Nucleic acid-binding properties of low-density lipoproteins: LDL as a natural gene vector.

The role of apo B-100 as a transcription factor is indicated by the presence of regions in its primary structure that are similar to the DNA-binding domains of the transcription factors ISGF3gamma, STATs, IRFs, and SREBPs as well as by the presence of 11 RNA-binding KH domains. The Apo B-100 sequence also contains numerous bipartite nuclear localization sequences (NLS). A modified gel shift assay was used to show binding of highly purified preparations of human LDLs to fragmented genomic DNA, plasmid DNA, synthetic oligonucleotides (ISRE, 5'-GGGAAACCGAAACTG and E/C, E-box motif and CCAAT, adipocyte-specific genes promoter site), and total RNA from human liver. LDL was observed to bind preferentially to plasmid DNA containing the hCMV IE2 promoter region. In experiments using human liver total RNA, RNA for five different genes was recovered from LDL and VLDL bands. Gene transfection experiments using human skin fibroblast cells were used to study the gene transfer capacity of LDL. Cells transfected with a pEGFP-N1 plasmid DNA and LDL expressed functional GFP, as indicated by fluorescence, at approximately 3 hrs after transfection. Our results strongly support an alternative role for apo B-100, in toto or perhaps as functional fragments, in the control of gene expression and as gene transfer vector.

[Clinical and anatomo-pathologic findings in a case of Cronkhite-Canada syndrome]. / Hallazgos clínicos y anatomopatológicos en un caso de síndrome de Cronkhite--Canadá.

We present a case of Cronkhite-Canada Syndrome with gastrointestinal polyposis, which is the first one to be reported in this country. We make a review of the literature and compare the clinical and anatomopathological finding in this case with others described.

Carbohydrate composition of protein and lipid components in sialic acid-rich and -poor low density lipoproteins from subjects with and without coronary artery disease.

Low density lipoprotein (LDL) from patients with coronary heart disease (CHD) caused 78-286% increase in accumulation of cholesterol in human aortic subendothelial cells compared to 2-17% caused by LDL from normal subjects. Ricin-Sepharose affinity chromatography was used to separate LDL into two subfractions, one sialic acid-rich (SAR) and the other sialic acid-poor (SAP). SAP-LDL from CHD patients caused 156-307% increase in accumulation of cellular cholesterol, whereas SAR-LDL from these patients caused only 14-21% increase. SAP-LDL from normal healthy subjects caused 50-86% increased accumulation, whereas their SAR-LDL induced only 2-12% increase. Carbohydrate analysis of SAP-LDL protein isolated from four CHD patients revealed mean values of 59, 25, 61, and 11 nmoles of N-acetyl glucosamine, galactose, mannose, and sialic acid per mg protein, respectively. Mean values for SAR-LDL protein from these patients were 59, 31, 77, and 24 nmol/mg protein, respectively. Analysis of SAP-LDL protein from four normal healthy subjects indicated respective mean values of 58, 29, 72, and 22 nmol/mg, whereas SAR-LDL protein from normals contained 59, 29, 72, and 29 nmol/mg. The carbohydrate content of LDL lipids represents about 25% of the total carbohydrate present in the lipoprotein. The mean values for SAP-LDL lipids from four CHD patients were about 2, 2, 18, 18, and 2 nmol/mg protein for N-acetyl galactosamine, N-acetyl glucosamine, galactose, glucose, and sialic acid, respectively. The mean values for SAR-LDL lipids from these patients were 3, 4, 34, 41, and 5 nmol/mg, respectively. Analysis of SAP-LDL lipids from four normal healthy subjects indicated respective mean values of 4, 6, 30, 31, and 3 nmol/mg, whereas SAR-LDL lipids from these subjects contained 6, 9, 41, 46, and 7 nmol/mg. These results suggest that the different biological properties of SAR-LDL and SAP-LDL are related to their different carbohydrate compositions.

Relation between Glutamine Synthetase and Nitrogenase Activities in the Symbiotic Association between Rhizobium japonicum and Glycine max.

The activity and extent of adenylylation of glutamine synthetase was examined in both free-living and bacteroid forms of Rhizobium japonicum in the presence of excess ammonia. Ammonia caused an apparent repression of glutamine synthetase in free-living R. japonicum and adenylylation of the enzyme was also increased. In contrast, neither the activity nor the extent of adenylylation of the bacteroid enzyme was consistently affected by ammonium treatment of bacteroid suspensions. Similar results were obtained after ammonium treatment of soybean plants even though nitrogenase activity was reduced markedly. We have been unable to demonstrate ammonium repression of nitrogenase activity in R. japonicum-Glycine max symbiotic association that is mediated through bacteroid glutamine synthetase. This result is in contrast to the situation in nitrogen-fixing strains of Klebsiella where a role of glutamine synthetase in the regulation of nitrogenase has been reported.

The purification and properties of the glutamine synthetase from the cytosol of Soya-bean root nodules.

The major portion of glutamine synthetase activity in root nodules of soya-bean plants is associated with the cytosol rather than with Rhizobium japonicum bacteroids. Glutamine synthetase accounts for about 2% of the total soluble protein in nodule cytosol. Glutamine synthetase from nodule cytosol has been purified by a procedure involving fractionation with protamine sulphate, ammonium sulphate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5m and affinity chromatography on glutamate-agarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a mol. wt. of about 376000 was determined for the native enzyme and 47300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, we have concluded that glutamine synthetase from nodule cytosol consists of eight subunits arranged in two sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 A) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains four half-cystine molecules per subunit, which is in contrast with two reported for the enzyme from pea seed. Enzyme activity is striking influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge.