Highly-expressed S100A3, a calcium-binding protein, in human hair cuticle.

Analyses on sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that the human hair cuticle extracts mainly consist of a 7-kDa component and keratin proteins. The S-carboxymethylation of the cuticle extracts made the 7-kDa band shift to the 15-kDa position. After electroblotting of the S-carboxymethyl derivative, the membrane pieces carrying the 15-kDa band were treated with trypsin and the released peptides were separated by reverse-phased HPLC. Amino acid sequence analyses revealed that the peptides corresponded to the partial sequences deduced from human genome coding for S100A3, a cysteine-rich calcium binding protein. The anti S100A3 serum, prepared by immunizing a synthetic peptide antigen, reacted with the 7-kDa and 15-kDa bands in immunoblotting analyses. Immunofluorescence microscopy showed intense labeling to the cuticular layer with the anti S100A3 serum. These results indicated that S100A3 was highly expressed in the human hair cuticle.

Expression of calcium-binding S100 proteins A4 and A6 in regions of the epithelial sac associated with the onset of hair follicle regeneration.

In order to elucidate the onset mechanisms of hair regrowth, we characterized the expression of metastasis and cell-growth-associated calcium-binding S100 proteins in the regenerating follicular epithelium. Hair-cycle-dependent expression of S100A4 and S100A6 was found in the epithelial sac regions (bulge area and hair germ) of mouse pelage follicles. Protein localization of S100A4 was confined to the bulge area, the region where the presumed follicular stem cells reside, during the catagen-telogen-anagen transitional periods, whereas S100A6 protein was distributed throughout the epithelial sac regions during anagen phase. Prior to entering anagen phase, however, both S100 mRNAs were upregulated in the epithelial sac. Despite the induction of extensive cell death in the bulge region after plucking, a new hair cycle was initiated following transcription of S100A6 in the hair germ. Upon wounding stimuli, both the outer root sheath and the basal layer of epidermis expressed S100A6 mRNA prior to hyper-proliferation. Once the epithelial sac was induced to transcribe both S100 genes, resting follicles were concomitantly rejuvenated. These results suggest that S100A4 and S100A6 might play important roles in the activation of stem cells at the onset of follicle regeneration.

Gene expression of mouse S100A3, a cysteine-rich calcium-binding protein, in developing hair follicle.

We have previously identified a cysteine-rich calcium binding protein S100A3 present in the cuticle of human hair fiber. In this study, we cloned a cDNA for mouse S100A3, identified its gene location, and elucidated the expression profile throughout hair follicle development. The mouse S100A3 gene was clustered with other S100 family members on chromosome 3, and specifically expressed in dorsal skin containing hair follicles. The level of S100A3 mRNA was elevated during the anagen phase of the hair growth cycle, and sharply declined from the regression phase on. In situ hybridization revealed that the S100A3 gene was prominently expressed in cuticular cells of the hair follicle, and mRNA levels were highest in the keratogenous zone over the entire cuticular layer. Expression was also observed to a lesser extent in differentiated cortical cells; however, expression was not observed in any other component of the hair follicle or dorsal tissues. Immunohistochemical analysis showed that the S100A3 protein accumulated in cuticular and cortical cells undergoing terminal differentiation. These results indicate that the S100A3 gene is exclusively expressed, and the translation product retained, in follicular cells differentiating into major components of the hair shaft. It seems likely that S100A3 plays an important role in calcium-dependent processes leading to hair shaft formation.

Ultrastructural localization of S100A3, a cysteine-rich, calcium binding protein, in human scalp hair shafts revealed by rapid-freezing immunocytochemistry.

We have characterized the subcellular distribution of S100A3, a cysteine-rich calcium binding protein, in human scalp hair shaft. This was accomplished using rapid-freezing immunocytochemistry, a technique that combines rapid-freezing, freeze-substitution fixation without chemical fixatives, and subsequent electron microscopic detection of immunocytochemical labeling. This technique preserves both the antigenicity and the ultrastructural integrity of fully keratinized tissues, which are highly unmanageable when prepared for immunoelectron microscopy. In the hair shaft, S100A3 was primarily identified in the endocuticle and was also present in the intermacrofibrillar matrix surrounding macrofibril bundles of intermediate filament keratins in cortex cells. Double immunolabeling of S100A3 and hair keratins revealed the in situ spatial relationship between them. In the endocuticle, S100A3 was present on the inner portion of the endocuticle adjacent to the cell membrane complex, whereas hair keratins were present on the outer portion. These results provide the first ultrastructural evidence that an S100 protein is localized in specific subcompartments in human hair cells. (J Histochem Cytochem 47:525-532, 1999)

X-ray study of baker's yeast lipoamide dehydrogenase at 4.5 A resolution by molecular replacement method.

The molecular structure of lipoamide dehydrogenase from baker's yeast has been determined at 4.5 A resolution by molecular replacement techniques using the known structure of human erythrocyte glutathione reductase as a starting model. The enzyme crystallizes in the space group P2(1)2(1)2(1) with a = 98.6(2), b = 162.0(2), c = 69.4(2) A. There is one molecule per asymmetric unit. The enzyme is a dimeric protein of identical subunits related by a local two-fold symmetry. Comparison of the tertiary structures between glutathione reductase and the present enzyme shows that the folding is almost the same except for the N and C termini, although some slight shortening or shifting of alpha-helices was found in the electron density map. FAD molecules are found at similar positions to those of glutathione reductase. Since the amino acid residues around FAD and NAD binding sites and at the reaction centers of the two enzymes are strongly conserved, the lipoamide dehydrogenase may catalyze the opposite reaction through a similar mechanism to that proposed for glutathione reductase. The newly found C terminus is located near the edge of a deep cave at the interface between the two subunits. These additional 18 residues form a narrow entrance to the cave, in which the long chain of the dihydrolipoyl moiety of lipoate acetyltransferase will be bound.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 19). Effects of two-week repeated dosing of enoxacin on the male reproductive organs.

The toxicity of Enoxacin (ENX), a fluoroquinolone antibacterial agent, on the testis and epididymis was studied in rats. ENX was administered to 5 male rats orally once daily for 2 weeks at the dose level of 3000 mg/kg/day. ENX-treated rats showed a marked decrease in body weight, and two of them died on Day 10. At the end of the dosing period, absolute weights of the epididymis were decreased; in contrast, relative weights of testis were increased in the ENX-treated group. On histopathological examination, testis of ENX-treated rats exhibited the following regressive changes: degeneration of spermatids and spermatocytes, retention of Step 19 spermatids, chromatin margination in nuclei of spermatids, multinucleated giant cell formation, and/or vacuolar degeneration of Sertoli cells. Additionally, desquamated cell debris was observed in the epididymis. Degenerative spermatids and spermatocytes were strongly positive by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). From these results, it is concluded that a 2-week treatment is sufficient to detect toxic effects of ENX on reproductive organs in male rats, and that testicular toxicity induced by ENX is associated with germ cell apoptosis.

[Nephrotoxicity of piperacillin combined with furosemide in rats].

Nephrotoxicity of piperacillin (PIPC) was evaluated in rats after combined administration with furosemide. After intravenous administration of PIPC (1600 mg/kg), the rats showed no change in urinalysis, biochemical analysis of plasma and histopathological analysis. The rats receiving furosemide (100 mg/kg) showed elevation of urinary NAG, BUN and creatinine concentrations, and showed slight degeneration of the renal proximal tubules. The rats receiving PIPC (1600 mg/kg) and furosemide (100 mg/kg) showed elevation of BUN and creatinine concentrations, and showed slight degeneration of the proximal tubules. These changes were comparable to those in rats receiving furosemide alone. The rats receiving cephaloridine (1600 mg/kg) showed elevation of urinary protein, BUN and creatinine concentrations, and showed moderate degeneration and necrosis of the proximal tubules. The nephrotoxicity was enhanced by combination with furosemide. In conclusion, no enhanced effect of nephrotoxicity was observed by combination of PIPC with furosemide.

Expression and translocation of aquaporin-2 in the endolymphatic sac in patients with Meniere's disease.

Meniere's disease, characterised by episodic vertigo, fluctuating hearing loss and tinnitus, can occur under conditions of stress. Its pathology was first revealed to be inner ear hydrops through temporal bone studies in 1938. Although its pathogenesis has been proposed to be a disorder of water transport in the inner ear, subsequently, it remains unsolved, until now. A recent study revealed that both plasma stress hormone, vasopressin (pAVP) and its receptor, V2 (V2R) expression in the inner ear endolymphatic sac were significantly higher in Meniere's patients. In the present study, to link V2R-related molecules and inner ear hydrops, we examined V2R-linked water channel molecule, aquaporin-2 (AQP2) expression and translocation in human endolymphatic sac. AQP2 mRNA expression in the endolymphatic sac was significantly higher in Meniere's patients by using real-time polymerase chain reaction, as further confirmed by western blotting. AQP2-like immunoreactivity (-LIR) was translocated from luminal to basolateral side with endosomal trapping in the endolymphatic sac at the time of AVP exposure in human endolymphatic sac tissue culture. The similar AQP2-LIR translocation was also demonstrated by forskolin and blocked by vasopressin/V2R specific antagonist, OPC31260 and protein kinase A (PKA) specific antagonists, H-89 and KT-5720. We concluded that in the pathogenesis of inner ear hydrops resulting in Meniere's attacks, pAVP elevation as a result of stress and subsequent V2R-cAMP-PKA-AQP2 activation and endosomal trapping of AQP2 in the endolymphatic sac, might be important as a basis of this disease. Further experimental and clinical studies are needed to better clarify the neuroscientific relationship between stress and Meniere's disease.

Behavioral assessment and identification of a molecular marker in a salicylate-induced tinnitus in rats.

Tinnitus is a non-observable phantom sensation. As such, it is a difficult condition to investigate and, to date, no effective treatment has been developed. To approach this phantom sensation, we aimed to develop a rat behavioral model of tinnitus using salicylate, an active component of aspirin known to induce tinnitus. We also aimed to establish a molecular marker of tinnitus by assessing the expression of transient receptor potential cation channel superfamily V-1 (TRPV1) in the rat auditory pathway during salicylate-induced tinnitus. Animals were trained to perform "an active avoidance task": animals were conditioned by electrical footshock to move to the other side of the conditioning box when hearing a sound. Animals received a single injection of saline or salicylate (400 mg/kg i.p.) and false positive responses were measured 2 h after injection as the number of movements during a silent period. The number of responses in salicylate-treated animals was highest when the conditioned stimulus was 60 dB sound pressure level (SPL) and 16 kHz. This indicates that animals could feel tinnitus 2 h after salicylate injection, equivalent to that induced by 60 dB SPL and 16 kHz. By means of real-time PCR and western blot analysis, TRPV1 expression was significantly upregulated in spiral ganglion cells 2 h after salicylate injection and this upregulation together with the increase in the number of false positive responses was significantly suppressed by capsazepine (10 mg/kg i.p.), a specific antagonist of TRPV1. This suggests that salicylate could induce tinnitus through activation of TRPV1 in the rat auditory pathway.

Meniere's attacks occur in the inner ear with excessive vasopressin type-2 receptors.

Meniere's disease is peculiar to humans and is characterised by episodic vertigo, fluctuating hearing loss and tinnitus, and attacks of the affliction occurring under conditions of stress. Its pathology was first revealed to be inner ear hydrops through temporal bone studies in 1938. Although subsequently proposed as a disorder of water metabolism in the inner ear, its pathogenesis remains unsolved. The present study aimed to assess the link between the inner ear pathology in Meniere's disease and vasopressin, an anti-diuretic stress hormone with a potential role in inner ear fluid homeostasis. Blood samples were obtained from Meniere's disease patients in the morning, before any surgical treatment, to examine plasma vasopressin (pAVP) levels, and then from inner ear tissue during surgical treatment, to examine vasopressin type-2 receptor (V2R) in the endolymphatic sac. pAVP and the relative V2R mRNA expression in the endolymphatic sac were examined using a real-time polymerase chain reaction. Relative cAMP activity in the endolymphatic sac was also examined using tissue culture and cAMP assay. Both pAVP (1.6-fold versus controls; P = 0.048) and inner ear V2R mRNA expression (41.5-fold versus controls; P = 0.022) were significantly higher in Meniere's patients. cAMP activity was basally up-regulated (2.1-fold versus controls) and cAMP sensitivity to vasopressin application was largely elevated (4.9-fold versus controls) in Meniere's patients. We conclude that, in the pathogenesis of inner ear hydrops, resulting in Meniere's attacks, elevation of pAVP levels (probably as a result of stress) may present as a matter of consequence, but susceptibility of the V2R-overexpressed and cAMP-hypersensitized inner ear to pAVP elevation might be essential as the basis of this disease. Further experimental and clinical studies are needed to better clarify the relationship between Meniere's disease and stress.

Aberrantly differentiated cells in benign pilomatrixoma reflect the normal hair follicle: immunohistochemical analysis of Ca-binding S100A2, S100A3 and S100A6 proteins.

BACKGROUND: Pilomatrixoma is a common benign cutaneous tumour containing differentiated hair matrix cells. This tumour is mainly composed of basophilic, transitional, shadow and squamoid cells. Although some S100 proteins are expressed in a tissue-specific manner in the hair follicle (e.g. S100A2 in the outer root sheath, S100A3 in the cortex and cuticle, and S100A6 in the inner root sheath), little information is available concerning their distribution in the aberrantly differentiated tissues of pilomatrixoma. OBJECTIVES: To characterize the disordered epithelial elements of pilomatrixoma by localizing S100A2, S100A3 and S100A6 proteins. METHODS: Immunohistochemistry and dual-immunofluorescence microscopy were performed on 22 pilomatrixoma specimens using antibodies specific to the three proteins. RESULTS: Tissue-specific distribution of the S100 proteins investigated was preserved in the morphologically disordered tumour tissues. Anti-S100A2 antibody stained squamoid cells and putative outer root sheath cells; basophilic and potential hair matrix cells were occasionally stained. S100A3 staining was found in transitional cells and putative cortical cells, and was strong in both dispersed cells and hair-like structures surrounding cells which were presumably cuticular cells. Anti-S100A6 antibody labelled some S100A3-negative transitional cell strands, potentially inner root sheath cells. CONCLUSIONS: The epithelial elements of pilomatrixoma can be characterized using S100 proteins as biochemical markers. Our results show that pilomatrixomas retain a certain degree of differentiation indicative of distinct hair-forming cells.

Calmodulin-binding peptides isolated from alpha-casein peptone.

Peptides that inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase were isolated from a pepsin digest of alpha-casein. Analysis of these peptides showed that they corresponded to the alpha S2-casein sequences 164-179 (Leu-Lys-Lys-Ile-Ser-Gln-Arg-Tyr-Gln-Lys-Phe-Ala-Leu-Pro-Gln-Tyr). 183-206 (Val-Tyr-Gln-His-Gln-Lys-Ala-Met-Lys-Pro-Trp-Ile-Gln-Pro-Lys-Thr-Lys-Val -Ile- Pro-Tyr-Val-Arg-Tyr) and 183-207 (C-terminus, Val-Tyr-Gln-His-Gln-Lys-Ala-Met-Lys-Pro-Trp-Ile- Gln-Pro-Lys-Thr-Lys-Val-Ile-Pro-Tyr-Val-Arg-Tyr-Leu). These peptides inhibited calmodulin-induced cyclic nucleotide phosphodiesterase activity over the range 1-50 microM without affecting the basal enzyme activity. These results demonstrated that the affinities of these peptides for calmodulin are comparable to the affinities of certain endogenous neurohormones and proteins that interact with calmodulin.

Interactions of amphiphilic peptides derived from alpha s2-casein with calmodulin.

Calmodulin-binding peptides, which had previously been isolated from a pepsin digest of alpha-CN, were synthesized and then examined for their inhibitory effects on the activation of cyclic nucleotide phosphodiesterase that was induced by calmodulin. The concentrations of the synthetic peptides corresponding to 164-179, LKKISQRYQKFALPQY; 183-206, VYQHQKAMKPWIQPKTKVIPYVRY; and 183-207, VYQHQKAMKPWIQPKTKVIPYVRYL, of alpha s2-CN that gave half-maximal inhibition were 65, 7.0, and 2.6 microM, respectively. These inhibitory effects were reversed by increasing the amount of calmodulin. Fragments and analogs were prepared to study the interactions of the peptides with calmodulin in more detail. The results indicated that modification of the carboxyl terminus enhanced the affinities of the three peptides for calmodulin, and a region involved in the inhibition by alpha s2-CN (f183-207) was located at the carboxyl terminus 191-207. Two predicted calmodulin-binding sequences, 164-179 and 191-207 of alpha s2-CN, despite rather divergent primary structures, shared the structural motif common to the calmodulin-binding domains of the target proteins in the previously proposed complex model.

Characterization of soluble protein extracts from keratinized tissues: identification of ubiquitin universally distributed in hair, nail, and stratum corneum.

Partial protein extracts were prepared from hair, nail, and stratum corneum in the absence of urea and interfacial surfactant. Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoreses of these extracts showed low-molecular weight protein-rich patterns apparently different from those of whole protein extracts, which mainly consist of keratin bands. Several protein bands characterized each keratinized tissue or its derived species. In addition, we identified a major band of approximately 7 kDa as ubiquitin, a ubiquitously distributed protein that mediates non-lysosomal protein degradation, through direct amino acid sequence analysis of the electro-blotted protein band. The partial extraction is useful for investigation of soluble proteins retained in the keratinized tissues.

Suppression of melanogenesis by induction of endogenous intracellular metallothionein in human melanocytes.

Nitric oxide (NO) is a potent intercellular mediator of melanogenesis, whereas metallothionein (MT) is an inducible intracellular antioxidant that has been reported to scavenge NO. We investigated the existence and induction of MT in melanocytes, and its inhibitory effect on NO-induced melanogenesis. The expression of MT was detected in melanocytes, however, at a lower level than in keratinocytes, and its induction was possible by the addition of zinc chloride. Further, an NO-stimulated increase of tyrosinase activity in melanocytes was remarkably suppressed, when MT was induced prior to NO stimulation. Melanogenesis was also suppressed, when dexamethasone was used to induce MT. However, an NO-stimulated increase of tyrosinase expression was not suppressed at the gene and protein level, when MT was induced in melanocytes. The same suppressive effect of melanogenesis was also observed, when alpha-melanocyte-stimulating hormone or endothelin-1 was used as a stimulator. Because these results implied a mechanism other than NO scavenging to explain the suppressive effect of MT induction on melanogenesis, the direct inhibition of tyrosinase by MT was examined. Melanosome fractions were prepared from melanocytes, whose melanogenesis was suppressed by the induction of MT. Tyrosinase suppression was observed in the melanosome fractions, which was neutralized by the addition of anti-MT antibody. These results suggest that MT induction may be effective to suppress melanogenesis stimulated by NO as well as other melanogens, and these suppressive effects might be due to a direct inhibition of tyrosinase activity in melanosome and not a scavenging effect of NO.

Inhibitory effects of an anti-rheumatic agent T-614 on immunoglobulin production by cultured B cells and rheumatoid synovial tissues engrafted into SCID mice.

OBJECTIVE: To clarify the pharmacological action of an anti-rheumatic agent T-614, we investigated its effects on immunoglobulin (Ig) production by cultured B cells and Ig secretion from synovial tissues of patients with rheumatoid arthritis (RA) using SCID mice engrafted with human RA tissue (SCID-HuRAg). METHODS: Murine B cells were prepared from mouse spleen by a T-cell depletion method. The cells were cultured with lipopolysaccharide (LPS) and/or interleukin 4 (IL-4) in the absence or presence of T-614. Human B cells were isolated from peripheral blood of healthy donors and the Ig production was induced by co-culture with autologous T cells and anti-CD3 antibody. SCID-HuRAg was prepared according to our previous method. T-614 was orally administered to the mice once daily for 4 weeks starting on the fourth week after the implantation. Then, peripheral blood was obtained and the implanted tissues were removed. Igs in the culture media or the sera were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: In murine B-cell cultures, T-614 significantly decreased not only the IgM production stimulated with LPS but IgG1 production induced by LPS and IL-4. Regarding human B cells stimulated with T cells, it also inhibited IgM and IgG production. In SCID-HuRAg mice, high concentrations of polyclonal human IgG were detectable in the sera of all mice. A significant decrease in the IgG level was observed in the T-614-treated group compared with the control group. CONCLUSIONS: We showed that T-614 inhibited Ig production by the cultured B cells and also decreased the high level of human IgG observed in SCID-HuRAg mice. These results may support its effect on plasma Ig in RA patients and provide insights into the mechanisms of its anti-rheumatic effect.

In vitro and in vivo efficacies of T-3811ME (BMS-284756) against Mycoplasma pneumoniae.

T-3811, the free base of T-3811ME (BMS-284756), a new des-F(6)-quinolone, showed a potent in vitro activity (MIC at which 90% of the isolates tested are inhibited [MIC(90)], 0.0313 microg/ml) against Mycoplasma pneumoniae. The MIC(90) of T-3811 was 4-fold higher than that of clarithromycin but was 4- to 8-fold lower than those of trovafloxacin, gatifloxacin, gemifloxacin, and moxifloxacin and was 16- to 32-fold lower than those of levofloxacin, ciprofloxacin, and minocycline. In an experimental M. pneumoniae pneumonia model in hamsters, after the administration of T-3811ME (20 mg/kg of body weight as T-3811, once daily, orally) for 5 days, the reduction of viable cells of M. pneumoniae in bronchoalveolar lavage fluid was greater than those of trovafloxacin, levofloxacin, and clarithromycin (20 and 40 mg/kg, orally) (P < 0.05).

Some chemical properties of the HCl-methanol extract from the puparial cuticle of Drosophila melanogaster.

1. The HCl-methanol (HCl-MeOH) soluble fraction from the puparial cuticle of yellow, black and ebony of D. melanogaster was hydrolyzed in hydrochloric acid and examined for beta-alanine, ketocatechol, and acetic acid. 2. Between beta-alanine and ketocatechol and between beta-alanine and acetic acid, a quantitatively inverse relationship was found, respectively. The former relationship was further confirmed by the feeding experiment of beta-alanine to black. 3. Of total beta-alanine in the HCl-MeOH extract, the proportion of those having free amino group was 74.8 per cent. 4. All these results indicate that the HCl-MeOH soluble fraction of the puparial cuticle may be useful for investigating the cross-link structure of the cuticle.

HMG-CoA reductase inhibitor affects blood pressure and vascular reactivity.

1. To investigate the effect of endogenous cholesterol synthesis on blood pressure and vascular response, a HMG CoA reductase inhibitor, pravastatin (1 or 10 mg/kg) was administered orally for 2 or 4 weeks to 8-13 week old spontaneously hypertensive rats (SHR/Izm) and normotensive Wistar-Kyoto (WKY/Izm) rats. 2. Blood pressure was significantly increased in the pravastatin-treated groups of both strains, but the elevation was observed in WKY after a longer treatment than in SHR. 3. After the thoracic aorta from 10-12 week old SHR and WKY was pretreated with pravastatin (10(-4) mol/L), the vascular response to norepinephrine was increased in pravastatin-treated SHR aorta but not in the WKY aorta in both contractivity and sensitivity. 4. These experiments suggest that the vascular response is affected by intracellular cholesterol synthesis pathway.