Ran/TC4: a small nuclear GTP-binding protein that regulates DNA synthesis.

Ran/TC4, first identified as a well-conserved gene distantly related to H-RAS, encodes a protein which has recently been shown in yeast and mammalian systems to interact with RCC1, a protein whose function is required for the normal coupling of the completion of DNA synthesis and the initiation of mitosis. Here, we present data indicating that the nuclear localization of Ran/TC4 requires the presence of RCC1. Transient expression of a Ran/TC4 protein with mutations expected to perturb GTP hydrolysis disrupts host cell DNA synthesis. These results suggest that Ran/TC4 and RCC1 are components of a GTPase switch that monitors the progress of DNA synthesis and couples the completion of DNA synthesis to the onset of mitosis.

Characterization of four novel ras-like genes expressed in a human teratocarcinoma cell line.

A mixed-oligonucleotide probe was used to identify four ras-like coding sequences in a human teratocarcinoma cDNA library. Two of these sequences resembled the rho genes, one was closely related to H-, K-, and N-ras, and one shared only the four sequence domains that define the ras gene superfamily. Homologs of the four genes were found in genomic DNA from a variety of mammals and from chicken. The genes were transcriptionally active in a range of human cell types.

Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation.

Although the human Ras proteins are members of a large superfamily of Ras-related proteins, to date, only the proteins encoded by the three mammalian ras genes have been found to possess oncogenic potential. Among the known Ras-related proteins, TC21/R-Ras2 exhibits the most significant amino acid identity (55%) to Ras proteins. We have generated mutant forms of TC21 that possess amino acid substitutions analogous to those that activate Ras oncogenic potential [designated TC21(22V) and TC21(71L)] and compared the biological properties of TC21 with those of Ras proteins in NIH 3T3 and Rat-1 transformation assays. Whereas wild-type TC21 did not show any transforming potential in vitro, both TC21(22V) and TC21(71L) displayed surprisingly potent transforming activities that were comparable to the strong transforming activity of oncogenic Ras proteins. Like Ras-transformed cells, NIH 3T3 cells expressing mutant TC21 proteins formed foci of morphologically transformed cells in monolayer cultures, proliferated in low serum, formed colonies in soft agar, and developed progressive tumors in nude mice. Thus, TC21 is the first Ras-related protein to exhibit potent transforming activity equivalent to that of Ras. Furthermore, mutant TC21 proteins also stimulated constitutive activation of mitogen-activated protein kinases as well as transcriptional activation from Ras-responsive promoter elements (Ets/AP-1 and NF-kappa B). We conclude that aberrant TC21 function may trigger cellular transformation via a signal transduction pathway similar to that of oncogenic Ras and suggest that deregulated TC21 activity may contribute significantly to human oncogenesis.

A T42A Ran mutation: differential interactions with effectors and regulators, and defect in nuclear protein import.

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran.GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin beta (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Identification and characterization of a human homolog of the Schizosaccharomyces pombe ras-like gene YPT-3.

The Polymerase Chain Reaction was used to amplify ras and ras-like sequences from two human cDNA libraries. Members corresponding to each of the three major ras-subfamilies (ras, rho, and rab/YPT) were identified. The one homologous to rab/YPT, referred to here as YL8, appears to be the human homolog of the recently reported Schizosaccharomyces pombe YPT3 gene. The YL8 gene could encode a guanine nucleotide binding protein of 216 amino acids with about 70% amino acid sequence identity to S. pombe YPT3, and is transcriptionally active in a variety of human cell lines.

Reticuloendothelial cell dysfunction in diabetes and hyperlipidemia.

Using clearances of microaggregated iodinated human serum albumin in a serial 'stress test,' defective phagocytosis by the Kupffer cells has been demonstrated in diabetic patients with K.W. nephropathy and proliferative retinopathy. This indicates a disturbance of phagocytic cell function that has implications for the cellular pathology of microangiopathy. The effect is not due to uremia, but could be due to T3 deficiency or lipid deposition. In hypothyroidism, there is defective RES phagocytosis, and alcoholics with hyperlipidemia can have impaired clearances. Hence, patients with advanced diabetes, hypothyroidism, and some alcoholics are at risk from infection.

Altered reticuloendothelial function in long-term renal allograft recipients.

Using the clearance of microaggregated iodinated human serum albumin reticuloendothialial system (RES) phagocytic function was tested in 48 long-term renal allograft recipients and was found to be defective at the time of testing in 70%. Depression of RES phagocytosis could be related to total steroid dosage in the previous year and to the patients liability to bacterial infections. Evidence from this test does not suggest an immunosuppressive effect of cytomegalovirus. However, three patients are discussed who have developed chronic active hepatitis that is not due to type B virus.

The erythrocyte protoporphyrin in chronic renal failure.

The erythrocyte protoporphyrin concentration in 63 patients suffering from chronic renal failure was estimated and found to be significantly decreased when compared to healthy subjects. There was a negative correlation between the decrease of the erythrocyte protoporphyrin and the blood urea and haematocrit. Following haemodialysis the concentration of erythrocyte protoporphyrin was reduced but not significantly so. It is proposed that uraemia or insufficiency of erythropoietin could affect erythroblasts and young red cells impairing haem synthesis.

Immobilization hypercalcaemia in patients on regular haemodialysis.

Immobilization of normal people causes reabsorption of calcium from bone, a small rise in serum ionized calcium, and, rarely, frank hypercalcaemia. The hazard is increased when patients with renal osteodystrophy are immobilized because of pathological fractures.

Study of reticuloendothelial phagocytic capacity in patients with cholestasis.

The phagocytic capacity of the reticuloendothelial system was studied by the clearance from the plasma of microaggregated iodinated human serum albumin. About half of the patients with cholestasis showed impaired Kupffer-cell phagocytosis. The finding may be relevant to the occurrence of surgical complications in patients with obstructive jaundice.

The small nuclear GTPase Ran: how much does it run?

Ran is one of the most abundant and best conserved of the small GTP binding and hydrolyzing proteins of eukaryotes. It is located predominantly in cell nuclei. Ran is a member of the Ras family of GTPases, which includes the Ras and Ras-like proteins that regulate cell growth and division, the Rho and Rac proteins that regulate cytoskeletal organization and the Rab proteins that regulate vesicular sorting. Ran differs most obviously from other members of the Ras family in both its nuclear localization, and its lack of sites required for post-translational lipid modification. Ran is, however, similar to other Ras family members in requiring a specific guanine nucleotide exchange factor (GEF) and a specific GTPase activating protein (GAP) as stimulators of overall GTPase activity. In this review, the multiple cellular functions of Ran are evaluated with respect to its known biochemistry and molecular interactions.

Erythrocyte ferritin in patients with chronic renal failure and heterozygous beta-thalassemia.

AIM: The aim of this research is to study the variance of erythrocyte ferritin (EF) in patients with chronic renal failure (CRF) and heterozygous beta-thalassemia (beta-TA), as well as the use of EF as a more reliable index for assessing the body iron status. METHODS: We studied 63 subjects with CRF, 40 subjects with heterozygous beta-TA, 53 subjects with CRF and heterozygous beta-TA and 24 normal subjects. In 11 patients with CRF and heterozygous beta-TA, sternal bone marrow aspiration was performed to evaluate iron stores in the bone marrow. EF was determined in the hemolysate of washed erythrocytes by a radioimmunoassay. RESULTS: EF showed the strongest correlation with bone marrow iron (p < 0.001) in comparison with the remaining hematological parameters that were examined. Patients with CRF without heterozygous beta-TA showed an increase in serum ferritin (SF), even in cases of iron deficiency. In the group of heterozygous beta-TA without renal failure, 22.5% of patients showed an increased EF content up to 150 ag/cell and a tendency for iron overload. Patients with CRF and heterozygous beta-TA showed a high value of EF, up to 200 ag/cell, and iron overload in 22.6%, almost the same proportion as in the previous group. It was also observed that a high value of SF does not indicate iron overload for these patients. In the group of hemodialysis, patients without heterozygous beta-TA who were under erythropoietin (EPO) treatment presented iron deficiency. Many patients with CRF and heterozygous beta-TA who were taking EPO presented iron overload, while very few of them presented iron deficiency. CONCLUSION: These findings suggest that EF is a reliable index for assessing the iron status in patients with CRF and heterozygous beta-TA.

Reticuloendothelial function in renal allograft recipients.

The phagocytic capacity of the reticuloendothelial system (R.E.S.) was assessed in patients with chronic renal failure and in renal transplant recipients. R.E.S. phagocytosis was increased in the former group. Soon after transplantation R.E.S. phagocytosis was moderately reduced (through levels were comparable with those of normal controls) but was particularly reduced after high-dose corticosteroid treatment for rejection. In long-term allograft recipients R.E.S. phagocytosis was also depressed though steroid maintenance doses were small.

A simultaneous study of the polymorphism of five proteins in the serum and the urine of nephrotic patients.

The phenotypes of the haptoglobin (Hp), ceruloplasmin (Cp), group-specific component (Gc), transferrin (Tf), and third component of complement (C3) were determined simultaneously in the serum and urine of patients with proteinuria secondary to nephrotic syndrome of various types. In a large number of cases the patterns of Hp, Cp, and C3 phenotypes in the urine showed marked deviations from those in the corresponding serum either in the mobility or the number of their electrophoretic bands. The monomeric Hp and the Cp were found to have a very augmented urine/serum ration in some cases. Such differences were not detected in the electrophoretic appearance of the Gc and Tf phenotypes. Our results imply that in the proteinuria of the nephrotic syndrome, factors other than molecular weight interfere in the passage of proteins through the glomerular wall.