Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Evidence for a role of dipeptidyl peptidase IV in fibronectin-mediated interactions of hepatocytes with extracellular matrix.

Dipeptidyl peptidase IV (DPP IV) is a cell surface glycoprotein which has been implicated in hepatocyte-extracellular matrix interactions [Hixson, DeLourdes, Ponce, Allison & Walborg (1984) Exp. Cell Res. 152, 402-414; Walborg, Tsuchida, Weeden, Thomas, Barrick, McEntire, Allison & Hixson (1985) Exp. Cell Res. 158, 509-518; Hanski, Huhle & Reutter (1985) Biol. Chem. Hoppe-Seyler 366, 1169-1176]. However, its proteolytic substrate(s) and/or binding protein(s) which mediate this influence have not been conclusively identified. Nitrocellulose binding assays using 125I-labelled DPP IV that was purified to homogeneity from rat hepatocytes revealed a direct interaction of DPP IV with fibronectin. Although fibronectin could mediate an indirect binding of DPP IV to collagen, no evidence was found for a direct binding of DPP IV to native or denatured Type I collagen. Fibronectin appeared to bind DPP IV at a site distinct from its exopeptidase substrate recognition site since protease inhibitors such as competitive peptide substrates and phenylmethanesulphonyl fluoride enhanced binding, possibly as a result of an altered conformation of DPP IV. To determine if fibronectin binding to DPP IV is involved in the interaction of fibronectin with the hepatocyte surface, the effect of various DPP IV inhibitors on 125I-fibronectin binding to isolated hepatocytes in suspension was examined. Kinetic studies revealed that inhibitors of DPP IV which enhanced fibronectin binding in vitro accelerated the initial binding of fibronectin to the cell surface where it was subsequently cross-linked (presumably by tissue transglutaminase) to as yet undefined components. Immunolocalization of fibronectin and DPP IV in normal rat liver sections showed that both proteins were present along the hepatocyte sinusoidal membrane. These observations, coupled with previous results showing that DPP IV is tightly bound to biomatrix isolated from rat liver (Hixson et al., 1984; Walborg et al., 1985), suggest that DPP IV binding to fibronectin may play a role in interactions of hepatocytes with extracellular matrix in vivo and possibly in matrix assembly.     

Photochemical labeling of bovine pancreatic ribonuclease A with 8-azidoadenosine 3',5'-bisphosphate

A simple method has been developed for the preparation of 5'-32P-labeled 8-azidoadenosine 3',5'-bisphosphate (p8N3Ap) for use in photoaffinity labeling studies. Irradiation of a complex between p8N3Ap and bovine pancreatic ribonuclease A (RNase A) with light of 300-350 nm led to the covalent attachment of the nucleotide to the enzyme. RNase A could also be labeled in the dark with prephotolyzed p8N3Ap. In either case, the nucleotide reacted with the same tryptic peptide, encompassing amino acids 67-85 of the protein. The site of labeling was determined to be either Thr-78 or Thr-82, both of which are close to or at the pyrimidine binding site of the enzyme. This result is consistent with recent nuclear magnetic resonance and X-ray studies which indicate that 8-substituted adenine nucleotides interact with the pyrimidine binding site of RNase A.     

A comparison of the small ribosomal RNA genes from the mitochondrial DNA of the great apes and humans: sequence, structure, evolution, and phylogenetic implications

Restriction endonuclease fragments produced by EcoRI/AvaI or KpnI digestion and containing the small (12S) ribosomal RNA (rRNA) genes from the mitochondrial DNAs (mtDNAs) of the common chimpanzee, pygmy chimpanzee, gorilla, and orangutan were inserted into the plasmids pBR322 or pADD1. After species verification the inserted fragments were digested with SauIIIA, subcloned into M13mp7 vectors, and sequenced. The small rRNA gene sequences were compared with each other and with the published human sequence (Anderson et al. 1981). Substitutions were detected at 118 of the 955 nucleotide positions compared. Pairwise, the sequence differences ranged from 1% (between the chimpanzee species) to 9% (comparisons involving the orangutan); the proportion that were transitions ranged from 87% to 100%. Deletions and/or additions were noted at seven locations. With respect to evolutionary sequence lability, kinetic analysis indicated the presence of at least two classes of nucleotide positions; the more labile class occurs in sequences thought to form self-complementary duplexes (stems) in the mature rRNA. The high frequency of compensating substitutions, which maintain base-pairing within these sequences, corroborates their inferred structure. Phylogenetic inferences drawn from the sequence comparisons support the notion of an approximately equidistant relationship among chimpanzees, gorilla, and man, with the orangutan much less closely related. However, inference from a shared deletion suggests that the gorilla and the chimpanzees may be more closely related to one another than they are to man.     

Evidence suggesting that virulence maps to the P1 region of the coxsackievirus B4 genome.

A chimeric virus containing the P1 region of a virulent variant of coxsackievirus B4 and the P2 and P3 regions of a nonvirulent strain was constructed from cDNA clones. The chimeric virus induced pancreatitis with concurrent hypoglycemia similar to that observed in mice infected with the virulent variant.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Simultaneous analysis of substrates,products, and inhibitors of xanthine oxidase by high-pressure liquid chromatography and gas chromatography

Procedures are presented for the simultaneous analysis of hypoxanthine, xanthine, allopurinol, oxipurinol, and uric acid in standard mixtures and physiological fluids using gas chromatography (gc) or high-pressure liquid chromatography (hplc). Excellent correlation was obtained between the two methods for hypoxanthine, xanthine, oxipurinol, and uric acid. There are advantages and disadvantages to both methods. hplc requires no prior derivatization, uses isocratic elution with a buffer containing no organic solvent, and has 50- to 100-fold greater sensitivity than gc. Simpler methods of prepurification, readily adapted to clinical laboratories, can be used for hplc analysis. Although substances that are found in some urine samples from cancer patients interfere with hplc, separations by gc are not affected by these substances.     

Restriction isotyping of human apolipoprotein A-IV: rapid typing of known isoforms and detection of a new isoform that deletes a conserved repeat.

Genetic polymorphisms of apolipoprotein A-IV (apoA-IV) have been detected by isoelectric focusing of serum proteins. Because genetic variation in apoA-IV has significant effects on lipid risk factors, we used restriction enzyme isoform genotyping (restriction isotyping) to determine apoA-IV isoform genotypes at the DNA level for a large population (n = 509). In contrast to isoelectric focusing methods, restriction isotyping relies on nucleotide differences, enabling unambiguous typing of known isoforms and detection of new alleles that mimic other isoforms with shared charge properties. To determine genotypes for the common A-IV-1 isoform (Gln at aa position 360) and A-IV-2 isoform (360His), we used a mismatched primer for polymerase chain reaction (PCR) to introduce a restriction site (PvuII) that distinguishes each isoform. Using a portion of the same PCR reaction, we used HinfI to distinguish isoforms with Thr at position 347 (347Thr) versus Ser (347Ser). In surveys for these common genotypes, we detected heterozygotes for an allele with an insertion of 12 bp. Nucleotide sequencing showed that this allele is identical to the A-IV-0 isoform that inserts a hydrophilic repeat (Glu Gln Gln Gln) in a conserved region near the carboxy terminus. In addition, we discovered a new allele with a 12 bp deletion that removes a repeat (Glu Gln Gln Gln) from the same region. Nucleotide sequencing showed that this allele removes an acidic charge relative to A-IV-1, so we have named this isoform A-IV-2*. This isoform has not been discovered at the protein level, perhaps due to shared charge properties with A-IV-2 isoforms.