Effect of head lift exercise on kinematic motion of the hyolaryngeal complex and aspiration in patients with dysphagic stroke.

The head lift exercise (HLE) is a head-raising workout performed in a supine position. This exercise facilitates activation of the submental muscles located above the hyoid bone in front of the neck. HLE is a potential method to improve the movement of the hyolaryngeal movement and swallowing functions. The purpose of this study was to investigate the effect of HLE on the hyolaryngeal movement and aspiration in patients with dysphagic stroke. A total of 27 patients with stroke were randomly assigned either into the experimental (n = 13) or the control group (n = 14). The experimental group performed HLE 5 days a week for 4 weeks (a total of 20 sessions). Both groups received the same conventional dysphagia therapy. Two-dimensional analysis of the hyolaryngeal movement was carried out using Image J program based on a videofluoroscopic swallowing study. Penetration-aspiration was assessed using Penetration-Aspiration Scale (PAS). The experimental group showed a significant increase in the only superior movements of the hyoid bone compared with the control group (P = 0·033). Aspiration in liquid also exhibited a significant decrease in the experimental group compared with the control group (P = 0·044). Findings from this study confirmed that HLE is an effective intervention to improve hyoid movement and decrease aspiration.

Survival and expression of acid resistance genes in Shiga toxin-producing Escherichia coli acid adapted in pineapple juice and exposed to synthetic gastric fluid.

AIMS: The aim of this research was to determine the ability of non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups to survive with exposure to synthetic gastric fluid (SGF) after adaptation to pineapple juice (PJ) at room and refrigerated temperatures compared to E. coli O157:H7 and to examine the relative transcriptional expression of acid resistance (AR) genes, rpoS, gadA and adiA. METHODS AND RESULTS: Resistant and sensitive strains belonging to five different STEC serogroups (O26, O103, O104, O111 and O157; n = 10) were used in this study. All strains were adapted in PJ (pH 3·8) stored at 4 and 20°C for 24 h, and then the relative transcription levels of genes in all strains were quantified using a real-time quantitative-PCR assay. After adaptation in PJ, the STEC strains were exposed to SGF (pH 1·5 and 2·0) at 37°C for 2 h. Generally, the STEC adapted in PJ at 4°C displayed enhanced survival compared to acid adaptation in PJ at 20°C and nonadapted controls with exposure to SGF (P < 0·05). Moreover, resistant strains exhibited higher survival rates compared to sensitive strains (P < 0·05). Overall, adaptation at 4°C resulted in significantly (P < 0·05) enhanced gene expression levels in PJ, and transcript levels of gadA were higher than those of the rpoS and adiA genes. CONCLUSIONS: The up-regulation of AR genes due to adaptation in PJ at low temperature may increase STEC survival in acidic environments such as the gastrointestinal tract. Some non-O157 STEC strains, including serotypes O103:H2 and O111:H8, showed relatively high AR levels similar to those of STEC O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Induction of AR genes in acidic fruit juice, and potentially in other acidic foods may increase the risk of foodborne illness by non-O157 STEC serogroups.

Effects of neuromuscular electrical stimulation combined with effortful swallowing on post-stroke oropharyngeal dysphagia: a randomised controlled trial.

Neuromuscular electrical stimulation (NMES) has been used as a therapeutic intervention for dysphagia. However, the therapeutic effects of NMES lack supporting evidence. In recent years, NMES combined with traditional swallowing therapy has been used to improve functional recovery in patients with post-stroke dysphagia. This study aimed to investigate the effects of effortful swallowing combined with neuromuscular electrical stimulation on hyoid bone movement and swallowing function in stroke patients. Fifty stroke patients with mild dysphagia who were able to swallow against the resistance applied by using NMES and cooperate actively in training were included. This study was designed as a 6-week single-blind, randomised, controlled study. In the experimental group, two pairs of electrodes were placed horizontally in the infrahyoid region to depress the hyoid bone. The NMES intensity was increased gradually until the participants felt a grabbing sensation in their neck and performed an effortful swallow during the stimulation. In the placebo group, the same procedure was followed except for the intensity, which was increased gradually until the participants felt an electrical sensation. All participants underwent this intervention for 30 min per session, 5 sessions per week, for 6 weeks. Videofluoroscopic swallowing studies (VFSS) were carried out before and after the intervention and kinematics of the hyoid bone and swallowing function were analysed based on the VFSS. The experimental group revealed a significant increase in anterior and superior hyoid bone movement and the pharyngeal phase of the swallowing function. This intervention can be used as a novel remedial approach in dysphagic stroke patients.

Effects of expiratory muscle strength training on oropharyngeal dysphagia in subacute stroke patients: a randomised controlled trial.

Expiratory muscle strength training (EMST) involves forcible blowing as a means of generating high expiratory pressure, against adjustable resistance. EMST has recently been introduced as a potential treatment for dysphagia. This study was performed to investigate the effects of EMST on the activity of suprahyoid muscles, aspiration and dietary stages in stroke patients with dysphagia. Twenty-seven stroke patients with dysphagia were randomly divided into two groups. The experimental group performed EMST with a 70% threshold value of maximal expiratory pressure, using an EMST device, 5 days a week for 4 weeks. The placebo group trained with a sham device. The EMST regime involved 5 sets of 5 breaths through the EMST device for a total of 25 breaths per day. Activity in the suprahyoid muscle group was measured using surface electromyography (sEMG). Further, the penetration-aspiration scale (PAS) was used to assess the results of the videofluoroscopic swallowing study (VFSS). In addition, dietary stages were evaluated using the Functional Oral Intake Scale (FOIS). The experimental group exhibited improved suprahyoid muscle group activity and PAS results, when compared to the placebo group. Following intervention, statistical analysis indicated significant differences in measured suprahyoid muscle activity (P = 0·01), liquid PAS outcomes (P = 0·03) and FOIS results (P = 0·06), but not semisolid type PAS outcomes (P = 0·32), between the groups. This study confirms EMST as an effective treatment for the development of suprahyoid muscle activity in stroke patients with dysphagia. Additionally, improvements in aspiration and penetration outcomes were observed.

An experimental validated in silico model to assess Staphylococcus aureus growth kinetics on different pork products.

AIMS: To gain an understanding of the growth kinetics of Staphylococcus aureus to ensure the safety of pork, and to develop a predictive growth model of Staph. aureus in raw pork, ham and sausage pork under specific storage time and temperature conditions. METHODS AND RESULTS: Growth of Staph. aureus was evaluated at 5-40°C using 5-6 replicates in each sampling time in order to capture experimental variability. Growth curves were fit to Baranyi model to estimate Lag time (λ) and maximum growth rate (µmax). The effect of temperature on λ and µmax was modelled using natural logarithm of λ and square root of µmax. The variability between repetitions was higher at 15, 20 and 40°C than observed at 25, 30 and 35°C. After only 3 and 2 days of storage at 5 and 10°C, respectively, Staph. aureus populations decreased to limit of detection (≤1 log CFU g(-1) ). Staphylococcus aureus populations on ham presented higher µmax in comparison to those grown on raw pork and sausage. Linear regression lines showed that Staph. aureus populations in ham grew faster than those observed in sausage and raw pork. Staph. aureus presented a theoretical minimum growth temperature (Tmin ) depending on the processed pork products, which were different from in raw pork. CONCLUSION: Staphylococcus aureus model predicts faster growth in ham compared to raw pork and sausage. The validation of models showed good predictions, suggesting that the developed models are useful in estimating growth kinetics of Staph. aureus in different pork products. SIGNIFICANCE AND IMPACT OF THIS STUDY: Established here is a data-driven, in silico model on Staph. aureus growth kinetics on different pork products. The model is extensively validated by experiments and simulations and further supported by comparisons to previous modelling efforts in other species.

Voriconazole N-oxide and its ultraviolet B photoproduct sensitize keratinocytes to ultraviolet A.

BACKGROUND: The antifungal agent, voriconazole, is associated with phototoxicity and photocarcinogenicity. Prior work has indicated that voriconazole and its hepatic N-oxide metabolite do not sensitize keratinocytes to ultraviolet B (UVB). Clinical observations have suggested that ultraviolet A (UVA) may be involved. OBJECTIVES: To determine the photochemistry and photobiology of voriconazole and its major hepatic metabolite, voriconazole N-oxide. MATERIALS AND METHODS: Voriconazole and voriconazole N-oxide were spectrophotometrically monitored following various doses of UVB. Cultured human keratinocytes were treated with parental drugs or with their UVB photoproducts, and survival following UVA irradiation was measured by thiazolyl blue metabolism. Reactive oxygen species (ROS) and 8-oxoguanine were monitored by fluorescence microscopy. RESULTS: Voriconazole and voriconazole N-oxide have varying UVB absorption but do not acutely sensitize cultured human keratinocytes following UVB exposure. However, sustained UVB exposures produced notable dose- and solvent-dependent changes in the absorption spectra of voriconazole N-oxide, which in aqueous solution acquires a prominent UVA absorption band, suggesting formation of a discrete photoproduct. Neither the parental drugs nor their photoproducts sensitized cells to UVB although all but voriconazole N-oxide were moderately toxic to cells in the dark. Notably, both voriconazole N-oxide and its UVB photoproduct, but not voriconazole or its photoproduct, additionally sensitized cells to UVA by greater than three-fold relative to controls in association with UVA-induced ROS and 8-oxoguanine levels. CONCLUSIONS: Voriconazole N-oxide and its UVB-photoproduct act as UVA-sensitizers that generate ROS and that produce oxidative DNA damage. These results suggest a mechanism for the phototoxicity and photocarcinogenicity observed with voriconazole treatment.

Synergetic effect of combined fumaric acid and slightly acidic electrolysed water on the inactivation of food-borne pathogens and extending the shelf life of fresh beef.

AIMS: To evaluate synergetic effect of slight acidic electrolysed water (SAEW) and fumaric acid (FA) on inactivation of total viable count (TVC) and Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium in fresh beef and to study shelf life and sensory quality of beef. METHODS AND RESULTS: Inoculated samples was dipped for 1, 3 and 5 min and immersed at 25, 40 and 60°C in SAEW, strong acidic electrolysed water (StAEW) and SAWE + FA. Treated meat was air-packaged and stored at 4 or 10°C. During storage, sampling was performed at 2-day intervals for microbiological and sensory changes. TVC was decontaminated at 40°C for 3 min by more than 3·70 log CFU g(-1) , and examined pathogens were reduced by more than 2·60 log CFU g(-1) with SAEW + FA treatment. This treatment prolonged shelf life of beef meat up to 9 and 7 days when stored at 4 and 10°C, respectively. CONCLUSION: The combined treatment of SAEW + FA showed greater bactericidal effect and prolonged shelf life compared with individual treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined treatment of SAEW and FA can be a suitable hurdle technology reducing bacteria in fresh beef, substantially enhancing their microbial safety and decreasing pathogens growth during storage.

Effects of dietary supplementation of citrus by-products fermented with a probiotic microbe on growth performance, innate immunity and disease resistance against Edwardsiella tarda in juvenile olive flounder, Paralichthys olivaceus (Temminck & Schlegel).

Two consecutive studies were conducted to evaluate the dietary supplementation of citrus by-products (CB) fermented with probiotic bacteria on growth performance, feed utilization, innate immune responses and disease resistance of juvenile olive flounder. In Experiment I, five diets were formulated to contain 0% (control) or 3% four different CB fermented with Bacillus subtilis (BS), Enterococcus faecium (EF), Lactobacillus rhamnosus (LR) and L. plantarum (LP) (designated as CON, CBF-BS, CBF-EF, CBF-LR and CBF-LP, respectively). During 10 weeks of a feeding trial, growth performance and feed efficiency were not significantly different among all the fish groups. However, fish fed CBF containing diets had significantly higher survivals than the CON group. Disease resistance of fish against Edwardsiella tarda was increased by the fermentation of CB. In Experiment II, we chose the BS as a promising probiotic and formulated five diets to contain 0%, 2%, 4%, 6% and 8% CBF-BS. Growth performance was not significantly affected by the CBF-BS supplementation during 6 weeks of a feeding trial. Innate immunity of fish was significantly enhanced by CBF-BS supplementation. Myeloperoxidase and lysozyme activities were increased in a dose-dependent manner by dietary CBF-BS inclusions. In a consecutive challenge test against E. tarda, an increased disease resistance was found by CBF-BS supplementation. These studies indicate that the fermentation process of CB with probiotic has beneficial effects on innate immunity and thereby increases disease resistance of olive flounder against E. tarda. Bacillus subtilis can be used as a promising probiotic microbe for by-product fermentation in fish feeds.

Precuneus hypoperfusion plays an important role in memory impairment of patients with systemic lupus erythematosus.

The present study was designed to identify regional cerebral blood flow (rCBF) abnormalities in systemic lupus erythematosus (SLE) patients with memory impairments. Nineteen SLE patients (mean age 36.1 ± 8.6 years, range 17-47) with subjective memory complaints underwent brain single-photon emission computed tomography (SPECT). The Korean Wechsler Adult Intelligence Scale (K-WAIS) and the Rey-Kim Memory Test (RKMT) were used objectively to evaluate cognitive functions in these patients. On the basis of the Intelligence Quotient-Memory Quotient (IQ-MQ) difference score, patients were classified into two groups: those with below one standard deviation (SD) from the mean for normal subjects of comparable age and education (memory impairment, n = 6) and those with without memory impairment (non-memory impairment, n = 13). Their brain SPECT images were analyzed by statistical parametric mapping (SPM) for group comparisons. The group of SLE patients with memory impairment showed significant hypoperfusion in the right precuneus compared with those with non-memory impairment (p < 0.001). Hypoperfusion of the precuneus may play a significant role in the memory function of SLE patients. SPM analysis of brain SPECT images could be a useful and objective tool for identifying abnormal rCBF in SLE patients with memory impairment.

Irradiation-induced shrinkage and expansion mechanisms of SiO2 circle membrane nanopores.

20 nm diameter SiO(2) nanopore arrays on gradient-thickness membranes were formed by a focused electron beam with in situ transmission electron microscopy (TEM). Nanopore shrinkage was seen in nanopores on thicker membranes, with the rate of diameter change remaining constant during the shrinkage process. In contrast, pore expansion was observed in thinner membranes, with the expansion rate being constant at the initial stage but with a slight increase at the later stage. The geometry model of shrinkage and expansion of the nanopores in relation to the electron irradiation time was investigated by utilizing the TEM tilting method.

Diversity of HLA-DQA1 gene in the Korean population.

Genotype of the human leukocyte antigen (HLA)-DQA1 locus was analyzed in Koreans (n= 467) using the 14th International Workshop protocol established to characterize the sequences of exons 1-4 of the gene. Unexpectedly, it appeared that the DQA1 (19 alleles) was more diverse than DQB1 (15 alleles) in the study population. DQA1*010201, DQA1*0303, DQA1*0103, and DQA1*0302 appeared to be major alleles exhibiting more than 10%. Among six allele groups, DQA1*01-*06, DQA1*01 showed highest diversity exhibiting seven different alleles. Analysis using maximum likelihood method showed numerous multi-locus HLA haplotypes. High relative linkage disequilibrium values (RLD) of the two-locus haplotypes and exclusive association of a specific DQA1 allele with a specific DRB1 and/or DQB1 alleles suggested tight linkage of DQA1 to DRB1 and DQB1. In HLA-matching process for hematopoietic stem cell transplantation, however, DQA1 typing would be informative for individuals carrying specific DRB1 allele (DRB1*0802, DRB1*1201, or DRB1*1403) that could be associated with multiple DQA1 alleles in the study population. Information obtained in this study will be useful in medical and forensic areas as well as in anthropology.

Four novel human leukocyte antigen-DQA1 alleles identified in the Korean population.

Four novel human leukocyte antigen (HLA)-DQA1 alleles have been characterized by direct DNA sequencing of coding exons 1-4. All the novel alleles exhibited a single nucleotide substitution either in exon 3 or in exon 4 when compared with previously defined alleles. Thus, it is likely that alleles were generated by point mutation from pre-existing alleles in the population. Substitutions resulted in either a silent (DQA1*010203) or an amino acid change (DQA1*0506, DQA1*0507, and DQA1*0508). The substituted sites were both previously known polymorphic and conserved positions. Putative haplotypes associated with the novel alleles were deduced based on the HLA types shared by the individuals carrying a novel allele or from previously reported population data.

Allelic and haplotypic diversity of HLA-A, -B, -C, -DRB1, and -DQB1 genes in the Korean population.

High-resolution human leukocyte antigen (HLA) typing exposes the unique patterns of HLA allele and haplotype frequencies in each population. In this study, HLA-A, -B, -C, -DRB1, and -DQB1 genotypes were analyzed in 485 apparently unrelated healthy Korean individuals. A total of 20 HLA-A, 43 HLA-B, 21 HLA-C, 31 HLA-DRB1, and 14 HLA-DQB1 alleles were identified. Eleven alleles (A*0201, A*1101, A*2402, A*3303, B*1501, Cw*0102, Cw*0302, Cw*0303, DQB1*0301, DQB1*0302, and DQB1*0303) were found in more than 10% of the population. In each serologic group, a maximum of three alleles were found with several exceptions (A2, B62, DR4, DR14, and DQ6). In each serologic group exhibiting multiple alleles, two major alleles were present at 62-96% (i.e. A*0201 and A*0206 comprise 85% of A2-positive alleles). Multiple-locus haplotypes estimated by the maximum likelihood method revealed 51 A-C, 43 C-B, 52 B-DRB1, 34 DRB1-DQB1, 48 A-C-B, 42 C-B-DRB1, 46 B-DRB1-DQB1, and 30 A-C-B-DRB1-DQB1 haplotypes with frequencies of more than 0.5%. In spite of their high polymorphism in B and DRB1, identification of relatively small numbers of two-locus (B-C and DRB1-DQB1) haplotypes suggested strong associations of those two loci, respectively. Five-locus haplotypes defined by high-resolution DNA typing correlated well with previously identified serology-based haplotypes in the population. The five most frequent haplotypes were: A*3303-Cw*1403-B*4403-DRB1*1302-DQB1*0604 (4.2%), A*3303-Cw*0701/6-B*4403-DRB1*0701-DQB1*0201/2 (3.0%), A*3303-Cw*0302-B*5801-DRB1*1302-DQB1*0609 (3.0%), A*2402-Cw*0702-B*0702-DRB1*0101-DQB1*0501 (2.9%), and A*3001-Cw*0602-B*1302-DRB1*0701-DQB1*0201/2 (2.7%). Several sets of allele level haplotypes that could not be discriminated by routine HLA-A, -B, and -DRB1 low-resolution typing originated from allelic diversity of A2, B61, DR4, and DR8 serologic groups. Information obtained in this study will be useful for medical and forensic applications as well as in anthropology.

Is slow nail growth a risk factor for onychomycosis?

This study was carried out to determine whether slow nail growth is a predisposing factor for onychomycosis or if onychomycosis results in slow nail growth. Forty-nine patients with unilateral onychomycosis of the great toenail were enrolled and classified in two groups according to the size of affected area, i.e. more than half or less than half of the toenail. The growth rates of affected and unaffected great toenails of all patients were measured. Before a normal appearance was reached, the growth rates of affected great toenails, when the affected area occupied more than half of total nail plate, was slower than that of the unaffected great toenails. After a normal appearance was achieved, there were no differences in growth rates between affected and unaffected great toenails. Therefore, this study of patients with unilateral toenail onychomycosis did not support the hypothesis that slow nail growth rate is a predisposing factor for onychomycosis.

Spatially localized generation of nucleotide sequence-specific DNA damage.

Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen-DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320-400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA-psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen-TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues.

The personal internetworked notary and guardian.

In this paper, we propose a secure, distributed and scaleable infrastructure for a lifelong personal medical record system. We leverage on existing and widely available technologies, like the Web and public-key cryptography, to define an architecture that allows patients to exercise full control over their medical data. This is done without compromising patients' privacy and the ability of other interested parties (e.g. physicians, health-care institutions, public-health researchers) to access the data when appropriately authorized. The system organizes the information as a tree of encrypted plain-text XML files, in order to ensure platform independence and durability, and uses a role-based authorization scheme to assign access privileges. In addition to the basic architecture, we describe tools to populate the patient's record with data from hospital databases and the first testbed applications we are deploying.

Transverse myelitis in a patient with long-standing ankylosing spondylitis.

Ankylosing spondylitis is reported to involve not only the joints but other organs as well. Among these extra-articular involvements, uncommon complications associated with nervous system such as single root lesions, compression of the myelum and cauda equina syndrome have also been documented. Here we present a patient with long-standing ankylosing spondylitis who developed spastic paraparesis. Extensive study to find the cause of a spastic paraparesis failed and therefore led to the conclusion that this patient was suffering from transverse myelitis. Similar reports in the past have been attributed to an association with multiple sclerosis; however, we suggest that the findings support the diagnosis of a rare complication of ankylosing spondylitis with an unknown etiology.

Incidence and characterization of Listeria spp. from foods available in Korea.

A total of 410 domestic Korean food samples were analyzed for the presence of Listeria spp. by the conventional U.S. Department of Agriculture protocol, and presumptive strains were identified by morphological, cultural and biochemical tests according to Bergey's manual and confirmed by API-Listeria kit. Among the total 410 food samples, 46 samples (11.2%) were found to be contaminated with Listeria species. Among the 46 strains of Listeria spp. isolates, 8 strains (17.42%) for Listeria monocytogenes, 3 strains (6.5%) for Listeria seeligeri, 33 strains (71.7%) for Listeria innocua, and 2 strains (4.4%) for Listeria welshimeri were identified, respectively. Also, only beef, chicken, pork, frozen foods, and sausage were contaminated with L. monocytogenes, and the other products were free of L. monocytogenes. Of 46 Listeria spp. isolates, L. innocua (71.7%) was the most predominantly isolated in a variety of foods compared to other Listeria spp. An in vitro virulence assay for Listeria spp. using myeloma and hybridoma cells from murine and human sources was performed. The result showed that only L. monocytogenes killed approximately 95 to 100% hybridoma cells after 6 h and the other Listeria species, such as L. innocua, L. seeligeri, and L. welshimeri strains had about 0 to 10% lethal effect on hybridoma cells. Also, an antibiotic susceptibility test showed that Listeria spp. isolates were very susceptible to the antibiotics tested, except for nalidixic acid. Also, serotyping results showed 75% of L. monocytogenes isolates from beef, chicken, and frozen pizza belonged to serotype 1 and 25% from sausage were type 4.

Binding and photoreactivity of psoralen linked to triple helix-forming oligonucleotides.

Triple helix-forming oligonucleotides conjugated to a psoralen (psoTFO) have been designed to bind to three distinct purine-rich sequences within the human interstitial collagenase (MMP1) gene. Gel mobility shift assays indicate that these psoTFO bind to and photoreact with model target DNA sequences following ultraviolet A (UVA) irradiation. The dissociation constants for binding of the psoTFO to their targets range from 0.3 to 4 microM. Psoralen monoadducts with the purine-rich target strand and interstrand crosslinks are efficiently formed on targets containing either 5'-ApT-3' or 5'-TpA-3' sequences adjacent to the TFO binding sequence. The dependence of adduct formation on UVA dose has provided quantitative estimates of the overall rate constants for psoralen monoadduct and crosslink formation in the presence of a TFO. When psoralen is tethered to a TFO, the rate of monoadduct formation exceeds that of crosslinking for all sequences studied. This contrasts with the relatively low rate of monoadduct formation that has been reported for free psoralens, suggesting that the bound TFO facilitates the initial photochemistry that generates monoadducts, but does not significantly affect interstrand crosslink formation. psoTFO and UVA treatment inhibit DNA cleavage by a restriction endonuclease when the psoralen covalently reacts directly at the endonuclease site. The particular TFO studied do not completely inhibit endonuclease activity when they are noncovalently bound or when the covalent psoralen adduct does not coincide with the endonuclease site. Our findings confirm that TFO are capable of directing psoralen photoadducts to specific DNA targets and suggest that TFO can significantly modulate psoralen photoreactivity and DNA-protein interactions.

Isolation of a cDNA encoding a 31-kDa, pathogenesis-related 5/thaumatin-like (PR5/TL) protein abundantly expressed in apple fruit (Nalus domestica cv. Fuji).

A fruit-specific and pathogenesis-related 5/thaumatin-like (PR5/TL), 31-kDa protein was isolated by 2D-PAGE from fully-grown apples (Malus domestica cv. Fuji) and named Mdtl1 (Malus domestica thaumatin-like protein 1). Using the N-terminal sequence of the protein, the full-length cDNA encoding Mdtll was isolated. The cDNA clone (Mdtl1) consists of 944 bp with an open reading frame (ORF) of 744 bp encoding a protein of 247 amino acids. The deduced amino acid sequence of Mdtl1 shows high similarity to the sequences of PR5/TL proteins. Mdtl1 is a slightly acidic protein with a putative signal peptide and a putative N-glycosylation site, and lacks a C-terminal extension. This suggests that Mdtl1 is an apoplastic glycoprotein. Results of northern blotting indicated that expressions of Mdtl1 are developmentally regulated. Southern blot analysis showed that Mdtl1 may be present as a single copy, and there exist other genes closely related to Mdtl1 in the apple genome.