No abstract

Turgor pressure, the difference in osmotic pressure across the inner membrane, has been found to regulate expression of the kdp operon in Escherichia coli. The kdp operon codes for a high-affinity repressible transport system for the uptake of potassium. We have studied the regulation of Kdp expression in a strain in which the gene for fi-galactosidase, lAcZ, was placed under control ofthe kdp promotor. Neither internal nor external K+ concentrations directly controlled Kdp expression. Only when the external K+ concentration was reduced to the point of limiting growth was the kdp operon expressed. An increase in external osmolarity at constant K+ concentration, a procedure that reduces turgor pressure, caused expression of the kdp operon. As the magnitude ofthe osmotic shift was increased, corresponding to greater decreases in turgor pressure, the amount of Kdp expression also increased. The kdp operon thus appears to be controlled by changes in a physical force, the turgor pressure.Bacteria maintain an internal osmolarity significantly higher than that outside the cell. This positive difference between the internal and the external osmolarity, known as the turgor pressure, is required for growth and division. In Escherichia coli, the turgor pressure is maintained at approximately 3 atm (1 atm = 101 kPa) (1) by the accumulation of K+ and a variety of metabolically produced anions (2). The primary role of K+ in E. coli is the' regulation of osmolarity (3), although it also serves to activate many cellular enzymes. The internal K+ concentration is determined by the osmolarity of the medium and ranges from 0.1 M in very dilute media to 0.6 M in 1200 mosM media (3). Activation of cellular enzymes requires K+ concentrations of less than 0.1 M (4), which is less than the lowest concentration ever found in E. coli.To regulate internal osmolarity, E. coli accumulate K+ by two distinct transport systems (5). Under most growth conditions, K+ is taken up by the constitutive TrkA system, which has a high rate oftransport and a low affinity for K+ (5). At very low external K+ concentrations or when the TrkA function has been impaired through mutation, K+ is taken up by the high-affinity Kdp system, which has a K., of2 ,uM (5). This repressible system is ATP driven (6) and requires the expression of four closely linked genes (7,8). Three of these genes, kdpA, kdpB, and kdpC, form an operon that codes for three inner membrane proteins (9). The fourth gene, kdpD, codes for a positive regulator and is located at the promoter-distal end of the kdpABC operon (8).The kdp operon is repressed by growth in media of high K+ concentration, and the K+ concentration at which derepression occurs depends on the activity of the TrkA system. Growth in 5 mM K+ medium represses the kdp operon in a wild-type strain but results in partial derepression in a strain that has an impaired TrkA function (5). These observations suggest -that the ability to meet K+ requirements can be detected and, translated into a signal controlling Kdp expres...

Analysis of K transport mutants indicates the existence of four separate K uptake systems in Escherichia coli K-12. A high affinity system called Kdp has a Km of 2 muM, and Vmax at 37 degrees C of 150 mumol/g min. This system is repressed by growth in high concentrations of K. Two constitutive systems, TrkA and TrkD, have Km's of 1.5 and 0.5 mM and Vmax's of 550 and 40 at 37 and 30 degrees C, respectively. Mutants lacking all three of these saturable systems take up K slowly by a process, called TrkF, whose rate of transport is linearly dependent on K concentration up to 105 mM. On the whole, each of these systems appears to function as an independent path for K uptake since the kinetics of uptake when two are present is the sum of each operating alone. This is not true for strains having both the TrkD and Kdp systems, where presence of the latter results in K uptake which saturates at a K concentration well below 0.1 mM. This result indicates some interaction between these systems so that uptake now has the affinity characteristic of the Kdp system. All transport systems are able to extrude Na during K uptake. The measurements of cell Na suggest that growing cells of E. coli have very low concentrations of Na, considerably lower than indicated by earlier studies.

Although activation of c-myc is a critical step in the development of lymphomas and other tumors, Its normal fumction(s) in cefl growth remain obscure because few mycregulated genes are known. myc expression normally ncrases in response to mitogens and peaks in GI when additional protein synthesis is required for cell-cycle progression. Protein synthesis jB controlled by the availability of translation initiation factors, including the mRNA cap binding protein (eIF-4E) and the a subunit of the eIF-2 complex that binds the initiator Met-tRNA. Consequently we examined eIF-4E and eIF-2a for evidence of regulation by c-myc. Expression of eIF-4E and eIF-2a correlated with c-myc expression in fibroblasts after growth stimulation. In addition, expression of eIF-4E and eIF-2a was increased in myc-transformed rat embryo fibroblasts but was not increased in iws-transformed cells. Transcription rates ofeIF-4E and eIF-2a mRNAs were regulated by c-myc in cells expressing an estrogen receptor-Myc fusion protein. Finally, electrophoretic mobiblty-shift assays Identified a sequence element in the eIF-2a promoter, TCCGCAL-GCGCG, which was speciflcally retarded by extracts of mycexpressing cells. c-myc is thought to deregulate the growth of cancer cells by activating btanwription, suggesting that specific genes regulated by c-myc should also function as oncogenes. In previous studies these translation initiation factors could induce neoplastic growth because overexpression of eIF-4E-transformed cells and inhibition of a suppressor of eIF-2a (eIF-2a kinase) also caused malignant transformation. Our studies suggest that one important biological function of c-myc may be to increase cell growth by increasing expression of eIF-4E and eIF-2a.

No abstract

Hepatocyte growth factor (HGF) is a potent mitogen for primary hepatocytes. Therefore, we examined HGF as a possible autocrine growth factor in hepatocellular carcinoma (HCC). We introduced an albumin-HGF expression vector into Fao HCC cells and transgenic mice. Expression of the albumin-HGF vector in Fao HCC cells inhibited their growth in vitro. In vivo, FaoHGF cells produced tumors that averaged 10% of the sizes of G418-resistant controls when transplanted into nude mice. In contrast, hepatocytes from transgenic mice expressing HGF grew more rapidly than did those from normal siblings. Further, growth of eight additional HCC cell lines was inhibited by the addition of recombinant HGF. Finally, of 35 tumor cell lines surveyed, only 6 cell lines expressed HGF mRNA, and no HCC cell line expressed HGF. Although HGF stimulates normal hepatocytes, it is a negative growth regulator for HCC cells.

Claudin-2 is a structural component of tight junctions in the kidneys, liver, and intestine, but the mechanisms regulating its expression have not been defined. The 5-flanking region of the claudin-2 gene contains binding sites for intestine-specific Cdx homeodomain proteins and hepatocyte nuclear factor (HNF)-1, which are conserved in human and mouse. Both Cdx1 and Cdx2 activated the claudin-2 promoter in the human intestinal epithelial cell line Caco-2. HNF-1␣ augmented the Cdx2-induced but not Cdx1-induced transcriptional activation of the human claudin-2 promoter. In mice, HNF-1␣ was required for claudin-2 expression in the villus epithelium of the ileum and within the liver but not in the kidneys, indicating an organ-specific function of HNF-1␣ in the regulation of claudin-2 gene expression. Tight junction structural components, which determine epithelial polarization and intestinal barrier function, can be regulated by homeodomain proteins that control the differentiation of the intestinal epithelium.

Regulation of D-glucose transport in the porcine kidney epithelial cell line LLC-PK, was examined. To identify the sodium-coupled glucose transporter (SGLT), we cloned and sequenced several partial cDNAs homologous to SGLT1 from rabbit small intestine (M. A. Hediger, M. J. Coady, T. S. Ikeda, and E. M. Wright, Nature (London) 330: [379][380][381] 1987). The extensive homology of the two sequences leads us to suggest that the high-affinity SGLT expressed by LLC-PK1 cells is SGLT1. SGLT1 mRNA levels were highest when the D-glucose concentration in the culture medium was 5 to 10 mM. Addition of D-mannose or D-fructose, but not D-galactose, in the presence of 5 mM D-glucose suppressed SGLT1 mRNA levels. SGLT1 activity, measured by methyl a-D-glucopyranoside uptake, paralleled message levels except in cultures containing D-galactose. Therefore, SGLT1 gene expression may respond either to the cellular energy status or to the concentration of a hexose metabolite(s). By isolating several cDNAs homologous to rat GLUT-1, we identified the facilitated glucose transporter in LLC-PK, cells as the erythroid/brain type GLUT-1. High-stringency hybridization of a single mRNA transcript to the rat GLUT-1 cDNA probe and failure to observe additional transcripts hybridizing either to GLUT-1 or to GLUT-2 probes at low stringency provide evidence that GLUT-1 is the major facilitated glucose transporter in this cell line. LLC-PK, GLUT-1 mRNAs were highest at medium nglucose concentrations of s2 mM. D-Fructose, D-mannose, and to a lesser extent n-galactose all suppressed GLUT-1 mRNA levels. Since the pattern of SGLT1 and GLUT-1 expression differed, particularly in low Dglucose or in the presence of D-galactose, we suggest that the two transporters are regulated independently.