Evaluation of serum IgE in peach-allergic patients with systemic reaction by using recombinant Pru p 7 (gibberellin-regulated protein)

BACKGROUND: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. METHODS: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). RESULTS: Peach-allergic patients (n = 27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n = 17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P<0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P < 0.05). CONCLUSIONS: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction

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Evaluation of serum IgE in peach-allergic patients with systemic reaction by using recombinant Pru p 7 (gibberellin-regulated protein).

BACKGROUND: Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy. METHODS: Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7). RESULTS: Peach-allergic patients (n=27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n=17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P<0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P<0.05). CONCLUSIONS: We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction.

A multi-throughput multi-organ-on-a-chip system on a plate formatted pneumatic pressure-driven medium circulation platform.

This paper reports a multi-throughput multi-organ-on-a-chip system formed on a pneumatic pressure-driven medium circulation platform with a microplate-sized format as a novel type of microphysiological system. The pneumatic pressure-driven platform enabled parallelized multi-organ experiments (i.e. simultaneous operation of multiple multi-organ culture units) and pipette-friendly liquid handling for various conventional cell culture experiments, including cell seeding, medium change, live/dead staining, cell growth analysis, gene expression analysis of collected cells, and liquid chromatography-mass spectrometry analysis of chemical compounds in the culture medium. An eight-throughput two-organ system and a four-throughput four-organ system were constructed on a common platform, with different microfluidic plates. The two-organ system, composed of liver and cancer models, was used to demonstrate the effect of an anticancer prodrug, capecitabine (CAP), whose metabolite 5-fluorouracil (5-FU) after metabolism by HepaRG hepatic cells inhibited the proliferation of HCT-116 cancer cells. The four-organ system, composed of intestine, liver, cancer, and connective tissue models, was used to demonstrate evaluation of the effects of 5-FU and two prodrugs of 5-FU (CAP and tegafur) on multiple organ models, including cancer and connective tissue.

Usefulness of dyssynchrony indices based on two-dimensional speckle tracking echocardiography in a canine model of left bundle branch block.

INTRODUCTION: Determine the usefulness of dyssynchrony indices derived from two-dimensional speckle tracking echocardiography for the detection of mechanical dyssynchrony in a canine model of left bundle branch block. ANIMALS: Ten healthy beagles. MATERIALS AND METHODS: Segmental, time-radial strain curves were obtained using two-dimensional speckle tracking echocardiography. The maximum difference and standard deviation of the time to peak radial strain for six predefined segments (MaxD-TpSR and 6SD-TpSR) were calculated, together with the left ventricular dyssynchrony by radial strain (DysSR), before and after ablation of the left bundle branch block. Receiver operating characteristic curve analysis was performed using dogs after ablation as positive controls. RESULTS: After ablation, all dogs showed multiple peaks in at least one segment on the time-radial strain curve, while all dyssynchrony indices increased significantly (MaxD-TpSR from 16.25 ± 16.04 [mean ± standard deviation] to 44.4 ± 26.18 ms, 6SD-TpSR from 7.59 ± 7.40 to 19.62 ± 11.91 ms, and DysSR from 4.20 ± 2.12 to 10.87± 2.92%, p<0.05). In receiver operating characteristic curve analysis, the areas under the curve for MaxD-TpSR, 6SD-TpSR, and DysSR were 0.825, 0.800, and 0.980, respectively. CONCLUSIONS: Left ventricular dyssynchrony by radial strain can detect mechanical dyssynchrony with higher sensitivity and specificity than dyssynchrony indices, based on the time to peak radial strain.

A pneumatic pressure-driven multi-throughput microfluidic circulation culture system.

Here, we report a pneumatic pressure-driven microfluidic device capable of multi-throughput medium circulation culture. The circulation culture system has the following advantages for application in drug discovery: (i) simultaneous operation of multiple circulation units, (ii) use of a small amount of circulating medium (3.5 mL), (iii) pipette-friendly liquid handling, and (iv) a detachable interface with pneumatic pressure lines via sterile air-vent filters. The microfluidic device contains three independent circulation culture units, in which human umbilical vein endothelial cells (HUVECs) were cultured under physiological shear stress induced by circulation of the medium. Circulation of the medium in the three culture units was generated by programmed sequentially applied pressure from two pressure-control lines. HUVECs cultured in the microfluidic device were aligned under a one-way circulating flow with a shear stress of 10 dyn cm(-2); they exhibited a randomly ordered alignment under no shear stress and under reciprocating flow with a shear stress of 10 dyn cm(-2). We also observed 2.8- to 4.9-fold increases in expression of the mRNAs of endothelial nitric oxide synthase and thrombomodulin under one-way circulating flow with a shear stress of 10 dyn cm(-2) compared with conditions of no shear stress or reciprocating flow.

The human ether-a-go-go-related gene (hERG) current inhibition selectively prolongs action potential of midmyocardial cells to augment transmural dispersion.

The majority of drug induced arrhythmias are related to the prolongation of action potential duration following inhibition of rapidly activating delayed rectifier potassium current (I(Kr)) mediated by the hERG channel. However, for arrhythmias to develop and be sustained, not only the prolongation of action potential duration but also its transmural dispersion are required. Herein, we evaluated the effect of hERG inhibition on transmural dispersion of action potential duration using the action potential clamp technique that combined an in silico myocyte model with the actual I(Kr) measurement. Whole cell I(Kr) current was measured in Chinese hamster ovary cells stably expressing the hERG channel. The measured current was coupled with models of ventricular endocardial, M-, and epicardial cells to calculate the action potentials. Action potentials were evaluated under control condition and in the presence of 1, 10, or 100 µM disopyramide, an hERG inhibitor. Disopyramide dose-dependently increased the action potential durations of the three cell types. However, action potential duration of M-cells increased disproportionately at higher doses, and was significantly different from that of epicardial and endocardial cells (dispersion of repolarization). By contrast, the effects of disopyramide on peak I(Kr) and instantaneous current-voltage relation were similar in all cell types. Simulation study suggested that the reduced repolarization reserve of M-cell with smaller amount of slowly activating delayed rectifier potassium current levels off at longer action potential duration to make such differences. The action potential clamp technique is useful for studying the mechanism of arrhythmogenesis by hERG inhibition through the transmural dispersion of repolarization.

Utilizing stomach content and faecal DNA analysis techniques to assess the feeding behaviour of largemouth bass Micropterus salmoides and bluegill Lepomis macrochirus.

In this study, the feeding behaviour of the non-native invasive predatory fishes largemouth bass Micropterus salmoides and bluegill Lepomis macrochirus was studied in the Ezura River, a northern tributary of Lake Biwa, Japan. Prey composition was estimated based on visual examination of stomach contents and faecal DNA analysis to determine feeding habits of these predatory fishes. Stomach content analysis showed that native fishes (e.g. ayu Plecoglossus altivelis and gobies Rhinogobius spp.) and shrimps (e.g. Palaemon paucidens) were the major prey items for M. salmoides, while snails, larval Chironomidae and submerged macrophytes were the dominant prey items of L. macrochirus. Micropterus salmoides tended to select larger fish in the case of crucian carp Carassius spp., but smaller fishes in the case of P. altivelis and Rhinogobius spp. Faecal DNA analyses revealed prey compositions similar to those identified in predator stomach contents, and identified additional prey species not detected in stomach content inspection. This study demonstrated that both stomach content inspection and DNA-based analysis bear several inherent shortcomings and advantages. The former method is straightforward, although identification of species can be inaccurate or impossible, whereas the latter method allows for accurate species identification, but cannot distinguish prey size or stage. Hence, integration of morphology-based and DNA-based methods can provide more reliable estimates of foraging habits of predatory fishes.

Polymorphisms in genes involved in the free-radical process in patients with sudden sensorineural hearing loss and Ménière's disease.

The etiologies of idiopathic sudden sensorineural hearing loss (SSNHL) and Ménière's disease remain unclear. Recently, accumulating evidence has demonstrated that free radicals are related to the pathology of inner ear disease. Because genetic factors may contribute partly to the etiologies of SSNHL and Ménière's disease, we investigated the association between genetic polymorphisms located in genes related to the free-radical process and susceptibility to SSNHL and Ménière's disease. We compared 83 patients affected by SSNHL and 83 patients affected by Ménière's disease with 2048 adults (for SSNHL) and 1946 adults (for Ménière's disease) who participated in the National Institute for Longevity Sciences, Longitudinal Study of Aging. Multiple logistic regression was used to calculate odds ratios (ORs) for SSNHL and Ménière's disease in individuals with polymorphisms in the genes: methionine synthase (MTR; rs1805087); methionine-synthase reductase (MTRR; rs1801394); nitric oxide synthase 3 (NOS3; rs1799983); caveolin 1 (Cav1; rs3840634); melatonin receptor 1B (MTNR1B; rs1387153); NAD(P)H oxidase p22(phox) subunit (NADH/NADPHp22phox; rs4673); and mitochondria 5178 (MT5178; rs28357984). The NOS3 polymorphism was significantly associated with a risk of SSNHL; in addition, the OR for the NOS3 polymorphism and SSNHL risk was 2.108 (CI, 1.343-3.309) with adjustment for age and sex. The Cav1 polymorphism was significantly associated with a risk of Ménière's disease; moreover, the OR for the Cav1 polymorphism and Ménière's disease risk was 1.849 (CI, 1.033-3.310) with adjustment for age and sex. In conclusion, the NOS3 and Cav1 polymorphisms were significantly associated with the risk of SSNHL and Ménière's disease, respectively.

Immunosuppressive effect of prolactin-induced protein.

Prolactin-induced protein (PIP) has been shown to bind to CD4 and is speculated to block CD4-HLA-DR interaction. However, the immunomodulatory effect of PIP on chronic allergic contact dermatitis (ACD) remains to be elucidated. The aim of this work was to define the role of PIP during the immunoresponse. Using an oxazolone-induced mouse chronic ACD model, expression of PIP was immunohistologically examined. Furthermore, effects of continued exposure of a peptide mimicking the major binding site of PIP (amino acids 106-132) for CD4 was examined in a mouse chronic ACD model. We clarified that keratinocytes and dermal infiltrating cells are positively stained with anti-PIP antibody. The PIP peptide significantly downregulated oxazolone-induced mouse ACD compared to the controls. We also found that inflammation of PIP-non-applied control ear was also suppressed in a synchronized manner in the late phase of the PIP peptide applied mouse. These findings suggest that PIP might have an immunosuppressive effect in mouse chronic ACD.

Simultaneous combined intravenous recombinant tissue plasminogen activator and endovascular therapy for hyperacute middle cerebral artery m1 occlusion.

We investigated the efficacy and safety of combined intravenous (IV) recombinant tissue plasminogen activator (rtPA) and simultaneous endovascular therapy (ET) for hyperacute middle cerebral artery (MCA) M1 occlusion. Between October 2005 and April 2007, in the combined group, 22 patients eligible for IV rtPA, who were diagnosed as having MCA M1 occlusion, were treated with IV rtPA and simultaneous ET was initiated as soon as possible. The other patients were treated with IV rtPA alone (IV group A: n = 11). Between May 2007 and November 2008, all patients eligible for IV rtPA, who were diagnosed as having MCA M1 occlusion, underwent thrombolysis by IV rtPA alone (IV group B: n = 24). The improvement of the National Institutes of Health Stroke Scale score at 24 hours was highest in the combined group (10 ± 4.1). In contrast, it was 5.1 ± 4.7 in the IV group A (P = 0.017) and 5.6 ± 5.6 in IV group B (P = 0.006). In the combined group, successful recanalization was observed in 18 of 22 patients with one symptomatic intracranial hemorrhage. The rate of mRS0-2 at three months was highest in the combined group, 36% in the IV group A and 33% in the IV group B (P = 0.008).Simultaneous treatment with IV rtPA and ET improved the clinical outcome of MCA M1 occlusion without a significant increase of adverse effects in our study.

Association of interleukin-1 gene polymorphisms with sudden sensorineural hearing loss and Ménière's disease.

Sudden sensorineural hearing loss (SSNHL) and Ménière's disease are the most common inner ear diseases in which the causes are unknown. As recent magnetic resonance imaging has demonstrated disruption of the blood-labyrinth barrier in these inner ear diseases, inflammatory reaction associated with increased permeability of the blood vessels may be involved. The genotypes of interleukin 1A (IL1A) (-889C/T; rs1800587) and interleukin 1B (IL1B) (-511C/T; rs16944) were determined using an allele-specific primer-polymerase chain reaction method in 72 patients with SSNHL, 68 patients with Ménière's disease, and 2202 control subjects living almost in the same area as the patients. A significantly higher prevalence of the IL1A-889T allele was observed in SSNHL and Ménière's disease compared with controls, although no significant difference in distribution of IL1B-511C/T genotypes was observed between the patients and controls. Adjusted odd ratios for SSNHL and Ménière's disease risks in the -889TT genotypes were 25.89 (95% confidence interval (CI) 12.19-54.98) and 18.20 (95% CI 7.80-42.46), respectively, after age and gender were taken as moderator variables. Our results suggested that IL1A is closely associated with susceptibility of SSNHL and Ménière's disease.

Immunosuppressive effect of prolactin-induced protein: a new insight into its local and systemic role in chronic allergic contact dermatitis.

BACKGROUND: Prolactin-induced protein (PIP) has been shown to bind to CD4 and is speculated to block CD4-HLA-DR interaction. However, the immunomodulatory effect of PIP on chronic allergic contact dermatitis (ACD) remains to be elucidated. OBJECTIVES: To define the role of PIP during the immunoresponse. METHODS: Using a low-dose oxazolone-induced mouse chronic ACD model, expression of PIP was examined immunohistologically. Furthermore, effects of continued exposure to a peptide mimicking the major binding site of PIP (amino acids 106-132) for CD4 was examined in a mouse chronic ACD model. RESULTS: We clarified that keratinocytes, dermal infiltrating cells and spleen infiltrating mononuclear cells are positively stained with anti-PIP antibody. The PIP peptide significantly downregulated oxazolone-induced mouse ACD compared with controls. We also found that inflammation of the control ear, to which the PIP peptide had not been applied, was also suppressed in a synchronized manner in the late phase of ACD. CONCLUSIONS: These findings suggest that PIP might have a local and systemic immunosuppressive effect in mouse chronic ACD.

Simultaneous treatment with intravenous recombinant tissue plasminogen activator and endovascular therapy for acute ischemic stroke within 3 hours of onset.

BACKGROUND AND PURPOSE: Because intravenous (IV) recombinant tissue plasminogen activator (rtPA) does not always lead to a good outcome in a considerable proportion of patients, combined IV rtPA and rescue endovascular therapy (ET) have been performed in several recent studies. However, rescue therapy after completion of IV rtPA often results in late ineffective recanalization. We examined the efficacy and safety of combined IV rtPA and simultaneous ET as primary rather than rescue therapy for hyperacute middle cerebral artery (MCA) occlusion. MATERIALS AND METHODS: A total of 29 patients eligible for IV rtPA, who were diagnosed as having MCA (M1 or M2) occlusion within 3 hours of onset, underwent thrombolysis. In the combined group, patients were treated by IV rtPA (0.6 mg/kg for 60 minutes) and simultaneous ET (intra-arterial rtPA, mechanical thrombus disruption with microguidewire, and balloon angioplasty) initiated as soon as possible. In the IV group, patients were treated by IV rtPA only. RESULTS: The improvement of the National Institutes of Health Stroke Scale (NIHSS) score at 24 hours was 11 +/- 4.8 in the combined group versus 5 +/- 4.3 in the IV group (P < .001). In the combined group, successful recanalization was observed in 14 (88%) of 16 patients with no symptomatic intracranial hemorrhage, and 10 (63%) of 16 patients had favorable outcomes (modified Rankin Scale [mRS] 0, 1) at 3 months. CONCLUSIONS: Aggressive combined therapy with IV rtPA and simultaneous ET markedly improved the clinical outcome of hyperacute MCA occlusion without significant adverse effect. Additional randomized study is needed to confirm our results.

Salmonid microarrays identify intestinal genes that reliably monitor P deficiency in rainbow trout aquaculture.

Nutrient-responsive genes can identify important metabolic pathways and evaluate optimal dietary levels. Using a 16K Salmo salar microarray, we identified in rainbow trout (Oncorhynchus mykiss) 21 potential phosphorus (P)-responsive genes, mainly involved in immune response, proteolysis or transport, whose expression levels changed in the intestine after 5 days of feeding a low-P (LP) diet. Diet-induced changes in the expression levels of several genes in each fish were tightly correlated with changes in serum P, and the changes persisted for an additional 15 days after dietary P deficiency. We then evaluated these and previously identified P-responsive genes under simulated farm conditions, and monitored the intestinal gene expression from 6 h to 7 days after the trout were switched from a sufficient-P (SP) diet to a LP diet (SP-->LP), and from a LP diet to a SP diet (LP-->SP). After 7 days, mean serum P decreased 0.14 mM/day for SP-->LP and increased 0.10 mm/day for LP-->SP. The mRNA abundance of the metalloendopeptidase meprin 1alpha (MEP1alpha), the Na(+)-dependent phosphate co-transporter (NaPi2b,SLC34A2), the sulfotransferase SULT2beta1 and carbonic anhydrase XIII genes all increased after SP-->LP and decreased after LP-->SP, suggesting that adaptive expression is reversible and correlated with dietary P. The duration of change in gene expression in response to SP-->LP was generally shorter than that of LP-->SP, suggesting potentially different mechanisms of adaptation to deficiency as opposed to excess. Diet-induced changes in mRNA abundance of other genes were either transient or modest. We identified, by heterologous microarray hybridization, new genes sensitive to perturbations in dietary P, and then showed that these genes can reliably monitor P deficiency under field conditions. Simultaneous changes in the expression of these P biomarkers could predict either P deficiency (to prevent economic losses to the farmers) or P excess (to prevent inadvertent pollution of nearby waters).

Identification and phylogenetic analysis of hepatitis C virus in forensic blood samples obtained from injecting drug users.

Injecting drug users (IDUs) are a high-risk group for contracting hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) infections. In Japan, data on the prevalence of those blood-borne viruses among IDUs are very limited. Blood samples were obtained from 12 cadavers of IDUs sent to Nagoya City University for the purpose of judicious autopsy and two alive IDUs with hepatitis C referred to a local hospital at the same period. The viruses were detected by polymerase chain reaction and phylogenetic analysis was performed. Two (16.6%) of the 12 autopsy cases were positive for HCV, but no case was positive for either HBV or HIV. Phylogenetic analysis of the two HCV isolates revealed that one was classified into genotype 1b and another was genotype 2b. Furthermore, nucleotide sequences of two isolates recovered from IDUs with hepatitis C were identical, that indicated the transmission of HCV between them, and those HCV were phylogenetically classified into genotype 2a. The prevalence of HCV infection among IDUs in Japan, despite the case of judicious autopsy, seems to be high, but HIV infection seems to be rare. The transmission of HCV between IDUs was demonstrated, and this indicates that phylogenetic analysis would applicable to also forensic analysis. HCV isolates identified in this study did not phylogenetically segregate, thus multiple transmission route of HCV among IDUs seems be exist in Japan.

The pathology of phosphorus deficiency in fish--a review.

Phosphorus (P) is an essential component of fish diets. Its deficiency affects not only hard tissues, where it is responsible for rickets, leading to skeletal malformation, but also influences various aspects of intermediary metabolism, and thus growth and feed conversion. Therefore, optimizing the dietary inclusion level is critical at all times. As the aquaculture industry has expanded, so the effects of P in farm effluents, derived from metabolic and uneaten food sources, have also become recognized. Diets are increasingly formulated on a basis that will not only provide adequate P for fish needs, but also endeavour to ensure minimal acceptable P levels in effluents at the same time. Many variables influence P requirements and P availability in fish diets, so it is inadvisable to feed diets formulated to an assumed minimum dietary requirement level, irrespective of the advantages that such a formulation may provide to environmental impact.

Acute and chronic smooth muscle cell apoptosis after mechanical vascular injury can occur independently of the Fas-death pathway.

Vascular smooth muscle cell (VSMC) apoptosis has been demonstrated in vascular lesions, such as atherosclerotic and postangioplasty restenotic lesions. Balloon injury also induces VSMC apoptosis. Fas is a death factor that mediates apoptosis when it is activated by its ligand, FasL. Fas-mediated apoptosis was found to be implicated in the pathogenesis of vascular diseases in which Fas/FasL expression was detected. We investigated whether the Fas/FasL interaction mediated acute and chronic VSMC apoptosis and lesion formation in a vascular injury model that may resemble balloon angioplasty. A large spring wire was inserted into the femoral artery of C3H/HeJ (wild-type), C3H-gld (Fas ligand-/-), and C3H-lpr (Fas-/-) mice. The wire was left in place for 1 minute to denude and expand the artery. Massive apoptosis was observed in medial VSMCs from 1 to 7 hours later. There was no difference in the number of apoptotic cells among the 3 groups of mice 4 hours after injury. At 4 weeks, the injured arteries presented signs of concentric neointimal hyperplasia composed exclusively of VSMCs. There was no difference in the degree of neointima hyperplasia (intima/media ratios were as follows: wild type 1.4+/-0.3, gld 1.0+/-0.2, and lpr 1.3+/-0.2) or in the number of apoptotic nuclei among the 3 groups. These findings suggest the existence of other signaling pathways for acute and chronic VSMC apoptosis, at least that induced by mechanical vascular injury.

In situ visualization of ultraviolet-light-induced DNA damage repair in locally irradiated human fibroblasts.

We have developed a novel method that uses a microfilter mask to produce ultraviolet-induced DNA lesions in localized areas of the cell nucleus. This technique allows us to visualize localized DNA repair in situ using immunologic probes. Two major types of DNA photoproducts [cyclobutane pyrimidine dimers and (6-4) photoproducts] were indeed detected in several foci per nucleus in normal human fibroblasts. They were repaired at those localized sites at different speeds, indicating that DNA photoproducts remain in relatively fixed nuclear positions during repair. A nucleotide excision repair protein, proliferating cell nuclear antigen, was recruited to the sites of DNA damage within 30 min after ultraviolet exposure. The level of proliferating cell nuclear antigen varied with DNA repair activity and diminished within 24 h. In contrast, almost no proliferating cell nuclear antigen fluorescence was observed within 3 h in xeroderma pigmentosum fibroblasts, which could not repair either type of photolesion. These results demonstrate that this technique is useful for visualizing the normal nucleotide excision repair process in vivo. Interestingly, however, in xeroderma pigmentosum cells, proliferating cell nuclear antigen appeared at ultraviolet damage sites after a delay and persisted as late as 72 h after ultraviolet exposure. This result suggests that this technique is also valuable for examining an incomplete or stalled nucleotide excision repair process caused by the lack of a single functional nucleotide excision repair protein. Thus, the technique provides a powerful approach to understanding the temporal and spatial interactions between DNA damage and damage-binding proteins in vivo.