A new quantitative evaluation method for age-related changes of individual pigmented spots in facial skin.

BACKGROUND: Facial skin pigmentation is one of the most prominent visible features of skin aging and often affects perception of health and beauty. To date, facial pigmentation has been evaluated using various image analysis methods developed for the cosmetic and esthetic fields. However, existing methods cannot provide precise information on pigmented spots, such as variations in size, color shade, and distribution pattern. The purpose of this study is the development of image evaluation methods to analyze individual pigmented spots and acquire detailed information on their age-related changes. METHODS: To characterize the individual pigmented spots within a cheek image, we established a simple object-counting algorithm. First, we captured cheek images using an original imaging system equipped with an illumination unit and a high-resolution digital camera. The acquired images were converted into melanin concentration images using compensation formulae. Next, the melanin images were converted into binary images. The binary images were then subjected to noise reduction. Finally, we calculated parameters such as the melanin concentration, quantity, and size of individual pigmented spots using a connected-components labeling algorithm, which assigns a unique label to each separate group of connected pixels. RESULTS: The cheek image analysis was evaluated on 643 female Japanese subjects. We confirmed that the proposed method was sufficiently sensitive to measure the melanin concentration, and the numbers and sizes of individual pigmented spots through manual evaluation of the cheek images. The image analysis results for the 643 Japanese women indicated clear relationships between age and the changes in the pigmented spots. CONCLUSION: We developed a new quantitative evaluation method for individual pigmented spots in facial skin. This method facilitates the analysis of the characteristics of various pigmented facial spots and is directly applicable to the fields of dermatology, pharmacology, and esthetic cosmetology.

Influence of fetal hiccups on Doppler blood flow waveform of fetal arteries: Difference among arteries.

BACKGROUND: A few studies have been reported on the influence of fetal hiccups on umbilical artery. The aim of this study is to clarify the influence of fetal hiccups on Doppler blood flow waveform (DBFW) of some fetal arteries, and to show the difference in these influences among fetal arteries. OBJECTIVE: DBFW of umbilical artery, descending aorta, and middle cerebral artery were recorded at hiccups in normal fetuses between 34th and 40th gestational weeks. The changes on DBFW were classified into three shapes by the direction and the size of the changes. Shape 1: sharp decrease but not to the baseline, Shape 2: sharp decrease to the baseline (absence), and Shape 3: reverse flow. RESULTS: At all hiccups, the changes on DBFW of these arteries were observed. These changes were classified into three shapes. Changes of umbilical artery were widely distributed in three shapes depending on when hiccup occurred during cardiac cycle. On the other hand, most changes of the descending aorta and middle cerebral artery were Shape 3 whenever the hiccup occurred during cardiac cycle. CONCLUSION: The changes on DBFW of fetal arteries were observed at all hiccups. Changes of umbilical artery were widely distributed in three shapes depending on when hiccup occurred during cardiac cycle. On the other hand, most changes of descending aorta and middle cerebral artery were Shape 3. This is the first study clarified the influence of fetal hiccups on DBFW of some fetal arteries, and showed the difference in these influences among fetal arteries.

Causal involvement of mammalian-type cryptochrome in the circadian cuticle deposition rhythm in the bean bug Riptortus pedestris.

Mammalian-type CRYPTOCHROME (CRY-m) is considered to be a core repressive component of the circadian clock in various insect species. However, this role is based only on the molecular function of CRY-m in cultured cells and it therefore remains unknown whether CRY-m is indispensable for governing physiological rhythms at the organismal level. In the present study, we show that RNA interference (RNAi) targeting of cry-m in the bean bug Riptortus pedestris disrupts the circadian clock governing the cuticle deposition rhythm and results in the generation of a single cuticle layer. Furthermore, period expression was induced in cry-m RNAi insects. These results verified that CRY-m functions as a negative regulator in the circadian clock that generates physiological rhythm at the organismal level.

Chromosome condensation in Xenopus mitotic extracts without histone H1.

The contribution of histone H1 to mitotic chromosome condensation was examined with the use of a cell-free extract from Xenopus eggs, which transforms condensed sperm nuclei into metaphase chromosomes. When H1 was removed from the extract, the resultant metaphase chromosomes were indistinguishable from those formed in complete extract. Nucleosomal spacing was the same for both. Thus, H1 is not required for the structural reorganization that leads to condensed metaphase chromosomes in this egg extract.

Characterization of the hatching enzyme from embryos of an anuran amphibian, Rana pirica.

The culture medium in which prehatching embryos of the frog, Rana pirica, were cultured (hatching medium) solubilized the vitelline coat (VC) of unfertilized eggs and contained molecules reactive to antibodies (anti-UVS.2) against the Xenopus hatching enzyme (HE). The hydrolyzing activity of the hatching medium against Pro-Phe-Arg-MCA was inhibited dose-dependently by the same antibodies. Using anti-UVS.2 as a probe, we purified two distinct 56 kDa molecules exhibiting Pro-Phe-Arg-MCA hydrolyzing activity. These 56 kDa molecules, which were separable on anion exchange chromatography, were the same with respect to VC solubilizing activity and a substrate specificity for various MCA-peptides, and they were regarded as charge isomers that function as the HE. The hydrolyzing activity against Pro-Phe-Arg-MCA of HE was optimal at pH of 7.6, with the apparent Km value of 250 microM at 30 degreesC. The activity was strongly inhibited by DFP and EDTA, and was accelerated by extremely low concentrations of Mg2+ and Zn2+, indicating the serine protease and metalloprotease nature of the HE. The HE was glycosylated and was present as a putative proenzyme form of 63 kDa.

Structure of genes for sperm-specific nuclear basic protein (SP4) in Xenopus laevis.

Nuclear basic proteins in sperm of Xenopus laevis consist of 6 sperm-specific proteins (SPs1-6) in addition to somatic core histones. Using a cDNA for SP4 as a probe, we cloned genomic DNA containing SP4 genes from a genomic library constructed from recombinant lambda bacteriophage containing 12.0 kbp-EcoRI digests of J-strain X. laevis liver DNA. Construction of restriction maps based on Southern blot analysis revealed the existence of a total of five SP4 genes which are arranged in a tandemly repeated array forming a cluster of simple multigenes per haploid genome, over a range of 18 kbp. Among these genes, the one located at the most upstream position differed from others in possessing a single base substitution which gave rise to a replacement of one out of 78 amino acid residues. The DNA containing the second to the fourth SP4 genes, arranged at about 3 kbp intervals each, was totally sequenced for 10,165 bp. Each gene was found to contain one intron, typical TATA and CCAAT boxes in the 5'-flanking region, and a polyadenylation signal in the 3'-flanking region. Comparative sequence analyses revealed three regions of extensive homology within the upstream non-coding region among three genes, suggesting a possible relevance to their expression at a particular phase of spermatogenesis and/or in testis.

Remodeling of sperm chromatin induced in egg extracts of amphibians.

Sperm nuclear basic proteins of Bufo japonicus consist of 2 distinct protamines, whereas those of Xenopus laevis consist of 6 sperm-specific basic proteins (SP1-6) in addition to H3, H4 and smaller amounts of H2A and H2B. Cloning of pertinent cDNAs and partial amino acid sequence studies suggested that these 6 sperm-specific proteins of Xenopus are encoded by 3 distinct genes. Despite differences in their initial compositions of chromatin, sperm nuclei exposed to amphibian egg extracts rapidly decondense, lose sperm-specific basic proteins, and concomitantly form an ordinary nucleosome core consisting of H2A, H2B, H3, H4, and cleavage-stage specific subtype H1X. In this remodeling process, nucleoplasmin plays dual roles as a molecular chaperone, selectively removing sperm-specific basic proteins from, and bringing H2A and H2B to, sperm DNA. Thus remodeling of chromatin is induced even in mammalian (human) sperm nuclei under defined conditions including nucleoplasmin and exogenous histones.

Molecular cloning of Xenopus hatching enzyme and its specific expression in hatching gland cells.

UVS.2 has been known as a cloned cDNA expressed selectively in the hatching gland cells of Xenopus laevis. To determine the molecular identity and function of UVS.2-encoded proteins, antibodies were raised against a bacterially-expressed fusion protein comprising glutathione-S-transferase (GST) and UVS.2. Anti-GST-UVS.2 antibodies inhibited the vitelline envelope digesting activity of the medium (hatching medium) in which dejellied prehatching embryos were cultured. On Western blotting, hatching medium contained 60 kDa and 40 kDa molecules reactive with these antibodies. Whole-mount immunostaining showed a specific localization of UVS.2 protein in the hatching gland cells which appeared first at stage 20, increased in number and intensity to stage 31 then decreased gradually thereafter. Immunoelectron microscopy revealed that UVS.2 protein is localized exclusively in the secretory granules in the hatching gland cells. A cDNA library from the dorsoanterior portion of stage 25 embryos was screened with UVS.2, and a 1.8 kb insert thus cloned contained additional 619bp and 204bp at the 5' and 3' ends of UVS.2, respectively. This clone, designated XHE, contained an open reading frame encoding 514 amino acids including both signal and propeptide sequences. The predicted mature enzyme comprising 425 amino acids consists of about 200 amino acid-long metalloprotease sequence of astacin family at the N-terminus, followed by two repeats of CUB domain each 110 amino acid-length. We conclude that UVS.2 represents an approximately 3/4 C-terminal portion of the hatching enzyme.

Epidermal steroid sulfatase and cholesterol sulfotransferase are regulated during late gestation in the fetal rat.

Lipids in the stratum corneum (SC) are organized into lamellar membrane unit structures that provide the permeability barrier. Cholesterol sulfate, a SC membrane lipid, is synthesized by cholesterol sulfotransferase (CSTase) in the lower epidermis and hydrolyzed to cholesterol by steroid sulfatase (SSase) in the SC. To determine whether these enzymes are induced during barrier ontogenesis, we examined their activity in epidermis of fetal rats before (gestational day 17), during (day 19), and after (day 21) barrier formation. CSTase activity increased approximately 10-fold between day 17 and day 19, then declined between day 19 and day 21. In contrast, SSase activity reached its peak activity on day 21, increasing >5-fold. Fetal rat skin explants develop a SC and barrier over the same time course in vitro as in utero. Likewise, CSTase and SSase activities during in vitro ontogenesis precisely mirrored those obtained in utero. Moreover, hormones that accelerate barrier ontogenesis (e.g. glucocorticoids, thyroid hormone, and estrogen) accelerated the increase in CSTase and SSase activities during in vitro ontogenesis. mRNA levels of SSase increased in parallel with enzymatic activity, suggesting that these developmental changes are regulated at the genomic level. Finally, addition of exogenous cholesterol sulfate to explants in vitro did not accelerate either SC development or barrier formation. These studies suggest that induction of the cholesterol sulfate cycle enzymes during SC ontogenesis is a component of the fetal epidermal differentiation program and that the synthetic and degradative enzymes of this pathway are differentially regulated.

Induction of selected lipid metabolic enzymes and differentiation-linked structural proteins by air exposure in fetal rat skin explants.

The epidermal permeability barrier of premature infants matures rapidly following birth. Previous studies suggest that air exposure could contribute to this acceleration, because: (i) development of a structurally and functionally mature barrier accelerates when fetal rat skin explants are incubated at an air-medium interface, and (ii) occlusion with a water-impermeable membrane prevents this acceleration. To investigate further the effects of air exposure on epidermal barrier ontogenesis, we compared the activities of several key enzymes of lipid metabolism and gene expression of protein markers of epidermal differentiation in fetal rat skin explants grown immersed versus air exposed. The rate-limiting enzymes of cholesterol (HMG CoA reductase) and ceramide (serine palmitoyl transferase) synthesis were not affected. In contrast, the normal developmental increases in activities of glucosylceramide synthase and cholesterol sulfotransferase, responsible for the synthesis of glucosylceramides and cholesterol sulfate, respectively, were accelerated further by air exposure. Additionally, two enzymes required for the final stages of barrier maturation and essential for normal stratum corneum function, beta-glucocerebrosidase, which converts glucosylceramide to ceramide, and steroid sulfatase, which desulfates cholesterol sulfate, also increased with air exposure. Furthermore, filaggrin and loricrin mRNA levels, and filaggrin, loricrin, and involucrin protein levels all increased with air exposure. Finally, occlusion with a water-impermeable membrane prevented both the air-exposure-induced increase in lipid enzyme activity, and the expression of loricrin, filaggrin, and involucrin. Thus, air exposure stimulates selected lipid metabolic enzymes and the gene expression of key structural proteins in fetal epidermis, providing a biochemical basis for air-induced acceleration of permeability barrier maturation in premature infants.

Elicitation of weak immune response in larval and adult Xenopus laevis by allografted pituitary.

To test the claim that tolerance to organ-specific self antigens is established during the ontogenic development of the immune system, pituitary anlagen were removed from early tailbud embryos of Xenopus laevis, and the resulting hypophysectomized tadpoles were grafted with pituitaries after the tadpoles had become immunocompetent. The result was that none of the histocompatible or allogeneic pituitary grafts was rejected throughout an observation period of 100 days. Subsequent experiments revealed that hypophysectomy does not affect the development of immune responsiveness. In addition, allogeneic pituitary grafts were usually not rejected by unoperated, normal tadpoles or froglets, although they suffered lymphoid invasion to various degrees. However, a significant number of allogeneic pituitaries was rejected when they were grafted, either after or at the same time as the grafting of skin from the pituitary donors. We conclude that the pituitary of Xenopus possesses weak transplantation antigens or expresses them in ways that make them less accessible to the immune surveillance system of the allogeneic host.

Response to skin grafts exchanged among siblings of larval and adult gynogenetic diploids in Xenopus laevis.

Gynogenetic diploid individuals were produced in an anuran amphibian, Xenopus laevis, and their response to skin grafts exchanged among siblings was studied. All skin grafts exchanged among nongynogenetic sibling froglets, as well as those from genetically unrelated donors, were rejected within 30 days. More than half (57%) of the gynogens that received grafts from sibling partners exhibited a prolonged survival (over 30 days), including long-term survival of over 120 days in 13%. The skin grafted from genetically unrelated froglets onto Nieuwkoop and Faber stage 42-56 larvae and onto perimetamorphic stage 58-65 animals was rejected within 30 days. Similarly, most (96%) of the skin grafts from outbred sibling froglets onto larvae at these stages were rejected acutely or subacutely (12-39 days). However, the skin grafted from sibling froglets to gynogens at larval stage 42-56 and perimetamorphic stage 58-61 enjoyed a long-term survival significantly more frequently (81%) than that in the final metamorphic (stage 64-65) counterparts (57%). These results support the view that in the adult Xenopus allograft responses are reactions to a single MHC as well as to cumulative, multiple minor H-locus barriers. The results also suggest that in larval stages the responses against minor H-locus barriers are generated only mildly.

Analysis of allotolerance in thymectomized Xenopus restored with semiallogeneic thymus grafts.

The triploid J strain Xenopus laevis (MHC haplotype, j/j/j) were thymectomized as early larvae, and in the adult stage each animal was given a pair of thymuses or lymphocytes from semiallogeneic diploid donor frogs (j/k). The alloreactivity of host frogs was restored to third-party donors in terms of skin graft rejection and mixed leukocyte reaction, but there was specific unresponsiveness against the k haplotype. Grafting of heavily irradiated (10,000 rads) thymuses also restored host reactivity with induction of tolerance to the k haplotype. In the latter frogs, all thymic and splenic lymphocytes were of host origin. Injection of splenocytes from the restored frogs into secondary thymectomized J frogs not only restored immunocompetence but also transferred specific tolerance to the k haplotype. Injection of lymphocytes from the restored frogs to normal frogs failed to transfer specific tolerance by both in vivo and in vitro immune tests. The results suggest that a selective deletion of the T cell population reactive to the k haplotype was maintained in the T-cell-restored frogs.

Oviducal Contribution to Alteration of the Vitelline Coat in the Frog, Rana japonica. An Electron Microscopic Study.

The vitelline coat (VC) surrounding coelomic eggs of the frog, Rana japonica, comprises bundles of filaments running both parallel and perpendicular to the egg surface. The coat gives little or no staining reaction with PA-CrA-Silver methenamine. In contrast, in the VC of uterine eggs the filament bundles are less conspicuous. and the interstices between the filament bundles stain strongly for carbohydrate. This alteration occurs during passage of the eggs down the first 1/20 th of the oviduct, the pars recta. The epithelium of the p. recta contains secretory cells, which contain electron-dense granules distinct from those in the jelly-secreting cells in more caudal portions of the oviduct. Treatment of coelomic eggs with an extract of p. recta followed by exposure to a sperm suspension resulted in marked swelling and softening of the VC. These results indicate that the contents of the granules secreted from the epithelial cells in the p. recta are deposited in the VC to increase its susceptibility to a fertilizing sperm.

MICROINJECTION OF TOAD SPERM INTO OOCYTES UNDERGOING MATURATION DIVISION.

Spermatozoa of Bufo bufo japonicus were briefly treated with Triton X-100 to remove their plasma membrane, and were injected into oocytes at various stages of maturation division. All the sperm injected into mature coelomic eggs transformed into pronuclei and synthesized DNA, as a normally fertilizing sperm does. The sperm injected into oocytes at the germinal vesicle (GV) stage did not show any change as long as the GV remained intact. In the oocytes which were induced to mature by progesterone, the injected sperm displayed characteristic features in synchrony with those of the resident female nucleus. These included the formation of several sperm-derived chromosomes in association with multipolar spindles in the oocytes from the stage of the germinal vesicle breakdown to the first polar spindle; the appearance of swollen, vesicular nuclei without concomitant DNA synthesis in those at the stage of the first polar body emission; and the reappearance of the condensed chromosomes with giant spindles in those at the stage of the second meiotic metaphase. Pricking of these last oocytes induced the formation of several male pronuclei and DNA synthesis. These results prove that the injection of detergent-treated sperm employed here provides an excellent means of studying the cytoplasmic state that regulates nuclear behavior.

Treatment of low density lipophorin with lipoprotein lipase: diacylglycerol content has no effect on dissociation of apolipophorin III from low-density lipophorin.

The mechanism of the conversion of low-density lipophorin (LDLp) to high-density lipophorin (HDLp) in long-distance flight insects was investigated using a lipoprotein lipase from a bacterium, Alcaligenes sp. Diacylglycerol of LDLp was steadily hydrolyzed in vitro by the lipase, resulting in a 90% loss of diacylglycerol from LDLp during incubation. The "lipase-treated LDLp" thus obtained still contained associated apolipophorin-III (apoLp-III). These data suggest that the dissociation of apoLp-III is independent of the depletion of diacylglycerol from LDLp, and that the decrease in particle diameter caused by the depletion of diacylglycerol does not force the dissociation of apoLp-III from the lipophorin particle. Some physico-chemical properties of the lipase-treated LDLp were measured.

Increases in protein phosphatase 2B activity in lymphoid tissues and T-lymphocytes of autoimmune MRL/MpJ-lpr/lpr mice.

The assay conditions for protein phosphatase 2B (PP2B) in crude extracts from mouse lymphoid tissues and lymphocytes were extensively investigated. Under the conditions elucidated, the PP2B activity was measured in autoimmune-prone MRL/MpJ-lpr/lpr mice (MRL/lpr mice) and two control strains, MRL/MpJ- +/+ mice (MRL/+/+ mice) and C3H/HeJ mice. In the control mice, PP2B activity was distinctly higher in spleen and thymus than brain and liver. PP2B activity was further elevated in spleen of MRL/lpr mice than in the controls. Furthermore, we observed a specific increase in PP2B activity in T lymphocytes from MRL/lpr mice as compared with in those from control mice. On the other hand, no alteration was observed in PP2B activity in B lymphocytes. These results suggest the involvement of PP2B in the abnormal signal transduction and proliferation of T lymphocytes in MRL/lpr mice.

Altering the timing of human chorionic gonadotropin injection according to serum progesterone (P) concentrations improves embryo quality in cycles with subtle P rise.

OBJECTIVE: To investigate the effect of earlier administration of hCG according to serum concentrations of P on the outcome of IVF-ET in cycles in which a subtle rise in serum P (1.0 to 2.0 ng/mL; conversion factor to SI unit, 3.180) occurred during the follicular phase. DESIGN: Retrospective study. PATIENTS: A total of 110 infertile women underwent 124 cycles of IVF-ET at Tottori University Hospital. MAIN OUTCOME MEASURES: Serum was obtained daily or every 12 hours from day 7 until the administration of hCG. Serum E2 and P concentrations were measured by RIA. In 19 of 36 subtle P rise cycles, hCG injection was given when the levels of serum P exceeded 1.0 ng/mL ("rescued" subtle P rise). Parameters of IVF outcomes for the no P rise, the subtle P rise, and the rescued subtle P rise cycles were compared. RESULTS: The mean day of hCG administration in the rescued cycles was 1 day earlier than those of the subtle P rise and no P rise cycles. The mean number of oocytes collected was significantly higher in the subtle P rise and rescued P rise cycles than in the no P rise cycles. The mean follicular diameter on the day of hCG administration was 13.9 mm in the rescued cycles, significantly smaller than those of the no P rise and subtle P rise cycles. However, there was no significant difference in the cleavage rates between the three groups. The rate of embryonic development beyond four-cell stage was increased significantly in the rescued cycles and no P rise cycles versus the subtle P rise cycles. Embryos obtained in the no P rise and rescued cycles were of better morphological quality than those obtained in the P rise cycles. The implantation rate was significantly higher in the rescued cycles than in the P rise cycles. CONCLUSION: The data suggest that, if hCG is administered when a subtle P rise is detected, embryo quality and subsequent implantation rate can be improved.

Some magnesium salts and a mixture of magnesium and calcium salts accelerate skin barrier recovery.

The effects of four different magnesium salts on the cutaneous barrier recovery rate after barrier disruption were evaluated. We spread an aqueous solution of each salt on the flank skin of hairless mice, occluded the area with a plastic membrane for 20 min, and then left the skin surface to dry. All of the magnesium salts, except magnesium bis(dihydrogen phosphate), accelerated barrier repair. We next estimated the effects of magnesium chloride aqueous solutions which contained calcium chloride at different molar ratios. When the calcium to magnesium ratio was lower than 1, the mixture accelerated barrier repair. The application of an aqueous solution of 10 mM magnesium chloride and 10 mM calcium chloride was found to hasten the barrier recovery more effectively than a solution of 10 mM magnesium chloride. These results suggest that the effects of these metal ions are different depending on the counter ion and/or the method of application.

Conformation of nucleoplasmin and its interaction with DNA-protamine complex as a simple model of fish sperm nuclei.

Nucleoplasmin was isolated from Xenopus laevis eggs and purified by an improved method using an open column. Its conformation was investigated spectrophotometrically by UV, CD and fluorescence. It was shown that alpha-helix content of nucleoplasmin was 30-40%, and one of the two tryptophan residues in nucleoplasmin located in the hydrophobic surroundings and the other in the relatively hydrophilic surroundings. The isolated nucleoplasmin was found to decondense sperm nuclei of salmon also, suggesting a possibility of the existence of nucleoplasmin-like protein in fish as well. Collapse of the protamine (salmine)-DNA complex as a simple model for fish sperm nuclei by nucleoplasmin was directly observed by measuring OD320 of aqueous protamine-DNA mixtures. This is a molecular level observation for the removal of protamine from DNA-protamine complex.