Attenuation of Gi- and Gq-mediated signaling by expression of RGS4 or GAIP in mammalian cells

Protein regulators of G protein signaling (RGS proteins) were discovered as negative regulators of heterotrimeric G protein-mediated signal transduction in yeast and worms. Experiments with purified recombinant proteins in vitro have established that RGS proteins accelerate the GTPase activity of certain G protein alpha subunits (the reaction responsible for their deactivation); they can also act as effector antagonists. We demonstrate herein that either of two such RGS proteins, RGS4 or GAIP, attenuated signal transduction mediated by endogenous receptors, G proteins, and effectors when stably expressed as tagged proteins in transfected mammalian cells. The pattern of selectivity observed in vivo was similar to that seen in vitro. RGS4 and GAIP both attenuated Gi-mediated inhibition of cAMP synthesis. RGS4 was more effective than GAIP in blocking Gq-mediated activation of phospholipase Cbeta.     

Rotavirus infection reduces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. These viruses infect mature enterocytes of the small intestine and cause structural and functional damage, including a reduction in disaccharidase activity. It was previously hypothesized that reduced disaccharidase activity resulted from the destruction of rotavirus-infected enterocytes at the villus tips. However, this pathophysiological model cannot explain situations in which low disaccharidase activity is observed when rotavirus-infected intestine exhibits few, if any, histopathologic changes. In a previous study, we demonstrated that the simian rotavirus strain RRV replicated in and was released from human enterocyte-like Caco-2 cells without cell destruction (N. Jourdan, M. Maurice, D. Delautier, A. M. Quero, A. L. Servin, and G. Trugnan, J. Virol. 71:8268-8278, 1997). In the present study, to reinvestigate disaccharidase expression during rotavirus infection, we studied sucrase-isomaltase (SI) in RRV-infected Caco-2 cells. We showed that SI activity and apical expression were specifically and selectively decreased by RRV infection without apparent cell destruction. Using pulse-chase experiments and cell surface biotinylation, we demonstrated that RRV infection did not affect SI biosynthesis, maturation, or stability but induced the blockade of SI transport to the brush border. Using confocal laser scanning microscopy, we showed that RRV infection induces important alterations of the cytoskeleton that correlate with decreased SI apical surface expression. These results lead us to propose an alternate model to explain the pathophysiology associated with rotavirus infection.     

Raf-1 protein expression in human breast cancer cells

BACKGROUND: The Raf-1 kinase, a 72-kDa cytoplasmic serine-threonine kinase, plays a central role as a second messenger in signal transduction. After ligand binding to a variety of transmembrane tyrosine kinase growth factor receptors including epidermal growth factor (EGF) receptor, the 72-kDa kinase is activated through phosphorylation to a 74-kDa phosphoprotein. The Raf-1 kinase is constitutively activated in many transformed cells either directly, by mutations within its amino-terminus regulatory region, or indirectly, due to overstimulation by autocrine growth factors or activated proximal oncogenes. The role of Raf-1 kinase in breast cancer has not been studied. METHODS: To investigate the role of Raf-1 kinase expression and its activation in breast cancer, we studied three human breast cancer cell lines expressing varying amounts of EGF receptor to determine the level of Raf-1 protein and the proportion expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on the Raf-1 protein were studied in T47D, BT474, and MDA-MB231 cells by precipitation of cell lysates with an anti-Raf-1 antibody followed by immunoblotting. [3H]Thymidine incorporation by these cells after EGF stimulation was also determined as a measure of DNA synthesis. RESULTS: In all three breast cancer cell lines studied, the Raf-1 protein was identified in a 70- and a 74-kDa form. The level of Raf-1 was similar in all three cell lines and appeared unrelated to EGF receptor expression on the cell surface. The majority of the protein was found in the 74-kDa form even after serum starvation. A minor shift from the lower to higher molecular weight form of Raf-1 was apparent in cells treated with EGF, and increased [3H] thymidine incorporation could be demonstrated in two of the cell lines after EGF stimulation. CONCLUSION: Baseline expression of the 74-kDa or activated form of the Raf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation. Further investigation into the role of Raf-1 protein in the pathogenesis of breast cancer is indicated.     

Significant expression of G-CSF-induced gene-1 (GIG-1) protein in myeloid cells and NK cells

25-Hydroxycholesterol negatively regulates cholesterol synthesis and activates cholesterol esterification in a variety of cultured cells. Concurrent with these effects, 25-hydroxycholesterol also stimulates the synthesis of sphingomyelin in Chinese hamster ovary (CHO)-K1 cells. The role of oxysterol binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol, in activation of SM synthesis was assessed by overexpression in CHO-K1 cells. When compared to mock transfected controls, three CHO-K1 clones overexpressing OSBP by 10- to 15-fold displayed a 2- to 3-fold enhancement of [3H]serine incorporation into sphingomyelin when treated with 25-hydroxycholesterol. Closer examination of one of these clones (CHO-OSBP cells) revealed a >8.5-fold stimulation of sphingomyelin synthesis after a 6-h treatment with 25-hydroxycholesterol compared to 3.5-fold in controls, slightly higher basal levels of sphingomyelin synthesis, and a more rapid response to 25-hydroxycholesterol. [3H]serine incorporation into phosphatidylserine, phosphatidylethanolamine, ceramide, or glucosylceramide was affected by <15%. Synthesis of sphingomyelin from exogenous [3H]sphinganine-labeled ceramide was enhanced in overexpressing cells treated with 25-hydroxycholesterol. However, in vitro activities of sphinganine N-acyltransferase, sphingomyelin synthase, and serine palmitoyltransferase were not affected by OSBP overexpression or 25-hydroxycholesterol. Overexpression of OSBP or 25-hydroxycholesterol did not significantly affect the ceramide content of Golgi-enriched fractions from control or overexpressing cells. However, diglyceride mass was reduced in Golgi-enriched fractions from overexpressing cells and by treatment with 25-hydroxycholesterol. Results from overexpressing cells show that OSBP potentiates the stimulatory effects of 25-hydroxycholesterol on sphingomyelin synthesis. 25-Hydroxycholesterol promotes translocation of OSBP to the Golgi apparatus where it appears to stimulate conversion of ceramide to sphingomyelin.     

The expression of steroidogenic acute regulatory protein (StAR) in bovine adrenocortical cells

StAR protein may facilitate rapid transfer of cholesterol from the outer to the inner mitochondrial membrane, the site at which cholesterol is converted to pregnenolone by the cholesterol side chain cleavage complex. We have studied the effect of ACTH treatment on StAR mRNA and protein levels in bovine adrenocortical cells in primary culture. Cells were initially cultured for 3 days after isolation, and then treated with ACTH (10(-8) M) for various times up to 24 hours. Northern analysis of total BAC mRNA, using a [alpha32P]-labelled cDNA probe encoding a 5' region of bovine StAR mRNA, revealed two principal hybridising species of 1.6 and 3.0 kb. Western immunoblot analysis revealed a principal band at 30 kDa. Levels of both StAR mRNA and protein showed an increase at 1 hour, reached a maximum at around 6 hours and declined to basal levels at 24 hours. Cortisol secretion (measured by RIA) showed a similar change over the same period. From these results it appears that StAR mRNA and protein levels in BAC are acutely regulated in concert with ACTH-stimulated cortisol secretion.     

Hormone-sensitive lipase expression and activity in relation to lipolysis in human fat cells

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in adipocyte lipolysis. The activity of HSL is thought to be primarily regulated by reversible phosphorylation. However, the regulation of HSL activity by pre-translational mechanisms has been poorly studied. The present studies were undertaken to explore the relationship between the levels of HSL protein and mRNA expressions and the lipolytic capacity. The study was performed in human abdominal subcutaneous adipocytes with identical sizes but having either a high (HL) or low (LL) lipolytic capacity (n = 16). Basal and maximal lipolysis induced by catecholamines, an adenylyl cyclase activator forskolin, and a cyclic AMP analogue dibutyryl cAMP were 50% lower in LL- in comparison with HL-fat cells (P < 0.05 or better). No differences in drug sensitivity were found. HSL activity and quantity were about 50% lower in LL- compared with HL-fat cells (P < 0.05). Moreover, the mRNA ratio between HSL and gamma-actin was 35% lower in LL- compared with HL-fat cells (P < 0.05). There was a strong linear correlation between the protein and enzymatic HSL measurements (r2 = 0.91). In addition, the maximum lipolytic capacity was significantly correlated with HSL activity (r2 = 0.75) and HSL protein amount (r2 = 0.64). It is concluded that hormone-sensitive lipase (HSL) expression, measured either as total HSL protein by Western blot analysis or as total amount of activatable HSL enzyme, is a major determinant of the maximum lipolytic capacity of human fat cells. In addition, HSL protein expression is at least, in part, determined by HSL mRNA expression.     

Elevated mitogen-activated protein kinase activity in estrogen-nonresponsive human breast cancer cells

The mitogen-activated protein kinase (MAPK) signal transduction pathway plays an essential role in cell cycle progression and can be activated by many growth factor/mitogen pathways including estrogen. MAPK has also been implicated in ligand-independent activation of estrogen receptor-alpha (ER-alpha). The development of estrogen-independent growth in breast cancer is likely a first step in progression to hormone independence and antiestrogen resistance. We examined MAPK expression and activity in T5-PRF and T5 human breast cancer cells. T5-PRF is an estrogen-nonresponsive cell line developed from T5 cells by chronically depleting the cells of estrogen in long-term culture. MAPK activity measured in vitro was significantly higher (P < 0.05) in T5-PRF compared with T5 cells. Western blot analyses showed increased levels of active dually phosphorylated MAPK in T5-PRF cell extracts compared with T5. The increased activity and expression of MAPK may contribute to the estrogen nonresponsive growth phenotype and ligand-independent activity of ER in T5-PRF cells.     

Apolipoprotein(a) induces monocyte chemotactic activity in human vascular endothelial cells

BACKGROUND: Elevated levels of lipoprotein(a) [Lp(a)] are associated with premature atherosclerosis; however, the mechanisms are not known. Recruitment of monocytes to the blood vessel wall is an early event in atherogenesis. METHODS AND RESULTS: This study has found that unoxidized Lp(a) induced human umbilical vein endothelial cells (HUVECs) to secrete monocyte chemotactic activity (MCA), whereas LDL under the same conditions did not. In the absence of HUVECs, Lp(a) had no direct MCA. Endotoxin was shown not to be responsible for the induction of MCA. Actinomycin D and cycloheximide inhibited the HUVEC response to Lp(a), indicating that protein and RNA synthesis were required. The apolipoprotein(a) [apo(a)] portion of Lp(a) was identified as the structural component of Lp(a) responsible for inducing MCA. Lp(a) and apo(a) also stimulated human coronary artery endothelial cells to produce MCA. Granulocyte-monocyte colony-stimulating factor (GM-CSF) antigen was not detected in the Lp(a)-conditioned medium, nor was monocyte chemoattractant protein-1 mRNA induced in HUVECs by Lp(a). CONCLUSIONS: These findings suggest that Lp(a) may be involved in the recruitment of monocytes to the vessel wall and provide a novel mechanism for the participation of Lp(a) in the atherogenic process.     

Cytotoxic and antitumor activity of a recombinant tumor necrosis factor-B1(Fv) fusion protein on LeY antigen-expressing human cancer cells

We have constructed a fusion protein composed of tumor necrosis factor alpha (TNF-alpha) fused at its COOH terminus to the scFv region of monoclonal antibody (mAb) B1, an antibody that recognizes LeY antigen present on many human cancer cells. Our rationale for fusing the scFv to the COOH terminus of TNF was to diminish the binding of the fusion protein to TNF receptors because the COOH terminus of TNF is involved in binding, and thus to partially inactivate (detoxify) the molecule. The Fv region should then target and accumulate the fusion protein on cancer cells, which should compensate for the reduced binding affinity of the TNF moiety and lead to selective killing of TNF-sensitive antigen-expressing cancer cells. The fusion protein was expressed in Escherichia coli and found in insoluble inclusion bodies. After refolding and purification by anion exchange, Ni-NTA affinity, and size-exclusion chromatography, we obtained monomeric TNF-B1(Fv). This molecule binds to LeY antigen on cancer cells with the same affinity as B1(scFv) and B1(scFv) immunotoxins but with significantly lower affinity to the TNF receptor compared to the TNF trimer. TNF-B1(Fv) is very toxic to LeY antigen-expressing cancer cells that are sensitive to TNF (e.g., MCF-7 breast or CRL-1739 gastric cancer cells). This cytotoxicity is antibody targeted and TNF mediated because it can be prevented (as shown on MCF-7 cells) by an antibody competing for LeY antigen binding and by an antibody that neutralizes TNF-alpha. TNF-B1(Fv) kills TNF-alpha-sensitive cells that do not express the target antigen only at much higher doses than TNF trimer, and it does not kill LeY-bearing but TNF-alpha-resistant cells. TNF-B1(Fv) can cause significant tumor regression of MCF-7 tumor xenografts in mice at doses that are not toxic to the mice. Thus, the reduced binding of the TNF moiety to TNF receptors, combined with binding of the B1(Fv) portion to LeY antigen, makes TNF-B1(Fv) an agent for selective killing of LeY-expressing TNF-sensitive cancer cells.     

Polymorphic protein expression (profile) in numigall

Ten protein and enzyme polymorphic systems, viz. haemoglobin, albumin, transferrin, adenosine deaminase, adenylate kinase phosphoglucomutase, esterase-D, glyoxalase, alkaline phosphatase and amylase were studied in numigall (guinea fowl x chicken hybrids) to assess structural gene expression and regulatory gene divergence between the parental species. The investigation revealed presence of both the maternal and paternal electrophoretic components in case of adenosine deaminase, alkaline phosphatase, amylase, albumin and transferrin although no clear differences could be identified for haemoglobin and glyoxalase. Esterase-D and adenylate kinase phenotypes showed a dominance of the chicken type.     

Secretion of human meprin from intestinal epithelial cells depends on differential expression of the alpha and beta subunits

Human meprin (N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase, EC 3.4.24.18), an astacin-type metalloprotease, is expressed by intestinal epithelial cells as a dimeric protein complex of alpha and beta subunits. In transfected cells, intracellular proteolytic removal of the membrane anchor from the alpha subunit results in its secretion, while the beta subunit and alpha/beta heterodimers are retained at the cell membrane. We investigated the consequence of differential intracellular processing of alpha and beta subunits in the human small and large intestine using subunit-specific immunohistochemistry, in situ hybridization and biosynthetic studies in organ culture. In the ileum, both subunits localize to the brush-border membrane of villus enterocytes. In contrast, the beta subunit is not expressed in the colon, which leads to the secretion of the alpha subunit. We conclude that differential expression of meprin alpha and beta subunits is a unique means of targeting the proteolytic activity of the alpha subunit either to the brush-border membrane in the ileum or to the lumen in the colon, suggesting dual functions of cell-associated and luminal meprin. Meprin alpha and beta subunits are also coexpressed in distinct lamina propria leukocytes, suggesting an additional role for this protease in leukocyte function in the intestinal mucosa.     

Effects of Cr(VI) on the expression of the oxidative stress genes in human lung cells

Intracellular metabolism of chromium(VI) [Cr(VI)] may lead to oxidative stress and this may account for the ability of Cr(VI) to act as a complete carcinogen. Therefore, we examined the effects of Cr(VI) treatment on the expression of oxidative stress genes in normal human lung LL 24 cells and human lung adenocarcinoma A549 cells. RT-PCR and northern blot analyses were used to determine the steady-state mRNA levels of catalase, glutathione S-transferase, glutathione reductase, Cu/Zn- and Mn-superoxide dismutases, glutathione peroxidase, NAD(P)H:quinone oxidoreductase, heme oxygenase and interleukin 8 in control cells and cells treated with 5-200 microM of Cr(VI). We found that only expression of the heme oxygenase gene is strongly elevated under the treatment with Cr(VI), and only in normal human lung LL 24 cells. Our data showed that even in the absence of Cr(VI) treatment, the level of heme oxygenase gene expression is much higher in A549 cells than in LL 24 cells. As glutathione is believed to play a protective role in cells against different forms of oxidative stress, we studied the correlation between intracellular glutathione levels and the inducibility of the heme oxygenase gene after treatment of cells with Cr(VI). Our results demonstrate that glutathione levels are increased by 35 % of control values in LL 24 cells treated with Cr(VI). The data obtained indicate that heme oxygenase, known to be a stress-inducible gene, may be involved in cellular pathways critical to the carcinogenic activity of Cr(VI) in normal human lung cells. Intracellular glutathione levels and reactive oxygen species do not appear to be primarily responsible for the stress response, induced by Cr(VI) in the studied human cells.     

Differential expression and androgen regulation of the human selenium-binding protein gene hSP56 in prostate cancer cells

Low levels of dietary selenium are associated with increased risk of malignancy of several organs, including the prostate. Using a subtractive approach called linker capture subtraction, we have found that the human selenium-binding protein gene hSP56 is differentially expressed by the relatively slow-growing, androgen-sensitive prostate cancer cell line LNCaP but not by the more rapidly growing androgen-insensitive lines PC-3 and DU145. We confirmed this differential expression by Northern blot analysis. Importantly, hSP56 expression by LNCaP cells was reversibly down-regulated by exogenous androgen in a concentration-dependent manner. Marked differences in steady-state hSP56 mRNA levels were found in a variety of normal and neoplastic human cells that were examined. hSP56 expression was especially high in normal tissues that appear to benefit from the cancer-protective action of dietary selenium and was low in many neoplastic cells. The results suggest that hSP56 may play a role in determining the neoplastic phenotype.     

Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart

BACKGROUND: Increased expression of Ca2+-sensitive protein kinase C (PKC) isoforms may be important markers of heart failure. Our aim was to determine the relative expression of PKC-beta1, -beta2, and -alpha in failed and nonfailed myocardium. METHODS AND RESULTS: Explanted hearts of patients in whom dilated cardiomyopathy or ischemic cardiomyopathy was diagnosed were examined for PKC isoform content by Western blot, immunohistochemistry, enzymatic activity, and in situ hybridization and compared with nonfailed left ventricle. Quantitative immunoblotting revealed significant increases of >40% in PKC-beta1 (P<0.05) and -beta2 (P<0.04) membrane expression in failed hearts compared with nonfailed; PKC-alpha expression was significantly elevated by 70% in membrane fractions (P<0.03). PKC-epsilon expression was not significantly changed. In failed left ventricle, PKC-beta1 and -beta2 immunostaining was intense throughout myocytes, compared with slight, scattered staining in nonfailed myocytes. PKC-alpha immunostaining was also more evident in cardiomyocytes from failed hearts with staining primarily localized to intercalated disks. In situ hybridization revealed increased PKC-beta1 and -beta2 mRNA expression in cardiomyocytes of failed heart tissue. PKC activity was significantly increased in membrane fractions from failed hearts compared with nonfailed (1021+/-189 versus 261+/-89 pmol. mg-1. min-1, P<0.01). LY333531, a selective PKC-beta inhibitor, significantly decreased PKC activity in membrane fractions from failed hearts by 209 pmol. min-1. mg-1 (versus 42.5 pmol. min-1. mg-1 in nonfailed, P<0.04), indicating a greater contribution of PKC-beta to total PKC activity in failed hearts. CONCLUSIONS: In failed human heart, PKC-beta1 and -beta2 expression and contribution to total PKC activity are significantly increased. This may signal a role for Ca2+-sensitive PKC isoforms in cardiac mechanisms involved in heart failure.     

Age-related alteration of proline hydroxylase and collagen-binding heat shock protein (HSP47) expression in human fibroblasts

It is now well recognized that a disorder of left ventricular filling can be sufficient to account for congestive heart failure. Furthermore, evaluation of heart disease would not be complete if it did not include assessment of left ventricular filling, improvement of which probably ensures better control of the heart disease. An efficient and reliable tool for the study of diastolic function is therefore essential. The authors review the current state of knowledge and the more recent developments in Doppler echocardiography in the evaluation of left ventricular diastolic function. After revising the pathophysiology, the methods of studying ventricular filling are described. The recording technique is described, taking into account recent developments in transthoracic and transoesophageal approaches. This investigation provides parameters allowing semiquantitative estimation of filling pressures (mean left atrial pressure, end-diastolic pressure) and reliable evaluation of overall diastolic performance.     

Cloning and squencing of human thrombopoietin (hTPO) cDNA and it''s expression in COS-7 cells]

Viliuisk encephalomyelitis (VE) is an unique neurological disease occurring in the Iakut (Sakha) people of Siberia. Evolution of the disease follows one of three broad clinical forms: subacute, slowly progressive or chronic. Death occurs within 3 to 6 months in subacute cases and within 6 years in the slowly progressive cases. Chronic cases lack a subacute phase but show a slowly progressive dementia associated with bradykinesia, dysarthria and spastic paraparesis that stabilizes late in the disease process. In subacute and slowly progressive cases, focal necrotizing encephalomyelitis is seen at necropsy. Chronic cases show multifocal areas of lysis with a gliotic margin, predominantly within grey matter, lacking associated chronic inflammatory changes seen in the other forms of the disease. Epidemiological studies are consistent with a disease of low-grade communicability, but laboratory studies have so far failed to reveal an infectious organism. The spectrum of neuropathological changes are reviewed in this examination of 11 cases. Although the aetiology of VE remains obscure, further studies are warranted since it may represent a novel disease process.     

Lipoxygenase products and expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human cultured synovial cells

Limb regeneration is a phenomenon occurring only in some urodeles. The process seems to be initiated by the dedifferentiation of the terminally differentiated cells. These cells differentiate, subsequently, to the tissues that comprise the limb, thus reconstructing the pattern of the missing limb part. In this paper we review and present evidence that certain cell types of the limb have the capacity to differentiate to different cell types than their original one by cellular metaplasia. This switch is called transdifferentiation. The focus of this review is the process of dedifferentiation which is the necessary prerequisite for differentiation, and the possible mechanisms involved.