Impact of microwave thermolysis energy levels on patient-reported outcomes for axillary hyperhidrosis and osmidrosis.

OBJECTIVE: Microwave thermolysis (MWT) is an emerging treatment for axillary hyperhidrosis reducing both sweat and odor. No prior studies have investigated and compared the different available energy settings of the MWT device. This study evaluated patient-reported outcome measures (PROMs) for axillary hyperhidrosis and osmidrosis following MWT treatment with two different energy levels. METHODS: Twenty adults with axillary hyperhidrosis and osmidrosis reported sweat on Hyperhidrosis Disease Severity scale (HDSS: 1-4) and odor on Odor scale (OS: 1-10), respectively, supplemented by overall Dermatology Life Quality Index (DLQI: 0-30). This was a prospective, randomized, patient-blinded and intraindividually controlled study with 3 months follow-up (FU). Randomization comprised MWT treatment of one axilla with a standard medium energy setting (energy level 3) and the contralateral axilla with a standard high energy setting (energy level 5). RESULTS: At baseline, patients reported substantial sweat and odor, negatively affecting their quality of life. At 3 months FU, PROMs showed improved quality of life with significantly reduced odor and sweat. Overall DLQI was reduced from a median of 10 to 4, with a median 6.5-point reduction (p = 0.0002). HDSS was reduced from a median of 4 to 2 on both sides, with a median reduction of 1 for medium energy level and 2 points for high energy level (p = 0.014). OS was reduced from a median of 8 to 3 for both energy levels, with a median reduction of 3.5 and 4.5 points for the medium and high energy level, respectively (p = 0.017). Local skin reactions were mild and transient, but slightly more pronounced following treatment with the high energy level. CONCLUSION: MWT effectively improved patients' quality of life, axillary sweat, and odor 3 months after on baseline treatment. Treatment with the high energy level presented a subtle but significant increase of efficacy based on PROMs for both sweat and odor. Patients were willing to accept a higher amount of temporary local skin reactions from a higher energy setting when experiencing greater odor and sweat reduction.

Detection and analysis of mycovirus-related RNA viruses from grape powdery mildew fungus Erysiphe necator.

The fungus, Erysiphe necator Schw., is an important plant pathogen causing powdery mildew disease in grapevines worldwide. In this study, high-throughput sequencing of double-stranded RNA extracted from the fungal tissue combined with bioinformatics was used to examine mycovirus-related sequences associated with E. necator. The results showed the presence of eight mycovirus-related sequences. Five of these sequences representing three new mycoviruses showed alignment with sequences of viruses classified in the genus Alphapartitivirus in the family Partitiviridae. Another three sequences representing three new mycoviruses showed similarity to classifiable members of the genus Mitovirus in the family Narnaviridae. These mycovirus isolates were named Erysiphe necator partitivirus 1, 2, and 3 (EnPV 1-3) and Erysiphe necator mitovirus 1, 2, and 3 (EnMV 1-3) reflecting their E. necator origin and their phylogenetic affiliation with other mycoviruses.

Sunscreen formulations do not interfere with sweat cooling during exercise.

OBJECTIVE: Sweating plays a critical role in maintaining thermal balance and keeping skin cool during exercise. People often wear sunscreens on hot summer days for sun protection. Most recreational sunscreens are designed to be water- and sweat-resistant, so that sweating will not remove or compromise the protection. The objective of this study was to determine whether wearing sweat-resistant sunscreen might impede natural sweating, potentially interfering with thermal regulation and resulting in the elevation of skin temperature. METHODS: We conducted a controlled, randomized, split-face and split-arm clinical study with 24 female subjects wearing an SPF 70 lotion sunscreen on half of the face and an SPF 70 spray sunscreen on one of the forearms at a dosage of 2 mg cm-2 . Following sunscreen application, subjects participated in two sessions of indoor exercise to induce clearly visible sweating. RESULTS: We found that both skin temperatures and sweat evaporation rates were significantly elevated after each session, yet there were no significant differences in either skin temperatures or sweat rates between the treated and untreated control sites at any time point for any of the skin sites measured. CONCLUSION: We conclude that the application of tested sweat-resistant sunscreen lotions and sprays does not have any measurable effects on skin cooling by natural sweating process. People should continue to use these sunscreens during recreational activities.

Shadow analysis via the C+K Visioline: A technical note.

BACKGROUND/PURPOSE: This research investigated the ability of shadow analysis (via the Courage + Khazaka Visioline and Image Pro Premiere 9.0 software) to accurately assess the differences in skin topography associated with photo aging. METHODS: Analyses were performed on impressions collected from a microfinish comparator scale (GAR Electroforming) as well a series of impressions collected from the crow's feet region of 9 women who represent each point on the Zerweck Crow's Feet classification scale. Analyses were performed using a Courage + Khazaka Visioline VL 650 as well as Image Pro Premiere 9.0 software. RESULTS: Shadow analysis showed an ability to accurately measure the groove depth when measuring impressions collected from grooves of known depth. Several shadow analysis parameters showed a correlation with the expert grader ratings of crow's feet when averaging measurements taken from the North and South directions. The Max Depth parameter in particular showed a strong correlation with the expert grader's ratings which improved when a more sophisticated analysis was performed using Image Pro Premiere. CONCLUSION: When used properly, shadow analysis is effective at accurately measuring skin surface impressions for differences in skin topography. Shadow analysis is shown to accurately assess the differences across a range of crow's feet severity correlating to a 0-8 grader scale. The Visioline VL 650 is a good tool for this measurement, with room for improvement in analysis which can be achieved through third party image analysis software.

Powdery Mildew Outbreaks caused by Podosphaera macularis on Hop Cultivars Possessing the Resistance Gene R6 in the Pacific Northwestern United States.

Resistant cultivars of hop (Humulus lupulus) have been grown, with the aim of helping to manage powdery mildew in the Pacific Northwest since the first report of the disease in the field in 1997 (4). A major objective of many breeding programs is development of resistance to powdery mildew, and this has generally been achieved by single resistance genes (qualitative resistance). One such gene, R6 (3), has been utilized extensively in new cultivars and has prevented epidemics of the disease in those cultivars across the Pacific Northwestern United States for approximately 15 years. In 2011, a grower in Washington State reported outbreaks of powdery mildew on cv. Apollo, which is thought to possess powdery mildew resistance derived from R6. Fungicides and cultural control measures were applied, and the grower reported no substantial crop damage from the disease. During the winter of 2012, the same grower planted rhizomes of cv. Apollo in a greenhouse in the Yakima Valley of Washington State and later found the plants to be affected by powdery mildew. Affected leaves from plants of cvs. Apollo, Newport, and Nugget (all reported [3] or assumed to possess R6 based on pedigree) grown in the same greenhouse were later provided to the authors. Conidia obtained from each affected plants were transferred to plants of the highly susceptible cv. Symphony, which is not known to contain any resistance genes. After 10 to 14 days of incubation, resultant conidia from each cultivar above (total of three isolates) were transferred to greenhouse grown plants of cvs. Nugget and Symphony and incubated at 18°C. Within 7 days, all three isolates produced powdery mildew colonies characteristic of P. macularis (2) on both cultivars. Cleistothecia did not develop in any colonies. In addition, Nugget and Symphony plants were inoculated with a field population of P. macularis originating from cultivars lacking R6 in Oregon. These inoculations on Nugget did not develop powdery mildew whereas Symphony plants did. Non-inoculated controls remained free of powdery mildew. Results were identical in two additional experiments. The sequence of the mating type idiomorph, MAT1-1, was obtained to confirm identity of the pathogen as P. macularis as described previously (1). The sequences were identical among the three isolates obtained from the greenhouse in Washington and isolates of P. macularis obtained previously from Oregon and Washington. MAT1-2 idiomorph was not detected in the isolates collected. While R6-virulent strains have been detected previously in race characterization experiments, these strains have not caused widespread epidemics of powdery mildew. The increasing prevalence of virulent strains of P. macularis and outbreaks of powdery mildew on formerly resistant cultivars necessitates changes in breeding strategies and disease management efforts to minimize damage resulting from the disease. The distribution of virulent strains of the pathogen and susceptibility of formerly resistance cultivars to powdery mildew are currently under investigation. References: (1) B. Asalfet et al. Phytopathology 103:717, 2013. (2) R. Bélanger et al. The Powdery Mildews: a Comprehensive Treatise. APS Press, St. Paul, MN, 2002. (3) P. Darby. Brew Hist. 121:94, 2005. (4) C. Ocamb et al. Plant Dis. 83:1072, 1999.

Grey hair: clinical investigation into changes in hair fibres with loss of pigmentation in a photoprotected population.

Loss of pigmentation in hair fibres is one of the most obvious phenotypic changes with ageing and has been a topic of increasing interest in the study of follicle biology. The onset of greying brings cosmetic complaints that grey fibres are wild or difficult to manage. Of course, these perceptions may be the consequence of visual obviousness rather than underlying physical or chemical differences. Although several studies have compared pigmented and unpigmented fibres, few have tried to control genetic and ethnic difference as well as extrinsic factors such as photoexposure and chemical treatment. We have recruited subjects with salt-and-pepper hair from a population of Old Order Mennonites who, for cultural reasons, are not only prohibited from chemically treating their hair but also limit their exposure to sunlight. Hair samples were examined for elemental composition, surface energy, Young's modulus, break stress, bending modulus, shear modulus and water sorption/desorption isotherm. The parameters were evaluated statistically for global differences, individual differences and typical individual differences. Consistent with previous published literature, few global differences were found between pigmented and unpigmented hair across the population. We do find that many individual subjects had differences between pigmented and unpigmented fibres. These differences tend to be more pronounced in bulk than in surface properties. The small differences in mechanical properties and moisture uptake and loss lend support to the perception by consumers that grey hair is wilder, drier and less manageable.

An RCT on the effects of topical CGP on surgical wound appearance and residual scarring in bilateral total-knee arthroplasty patients.

OBJECTIVE: To test the hypothesis that topically applied calcium glycerophosphate (CGP) would improve the appearance of the wound following bilateral knee replacement. METHOD: Healthy patients, aged 45-75 years, scheduled for bilateral total-knee replacement surgery were recruited into the study. One knee was randomly assigned to the treatment group, while the contralateral knee was designated the control (standard care). Subjects were instructed to apply a preparation of 10% CGP in an aqueous lotion to the treated knee once daily for 42 days, starting at the third postoperative day. Functional sealing and cosmetic appearance of the incision were evaluated by two surgeons by direct examination of the patient and then by two experienced assessors from photographs. The investigators qualitatively scored the intensity and extent of erythema along the incision and over the entire knee, the appearance of visible oedema along the incision and over the knee, and the overall clinical impression of wound healing. All four assessors were blinded to the subjects' allocation and the latter two assessors to the initial investigators' assessments. Subjects were also followed up for an additional 46 weeks, giving a total study duration of 12 months. RESULTS: Twenty patients completed the study. Statistical analysis showed that both the area and intensity of erythema along the incision were significantly reduced in the treated vs untreated knee over the entire study period. The analysis further showed that treatment significantly reduced oedema, both along the incision and across the entire knee. The differences were most marked at the seventh postoperative day and diminished with time. No adverse effects were observed for any patient, in either treated or untreated knees. CONCLUSION: These data demonstrate that postoperative application of 10% CGP could improve the appearance of the wound following total knee arthroplasty.

Perennation of Uncinula necator in Vineyards of Eastern Washington.

Studies on the mode of perennation of Uncinula necator in Eastern Washington were conducted over a 4-year period. Evidence of perennation of U. necator in infected dormant buds was not evident during vineyard surveys conducted over the period. Cleistothecia retrieved from bark fissures and senesced leaves contained viable ascospores at bud burst and later. The proportion of cleistothecia retrieved from bark that contained viable ascospores at bud burst ranged from 0.19 to 0.48, 0.09 to 0.72, 0.18 to 0.22, and 0.48 to 0.67 in 1998, 1999, 2000, and 2001, respectively. Viability of cleistothecia retrieved from senesced leaves in two vineyards at bud burst was 0.41 and 0.40 in 1998 and was 0.5 and 0.4 in 1999. Ascospore release in lab studies occurred from the late-dormant stage through the prebloom and (in some cases) the bloom stages. The initial ascosporic infection of Chardonnay leaves began at the late-dormant stage; colony numbers then declined through the prebloom and bloom stages. In vineyard studies, ascospores were trapped as late as 70 days after bud burst during rain events of 3.9 to 9.6 mm. Detection of ascospores in vineyard air preceded the initial occurrence of powdery mildew symptoms and signs and the occurrence of conidia in volumetric spore traps by several days. Cleistothecia are the only known source of primary inoculum in the grape-production regions of Eastern Washington.

Scrotal temperatures do not differ among young boys wearing disposable or reusable diapers.

BACKGROUND/AIMS: This study investigated the effect of specific, commonly used diaper types on scrotal temperatures in normal healthy, young boys. These included both modern disposable and reusable diapers as well as various types of protective outer coverings that are in common use in both North America and Europe METHODS: Scrotal and skin surface temperatures were continuously monitored in healthy, young males using a computerized data-logging system based on temperature probes specifically designed for paediatric studies. These systems could be used either tethered to the PC or made completely portable depending upon the age and activity of the child being measured. Based on our results from several pilot studies, it became clear that the best way to determine if disposable and reusable diapers differ with regard to their impact on scrotal temperatures is to run these comparisons under controlled laboratory conditions where "diaper type" was the primary variable. A 2-h time period was chosen to ensure that sufficient time had elapsed for thermal equilibrium to be established under the diapers. We also felt it necessary to study the impact of urination and simulated this condition over the last 15 min using standardized methods. In addition to the skin surface temperatures, we also measured the temperature of the tympanic membrane using an infrared thermometer as an estimate of "core" temperature for each individual at various times during the session. RESULTS AND CONCLUSIONS: In this study, we have clearly shown that scrotal temperatures are the same whether the child is wearing disposable or reusable cloth diapers with a protective cover. The only situation in which scrotal temperatures were found to be lower is when the cloth diaper is used alone without a protective cover but this is not representative of how these products are actually used. We also found that on average scrotal temperatures are significantly lower than core for each diaper type. Occasionally, we did see individuals in which the maximal scrotal temperatures approached core temperatures but in every case the thermal sensors were soiled by a bowel movement. We also found that skin surface temperatures increased not only when covered by a diaper but also due to the thermal insulation provided by outer garments and blankets.

Occurrence and Identification of Phytophthora spp. Pathogenic to Pear Fruit in Irrigation Water in the Wenatchee River Valley of Washington State.

ABSTRACT Seven hundred forty-nine isolates of Phytophthora spp. were obtained from irrigation canals in eastern Washington State during the 1992 to 1995 and 1999 growing seasons. Isolates were retrieved using pear baiting techniques. All isolates were pathogenic to pear and were present in irrigation water beginning early in fruit development. Over the course of the 5 year study, 10 and 5% of isolates were identified as P. cactorum and P. citricola, respectively, using morphological criteria. The remaining isolates could not be identified using morphological criteria. Colony morphology of these isolates was characterized during all years of the study. In 1999, more detailed studies utilizing polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of entire internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) of ribosomal DNA for 180 isolates, and sequence analysis of ITS2 for 50 isolates, were used to investigate genetic variation and phylogenetic relationships among isolates. Isolates were divided into 12 groups based on their growth type on corn meal agar. Restriction digestion of the entire ITS region with three enzymes revealed 11 restriction digestion patterns among 180 isolates. PCR-RFLP and sequence data were obtained for 12 reference Phytophthora spp. (two species in each of Waterhouse's six morphological groups). Phylogenetic analysis of ITS2 regions revealed nine clades, each with strong bootstrap support. Molecular analyses revealed 23 isolates that were in the P. gonapodyides clade, 9 in the P. parasitica clade, 1 in the P. cactorum clade, 7 in the P. citricola/capsici clade, and 4 in the P. cambivora/pseudotsugae clade. The three isolates comprising clade 5 were significantly distinct from all other Phytophthora spp. in the databases and may represent a new Phytophthora sp. Colony morphology was not consistently correlated to PCR-RFLP pattern or ITS2 phylogeny, suggesting that the former criterion is insufficient for species identification. The results of this study indicate that at least nine phylogenetically distinct taxa of Phytophthora pathogenic to pear are present in irrigation water in North Central Washington.

First Report of Phytophthora syringae Causing Rot on Apples in Cold Storage in the United States.

A decay of 'Granny Smith' apples (Malus domestica Borkh.) was observed in 1988, 1990, and 1991 on fruit grown in the lower Hood River Valley of Oregon and stored at 0°C. Harvested fruit were drenched with thiabendazole and stored in October in all years. In mid-November, fruit were sized, drenched with sodium hypochlorite, and returned to cold storage. Decay was observed in January when fruit were removed from cold storage, sorted, and packed. Decayed areas were light brown and firm with a slightly indefinite margin. Losses were less than 1% of fruit packed. Diseased fruit were surface-disinfested with 95% ethanol, and tissue pieces were transferred aseptically to potato dextrose agar acidified with lactic acid and incubated at approximately 22°C. The fungus consistently isolated was identified as Phytophthora syringae (Kleb.) Kleb. based on morphological characters (3). Sporangia were persistent and averaged 60 µm long (range 59 to 69) × 40 µm wide (range 37 to 43). Antheridia were paragynous, and oospores averaged 37 µm (range 31 to 46). 'Golden Delicious', 'Granny Smith', and 'Gala' apples were inoculated with mycelial plugs from a 7-day-old culture of P. syringae and incubated 12 days at 5°C and 7 to 12 days at 22°C. Twenty fruit of each cultivar were used-ten were inoculated, and ten uninoculated fruit served as controls. Lesions developed on all inoculated fruit but not on uninoculated controls. Lesions were spherical, chocolate brown, and firm with no evidence of external mycelia. Lesion morphology was similar on all cultivars. P. syringae was reisolated from lesion margins of all infected fruit. This postharvest decay of apples has not been observed in the Hood River Valley since 1991. Fruit rot of apples caused by P. syringae is known in Canada (1) and is common in the United Kingdom (2), but has not been reported previously in the United States. To our knowledge, this is the first report of postharvest decay of apples by P. syringae in the United States. References: (1) R. G. Ross and C. O. Gourley. Can. Plant Dis. Surv. 49:33, 1969. (2) A. L. Snowdon. A Color Atlas of Postharvest Diseases. CRC Press, Inc., Boca Raton, FL, 1990. (3) G. M. Waterhouse. The Genus Phytophthora. Misc. Publ. 12. The Commonwealth Mycological Institute, Kew, Surrey, England, 1956.

Methods for evaluating changes in skin condition due to the effects of antimicrobial hand cleansers: two studies comparing a new waterless chlorhexidine gluconate/ethanol-emollient antiseptic preparation with a conventional water-applied product.

BACKGROUND: Hand-cleansing products that are milder to the skin of health care personnel are being developed, but the available methodologies to appropriately evaluate these products and quantify differences are not generally being applied in well-controlled studies. METHODS: Two randomized, blinded, bilateral comparison studies evaluated skin condition during use of 2 antiseptic hand preparation products: a new 1% chlorhexidine gluconate (CHG)/61% wt/wt ethanol antiseptic hand preparation in a unique emollient system for waterless/brushless application and a conventional 4% CHG antimicrobial product that is applied with water and a scrub brush. Trained technicians applied treatments 6 times (for a surgical scrub study) or 24 times (for a personnel handwash study) daily to the hands of healthy volunteers during 5 days of controlled washing. An expert grader evaluated skin for dryness, erythema, and roughness. Subjects completed a self-assessment questionnaire on skin condition. Transepidermal water loss was measured by an evaporimeter, and the skin surface hydration level was measured by an electrical conductance meter. RESULTS: Fifty-eight subjects were enrolled in the 2 studies and received both treatments. In general, skin treated with the waterless CHG/ethanol product scored significantly (P <.004) better on evaluations of visual dryness and erythema and showed greater improvement in the level of hydration (P <.003). In the health care personnel handwash study, transepidermal water loss was less than that for skin treated with the conventional CHG product (P <.002). Subject assessments showed similar results (total score, P <.007). CONCLUSIONS: All 3 approaches of expert grader evaluation, subject assessment, and instrumentation were in concordance, demonstrating that the waterless CHG/ethanol product was gentler to skin than the conventional CHG product.

Randomized facial tolerability studies comparing gel formulations of retinoids used to treat acne vulgaris.

Two double-blind, randomized, split-face studies have been performed to compare the facial tolerability of topical retinoids in volunteers with sensitive skin. In one study, subjects applied tazarotene 0.1% gel to one side of their face and tretinoin 0.1% gel microsponge, tretinoin 0.025% gel, or adapalene 0.1% gel to the other side of their face, for up to 29 days. Increases in facial dryness and erythema were comparable among all retinoids. Some subjects in each treatment group experienced levels of retinoid-associated irritation that required temporary suspension of, or reduction in, treatment. Facial dryness and erythema tended to be greater in these subjects than in those who tolerated the regimen without change, suggesting that the need to discontinue or modify treatment depends more on the individual than on any major inherent differences in the irritant potential of these retinoids. A second study compared once-daily versus alternate-day tazarotene 0.1% gel therapy. Tolerability was superior when initiating therapy with the alternate-day regimen.

Whole body protein turnover can be measured non-invasively in women using the end product method with [15N]glycine to show changes with the menstrual cycle and pregnancy.

OBJECTIVES: To assess the effect of the menstrual cycle and pregnancy on whole body protein turnover. DESIGN AND SUBJECTS: Whole body protein turnover was determined using oral [15N]glycine in normal women from enrichment measured in urine at days 7 and 14 of the menstrual cycle and in pregnant women at 17-20 weeks and 30-32 weeks gestation. RESULTS: Nitrogen flux was 38.2 (5.8) mg N/kg/h using a single dose and 31.1+/-4.1 mg N/kg/h with prime-intermittent doses-the same as in men using the same methods. Nitrogen flux around the time of ovulation, 32.3 (8.2) mg N/kg/h, was greater than at day 7, 26.8 (3.0) mg N/kg/h (P<0.05). For women taking the contraceptive pill flux at day 7, 22.7 (0.2) mg N/kg/h, and day 14, 21.3 (2.1) mg N/kg/h, were similar, but were lower at day 14 than for women not taking the pill (P<0.05). Protein synthesis increased between mid and late pregnancy (1.7-2.0 g N/h; P=0.012), but not when expressed in relation to body weight CONCLUSIONS: Protein turnover can be measured reproducibly in women using a non-invasive method, provided care is taken to standardize the conditions, and especially the duration over which urine is collected. There is an increase around the time of ovulation when hormonal levels are at their highest and during pregnancy the results obtained are similar to those reported using [13C]leucine. SPONSORSHIP: University of Southampton and Medical Research Council.

A pilot study of noninvasive methods to assess healed acute and chronic wounds.

BACKGROUND: A variety of instruments are available that can objectively assess physical parameters of the skin such as strength, firmness, elasticity, hydration, and color, often undetected by clinical assessment. OBJECTIVE: To assess the physical properties of healed acute and chronic wounds using several noninvasive instruments. METHODS: Four patients with healed acute wounds and four patients with healed chronic wounds were studied using ballistometric, impedance, levarometric, and spectrophotometric measurements. RESULTS: In general, scars were harder, less elastic, dryer, and more erythematous than control skin. These differences were more pronounced in healed chronic wounds. CONCLUSION: A scar from an acute surgical wound becomes softer, more elastic, dryer, less erythematous, and less pigmented as it ages. In contrast, chronic wound scars become harder as they age. These different properties of healed acute wounds and healed chronic wounds may be a result of the different healing processes in each wound type.

A hand immersion test under laboratory-controlled usage conditions: the need for sensitive and controlled assessment methods.

Exaggerated test conditions were frequently used to investigate the cutaneous tolerance of detergent products in the past. As the sensitivity of newly designed biometric methods is steadily improving, the trend towards more realistic test conditions should be encouraged. A hand immersion test under laboratory-controlled usage conditions is presently described, fulfilling such principles. Panelists soaked their hands in 2 different hand dishwashing liquids, 2x daily for 10 min each (with successive in-solution/out-of-solution cycles) for 4 consecutive days. Products were at usual dilution for dishwashing liquids and were randomized between the dominant and non-dominant hands of panelists. Visual scoring of erythema and dryness developing on the whole hands (scoring scales including interdigital areas and joints) during the week did not allow discrimination between the 2 products. However the dominant hands were significantly more susceptible to alterations than the non-dominant hands, regardless of product attribution. In contrast, skin electrical measurements (Corneometer CM800 and Skicon 200) on the dorsum of the hands (muscle mass between thumb and index) and squamometry analysis of tape stripping (harvested from the same site) yielded significant differences between the 2 products. In conclusion, a hand immersion test under realistic conditions has been described, which discriminates between products when sensitive assessment methods are used to explore skin sites partially protected from daily-life skin aggressions.

Calcium binding studies of photosystem II using a calcium-selective electrode.

The identification of Ca2+ as a cofactor in photosynthetic O2 evolution has encouraged research into the role of Ca2+ in photosystem II (PSII). Previous methods used to identify the number of binding sites and their affinities were not able to measure Ca2+ binding at thermodynamic equilibrium. We introduce the use of a Ca2(+)-selective electrode to study equilibrium binding of Ca2+ to PSII. The number and affinities of binding sites were determined via Scatchard analysis on a series of PSII membrane preparations progressively depleted of the extrinsic polypeptides and Mn. Untreated PSII membranes bound approximately 4 Ca2+ per PSII with high affinity (K = 1.8 microM) and a larger number of Ca2+ with lower affinity. The high-affinity sites are assigned to divalent cation-binding sites on the light-harvesting complex II that are involved in membrane stacking, and the lower-affinity sites are attributed to nonspecific surface-binding sites. These sites were also observed in all of the extrinsic polypeptide- and Mn-depleted preparations. Depletion of the extrinsic polypeptides and/or Mn exposed additional very high-affinity Ca2(+)-binding sites which were not in equilibrium with free Ca2+ in untreated PSII, owing to the diffusion barrier created by the extrinsic polypeptides. Ca2(+)-depleted PSII membranes lacking the 23 and 17 kDa extrinsic proteins bound an additional 2.5 Ca2+ per PSII with K = 0.15 microM. This number of very high-affinity Ca2(+)-binding sites agrees with the previous work of Cheniae and co-workers [Kalosaka, K., et al. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) pp 721-724, Kluwer, Dordrecht, The Netherlands] whose procedure for Ca2+ depletion was used. Further depletion of the 33 kDa extrinsic protein yielded a sample that bound only 0.7 very high-affinity Ca2+ per PSII with K = 0.19 microM. The loss of 2 very high-affinity Ca2(+)-binding sites upon depletion of the 33 kDa extrinsic protein could be due to a structural change of the O2-evolving complex which lost 2-3 of the 4 Mn ions in this sample. Finally, PSII membranes depleted of Mn and the 33, 23, and 17 kDa extrinsic proteins bound approximately 4 very high-affinity Ca2+ per PSII with K = 0.08 microM. These sites are assigned to Ca2+ binding to the vacant Mn sites.