Classification of the pathohistology of diseases of the salivary glands - review of 2,600 cases in the Salivary Gland Register. Three major disease groups emerge from a classification of the pathohistology of 2,600 salivary gland preparations in the salivary gland register (from 1965 to 1974): salivary gland tumors (41%), salivary gland inflammations (26%), and sialadenosis (6%). Mucoceles (2%) and salivary gland cysts (1.5%) are seen less often. It is diagnostically important that lymph node changes in the area of the salivary glands (lymphadenitis, lymphomas, etc.) are worked up under suspicion of a primary salivary gland disease. Sialadenosis involves a primary, vegetative neuropathy, which is accompanied by enlargement of acinar cells. Under the heading of chronic sialdenitis, we fine 50% socalled Küttner tumors of the submandibular gland, 21% chronic relapsing parotitis, 14% chronic inflammations of the minor salivary glands and the sublingual gland, and 12% immune sialadenitis (myoepithelial sialadenitis in Sjögren's syndrome and epitheloid cell sialadenitis in Heerfordts syndrome). A more than average number of immune sialadenitis cases develop into malignant lymphomas (4 cases in the salivary gland register). The 1,067 tumor cases containing of 929 epithelial tumors (87%), 89 mesenchymal tumors (hemangiomas, lymphangiomas, lipomas, neurofibromas, sarcomas) and 49 metastases or periglandular tumors. Epithelial tumors were grouped on the basis of the World Health Organization (WHO) classification. Benign tumors (71.5%) consisted of pleomorphic adenomas (51.5%) and monomorphic adenomas (50%). After the pleomorphic adenomas, cystadenolymphomas (over 10%) represented the most frequent benign salivary gland tumor. On the basis of epithelial and stromal differentiation, pleomorphic adenomas were subdividied into four subtypes (stroma-rich and stroma-poor adenomas). The malignant tumors consisted of acinar cell tumors (2.5%), mucoepidermoid tumors (6%), and carcinomas at various levels of differentiation (adenoid-cystic carcinomas, 6.5%; adenocarcinomas, 2%; squamous cell carcinomas, 2%; carcinomas in pleomorphic adenomas, 4%; miscellaneous carcinomas - salivary duct carcinomas, clear-cell carcinomas, undifferentiated carcinomas, 5.5%). Differences in age and sex distribution, localization, malignity metastases, recidivism, et


The effect of intrauterinely injected lymphocytes prepared from sensitized or non-sensitized guinea-pigs on the ultrastructure of rat fetal membranes (visceral yolk sac). The susceptibility of rat fetal membranes (visceral yolk sac) to cell-mediated immunity was studied electron microscopically at day 19 of pregnancy. For this purpose guinea-pigs were sensitized to rat placental antigens and the activated lymphocytes were injected intrauterinely. Simultaneously, the effect of lymphocytes from non-sensitized guinea-pigs was examined on the rat yolk sac. Lymphocytes from sensitized guinea-pigs revealed a clear cytotoxic effect on the visceral yolk sac, but the degree of cell destruction showed marked differences from one epithelial cell to the other. Furthermore, the yolk sac regionally displayed great differences in the intensity of immune cytolysis...


Ecology of microbial population of soils of North Bihar as affected by soil factors. The determination of the number of bacteria, actinomycetes, and fungi in 72 soil samples of different sugar cane soils in North Bihar (India) and the calculation of the correlations between these values and the content of organic carbon, total nitrogen, total soluble salts, available P2O5, and water-soluble K2O gave these results: 1. Significant and positive correlations were found between the content of organic carbon in the soil and the number of actinomycetes and fungi. 2. Significant and negative correlations were found between the content of water-soluble salts and the number of microorganisms. 3. There is a significant and positive correlatin between the number of bacteria and fungi and the total nitrogen content and and the available P2O5.


[Studies of climatic factors influencing the performance of cattle in the Syrian Arab Republic. 2. Assessment fo 1968/69 climatic factors]. On the basis of the values of temperature and moisture for one year, an experimental cowshed (80 cows) on the border of the Syriain semidesert was investigated using a new evaluation scheme. The assessment was made according to three ranges of physiological compatibility. Essential deficiencies were found in the construction and in the function of the cowshed. Using the relevant literature (see part 1) on this subject, the paper tries to show that such conditions must be detremental to the performance of European cattle breeds. If cattle stay for a long time in temperature regions above the physiological compatibility range, damages to the health must also be expected in non-adapted and local breeds. Suitable devices must be built into the cowsheds to enable release of warmth from the cattle kept there. Improvements in the climate of the cowshed are part of the complex measure for increasing the performance. The paper tries to stimulate more intensive studies of the climate and its effect on the performance in animal production for other regions and countries as well.


Light peak to dark trough ratio in clinical electro-oculography: influence of dark oscillations on the following light peak. In the clinical standard EOG procedure the light rise potential is influenced by the preceding dark troughs oscillation. This is apparent in the different configuration of the light rise potential when following a dark trough instead of a steady state. Uninfluenced responses can be obtained only under steady-state conditions. Recording of the light peak potential in this manner may be used on a trial basis for clinical routine examination. Experimental and more detailed clinical studies require separate recording of light peak and dark trough potential, each preceded by a steady state. The evaluation of these responses, expressing them in percent of the steady state, should reveal more information than can be expected from the standard light peak to dark trough ratio.


The value of electro-oculography for the diagnosis of tapeto-retinal degenerations. The particular value of the EOG for diagnosis and prognosis has been demonstrated with the findings and case histories of seven patients. We have shown (1) that the EOG in connection with the ERG has contributed decisively to the differentiation of numerous types of tapeto-retinal dystrophies; (2) that the EOG is, as a rule, very valuable in distinguishing between tapeto-retinal dystrophies and flourishing or healed chorioretinitis, and (3) the different values of the other examination methods (visual field, colour vision tests, dark adaptation, angiography) in comparison with the electrophysiological methods. We have used the same methods as were used in previous examinations.


Clinical importance of the electro-oculogram with special reference to the chloroquine retinopathy. Examination of the EOG is surely worthwhile in cases of generalized pigment alteration and in juvenile foveal dystrophies. Its value in the control of chloroquine treatment is still doubted, but the method will become better if an EOG is made before treatment is started. EOGs made during the treatment can then be compared with those made earlier. As a result, the large interindividual variation is replaced by the much smaller intraindividual variation. In a group of normals and patients, the intraindividual standard deviation appeared to be about 10% of the original value of the EOG. Using this criterion, we think the EOG to be a valuable tool in preventing chloroquine retinopathy, although it is not yet certain if static perimetry with red light is still a better method.


Subsite mapping of enzymes. Depolymerase computer modelling. We have developed a depolymerase computer model that uses a minimization routine. The model is designed so that, given experimental bond-cleavage frequencies for oligomeric substrates and experimental Michaelis parameters as a function of substrate chain length, the optimum subsite map is generated. The minimized sum of the weighted-squared residuals of the experimental and calculated data is used as a criterion of the goodness-of-fit for the optimized subsite map. The application of the minimization procedure to subsite mapping is explored through the use of simulated data. A procedure is developed whereby the minimization model can be used to determine the number of subsites in the enzymic binding region and to locate the position of the catalytic amino acids among these subsites. The degree of propagation of experimental variance into the subsite-binding energies is estimated. The question of whether hydrolytic rate coefficients are constant or a function of the number of filled subsites is examined.


Subsite mapping of enzymes. Application of the depolymerase computer model to two alpha-amylases. In the preceding paper (Allen and Thoma, 1976) we developed a depolymerase computer model, which uses a minimization routine to establish a subsite map for a depolymerase. In the present paper we show how the model is applied to experimental data for two alpha-amylases. Michaelis parameters and bond-cleavage frequencies for substrates of chain lengths up to twelve glucosyl units have been reported for Bacillus amyloliquefaciens, and a subsite map has been proposed for this enzyme [Thoma et al. (1971) J. Biol. Chem. 246, 5621-5635]. By applying the computer model to the experimental data, we have arrived at a ten-subsite map. We find that a significant improvement in this map is achieved by allowing the hydrolytic rate coefficient to vary as a function of the number of occupied subsites comprising the enzyme-binding region. The bond-cleavage frequencies, the enzyme is found to have eight subsites. A partial subsite map is arrived at, but the entire binding region cannot be mapped because Michaelis parameters are complicated by transglycosylation reactions. The hydrolytic rate coefficients for this enzyme are not constant.


A method for the determination of hyaluronate in the presence of other glycosaminoglycans and its application to human intervertebral disc. A highly sensitive proteoglycan-binding assay was developed for the detection and determination of small amounts of hyaluronate. Application of this assay to human intervertebral-disc tissue showed that the radial distribution of hyaluronate closely followed the distribution of proteoglycans.


Removal of a bound ligand from a macromolecule by gel filtration. If a solution of macromolecule of concentration c and volume a, initially saturated with a ligand of dissociation constant K, is gel filtered through a volume v of solution initially free of ligand, the saturation y with which it emerges is given by the equation y-lny-1=Kv/ca.


The pre-eminence of k(cat) in the manifestation of optimal enzymic activity delineated by using the Briggs-Haldane two-step irreversible kinetic model. The suggestion by Fersht [(1974) Proc. R. Soc. London Ser. B 187, 397-407] that enzymes that provide maximal rates of catalysis should be characterized by values of Ks, the dissociation constant of the enzyme-substrate complex, greater than 10 times the value of the ambient substrate concentration has been examined. 2. For such enzymes, Ks is not relevant, and attention is best focused on the relative numerical values of k(cat). (in units of s(-1) and the substrate molarity. It is necessary only that the former be about 10(10)-10(11) times the latter to ensure that the rate of product formation be diffusion-limited and thus maximal.


Algebraic methods for deriving steady-state rate equations. Practical difficulties with mechanisms that contain repeated rate constants. Methods of deriving rate equations that rely on repetition of terms for identification of redundant or invalid terms give incorrect results if used with mechanisms in which some rate constants appear more than once.


The proton and metal complexes of adenyl-5'-yl imidodiphosphate. The formation constants of the complexes of adenyl-5'-yl imidodiphosphate with H+ Mg2+, Ca2+ and a number of bivalent transition-metal ions were measured potentionmetrically. The complexes are generally a little more stable than the analogous complexes of ATP. By measuring the formation constants at two temperatures, this increase in stability was shown to result from an increased enthalpy change on complex-formation.


Alternative products in the reaction of 2-nitro-5-thiocyanatobenzoic acid with thiol groups. 2-Nitro-5-thiocyanatobenzoic acid has been proposed as a reagent for converting thiol groups in proteins into their S-cyano derivatives. Evidence was obtained for formation of both the S-cyano derivative and the mixed disulphide derivative. Formation of the S-cyano derivative can be promoted by addition of excess of CN-to the reaction mixture.


Molecular weight and subunit size of fatty acid synthetase from rabbit mammary gland. Fatty acid synthetase purified from the mammary gland of the rabbit has a mol. wt. of 968000 as determined by gel filtration. The enzyme gave one band, corresponding to a mol.wt. of approx. 35000, on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and phenylmethanesulphonyl fluoride.


Chicken muscle and fibroblast actin structure. Double labelling and the isolation of peptides specific to muscle actin indicates that completely homologous 20-residue peptides can be produced from the C-terminal regions of muscle and chicken-embryo fibroblast actins by treatment with CNBr. By quantification of the amount of this peptide that can be produced from acetone-dried powders by CNBr treatment, 6.8% of the protein of the fibroblasts has been estimated to be actin.


Stereospecific haem cleavage. A model for the formation of bile-pigment isomers in vivo and in vitro. A new approach is suggested for an explanation of sterospecific haem degradation to biliverdin and bilirubin. A model is proposed in which an oxygen molecule, bound to the haem iron atom, attacks a methene-bridge carbon atom in an intramolecular reaction. Specificity of macrocyclic ring cleavage is explained on the basis of the different accessibilities of the bound oxygen molecule to the four methene bridges. The consequences of these ideas are assessed in relation to coupled oxidation in model systems and to haem catabolism.


Isolation and characterization of an active three-chain molecular species of bovine thrombin. Partially purified bovine prothrombin was activated in half-saturated trisodium citrate seeded with thrombin, and the resulting thrombin was chromatographed on Amerblite IRC-50, followed by rechromatography on DEAE-Sephadex A-50. Five fractions, possessing both esterase and clotting activities, were partially isolated, but fraction VI was shown to be a pure three-chain active species with threonine, isoleucine and lysine, in 1:1:1 molar proportions as N-termini. The amino acid composition and C-termini of fraction VI were determinied. The molecular weights of the isolated chains, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were 7300, 12000 and 19500 respectively. These data, when taken together with the amino acid sequence of the two-chain thrombin reported by Magnusson et al. (1975) [in Prothrombin and related Coagulation Factors, (Hember, H. C. & Veltkamp, J. J., eds.), pp. 25-46, Leiden University Press, Leiden], indicated that proteolysis occurred at the Arg(78)-Lys(79) peptide bond of the B chain of a precursor molecular species, thus converting this two-chain species into the three-chain active form described here.


Mouse haemoglobin beta chains. Comparative sequence data on adult major and minor beta chains from two species, Mus musculus and Mus cervicolor. In inbred Mus musculus several different beta chains are known. In certain strains two beta chains are produced in unequal amounts by the two closely linked genes of the doublet breeding unit allele Hbb(d): Betadmaj and betadmin. One strain has a variant doublet allele, Hbb(p), which produces a variant minor beta chain, betapmin (the major beta chain, betapmaj, may not differ from betadmaj chain). Certain other strains have a singlet allele, Hbb(s), that produces only one beta chain, betas. Other species have different beta-chain patterns. In M. cervicolor two variant major beta chains are found, betacmaj (d-like) and betacmaj (s-like), both of which were found associated with minor beta chains. M. caroli has only one, 'Leporelike' beta chain, with structural features characteristic predominantly of betadmin chain in the N-terminal half and of betadmaj chain in the C-terminal half. The present paper presents sequence data on betas, betadmaj, betadmin, betapmin and betacmaj (d-like) chains. The data on betadmin chain cover almost the whole of that chain and show a minimum of nine differences from betadmaj chain and two from betapmin chain. It is suggested that the data on the beta chains of the various species show evidence for the past occurrence of double crossovers over regions within a gene coding for only one or a few amino acids, which events can be explained by the 'hybrid DNA' models of genetic recombination. Supplementary information on the amino acid sequence of the proteins has been deposited as Supplementary Publication SUP 50067 (36 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1976) 153, 5.


Studies on ligand binding to bovine liver uridine diphosphate glucose pyrophosphorylase. A procedure for the preparation of crystalline UDP-glucose pyrophosphorylase is described. K(s) values for UDP-glucose and UTP were determined as 7 and 20 muM respectively, the latter being confirmed by three methods. By assuming an octameric structure, 1 mol of enzyme subunit bound 1 mol of substrate. The metal-ion activator, Mg2+, did not affect the equilibrium between nucleotide and enzyme. A substrate analogue, alphabeta-methylene-UTP, was synthesized and had the same K(s) value as UTP. In its presence, the K(s) for glucose 1-phosphate decreased by two orders of magnitude, thus confirming a compulsory binding order and excluding an uridylated enzyme intermediate. The results are discussed with respect to their implications in vivo.


Delineation of the third antigenic site of lysozyme by application of a novel 'surface-simulation' synthetic approach directly linking the conformationally adjacent residues forming the site. We have previously shown that an antigenic site in native lysozyme resides around the disulphide bridge 30-115 and incorporates Lys-33 and Lys-116 and one or both of Tyr-20 and Tyr-23. These residues fall in an imaginary line circumscribing part of the surface of the molecule and passing through the spatially adjacent residues Tyr-20, Arg-21, Tyr-23, Lys-116, Asn-113, Arg-114, Phe-34 and Lys-33. The identity of the site was confirmed by demonstrating that the synthetic peptide Tyr-Arg-Tyr-Gly-Lys-Asn-Arg-Gly-Phe-Lys (which does not exist in lysozyme but simulates a surface region of it), and an analogue in which glycine replaced Tyr-23, possessed remarkable immuno-chemical reactivity that accounted entirely for the expected reactivity of the site in native lysozyme. Tyr-23 is not part of the site, and its contribution was satisfied by a glycine spacer. The novel approach presents a powerful technique for the delineation of antigenic (and other binding) sites in native proteins and confirms that these need not always comprise residues in direct peptide linkage.


Carboxymethylation of methionine residues in bovine pituitary luteinizing hormone and its subunits. Location of specifically modified methionine residues. Bovine lutropin (luteinizing hormone) was carboxymethylated at pH3.0 for 12 h at 37 degrees C with iodoacetic acid for specific modification of methionine residues. To facilitate the location of preferentially modified methionine residues, iodoE114C]acetic acid was added as tracer. The alpha and beta subunits of bovine lutropin were carboxymethylated with a 2- or 5-fold molar excess of iodoacetic acid either in the presence or absence of their counterpart subunits. The modified subunits were separated and isolated by counter-current distribution followed by gel filtration on Sephadex G-100. To locate the modified methiones, the isolated alpha or beta chain was reduced. S-carboxymethylated and subjected to tryptic hydrolysis. The tryptic peptides were fractionated by gel filtration on Bio-Gel P-10. From analyses of the purified 14C-labelled tryptic peptides, it was observed that methionine-8 and -33 in bovine lutropin alpha chain and methionine-52 in the beta chain were preferentially modified. Similar results were obtained when isolated alpha and beta subunits were individually carboxymethylated in the absence of their counterpart subunit under identical conditions. The fact that a recombinant of native human lutropin alpha chain, in which a valine residue is present in the position corresponding to methionine-8 of bovine lutropin alpha chain, and carboxymethylated bovine lutropin beta chain regenerated a substantial amount of receptor-site-binding activity indicated that methionine-8 in bovine alpha chain was biologically not essential. These studies showed clearly that both methionine-33 in the alpha chain and methionine-52 in the beta subunit were involved for optimum binding between bovine lutropin and its receptors for expression of hormonal activity.


Kinetics of rat liver glucokinase. Co-operative interactions with glucose at physiologically significant concentrations. The kinetics of glucokinase from rat liver were studied over wide ranges of glucose and MgATP2- concentrations. The initial rate shows a co-operative dependence on the glucose concentration, with Hill coefficients in the range 1.2-1.5. The degree of glucose co-operativity increases with the MgATP2- concentration, but no co-operativity was detected for the dependence of the rate on the MgATP2- concentration. The effects observed occur at physiologically reasonable concentrations of glucose and MgATP2- and are consistent with the presumed function of glucokinase in maintaining a constant concentration of glucose in the blood.


Selective modification of rabbit muscle pyruvate kinase by 5-chloro-4-oxopentanoic acid. Rabbit muscle pyruvate kinase was irreverisbly inactivated by 5-chloro-4-oxopentanoic acid with a pKa of 9.2. The inhibition was time-dependent and was related to the 5-chloro-4-oxopentanoic acid concentration. Analysis of the kinetics of inhibition showed that the binding of the inhibitor showed positive co-operativity (n = 1.5 +/- 0.2). Inhibition of pyruvate kinase by 5-chloro-4-oxopentanoic acid was prevented by ligands which bind to the active site. Their effectiveness was placed in the order Mg2+ greater than phosphoenolpyruvate greater than ATP greater than ADP greater than pyruvate. Inhibitor-modified pyruvate kinase was unable to catalyse the detritiation of [3-(3)H]pyruvate in the ATP-promoted reaction, but it did retain 5-10% of the activity with either phosphate or arsenate as promoters. 5-Chlor-4-oxo-[3,5-(3)H]pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoicheiometry of 1 mol of inhibitor bound per mol of pyruvate kinase protomer. The incorporation of the inhibitor and the loss of enzyme was proportional. These results are discussed in terms of 5-chloro-4-oxopentanoic acid alkylating a functional group in the phosphoryl overlap region of the active site, and a model is presented in which this compound alkylates an active-site thiol in a reaction that is controlled by a more basic group at the active site.


Delta8(14)-steroids in the bacterium Methylococcus capsulatus. The 4,4-dimethyl and 4alpha-methyl sterols of the bacterium Methylococcus capsulatus were identified as 4,4-dimethyl- and 4alpha-methyl-5alpha-cholest-8(14)-en-3beta-ol and 4,4-dimethyl- and 4alpha-methyl-5alpha-cholesta-8(14),24-dien-3beta-ol. Sterol biosynthesis is blocked at the level of 4alpha-methyl delta8(14)-sterols.


125I-labelling = 125I-Labelling.

125I-labelling = 125I-Labelling. Erythropoietin, the hormone that regulates erythropoiesis in mammals, was 125I-labelled by using the catalytic properties of lactoperoxidase and the H2O2-generating properties of glucose oxidase. This methodology, both rapid and simple, not only produced hormone preparations with high specific radioactivity but also did not substantially alter the biological integrity of erythropoietin when it was assayed in vivo.


Location of the carbohydrate groups of ovomucoid. Tryptic glycopeptides were purified from the sialic acid-free variant of ovomucoid, O1, and its CNBr fragments. The amino acid sequences adjacent to the four major sites of carbohydrate (Carb.) attachment were: (1), Phe-Pro-Asn(Carb.)-Ala-Thr-Asp-Lys-Glu-Gly-Lys; (2), Ala-Try-Ser-Ile-Glu-Phe-Gly-Thr-Asn (Carb.)-Ile-Ser-Lys; (3), Glu, Thr-Val-Pro-Met-Asn(Carb.)-cys-Ser; (4), Ser-Ser-Tyr-Ala-Asn (Carb.)-Thr-Thr-Ser-Glu-Asp-Gly-Lys, Glycosylated Asn residues were located at position 10, between residues 49 and 60, and at positions 69 and 75, in the primary sequence. All of these carbohydrate groups contained GlcNAc, Man and Gal in the approximate molar proprotions 5:3:0.5. A further glycopeptide containing His was isolated in low yield, suggesting that some carbohydrate is attached at a fifth site. Two of the carbohydrate-attachment sites (Asn-10 and Asn-75) occur in sequences that show internal homologies. These are presumed to have evolved as a consequence of partial gene duplication. Three of the carbohydrate-attachment sites occur in similar positions to the carbohydrate groups in quail ovomucoid [Laskowski (1976) Protides Biol. Fluids Proc. Colloq. 23, in the press]. Prediction of peptide conformation from the sequence data by the method of Chou & Fasman [(1974) Biochemistry 13, 222-225] indicated that four glycosylated Asn residues in hen ovomucoid are very close to groups of amino acids that occur with high frequency in beta-turns. The possible significance of peptide-chain conformation in the attachment of carbohydrate to glycoproteins is briefly discussed.


Amino acid sequence of a four-iron-four-sulphur ferredoxin isolated from Bacillus stearothermophilus. 1. The primary structure of a 4Fe-4S ferredoxin from Bacillus stearothermophilus was determined and shown to consist of a single polypeptide chain of 81 amino acid residues. The molecular weight of the holoprotein is about 9120. 2. There are only four cysteine residues in the molecule; three of these are located near the N-terminus as a Cys-X-X-Cys-X-X-Cys segment, and the fourth cysteine residue is followed by a proline and located in the C-terminal half. 3. The Fe-S chromophore in B. stearothermophilus ferredoxin was previously well characterized and was shown to consist of a single 4Fe-4S cluster. This ferredoxin sequence establishes for the first time the relative location of the four cysteine residues necessary to bind the 4Fe-4S cluster of a 4Fe ferredoxin, and is in agreement with the criteria for the relative positions of the cysteines proposed from X-ray-crystallographic studies on an 8Fe (two 4Fe-4S clusters) ferredoxin. 4. The sequence of B. stearothermophilus ferredoxin is homologous in many segments to that of other bacterial ferredoxins, the degree of homology being greater towards ferredoxins from Desulfovibrio gigas and photosynthetic bacteria than to Clostridial ferredoxins. 5. The presence of a relatively higher number of glutamic acid and lower number of cysteine residues in the molecule may explain the greater thermal stability and oxygen-insenstivity of this ferredoxin.


Thiol-protein disulphide oxidoreductases. Differences between protein disulphide-isomerase and glutathione-insulin transhydrogenase activities in ox liver. 1. Protein disulphide-isomerase and glutathione-insulin transhydrogenase activities were assayed in parallel through a conventional purification of protein disulphide-isomerase from ox liver. 2. Throughout a series of purification steps (differential centrifugation, acetone extraction, (NH4)2SO4 precipitation and ion-exchange chromatography), the two activities appeared in the same fractions but were purified to different extents. 3. The final sample was 143-fold purified in protein disulphide-isomerase but only 10-fold purified in glutathione-insulin transhydrogenase; nevertheless the two activities in this preparation were not resolved by high-resolution isoelectric focusing and both showed pI4.65. 4. In a partially purified preparation containing both activities, glutathione-insulin transhydrogenase was far more sensitive to heat denaturation than was protein disulphide-isomerase; conversely protein disulphide-isomerase was more sensitive to inactivation by deoxycholate. 5. The data are inconsistent with a single enzyme being responsible for all the protein disulphide-isomerase and glutathione-insulin transhydrogenase activity of ox liver. It is suggested that several similiar thiol-protein disulphide oxidoreductases of overlapping specificities may better account for the data.


Arylsulphatase and glycosulphatase of Charonia lampas. Substrate specificity towards sugar sulphate derivatives. The specificity of the arylsulphatase and glycosulphatase enzymes of the marine mollusc Charonia lampas towards a number of carbohydrate sulphate esters was examined. The results confirm that the relationship between the established nomenclature of the two enzymes and their respective specificities is inconsistent.


The lupus anticoagulant.

The lupus anticoagulant. The clinical and laboratory experience with the lupus anticoagulant was reviewed in 37 patients. The anti-coagulant is thought to act by blocking the activation of prothrombin by the prothrombin activator comlex of factors Xa, V, and phospholipid. Although the anticoagulant has been principally associated with diseases of immune origin, 14 of the present patients had disorders not thought to be immune in nature. Eighteen patients underwent twenty-one operative procedures with only a single episode of excessive bleeding. In the author's experience, the lupus anticoagulant is a rare cause of bleeding.


Frequency of neoplasia in systemic lupus erythematosus and rheumatoid arthritis. A patient population admitted to the hospital for either SLE or RA was surveyed for the subsequent development of neoplasms. The frequency of neoplasm in SLE patients appeared to be exaggerated, whereas the frequency of subsequent neoplasm in rheumatoid patients was unexpectedly low. A paucity of nephritis in the SLE group was noted. Further reports are encouraged so that the magnitude of the risk of malignancy developing with immunosuppressive therapy can be more precisely ascertained.


Coagulation abnormalities in rheumatoid disease. Forty-one patients with rheumatoid arthritis, including 6 with acute vasculitis, 13 with chronic vasculitis, and 22 without vasculitis, were studied for evidence of intravascular coagulation and fibrinolysis (ICF). The mean plasma fibrinogen levels were elevated in all groups. The fibrinogen, platelet count, and fibrin split products were usually elevated in acute vasculitis. Fewer patients on corticosteroids had abnormal coagulation tests. Active plasmin was detected in 12 patients primarily with chronic vasculitis. Plasminogen activator activity was not diminished in vascular endothelium of normal appearing skin of those patients with or without vasculitis. None of the patients demonstrated decompensated intravascular coagulation and fibrinolysis. The results suggest overcompensated ICF occurring in rheumatoid arthritis, but rheumatoid patients with vasculitis cannot be clearly distinguished from those without vasculitis on the basis of the usual tests performed for coagulation and fibrinolysis abnormalities.


Hyaluronic acid produced by human synovial fibroblasts. Effect of polyinosinic-polycytidylic acid (poly I:C) and interferon. Poly I:C (polyinosinic-polycytidylic acid) stimulated hyaluronic acid production by rheumatoid and non-rheumatoid human synovial fibroblasts. Stimulation was dose dependent and was inhibited by acetylsalicyclic acid and indomethacin. Poly I and Poly C, when separately added, had no stimulatory effect on hyaluronic acid production, and Poly A:U had only a slight effect on this parameter. Cells grown with Poly I:C were virus resistant and interferon was detected in their medium. Human interferon had also a dose-dependent stimulatory effect on hyaluronic acid production by synovial cells. A possible interferon-mediated relationship between virus infection and pathologic accumulation of joint fluid is suggested.


Aggregation of cartilage proteoglycans. II Evidence for the presence of a hyaluronate-binding region on proteoglycans from osteoarthritic cartilage. Proteoglycan aggregates isolated from normal bovine knee cartilage were larger than those from osteoarthritic cartilage of the same joints and appeared relatively more resistant to digestion with leech hyaluronidase. Incubation of proteoglycan subunits from the arthritic cartilage with hyaluronic acid resulted in marked aggregation, comparable in magnitude to that shown by subunits from normal cartilage. The results indicate that the hyaluronate-binding region of these proteoglycans was functionally intact and suggest that diminished aggregation of proteoglycans in osteoarthritic cartilage may be due to an abnormality in some other constituent of the aggregates.


Serum antibody levels against T mycoplasmas in two North American Indian populations predisposed to spondylitis. Serum antibody levels against T mycoplasmas (Ureaplasma urealyticum) were determined by the metabolic inhibition method in several populations. A higher prevalence of antibody was found in Haida Indians and Bella Coola Indians than in blood donors, patients with rheumatoid arthritis, and patients attending a VD clinic. Antibody levels did not correlate with the presence of spodylitis or the histocompatibility antigen HLA-B27, although both these Indian populations have a high prevalence of spondylitis and HLA-B27.


Measuring synovial fluid viscosity with a white blood cell diluting pipette. A simple, rapid,and reproducible method. A convenient and accurate way to measure the relative viscosity of synovial fluid is to use a white blood cell diluting pipette as a simple viscometer. The technique is reproducible with 2% accuracy and adds little to the time required for routine synovial fluid analysis. The magnitude of the variation of synovial fluid viscosity with temperature is demonstrated, and it is suggested that routine clinical measurements be made at room temperature. Clinical data confirm that a single viscosity measurement has little or no diagnostic value.


Carrageenin-induced arthritis. III. Proteolytic enzymes present in rabbit knee joints after a single intraarticular injection of carrageenin. A single intraarticular injection of carrageenin into the rabbit knee joint initiates an inflammatory reaction in the synovial tissues. the exudate from the joint was able to degrade proteoglycan at pH 5.2 and pH 7.2. Further characterization of proteolytic enzymes in the inflamed synovial tissues showed the presence of cathepsin D, a neutral protease, and cathepsin B1. Maximum activities of two lysosomal enzymes, acid phosphatase and cathepsin D, were observed within 7 days of injection. Most of this activity was found to be associated with cells in the synovial fluid.


Organic brain syndrome in rheumatoid arthritis following corticosteroid withdrawal. Six patients with seropositive nodule-forming rheumatoid arthritis developed severe central nervous system manifestations consistent with a diagnosis of organic brain syndrome. Organic brain syndrome occurred while 5 of these patients were undergoing corticosteroid withdrawal after prolonged administration. Neuropsychiatric symptoms rapidly cleared, responding to reinstitution of oral or parenteral corticosteroids in large doses in 4 patients, to increase in dosage in 1 patient, and to no drug therapy in the remaining 1. Marked reduction in rheumatoid factor in sera and demonstration of IgM deposits in the choroid plexus in 1 of the patients raised the possibility of immune complex-mediated central nervous system vasculitis.


Multielement and automated radiochemical separation procedures for activation analysis. In recent years the demand for information about the distribution of elements at trace concentration levels in high purity materials and in biological, environmental and geological specimens has increased greatly. Neutron activation analysis can play an important role in obtaining the required information. Radiochemical separations are required in many of the applications mentioned. A critical review of the progress made over the last 15 years in the development and application of radiochemical separation schemes for multielement activation analysis and in their automation is presented. About 80 radiochemical separation schemes are reviewed. Advantages and disadvantages of the automation of radiochemical separations are critically analysed. The various machines developed are illustrated and technical suggestions for the development of automated machines are given.


Amniotic fluid testosterone levels in midpregnancy. Amniotic fluid testosterone levels were measured on specimens obtained between 12 and 25 weeks' gestation from 58 male-fetus and 77 female-fetus pregnancies. For the male fetuses the mean +/- SE amniotic fluid testosterone level of 223 +/- 10 pg/ml was significantly higher (P less than 0.001) than the concentration found for the female fetuses (40 +/- 2 pg/ml). The ranges were 104-424 and 18-82 pg/ml, respectively, for the same fetuses. In the male fetuses the highest mean level was found during the 17th gestational week, but the mean level observed during any 1 week was not significantly differenf from any other week for both sexes. These data are consistent with the concept that amniotic fluid testosterone levels may be a rapid and effective method for establishing fetal sex in utero during midgestation.


Hypothyroidism presenting as a polymyositis-like syndrome. Report of two cases. Two patients with proximal muscle weakness and marked elevations of serum muscle enzymes were initially believed to have polymyositis; however electromyography and muscle biopsies were normal. Both patients were subsequently found to have hypothyroidism. Each regained her muscle strength, and serum enzymes normalized with thyroid hormone replacement. Because muscle weakness and an elevated creatine phosphokinase occur in most patients with hypothyroidism, thyroid dysfunction must be considered in the differential diagnosis of polymyositis.


Ultrastructural alterations of nonciliated cells after nitrosamine treatment and their significance for pulmonary carcinogenesis. Syrian golden hamsters received multiple subcutaneous injections of N-diethylnitrosamine (DEN) or N-dibutylnitrosamine (DBN). Ultrastructural examination of the sequential alterations of bronchial epithelia was performed. Starting from the third to fourth treatment, the formation of lamellated inclusion bodies (LBs) was observed in nonciliated cells of segmental bronchi and peripheral bronchioles. These organelles closely resembled those LBs normally found in alveolar epithelial cells Type 2, which are a characteristic feature of alveolar cell carcinomas.


[Ultrastructure of the granulomatous reaction against Descemet's membrane (author's transl)]. Ultrastructural findings in regard to the so-called granulomatous reaction against Descemet's membrane indicate that the membrane is phagocytized in the form of small clumps. Basically epithelial cells and, to a lesser extent giant cells, participate in the phagocytosis. In addition to the connective tissue, the epithelium is fairly actively involved in the regeneration of the cornea.


Gastric secretion and motility in duodenal ulcer: effect of current vagotomies. Proximal gastric vagotomy, total gastric vagotomy, and truncal vagotomy all decrease gastric secretion of acid and pepsinogen, increase the concentration of gastrin in the serum, impair gastric receptive relaxation and accommodation, and speed gastric emptying of liquids. Only proximal gastric vagotomy preserves antral motility, gastric emptying of solids, and the pyloric barrier to duodenal-gastric reflux.


A comparison in mice of naloxone and nalorphine as antagonists to neuroleptanalgesic drugs. The effectiveness of naloxone and nalorphine in antagonizing the effects of fentanyl and droperidol on the hot plate reaction time and the respiratory rate of the mouse has been compared. Naloxone was superior to nalorphine, being a more effective antagonist, and was also free from significant agonist effects. However, neither antagonist was completely effective against the respiratory rate depression produced by combinations of fentanyl and droperidol. It is suggested that the duration of the antagonist effects of naloxone are shorter against respiratory depression than against analgesia.


Neuromuscular sensitivity to tubocurarine. A comparison of 10 parameters. The pharmacometrics of neuromuscular block by tubocurarine was studied. Ten parameters were compared for the sensitivity, the scatter and the linearity of response at three levels of block in 11 cats. The fourth twitch of the train-of-four is the most sensitive parameter, followed in order of decreasing sensitivity by the train-of-four fade, the faded tetanus, the tetanus, the single twitch, the post-tetanic twitch and the tetanic fade. At the level of 50% block of the single twitch, all other parameters differ significantly (P less than 0.05) from the single twitch in sensitivity. With the exception of the tetanic fade, all parameters examined correlate with the single twitch (r xy = 0.42-0.77). This permits limited translation of results among various parameters. In cats the scatter of results is least for the most sensitive parameter, and vice versa. We conclude that the train-of-four is the most sensitive parameter, and that the single twitch is valid for an over-all estimation of the neuromuscular block induced by tubocurarine.


Investigations of the sensory blockade effect of perineurally injected ethanol on the tail nerve of the mouse. The effect of an alcohol block on the conduction of sensory stimuli in the tail nerve of the mouse was investigated using the perineural injection of solutions of ethanol (35, 40 and 45%). One hundred and fifty white mice of either sex were given 2 X 0.03 ml of the relevant alcohol solution into both sides of the tail. Before and after the injections repeated sensory conduction measurements were made using the rat tail method. Using 35% ethanol a temporary block of pain conduction could be achieved in both sexes. By increasing the concentration to 40 or 45%, a prolongation of the blocking effect and an increase in the accompanying increase of the pain threshold was observed in some animals. On the other hand, in some animals, temporary motor paralysis occurred following 35% ethanol, and this effect became more frequent and severe using 40 and 45% ethanol. A further increase in the alcohol concentration was not possible because of the occurrence of anatomical changes in the tails of the experimental animals.


Liver function tests following anaesthesia. Liver function tests were performed in 41 patients who required repeated anaesthetics for genito-urinary surgery, and who had received multiple halothane anaesthetics in the past, first following anaesthesia using halothane and then following an anaesthetic without halothane. There was a smaller frequency of disturbance of liver function after halothane than after the non-halothane anaesthetic. There was no obvious relationship between the number of anaesthetics, or the total duration of anaesthesia, and the disturbance of liver function tests.


A comparison of some cardiorespiratory effects of althesin and ketamine when used for induction of anaesthesia in patients with cardiac disease. Cardiorespiratory effects of ketamine and Althesin were measured in two groups of premedicated patients with cardiac disease. The drugs were given in clinically equivalent doses with a second dose administered about 10 min after induction. The first dose of ketamine caused a marked increase in systemic and pulmonary arterial pressure, heart rate, and central venous and wedge pressures and cardiac index. The first dose of Althesin caused a decrease in systemic arterial pressure, central venous pressure, cardiac index and heart work, but little change in heart rate. The second dose of induction agent was administered before the cardiorespiratory effects of the initial dose had resolved. The second dose of Althesin caused changes similar to those following the first dose, but less marked. The changes following the second dose of ketamine were opposite to those following the first dose.


The effect of althesin (CT 1341) on the oxyhaemoglobin dissociation curve in vitro. Blood from non-smoking donors was incubated with Althesin and the PO2 for half-saturation of haemoglobin at standard pH, PCO2 and temperature (P 50) was measured. The mean P 50 values of blood with blood concentrations of Althesin of 0.14 ml/dl and 0.42 ml/dl were greater (1.14 +/- 0.31 (SEM) mm Hg (P less than 0.005) and 2.22 +/- 0.35 (SEM) mm Hg (P less than 0.001), respectively) than that of control blood. These increases were dose-dependent (P less than 0.05). Erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentrations were not significantly different between the Althesin-treated and the control blood. Although a decrease in the affinity of haemoglobin for oxygen in vitro in the presence of Althesin was statistically significant, this would have no clinical significance if the drug was being used for the induction of anaesthesia only.


Plasma diazepam concentrations following prolonged administration. Plasma diazepam and N-desmethyl diazepam concentrations were measured in patients receiving diazepam 5 mg or 10 mg i.v. at 4-h intervals for periods of 6-22 days. At both doses there was an accumulation of both diazepam and its metabolite, the latter reaching concentrations of up to two to three times that of the parent drug. Plasma diazepam concentrations reached a plateau after 8 days while the concentration of N-desmethyl metabolite continued to increase throughtout the period of drug administration. On discontinuation of diazepam therapy both diazepam and N-desmethyl diazepam concentrations decreased slowly, the former with a half-life of 2-4 days and the latter with a half-life of 4-8 days.


Diazepam: routes of administration and rate of absorption. A study of women with pre-eclampsia. Plasma diazepam concentrations were determined following oral, i.m. and i.v. administration to a group of pregnant women with pre-eclampsia and a group of normal pregnant women. Diazepam concentrations were greater following oral as compared with i.m. administration in the control group, who received a single 5-mg dose, and this confirms previous similar reports. In the patients with pre-eclampsia, who were pre-loaded with diazepam, the i.m. route provided much higher plasma diazepam concentrations than did the oral route. This was probably a result of reduced gastric motility and gastric secretion caused by diazepam which affected the absorption of subsequent diazepam administered orally, and it is unlikely that pre-eclampsia contributed to these differences. Mist. magnesium trisilicate seemed to improve the rate of absorption of diazepam from the intestine in five patients studied, although this effect is unlikely to have much clinical importance.


Self-antagonism: a possible mechanism of tachyphylaxis in suxamethonium-induced neuromuscular block in man. Twelve adults receiving halothane-nitrous oxide anaesthesia for elective surgery were investigated during suxamethonium-induced neuromuscular block, using the train-of-four twitch for measurement of both the degree of block and the magnitude of fade (decrease in fourth-to-first ratio). The residual neuromuscular block resulting from prolonged exposure to suxamethonium could be antagonized by bolus injections of suxamethonium itself, in doses which increased the block in Phase I. When self-antagonism was demonstrated, larger doses were necessary to increase the residual block, but only after overcoming the self-antagonizing effect. A biphasic response, first antagonizing then increasing the block, was observed following injection of a single bolus of suxamethonium. The self-antagonizing effect was more obvious in the fourth twitch of the train-of-four, resulting in a marked decrease in train-fo-four fade (increase in train-of-four ratio). It was concluded that self-anatagonism may be an important cause of tachyphylaxis in suxamethonium-induced neuromuscular block in man.


Blunt trauma to the trachea.

Blunt trauma to the trachea. A 5-year-old child sustained blunt trauma to the cervical trachea. Diffuse subcutaneous emphysema of the neck and thorax developed rapidly. Anaesthesia was induced with halothane through a face-piece and the patient breathed spontaneously until the lesion has been evaluated. This course is advocated in the management of such injuries.


A colony-forming assay for human tumour xenografts using agar in diffusion chambers. A technique for growing colonies from single-cell suspensions of human tumour xenografts using agar in diffusion chambers is described. Modified Millipore diffusion chambers containing tumour cells in semi-solid agar-medium were implanted into the peritoneal cavity of pre-irradiated mice and provided standard culture conditions for the study of colony-forming cells. All 11 xenograft tumours so far studied produced colonies. The incubation period for colony growth ranged from 12 to 28 days and the plating efficiency ranged from 0-3% to 16% for different tumours, but both parameters were constant for each individual tumour. The reproducibility of the system provides a colony-forming assay which can be used to study the effects of irradiation and cytotoxic drugs on human tumour clonogenic cells and may therefore have some advantages over similar assays based on experimental animal tumours.


Complement activation in vivo in cancer patients receiving C. parvum immunotherapy. Serum complement levels were assayed in 26 patients with disseminated cancer, who received immunotherapy with infusion of C. parvum. Complement activation, indicated by the consumption of C3 or C4 or both, was found in 46% of the patients. Serum samples showed direct correlation between decreased C3 and conversion of C3 proactivator, whereas such conversion did not occur when C4 alone was decreased. It is concluded that the bypass (properdin) pathway was activated in patients in whom C3 consumption was detected, while the classical (C1) pathway was activated in the patients with C4 consumption unaccompanied by C3 decrease. Direct correlation was observed between delayed cutaneous hypersensitivity reactions to recall antigens and the incidence of C. parvum-associated complement activation.


Tumour-initiating activities on mouse skin of dihydrodiols derived from benzo[a]pyrene. Three dihydrodiols that are metabolites of benzo[a]pyrene and benzo[a]-pyrene itself have been tested in a comparative experiment for their activities as initiators of tumours in mouse skin. A single application (25 mug) of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, of 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, or of benzo[a]pyrene was made to the shaved dorsal skin of adult female CDI mice; this was followed 2 weeks later by multiple thrice-or twice-weekly applications (1 mug) of 12-O-tetradecanoyl-phorbol-13-acetate as promoting agent. A control group of 30 mice received the promoting agent alone. The experiments were terminated 52 weeks after initiation. At this stage, all the groups contained mice bearing skin papillomas, some of which had progressed to malignancy. Quantitatively the results show that the 7,8-dihydrodiol is almost as active an initiator of mouse skin tumours as benzo[a]pyrene itself; the 4,5- and 9,10-dihydrodiols were significantly less active. The significance of these results is discussed in relation to the hypothesis that diol-epoxides are important in the metabolic activation of polycyclic hydrocarbons like benzo[a]pyrene.


Effect of age and sex on lung-colony-forming efficiency of injected mouse tumour cells. The i.v. injection of a specified number of cells of either an Ehrlich ascites tumour (ELD) or spontaneous mouse mammary adenocarcinomas (MA) into C3H mice yielded a number of lung colonies which varied significantly with the age or sex of recipient mice. The yield was higher in mice of 71 weeks than in those of 15 weeks, except for MA cells injected into females, when the yield was higher in the younger mice. Sex did not influence very significantly the yield of colonies from ELD cells; in the case of MA cells the direction of sex differences depended on age. A difference in the effect of pre-immunization with age was not observed.


Modulation by lymphocytes of the vascular effects caused by inflammatory mediators and carrageenin in the rat. Inhibited permeability responses to intradermally injected histamine, serotonin and bradykinin were observed in leucopenic rats, when compared to those measured in normal animals. Significant reversal of the inhibited responses was seen when leucopenic rats were given suspensions of lymphocytes i.v. Suspensions of PMN granulocytes, however, were ineffective. In both cases, the volumes of the suspensions contained adequate quantities of the particular cells to counteract their deficiency. Histological changes provoked by carrageenin in the paws of leucopenic rats injected with suspensions of lymphocytes resembled those of normal rats, the main difference being that the number of cells which had emigrated into the affected tissues was reduced. In leucopenic controls or leucopenic animals injected with suspensions of PMN granulocytes, minimal histological alterations were observed. It is concluded that lymphocytes play a role in the development of acute inflammatory reactions.


An electron microscopic study of pig liver viability after extracorporeal storage. The effects of hyperbaric oxygenation and ambient pressure oxygenation on the viability of pig liver stored in the Vickers' transportable organ storage unit were assessed by electron microscopy. It was found that hyperbaric oxygenation resulted in marked liver damage in contrast to ambient pressure storage. Although electron microscopy proved to be a sensitive method for detecting damage induced by unsuitable storage protocols consideration is given to the possibility that other changes, which may not be detectable by electron microscopy, limit the success of liver storage.


Immunization of mice against Coxsackievirus B3 and prevention of foetal growth retardation. Coxsackievirus B3 infection in pregnant mice leads to a severe pancreatitis with a retardation of foetal growth and increased wastage. The present study demonstrates that animals may be immunized actively or passively against this infection to allow foetal development to proceed normally. Active immunization was achieved by injecting a low dose of live virus into 4-week-old animals. These mice were then mated at 10 weeks and given a high dose of virus on the eighth day of pregnancy. Examination at 18 days' gestation revealed that foetal growth was not significantly different from the controls injected with heat-killed virus, and pathological changes in the mothers were not seen. Animals were passively immunized against Coxsackievirus B3 in pregnancy by injecting serum from immunized animals 1 day before the high dose of live virus was given. This procedure also protected against the effects of the virus and litter sizes and foetal weights were normal.


Placental agenesis, embryonal hydraemia, embryolethality and acute hypervitaminosis A in rats. Acute maternal hypervitaminosis A established on Day 9 of gestation in Sprague-Dawley-derived rats caused a dose-related increase in the resorption of implants. The median embryolethal dose was 189,000 i.u./kg. In addition to suppression of the allantois leading to placental agenesis, damaged embryos showed retarded somatic development and hydraemia, all apparent 24 h after treatment. At about Day 11 the hydraemia involved the visceral wall of the yolk sac causing death of the embryo soon after. The fluid in the vitelline vessels continued to collect until Day 13 when it absorbed following necrosis of the wall of the yolk sac. Two mechanisms are suggested for the embryonal hydraemia: either the excess fluid resulted from a permeability disorder induced by the vitamin A; or it was retained metabolic water or water specifically absorbed to inflate the allantois and, being unused for this purpose, it pooled in the blood vessels of the embryo. The yolk sac hydraemia is more likely to have followed injury to the proximal endoderm.


Factors affecting the susceptibility of staphylococci to killing by the cationic proteins from rabbit polymorphonuclear leucocytes: the effects of alteration of cellular energetics and of various iron compounds. Anaerobiosis, various respiratory inhibitors and certain agents altering cellular energetics profoundly affect the staphylocidal action of the cationic proteins from rabbit polymorphonuclear leucocytes. It is suggested that sensitivity to these proteins depends on the structure of the cell membrane as influenced by (1) the oxidation level of the cytochrome chain and (2) its energized state. Agents such as amytal and rotenone, which cause a block at the beginning of the chain and would increase its oxidation level, enhance killing, whereas those causing a block in or at the end of the chain, such a 2-n-heptyl-4-hydroxyquinoline-N-oxide, cyanide and anaerobiosis, which would cause reduction of a part or whole of the chain, prevent killing. Among agents altering the energized state of the membrane, dicyclohexyl-carbodi-imide, an ATPase inhibitor, does not prevent killing, whereas 2,4-dinitrophenol, carbonylcyanide-trifluoromethoxy-phenylhydrazone and 5-Cl, 3-t-butyl, 2'-Cl, 4'-NO2-salicylanilide, all uncouplers and ionophores for a specific ion, do prevent killing, although gramicidin, a relatively nonspecific ionophore, does not. The paper also contains an extension of previous work on the effect of iron and haematin, to include various other iron compounds and haematin derivatives.


Ultrastructural changes in the isolated rat kidney induced by conjugated bilirubin and bile acids. The effects of bilirubin and bile acids on the ultrastructure of proximal renal tubules have been studied using an isolated rat kidney preparation, perfused with a protein-free dextran medium. Control kidneys perfused for 1 h had a normal glomerular filtration rate and effective renal plasma flow; the ultrastructure of proximal tubular cells was well preserved, with normal mitochondria, nuclear and plasma membranes, and microvilli of the brush border. When conjugated bilirubin, prepared from human hepatic bile, was added to the perfusion medium (5-0-7-5 mg/100 ml), marked alterations were observed in some cells, particularly with regard to the mitochondria and plasma membranes. These changes were greatly diminished by the inclusion of bovine albumin in the medium, indicating that the unbound fraction was primarily responsible for the tubular damage. The addition of taurocholate (450 muM), taurochenodeoxycholate (550 muM) or taurolithocholate (250 muM, bound to albumin) also produced plasma membrane changes, but only slight abnormalities were seen in the mitochondria and other structures. These ultrastructural observations support the concept that the elevated plasma levels of conjugated bilirubin and to a lesser extent bile acids are related to the renal failure associated with obstructive jaundice.


Conformational flexibility of the serum amyloid precursor SAA. SAA is a normal acute-phase serum protein and is thought to be the precursor of amyloid protein AA which is deposited as insoluble beta-pleated sheet fibrils in secondary amyloidosis. Native SAA has a molecular weight of 160,000 and has not been isolated; it has been most frequently purified as a species (designated SAAL) of 12,500 mol. wt. by gel filtration in dissociating solutions. The conformational properties of SAA proteins in patients with and without amyloidosis have been compared in an effort to determine the factors involved in the induction of the beta-pleated sheet conformation in the amyloid SAA protein prior to fibril deposition. Amyloid and nonamyloid SAA proteins are similar in that they readily undergo conformational changes which result in the formation of heterogenous mol. wt. SAA species and in an increased exposure of antigenic determinants which cross-react with AA fibril proteins. Amyloid and nonamyloid SAA are different, however, in that amyloid SAA is more resistant to dissociation to SAAL. Amyloid SAAL, while similar to nonamyloid SAAL in immunoreactivity, shows a greater tendency toward aggregation. The relative resistance of both amyloid SAA and SAAL to complete dissociation may play an important role in amyloid fibril formation from these precursors.


Effect of hepatic venous outflow obstruction on pores and fenestration in sinusoidal endothelium. The ultrastructure of pores and fenestrations in hepatic sinusoidal endothelial cells was examined following partial surgical occlusion of the suprahepatic portion of the inferior vena cava. Within 12 h after partial obstruction of hepatic venous outflow, endothelial pores (less than 0-1 mum in diameter) and sieve plates in the distal halves of sinusoids were greatly reduced in number or were totally absent, and they were replaced by large fenestration (less than 1-0 mum in diameter). These results suggest that pores forming sieve plates may fuse to form large fenestrations. The findings also indicate that sinusoidal hypertension and hypoxia associated with obstruction of hepatic venous outflow alter the distribution of pores and fenestrations in sinusoidal endothelium


Effect of different factors modifying the activity of some enzyme systems of the endoplastic reticulum on the sensitivity of cell organelles against the damaging action of chemical agents. I: Interrelations between the activity of some enzyme systems located in endoplasmic reticulum and hepatotoxicity of carbon tetrachloride. An increased stability of liver lysosomes and mitochondria of burned, dehydrated and tumour-bearing rats has been found in the present investigations. Using biochemical criteria we were able to show that the membranes of liver subcellular particles of these animals become more resistent to the labilizing action of CCl4. The latter effect was confirmed by electron microscopic data. The tumour growth, dehydration and burning of rats as well as sleep deprivation, overheating, lanthanum and ruthenium red have been shown to cause changes in the activity of some enzyme-systems located in endoplasmic reticulum. The protective action of these chemical, pathological and stress factors against CCl4 produced damage to the cell organelles studied is discussed in relation to the activity of the drug metabolizing pathways; biochemical (modifying of some enzyme activity) as well as pharmacological (duration of hexobarbital sleep) data were used as criteria for the activity of these pathways.


The giant cell in cholesterol resorption. Giant cells in the experimental cholesterol granuloma provided a convenient, recognizable cellular model in which to follow the transformation of crystalline cholesterol to liquid lipid. Crystals that were too large to be ingested by individual macrophages were incorporated into a multinucleate cell by a process of circumfusion, and were progressively converted to cholesterol esters within the giant cell. The liquid ester droplets seemed then to be mobilized by mononuclear cells until the entire implant had been resorbed.


Occupational and regional associations of death, disablement, and sickness absence among Post Office staff 1972-75. Rates of death in service and rates of premature retirement due to disability in an employed population of more than 400 000 people under the age of 60 years have been compared with duration rates of certified sickness absence for three years from April 1972. Standardized for age, rates of sickness absence and of death, combined with retirement (medical wastage) for men and women differed between 11 occupational groups by a factor of three. Significant rank correlations were observed between these two measures of ill health, rs = 0-827 for men, rs = 0-857 for women. Similar comparisons for 1974/75 of rates for staff employed in 10 geographical regions of the United Kingdom standardized for age and occupation, revealed twofold differences of rate, and rank correlation coefficients of + 0-794 for men and + 0-649 for women. These observations show that the duration of certified sickness absence is higher in groups of people showing other objective evidence of ill health and who are most in need of preventive health care. The need is discussed for a balanced view of the complex aetiology of sickness absence.


Sickness absence of lead workers and controls. Certificated sickness rates and levels of lead exposure of 955 men who worked in a lead accumulator factory during a period of seven years (1965-72) were examined. The men were divided by department into four exposure groups; a second division into three groups was made, based on the mean of each man's blood lead measurements during the study period. Absences showed no age pattern, but men who left during the seven years of observation (ex-workers) had had a higher absence rate (842 spells/1000 man years) than those still employed in 1972 (535 spells/1000 man years). There was no significant difference in absence rates or lengths of absences between differently exposed departments either for all causes or for a selected group of potentially lead-induced causes. However, the proportion of potentially lead-induced absences was significantly higher in ex-workers (12-2%) than in current workers (7-4%). Similar analysis showed no significant differences in absence rates of men with different blood lead levels. It was concluded that higher levels of lead exposure did not seem to be associated with higher rates of absence or longer absences either for all causes of absence combined or for those causes which might be attributable to lead.


Sickness absence caused by chest diseases in relation to smoking and chronic bronchitis symptoms. The relationship between sickness absence caused by chest diseases, smoking habit, and symptoms of chronic bronchitis among the workers of a fertilizer factory in Karaków is examined. The analysis of absence was based on the spells and days of absence registered during a period of six years. The results show that the group of workers with symptoms of chronic bronchitis had many more spells and days of absence caused by chest diseases than persons free from these symptoms. The activity and chronicity of bronchitis symptoms strongly influenced the level of absence, but the role of smoking was not so evident. From the results obtained it appears that analysis of absence can be a valid screening test in the detection of chronic chest diseases, the frequency of which can easily be estimated from absence data.


A survey of occupational cancer in the rubber and cablemaking industries: analysis of deaths occurring in 1972-74. The records of 40 867 men employed for at least one year in the rubber and cablemaking industries have now been observed for eight years. This analysis compares the mortality pattern for 1972-74 with that previously reported for 1968-71. It indicates a significant excess of deaths due to cancer of the bladder throughout the industry including men who had not been exposed to acknowledged bladder carcinogens. This excess is in deaths occurring in 1973 and 1974 in the 45-64 and 65 years plus age groups. The two sectors of the industry where this excess is significant are footwear and footwear supplies except adhesives, and the tyre sector. The excess of all cancers taken together previously noted throughout the study population for 1968-71 is confirmed for 1972-74 as is the excess for lung cancers. The greater excess in the tyre sector is also confirmed, particularly in those men in the 55-64 year age group and those who entered the industry between 1950 and 1960. While men employed in 1967 on moulding, press, autoclave, and pan curing, and workers in finished goods, stores, packaging, and despatch continue to have more lung cancer deaths than expected for 1972-74, the excess is no longer statistically significant. An excess of cancer of the stomach which was overlooked in 1968-71 is not confirmed in 1972-74 but is nevertheless high when the total period of study 1968-74 is considered. The limitations of the study are discussed with particular reference to extrapolating the results to the whole industry. We conclude that there is a higher rate of lung cancer in the tyre sector of the industry and that immediate investigations are required to test the hypothesis concerning the recent excess of bladder cancers. Attention should now be paid to the control of exposures to all potential hazards in the industry.


Farmer's lung disease in Somerset.

Farmer's lung disease in Somerset. A survey of laboratory records was made to assess the value of the precipitin test and isolation methods in the diagnosis of farmer's lung disease and also to determine its prevalence in the farming population of Somerset. A link was established between the clinical diagnosis as written on the form that accompanied the specimen and the actual number of positive laboratory diagnoses made. Fifty (43%) of the clinically diagnosed patients were serologically positive for farmer's lung during a four-year period. If the clinically diagnosed but serologically negative cases of farmer's lung disease are added to this number, a prevalence of about 23 per 1000 of the farming population of Somerset is obtained.


Excretion of 35S-Tobias acid (2-naphthylamino, 1-sulphonic acid) by the rat after oral and intravenous administration. Urinary and faecal excretion of radioactivity after either an intravenous or oral (1 mg/kg) dose of 35S-labelled Tobias acid (2-naphthylamino, 1-sulphonic acid), a dyestuff intermediate structurally similar to the powerful carcinogen 2-naphthylamine, was studied in rats. The Tobias acid was eliminated from the body within 24 hours of administration, almost exclusively through the urine. TLC-chromatography of faecal extracts and urine did not disclose the presence of excreted products other than unchanged Tobias acid and the search for inorganic 35SO4 in the urine by BaCl2 precipitation was negative. There was significant absorption from the gastrointestinal tract, but neither cleavage of the sulphonic group nor other biotransformation by the intestinal flora was apparent under the test condition. There was no evidence that the sulphonic group of Tobias acid is cleaved in the body to a significant extent to give 2-naphthylamine. This information should help in the evaluation of the occupational hazard potential of Tobias acid.


Dimers and trimers of immunoglobulin G covalently cross-linked with a bivalent affinity label. A bivalent affinity label, bis(alpha-bromoacetyl-epsilon-2,4-dinitrophenyllysylproline)ethylenediamine, has been synthesized. Treatment of anti-2,4-dinitrophenyl antibodies with this compound produces a mixture of covalently and noncovalently cross-linked material. Only specific antibodies are covalently cross-linked, suggesting that covalent attachment occurs in the variable regions. Covalently cross-linked dimers and trimers have been isolated from the reaction mixture in a high state of purity, in yields of about 12 and 4%, respectively. The complexes are stable in solutions containing 10(-4) M hapten and can therefore be used as sensitive probes of immune effector functions.


Abundance of tRNAPhe lacking the peroxy Y-base in mouse neuroblastoma. Affinity chromatography on anti-Y (Y is a tricyclic imidazopurine to which is attached a complex four-carbon side chain) antibody immobilized to Sepharose was used to determine the proportion of rat liver tRNAPhe species containing the peroxy Y-nucleoside. Unfractionated Unfractionated mammalian tRNA was aminoacylated with labeled phenylalanine. The phenylalanyl-tRNA was then chemically acetylated to yield N-acetylphenylalanyl-tRNA. When this preparation was applied to the antibody column, between 6-10% of the radioactivity was not bound to the column, indicating a deficiency of peroxy Y-nuceloside in a minor isoaccepting tRNAPhe species. In contrast to normal tissues (including embryonic tissue), about 85% of the tRNAPhe from mouse neuroblastoma C-1300 or N-18 tumors lack the peroxy Y-base, a property which is not affected by tumor age. Rat liver labeled N-acetylphenylalanyl-tRNA preparations were resolved on Plaskon chromatography (RPC-5) into two minor peaks closely followed by a mojor component. A high proportion of the two minor tRNAPhe species was unable to bind to anti-Y antibodies. Upon mild acid treatment, the minor and major tRNAPhe species eluted simultaneously from Plaskon columns, at a much reduced salt concentration. These results would indicate that the two minor tRNAPhe species from rat liver as well as the major component contain a tricyclic imidazopurine base that differs from each other in its side chain. About 85% of the N-acetylphenylalanyl-tRNA from neuroblastoma was resolved by Plaskon chromatography as an early eluting peak. The position of this major neuroblastoma tRNAPhe species was not altered by mild acid treatment, and its elution position from the column almost coincides with that of acid-treated normal rat liver tRNAPhe. The latter results would suggest that most of the tRNAPhe chains from neuroblastoma lack the tricyclic imidazopurine of normal rat liver tRNAPhe, but are very close if not identical in primary nucleotide sequence.


Isolation and characterization of purified rat casein messenger ribonucleic acids. Purification of casein messenger ribonucleic acids (mRNAs) from lactating rat mammary gland RNA has been accomplished by a combination of sizing techniques, including Sepharose 4B chromatography and preparative agarose-urea gel electrophoresis, and affinity chromatography of poly(adenylic acid)-containing mRNA on oligo(dT)-cellulose. The separation of the individual casein mRNAs into discrete molecular species free of apparent ribosomal RNA contaminants was facilitated by the use of denaturing conditions either prior to or during each of the fractionation procedures. Two casein mRNA fractions were isolated: (1) a 15S mRNA doublet which directed the synthesis of the two largest rat caseins in the wheat-germ, cell-free, translation assay, and (2) a 12S mRNA which migrated as a single species during agarose-urea gel electrophoresis and directed the in vitro synthesis of the smallest of three rat caseins. These mRNAs had apparent molecular weights of 450 000 +/- 30 000 and 320 000 +/- 25 000 and contained poly(adenylic acid) sequences at their 3' termini ranging from 15 to 150 residues with number average lengths of 42 and 38 adenosines, respectively. The purity of the isolated casein mRNA'S was determined both by agarose-urea gel electrophoresis and by a careful comparison of the total products synthesized in the wheat-germ translation assay with those recognized by a specific casein antibody using an indirect immunoprecipitation technique. The specificity of the indirect immunoassay procedure was demonstrated by the selective displacement by purified rat casein of greater than 95% of the radioactive product synthesized in the cell-free system. Under optimal translation conditions for casein mRNA, at least 90% of the released protein synthesized in response to the 15S casein mRNA was specifically immunoprecipitable, representing a 178-fold purification compared with the initial RNA extract. Using these techniques a comparable purification was also obtained for a 15S mouse casein mRNA fraction. Finally, an analysis by fluorography on 5-20% (w/v) polyacrylamide gradient slab gels of the total proteins synthesized in response to both the 15S and 12S casein mRNAs revealed a close correspondence with those proteins which were specifically immunoprecipitated.


Papain-catalyzed reactions at subzero temperatures. As a first step in the investigation of papain catalysis using subzero temperatures to detect, accumulate, and characterize enzyme-substrate intermediates, we have studied some potential cryosolvents and carried out preliminary intermediate trapping experiments. The effects of subzero temperatures and aqueous dimethyl sulfoxide solutions on the papain-catalyzed hydrolysis of Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester have been investigated in detail. At 0 degrees C, the value of kcat decreases with increasing dimethyl sulfoxide concentration, decreasing in proportion to the decreased water concentration; however, the value of Km increases exponentially. The effect on Km can be accounted for by a combination of both dielectric and competitive inhibition effects. The Arrhenius plot for the deacylation reaction in 7.65 M (60% v/v) dimethyl sulfoxide is linear over the temperature range 0 to -45 degrees C and extrapolates to a calculated value of kcat at 25 degrees C in excellent agreement with that obtained in the absence of organic solvent. The pH-rate profile is not substantially perturbed by the presence of 7.65 M dimethyl sulfoxide. At -45 degrees C and below, turnover occurs extremely slowly, and is essentially negligible, although acylation is still quite rapid. Consequently, the acyl enzyme, Na-carbobenzoxy-L-lysyl-papain, can be readily accumulated and trapped at temperatures below -50 degrees C. At these low temperatures, under conditions of excess substrate, the amount of p-nitrophenol liberated in the acylation reaction is equivalent to the active-site normality of the enzyme, indicating a 1:1 stoichiometry in formation of the acyl enzyme. The effect of dimethyl sulfoxide up to 7.65 M, on the intrinsic ultraviolet, fluorescence, and circular dichroic properties of the enzyme shows no evidence of any solvent-induced structural changes. All experimental observations are consistent with the conclusion that 7.65 M dimethyl sulfoxide and subzero temperatures have no deleterious effects on papain-catalyzed reactions. A related series of experiments indicate that aqueous ethanol cryosolvents up to 13.7 M (80% v/v) are also suitable. Preliminary experiments at subzero temperatures using Na-carbobenzoxy-L-lysine methyl ester suggest the existence of three enzyme-substrate intermediates which can be detected and accumulated.


Studies on DNA alpha-polymerase of mouse myeloma: partial purification and comparison of three molecular forms of the enzyme. Activity of DNA alpha-polymerase in extracts from MOPC-104E was not associated with a single protein molecule, but with several molecular species that differed in isoelectric point. The three most abundant of these enzyme species were first separated from other DNA polymerases and then resolved from each other by repeated chromatography on diethylaminoethylcellulose columns. Next, with the use of glycerol gradient centrifugation and DNA-cellulose column chromatography, the three species were further purified to a state representing more than 5000-fold purification over the crude extract. These three highly purified enzyme species exhibited very similar catalytic properties, and the main activity of each species sedimented at the same rate (6-7S) in glycerol gradients containing 0.5 M KCl. Analysis of the polypeptide content of each species revealed that polypeptides of about 150 000 and 60 000 daltons cofractionated with the DNA polymerase activity. The multiple alpha-polymerase species did not appear to result from in vitro proteolytic cleavage, since multiple species were observed in extracts prepared under several different types of conditions, including the presence of the protease inhibitors, phenylmethanesulfonyl fluoride, or trasylol. The three species were recovered in about the same relative amounts from both the nuclear and cytoplasmic fractions of MOPC-104E, and it appeared that multiple species of alpha-polymerase were also present in extracts from fetal bovine liver.


Characterization of the cyanogen bromide fragments of the beta chain of human haptoglobin. Characterization of the cyanogen bromide (CNBr) fragments of the beta chain of human haptoglobin revealed five major fragments resulting from cleavage of four methionyl residues. The fragments were isolated by gel filtration in guanidine-HCl on Sepharose 6B and Bio-Gel P10 and P60. Compositional analyses of the five cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact beta chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin beta chain and cyanogen bromide fragments identified 139 residues, or about 55% of the beta-chain molecule. The placement of the fragments within the beta-chain molecule was established by sequence analysis of whole beta chain and a plasmin cleavage fragment. The position of CNBr V was confirmed by the absence of homoserine or homoserine lactone. Cyanogen bromide reaction of intact haptoglobin 1-1 resulted in the isolation of a beta-chain fragment, CNBr III, covalently attached to the intact alpha1 chain by a single disulfide bond. The beta chain was shown to have primary structural similarities to the chymotrypsin family of serin eproteases. Partial sequence analysis of CNBr V established the region which is comparable to the serine-195 active-site region: /Asp-Thr-Cys-Tyr-Gly-Asp-Ala-Gly-Ser-Ala-Phe/ (residues 189-199, chymotrypsinogen A numbering). The active-site serine-195 is replaced by alanine; however, the specificity residue of the trypsin-like enzymes, Asp-189, is preserved. Several minor cyanogen bromide cleavage products were also identified in yields of up to 15%. These minor cleavage products give evidence that tryptophanyl residues in proteins, or glycoproteins, are also susceptible to cyanogen bromide cleavage.


Carbon monoxide binding properties of hemoglobin M Iwate. The kinetic and equilibrium CO binding properties of hemoglobin (Hb) M Iwate (alpha2 87 His leads to Tyr beta2) have been investigated. The results show that the alpha(Met) beta2 (CO) tetramer of this protein has a low affinity for CO, as indicated by the stopped-flow and flash-photolysis kinetic, as well as the CO binding equilibrium, measurements. However, it has been found that the phosphate-free alpha2(Met)beta2(CO) tetramer does tend to dimerize extensively (K4.2 = 55 muM). The high-affinity forms seen in earlier kinetic measurements may be explained by this fact. When dimers are accounted for in the functional studies, the results show that the tetramer binds CO noncooperatively either with or without the allosteric cofactor, inositol hexaphosphate (IHP). IHP appears to influence the functional properties of a solution of Hb M Iwate by stabilizing the tetrameric state of aggregation, thereby greatly reducing the population of high-affinity dimers. When the CO "off" rate with IHP present (0.23 s-1) and the CO "on" rate to the tetramer either with or without IHP (1.9 X 10(5) M-1 s-1) at 25 degrees C are sued to calculate the equilibrium constant, the value obtained (8.3 X 10(5) M-1 s-1) is similar to that in equilibrium binding measurements on the phosphate-free tetramer (9.5 X 10(5) M-1) estimated from the observed P 1/2 value at 0.48 mM total heme concentration. By showing that dimers account for the high-affinity component seen in earlier kinetic experiments with Hg M Iwate, we can now more strongly suggest that cooperative CO binding to this tetramer is minimal or absent, with both of the active beta-hemes presenting a very low affinity.


Oxygenation-linked subunit interactions in human hemoglobin: experimental studies on the concentration dependence of oxygenation curves. An experimental study on the concentration dependence of oxygenation curves for human hemoglobin has been carried out between 4 X 10(-8) M heme and 5 X 10(-4) M heme in 0.1 M tris(hydroxymehtyl)aminomethane hydrochloride, 0.1 M NaCl, 1 mM disodium ethylenediaminetetraacetic acid, pH 7.4, 21.5 degrees C. With decreasing hemoglobin concentration the curves show pronounced shifts in position and shape, consistent with dissociation of tetrameric hemoglobin into dimeric species of high affinity and low cooperativity. Combination of these data with independently determined values of dissociation constants for unliganded and fully liganded hemoglobin permits a resolution of the seven parameters necessary to define the linked binding and subunit association processes. The total oxygenation-linked subunit dissociation energy (6.34 kcal) was resolved into intersubunit contact energy changes between alphabeta dimers in tetrameric hemoglobin which accompany binding of the first, middle two, and last oxygen molecules. The resolution is accurate to within approximately +/-0.3 kcal. To within this limit the isolated dimers are found to bind oxygen noncooperatively and with the same affinity as isolated alpha and beta chains. Equally good fits to the data are obtained when dimers are slightly anticooperative. At least three major energetic states are apparently assumed by hemoglobin tetramers, with respect to the alpha1beta2 contact region, corresponding to (a) unliganded, (b) singly liganded, (c) triply and quadruply liganded species. The results do not establish whether these states may be assumed by a single molecule, or whether they arise as averages over a distrubution of conformational states. They do provide unequivocal evidence against a concerted transition at any particular binding step in a system with only two energetic states of tetramer (i.e., an all or none switchover between T and R states at a particular binding step).


Oxygenation-linked subunit interactions in human hemoglobin: analysis of linkage functions for constituent energy terms. Resolution of the linkage functions between oxygenation and subunit association-dissociation equilibria in human hemoglobin into the constituent microscopic terms has been explored by numerical simulation and least-squares analysis. The correlation properties between parameters has been studied using several choices of parameter sets in order to optimize resolution. It is found that, with currently available levels of experimental precision and ranges of variables, neither linkage function can provide sufficient resolution of all the desired energy terms. The most difficult quantities to resolve always include the dimer-tetramer association constant for unliganded hemoglobin and the oxygen binding constants to alphabeta dimers. A feasible experimental strategy for overcoming these difficulties lies in independent determination of the dimer-tetramer association constants for unliganded and fully oxygenated hemoglobin. These constants, in combination with the median lignad concentration, provide an estimate of the energy for total oxygenation  of tetramers which is essentially independent of the other constituent energies. It is shown that if these separately determinable parameters are fixed, the remaining terms may be estimated to good accuracy using data which represents either linkage function. In general it is desirable to combine information from both types of experimental quantities. A previous paper (Mills, F.C., Johnson, M.L., and Ackers, G.K. (1976), Biochemestry, 15, the preceding paper in this issue) describes the experimental implementation of this strategy.


Structure of gene 5 protein-oligodeoxynucleotide complexes as determined by 1H, 19F, and 31P nuclear magnetic resonance. 1H nuclear magnetic resonance (NMR) spectra at 270 MHz of gene 5 protein from bacteriophage fd and its complexes with tetra- and octadeoxynucleotides show that approximately 12 of the 37 aromatic protons of the protein undergo upfield shifts upon nucleotide binding. In the complex with d(pT)8, the upfield shifts of the aromatic protons average approximately 0.3 ppm, while in the d(pA)8 complex the same resonances (assigned to tyrosyl protons) shift upfield approximately 0.8 ppm. These are interpreted as ring current shifts induced by stacking of the phenyl rings of three of the five tyrosyl residues with the bases of the nucleotides. 19FNMR of m-fluorotyrosyl gene 5 protein shows five separate resonances: two downfield from m-fluorotyrosine corresponding to "buried" tyrosyls and three near m-fluorotyrosine corresponding to "surface" tyrosyls. The latter (assigned to Tyr-26, -41, and -56, shown by chemical modification to be exposed to solvent) move upfield on nucleotide binding. The downfield 19F resonances are unaffected. Thus the aromatic protons shifted upfield on nucleotide binding appear to be those of Tyr-26, -41, and -56. In contrast to tetra-, octanucleotide binding to gene 5 protein induces large changes in the 1H resonances of the -CH3 groups of the Val, Leu, and Ile side chains. These may reflect conformational changes induced by protein-protein interactions between two monomers bound to the octanucleotide. 1H resonances of the epsilon-CH2 groups of the lysyl residues in the protein and the complexes with nucleotides are narrow with long T2 values, suggesting considerable rotational motion. Thus epilson-NH3+-phosphate interactions, if they occur, are on the surface of the complex and allow the epsilon-CH2 groups to retain considerable rotational freedom. 31P NMR of the bound nucleotides shows large decreases in T1 for the 3'-5' diesters, but little chemical shift suggesting no unusual distortion of the nucleotide backbone on binding to gene 5 protein. A three-dimensional model of a gene 5 protein-octanucleotide complex has been built based on predictions of the secondary structure from the amino acid sequence (87 AA) and tertiary folding dictated by known chemical and NMR features of the complex.


The salt-induced destacking of purine in aqueous NaCl systems and its implications on life at elevated temperatures. Aqueous purine solutions in the absence and presence of NaCl have been studied by vapor-pressure osmometry and high-resolution proton-magnetic-resonance spectroscopy. The presence of NaCl causes a marked change in the temperature dependence of the molal osmotic coefficient and the chemical shift of the purine resonances when compared with the corresponding quantities in purine solutions containing no added salt. In the absence of salt, the destacking of the purine is gradual as the temperature is increased, whereas there is a sharp decrease in purine stacking at approximately 42 degrees C in the presence of NaCl. This salt-induced destacking of purine is consistent with the previously noted salt-induced bulk water destructuring at temperatures above approximately 42 degrees C. This effect has been deduced from considerations of species profiles in acid-water systems as a function of temperature (Leifer, L., and Inoue, K., manuscript in preparation). The implications of this phenomenon on biological systems at elevated temperatures will be discussed.


Effects of podophyllotoxin and VP-16-213 on microtubule assembly in vitro and nucleoside transport in HeLa cells. VP-16-213, a semisynthetic derivative of podophyllotoxin, is an active antitumor agent. In this paper, the effects of VP-16-213 and podophyllotoxin on microtubule assembly in vitro and nucleoside transport in HeLa cells are compared. At 100 muM, VP-16-213 does not inhibit microtubule assembly in vitro, while 5 muM podophyllotoxin completely prevents the formation of microtubules. The presence of the glucoside moiety in VP-16-213 is responsible for the inactivity of VP-16-213 in this system because 4'-demethylepipodo-phyllotoxin, the nonglucoside congener of VP-16-213, inhibits microtubule assembly. In HeLa cells, VP-16-213 and podophyllotoxin share a common biological property; both agents inhibit the uptake of thymidine and uridine into cells by inhibiting the facilitated diffusional component of nucleoside transport. The conncentrations of drug necessary to inhibit thymidine and uridine uptake into HeLa cells by 50% are 10 and 5 muM, respectively, for podophyllotoxin, and 25 and 20 muM for VP-16-213. The action of podophyllotoxin on nucleoside transport appears unrelated to its effect on microtubule assembly, since VP-16-213, which does not inhibit microtubule assembly, inhibits nucleoside transport.


Effect of VP-16-213 on the intracellular degradation of DNA in HeLa cells. The effect of VP-16-213 on cellular DNA was studied by following the sedimentation profiles of radioactive DNA in HeLa cells on alkaline sucrose gradients. In VP-16-213 treated cells, high-molecular-weight DNA is converted to a lower molecular-weight form in a dose-dependent, temperature-dependent reaction. The effect of VP-16-213 on cellular DNA is reversed after the drug has been removed from the growth medium for 150 min. These results suggest that VP-16-213 induces single-stranded breaks in DNA in HeLa cells and that HeLa cells can repair these breaks within 150 min. The nonglucoside derivative of VP-16-213, 4'-demethylepipodophyllotoxin, also induces the cleavage of cellular DNA but podophyllotoxin has no effect on DNA. A structure-activity relationship study, in which the effects of various VP-16-213 and podophyllotoxin congeners were tested for their ability to cleave cellular DNA,revealed that an hydroxyl group at the C-4' position is required for activity and that the configuration of the C-4 carbon influences the activity of a congener. These results may offer insights into the mechanism of action of VP-16-213 as an antitumor agent.


Sulfated glycoproteins, glycolipids, and glycosaminoglycans from synaptic plasma and myelin membranes: isolation and characterization of sulfated glycopeptides. In this report we provide biochemical evidence that a highly purified synaptic plasma membrane fraction derived from rat brain, after intraventricular injection of 35S-labeled sodium sulfate, is enriched in a number of large sulfated glycoproteins compared with a purified myelin fraction studied concurrently. A fraction of the detergent-solubilized sulfated glycoprotein bound specifically to concanavalin A-Sepharose. In addition, we have identified the 35S-labeled lipid-soluble material in these membrane fractions as cerebroside sulfate. The sulfated protein in the lipid-extracted membranes was shown to consist predominantly of a class of glycoproteins containing sulfate in ester linkage to oligosaccharide chains, which are differentiated structurally from the sulfated glycosaminoglycans of brain. These two classes of sulfated macromolecules were distinguished from one another by several chemical and physical parameters. We present the chemical characterization of the sulfated glycopeptides derived from synaptic plasma and myelin membranes by extensive proteolytic digestion after quantitative removal of cerebroside sulfate. Membrane-associated glycosaminoglycans, either specifically or adventitously associated with these neuronal membranes, were quantitatively precipitated and identified.


Differential translation of mouse myeloma messenger RNAs in a wheat germ cell-free system. Translation of the polysomal mRNA of mouse myeloma cells in a wheat germ cell-free system leads to the immunoglobulin (Ig) light-chain precursor as the major product. Excess polysomal RNA causes strong inhibition of polypeptide synthesis, but has little effect of light-chain precursor synthesis. The inhibitory effect of excess RNA is avoided when the poly(A)-containing RNA fraction is used. With nearly saturating amounts of the latter RNA, light-chain recursor synthesis becomes more predominant, possibly as a result of competition between different mRNA species. High levels of potassium acetate cause strong inhibition of overall translation, but do not inhibit light-chain precursor synthesis. Addition of poly(A) to the cell-free system also causes inhibition, presumably through interference with the intiation process. Again, light-chain precursor synthesis is relatively resistant. Ig heavy-chain synthesis is relatively inefficent, but its resistance to the inhibitors tends to be nearly as great as that of the light-chain precursor. The results indicate that the Ig mRNAs are particulary efficient in initiating translation. This characteristic may account for certain features of the regulation of Ig synthesis in intact myeloma cells.


Isolation and in vitro translation of zein messenger ribonucleic acid. Zein messenger RNA was isolated from membrane-bound polyribosomes of developing maize kernels by oligo(dT)-cellulose chromatography. Translation of the mRNA in vitro yielded protein similar to native zein in amino acid content, ethanol solubility, and mobility on sodium dodecyl sulfate- polyacrylamide gels. The zein mRNA sedimented as a homogeneous peak on sucrose gradients and contained a poly(A)-rich region based upon hybridization to [3H]poly(U). The mRNA had an apparent molecular weight of 540 000 on agarose-acrylamide gels. It synthesized both 21 800 and 19 000 molecular weight zein components in the wheat-germ cell-free protein synthesis system. The possibility of a polycistronic mRNA or two mRNAs of similar molecular weight is considered.


13C nuclear magnetic resonance study of molecular motions and conformational transitions in muscle calcium binding parvalbumins. 13C nuclear magnetic resonance is used to detect the Ca2+ ion controlled conformational transition in muscle calcium binding parvalbumin and to study its intramolecular motions. Nuclear relaxation parameters are used to evaluate the reorientation rates of the protein and some of the amino acid side chains. While peripheral residues exhibit greater motional freedom than the protein interior, an interesting finding is that significant rapid internal motion is present in the phenylalanine rings comprising the hydrophobic core of the protein.