The 67-kDa laminin-binding protein is involved in shear stress-dependent endothelial nitric-oxide synthase expression.

It has been suggested that the mechanical forces acting on endothelial cells may be sensed in part by cell-matrix connections. We therefore studied the role of different matrix proteins, in particular laminin I, on a shear stress-dependent endothelial response, namely nitric-oxide synthase (eNOS) expression. Primary porcine aortic endothelial cells were seeded onto glass plates either noncoated (NC cells) or precoated with fibronectin (FN cells), laminin (LN cells), or collagen I (CN cells). Western blots were used to detect differences in the final matrix composition of these cells. A shear stress of 16 dyn/cm2 was applied for 6 h. Only LN cells showed detectable amounts of laminin I in their underlying matrix when they reached confluence. They reacted with a 2-fold increase of eNOS expression (n = 16, p < 0.001) to the exposure of shear stress, which went along with enhanced eNOS protein and NO release. In contrast, neither FN cells (n = 9) nor NC cells (n = 13) showed a significant increase of eNOS expression under shear stress. The increase in CN cells was borderline (1.4-fold; n = 9, p < 0.05) and was not associated with an increase of eNOS protein. The shear-induced increase in eNOS expression of LN cells was abolished by the peptide YIGSR, which blocks the cellular binding to laminin I via a 67-kDa laminin-binding protein, whereas a control peptide (YIGSK) had no effect. The induction of eNOS expression by shear stress is stimulated by an interaction of endothelial cells with laminin which is, at least in part, mediated by a 67-kDa laminin-binding protein.     

甲状腺癌患者血清促甲状腺激素和甲状腺激素表达水平及临床意义

目的:探讨甲状腺癌患者血清促甲状腺激素和甲状腺激素表达水平及临床意义。方法:应用电化学发光方法检测甲状腺癌组、甲状腺良性病变组和正常对照组血清促甲状腺激素(TSH)和甲状腺激素(TT3、FT3、TT4、FT4)水平。结果:①血清TSH在三组中比较有统计学意义(P〈0.001),甲状腺癌组血清TSH水平(3.56±0.93ulU/ml)明显高于甲状腺良性病变组(2.82±0.70ulU/ml)和正常对照组(2.04±0.56ulU/ml);TSH与肿瘤病理分期和肿瘤大小呈正相关(P<0.05)。②血清FT3、FT4水平在三组中有统计学意义(均P〈0.001),甲状腺癌组FT3、FT4水平处于较低水平,二者均明显低于甲状腺良性病变组和正常对照组(P<0.001);FT3与肿瘤病理分期和淋巴结转移呈负相关(P<0.05)。③TT3和TT4水平在三组之间比较均无统计学意义(P>0.05)。结论:高水平TSH可增加甲癌复发的危险性。低甲状腺激素水平在甲状腺癌形成中可能起到一定的作用,因此可以将其作为预测甲癌复发的重要指标之一。     

Differential tissue expression of three 35-kDa annexin calcium-dependent phospholipid-binding proteins

We have purified three 35-kDa calcium- and phospholipid-binding proteins from rat liver. These three calcimedins bind to phosphatidylserine in a calcium-dependent manner and have been termed 35 alpha, 35 beta, and 35 gamma based on their relative charge as determined by isoelectric focusing. Purification of the three 35-kDa calcimedins is achieved by phenyl-Sepharose, ion exchange, and gel filtration chromatography. Antibody was produced against the annexin consensus peptide, Lys-Ala-Met-Lys-Gly-Leu-Gly-Thr-Asp-Glu, which was derived from the sequence of several Ca2+/phospholipid-binding proteins including calpactin, lipocortin, endonexin II, 67-kDa calelectrin, lymphocyte 68-kDa protein, and protein II. Recognition of each 35-kDa calcimedin by anticonsensus sequence antibody places them in this protein family. Antibodies against each 35-kDa calcimedin were raised and purified by antigen-affinity chromatography. Each antibody is monospecific for the respective 35-kDa calcimedin. Immunological cross-reactivity defines 35 alpha, 35 beta, and 35 gamma as lipocortins III, IV, and V, respectively. Surveys by immunoblot analysis using these monospecific antibodies demonstrate a markedly different tissue expression pattern for each 35-kDa calcimedin. Furthermore, the levels of 35 alpha, 35 beta, and 35 gamma are differentially regulated in maturing rat ovary and uterus. Each calcimedin has been localized by indirect immunofluorescence within specific cell types. These results support the concept that mediation of the intracellular calcium signal can occur via multiple pathways through several related yet independent mediator proteins.     

Modulation of a major 30-kDa skeletal muscle protein by thyroid hormone

Thyroidectomy results in the transformation of type II fibres to type I in rat soleus muscle. In vitro translations containing polyribosomes indicate that the template activity of mRNA coding for a 30-kDa protein is increased in hypothyroid (6 months) rats. The cellular content of this protein is also increased in hypothyroid rats. The in vitro synthesis of the 30-kDa protein is not observed in thyroidectomized (10 weeks) rats that have been treated with triiodothyronine. The synthesis and accumulation of this protein are directly related to the proportion of type I fibres in rat skeletal muscle and appear to be modulated by thyroid hormone.     

Effects of thyroid state on the expression of hepatic thyroid hormone transporters in rats

Liver uptake of thyroxine (T4) is mediated by transporters and is rate limiting for hepatic 3,3',5-triiodothyronine (T3) production. We investigated whether hepatic mRNA for T4 transporters is regulated by thyroid state using Xenopus laevis oocytes as an expression system. Because X. laevis oocytes show high endogenous uptake of T4, T4 sulfamate (T4NS) was used as an alternative ligand for the hepatic T4 transporters. Oocytes were injected with 23 ng liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats, and after 3-4 days uptake was determined by incubation of injected and uninjected oocytes for 1 h at 25 degrees C or for 4 h at 18 degrees C with 10 nM [125I]T4NS. Expression of type I deiodinase (D1), which is regulated by thyroid state, was studied in the oocytes as an internal control. Uptake of T4NS showed similar approximately fourfold increases after injection of liver mRNA from euthyroid, hypothyroid, or hyperthyroid rats. A similar lack of effect of thyroid state was observed using reverse T3 as ligand. In contrast, D1 activity induced by liver mRNA from hyperthyroid and hypothyroid rats in the oocytes was 2.4-fold higher and 2.7-fold lower, respectively, compared with euthyroid rats. Studies have shown that uptake of iodothyronines in rat liver is mediated in part by several organic anion transporters, such as the Na+/taurocholate-cotransporting polypeptide (rNTCP) and the Na-independent organic anion-transporting polypeptide (rOATP1). Therefore, the effects of thyroid state on rNTCP, rOATP1, and D1 mRNA levels in rat liver were also determined. Northern analysis showed no differences in rNTCP or rOATP1 mRNA levels between hyperthyroid and hypothyroid rats, whereas D1 mRNA levels varied widely as expected. These results suggest little effect of thyroid state on the levels of mRNA coding for T4 transporters in rat liver, including rNTCP and rOATP1. However, they do not exclude regulation of hepatic T4 transporters by thyroid hormone at the translational and posttranslational level.     

Stromal expression of tenascin is inversely correlated to epithelial differentiation of hormone dependent tissues

We were previously investigating the expression of the extracellular matrix glycoprotein tenascin in normal and malignant endometrial tissues of humans and rodents. These studies suggested that the expression of tenascin was induced by proliferating epithelia (normal and particularly malignant) and was downregulated with their differentiation. The aim of this study was to investigate the hormone dependency of tenascin expression in (a) the transplantable EnDA endometrial tumor model with or without estrogen deprivation (overiectomy) of the animals, (b) DMBA-induced rat mammary tumors with or without a hormonal treatment of the animals [ovariectomy, antiestrogen (tamoxifen) or antiprogestin (ZK 98299) treatment] and (c) in the rat prostate of untreated or androgen deprived animals (orchiectomy, flutamide-, casodex- or cyproterone acetate (CPA)- treatment). 1. Estrogen withdrawal by ovariectomy did not affect tenascin expression in transplantable EnDA endometrial adenocarcinoma, meaning the entire extracellular space of the stromal mesenchyme was decorated by tenascin immunoreactivity. 2. In untreated DMBA-induced rat mammary tumors almost the entire extracellular space of the stroma was stained by tenascin immunoreactivity. Ovariectomy and antiestrogen treatment did not affect tenascin expression. In contrast, antiprogestin treatment induced terminal differentiation of mammary tumor cells and in parallel downregulated tenascin expression. 3. In the normal rat prostate no tenascin was detectable by immunocytochemistry. However, following androgen deprivation we found tenascin expression in the stroma of the prostate. The most prominent expression was observable after CPA-treatment, possibly due to its progestagenic potency. In conclusion, the hormones and antihormones tested show no direct effect on the stromal expression of tenascin. However, proliferative activity and a low degree of differentiation of the epithelium induces tenascin expression, whereas epithelial differentiation apparently shuts down tenascin expression. Preliminary in vitro studies suggest that paracrine acting growth factors trigger the hormonal regulation of tenascin expression.     

A unique protein-kinase activity is responsible for the phosphorylation of the 26- and 14-kDa proteins but not of the 67-kDa protein in the chloroplast envelope membranes of spinach

Protein-phosphorylation activity has been reported in chloroplast envelope membranes of several species. In spinach (Spinacia oleracea L.), we found three major phosphoproteins after incubation in vitro of envelope membranes in the presence of [-³²P]ATP. A 67-kDa phosphoprotein was associated with both inner and outer envelope membranes whereas 26- and 14-kDa proteins were observed in the inner membrane. Although the phosphorylation of the 67-kDa protein is likely to take place via its phosphoglucomutase activity (Salvucci et al., 1990, Plant Physiol. 93, 105–109), the mechanism by which ³²P is incorporated into the 26- and 14-kDa proteins remains to be elucidated. To this aim, we have compared the conditions under which phosphorylation occurs in these three proteins. The effects of Mg²⁺, Ca²⁺, pH, ATP and H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], a specific inhibitor of protein-kinase C, as well as pulse-chase experiments with cold ATP, showed that the phosphorylation mechanism was identical for the 26- and 14-kDa proteins but quite different for the 67-kDa one. The protein kinase involved in the phosphorylation of the 26- and 14-kDa proteins was Ca²⁺-dependent, which was not the case of the 67-kDa protein. In addition, the use of a Triton X-114 phase-separation treatment indicated that both the 26- and 14-kDa proteins exhibited strong hydrophobic properties, in contrast to the hydrophilic character of the 67-kDa phosphoprotein. As indicated by analyses of phosphoamino acids, the three proteins were exclusively phosphorylated on serine residues. Furthermore, a treatment of envelopes by phospholipase C prior to the phosphorylation process inhibited ³²P incorporation into the three phospho-proteins to different extents (61%, 50% and 29% inhibition for the 67-, 14- and 26-kDa proteins, respectively). These results show that phosphatidylcholine and — or phosphatidylglycerol but not phosphatidylinositol were involved in this phosphorylation process.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SDS sodium dodecyl sulfate The authors are grateful to Mrs. Delphine Herrmann and Mr. Daniel Leemann for their skillful technical assistance. This study was supported by the Swiss National Science Foundation (Grant No. 31.26386.89). This work is part of a doctoral program which is carried out by L.B. in the Laboratoire de Physiologie végétale, Université de Neuchâtel.     

Invasion of tumorigenic HT1080 cells is impeded by blocking or downregulating the 37-kDa/67-kDa laminin receptor

The 37-kDa/67-kDa laminin receptor precursor/laminin receptor (LRP/LR) acting as a receptor for prions and viruses is overexpressed in various cancer cell lines, and their metastatic potential correlates with LRP/LR levels. We analyzed the tumorigenic fibrosarcoma cell line HT1080 regarding 37-kDa/67-kDa LRP/LR levels and its invasive potential. Compared to the less invasive embryonic fibroblast cell line NIH3T3, the tumorigenic HT1080 cells display approximately 1.6-fold higher cell-surface levels of LRP/LR. We show that anti-LRP/LR tools interfere with the invasive potential of HT1080 cells. Anti-LRP/LR single-chain variable fragment antibody (scFv) iS18 generated by chain shuffling from parental scFv S18 and its full-length version immunoglobulin G1-iS18 reduced the invasive potential of HT1080 cells significantly by 37% and 38%, respectively. HT1080 cells transfected with lentiviral plasmids expressing small interfering RNAs directed against LRP mRNA showed reduced LRP levels by approximately 44%, concomitant with a significant decrease in the invasive potential by approximately 37%. The polysulfated glycans HM2602 and pentosan polysulfate (SP-54), both capable of blocking LRP/LR, reduced the invasive potential by 20% and 35%, respectively. Adhesion of HT1080 cells to laminin-1 was significantly impeded by scFv iS18 and immunoglobulin G1-iS18 by 60% and 68%, respectively, and by SP-54 and HM2602 by 80%, suggesting that the reduced invasive capacity achieved by these tools is due to the perturbation of the LRP/LR-laminin interaction on the cell surface. Our in vitro data suggest that reagents directed against LRP/LR or LRP mRNA such as antibodies, polysulfated glycans, or small interfering RNAs, previously shown to encompass an anti-prion activity by blocking or downregulating the prion receptor LRP/LR, might also be potential cancer therapeutics blocking metastasis by interfering with the LRP/LR-laminin interaction in neoplastic tissues.     

The effect of hormone therapy on olfactory sensitivity is dependent on apolipoprotein E genotype

Patients with Alzheimer's disease (AD) show a deficit in olfactory threshold sensitivity. The Apolipoprotein E (ApoE) 4 allele is associated with increased risk of AD and earlier symptom onset. Hormone therapy (HT) may exert neuroprotective effects on brain areas affected by AD. The current study investigated the effect of HT on performance on an olfactory threshold test in 4 positive and 4 negative non-hysterectomized, non-demented, elderly females and AD patients. Among the non-demented participants, 4 positive females who had received HT performed 1) significantly better than those without HT, and 2) at levels similar to those of 4 negative females. In contrast, those without HT who were 4 positive performed significantly worse than those who were 4 negative. HT had no effect on performance in AD patients regardless of 4 status. These results suggest that HT may offer protection against loss of olfactory function in 4 positive individuals in preclinical stages of AD. Future research is warranted in order to investigate further the neuroprotective role of HT on sensory and cognitive functions in non-demented aging individuals.     

Thyroid hormone induces the expression of 4-1BB and activation of caspases in a thyroid hormone receptor-dependent manner.

Thyroid hormone has various effects on cell proliferation, growth and apoptosis. To gain more insight into the molecular dynamics caused by thyroid hormone, gene expression in HeLaTR cells that constitutively overexpressed the thyroid hormone receptor (TR) was analyzed. Gene expression profiling of the HeLaTR cells with an oligonucleotide microarray yielded 229 genes whose expression was significantly altered by T3. Among these genes, the expression of 4-1BB, which is known to initiate a signal cascade activating NF-kappaB, was significantly up-regulated by T3. Although treatment of the HeLaTR cells with T3 did not induce expression of NF-kappaB reporter luciferase, even in the presence of the 4-1BB-Ligand, it increased the caspase activities. An increase in the caspase activities was also observed in the HeLaTR cells transfected with 4-1BB cDNA, and the 4-1BB-Ligand further increased the caspase activities of the HeLaTR cells overexpressing the 4-1BB. Furthermore, up-regulation of 4-1BB and an increase in caspase activities also occurred in the rat FRTL cells that expressed only authentic TR. These results demonstrate that the expression of 4-1BB serves as the mediator of signals from T3 to activate caspases.     

The 35- and 36-kDa beta subunits of GTP-binding regulatory proteins are products of separate genes

The wide range of functions attributed to GTP-binding regulatory proteins (G proteins) is reflected in the structural diversity which exists among the alpha, beta, and gamma subunits of G proteins. Recently two cDNA clones encoding beta subunits, beta 1 and beta 2, were isolated from bovine and human cDNA libraries. We report here that the beta 2 gene encodes the 35-kilodalton (kDa) component of the beta 35/beta 36 subunit of G proteins and that the beta 1 gene encodes the 36-kilodalton component. The in vitro translation product of the beta 2 cDNA co-migrates with the 35-kDa beta subunit (beta 35), while the in vitro product of the beta 1 cDNA co-migrates with the 36-kDa beta subunit (beta 36) on denaturing polyacrylamide gels. In addition, antisera generated against synthetic beta 2 peptides bind specifically to the beta 35 component of isolated G proteins and to a 35-kDa protein in myeloid cell membranes. Our results suggest that the two beta subunits could serve distinct functions, as they are derived from separate genes which have been highly conserved in evolution.     

Functional domains of the 67-kDa laminin receptor precursor

We report the characterization of two functional domains of the metastasis-associated 67-kDa laminin receptor (67-LR). Using synthetic peptides deduced from the cDNA sequence of the 37-kDa precursor of the laminin receptor (37-LRP) as well as their corresponding affinity-purified polyclonal antibodies, we identified a unique laminin binding site as well as a membrane-associated domain of the receptor. In laminin dot blot and solid phase radioligand assays, a 20 amino acid synthetic peptide (IPCNNKGAHSVGLMWWMLAR, amino acid residues 161-180, designated peptide G) specifically bound to laminin with high affinity (Kd = 5 x 10(-8) M). Peptide G also specifically eluted the 67-LR from a laminin affinity column. Peptide G and laminin reacted with a 1:1 stoichiometry, suggesting that there is one recognition site on laminin for the peptide G domain. Immunofluorescence studies, performed on permeabilized and nonpermeabilized human A2058 melanoma cells using 10 different affinity-purified antibodies to distinct regions of the 37-LRP, identified an unusually short membrane-associated domain that was consistent with a computer predicted transmembrane domain (residues 86-101). Our data demonstrate for the first time that the 37-LRP has two functional domains consistent with the characteristics of the mature 67-LR. Furthermore, we propose peptide G as a potential inhibitor of tumor cell interactions with laminin.