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Julie M Bianchini,1 Qihong Zhang,2 Gabriel Hanna,2 Carol R Flach,2 Hequn Wang,1 Michael D Southall,1 Richard Mendelsohn,2 Manpreet Randhawa11Johnson and Johnson Consumer Companies, Inc., Skillman, NJ, USA; 2Department of Chemistry, Rutgers University, Newark, NJ, USAIntroduction: As skin ages, it loses its ability to retain moisture and becomes rough and dry. This results in a clinically dull appearance with a loss of radiance, firmness, and suppleness. Symptoms can be improved with use of a moisturizer that builds and maintains skin hydration over time; however, most moisturizers that occlude the skin surface are perceived as heavy and greasy and are not consumer preferred.Methods: A unique, consumer-preferred gel matrix formula was developed by combining liquid crystal structures, which mimic skin barrier lipid assembly, with specific emulsifiers that deliver water deep into skin. Ex vivo studies were conducted to investigate the superior hydrating effects of the gel matrix formula. Confocal Raman microscopy studies assessed the spatial distribution of water in ex vivo skin after application of the gel matrix formula. To determine the effects of the gel matrix formula on dry facial skin, a 12-week clinical study was conducted with subjects with self-perceived skin dryness and dullness.Results: The formulation significantly increased the relative water content throughout epidermal regions, which was not observed with the application of a competitive gel formula. Instrumental measurements assessed improvements in skin surface moisturization and barrier function. Clinical grading showed significant improvements in hydration-related endpoints including radiance, clarity, and texture. Subject self-agree assessment demonstrated that subjects observed improvements in the appearance of their facial skin.Conclusion: These studies demonstrated that the gel matrix formula increased skin water content in deeper layers, and resulted in significant clinical improvements in hydration, barrier function, and clinical appearance of radiance.Keywords: confocal Raman microscopy, skin radiance, moisturization, dynamic skin barrier, skin hydration

Qihong Zhang,1 Carol R Flach,1 Richard Mendelsohn,1 Guangru Mao,2 Apostolos Pappas,2 M Catherine Mack,2 Russel M Walters,2 Michael D Southall2 1Department of Chemistry, Rutgers University, Newark, 2Johnson & Johnson Consumer Companies, Inc., Skillman, NJ, USAAbstract: Ceramides (CERs), structural components of the stratum corneum (SC), impart essential barrier properties to this thin outer layer of the epidermis. Variations in CER species within this layer have been linked to several skin diseases. A recent proliferation of CER-containing topical skin-care products warrants the elucidation of CER penetration profiles in both healthy and diseased skin. In the current study, the spatial distributions of CER concentration profiles, following topical application of two species of CER, were tracked using infrared imaging. Suspensions of single-chain perdeuterated sphingosine and phytosphingosine CER in oleic acid were applied, in separate experiments, to the surface of healthy intact ex vivo human skin using Franz diffusion cells. Following either a 24- or 48-hour incubation period at 34°C, infrared images were acquired from microtomed skin sections. Both CER species accumulated in glyph regions of the skin and penetrated into the SC, to a limited extent, only in these regions. The concentration profiles observed herein were independent of the CER species and incubation time utilized in the study. As a result, a very heterogeneous, sparse, spatial distribution of CERs in the SC was revealed. In contrast, oleic acid was found to be fairly homogeneously distributed throughout the SC and viable epidermis, albeit at lower concentrations in the latter. A more uniform, lateral distribution of CERs in the SC would likely be important for barrier efficacy or enhancement.Keywords: stratum corneum, infrared imaging, topical delivery, oleic acid

The sterol and fatty acid compositions of Epifagus virginiana, which is a nonphotosynthetic flowering plant, have been examined by combined gas liquid chromatography-mass spectrometry (GLC-MS) and nuclear magnetic resonance (NMR). The organism exhibited a typical higher plant Δ-sterol profile with the C-24 alkyl group oriented 'α' for the major sterol Trace amounts of 24-methylenecycloartanol and cycloeucalenol established connection with the first cyclic intermediate (cycloartenol) in photosynthetic plant sterol biosynthesis. In a separate in vitro experiment, excised root and stem tissue were incubated with two labeled triterpenoid precursors, [2-H] lanosterol and [2-H]cycloartenol. Both the radioactive compounds were converted to labeled [H]sitosterol indicating the presence of an active biosynthetic pathway in Epifagus. Characteristic photosynthetic higher plant fatty acids were also present, and the polyenic acid was identified as α-linolenic acid. [ABSTRACT FROM AUTHOR]

Background: The loss of structural elastin due to intrinsic and extrinsic ageing results in the skin's inability to stretch and recoil (decrease in elasticity) and manifests as loss of skin firmness and sagging. While other extracellular matrix (ECM) components such as collagen and hyaluronic acid are continually synthesized and assembled through life, elastic fibres are not. Elastic fibre assembly and functionality require fibre cross-linking, induced by the lysyl oxidase-like (LOXL) enzymes, which sharply decrease during ageing., Objective: To evaluate the enhanced elastogenic effect of a blackberry-dill extract combination, which was hypothesized to induce elastin fibre component synthesis, fibre cross-linking and reduce elastin fibre degradation., Methods: The blackberry and the dill extracts were tested separately and in combination to confirm single ingredient bioactivity and synergistic benefits. Human skin explants, dermal fibroblasts, elastase assays, ELISAs, quantitative real-time PCRs and spectrofluorometer measurements were used. Moreover, a double-blinded, placebo-controlled clinical study was carried out to assess skin elasticity using Cutometer and histologically from biopsies., Results: The blackberry extract induced elastin gene expression, elastin promoter activity and inhibited elastic fibre degradation by matrix metalloproteinases (MMPs) 9 and 12. The dill extract induced elastin, collagen and LOXL1 gene expression, resulting in enhanced fibre cross-linking in human skin explants. Clinically, the blackberry and dill combination treatment displayed synergistic pro-elasticity activity as compared to each ingredient alone and placebo., Conclusion: Taken together, these results demonstrated the two multimodal plant-based extracts complemented each other in terms of bioactivity and resulted in a synergistic elastogenesis induction., (© 2020 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Background and Objective: Recent advances in low-level light devices have opened new treatment options for mild to moderate acne patients. Light therapies have been used to treat a variety of skin conditions over the years but were typically only available as treatments provided by professional clinicians. Clinical application of blue light has proven to be effective for a broader spectral range and at lower fluences than previously utilized. Herein, we tested the hypothesis that sub-milliwatt/cm 2 levels of long-wave blue light (449 nm) effectively kills Propionibacterium acnes, a causative agent of acne vulgaris, in vitro., Materials and Methods: Two types of LED light boards were designed to facilitate in vitro blue light irradiation to either six-well plates containing fluid culture or a petri plate containing solid medium. P. acnes. Survival was determined by counting colony forming units (CFU) following irradiation. P. acnes was exposed in the presence and absence of oxygen. Coproporphyrin III (CPIII) photoexcitation was spectrophotometrically evaluated at 415 and 440 nm to compare the relative photochemical activities of these wavelengths., Results: 422 and 449 nm blue light killed P. acnes in planktonic culture. Irradiation with 449 nm light also effectively killed P. acnes on a solid agar surface. Variation of time or intensity of light exposure resulted in a fluence-dependent improvement of antimicrobial activity. The presence of oxygen was necessary for killing of P. acnes with 449 nm light. CPIII displayed clear photoexcitation at both 415 and 440 nm, indicating that both wavelengths are capable of initiating CPIII photoexcitation at low incident light intensities (50 uW/cm 2 )., Conclusion: Herein we demonstrate that sub-milliwatt/cm 2 levels of long-wave blue light (449 nm) effectively kill P. acnes. The methods and results presented allow for deeper exploration and design of light therapy treatments. Results from these studies are expanding our understanding of the mode of action and functionality of blue light, allowing for improved options for acne patients. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Background: Retinol, a precursor of retinoic acid, has great potentials as a topical anti-aging molecule; however, only a handful of clinical investigations have been published to date., Objective: This study aimed to assess the efficacy and safety of 0.1% stabilized retinol on photodamaged skin during a one-year treatment., Methods: The investigation included two 52-week, double-blind, vehicle-controlled studies. In the main study, 62 subjects applied either a stabilized retinol formulation or its vehicle to the full face. A second exploratory study evaluated histological/histochemical markers in 12 subjects after 52 weeks of either retinol or vehicle use on contralateral dorsal forearms., Results: The retinol group showed significant photodamage improvement over vehicle at all timepoints during the study. After 52 weeks, retinol had improved crow's feet fine lines by 44%, and mottled pigmentation by 84%, with over 50% of subjects showing +2 grades of improvement in many parameters. Additionally, at week 52, histochemical data confirmed the clinical results, showing increased expression of type I procollagen, hyaluronan, and Ki67 as compared to vehicle., Conclusion: This study confirms that a stabilized retinol (0.1%) formulation can significantly improve the signs of photoaging, and improvements in photodamage continue with prolonged use.

Skin chronically exposed to sun results in phenotypic changes referred as photoaging. This aspect of aging has been studied extensively through genomic and proteomic tools. Metabolites, the end product are generated as a result of biochemical reactions are often studied as a culmination of complex interplay of gene and protein expression. In this study, we focused exclusively on the metabolome to study effects from sun-exposed and sun-protected skin sites from 25 human subjects. We generated a highly accurate metabolomic signature for the skin that is exposed to sun. Biochemical pathway analysis from this data set showed that sun-exposed skin resides under high oxidative stress and the chains of reactions to produce these metabolites are inclined toward catabolism rather than anabolism. These catabolic activities persuade the skin cells to generate metabolites through the salvage pathway instead of de novo synthesis pathways. Metabolomic profile suggests catabolic pathways and reactive oxygen species operate in a feed forward fashion to alter the biology of sun exposed skin.

Background: Environmental factors such as solar ultraviolet (UV) radiation and other external aggressors provide an oxidative challenge that is detrimental to skin health. The levels of endogenous antioxidants decrease with age, thus resulting in less protection and a greater potential for skin damage. The NF-E2-related factor-2 (Nrf2) - antioxidant response element (ARE) pathway is a primary defense mechanism against oxidative stress, and induces the expression of antioxidant, detoxification and repair genes. Activation of ARE-Nrf2 can help restore oxidative homeostasis of the skin and play a role in inflammatory response and DNA repair mechanisms., Objective: To evaluate the role of a purified parthenolide-depleted Feverfew (PD-Feverfew) extract on the ARE-Nrf2 pathway and DNA repair in skin cells., Methods: These studies were undertaken in primary human keratinocytes or KB cells using Luciferase Promoter assay, siRNA transfection studies, Western blot analyses, Immunofluorescence microscopy, comet assay and quantitative real-time PCR., Results: PD-Feverfew was found to induce Nrf2 nuclear translocation and to increase ARE activity in a dose dependent manner. Furthermore, knockdown of Nrf2 resulted in suppression of PD-Feverfew-induced ARE activity. PD-Feverfew was also found to induce phosphorylation of Akt, a kinase downstream of PI3K. Inhibition of PI3K via pre-treatment with the selective pharmacological inhibitor, LY294002, abolished PD-Feverfew-induced Nrf2/ARE activation. PD-Feverfew also reduced UV-induced DNA damage in a PI3K and Nrf2-dependent manner., Conclusions: Therefore, by increasing endogenous defense mechanisms and aid in DNA repair of damaged skin cells via activation of a PI3K-dependent Nrf2/ARE pathway, PD-Feverfew may help protect the skin from numerous environmental aggressors., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

It is very well known that exposure of skin to sun chronically accelerates the mechanism of aging as well as making it more susceptible toward skin cancer. This aspect of aging has been studied very well through genomics and proteomics tools. In this study we have used a metabolomic approach for the first time to determine the differences in the metabolome from full thickness skin biopsies from sun exposed and sun protected sites. We have primarily investigated the energy metabolism and the oxidative pathway in sun exposed skin. Biochemical pathway analysis revealed that energy metabolism in photoexposed skin is predominantly anaerobic. The study also validated the increased oxidative stress in skin.

Background: While skin color is the most notable difference among ethnic skins the current knowledge on skin physiological and aging properties are based mainly on Caucasian skin studies., Objective: To evaluate histological differences in elastin fiber network and differential responsiveness to TGF-β in skin of Caucasians and African Americans., Methods: These studies were undertaken using human skin biopsies, primary dermal fibroblasts, Western blot analyses, immunofluorescence microscopy, cDNA array and quantitative real-time PCR., Results: In Caucasian subjects, tropoelastin expression and elastin fibers in photoprotected skin was substantially less than in age-matched African American skin. Expression of tropoelastin in photoexposed skin of African American was similar to their photoprotected skin, suggesting that photoexposure did not affect elastin fibers in African American skin to the same extent as Caucasian skin. An elevated level of TGF-β1 present in media from dermal fibroblasts derived from African American skins correlated well with the higher levels of TGF-β mRNA in African American skins analyzed by cDNA array. Treatment with TGF-β1 resulted in a considerably higher induction of elastin mRNA in dermal fibroblasts from African Americans than from Caucasian fibroblasts, indicative of enhanced TGF-β signaling in African American skins. Furthermore, UVA exposure decreased levels of elastin mRNA in Caucasian fibroblasts compared to African Americans fibroblasts., Conclusion: These results suggest that there are ethnic differences in the elastin fiber network and in TGF-β signaling in African American and Caucasian skin, and that African American have less UV dependent loss of elastin than Caucasian which may contribute to the different perceived aging phenotypes., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Three-dimensional in vitro skin models provide an alternative to animal testing for assessing tissue damage caused by chemical or physical agents and for the identification and characterization of agents formulated to mitigate this damage. The StrataTest® human skin model made with pathogen-free NIKS® keratinocyte progenitors is a fully-stratified tissue containing epidermal and dermal components that possesses barrier function as determined by measurements of electrical impedance. Independent batches of skin tissues responded consistently to known chemical irritants even after refrigerated storage for up to 7 days. Reactive oxygen species (ROS) were detected after exposure of skin tissues to ozone, cigarette smoke or ultraviolet (UV) irradiation. Pretreatment with the antioxidant parthenolide-depleted (PD)-Feverfew extract prevented cigarette smoke-induced or UV irradiation-mediated increases in ROS. Interleukin (IL)-1α and IL-1 receptor antagonist (IL-1RA) secretion increased in a dose dependent manner following UV irradiation but cytokine release was abrogated by pretreatment with a UVA/UVB sunscreen. Similarly, immunohistochemical detection showed increased thymidine dimer formation in UV-irradiated skin tissue that was prevented with sunscreen pretreatment. These results demonstrate that the StrataTest® human skin model is broadly applicable to a wide range of in vitro toxicological assays., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The skin is under continual assault from a variety of damaging environmental factors such as ultraviolet irradiation and atmospheric pollutants, and as organisms age the cumulative damage exceeds the capacity of endogenous antioxidant defenses resulting in chronic inflammation and premature aging. Botanical extracts such as Feverfew containing naturally occurring antioxidants could replenish the depleted cutaneous stores and perhaps forestall these degenerative changes. A parthenolide-depleted extract of Feverfew (PD-Feverfew), which was free of sensitization potential, was found to possess free radical scavenging activity against a wide range of reactive oxygen species and with greater activity than Vitamin C. In vitro, PD-Feverfew restored cigarette smoke-mediated depletion of cellular thiols, attenuated the formation of UV-induced hydrogen peroxide and reduced pro-inflammatory cytokine release. In vivo, topical PD-Feverfew reduced UV-induced epidermal hyperplasia, DNA damage and apoptosis. In a clinical study PD-Feverfew treatment significantly reduced erythema versus placebo 24 h post-UV exposure. Through the ability to scavenge free radicals, preserve endogenous antioxidant levels, reduce DNA damage and induce DNA repair enzymes, which can help repair damaged DNA, parthenolide-depleted extract of Feverfew may protect skin from the numerous external aggressions encountered daily by the skin and reduce the damage to oxidatively challenged skin.

Tissue inflammation is often accompanied by local interstitial fluid accumulation expressed as edema. Edema can be the manifestation of infection, lymphatic blockage, wound healing, or even cancer, and is typically graded visually. Here we demonstrate that the edema reaction can be objectively quantitated in vivo by the use of spectral imaging. To this end we applied the method on a histamine-induced cutaneous edema model. Apparent concentrations of oxy-hemoglobin, deoxy-hemoglobin, and water were calculated for each pixel of a spectral image stack. These values were used to construct concentration maps for each of these molecules as well as an intensity map of an optical tissue-scattering parameter. The oxy-hemoglobin and the tissue water maps are two-dimensional quantitative representations of the skin areas involved in erythema and edema, respectively. These maps demonstrated characteristics of the wheal-and-flare reaction and their gray-level intensities were dependent on the applied histamine dose. We conclude that spectral imaging can be a valuable noninvasive tool in the study of edema pathology and can be used to monitor the edema reaction in vivo or follow the efficacy of treatments in a clinical setting.

Topical administration of chemicals such as methyl nicotinate that induce erythema have been employed to measure the effectiveness of formulations containing anti-inflammatory agents. Prior studies have utilized a single concentration of methyl nicotinate, between 36.5 and 100 mM, for all test subjects in evaluations of topical formulations. However, individuals have different thresholds of response to methyl nicotinate; thus, a single concentration may not be appropriate for all individuals and could result in the apparent lack of anti-inflammatory activity of the formulation being evaluated. In the current study, we evaluated the use of a minimal erythema concentration (MEC) of methyl nicotinate, defined as the lowest concentration that produces a complete and even erythema at the test site, compared with a 36.5-mM concentration of methyl nicotinate. Hydroalcoholic gels containing the nonsteroidal anti-inflammatory drug ibuprofen were compared with placebo. Diffuse reflectance spectroscopy was employed to measure differences in cutaneous inflammatory response between the control (placebo)-treated group and the ibuprofen-treated group. When chemical erythema was induced using an MEC of methyl nicotinate, greater reductions in erythema were seen in ibuprofen-treated sites compared with sites treated with a 36.5-mM concentration of methyl nicotinate. In conclusion, for an accurate assessment method of erythema induced by methyl nicotinate, consideration should be given to determining the extent of response of an erythema-producing agent on an individual basis. An MEC of methyl nicotinate should be determined and employed for each individual to obtain more consistent and reliable efficacy results of anti-inflammatory activity., (2006 S. Karger AG, Basel)

A number of chemical mediators can induce human keratinocytes and epidermal-derived carcinomas to undergo apoptosis, or programmed cell death. Recent evidence suggests pro-inflammatory cytokines, such as interleukin-1 beta or transforming growth factor alpha, protects carcinomas from numerous pro-apoptotic stimuli. Platelet-activating factor (1-alkyl-2-acetyl-3-glycerophosphocholine; PAF) is a lipid mediator with pro-inflammatory effects on numerous cell types. Although PAF can be metabolized to other bioactive lipids, the majority of PAF effects occur through activation of a G protein-coupled receptor. Using a model system created by retroviral transduction of the PAF receptor (PAF-R) into the PAF-R-negative human epidermal cell line KB and the PAF-R-expressing keratinocyte cell line HaCaT, we now demonstrate that activation of the epidermal PAF-R results in protection from apoptosis induced by tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. The PAF-mediated protection was inhibited by PAF-R antagonists, and protection did not occur in PAF-R-negative KB cells. Additionally, we show protection from TNFalpha- or TRAIL-induced apoptosis by PAF-R activation is dependent on the transcription factor nuclear factor (NF)-kappa B, because PAF-R activation-induced NF-kappa B and epidermal cells transduced with a super-repressor form of inhibitor kappa B were not protected by the PAF-R. These studies provide a mechanism whereby the epidermal PAF-R, and possibly other G protein-coupled receptors, can exert anti-apoptotic effects through an NF-kappa B-dependent process.

Although a number of prostaglandin E(2) (PGE(2)) receptor subtypes have been cloned, limited studies have been performed to elucidate subtypes that subserve specific actions of this eicosanoid, in part because of a paucity of selective receptor antagonists. Using reverse transcription-polymerase chain reaction (PCR) and antisense oligonucleotides, we examined which prostaglandin E(2) receptor (EP receptor) subtypes are expressed in sensory neurons and which mediate the PGE(2)-induced increase in cAMP production and augmentation of peptide release. Reverse transcription-PCR of cDNA isolated from rat sensory neurons grown in culture revealed PCR products for the EP1, EP2, EP3C, and EP4 receptor subtypes but not the EP3A or EP3B. Preexposing neuronal cultures for 48 h to antisense oligonucleotides of EP3C and EP4 mRNA diminished expression of the respective receptors by approximately 80%, abolished the PGE(2)-stimulated production of cAMP, and blocked the ability of PGE(2) to augment release of immunoreactive substance P and calcitonin gene-related peptide. Pretreating with individual antisense against the EP2, EP3C, or EP4 receptors or combinations of missense oligonucleotides had no effect on PGE(2)-induced activity. Treatment with antisense to EP3C and EP4 receptor subtypes did not alter the ability of forskolin to increase cAMP or enhance peptide release. These results demonstrate that sensory neurons are capable of expressing multiple EP receptor subtypes but that only the EP3C and EP4 receptors mediate PGE(2)-induced sensitization of sensory neurons.

Isoprostanes are a novel class of eicosanoids primarily formed by peroxidation of arachidonic acid. Because of their potential as inflammatory and/or hyperalgesic agents whose formation is largely independent of cyclooxygenases, we examined whether 8-iso prostaglandin E(2) (8-iso PGE(2)) or 8-iso prostaglandin F(2alpha) (8-iso PGF(2alpha)) reduces mechanical and thermal withdrawal threshold in rats, and whether they sensitize rat sensory neurons. Injection of 1 microg of 8-iso PGE(2) (in 2.5 microl) into the hindpaw of rats significantly reduced mechanical and thermal withdrawal thresholds, whereas 1 microg of 8-iso PGF(2alpha) elicited a transient decrease in only the mechanical withdrawal threshold. Both isoprostanes enhanced the firing of C-nociceptors in a concentration-dependent manner when injected into peripheral receptive fields. Exposing sensory neurons grown in culture to 1 microM 8-iso PGE(2) or 8-iso PGF(2alpha) augmented the number of action potentials elicited by a ramp of depolarizing current. In contrast, 8-iso PGE(2) but not 8-iso PGF(2alpha) enhanced the release of substance P- and calcitonin gene-related peptide-like immunoreactivity from isolated sensory neurons. Ten micromolar 8-iso PGE(2) stimulated peptide release directly, whereas treatment with 1 microM 8-iso PGE(2) augmented the release evoked by either bradykinin or capsaicin. Pretreating neuronal cultures with the nonsteroidal anti-inflammatory drug ketorolac did not alter the sensitizing action of 8-iso PGE(2) on peptide release, suggesting that this action of the isoprostane was not secondary to the production of prostaglandins via the cyclooxygenase pathway. These data support the notion that isoprostanes are an important class of inflammatory mediators that augment nociception.

Age related changes in dopaminergic and serotonergic receptors were examined in Aplysia californica. In this study dopamine (DA) and serotonin (5-HT) receptor levels were examined for animals belonging to 4-, 5-, 6-, 8-, 9- and 12-month age groups. Receptors analysis was performed using radio-labeled d-[3H] lysergic acid diethylamide (LSD) as the specific ligand. Specific binding for 5-HT was found to be significantly greater than that for DA in the young (4-month post-hatch) animals. The total DA and 5-HT receptor levels changed significantly with age. Dopamine levels increased from 5.34 fmol/mg of protein at 4 months to 19.11 fmol/mg at 12 months. Serotonin receptor levels increased from 7.35 fmol/mg at 4 months to 20.45 at 12 months.

Several lines of evidence point to the involvement of the proto-oncogene c-raf-1 in the development of lymphoma and leukemia and in a previous study we found variants of this gene in 3 lymphoma patients. To see whether variation at this locus plays a role in predisposition to these cancers, we have examined the frequency of an unusual EcoRI allele of c-raf-1 in 99 non-Hodgkin's lymphoma and 28 acute lymphoblastic leukemia patients, and in 182 controls. To optimize the chance of detecting a difference, we selected 113 of the controls from geriatric patients with no family history of cancer. No difference in the frequency of the allele was found between patients and controls, thereby refuting our hypothesis that this polymorphism of the c-raf-1 locus contributes to genetic susceptibility to these two related cancers. We estimate that the frequency of the rarer b EcoRI allele of this locus is 0.091 +/- 0.012 in the Australian Caucasian population.