Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and secretion of alpha-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata, and its synthesis by hepatocytes seeded onto a mixed substratum of laminin and fibronectin is down-regulated by fibronectin in a dose-related manner. Similarly, type IV collagen synthesis is less when the cells are seeded on the homologous matrix protein substratum than on heterologous substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as alpha-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.     

Modulation of endothelial nitric oxide synthase by fibronectin

Nitric oxide (NO) produced by the action of endothelial nitric oxide synthase (eNOS) plays an important role in the regulation of vascular tone, cell survival, and angiogenesis. Interaction of endothelial cells (ECs) with a fibronectin (FN) rich matrix is important in the regulation of EC function and survival during angiogenesis. The present study was carried out to examine if FN can regulate eNOS and thereby NO levels in ECs. The activity and the levels of mRNA and protein of eNOS were significantly low in HUVECs maintained in culture on FN. Inhibition of p38 MAPK and blocking the interaction of FN with α₅β₁ integrin using antibody caused the reversal of the FN effect. Immunoblot analysis of Ser/Thr phosphorylation of purified eNOS suggested that FN downregulates post-translational phosphorylation of eNOS at Ser residues. These results suggest that FN negatively modulates eNOS in an α₅β₁ integrin-p38 MAPK-dependent pathway.     

Metalloids in plants: A systematic discussion beyond description

Metalloids represent a wide range of elements with intermediate physiochemical properties between metals and non-metals. Many of the metalloids, like boron, selenium, and silicon are known to be essential or quasi-essential for plant growth. In contrast, metalloids viz. arsenic and germanium are toxic to plant growth. The toxicity of metalloids largely depends on their concentration within the living cells. Some elements, at low concentration, may be beneficial for plant growth and development; however, when present at high concentration, they often exert negative effects. In this regard, understanding the molecular mechanisms involved in the uptake of metalloids by roots, their subsequent transport to different tissues and inter/intra-cellular redistribution has great importance. The mechanisms of metalloids' uptake have been well studied in plants. Also, various transporters, as well as membrane channels involved in these processes, have been identified. In this review, we have discussed in detail the aspects concerning the positive/negative effects of different metalloids on plants. We have also provided a thorough account of the uptake, transport, and accumulation, along with the molecular mechanisms underlying the response of plants to these metalloids. Additionally, we have brought up the previous theories and debates about the role and effects of metalloids in plants with insightful discussions based on the current knowledge.     

Actin binds to the cytoplasmic tail of alpha 1 subunit of integrin in hepatocytes

alpha 1 beta 1-Integrin is a common receptor for laminin and collagen IV on hepatocytes. The interactions of intracellular domain of integrins with cytoplasmic elements are critical in the initiation and transduction of signals. In order to understand the nature of cytoplasmic components that can interact with cytoplasmic domain of alpha 1 integrin, cytoplasmic extracts of monolayers of rat hepatocytes were subjected to chromatography over an affinity column prepared by coupling a 60-mer synthetic cytoplasmic tail of alpha 1 subunit. SDS-PAGE analysis of the eluate showed the presence of a 47 kDa protein. Dot-Blot assay using radio-iodinated 47 kDa protein showed the binding of the protein to 60-mer C tail in a concentration dependent manner. Immunoblot analysis using specific antibodies showed that the 47 kDa protein is actin.     

Matrix Metalloproteinase in Mammary Gland Remodeling-Modulation by Glycosaminoglycans

Mammary gland which undergoes proliferation, differentiation and involution in adult life is a useful model system to study the role of extracellular matrix (ECM) in regulating tissue specific functions. The involution that follows weaning results in the suppression of casein gene expression, collapse of alveolar structures and degradation of basement membrane as evidenced by biochemical analysis of matrix components like proteoglycans and collagen. Differential expression of three different MMPs viz. 130 K, 68 K and 60 K with varying specificity to Col IV of basement membrane and Col I of stroma, their selective inhibition by TIMP and proteoglycans and modulation by estrogen highlight the importance of these in the remodeling of the ECM in the mammary gland. The inhibition of these MMPs by glycosaminoglycans, particularly CS and change in the concentration of CS at different stages of mammary gland development suggests the existence of a novel mechanism for the regulation of the activity of MMPs at extracellular sites.