detail, a method based on immunoprecipitation was developed to assess the relative affinity of XPC-HR23B for defined lesions on DNA. Here we show that XPC-HR23B preferentially binds to UV-induced (6-4) photoproducts (6-4PPs) as well as to cholesterol, but not to the cyclobutane pyrimidine dimer (CPD), 8-oxoguanine (8-oxo-G), O6-methylguanine (O6-Me-G), or a single mismatch. Human whole cell extracts could efficiently excise 6-4PPs and cholesterol in an XPC-HR23B-dependent manner, but not 8-oxo-G, O6-Me-G or mismatches. Thus, there was good correlation between the binding specificity of XPC-HR23B for certain types of lesion and the ability of human cell extracts to excise these lesions, supporting the model that XPC-HR23B initiates global genomic NER. Although, XPC-HR23B does not preferentially bind to CPDs, the excision of CPDs in human whole cell extracts was found to be absolutely dependent on XPC-HR23B, in agreement with the in vivo observation that CPDs are not removed from the global genome in XP-C mutant cells. These results suggest that, in addition to the excision repair pathway initiated by XPC-HR23B, there exists another sub-pathway for the global genomic NER that still requires XPC-HR23B but is not initiated by XPC-HR23B. Possible mechanisms will be discussed.

Reconstitution of damage DNA excision reaction from SV40 minichromosomes with purified nucleotide excision repair proteins.

We previously constructed the cell-free nucleotide excision repair (NER) assay system with UV-irradiated SV40 minichromosomes to analyze the mechanism of NER reaction on chromatin DNA. Here we investigate the factor that acts especially on nucleosomal DNA during the damage excision reaction, and reconstitute the damage excision reaction on SV40 minichromosomes. NER-proficient HeLa whole cell extracts were fractionated, and the amounts of known NER factors involved in the column fractions were determined by immunoblot analyses. The column fractions were quantitatively and systematically replaced by highly purified NER factors. Finally, damage DNA excision reaction on SV40 minichromosomes was reconstituted with six highly purified NER factors, XPA, XPC-HR23B, XPF-ERCC1, XPG, RPA and TFIIH, as those essential for the reaction with naked DNA. Further analysis showed that the damages on chromosomal DNA were excised as the same efficiency as those on naked DNA for short incubation. At longer incubation time, however, the damage excision efficiency on nucleosomal DNA was decreased whereas naked DNA was still vigorously repaired. These observations suggest that although the six purified NER factors have a potential to eliminate the damage DNA from SV40 minichromosomes, the chromatin structure may still have some repressive effects on NER.

Postnatal development of the masseter muscles in the Japanese field vole Microtus montebelli, with special attention to differentiation of the fast-twitch oxidative fiber.

Postnatal development and differentiation of the masseter muscles consisting only of fast-twitch oxidative (FO) fibers in the adult Japanese field vole Microtus montebelli were studied using histochemical and electron microscopic techniques. The masseter muscles were composed of myotubes and muscle fibers at day 0 (birth day). Most muscle cells showed the strong reaction for myosin ATPase after both alkaline and acid preincubations. For NADH-dehydrogenase (NADH-DH), small granular diformazan deposits were recognized in the sarcoplasm. Afterwards, the masseter muscles consisted of myofibers and satellite cells at day 5. For myosin ATPase, weakly-reactive fibers after acid preincubation (fast-twitch fibers) increased in number. For NADH-DH, granular diformazan deposits in all the myofibers increased in size. Since all the myofibers had numerous sarcoplasmic reticula, and they reacted strongly after alkaline preincubation and weakly after acid preincubation for myosin ATPase at day 10 when the young start to take solid food, it seems that the masseter muscles become contractive fast. At day 15 (before weaning), all the myofibers showed the adult-like strong reaction for NADH-DH and had numerous well-developed mitochondria, thus they acquired the ability of the fast and sustained contraction. It is accordingly considered that the masseter muscles of the vole mature in a short time after birth because of adaptation for herbivorous food habit.

Effects of a soft diet and hypothyroidism on the oxidative capacity of the masseter muscle fibers of the young Japanese field vole Microtus montebelli.

Effects of a soft diet (reduction in mastication activity: an exogenous factor) and hypothyroidism (endogenous factors) on the oxidative capacity of the masseter muscles in the young Japanese field vole Microtus montebelli, consisting only of fast-twitch oxidative (FO) fibers in the adult vole, was investigated histochemically and electron microscopically. Oxidative enzyme activity and mitochondrial development in the muscle fibers were not affected by a soft diet, while they were suppressed by doses of propylthiouracil (PTU) (hypothyroidism). Thus, it was suggested that acquisition of the sustained contraction ability in the masseter muscles of the young vole is induced by the endogenous factors rather than the exogenous factor.

Neuro-otological findings after radiosurgery for acoustic neurinomas.

OBJECTIVE: To evaluate the neuro-otological complications in patients after radiosurgery for acoustic neurinomas. DESIGN: Inception cohort, retrospective study. SETTING: University hospital. PATIENTS: A consecutive sample of 46 patients with acoustic neurinomas who underwent unilateral gamma knife radiosurgery at the University of Tokyo, Japan, between June 1990 and June 1994 were followed up by otolaryngologists for more than 3 months. INTERVENTION: Gamma knife stereotactic radiosurgery. MAIN OUTCOME MEASURES: Neuro-otological examinations including pure tone audiometry, auditory brain stem response, and caloric test. RESULTS: Tumor growth occurred in 2 patients (4.3%). Seven (18%) of the 38 patients with preserved hearing of any extent became deaf within 1 year. In cases of gradual hearing loss, the average deterioration rate was approximately 8 dB per year. Abnormalities of auditory brain stem response preceded deafness in 2 patients. Caloric response, preserved before treatment in 13 patients, disappeared 4 to 13 months after treatment (median, 8 months) in 9 (69%) of them, whereas their hearing was preserved. Delayed facial palsy and persistent trigeminal neuropathy occurred in 10 (22%) and 7 (15%) of the 46 patients, respectively. Severe facial palsy tended to persist. CONCLUSIONS: The rates of neuro-otological complications of radiosurgery are almost comparable with those previously reported from other institutions. The deafness within 1 year after treatment might be attributed to a lesion in the cochlear nerve. Hearing loss did not parallel vestibular function loss. The persistent severe facial palsy contrasts with previously reported findings. Considering the serious facial nerve complications that occurred in some of our patients, further study to disclose the risk factors for neurological dysfunction would be needed for radiosurgery to become a true, safe alternative to microsurgery.

[Effects of corticosteroid on porcine retinal pigment epithelial cells in culture--2. Effects on phagocytosis and lysosomal activity].

PURPOSE: The effects of a corticosteoid on the phagocytosis and lysosomal activity of cultured porcine retinal pigment epithelium (RPE) cells were investigated. METHODS: After exposing cultured RPE cells to various concentrations (10, 50, 100, 500, 1,000 nM) of betamethasone sodium phosphate (betamethasone), the cells were incubated with latex microspheres for 6 hours. RESULTS: The number of latex microspheres phagocytized by the cultured RPE cells was inhibited by 50 nM betamethasone within 24 hours. Ten-nM betamethasone did not inhibit the proliferation of cultured RPE cells, but ingestion of latex microspheres by the cells was inhibited after 3 days. Lysosomal activity (acid phosphatase, beta-glucuronidase) of RPE cells was inhibited by a high concentration (500 nM) of betametasone. CONCLUSION: These results suggest that corticosteroid inhibits the phagocytosis and lysosomal activity of cultured RPE cells.

[Effects of corticosteroid on porcine retinal pigment epithelial cells in culture--1. Inhibitory effect on cell proliferation].

PURPOSE: Proliferation is an important function of the retinal pigment epithelial (RPE) cells. The effect of a corticosteroid on the proliferation of cultured porcine RPE cells was investigated. METHODS: After administration of various concentrations (10-1,000 nM) of betamethasone sodium phosphate (betamethasone), we counted RPE cell numbers at 1, 3, 6, 9, and 14 days. RESULTS: Betamethasone administration resulted in a dose-dependent decrease in RPE cell proliferation. The proliferation of the cultured RPE cells was significantly inhibited by betamethasone, at a dose of 300 nM in 9 days. Ten-nM betamethasone neither inhibited nor promoted the RPE cell proliferation in culture. CONCLUSIONS: These results suggest that corticosteroids inhibit proliferation of cultured porcine RPE cells.

[Immunohistological study in Bruch's membrane of senescence accelerated mouse].

Age-related macular degeneration is one of the major causes of severe visual loss is elderly individuals. However, relatively little is known about its etiology. The disease may be associated with senescence. Ultrastructural and immunohistochemical studies on SAM (senescence accelerated mouse) eyes were carried out to learn details of aging changes in the retinal pigment epithelium (RPE) and Bruch's membrane. SAM P 1 mice aged 2, 10, 14 months were examined in this study. The eyes were analysed for type IV collagen and heparan sulfate proteoglycan (HSPG) by the avidin-biotin-peroxidase complexes (ABC) method and post-embeddig immunolocalization with colloidal gold. With the ABC method, the basement membranes of both the RPE and the choriocapillaris showed markedly positive staining when treated with anti-type IV collagen antibody and moderately positive staining when treated with anti-HSPG antibody. In ultrastructural immunolocalization, both basement membranes showed fairly heavy labeling in response to the antibodies to type IV collagen, and moderate labeling in response to the antibodies to HSPG. With aging, the thickness of the basement membrane of the choriocapillaris and gold particle labeling by the antibodies to type IV collagen increased. The gold particle labeling by the antibodies to HSPG increased slightly, but was distributed sparsely. These results showed the advancing process of senescence changes in Bruch's membrane.