Preconditioning with subneurotoxic allyl nitrile: protection against allyl nitrile neurotoxicity.

High-dose cruciferous allyl nitrile can induce behavioral abnormalities in rodents, while repeated exposure to allyl nitrile at subneurotoxic levels can increase phase 2 detoxification enzymes in many tissues, although the brain has not been investigated yet. In the present study, we examined the effect of 5 days repeated exposure to subneurotoxic allyl nitrile (0-400 micromol/kg/day) on the brain. Elevated glutathione S-transferase activity was recorded in the striatum, hippocampus, medulla oblongata plus pons, and cortex. Enhancement of quinone reductase activity was observed in the medulla oblongata plus pons, hippocampus, and cortex. In the medulla oblongata plus pons, elevated glutathione levels were recorded. Following repeated subneurotoxic allyl nitrile exposure (0-400 micromol/kg/day), mice were administered a high-dose allyl nitrile (1.2 mmol/kg) which alone led to appearance of behavioral abnormalities. Compared with the 0 micromol/kg/day group, animals in the 200 and 400 micromol/kg/day pre-treatment groups exhibited decreased behavioral abnormalities and elevated GABA-positive cell counts in the substantia nigra pars reticulata and the interpeduncular nucleus. These data suggest that repeated exposure to subneurotoxic levels of allyl nitrile can induce phase 2 enzymes in the brain, which together with induction in other tissues, may contribute to protection against allyl nitrile neurotoxicity.

Aberrant human tissue kallikrein levels in the stratum corneum and serum of patients with psoriasis: dependence on phenotype, severity and therapy.

BACKGROUND: Human tissue kallikreins (KLKs) are a family of 15 trypsin-like or chymotrypsin-like secreted serine proteases (KLK1-KLK15). Multiple KLKs have been quantitatively identified in normal stratum corneum (SC) and sweat as candidate desquamation-related proteases. OBJECTIVES: To quantify KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of patients with psoriasis, and their variation between lesional and nonlesional areas and with phenotype, therapy and severity. The overall SC serine protease activities were also measured. METHODS: Enzyme-linked immunosorbent assays and enzymatic assays were used. RESULTS: The lesional SC of psoriasis generally contained significantly higher levels of all KLKs. KLK6, KLK10 and KLK13 levels were significantly elevated even in the nonlesional SC. The overall trypsin-like, plasmin-like and furin-like activities were significantly elevated in the lesional SC. Plasmin-like activity was significantly elevated also in the nonlesional SC. The SC chymotrypsin-like activity was only slightly elevated in psoriasis. KLK7 serum levels did not differ between normal volunteers and patients with psoriasis. Serum KLK6, KLK8, KLK10 and KLK13 levels in patients with untreated psoriasis significantly correlated with Psoriasis Area and Severity Index score. Serum KLK5 and KLK11 levels decreased in patients with psoriasis after therapy, especially with etretinate. Patients with erythrodermic psoriasis exhibited significantly higher serum KLK levels than normal subjects or patients with psoriasis vulgaris or arthropathic psoriasis. CONCLUSIONS: We found aberrant KLK levels in the SC and serum of patients with psoriasis and suggest that KLKs might be involved in the pathogenesis of this disease.

Multiple tissue kallikrein mRNA and protein expression in normal skin and skin diseases.

BACKGROUND: Human tissue kallikreins are a gene family (KLK1-KLK15) encoding for 15 secretory serine proteases (hK1-hK15). Two tissue kallikrein proteins, hK5 and hK7, were previously found in the stratum corneum (SC), stratum granulosum (SG) and appendages. hK8 was also shown to be secreted via lamellar granules and numerous KLK mRNAs were previously identified. KLKs are believed to be responsible for desquamation of corneocytes and sebum, sweat and hair maturation. OBJECTIVES: To demonstrate immunohistochemically the expression of hK6, hK8 and hK13 in normal skin tissue and to show an increased cell number expressing kallikrein mRNAs and proteins in psoriasis vulgaris (PV) and atopic dermatitis (AD). METHODS: Samples of normal, PV and AD skin were obtained. hK6-, hK8- and hK13-specific antibodies were produced and used for immunohistochemical analysis. Multiple KLK mRNAs were synthesized and used for in situ hybridization study. RESULTS: Three other hKs, namely hK6, hK8 and hK13, were immunohistochemically identified as new skin serine proteases in the whole SC, SG, sebaceous glands, eccrine sweat glands, hair follicles and nerves. We also demonstrated an increased number of cells expressing KLK mRNAs and hKs in PV and AD. In PV, KLK mRNAs/hKs were predominantly expressed in the upper epidermis. In AD, hK distribution was rather diffuse and expanded into the lower epidermis. CONCLUSIONS: The colocalization of various hKs seems to be essential for the regulation of serine protease activity in skin and for steady desquamation and skin barrier function. Moreover, the increased number of cells expressing multiple KLK mRNA and hK in PV and AD could be a clue to elucidate their pathogenesis.

Allylnitrile: generation from cruciferous vegetables and behavioral effects on mice of repeated exposure.

The objective of the present study was to examine the possible generation of allylnitrile from commonly consumed cruciferous vegetables, and to determine the long-term behavioral effects of its oral administration at levels comparable to or greater than human dietary exposures. On the basis of gaschromatographic and mass spectrometric analyses, allylnitrile generation was observed in eight out of twelve vegetables, broccoli, broccoli (young stems and leaves), brussels sprouts, cabbage, cauliflower, chinese cabbage, komatsuna and kaiware-daikon (young stems and leaves). The daily dietary intake of allylnitrile was estimated to be at least 0.12 micromol/kg body weight for Japanese, based on its generation from the vegetables, broccoli, cabbage, cauliflower and Chinese cabbage and their daily dietary consumption. Mice received oral doses of 2, 20, 200, 500 and 1,100 micromol/kg allylnitrile once a day, 5 days per week for 13 weeks. Mice in the lower dosage groups of 2, 20 and 200 micromol/kg exhibited no behavioral changes. Mice dosed at the level of 500 micromol/kg showed restlessness, and one of them displayed alteration in tail hanging. These abnormalities were seen around seven days following the beginning of the treatment period. Animals in the highest dosage group elicited behavioral abnormalities, and their degree increased with increasing dosage. These results suggest that allylnitrile intake levels through daily vegetable consumption is below the level producing behavioral abnormalities.

Reduction in the local expression of complement component 6 (C6) and 7 (C7) mRNAs in oesophageal carcinoma.

Differential displays of tumour/normal pair specimens of human oesophagus identified complement component 7 (C7) as being enhanced in normal tissues, but remarkably reduced in carcinoma tissues. In situ hybridisation confirmed the localisation of C7 mRNA in normal oesophageal epithelial cells and its disappearance in tumour cells. When mRNA expressions of other components were examined by means of semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 10 tumour/normal pair specimens, significant reductions in C6 and C7 mRNAs were observed, while C3 and C5 mRNAs were enhanced in both normal and tumour tissues. A similar reduction was observed in colon and kidney cancers using the tumour/normal expression array analysis. Gene deletion of C7 was not found in the cell lines by Southern blot analysis. Our findings suggest a possible relationship between oesophageal tumorigenesis and reduced expression of C6 and C7 mRNAs, which is probably caused by a change in gene expression regulation and not by genetic loss of the locus.

Overexpression of latent transforming growth factor-beta 1 (TGF-beta 1) binding protein 1 (LTBP-1) in association with TGF-beta 1 in ovarian carcinoma.

Using the differential display method, latent transforming growth factor-beta 1 (TGF-beta 1) binding protein 1 (LTBP-1) mRNA was identified as one of the enriched mRNAs in ovarian carcinoma tissues after isolation of genes responsible for the development of ovarian cancer. Semi-quantitative reverse transcription (RT)-PCR analysis showed that expression of LTBP-1 and TGF-beta 1 mRNAs was much higher in both serous and mucinous adenocarcinomas than in their benign counterparts, including serous and mucinous cystadenomas and cystadenomas of low malignant potential (LMPs). Immunohistochemical analysis demonstrated that only proliferating benign adenoma cells were immunoreactive for both LTBP-1 and TGF-beta 1 proteins. In contrast, most serous and mucinous adenocarcinoma cells and their surrounding stroma were intensely immunoreactive for LTBP-1 and TGF-beta 1. LTBP-1 and TGF-beta 1 proteins, and their complex forms were identified in ovarian carcinoma cell lines and in their culture media by western blot analysis, suggesting these products were produced in ovarian carcinoma cells. RT-PCR analysis demonstrated that LTBP-1L, one of the LTBP-1 transcripts that has a strong activity in targeting the latent form of TGF-beta 1 to extracellular matrix (ECM), was predominantly expressed in ovarian carcinomas. Taken together, the results suggest that upregulation of LTBP-1 in ovarian carcinoma cells may have an important role in distributing TGF-beta1 in the stromal tissues surrounding carcinoma cells.

Fos induction in the brain of mice exhibiting behavioral abnormalities following administration of allylnitrile or crotononitrile.

Allylnitrile and crotononitrile induce behavioral abnormalities in mice. To explore the possible involvement of the vestibular system in these behavioral abnormalities, the expression of Fos protein, used as an indicator of neuronal activity, was examined within various brain structures in allylnitrile-, crotononitrile- and vehicle-treated mice. In each nitrile-treated mouse, Fos expression was observed in brain structures, which were divided into two groups. The structures in group 1 showed Fos expression between 1.5 h and 2 days postdosings, and in those in group 2 expression remained for up to 30 days postdosing. As most of these structures, especially in group 2, were identical to some Fos-positive structures observed after unilabyrinthectomy, the present results indicate that each nitrile induces Fos expression by causing a change in the peripheral vestibular system, resulting in behavioral abnormalities.

Clinicopathological significance of fragile histidine triad transcription protein expression in endometrial carcinomas.

Abnormalities in structure and expression of the fragile histidine triad transcription (FHIT) gene have been reported in a variety of cancers, including endometrial cancers. A good correlation between FHIT gene alteration and loss of Fhit expression was observed in endometrial cancers, although those are the selected cases. Therefore, we investigated the association of Fhit expression with clinicopathological features in 111 cases of endometrial cancer. Loss of Fhit expression was associated with high malignant potential, including extensive muscular invasion, advanced surgical stage, high histological grade, nonendometrioid types of adenocarcinoma, negative estrogen receptor status, and p53 overexpression. The presence of personal cancer history was also related to the loss of Fhit with a marginal significance. Survival curves determined by the Kaplan-Meier method and univariate analysis demonstrated that decreased expression of Fhit was associated with a poor outcome. However, multivariate analysis using the stepwise Cox proportional hazard model showed that whereas lymph node metastasis, advanced stage, and high tumor grade were related to poor survival rates, loss of Fhit expression was not. Consequently, loss of Fhit expression is associated with advanced surgical stage and does not appear to be an independent prognostic factor in endometrial cancers, although a still larger sample of patients will be required to asses this issue definitively.

Differential expression of neuroendocrine-specific protein in form-deprived chick eyes.

PURPOSE: To identify genes that are highly expressed in form-deprived retina-retinal pigment epithelium-choroid tissues. Neuroendocrine-specific proteins were found to be highly expressed. METHODS: mRNAs enriched in retina-retinal pigment epithelium-choroid tissues from 3-, 7-, and 14-day form-deprived chick eyes were isolated by differential display technique with cDNA library screening. Neuroendocrine-specific protein A and C were cloned in control and form-deprived eyes. mRNA and protein levels, with respective regional localizations, were examined by Northern blot, Western blot, and immunohistochemical analyses, respectively. RESULTS: The isolated clone included an insert with a sequence homologous to both chick neuroendocrine-specific proteins A and C. The increases in mRNA and protein levels were confirmed by Northern and Western blot analyses, respectively. Immunohistochemical localization of neuroendocrine-specific proteins A and C was detected in the layer of photoreceptor inner segments, presumably in the cone cells. Northern blot analysis using negative lenses showed that levels of neuroendocrine-specific protein A and C mRNAs were not altered using negative lenses. CONCLUSIONS: The expression of both neuroendocrine-specific proteins A and C mRNAs in cone photoreceptor cells was upregulated within 14 days of form deprivation, but not in response to negative spectacle lenses. These data suggest that the increase in induction of neuroendocrine-specific proteins is not a secondary consequence of ocular elongation or myopic refraction. Induction of neuroendocrine-specific proteins in form-deprived eyes may be causally related to the development of myopia or may be an unrelated effect of form deprivation.

Involvement of GABA neurons in allylnitrile-induced dyskinesia.

Nitriles are a class of compounds with potential relevance to human health. Allylnitrile, one of nitriles, induces persistent behavioral abnormalities in mice. To explore what type of neuronal system is involved in these behavioral abnormalities, five neuronal markers, gamma-aminobutyric acid (GABA), tyrosine hydroxylase, serotonin, the serotonin transporter and choline acetyltransferase were immunohistochemically examined within various brain structures in allylnitrile and vehicle-treated mice. Allylnitrile induced changes in the immunolabelling of GABA in the medial habenula, interpeduncular nucleus, substantia nigra, dorsal raphe nucleus and median raphe nucleus; the amount of immunolabelling decreased in all of these brain structures except the medial habenula at 2 days postdosing, and increased in all of these structures at 14 days postdosing. Allylnitrile also induced changes in the amount of immunolabelling of tyrosine hydroxylase in the arcuate nucleus, substantia nigra pars compacta, locus coeruleus and caudoventrolateral reticular nucleus at either 2 or 14 days postdosing, depending on the structures. No immunohistochemical change was seen for serotonin, serotonin transporter and choline acetyltransferase. The present results suggest that the GABAergic systems through the medial habenula-interpeduncular nucleus-ascending raphe nuclei relay and through the substantia nigra may be involved in allylnitrile-induced behavioral abnormalities.

[Allylnitrile-induced behavioral abnormalities and findings relating to the mechanism underlying behavioral abnormalities].

Nitriles are widely used in industry as plastics, solvents, and synthetic intermediates. It has been shown that the thermal degradation of acrylonitrile-based plastics leads to the emission of a great variety of nitriles. Exposure of humans and experimental animals to some nitriles has been shown to lead to disorders of the central nervous, hepatic, cardiovascular, renal, and gastrointestinal systems. Iminodipropionitrile has long been known to induce in experimental animals behavioral syndromes that other nitriles have not been reported to induce. Recently, we have found that a single administration of allylnitrile, an analog of acrylonitrile, induces in rodents behavioral abnormalities including head twitching, head weaving, random circling, increased locomotor activity, backward pedaling, pivoting, and somersaulting. The induced abnormalities were persistent. Crotononitrile and 2-pentenenitrile also are able to produce behavioral abnormalities. Thus, the nitriles appear as a new class of neurotoxic compounds with potential relevance to the human health. The mechanism by which allylnitrile induces and maintains the behavioral abnormalities is summarised below. 1. Allylnitrile activates the serotonin (5-HT) system in the central nervous system, and as a consequence activation of 5-HT-2 receptors due to increased 5-HT may lead to induction of head twitching. 2. Although the data available indicate that the dopamine (DA) system may be involved in allylnitrile-induced behavioral abnormalities, it remains unknown how the DA system relates to the abnormalities. 3. Allylnitrile decreases the noradrenaline level in the central nervous system, which is thought to be secondary to the 5-HT system activation mentioned above. The allylnitrile-induced head twitching, however, may occur in consequence to both enhanced beta-adrenoceptor stimulation and to the removal of tonic inhibitory control by alpha-2-adrenoceptors. 4. The neuropathological data indicate an important role of the medial habenular and raphe nuclei in allylnitrile-induced behavioral abnormalities. Onset of the behavioral abnormalities appears to be associated with the impairment in the medial habenulo-raphe relay owing to activation of apoptotic cascade in neurons. 5. On the basis of the findings with iminodipropionitrile and crotononitrile, allylnitrile might produce pathological changes in the vestibular sensory hair cells. Further studies are needed to explore the mechanism underlying the allylnitrile-induced syndromes.

Behavioral abnormalities and apoptotic changes in neurons in mice brain following a single administration of allylnitrile.

A single dose of allylnitrile in mice might induce persistent behavioral abnormalities, of which the mechanism is not yet known. The present study was undertaken to explore the relationship between behavioral abnormalities and pathological changes in the brain of mice following exposure to allylnitrile. Exposure to allylnitrile (63, 84, and 112 mg/kg, p.o.) resulted in dose-dependent changes in behavioral abnormalities, including increased locomotor activity, circling, retropulsion, head twitching, and alteration in reflexive behavior, which appeared at day 2 postdosing and were persistent throughout the experimental period (60 days) at the higher dose levels. Allylnitrile produced neuronal retraction including hyperchromasia of the nuclei in the raphe nuclei, cerebral cortex, hypothalamus, hippocampal CA1 and dentate gyrus later than 30 days. No gliosis was observed in these regions. Not all but a significant number of neurons in the hippocampal CA1, medial habenula and raphe nuclei were immuno-reactive to CPP32 (Caspase-3) even at day 2. These neurons were also positive to Hoechst 33258 staining, indicating allylnitrile caused apoptotic changes in specific neurons when neuronal behaviors became apparent. These apoptotic changes were persistent even in the area without neuronal contraction such as medial habenula. However, almost all neurons in these areas were also positive to terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL). It is conceivable that allylnitrile caused apoptotic changes in neurons but did not always lead them to cell death immediately. Moreover, even when neuronal contraction resulted in retention of behavioral abnormalities, onset of these abnormalities seems to be associated with the impairment in the habenulo-raphe relay due to activation of apoptotic cascade in neurons.

Differential expression of nitric oxide synthase isoforms in form-deprived chick eyes.

PURPOSE: To clarify whether nitric oxide synthase (NOS) is involved in development of myopia, we examined the influence of form deprivation on the expressions of NOS isoform mRNA. METHODS: NOS isoform cDNAs were amplified from total RNA extracted from control and 7-day-form-deprived chick retina-RPE (retinal pigment epithelium)-choroid, using competitive RT-PCR (reverse-transcription polymerase chain reaction). Each NOS isoform protein was also analyzed by Western blotting and immunohistochemistry. RESULT: Expression of inducible NOS (iNOS) mRNA was highest in the control chick retina-RPE-choroid, followed by the expression of brain NOS (bNOS) mRNA. Expression of endothelial NOS (eNOS) mRNA was faint. The iNOS protein level, however, was only slightly higher than the levels of the bNOS and eNOS proteins and was found mainly in the outer part of the photoreceptor layer and inner and outer parts of RPE and choroid. bNOS alone was found in the outer nuclear layer. Although form deprivation reduced the iNOS and bNOS mRNA expressions, only the iNOS protein showed significant reduction. CONCLUSION: All three NOS isoforms were expressed in chick retina-RPE-choroid. Predominant expression of iNOS, instead of bNOS and eNOS, suggested the existence of ocular tissue-specific regulation of the iNOS gene. In addition to differences in expression level, bNOS displayed regional differential expression. Moreover, only iNOS was reduced in response to form deprivation. It is suggested that NOS isoforms may be differentially involved in the mechanisms regulating the posterior eye tissues, including myopic eye growth.

Immunodeficiency induced by child abuse.

We report the case of a 9-year-old girl who died from sepsis from cellulitis of the neck caused by a right ear injury. The autopsy findings showed severe involution of the thymus and atrophy of lymphoid tissues. The impairment of T- and B-cell functions was demonstrated both histologically and immunohistologically. Thymic involution caused by child abuse might lead to secondary immunodeficiency.

Cloning of mouse diastrophic dysplasia sulfate transporter gene induced during osteoblast differentiation by bone morphogenetic protein-2.

Although intensive studies have been directed at understanding osteoblastic differentiation, the molecular mechanisms are still unclear. In this study, we describe a cDNA that encodes a sulfate transporter that was cloned as a gene induced in osteoblast precursor cells in association with osteoblastic differentiation. Based on the fact that bone morphogenetic protein-2 (BMP-2) induces osteoblastic phenotypes in immature mouse fibroblastic C3H10T1/2 cells, we performed a subtraction hybridization between BMP-2-treated and untreated cells, and have isolated one clone (designated as st-ob for sulfate transporter in osteoblast) induced by BMP-2 that is constantly expressed in osteoblastic cells. The deduced amino acid sequence and proposed structure of st-ob are mostly identical to those of the human diastrophic dysplasia sulfate transporter gene product (DTDST). St-ob mRNA was abundantly expressed in the thymus, testis, calvaria and osteoblastic MC3T3-E1 cells, whereas its expression was faint in C3H10T1/2 cells. Expression of st-ob in C3H10T1/2 cells was increased by transforming growth factor-beta1 (TGF-beta1), retinoic acid and dexamethasone as well as BMP-2. Furthermore, BMP-2 increased sulfate incorporation in C3H10T1/2 cells about twice as high as the baseline level. Osteoblasts actively take up sulfate to synthesize proteoglycans, which are one of the major components of the extracellular matrix of bone and cartilage. The present study demonstrates that st-ob induced during osteoblastic differentiation is an important phenotype of osteoblasts for characterizing their function.

Analysis of bovine selenoprotein P-like protein gene and availability of metal responsive element (MRE) located in its promoter.

Selenoprotein P-like protein, similar to selenoprotein P, uses multiple TGAs for incorporation of selenocysteines but not as stop codons. It is also characterized by having a His-Pro-rich domain and a regionally differential expression pattern. Hence, in addition to selenium metabolism, this protein is considered to have a developmental function. In the present study, the structure of the selenoprotein P-like protein gene was analyzed. The gene consisted of five exons, and the 5'-flanking region contained a TATA box, TCF-1-CS, bHLH-CS, gamma-IRE-CS, c-Myb-CS, C/EBP-CS, HNF-5-CS, MRE2-CS, etc. The presence of motifs like TCF-1-CS, c-Myb-CS, etc. supports the suggestion that this protein is involved in cellular maturation. Since the presence of MRE2-CS suggests that this protein is related to the antidote effect of selenium against heavy metal intoxication, the availability of this motif was examined using bovine kidney cell lines, CKT-1 and MDBK. Metallothionein mRNA markedly increased 6 h after administration of 10(-6) M CdCl2 and ZnCl2 in both cell lines. No significant alteration was observed in selenoprotein P-like protein mRNA, whereas its basal expression was high, indicating that this protein is constitutively expressed. Thus, it is still possible that this protein acts as an antidote, even though it is not inducible by heavy metals.