Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.     

Existence of ghrelin-immunopositive and -expressing cells in the proventriculus of the hatching and adult chicken

Ghrelin was isolated from the rat stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and has been found in the gastrointestinal tract of many vertebrates. Although the sequence and structure of chicken ghrelin has recently been determined, morphological characteristics of ghrelin cells in the chicken gastrointestinal tract are still obscure. In this study, we investigated ghrelin expression and distribution of ghrelin-producing cells in the hatching and adult chicken gastrointestinal tract by RT-PCR, immunohistochemistry and in situ hybridization. Ghrelin mRNA expression was observed mainly in the proventriculus in the hatching chicken and in the proventriculus, pylorus and duodenum of the adult chicken by RT-PCR. Ghrelin-immunopositive (ghrelin-ip) cells in the proventriculus were located at the mucosal layer but not in the myenteric plexus or smooth muscle layer. The number of ghrelin-ip cells in the adult chicken was greater than that in the hatching chicken. Interestingly, in the adult chicken, the number of ghrelin-ip cells were almost the same as that of ghrelin mRNA-expressing (ghrelin-ex) cells; however, in the hatching chicken, the number of ghrelin-ex cells was greater than that of ghrelin-ip cells. These results clearly demonstrate that ghrelin-producing cells exist in the chicken gastrointestinal tract, especially in the proventriculus, from hatching to adult stages of development, as well as in mammals.     

Molecular characterization of the essential response regulator protein YycF in Bacillus subtilis

The response regulator YycF is essential for cell growth in gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae. To study the function of YycF in the essential process, we characterized a YycF (H215P) mutation that caused temperature-sensitive growth in B. subtilis. The response regulators YycF and YycF (H215P) were analyzed using circular dichroism spectroscopy, whose T(m) values were 56.0 and 45.9 degrees C, respectively, suggesting that YycF (H215P) significantly affects the protein structure with an increase in temperature. Furthermore, using the gel mobility shift assay and DNase I footprinting, we investigated the effect of YycF (H215P) on binding to the YycF box of ftsAZ operon of B. subtilis. The replacement of the histidine 215 with proline resulted in a decrease of the DNA-binding ability of YycF in vitro. In vivo, using Escherichia coli two-hybrid and homodimerization assays, we clarified that His 215 of YycF plays a crucial role in the homodimerization of the protein. Thus the essential genes involved in growth of B. subtilis appear to be regulated by the homodimer of YycF. These results suggest that the YycF dimerization is an excellent target for the discovery of novel antibiotics.     

Laryngeal endocrine cells: topographic distribution and adaptation to chronic hypercapnic hypoxia

The morphology, topographic distribution, effects of denervation, and exposure to hypercapnic hypoxia of endocrine cells were examined in rat larynx. The endocrine cells, which were immunoreactive for protein gene product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP), were observed within the epithelial layer of the laryngeal cavity and in the laryngeal gland, while solitary endocrine cells with apical and/or basal cytoplasmic processes appeared near the glottis. After denervation of the left cervical vagosympathetic trunk and the superior laryngeal nerve, the number of mucosal endocrine cells in the denervated side was not significantly different from that in the intact side. After exposure to hypercapnic hypoxia for 3 months, the number of endocrine cells with PGP 9.5 and CGRP was markedly increased. In conclusion, the secretion of laryngeal endocrine cells may be stimulated by CO2 rather than O2. Furthermore, the endocrine cells and the sensory and autonomic nervous system may regulate each other by an axon reflex mechanism. Endocrine cells appear to play a very important role in the local regulation of the laryngeal mucosa.     

Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958^T, P. bryantii B₁4^T, P. albensis M384^T, and P. brevis ATCC19188^T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species. Received: 29 November 1999 / Accepted: 1 February 2000     

Pain threshold, learning and formation of brain edema in mice lacking the angiotensin II type 2 receptor

The main biological role of angiotensin II type 2 receptor (AT2) has not been established. We made use of targeted disruption of the mouse AT2 gene to examine the functional role of the AT2 receptor in the central nervous system (CNS). We have previously shown that AT2-deficient mice displayed anxiety-like behavior in comparisons with wild-type mice. In the present study, we analyzed the pain threshold, learning behavior and brain edema formation using the tail-flick test, the tail-pinch test, the passive avoidance task and cold injury, respectively. In the passive avoidance task and cold injury, no differences were found between wild-type mice and AT2-deficient mice. In contrast, the pain threshold was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. The immunohistochemical distribution of beta-endorphin in the brain was analyzed quantitatively in AT2-deficient mice and wild-type mice, using microphotometry. The fluorescence intensity of beta-endorphin in the arcuate nucleus of the medial basal hypothalamus (ARC) was significantly lower in AT2-deficient mice, compared with findings in wild-type mice. We found that the AT2 receptor does not influence learning behavior and brain edema formation. As AT2-deficient mice have increased sensitivity to pain and decreased levels of brain beta-endorphin, AT2 receptors may perhaps mediate regulation of the pain threshold.     

Treatment of Irradiated Mice with High-Dose Ascorbic Acid Reduced Lethality

Ascorbic acid is an effective antioxidant and free radical scavenger. Therefore, it is expected that ascorbic acid should act as a radioprotectant. We investigated the effects of post-radiation treatment with ascorbic acid on mouse survival. Mice received whole body irradiation (WBI) followed by intraperitoneal administration of ascorbic acid. Administration of 3 g/kg of ascorbic acid immediately after exposure significantly increased mouse survival after WBI at 7 to 8 Gy. However, administration of less than 3 g/kg of ascorbic acid was ineffective, and 4 or more g/kg was harmful to the mice. Post-exposure treatment with 3 g/kg of ascorbic acid reduced radiation-induced apoptosis in bone marrow cells and restored hematopoietic function. Treatment with ascorbic acid (3 g/kg) up to 24 h (1, 6, 12, or 24 h) after WBI at 7.5 Gy effectively improved mouse survival; however, treatments beyond 36 h were ineffective. Two treatments with ascorbic acid (1.5 g/kg × 2, immediately and 24 h after radiation, 3 g/kg in total) also improved mouse survival after WBI at 7.5 Gy, accompanied with suppression of radiation-induced free radical metabolites. In conclusion, administration of high-dose ascorbic acid might reduce radiation lethality in mice even after exposure.     

Endurance Training-Induced Increase in Circulating Irisin Levels Is Associated with Reduction of Abdominal Visceral Fat in Middle-Aged and Older Adults

To elucidate the effects of endurance training on circulating irisin levels in young and middle-aged/older adults, and to determine the association between endurance training-induced alteration of irisin and reduction in body fat. Twenty-five healthy young (age 21 ± 1 years; 16 men, 9 women) and 28 healthy middle-aged/older adults (age 67 ± 8 years; 12 men, 16 women) participated in the study. Each age cohort was divided into two groups: the endurance-training group (14 young, 14 middle-aged/older) and the control group. Subjects in the training groups completed an 8-week endurance-training program (cycling at 60-70% peak oxygen uptake [V˙O_2peak] for 45 min, 3 days/week). Before and after the intervention, we evaluated serum irisin level, V˙O_2peak, and body composition. The increase in V˙O_2peak in the young and middle-aged/older training groups after the intervention period was significantly greater than those in the young and middle-aged/older control groups (P < 0.05). Serum irisin level was significantly increased in the middle-aged/older training group after the intervention period (P < 0.01), but not in the young training group. Furthermore, in the middle-aged/older training group, the endurance training-induced reduction in visceral adipose tissue area was negatively correlated with the change in serum irisin level (r = −0.54, P < 0.05). These results suggest a possible role for secreted irisin in the exercise-induced alteration of abdominal visceral fat in middle-aged and older adults.     

Cumulative Incidence and Predictors of Progression in Corticosteroid-Na?ve Patients with Sarcoidosis

Background

Assessment of the clinical course of sarcoidosis requires long-term observation. However, the appropriate period of follow-up for sarcoidosis remains unclear, especially in patients without indication of corticosteroid therapy at the time of diagnosis.

Objective

This study aimed to clarify the cumulative incidence and identify risk factors for disease progression in corticosteroid-naïve sarcoidosis patients.

Methods

The clinical courses of 150 Japanese patients with sarcoidosis, who were followed for more than 2 years and had no indication for corticosteroid therapy at diagnosis, were retrospectively reviewed. Disease progression was defined as worsening of pulmonary sarcoidosis, development of new organ involvement, or extrapulmonary organ damage. The cumulative incidence of progression was estimated by generating a cumulative incidence curve with the Fine and Gray method.

Results

The median follow-up duration was 7.7 years (interquartile range, 4.7–13.6 years). Thirty-two (21%) patients experienced disease progression. New organ involvement appeared in 16 patients (11%). The 6-month, and 1-, 5-, 10-, and 15-year cumulative incidence of progression was 2%, 5%, 15%, 28%, and 31%, respectively. The number of organs involved at diagnosis was an independent predictor for progression with a multifactorial adjusted hazard ratio of 1.71 (95% confidence interval, 1.11–2.62). The optimal cut-off of the number of organs involved at diagnosis to identify future progression was three.

Conclusions

In corticosteroid-naïve sarcoidosis patients, the risks of disease progression are comparable from 0–5 years and 5–10 years after diagnosis. The number of organs involved at diagnosis is a useful predictor for progression of sarcoidosis.