A distance geometry program for determining the structures of small proteins and other macromolecules from nuclear magnetic resonance measurements of intramolecular1H−1H proximities in solution

DISGEO is a new implementation of a distance geometry algorithm which has been specialized for the calculation of macromolecular conformation from distance measurements obtained by two-dimensional nuclear Overhauser enhancement spectroscopy. The improvements include (1) a decomposition of the complete embedding process into two successive, more tractable calculations by the use of “substructures”, (2) a compact data structure for storing incomplete distance information on a structure, (3) a more efficient shortest-path algorithm for computing the triangle inequality limits on all distances from this information, (4) a new algorithm for selecting random metric spaces from within these limits, (5) the use of chirality constraints to obtain good covalent geometry without the use ofad hoc weights or excessive optimization. The utility of the resultant program with nuclear magnetic resonance data is demonstrated by embedding complete spatial structures for the protein basic pancreatic trypsin inhibitor vs all 508 intramolecular, interresidue proton-proton contacts shorter than 4.0 Å that were present in its crystal structure. The crystal structure could be reproduced from this data set to within 1.3 Å minimum root mean square coordinate difference between the backbone atoms. We conclude that the information potentially available from nuclear magnetic resonance experiments in solution is sufficient to define the spatial structure of small proteins.     

Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

The binding of selenium to the lipids of two unicellular marine algae

Axenic cultures of the green algae $\text{Dunaliella}\text{primolecta}$ and red algae $\text{Porphyridium}\text{cruentum}$ were grown in the presence of sublethal quantities of selenite. All purified lipids from both algae were found to contain bound selenium, except for saturated hydrocarbons. Of the lipids which contain selenium, carotenoid pigments contain the greatest concentrations. Lipid-associated selenium is not metabolically incorporated. The selenium is probably non-covalently bound to the lipids.     

Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Evidence for lysosomal enzyme recognition by human fibroblasts via a phosphorylated carbohydrate moiety

Adsorptive endocytosis of five different lysosomal enzymes from various human and non-human sources was susceptible to inhibition by mannose and l-fucose, methyl α-d-mannoside, α-anomeric p-nitrophenyl glycosides of mannose and l-fucose, mannose 6-phosphate and fructose 1-phosphate. A few exceptions from this general scheme were observed for particular enzymes, particularly for β-glucuronidase from human urine. The inhibition of α-N-acetylglucosaminidase endocytosis by mannose, p-nitrophenyl α-d-mannoside and mannose 6-phosphate was shown to be competitive. The loss of endocytosis after alkaline phosphatase treatment of lysosomal enzymes supports the hypothesis that the phosphorylated sugars compete with a phosphorylated carbohydrate on the enzymes for binding to the cell-surface receptors [Kaplan, Achord & Sly (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2026–2030]. Endocytosis of `low-uptake' forms of α-N-acetylglucosaminidase and α-mannosidase was likewise susceptible to inhibition by sugar phosphates and by alkaline phosphatase treatment, suggesting that `low-uptake' forms are either contaminated with `high-uptake' forms or are internalized via the same route as `high-uptake' forms. The existence of an alternative route for adsorptive endocytosis of lysosomal enzymes is indicated by the unaffected adsorptive endocytosis of rat liver β-glucuronidase in the presence of phosphorylated sugars and after treatment with alkaline phosphatase.     

The paramedial neurosecretory cells of the suboesophageal ganglion of the cricket,Teleogryllus commodus (Walk.)

Summary Ovariectomy, performed immediately after the final hatch, caused a reduction of stainable (neurosecretory?) material in the paramedial neurosecretory cells (PNC) (A-type) of the suboesophageal ganglion in 10 day-old females of Teleogryllus commodus (Walk.). A concomitant increase in nuclear volume and in the incorporation of ³⁵S-cysteine indicates increased synthesis of neurosecretory material. From these findings it is concluded that more stainable material is secreted in the cerebral neurohaemal organ after Ovariectomy. A functional relationship between the PNC and the ovaries is suggested.     

Anencephaly in trisomy 18 associated with elevated alpha-1-fetoprotein in amniotic fluid

Summary Elevated levels of alpha-1-fetoprotein (AFP) were found in the amniotic fluid of a 36-year-old woman in the 15th week of gestation. Because of this and the results of repeated ultrasonography, abortion was induced. An anencephalic fetus with trisomy 18 was delivered. The possible correlation among neural-tube defects, chromosomal abnormalities, and level of AFP is discussed.     

A comparative study on the influence of cysteamine and metyrapone on mixed-function oxygenase activities in variously pretreated liver microsomes from rats and mice.

It has been found that metyrapone can inhibit both type I and type II mixed-function oxygenase reactions, while cysteamine inhibits only type I activity in this mammalian system. Following pretreatment with phenobarbital and 3-methylcholanthrene the half-maximal inhibiting concentrations for the O-demethylation of paranitranisol are increased for cysteamine and decreased for metyrapone. Both cysteamine and metyrapone give type II binding spectra with oxidized cytochrome P-450. The negative and positive peaks are at 393 and 426 nm respectively for metyrapone, and 410 and 434 nm for cysteamine. Cysteamine showed no binding comparable to that of metyrapone for reduced cytochrome P-450. Metyrapone showed little or no inhibition of the NADH cytochrome-c reductase (EC 1.6.1.1) or NADPH (EC 1.6.2.3) cytochrome-c reductase while cysteamine had a more or less strong inhibiting effect depending on the pretreatment of animals. Neither the binding to P-450 heme nor the inhibition of NADH and NADPH cytochrome-c reductase correlates well with cysteamine inhibition of total activity. It is therefore suggested that cysteamine reacts with an intermediate electron carrier of non-heme iron or glycoprotein character thus inhibiting mixed-function oxygenase activity.     

Characterization of microsomal electron transport components from control, phenobarbital- and 3-methylcholanthrene-treated mice. II. Improved resolution and quantitation of major components in ammonium sulfate fractions from total liver microsomes.

Quantitation of microsomal components in ammonium sulfate fractions using a high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and a comparison of these results with those from similar experiments on total liver microsomes has enabled us to identify and better characterize the interactions between microsomal electron transport components. It was found that: (1) phenobarbital decreased the amount of one protein component of approximately 50 000 molecular weight while increasing a component of very similar molecular weight; (2) only two proteins appeared to be associated with CO binding; (3) another protein of approximately 68 000 molecular weight, one of the glycoproteins found in liver microsomes, appears to be induced by phenobarbital pretreatment; (4) the induction of NADPH-cytochrome c reductase activity after phenobarbital pretreatment is not dependent on an increase in the known NADPH-dependent flavoprotein, but rather on the increase in some component found predominately in our most soluble sub-microsomal fraction. A very good separation of the above components was achieved by ammonium sulfate fractionation, e.g. simply on the basis of their solubility. This and the fact that the more-or-less soluble proteins were induced by phenobarbital or 3-methylcholanthrene respectively indicate that the solubility of membrane proteins plays a major role in the structure and function of microsomal membranes.