Assessment of metallothionein and antibodies to metallothionein in normal and autistic children having exposure to vaccine-derived thimerosal

Allergic autoimmune reaction after exposure to heavy metals such as mercury may play a causal role in autism, a developmental disorder of the central nervous system. As metallothionein (MT) is the primary metal-detoxifying protein in the body, we conducted a study of the MT protein and antibodies to metallothionein (anti-MT) in normal and autistic children whose exposure to mercury was only from thimerosal-containing vaccines. Laboratory analysis by immunoassays revealed that the serum level of MT did not significantly differ between normal and autistic children. Furthermore, autistic children harboured normal levels of anti-MT, including antibodies to isoform MT-I (anti-MT-I) and MT-II (anti-MT-II), without any significant difference between normal and autistic children. Our findings indicate that because autistic children have a normal profile of MT and anti-MT, the mercury-induced autoimmunity to MT may not be implicated in the pathogenesis of autism.     

Perturbation of copper (Cu) homeostasis and expression of Cu-binding proteins in cadmium-resistant lung fibroblasts.

To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.     

ApoMT对CdMT肾毒性保护作用研究

本文研究了去除金属离子的金属硫蛋白（ApoMT）对镉金属硫蛋白（CdMT）肾毒性作用的影响。结果发现，与单独给予CdMT比较，ApoMT能降低尿蛋白量和尿碱性磷酸酶（AKP）活性，并能促进尿Cd的排泄。肾组织形态学结果显示肾近曲小管损伤程度明显减轻。提示ApoMT对CdMT肾小管损伤具有保护作用。     

The major 40-kDa glycoprotein in human prostatic fluid is identical to Zn-alpha 2-glycoprotein

A major 40-KDA protein secreted by human prostate was isolated from whole seminal plasma by sequential column chromatography on DEAE-Sepharose CL-6B, concanavalin A(Con A)-Sepharose, and Sephadex G-100. Although the purified preparation still contained minor contaminants, its amino acid composition was found to be identical to the one of a glycoprotein isolated previously from seminal plasma by Lin et al (1983). Antibodies against this protein were produced in rabbits and their use in immunoblotting experiments revealed the presence of the antigen in several tissues including the prostate, the liver, the heart, the kidney, the epididymis, and the testis. A radioimmunoassay confirmed these results and showed that blood serum concentrations of this protein were relatively high in men (81 +/- 3 micrograms/ml), women (68 +/- 3 micrograms/ml), and cord blood of newborns (32 +/- 1 micrograms/ml). The serum concentrations of this protein along with its physicochemical characteristics suggested that it could be identical to Zn-alpha 2-glycoprotein, a human serum protein previously isolated by Burgi and Schmid (1961). This hypothesis was confirmed by a double immunodiffusion analysis using a commercial anti-Zn-alpha 2-glycoprotein antiserum. Finally, in vitro translation of prostatic poly(A) + RNA in rabbit reticulocyte lysate in the presence of canine pancreatic microsomal membranes resulted in the formation of an immunoprecipitable 42-kDa band. These results show that Zn-alpha 2-glycoprotein can be synthesized in the prostate itself. The demonstration of high concentrations of this protein in prostatic tissue and prostatic secretion should facilitate the elucidation of its role in the prostate and in other tissues.     

Atrophic variants of dermatofibroma and dermatofibrosarcoma protuberans

Dermal atrophy of more than 50% of the locoregional dermis may be the predominant histopathological feature in dermatofibroma and dermatofibrosarcoma protuberans. This may cause diagnostic difficulties. In the present study 26 cases of atrophic dermatofibroma were compared with three cases of atrophic dermatofibrosarcoma protuberans. Clinically, both conditions mostly occurred on the (upper) trunk of females. While atrophic dermatofibroma usually presented as a reddish, umbilicated lesion (0.5–1-cm), often suspected to be a basal cell carcinoma, atrophic dermatofibrosarcoma protuberans showed irregularly arranged tan-brown plaques (3–6 cm). Histologically, atrophic dermatofibroma showed a regular silhouette with a smooth nodular (9/26) or scalloped lower margin with an intervening lace-like pattern of superficial fatty tissue infiltration (17/26) and variable sclerosis; atrophic dermatofibrosarcoma protuberans showed a deep, irregular infiltration of fatty tissue in a lacelike/honeycomb and/ or multilayered pattern, but no sclerosis. Immunohistochemically, atrophic dermatofibroma was mostly negative with QBEnd 10 (CD34; 24/26), variably positive for factor XIIIa (20/26) and metallothionein (11/26). Labelling for factor XIIIa and metallothionein was usually seen in 'early' (metabolically active) lesions, while 'late' sclerotic ones were negative. In contrast to atrophic dermatofibroma all three atrophic dermatofibrosarcoma protuberans showed a consistently uniform profile: CD34 positive, factor XIIIa and metallothionein negative. Our study delineates atrophic dermatofibroma and atrophic dermatofibrosarcoma protuberans as distinct entities clearly distinguishable from each other by clinicopathologic criteria.     

Expression of metallothionein in lung carcinoma: correlation with histological type and grade

AIMS: Over-expression of cellular metallothionein occurs frequently in human tumours but the underlying mechanism remains unknown. The aim of this study was to assess metallothionein expression in cases of lung carcinoma and to correlate it with histopathological parameters. METHODS AND RESULTS: Tumour tissue samples from 89 patients with lung carcinoma were immunostained by the streptavidin-biotin-peroxidase technique, using a monoclonal antibody against both metallothionein-1 and -2 isoforms. Positive matallothionein immunostaining was prominent in 44 out of 89 (49%) and negative in 45 out of 89 (51%) cases of lung carcinoma examined. Metallothionein positivity was prominent in 32 out of 43 (74%) cases of squamous cell lung carcinoma, and in 12 out of 35 (34%) cases of adenocarcinoma, while it was negative in all 11 cases of small-cell lung carcinoma examined, presenting a statistically significant difference between the different histological types. The intensity of metallothionein staining revealed a statistically significant difference between the squamous cell and adenocarcinoma cases examined. The pattern and extent of metallothionein staining in tumour cells and the expression of metallothionein in stromal cells were not correlated with histopathological parameters (type and grade) in metallothionein-positive cases of lung carcinoma examined. No association was found between metallothionein expression and lymph node status in the examined cases of lung carcinoma. CONCLUSIONS: Our findings indicate that expression of metallothionein was evident in squamous cell lung carcinoma and adenocarcinoma, but absent in small-cell lung carcinoma, supporting evidence for participation of this protein in the biological mechanisms underlying the carcinogenic evolution in the lung.     

Imbalanced secondary mucosal antioxidant response in inflammatory bowel disease

Intestinal mucosal damage in the inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC) involves reactive oxygen metabolites (ROMs). ROMs are neutralized by endogenous antioxidant enzymes in a carefully balanced two-step pathway. Superoxide dismutases (SODs) convert superoxide anion to hydrogen peroxide (H(2)O(2)), which is subsequently neutralized to water by catalase (CAT) or glutathione peroxidase (GPO). Remarkably changed expression levels of the three isoforms of SOD in paired non-inflamed and inflamed mucosae from CD and UC patients have been previously reported in comparison to normal control mucosa. Most notable was the strong up-regulation of Mn-SOD in inflamed epithelium. It was hypothesized that in order to provide optimal protection against ROM-mediated damage, these changes should be coordinately counterbalanced by an increased H(2)O(2)-neutralizing capacity. Therefore, the same tissue samples were used to assess the levels, activities, and/or localization of the most prominent mucosal H(2)O(2)-related antioxidants CAT, GPO, glutathione (GSH), myeloperoxidase (MPO), and metallothionein (MT). Quantitative measurements showed that in both CD and UC patients, intestinal inflammation was associated with increased activities of CAT, GPO, and MPO, whereas the mucosal GSH content was unaffected and the concentration of MT was decreased. Despite this overall increase in mucosal H(2)O(2)-metabolizing enzyme capacity, immunohistochemical analysis revealed a differentially disturbed antioxidant balance in IBD epithelium and lamina propria. In the lamina propria, the risk of direct H(2)O(2)-mediated damage seemed to be restrained by the increasing numbers of CAT- and MPO-positive monocytes/macrophages and neutrophils that infiltrated the inflamed areas. On the other hand, MPO overexpression might increase the lamina propria levels of hypochlorous acid, a stable ROM with multiple pro-inflammatory effects. In the epithelium, the number of cells that expressed CAT remained unchanged during inflammation and GPO was found in only a very low and constant number of epithelial cells. In addition, the inflamed epithelium displayed decreased expression of the hydroxyl radical (OH(*)) scavenger MT. In view of the high epithelial SOD levels in inflamed IBD epithelium, it is speculated that the efficient removal of excess H(2)O(2) is hampered in these cells, thereby increasing not only the risk of detrimental effects of H(2)O(2) directly, but also those of its extremely reactive derivatives such as OH(*). Taken together, the results suggest an imbalanced and inefficient endogenous antioxidant response in the intestinal mucosa of IBD patients, which may contribute to both the pathogenesis and the perpetuation of the inflammatory processes.     

大鼠胸主动脉球囊拉伤后金属硫蛋白及丙二醛含量变化

目的 :观察大鼠胸主动脉球囊拉伤后血浆及组织金属硫蛋白 (MT)及丙二醛含量变化 ,探讨再狭窄与氧化防御系统的关系。方法 :对照组常规饲养 2 1d ;球囊导管拉伤大鼠胸主动脉 3d ,10d ,2 1d后 ,分别取大鼠血浆及心、肝和肾组织 ,以10 9Cd血红蛋白饱和法测定大鼠血浆及组织MT含量 ,以硫代巴比妥酸钠法测定丙二醛 (MDA)含量。结果 :与对照组比较 ,球囊拉伤 3d组大鼠肝、心和肾组织及血浆MT含量分别升高 5 5 .8% ,73 .5 % ,49.9% ,5 6 .2 % ,MDA含量分别升高 94.0 % ,49 1% ,31 4% ,141 1% ;球囊拉伤 10d组大鼠肝、心、肾组织及血浆MT含量分别升高 31.8% ,6 8 2 % ,5 8.0 % ,41 8% ,MDA含量分别升高 148.0 % ,41.6 % ,2 9.1% ,83.8% ;球囊拉伤 2 1d组大鼠肝、心、肾组织及血浆MT含量分别升高 5 6 .8% ,38.8% ,39.4% ,48.1% ,MDA含量分别升高 98.0 % ,48.4% ,44 .8% ,49.2 %。结论 :大鼠胸主动脉球囊拉伤后MT产生增多与脂质过氧化损伤有关     

转小鼠金属硫蛋白基因集胞藻6803及其野生藻培养

通过摇瓶培养考察转小鼠金属硫蛋白基因集胞藻６８０３及其野生藻的光合自养培养过程中碳源浓度、氮源浓度和光照度对这两种藻生长的影响。野生藻的最适生长碳源浓度（０．０５－０．５ｇ／Ｌ）比转基因藻的最适生长碳源浓度（０．０２－０．０５ｇ／Ｌ）高。氮源浓度对转基因藻和野生藻的影响趋势相同。在０．３－４．５ｇ／Ｌ范围内,随着氮源浓度的增高,藻体生长的最大细胞浓度降低。转基因藻生长的最佳光照度为１５００ｌｘ,野     

Cadmium pathways during gestation and lactation in control versus metallothoinein 1,2-knockout mice.

Effects of metallothionein (MT) on cadmium absorption and transfer pathways during gestation and lactation in mice were investigated. Female 129/SvJ metallothionein-knockout (MT1,2KO) and metallothionein-normal (MTN) mice received drinking water containing trace amounts of (109)CdCl(2) (0.15 ng Cd/ml; 0.074 micro Ci (109)Cd/ml). (109)Cd and MT in maternal, fetal, and pup tissues were measured on gestation days 7, 14, and 17 and lactation day 11. In dams, MT influenced both the amount of (109)Cd transferred from intestine into body (two- to three-fold higher in MT1,2KO than MTN dams) and tissue-specific (109)Cd distribution (higher liver/kidney ratio in MT1,2KO dams). Placental (109)Cd concentrations in MT1,2KO dams were three- and seven-fold higher on gestation days 14 and 17, respectively, than in MTN dams. Fetal (109)Cd levels were low in both mouse types, but at least 10-fold lower in MTN fetuses. MT had no effect on the amount of (109)Cd transferred to pups via milk; furthermore, 85-90% of total pup (109)Cd was recovered in gastrointestinal tracts of both types, despite high duodenal MT only in MTN pups. A relatively large percentage of milk-derived intestinal (109)Cd was transferred to other pup tissues in both MT1,2KO and MTN pups (14 and 10%, respectively). These results demonstrate that specific sequestration of cadmium by both maternal and neonatal intestinal tract does not require MT. Although MT decreased oral cadmium transfer from intestine to body tissues at low cadmium exposure levels, MT did not play a major role in restricting transfer of cadmium from dam to fetus via placenta and to neonate via milk.