AKT1-mediated Lamin A/C degradation is required for nuclear degradation and normal epidermal terminal differentiation.

Nuclear degradation is a key stage in keratinocyte terminal differentiation and the formation of the cornified envelope that comprises the majority of epidermal barrier function. Parakeratosis, the retention of nuclear material in the cornified layer of the epidermis, is a common histological observation in many skin diseases, notably in atopic dermatitis and psoriasis. Keratinocyte nuclear degradation is not well characterised, and it is unclear whether the retained nuclei contribute to the altered epidermal differentiation seen in eczema and psoriasis. Loss of AKT1 function strongly correlated with parakeratosis both in eczema samples and in organotypic culture models. Although levels of DNAses, including DNase1L2, were unchanged, proteomic analysis revealed an increase in Lamin A/C. AKT phosphorylates Lamin A/C, targeting it for degradation. Consistent with this, Lamin A/C degradation was inhibited and Lamin A/C was observed in the cornified layer of AKT1 knockdown organotypic cultures, surrounding retained nuclear material. Using AKT-phosphorylation-dead Lamin A constructs we show that the retention of nuclear material is sufficient to cause profound changes in epidermal terminal differentiation, specifically a reduction in Loricrin, Keratin 1, Keratin 10, and filaggrin expression. We show that preventing nuclear degradation upregulates BMP2 expression and SMAD1 signalling. Consistent with these data, we observe both parakeratosis and evidence of increased SMAD1 signalling in atopic dermatitis. We therefore present a model that, in the absence of AKT1-mediated Lamin A/C degradation, DNA degradation processes, such as those mediated by DNAse 1L2, are prevented, leading to parakeratosis and changes in epidermal differentiation.

A heterozygous null mutation combined with the G1258A polymorphism of SPINK5 causes impaired LEKTI function and abnormal expression of skin barrier proteins.

BACKGROUND: Loss-of-function mutations in the Kazal-type serine protease inhibitor, LEKTI, encoded by the SPINK5 gene cause the rare autosomal recessive skin disease Netherton syndrome (NS). G1258A polymorphism in SPINK5 may be associated with atopic dermatitis, which shares several clinical features with NS. OBJECTIVES: To determine if the phenotype of NS can be caused by a single null mutation in SPINK5 combined with the homozygous G1258A polymorphism. METHODS: We screened mutations in the gene SPINK5 by direct DNA sequencing and position cloning and examined the expressions of the SPINK5-encoded protein LEKTI and other relevant proteins by immunostaining and immunoblot. RESULTS: We describe here a patient who was clinically diagnosed with NS and carried a single null mutation in SPINK5 combined with the homozygous G1258A polymorphism. SPINK5 mRNA was present at normal levels and LEKTI was expressed in the epidermis. Nonetheless, the putative downstream LEKTI substrates stratum corneum trypsin-like enzyme (SCTE), desmoglein 1 and protein markers of keratinocyte differentiation were expressed abnormally, similar to that seen in NS if two null mutant alleles are present. CONCLUSION: This finding indicates that haploinsufficiency of SPINK5 can cause the NS phenotype in the presence of one null mutation with homozygous G1258A polymorphisms in SPINK5, and this could impair the function of LEKTI and therefore acts as a true mutation.

Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing.

Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein-protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels.

Aberrant gating, but a normal expression pattern, underlies the recessive phenotype of the deafness mutant Connexin26M34T.

Mutations in the gene GJB2, encoding the gap junction protein Connexin26 (Cx26), are the most prevalent cause of inherited hearing loss, and Cx26M34T was one of the first mutations linked to deafness (Kelsell et al., 1997; Nature 387, 80-83). We report the first characterization of the gating properties of M34T, which had previously been reported to be nonfunctional. Although homotypic mutant channels did not produce detectable currents, heterotypic pairings with wtCx26 confirmed that M34T formed intercellular channels, although the gating properties were altered. Cx26M34T displayed an inverted response to transjunctional voltage (Vj), mediating currents that activate in a time- and Vj-dependent manner. These characteristics suggest that the channel population is only partially open at rest, consistent with previous reports that dye transfer in M34T-expressing cells is reduced or abolished (e.g., Thonnissen et al., Human Genet. 111, 190-197). To investigate the controversial recessive/dominant behavior of this mutant, we coexpressed M34T with wtCx26 RNA at equimolar levels, mimicking the situation in heterozygotic individuals. Under these conditions, M34T did not significantly reduce Cx26/Cx26 coupling, or alter the electrophysiological properties of the wt channels, consistent with the recessive nature of the allele. Overexpression of the mutant did have some inhibitory effects on conductance, possibly explaining some of the previous reports in exogenous expression systems and some patients. Consistent with its electrophysiological behavior, we also show that M34T localizes to cell junctions in both transfected HeLa cells and patient-derived tissue.

Multiple epidermal connexins are expressed in different keratinocyte subpopulations including connexin 31.

Recent genetic studies have demonstrated the importance of epidermal gap junctions with mutations in four beta-connexins associated with autosomal dominant epidermal disease. One of these disorders, erythrokeratoderma variabilis, is associated with germline mutations in the genes encoding connexins (Cx) Cx31 and Cx30.3. Towards understanding the functional mechanism of Cx31 mutations in epidermal disease, we have developed and characterized a polyclonal antibody raised against human Cx31. Using this antibody to immunostain normal epidermis, Cx31 protein was found to be expressed predominately in the stratum granulosum with a punctate pattern of staining at the plasma membrane. In addition, we used reverse transcriptase polymerase chain reaction and, where reagents were available, immunocytochemistry to investigate which other connexins are expressed in the epidermis. Surprisingly, this analysis revealed that there are at least 10 connexins expressed with an overlapping distribution and localization to distinct keratinocyte subpopulations. These data provide additional evidence for multiple gap junction channel types in the human epidermis. Elucidation of this complexity of channel types with respect to specific permeabilities and function of each wildtype and mutant channel type in epidermal biology will require further investigations.

Oestriol and oestradiol increase cell to cell communication and connexin43 protein expression in human myometrium.

Oestradiol increases the protein expression of connexin43 (Cx43) gap junctions in myometrium but the effect of oestriol on gap junction expression has not been described previously. Oestriol is the most abundant free oestrogen in pregnant women and there is a marked surge in oestriol concentrations before term and idiopathic preterm labour. In order to determine whether oestriol may have a physiological action on the myometrium, cultured human myometrial cells obtained from non-pregnant hysterectomy specimens were exposed to 10 nmol/l oestradiol or oestriol. Intercellular communication between myometrial cells was investigated by microinjection of confluent cultured cells with the gap junction-permeant tracer Cascade Blue. There was a progressive increase in coupling after exposure to oestradiol or oestriol (P < 0.0005). An increase in Cx43 protein expression was demonstrated by immunocytochemistry after 1 h (P < 0.01) and 3 days (P < 0.01) exposure, and by Western blotting after 1 h (P < 0.01) and 3 days (P < 0.05) exposure, to both oestradiol and to oestriol. We conclude that oestriol increases gap junction communication in human myometrium by increasing gap junction expression. Elevated oestriol concentrations may thus play a role in the initiation of labour in women, by increasing cell-cell communication in the myometrium.

Genetic analysis of the connexin-26 M34T variant: identification of genotype M34T/M34T segregating with mild-moderate non-syndromic sensorineural hearing loss.

Mutations in the human gap junction beta-2 gene (GJB2) that encodes connexin-26 have been shown to cause non-syndromic sensorineural hearing loss (NSSNHL) at the DFNB1 locus on 13q11. Functional and genetic data regarding the disease causing potential of one particular GJB2 sequence variant, 101 T-->C (M34T), have proven contradictory. In this study, we found the prevalence of the M34T allele in a cohort of white sib pairs and sporadic cases with NSSNHL from the United Kingdom and Ireland to be 3.179% of chromosomes screened. Significantly, we identified the first M34T/M34T genotype cosegregating in a single family with mid to high frequency NSSNHL. Screening a control population of 630 subjects we identified 25 M34T heterozygotes; however, no M34T homozygotes were detected. Surprisingly, the majority of M34T alleles (88%) were in cis with a 10 bp deletion in the 5' non-coding sequence. This non-coding deletion was also homozygous in the homozygous M34T subjects. Microsatellite analysis of flanking loci in M34T heterozygotes and controls does not define an extensive ancestral haplotype but preliminary data suggest two common alleles in subjects with the M34T allele. In summary, we provide data that support M34T acting as a recessive GJB2 allele associated with mild-moderate prelingual hearing impairment.

Connexin 26 expression and mutation analysis in epidermal disease.

Gap junctional communication has a key role in the co-ordination of keratinocyte differentiation. Multiple connexins are expressed in the epidermis and mutations in four of these connexins are associated with disorders of keratinisation. Specific autosomal dominant Cx26 mutations have been associated with syndromes of skin disease and hearing loss. Here we describe the characterization of a new Cx26 polyclonal antibody raised against the cytoplasmic region of the protein. It has been used to investigate Cx26 protein localization in epidermal disease and in the study of mutant Cx26 proteins.

The pineal and extra-pineal origins of 5-sulphatoxy N-acetyl-serotonin in humans.

In humans 6-sulphatoxy melatonin (SaMT) is the principal metabolite of endogenous and exogenous melatonin. 5-sulphatoxy N-acetyl-serotonin (SNAS) is a minor metabolite of exogenous melatonin, but it has not been established whether the levels of endogenous SNAS in plasma derives principally from endogenous melatonin. We have developed the first radioimmunoassay (RIA) for SNAS and used it (together with RIAs for melatonin and SaMT) to determine whether endogenous SNAS derives from endogenous melatonin or from platelet serotonin. Our results show a) the values of endogenous SNAS, unlike endogenous SaMT, increased with blood collection procedures that increased the values of serotonin, b) the values of endogenous SNAS in urine or in platelet-poor plasma were approximately the same as those of endogenous SaMT, but, unlike SaMT, did not show a diurnal rhythm, and c) we confirmed that SNAS was a minor metabolite of orally ingested melatonin. Thus, our conclusion is that SNAS is a minor metabolite of exogenous melatonin, but is not a significant metabolite of endogenous melatonin. In all probability, endogenous SNAS is principally the metabolite of platelet serotonin.

Elevated nocturnal melatonin is a consequence of gonadotropin-releasing hormone deficiency in women with hypothalamic amenorrhea.

Elevated nocturnal melatonin is found in women with idiopathic hypogonadotropic hypogonadism (IHH), but it is not known whether this is implicated in the etiology of their GnRH deficiency. It is unlikely that nocturnal melatonin can be implicated in the etiology of the GnRH deficiency of Kallmann's syndrome (KS), because this condition is caused by defective neuronal migration in embryonic life. We therefore measured nocturnal melatonin in women with IHH and KS to determine whether it was elevated in one or both conditions and thereby to determine whether it was implicated as cause or consequence of GnRH deficiency. Four women with IHH, 3 women with KS, and 7 individually matched (age and body size) controls were recruited. Frequent day- and nighttime samples were taken for LH pulsatility studies. All patients showed absent or diminished LH pulsatility, compared with their respective controls. Samples were also taken over 24 h for melatonin and 6-sulphatoxymelatonin (the principle metabolite of melatonin and an independent marker of its secretion). Melatonin and 6-sulphatoxymelatonin levels were elevated in 6 of 7 patients (compared with their matched controls) and were significantly elevated in the KS group (compared with their controls). The finding of elevated nocturnal melatonin (and its metabolite) in GnRH-deficient women with KS (as well as IHH) suggests that nocturnal melatonin is elevated as a consequence of GnRH deficiency, irrespective of its etiology.

Radioimmunoassay of bound and free melatonin in plasma.

We describe a nonextraction procedure, and two extraction procedures, for RIA of melatonin in human plasma. All procedures showed a diurnal rhythm of melatonin in human subjects, with interindividual differences greater than interprocedure differences. However, further investigations demonstrated considerable variability of recovery in the nonextraction procedure, suggesting a variability of binding proteins between samples. Combining recovery and dialysis experiments in the extraction procedures, we demonstrated that chloroform was unable to extract albumin-bound melatonin from a human serum albumin solution but, paradoxically, was able to extract bound and free melatonin from a plasma sample. The methanol extraction procedure extracted free and bound melatonin from all sources. These results indicate that albumin binding can substantially affect the RIA procedures. We conclude that assays should be validated against free and bound melatonin and that the two forms should be independently investigated when assessing bioactivity.

An enzyme immunoassay for 6-sulphatoxy-melatonin in human urine.

We describe a newly developed enzyme immunoassay (EIA) for the determination of 6-sulphatoxy-melatonin (aMT6s) in human urine, using a aMT6s-bovine serum albumin-horseradish peroxidase (aMt6s-BSA-HRP) conjugate as the enzyme label. The assay incorporates a highly specific antibody raised in rabbits. The EIA has a sensitivity of 2 pg/well (40 pg/ml) with intraassay coefficients of variation of 2.3-6.1% in the range of the assay. The material with the highest level of cross-reactivity was N-acetyl serotonin sulphate, with a relative potency of 0.000078%. One hundred thirty-four urine samples from children and adults at different time points were assayed and the results compared with those from an established radioimmunoassay (RIA) and with a newly developed RIA using the same antibody as the EIA. The correlation coefficient, r, comparing the two RIA's was 0.9869, and the regression equation was log (kit) = 0.9340 log (new) + 0.1213. The correlation coefficient, r, comparing the EIA with the newly developed RIA, was 0.9686, and regression equation log (new) = 0.9674 log (EIA) + 0.0600. The EIA for the measurement of aMT6s in urine represents a new approach in the investigation of pineal function.

The purification and characterization of biological 6-sulphatoxymelatonin and comparison with synthetic 6-sulphatoxymelatonin.

We have purified the major metabolite of melatonin, 6-sulphatoxymelatonin, from urine and compared it to its synthetic counterpart. For preparation of the biological material, oral melatonin was administered to human volunteers and their urine extracted onto Amberlite XAD-2 resin to remove urea; the glucuronide metabolites of melatonin were removed by silica chromatography; and 6-sulphatoxymelatonin was separated from N-acetyl serotonin sulphate, the other sulphate metabolite of melatonin, by preparative thin-layer chromatography. Synthetic 6-sulphatoxymelatonin was produced by reacting 6-hydroxymelatonin with chlorosulphonic acid in dimethylformamide; the reaction mixture was purified on Florisil and preparative thin-layer chromatography was used to remove indolic by-products of the reaction. Elemental and X-ray microanalysis of the biological and synthetic products showed that classical methods used for their purification introduced inorganic impurities, such as silicon- and chlorine-containing compounds, which were not detectable by thin-layer chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, or gas chromatography-mass spectrometry. We introduced further purification steps to remove these inorganic impurities, monitoring the process using elemental and X-ray microanalysis. Extensive characterization of the resulting purified products showed that the biological and synthetic compounds were identical.

Evidence for the Bauman variant in Kallmann's syndrome.

BACKGROUND AND OBJECTIVE: Idiopathic hypogonadotrophic hypogonadism (IHH) is a condition of gonadotrophin releasing hormone (GnRH) deficiency. IHH associated with anosmia is Kallmann's syndrome. A variant has been described by Bauman where a patient with Kallmann's syndrome apparently regained normal hypothalamo-pituitary function 2 years after the initial diagnosis. GnRH secretory activity can be assessed by measuring LH pulsatility. Our objective was to define the pattern of LH pulsatility in men with IHH and Kallmann's syndrome compared with those of normal controls, and to determine whether there is evidence for a Bauman variant of Kallmann's syndrome. DESIGN: Patients with IHH and Kallmann's syndrome were recruited from the endocrine clinic. Long-term hormone replacement therapy was discontinued. LH pulsatility was determined. PATIENTS: Three men with IHH, 3 men with classical Kallmann's syndrome and 5 normal male volunteers. MEASUREMENTS: Baseline serum FSH, LH and testosterone. Intensive blood sampling every 10 minutes for serum LH from 1000 to 1600 h during the day and 2200 to 0400 h during the night to measure LH pulsatility. RESULTS: The volunteer group showed normal LH pulsatility. In the patient group, LH secretion was apulsatile in one, showed significantly diminished amplitude in four, and there was normal pulsatility in one patient which remained normal 5 months later. CONCLUSION: Three patients with idiopathic hypogonadotrophic hypogonadism and 2 with Kallmann's syndrome had variable degrees of GnRH deficiency. One patient with Kallmann's syndrome had apparently normal GnRH activity, which remained normal 5 months later. This patient appears to have the Bauman variant of Kallmann's syndrome.

HPLC assay of melatonin in plasma with fluorescence detection.

We report an HPLC assay for melatonin that incorporates automated injection, methanol/water mobile phase, and fluorescence detection. Plasma samples were extracted by solid and liquid phases. Recovery was > 70% for 1-10 mL of plasma extracted, approximately 40 pg-250 ng of melatonin. Samples were dried and reconstituted in 100 mL/L methanol. Injections were 25 microL or 150 microL, depending on sample concentration, and the melatonin peak was eluted in 380 mL/L methanol. The detection limit of the assay was 6 pg on the column, allowing a practical sensitivity in plasma of 11 pmol/L for 8-mL samples and 34 pmol/L for 2-mL samples. More than 100 plasma samples from volunteers and patients were assayed and the results compared with an established RIA. The mean daytime concentration of melatonin was 20.7 pmol/L (SEM = 1.2) and 18.5 pmol/L (SEM = 1.6) for HPLC and RIA, respectively, and the mean nighttime concentration was 82.4 pmol/L (SEM = 6.5) and 82.2 (SEM = 7.3), respectively.

[Effects of anisodamine on blood coagulation, fibrin and thrombosis in rabbits].

Anisodamine at dose of iv 10 and 20 mg/kg prolonged plasma prothrombin time, bleeding time and coagulation time, and showed positive reaction of plasma protamine paracoagulation test in conscious rabbits. The drug prolonged the thrombin time and reduced the contend of plasma fibrin. Anisodamine markedly inhibited blood platelet aggregation both in vitro and in vivo, dose-dependently. The extracorporeal thrombosis time was prolonged, the length and weight of thrombus was decreased after iv anisodamine 20 mg/kg. These results suggest that anisodamine has inhibitory action thrombosis and blood coagulation.