Abnormal copper metabolism in menkes cultured fibroblasts

Menkes fibroblast cultures were established and copper metabolism was investigated. Menkes fibroblasts contained four to six times higher concentrations of copper than control cells. In Menkes cells more than 90% of the intracellular copper was present in cytosol (105,000 x g supernatant fraction); in control fibroblasts the corresponding value was about 67%. During cultivation of fibroblasts in medium supplemented with 100 ng/ml of copper (270 ng CuCl₂·2H₂O /ml), the amount of copper increased continuously in Menkes cells, at least up to 4 days, while in control cells in reached a maximum after 24 h, followed by a gradual decrease. When the medium was replaced with one without copper chloride, copper concentrations in Menkes cells returned to the original level in three days, whereas those in control cells returned to the normal level in one day. Using Sephadex G-75 column chromatography of cytosol, two copper-containing peaks were observed (peaks 1 and 3, corresponding to the peaks from rat liver cytosol). Approximately 75% of the copper in the cytosol from Menkes cells was eluted in peak 3. The corresponding copper peak was very small in control cells. Copper peaks 1 and 3 from both cells increased after treating cells with copper chloride and the increase was inhibited by cycloheximide, an inhibitor of protein biosynthesis.Metallothionein purified from human kidneys was eluted as a single copper-protein from a Sephadex G-75 column in the same fractions as peak 3. SDS polyacrylamide-slab gel electrophoresis of the purified metallo-thionein and the material eluted in peak 3 from Menkes fibroblasts showed single peaks for copper at identical migration distances.     

Induction of hair growth by skin irritants and its relation to skin protein kinase C isoforms

Induction of hair growth by skin irritants and its relation to skin protein kinase C (PKC) isoforms were evaluated. Dorsal skin of BALB/c mice was shaved and anthralin (0.1% in corn oil) was applied on one side of the shaved backs in 20 mice daily for 5 days. The corresponding opposite side treated with corn oil served as a control. In another 20 mice, sodium dodecyl sulphate (SDS, 10% in water) was applied on one side of the shaved backs for 5 days by the same procedure as above and the corresponding opposite side treated with water served as control. Visible acceleration of hair growth on anthralin-treated skin was observed as early as 14 days after the application of anthralin and significant hair growth was observed at about 17-20 days. Enhancement of hair growth on SDS-treated skin was observed at about 3 weeks from the beginning of the treatment. None of the mice showed remarkable hair growth on the control side in either group. Mouse skin PKC isoforms levels detected by Western blot showed a similar pattern in both treatment groups. PKC alpha was downregulated initially, and was then elevated from 3 days after anthralin treatment and 14 days after SDS application. PKC beta was unchanged initially, decreased at 8 and 14 days after anthralin and SDS treatment, respectively, and reverted to the control level at 25 days after anthralin treatment, when it was still lower than the control in SDS-treated skin. PKC delta was also unchanged at first, but was elevated from 3 days after anthralin treatment and 14 days after SDS application. These results suggest that involvement of PKC may be a general phenomenon in irritant-induced hair growth in mice. Considering the stimulatory effect of PKC alpha and inhibitory effect of PKC delta on cell growth, we postulate that PKC alpha may be responsible for enhancement of hair growth while PKC delta may inhibit hair growth to keep the hair growth in balance.     

Abortive synaptogenesis as a factor in the inner hair cell degeneration in the Bronx Waltzer (bv) mutant mouse

The bronx waltzer (vb) mutation in the mouse results in the degeneration of most but not all of the primary auditory receptors, the inner hair cells, and their afferent neurons. We analyzed the ultrastructure of 94 inner hair cells in the intact postnatal mutant mouse and in neonatal cochleas in culture to understand the pathogenesis of hair cell death and to detect factors that may prevent it. The vb spiral neurons of the bronx waltzer display two distinctive features: some of them continue to divide mitotically for at least seven postnatal days, and the type I radial fibers that innervate inner hair cells display a deficiency in immunoexpression of GAD. The growing endings of spiral neurons converge around the inner hair cells or, in their absence, invade the outer hair cell region. Their profuse sprouting among inner spiral sulcus cells contributes to the characteristic ultrastructural picture of the bv cochlea. During the first three days after birth, 40% of the inner hair cells appear normal and innervated, 40% are mostly denervated and degenerating, and 20% are immature, with minimal or no neuronal appositions. However, in mutants 6 days and older only a few inner hair cells survive, and these show either normal or superfluous afferent innervation and axosomatic GABAergic efferent innervation. Degeneration of inner hair cells begins with a distention of the nuclear envelope and the ribosomal endoplasmic reticulum. The outer nuclear membrane eventually breaks, and exudate fills the cell interior. The cellular edema leads to cell death. We propose that success or failure in synaptic acquisition is a decisive factor in the survival or decline of the mutant inner hair cells. We also suggest that the developmental delay in maturation of the spiral ganglion neurons (type I) and the failure in their synaptogenesis may be caused by an impairment in neurotrophin (NT3/BDNF) synthesis by their mutant receptor cells.     

Hair analysis underestimates heroin use in prisoners

The value of hair analysis in measuring treatment outcome was examined in a randomised controlled trial (RCT) of an Australian state prison-based methadone programme between 1997 and 1998 (n = 382 male prisoners). Hair samples were analysed for morphine using immunoassay techniques. Agreement between hair analysis and self-report was tested using kappa, McNemar's test of symmetry and Pearson's correlation coefficient r. Hair analysis based on immunoassay was inadequate as the primary outcome measure for the RCT but had value in supplementing self-reported heroin use. There was a modest correlation (r = 0.31, p < 0.001) between self-reported frequency of heroin use and morphine concentrations in hair. Sectional hair analysis, a reflection of duration of drug use, was uninformative and generally impractical due to the length of hair sections needed.     

Cellular localization of voltage-gated calcium channels and synaptic vesicle-associated proteins in the guinea pig cochlea

The cellular localization of voltage-gated calcium channels (VGCCs) and synaptic vesicle-associated proteins, SV2, synapsin I, and vesicle-associated membrane protein (VAMP) (synaptobrevin), was investigated in the guinea pig cochlea using immunocytochemistry and confocal laser scanning microscopy. Reactivity, in guinea pig, of antibodies to the alpha1 subunits of L-type, alpha1C [Cav1.2] and alpha 1D [Cav1.3]; P/Q-type, alpha1A [Cav2.1]; and R-type, a1E [Cav2.3] high voltage-activated calcium channels, was determined by Western blotting and immunolabeling of cerebellum. In the cochlea the sensory inner hair cells of the organ of Corti displayed strong intracellular staining, predominantly localized to their basolateral poles, with an antibody directed against the alpha1C subunit. Some alpha1C labeling was also observed in the inner pillar cells, in cell bodies of afferent neurons in the spiral ganglion, and in the inferior region of the spiral ligament. The supporting pillar cells were strongly immunoreactive throughout for alpha1D, but no alpha1D labeling of the inner hair cells was seen. The alpha1A subunit showed a cytoplasmic distribution in all three rows of outer hair cells. alpha1E labeling localized to the outer hair cells, predominantly in the subcuticular plate region, and also to nerve fiber bundles beneath these hair cells. Strong immunoreactivity was consistently seen with antibodies directed against SV2 and synapsin I in neuronal structures surrounding the basolateral surfaces of both the inner and outer hair cells but was absent from the sensory cells themselves. VAMP labeling was found throughout the cytoplasm of the inner hair cells and in neuronal structures beneath the hair cells. These results reveal a differential distribution of VGCC-types in the sensory and nonsensory elements of the guinea pig cochlea, with the inner hair cells expressing alpha1C L-type channels and VAMP but not synapsin I or SV2.     

Mice lacking Dfna5 show a diverging number of cochlear fourth row outer hair cells

A complex mutation in DFNA5, resulting in exon 8 skipping, causes autosomal dominant hearing impairment, which starts in the high frequencies between 5 and 15 years of age and progressively affects all frequencies. To study its function in vivo, Dfna5 knockout mice were generated through the deletion of exon 8, simultaneously mimicking the human mutation. To test the hearing impairment, frequency-specific Auditory Brainstem Response (ABR) measurements were performed at different ages in two genetic backgrounds (C57Bl/6J and CBA/Ca), but no differences between Dfna5-/- and Dfna5+/+ mice could be demonstrated. Morphological studies demonstrated significant differences in the number of fourth row outer hair cells between Dfna5-/- mice and their wild-type littermates. These results were obtained in both genetic backgrounds, albeit with opposite effects. In contrast to the results obtained in Dfna5-/- zebrafish, we did not observe different UDP-glucose dehydrogenase and hyaluronic acid levels in Dfna5-/- mice when compared to Dfna5+/+ mice.     

Topical application of ketoconazole stimulates hair growth in C3H/HeN mice

Ketoconazole (KCZ) is an imidazole anti-fungal agent that is also effective in topical applications for treating seborrheic dermatitis and dandruff. Recently, topical use of 2% KCZ shampoo has been reported to have had a clinically therapeutic effect on androgenetic alopecia. The present study was conducted with the purpose of quantitatively examining the stimulatory effect of KCZ on hair growth in a mouse model. Coat hairs on the dorsal skin of seven week-old male C3H/HeN mice were gently clipped, and either 2% KCZ solution in 95% ethanol or a vehicle solution was topically applied once daily for three weeks. The clipped area was photographed, and the ratio of re-grown coat area was then calculated. The results demonstrated that 2% KCZ had a macroscopically significant stimulatory effect compared with the vehicle group (p<0.01, n=10). Repeated experiments showed similar effects, confirming the efficacy of KCZ as a hair growth stimulant. Although the therapeutic mechanism of topical KCZ for hair growth is unclear, our results suggest that topical applications of the substance are useful for treating seborrheic dermatitis accompanied by hair regression or male pattern hair loss.     

Structural optimization of pep7, a small peptide extracted from epimorphin, for effective induction of hair follicle anagen

Epimorphin is representative of a unique class of stromal membrane-anchored proteins that plays distinct functions depending on its membrane topology. When exposed extracellularly, this molecule acts as a morphoregulator for various tissues including hair follicle epithelia. Previous study identified its functional domain (the pep7 domain: SIEQSCDQDE) for hair follicular morphogenesis followed by the successful generation of a chemically modified active peptide. Here, we report optimization of this peptide by the introduction of sequential mutations and subsequent structural determination. We found that three residues from the C-terminus are dispensable, and alternation of the seventh amino acid to an Alanine residue enhanced activity. To favour the biologically active conformation, epsilon-Acp (NH(CH(2))(5)CO) linked to a Cysteine residue was connected at the N-terminus followed by the introduction of an intramolecular disulphide bridge, the modification process of which could be included in the peptide synthesis. The obtained modified peptide, termed 'EPM (epimorphin-derived) peptide', has a Mw of 950 Da and exerts an inductive effect on hair follicle regeneration at a concentration of approximately 0.00001% or even lower. The action of this EPM peptide was more apparent in mice treated with 1% minoxidil, suggesting its potential clinical benefit as a new type of hair-regenerating agent.     

Influence of prostaglandin F2alpha and its analogues on hair regrowth and follicular melanogenesis in a murine model

Latanoprost and isopropyl unoprostone, which are analogues of prostaglandin F2alpha (PGF2alpha), are promising drugs for the reduction of intra-ocular pressure. However, they have been reported to have side effects, including hypertrichosis and hyperpigmentation of the eyelashes and periocular skin, and occasionally poliosis. In order to investigate these effects further, PGF2alpha, latanoprost and isopropyl unoprostone were applied to the dorsal skin of 7-week-old C57BL/6 mice, and hair length was measured during the treatment. The three molecules all showed stimulatory effects on the murine hair follicles and the follicular melanocytes in both the telogen and anagen stages, and stimulated conversion from the telogen to the anagen phase. PGE2 is known to act synergistically with PGF2alpha, and hence the influence of PGE2 was also examined. PGE2 did not induce distinct telogen-to-anagen conversion, but showed moderate growth stimulatory effects on early anagen hair follicles. In addition, we observed a case of hypertrichosis and trichomegaly with an excess of melanogenesis, leading to the emergence of white hair, suggesting that poliosis can occur as a side effect of eye treatment with solutions of PGF2alpha analogues. The stimulatory effects of PGF2alpha and PGE2 on hair growth have been discussed with regard to the role of protein kinase C and mast cells.