Studies on rat liver catalase. IX. Role of methionine in polypeptide chain inhibition. Initiation with methionine of the synthesis of rat liver catalase [EC 1.11.1.6] has been investigated. Analysis of the N-terminal residue of nascent catalase peptides labeled in vivo with injected radioactive amino acids, including [3H]methionine, indicated a remarkably high content of methionine. By fractionating [3H]methionine-labeled nascent catalase according to chain length, it was found that peptides of shorter chain length contained more N-terminal methionine relative to total methionine incorporated. In addition, only a small amount of [3H]methionine was detected as the N-terminal amino acid when newly completed catalase was examined by Edman degradation. These results indicate that the synthesis of liver catalase is initiated with methionine, and suggest the presence of a mechanism for its subsequent removal from the N-terminal position. Catalase was also synthesized in a cell-free system directed by the catalase mRNA, using [3H]Met-tRNAf or [3H]Met-tRNAm. The results obtained in such in vitro experiments were in good agreement with those from in vivo studies, and further showed that the N-terminal methionine was provided by a specific initiator tRNA, i.e. tRNA Met f.


Synthesis of AMP analogs and their use for studies on the allosteric site of rabbit muscle glycogen phosphorylase b. In order to obtain a better understanding of the allosteric site of rabbit muscle phosphorylase b, nine AMP analogs having a bulky hydrophobic benzene ring were synthesized and tested for activity as activators or inhibitors. N6-Benzyl-AMP derivatives activated phosphorylase b to the same extent as AMP but bound to the enzyme more tightly than AMP. N6-p-nitrobenzyl-AMP had the highest affinity (Ka = 7.7 X 10(-7) M) for the AMP site. In an attempt to irreversibly modify the allosteric site of phosphorylase b, N6-p-bromoacetaminobenzyl-AMP was synthesized. Phosphorylase b was maximally activated upon incorporation of 1.0 mol of N6-p-bromoacetaminobenzyl-AMP per enzyme subunit, and its activity was approximately 90% of that of native phosphorylase b measured in the presence of AMP. The modified enzyme showed characteristics (e.g., kinetic parameters, stability, solubility, inhibition by glucose-6-phosphate, and state of aggregation) quite similar to those observed for the native enzyme in the presence of AMP. These results indicate that the AMP site of phosphorylase was specifically labeled by N6-p-bromoacetaminobenzyl-AMP. The nature of the allosteric  site of phosphorylase b is discussed based on the results obtained.


Macromolecular components of the vitelline membrane of hen's egg. III. Physicochemical properties of glycoprotein II. A glycoprotein fraction (GP-II) has been isolated from the vitelline membrane of hen's egg and its physicochemical properties clarified. GP-II is composed of polypeptide (92%), neutral sugar (4%), hexosamine (3.3%), and sialic acid (0.6%). The constituent neutral sugars of this glycoprotein are fucose, mannose, and galactose, in a molar ratio of 2:6:5. An interesting feature of the amino acid composition of GP-II is the high proportion of proline. GP-II exists in an aggregated form and is hydrophobic in nature. Upon velocity sedimentation in 0.5% SDS solution, it showed a hypersharp boundary with an apparent sedimentation coefficient of 5.6 S. Reduction of GP-II, however, gave a single component of 3.6 S which seems to be a subunit of GP-II.


Changes of DNA ligase in developing rat brain. DNA ligase was isolated from rat brain and characterized. In developing rat brain the DNA ligase activities of the cerebellum increased after birth, at least 5 fold more than those of non-cerebellar parts. The DNA ligase activity in the cerebellum reached a maximum about 6 days after birth and then decreased towards maturation. The DNA ligase from the cerebellum can be fractionated into three molecular forms. Aging of the extracts leads to the conversion of the high molecular weight form into smaller weight forms.


In vitro effects of streptomyces protease inhibitors on human kidney renin activity. The effects of several low molecular streptomyces protease inhibitors on human kidney renin activity have been observed. This study shows that, in addition to pepstatin A, leupeptin, antipain, and SP-I also inhibit human renin activity in vitro.


Kinetic studies on the chemical modification of lysozyme by N-bromosuccinimide and its protection by substrates and analogs. The chemical modification of tryptophan residues of hen egg-white lysozyme by N-bromosuccinimide (NBS) was studied kinetically by the stopped-flow method, monitoring changes in absorbance and fluorescence. One most rapidly reacting tryptophan residue, probably Trp 62, was clearly distinguished from four other residues in terms of rate of modification. This residue was protected by ethylene glycol chitin, N-acetyl glucosamine (NAG), and tri-NAG, but not by gluconolactone. The dissociation constant Kd of the enzyme-ligand complex was obtained from the protection effects. These results are in good agreement with results previously obtained.


Structure of a carbohydrate moiety of a unit A glycopeptide of calf thyroglobulin. The structure of the carbohydrate moiety of a unit A glycopeptide of calf thyroglobulin was studied by glycosidase digestion, acetolysis, and methylation analysis. Based on the results, the whole structure of a unit A glycopeptide with the largest carbohydrate moiety was determined as Man alpha 1 leads to 2Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn-peptide.


Cytolipin R from rat spleen.

Cytolipin R from rat spleen. Normal rat spleen contained a large amount of cytolipin R, N-acetylgalactosaminyl(beta1 leads to 3)-galactosyl(alpha1 leads to 3)galactosyl(beta1 leads to 4)glucosyl ceramide, together with a lesser amount of cytolipin K, N-acetylgalactosaminyl(beta1 leads to 3)galactosyl(alpha1 leads to 4)galactosyl(beta1 leads to 4)glucosyl ceramide, as in the case of rat kidney. Cytolipin R was confirmed to be not a rat tumor-dependent, but a species-specific glycolipid.


An improved estimation method for amino alkyl ligand immobilized on matrix beads. A direct spectrophotometric suspension method for the estimation of immobilized ligands on a gel matrix was developed. The method uses 0.05% agarose solution to prepare a practically stable suspension of various agarose beads. The quantity of agarose beads in the suspension was estimated turbidometrically from the attenuance at 650 nm. The determination of immobilized ligands on agarose beads was performed colorimetrically at the wavelength of the absorption maximum against a control suspension of a similar amount of underivatized agarose beads. This method is more sensitive and simpler than previously known methods. The method is applicable to the estimation of absorption spectra and the measurement of the time course of reactions on agarose beads.


Amino acid sequence of chromatium vinosum ferredoxin: revisions. Reexamination of the amino acid sequence of chromatium vinosum ferredoxin revealed that the original sequence proposed in 1970 (I) should be revised. Two segments in the sequence, residues 50-52 and residues 53-57, should be mutually displaced and therefore, the correct sequence in this region is concluded to be -Val-Glu-Val-Cys-Pro-Val-Asp-Cys-(residues 50-57). Another correction was the addition of one isoleucine residue between residues 57 and 58 and therefore, the total number of residues should be 82 instead of 81 originally counted. Sequence studies of other portions confirmed the previous results.


The mode of binding of pyridoxal 5'-phosphate in rabbit muscle glycogen phosphorylase b: circular dichroism and absorption studies. Large bands absorbing at 251 and 335 nm were observed in the difference circular dichroism and difference absorption spectra of the holo- versus the apo-enzyme of rabbit muscle phosphorylase b. In addition to the previously known band at 335 nm, the band at 251 nm is here assigned to the bound pyridoxal 5'-phosphate (PLP). These observed difference spectra are simulated by the optical properties of the enol-imine form of the Schiff base rather than those of the substituted aldimine. Pyridoxal and salicylaldehyde also bound to apophosphorylase in the same manner as PLP. Since analogs having no hydroxyl group at the ortho position to the aldehyde group did not bind to the apoenzyme, the hydroxyl group is thought to be important in stabilizing the interaction between apophosphorylase and PLP. The PLP bound at Site I of bovine serum albumin, which is known to be one of the PLP-binding proteins absorbing at 330-340 nm, showed two prominent bands at 253 and 335 nm. The circular dichroism and absorption spectra induced by the binding of PLP fit those of the enol-imine of the Schiff base, as in the case of phosphorylase.


Studies on the structure of rat liver ornithine aminotransferase. Two distinct types of apoenzyme from native ornithine aminotransferase [EC 2.6.1.13], named forms I and II, have been found to be composed of a single type of subunit by means of Sephadex G-100 column chromatography in the presence of sodium dodecylsulfate, DE 52 cellulose column chromatography in the presence of 8 M urea and urea-polyacrylamide gel disc electrophoresis. The amino-terminal residues of both apoenzymes were blocked, but several amino acids in sequence from the carboxylterminals were identical and the following sequence was found in both: Phe-Ser-Leu-IIe-Ala-(Val)-. In the native enzyme, 4 SH groups out of a total 20 SH groups/tetramer could be titrated without loss of enzyme activity, while in the apo-form I, 4 SH groups out of a total 20 SH groups/tetramer could be titrated with 80% loss of activity. In the apo-form II, in contrast, 6 SH groups out of a total 10 SH groups/dimer could be titrated and titration of the first 2 SH groups caused 100% loss of activity.


Purification and properties of hexokinase from the starfish, Asterias amurensis. Hexokinase from pyloric caeca of the starfish, Asterias amurensis, was purified to a specific activity of 148 units/mg protein. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The molecular weight determined by SDS polyacrylamide gel electrophoresis and Ultrogel AcA 34 gel filtration was about 50,000. The enzyme showed a broad pH optimum ranging from 7.4 to 9.5. The Km values for D-glucose, D-fructose, 2-deoxy-D-glucose, D-mannose, D-glucosamine and ATP were 0.045, 4, 0.21, 0.05, 0.35 and 0.3 mM, respectively. N-Acetyl-D-glucosamine, D-xylose and D-galactose were not phosphorylated. The enzyme was strongly inhibited by the reaction products, glucose 6-phosphate and ADP, but not by high levels of D-glucose. The starfish hexokinase thus resembled mammalian isozyme A with respect to kinetic properties.


Troponin binding region of tropomyosin.

Troponin binding region of tropomyosin. Four fragments of alpha-tropomyosin were prepared by specific cleavage at the Cys 190 by 2-nitro-5-thiocyanobenzoic acid and by tryptic digestion. These fragments were called the N-chain corresponding to residue 1 to 189 of the original chain, the C-chain from 190 to 284, the s-fragment from 13 to 149 and/or 128, and the p-fragment from 183 to 284, respectively. Fragments individually have little binding capacity to troponin as shown by gel electrophoresis. But a new band of the complex with troponin was detected using mixtures of the fragments, one from the N-terminal side and the other from the C-terminal side, i.e., the N- and C-chains, the s- and p-fragments, the N-chain and the p-fragment, and the s-fragment and the C-chain. Therefore, the troponin binding region of tropomyosin is thought to be located between residues 150 and 190.


gamma-Aminobutyric acid metabolism in subcellular particles of mouse brain and its relationship to convulsions. The in vivo effects of convulsant drugs (hydrazine and penicillamine) on the metabolism of gamma-aminobutyric acid (GABA) in subcellular fractions of mouse brain were studied. Both substances inhibited the activity of glutamic acid decarboxylase [EC 4.1.1.15] (GAD) in the synaptosomal fraction (nerve ending particles) and reduced the concentration of GABA in the same fraction at the onset of convulsions, though changes in the total GABA concentration in the brain did not correlate with the onset of convulsions. Therefore, it is suggested that the concomitant decrease of GAD activity and GABA concentration in the nerve endings, independently of the total GABA concentration, is probably an important factor in the onset of some kinds of convulsions.


Identification of acylcholine acyl-hydrolase with carboxylic ester-hydrolase in human serum. The relationship between pseudocholine esterase [acylcholine acyl-hydrolase, EC 3.1.1.8] and non-specific esterase [carboxylic ester-hydrolase, EC 3.1.1.1] in human serum was investigated. The purified preparation (purified 500-fold) which had both pseudocholine esterase and non-specific esterase activities, was found to give a single band with faint tailing on polyacrylamide gel electrophoresis. The ratio of the specific activity of pseudocholine esterase to that of non-specific esterase remained essentially the same during the purification procedures. Furthermore, the pseudocholine esterase was demonstrated to be identical with the non-specific esterase by immunochemical studies. All these results suggest that activities of pseudocholine esterase and non-specific esterase in human serum derive from the same enzyme molecule. Observation of Yoshida-cho in Ehime after the application of organophosphorus insecticide supported our results: the activity of pseudocholine esterase was found to be reduced with a concomitant decrease in the activity of non-specific esterase. Based on these results, the physiological significance of the esterase is discussed.


Analysis of the reactivity of microsomal cytochrome P-450 toward drugs and exogenous ligands. Three forms of cytochrome P-450 in rat liver microsomes (Comai, K. and Gaylor, J.L. (1973) J. Biol. Chem. 284, 4947-4955) were characterized as two types of P-450 in the membrane-bound state. One was reactive not only toward cyanide but also aniline, aminopyrine, and hexobarbital, and the other was less reactive toward cyanide and rather unreactive toward the drugs. Cyanide titrations of microsomes in the presence and absence of the drugs were performed spectrophotometrically for analysis of the interactions. The affinity of cyanide for the less reactive P-450 was about twenty times less than that for the reactive P-450. About 40% of P-450 in the microsomal membrane was reactive and the rest was the less reactive form. The reactive P-450 involved two forms having different affinities for cyanide. The ratio of their amounts was 1:2. Triton WR-1339 interfered with cyanide binding to the reactive P-450 mainly. The relative amount of each form of P-450 as well as the dissociation constant of cyanide could be estimated by the present method, and the modes of interaction of the membrane-bound P-450 with the drugs and exogenous ligands were deduced.


Horsetail (Equisetum telmateia) ferredoxins I and II. Amino acid sequences. Two ferredoxins were isolated from horsetail (Equisetum telmateia) and their amino acid sequences were determined by use of a sequence analyzer in combination with carboxypeptidase digestion and manual Edman degradation of tryptic peptides of carboxymethyl-ferredoxins. Ferredoxins I and II each had only four cysteine residues in a total of 95 and 93 residues, respectively. The amino-terminal residues of both ferredoxins were heterogeneous, but alanine was concluded to be their genuine terminal residue. The comparison of these isozymelike molecules showed 29 differences in amino acid residues with three inverted replacements. One gap was inserted in ferredoxin II at position 32 to align the ferredoxins with greatest homology. Despite the many differences in amino acid residues there was no difference in net charges of the two ferredoxins.


Horsetail (Equisetum arvense) ferredoxins I and II Amino acid sequences and gene duplication. Amino acid sequences of two ferredoxins isolated from Equisetum arvense were determined by conventional procedures. Ferredoxins I and II of E. arvense had 95 and 93 residues, respectively, and nearly identical sequences each with only one amino acid difference from ferredoxins. I and II of E. telmateia (1). The overall structural characteristics of these two ferredoxins were therefore very similar to those of E. telmateia ferredoxins. Ferredoxins I and II from E. arvense differ in 31 sites and those from E. telmateia in 29 sites from each other. These facts suggested that duplication of the ferredoxin gene in one organism occurred at an early evolutionary stage long before the divergence of the two horsetail species. The number of differences in amino acids between horsetail ferredoxins and other chloroplast-type ferredoxins indicated that the duplication occurred after divergence of horsetails from other plants. Comparing green plant ferredoxins, it was estimated that this gene duplication occurred about 250 million years ago. Some comments on the unique amino acid substitutions in horsetail ferredoxins are also presented.


Crystal structure of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor, at 2.3 angstrom resolution. The crystal structure of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor which strongly inhibits bacterial alkaline proteinases, was determined at 2.3 angstrom resolution. The subunit (molecular weight, 11,485) of this dimeric molecule has a unique fold of polypeptide chain with a five-fold anti-parallel beta-sheet structure (about 21% of the 113 amino acid residues) and two small segments of alpha-helices (about 16%). The region around the apparent reactive site, Met(73)-Val(74), is held tight by a combination of various structural features. The conformation of this region seems to have close similarity to that found in substrate analogues of low molecular weight bound to subtilisin BPN'.


Soluble proteins from fowl feather keratin. III. Isolation and characteristics of some calamus proteins. The main fraction, GF-3, separated from SCM-proteins of fowl feather calamus by gel filtration, was separated into seven peaks, C-1 to C-7, by ion exchange chromatography on a DEAE-cellulose column. Each fraction was found to contain two components by polyacrylamide gel electrophoresis. In order to separate these two components, recycling gel filtration on a Sephadex G-75 column was carried out. Two peaks separated from each of four fractions, C-3 to C-6, were termed C-3a and -3b, C-4a and -4b, and so on. Electrophoretic results showed that all the fractions were single protein components. Components a and b of a particular fraction had almost identical electric charge and slightly different molecular size. All components in Group a (C-3a to C-6a) had similar amino acid compositions except for SCM-cysteine content, and similar results were obtained for Group b (C-3b to C-7b). Groups a and b had significantly different amino acid compositions.


Interaction between xanthurenic acid-insulin complex and zinc ions. An equimolar mixture of a xanthurenic acid-insulin complex and ZnSO4 was separated into an insulin peak and a peak containing xanthurenic acid (XA) and Zn by Sephadex G-75 column chromatograpy. XA and di-[L-histidino]-zinc (II) readily combined to produce di-[L-histidino]-di-xanthurenato zinc (II) (His2-Zn2+-XA2). By increase in the concentration of Zn2+ ions, XA was removed from di-[L-histidino]-di-xanthurenato zinc (II) (His2-Zn2+-XA2) as XA-Zn2+. The XA-insulin complex showed dcreased relative intensity of fluorescence compared with the Zn-insulin when excited at a wavelength of 284 nm. The difference spectrum between native Zn-insulin and XA-insulin complexes showed a slight red shift. A diffference in the CD spectrum between native Zn-insulin and XA-insulin complexes was observed.


Purification of human fibroblast interferon by zinc chelate affinity chromatography. Zinc chelated by iminodiacetic acid linked to an insoluble matrix binds human fibroblast interferon quite selectively at neutral pH in 0.15 M NaCL. On reduction of the pH and increase of the ionic strength, the interferon is eluted. Using a pH gradient at constant high ionic strength, good purification of the interferon can be obtained, up to a final specific activity of 108.5 units/mg of protein. Approximately 60% of the applied activity can be recovered.


Regulation by glucagon and divalent cations of inhibition of hepatic adenylate cyclase by adenosine. Adenosine inhibits the rat liver adenylate cyclase system at a regulatory site that is distinct from the glucagon receptor, the guanine nucleotide regulatory site, and the active site involved in catalysis of ATP to cyclic AMP. The effects of the nucleoside are also independent of the concentration of uncomplexed ATP (ATP4-) in the assay medium. Glucagon, but not guanine nucleotides, sensitizes the system to inhibition by adenosine. Depending on assay conditions, the hormone can shift the concentration of adenosine required for 50% inhibition by as much as 10-fold. Under optimal conditions, the apparent Ki for adenosine is 25 micron. Both Mg2+ and Mn2+ increase adenylate cyclase activity and, in order of relative potency, increase the sensitivity of the enzyme to adenosine inhibition; Mn2+ is 50- to 100-fold more potent than Mg2+. The adenosine inhibitory site exhibits stringent structural requirements for nucleoside action. Most alterations of the purine ring result in loss of activity, whereas alterations in the ribose ring are tolerated, and some deoxyadenosine analogs are even more effective than adenosine. Naturally occurring nucleosides and nucleotides, such as inosine, guanosine, and 5'-AMP, are inactive. Analog studies reveal also that inhibition of the hepatic system occurs at a site which is clearly different from the sites through which adenosine activates other adenylate cyclase systems, and that the liver enzyme appears to have no site for activation by the nucleoside.


Chromatin subunits contain normal levels of major acetylated histone species. Chromatin subunits or nucleosomes prepared by micrococcal nuclease digestion of nuclei from trout testis have been examined for the presence of in vivo modified histone species. Both monomers and multimers are labeled when testis cells are incubated in the presence of [14C]acetate, but the level of radioactivity in the monomer fraction is 10 to 20% higher than in the multimer fraction. This difference is attributed to the trimming of nucleotides from the monomer DNA, associated with the loss of H1. The specific activities of the [14C]acetate label in histones H2A, H2B, H3, and H4 in monomer particles are similar to their respective values in whole chromatin. Starch gel electrophoresis of histone fractions derived from monomeric nucleosomes revealed the presence of monoacetylated and phosphorylated species of H2A, monoacetylated species of H2B and H3, and mono-, di-, tri-, and tetraacetylated species of H4. No differences in the content of these species between monomeric nucleosomes and whole chromatin could be discerned.


Cleavage of tryptophanyl peptide bonds in cytochrome b5 by cyanogen bromide. Quantitative cleavage of peptide bonds adjacent to tryptophanyl and methionyl residues in the polar moiety of cytochrome b5 was effected using cyanogen bromide in the presence of heptafluorobutyric and formic acids. Application of this method to native cytochrome b5 resulted in cleavage at tryptophanyl and methionyl residues in the polar and membranous segments in high yield. Amino acid analysis of peptides isolated from such digests indicated that tyrosine was modified and the derivative eluted in a position preceding lysine; however, the color constant with ninhydrin remained unchanged. Hydriodic acid hydrolysis of the phenylthiohydantoin derivative of the modified tyrosine regenerated the parent amino acid. Peptides containing the altered tyrosine remained susceptible to chymotryptic cleavage at this residue. Cleavage of methionyl bonds could be prevented by methylene blue-sensitized photooxidation prior to cyanogen bromide/anhydrous heptafluorobutyric acid treatment. The conditions employed for photooxidation were selective for methionyl residues, and the yield and specificity of tryptophanyl cleavage by the cyanogen bromide/anhydrous heptafluorobutyric acid method was unaffected.


Conversion of oxy- into methemerythrin in the presence of anions. The interaction of oxyhemerythrin (HrO2) with cyanate and azide ions (X-) is biphasic. The rapid loss of HrO2 is monitored at 500 nm using stopped flow. The dependence of the pseudo-first order rate constant on [HrO2], [X-], and [O2] can be interpreted by the scheme HrO2 in equilibrium Hr + O2 K1 Hr + X-in equilibrium HrX- k2, k-2, k2 At 25 degrees, pH = 6.3 and I = 0.15 M; for X- = CNO-, k2 = 59 M-1 S-1 and k-2 = 0.019 S-1; and for X- = N3-, k2 = 1.6 X 10(2) M-1 S-1 and k-2 = 0.10 S-1. The values of K1K2 from kinetics are in good agreement with those obtained from spectral and [O2] determinations of equilibrated mixtures of Hr, CNO-, and O2. The second step has been analyzed separately by flow observations of the small spectral changes accompanying addition of X- to deoxyhemerythrin. The slower, stoichiometric formation of methemerythrin cyanate or azide has also been studied and second order rate constants for reaction of HrO2 with X- determined (N3-, 0.34 M-1 S-1 and CNO-, 0.08 M-1 S-1 at 25 degrees and pH = 6.3). These are compared with those for accelerated formation of the methemerythrin ligand adduct in the same conditions in presence of NO2- (0.31 M-1 S-1), HCO2- (8.3 X 10(-3) M-1 S-1), F- (0.12 M-1 S-1), Cl- (1.4 X 10(-4) M-1 S-1), and imidazole (4 X 10(-3) M-1 S-1 at pH 8.0). No rapid pre-equilibria are observed in the case of the latter ligands. The effect of pH is examined.


Affinity labeling of human serum prealbumin with N-bromoacetyl-L-thyroxine. Affinity labeling of human serum prealbumin with N-bromoacetyl-L-thyroxine (BrAcT4) was used to investigate the binding domain for L-thyroxine (T4) on prealbumin. Fluorescence titration with 8-anilinonaphthalene-1-sulfonate revealed a strong and a weak binding site for BrAcT4 (K1 = 1 X 10(8) M-1; K2 = 1 X 10(6) M-1). The reaction of BrAcT4 with prealbumin to form a covalent bond was inhibited in the presence of T4 and binding of T4 to prealbumin was nearly abolished after affinity labeling with BrAcT4. Affinity labeling with 2 mol of BrAcT4/mol of prealbumin resulted in covalent binding of 1 mol of ligand. Acid hydrolysis of affinity-labeled prealbumin gave Nepsilon-carboxymethyllysine and iminodiacetic acid, the latter being derived from the NH2-terminal glycine. A combination of analytical procedures, including tryptic digestion after maleylation, cyanogen bromide cleavage, digestion with yeast protease C, and sequential Edman degradations, revealed that the Nepsilon-carboxymethyllysine was derived from lysine-9 and lysine-15 and that the affinity label had distributed itself among glycine-1, lysine-9, and lysine-15 in a ratio of 29:63:8.


Opposite effect of organic phosphates on hemoglobin oxidation by hydroxylamine under aerobic and anaerobic conditions. The effect of organic phosphates such as inositol hexaphosphate (IHP), ATP, and 2,3-diphosphoglycerate (2,3-DPG) on hemoglobin oxidation by hydroxylamine was studied under aerobic and anaerobic conditions. Under aerobic conditions, the oxidation rate of hemoglobin by hydroxylamine was accelerated as much as about 1.6 times, 1.4 times, and 1.4 times in the presence of IHP, ATP, and 2,3-DPG, respectively; however, under anaerobic conditions it was inhibited as much as 2.2 times, 2 times, and 2 times in the presence of these organic phosphates compared with the case for the absence of these organic phosphates. The effects of these organic phosphates on hemoglobin oxidation by hydroxylamine under aerobic conditions were elucidated by the two state models of hemoglobin including the R and the T state, however, the opposite effects under anaerobic conditions cannot be explained without the assumption of the existence of a third conformation of hemoglobin. The dissociation constant of IHP to deoxyhemoglobin was estimated from the rate of the hemoglobin oxidation by hydroxylamine in different concentrations of IHP under anaerobic conditions.


Purification and characterization of pig kidney pyruvate kinase (type A). Pyruvate kinase type A was purified from pig kidney with a yield of 9%. The final enzyme fraction had a specific activity of 500 units/mg of protein. The enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis in detergent and in ultracentrifugation experiments. The molecular weight of the enzyme was found to be 210,000 with the use of ultracentrifugation and 249,000 at gel chromatography. The sedimentation coefficient (S degrees 20, w) was calculated to be 9.8 S. For the reduced and alkylated pyruvate kinase, a molecular weight of 60,000 was found with the use of several methods. The Stokes radius for the enzyme was calculated to be 56 A. No NH2-terminal amino acid was detected in the enzyme, and the only findings in carbohydrate analyses of the kidney pyruvate kinase were trace amounts of glucose. The isoelectric point of the enzyme was estimated to be pH 5.6. Pig kidney pyruvate kinase type A was not phosphorylated on incubation with ATP and cyclic 3':5'-AMP-dependent protein kinase. The amino acid compositions of pig kidney and pig muscle pyruvate kinases were very similar and differed clearly from that of pig liver pyruvate kinase.


A novel chemical modification of delta 5-3-ketosteroid isomerase occurring during its 3-oxo-4-estren-17 beta-yl acetate-dependent photoinactivation. The chemical change responsible for the 3-oxo-4-estren-17 beta-yl acetate-dependent photoinactivation of delta 5-3-ketosteroid isomerase has been identified by amino acid analysis and amino acid sequencing. Amino acid analysis of the enzyme and its photoinactivated derivative shows that photoinactivation is accompanied by loss of nearly 1 residue of aspartic acid/polypeptide chain and an increase in nearly 1 residue of alanine. Edman degradation of a peptide comprising residues 31 to 48 from native isomerase showed the presence of aspartic acid at residue 38. When the corresponding peptide from photoinactivated enzyme was sequenced, residue 38 was revealed to be alanine.


A nicked form of kinetoplast DNA in Leishmania tarentolae. The mitochondrial DNA of the protozoan Leishmania tarentolae, known as kinetoplast DNA, contains thousands of minicircles linked in a two-dimensional network. When kinetoplast DNA from exponentially growing cells is centrifuged to equilibrium in a CsCl/ethidium bromide gradient, it is resolved into two discrete components, Form I and Form II. Nearly all of the minicircles in Form I networks are covalently closed and all of those in Form II networks are open. These forms are indistinguishable from each other when examined by electron microscopy and they appear identical when analyzed by gel electrophoresis after digestion with the restriction enzymes Hae III or Hpa II. However, Form II networks sediment roughly 50% faster than Form I networks on a neutral sucrose gradient, indicating that Form II networks are larger in size or more compact in conformation, or both. Analysis of denatured Form II DNA by sedimentation or electron microscopy indicates that nearly all of its minicircles have one or more interruptions in both strands. Since the majority of the Form II minicircles can be closed by DNA ligase, most of these interruptions must be nicks. Experiments with S1 nuclease indicate that some small gaps may also exist in Form II minicircles. 5'-Terminal nucleotide analysis of Form II kinetoplast DNA does not suggest that the interruptions are at specific locations in the minicircles. The significance of the two forms of kinetoplast DNA has not yet been determined, but it is possible that Form II is an intermediate in replication of this DNA.


Isolation and characterization of a novel trisialoganglioside, GT1a, from human brain. A novel trisialoganglioside has been isolated from normal adult human brain in a yield of 0.6% of the total gangkioside. By graded neuraminidase treatment, mild acid hydrolysis and periodate oxidation analysis, the ganglioside was identified as GT1a having the following structure: NeuAc(alpha, 2-8)NeuAc(alpha, 2-3)Gal(beta, 1-3)GalNAc(beta, 1-4) [NeuAc(alpha, 2-3)]Gal(beta, 1-4)Glc(1-1)ceramide.


Circular dichroism studies of fd coat protein in membrane vesicles. Circular dichroism studies on filamentous virus coat protein in lipid vesicles show that distinctive spectra result depending on the history of the protein and the method of vesicle preparation.


Isolation, crystallization, and primary amino acid sequence of human platelet factor 4. Human platelet factor 4 was purified by a method employing affinity chromatography on heparin/agarose. The amino acid sequence of the protein was determined by automatic Edman degradations and carboxypeptidase Y digestion. There are 70 amino acids in the protein with 5 of the 8 negatively charged residues clustered near the NH2 terminus and 10 of the 13 positively charged residues in clusters of 3 and 4 elsewhere in the protein. Small crystals have been obtained from ammonium sulfate solutions which give a promising preliminary x-ray diffraction pattern.


Micrococcus luteus correndonucleases. I. resolution and purification of two endonucleases specific for DNA containing pyrimidine dimers. Five peaks of endonuclease activity showing a preference for ultraviolet-damaged DNA have been chromatographically identified from extracts of Micrococcus luteus. They are numerically designated as I to V in order of their elution from phosphocellulose (Whatman P-11) columns. The first two of these peaks have been highly purified by a combination of gel filtration and affinity chromatography and are catalytically homogeneous judging from their effect on transforming DNAs. Peak I, which has an isoelectric point of 4.7, is heat-stable, requires high ionic strength for optimal activity, acts with equal facility on ultraviolet-irradiated native and denatured DNA, and has been designated as Py pyrimidine dimer Py correndonuclease I. Peak II which has a pI value of 8.7, is heat-labile, is inhibited by high ionic strength, acts on ultraviolet-irradiated native but not denatured DNA, and has been designated as Py pyrimidine dimer Py correndonuclease II. Both enzymes are inhibited by Ca2+ and Zn2+, do not show any cofactor or sulfhydryl requirement, act optimally between pH 7.0 and 7.4, and have molecular weights between 11,000 and 15,000. Py pyrimidine dimer Py correndonuclease I requires a dose about 1.6 times that for Py pyrimidine dimers Py correndonuclease II for incision saturation of irradiated phiX174 RFI DNA.


Micrococcus luteus correndonucleases. III. Evidence for involvement in repair in vivo of two endonucleases specific for DNA containing pyrimidine dimers. Involvement of Py pyrimidine dimers Py correnconucleases I and II in repair of ultraviolet radiation damage in vivo by Micrococcus luteus has been demonstrated by their absence in the ultraviolet-sensitive mutant DB-7 derived by treatment of the wild type parent with N-methyl-N'-nitro-N-nitrosoguanidine. The necessity for their combined action in DNA repair in M. luteus is shown by: (a) reactivation of ultraviolet-damaged phiX174 RFI DNA in incision-defective hosts after in vivo treatment with both enzymes, (b) correlation between survival after ultraviolet irradiation and the level of the two enzymes, and (c) increased levels of repair synthesis after ultraviolet irradiation of toluenized cells DB-400 with wild type correndonuclease levels when compared with the transformant DB-200 and the mutant DB-7, which lack one or both enzymes.


Properties of dihydroxyacetone phosphate acyltransferase in the harderian gland. We have used a microsomal preparation from the pink portion of the rabbit harderian gland to study the properties of dihydroxyacetone-P acyltransferase. The enzymatic activity in the microsomes is latent and is stimulated approximately 20-fold by the addition of detergent to the assay system. Both deoxycholate and cholate stimulated the enzyme at concentrations considerably below the critical micellar concentration of these detergents. The acyltransferase was not solubilized by treatment with these detergent concentrations. In addition, we have shown that the acyltransferase is sensitive to proteolytic digestion in the presence of deoxycholate concentrations that render the microsomal vesicles permeable to the protease, but is insensitive to the protease treatment in the absence of detergent. Collectively, these data indicate that the active site of dihydroxyacetone-P acyltransferase is exposed to the lumenal surface of the microsomal vesicles. This enzyme is distinct from the glycerol-P acyltransferase in that the dihydroxyacetone-P acyltransferase is sensitive to protease digestion only  in the presence of deoxycholate, is not inhibited by sulfhydryl reagents, and is not competitively inhibited by sn-glycerol-3-P.


Precursor of egg white lysozyme. Amino acid sequence of an NH2-terminal extension. Lysozyme mRNA was translated in a reticulocyte lysate with mixtures of radioactive amino acids. The in vitro product isolated by immunoprecipitation was shown by gel electrophoresis, peptide mapping, and sequence analysis to be larger than lysozyme synthesized in vivo. An NH2-terminal extension was completely sequenced by automated Edman degradation; the phenylthiohydantoins from each cycle were separated by high pressure liquid chromatography and quantitated by scintillation spectroscopy. The NH2-terminal sequence of pre-lysozyme is: (formula: see text) where lysine is the NH2 terminus of lysozyme. Sixteen of the eighteen residues in this sequence are hydrophobic and in this regard it resembles the partial sequences recently elucidated for other secretory proteins. The NH2-terminal methionine is donated by initiator Met-tRNAfMet; thus, this sequence represents the primary translation product. This 18-amino acid sequence is cleaved from lysozyme in vivo before the lysozyme molecules are completely synthesized.


Purification and mode of action of exoribonuclease from bovine brain. The exoribonuclease in bovine brain has been purified about 900-fold in 10% yield. The molecular weight is about 65,000. The enzyme is free from other ribonucleases of bovine brain. Studies of the mode of action of the enzyme show the following: (a) The enzyme cleaves both oligo- and polyribonucleotides exonucleolytically, initiating nucleolytic attack from the 5'-hydroxyl end to yield 3'-mononucleotides. The enzyme differs from spleen exonuclease in that it does not act on polydeoxyribonucleotides. (b) When the 5'-hydroxyl group is phosphorylated, the enzyme is inactive. (c) The enzymic action is processive in nature; the enzyme hydrolyzes one polynucleotide chain to completion before proceeding to the degradation of another chain.


Purification and properties of superoxide dismutase from a red alga, Porphyridium cruentum. The major superoxide dismutase of the unicellular red alga, Porphyridium cruentum, has been purified to homogeneity. This enzyme has a molecular weight of 40,000 and is composed of two subunits of equal size, which are joined by noncovalent interactions. Manganese constituted 0.13% of this superoxide dismutase. T,is is equivalent to 1 manganese atom/molecule of enzyme. Cyanide at 5 mM and H2O2 at 3 mM had no effect on the activity of this superoxide dismutase but 20 mM azide caused 50% inhibition. The isoelectric point, assessed by isoelectric focusing, is 4.2. The optical spectrum of this enzyme exhibited a maximum at 280 nm (Em = 49,000 M-1 cm-1) and a broad band centered at 450 nm (Em = 170 M-1 cm-1). Exposure to a pH of 3.8 in the presence of 8.0 M urea labilized the manganese and allowed the preparation of a colorless and inactive apoenzyme which could be reconstituted by subsequent treatment with MnCl2. The reconstituted enzyme was found to have regained both manganese and activity. The amino acid composition was also determined. The P. cruentum superoxide dismutase did not cross-react with antibody to the Escherichia coli manganese-containing superoxide dismutase.


A simple generalized equation for the analysis of multiple inhibitions of Michaelis-Menten kinetic systems. The summation of the effects of two or more reversible inhibitors of various types on the initial velocity of enzyme systems obeying Michaelis-Menten kinetics is described by the the general relation: (formula: see text) wherein v1,2,3...n is the velocity of reaction in the simultaneous presence of n inhibitors, vi is the velocity observed in the presence of each individual inhibitor, and v0 is the velocity in the absence of inhibition. The derivation is based on the assumption that each enzyme species can combine with no more than one of the inhibitors (i.e. the inhibitors are mutually exclusive). The above relationship holds irrespective of the number of inhibitors, the type of inhibition (competitive, noncompetitive, or uncompetitive), or the kinetic mechanism (sequential or ping-pong) of the enzyme reaction under consideration. Deviations from this equality define synergism or antagonism of inhibitors depending on whether the value of the left side of the above equation is greater or smaller than the right, respectively. Knowledge of the kinetic constants for substrates and inhibitors is not required. If two or more inhibitors act independently (i.e. are not mutually exclusive), their combined effects are necessarily synergistic. Under certain circumstances, described in the text, mutually nonexclusive inhibitors obey the fractional velocity product relationship: v1,2,3...n/v0 = (v1/v0) x (v2/v0) x (v3/v0)...(vn/v0).


Determination of the amino acid sequence of troponon C from rabbit skeletal muscle. The complete amino acid sequence of rabbit skeletal muscle troponin C (TnC) was determined from studies on overlapping peptides isolated from cyanogen bromide and tryptic digests. TnC was found to be a single polypeptide chain of 159 amino acid residues, including 2 residues of tyrosine and 1 each of cysteine, histidine, and proline. The amino end is acetylated, the calculated net charge at pH 7.0 is -29, and the calculated molecular weight is 17,965. There was no evidence for heterogeneity in the sequence. The previously proposed four apparent Ca2+ binding sites are located at residues 27 to 38, 63 to 74, 103 to 114, and 139 to 150.


Human ornithine transcarbamylase. Purification and characterization of the enzyme from normal liver and the liver of a Reye's syndrome patient. Ornithine transcarbamylase was purified and characterized from normal human liver. The properties of this enzyme were compared to those of ornithine transcarbamylase purified from the liver of a patient with Reye's syndrome. The enzyme isolated from both sources appeared virtually identical for a variety of biochemical characteristics. The native molecular weight of ornithine transcarbamylase is 110,000 as determined by gel filtration. Electrophoresis of the enzyme, dissociated by sodium dodecyl sulfate, indicated that the enzyme exists as a trimer of identical or similar subunits of 36,500 daltons. Ornithine transcarbamylase from normal liver has an isoelectric point of 7.95, and the value for the enzyme from the Reye's syndrome liver was 8.05. No evidence of multiple species was found during the purification or subsequent characterization of the enzyme. The enzyme exhibited normal Michaelia-Menten kinetics, and the apparent Michaelis constants for L-ornithine and carbamyl phosphate are 0.20 mM and 0.09 mM, respectively. Inhibitor studies established the structural requirements for L-ornithine antagonists. L-Norvaline is the best competitive inhibitor of the enzyme with respect to L-ornithine. This study indicated that the reduced level of ornithine transcarbamylase activity commonly observed in Reye's syndrome is not necessarily due to structural or functional alterations of the enzyme.


Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. A method is described for radioiodination to high specific activity of fixed and stained proteins within sodium dodecyl sulfate-polyacrylamide gels, without elution of the proteins from the gel. Following radioiodination, the proteins can be removed from the gel by trypsin treatment and the peptides analyzed. This procedure offers a means to structurally compare the proteins of multicomponent systems when purification of each component to homogeneity is unfeasible. Using this technique, we have compared the tryptic peptides of all the major protein components of Moloney and Rauscher leukemia viruses using only 50 to 100 microgram of total protein from each virus. Additionally, we have analyzed the membrane proteins of Dictyostelium discoideum at various stages in development. The validity of the technique and its value as a tool for comparative studies and identification of precursor-product relationships is discussed.


Localization of the sites along nucleosome DNA which interact with NH2-terminal histone regions. Trypsin digestion of HeLa nucleosomes produces the same series of discrete histone breakdown products observed previously by others during digestion of chromatin; thus, trypsin excises the NH2-terminal ends of the histones from the chromatin core particle. The resulting nucleoprotein complex sediments at 9 S, has an increased molecular ellipticity at 280 nm, and has DNase I-susceptible sites at 10 nucleotide intervals. Nucleosomes containing a 32P label at the 5'-DNA termini were digested sequentially with trypsin and DNase I. Following trypsin digestion, the segments of nucleosome DNA 20 to 35 and 60 to 80 nucleotides from the 5' end became more susceptible to DNase I, suggesting that these segments interact with the trypsin-sensitive regions of the histones.


A novel RNA affecting embryonic gene functions in early chick glastoderm. A low molecular weight RNA species (CEH-RNA Fraction 1a) of about 7 S size was isolated from the 16-day-old chick embryonic heart. The RNA was capable of inducing a specific mode of changes in the early embryonic cells of Stage 4 (definitive primative streak stage) chick blastoderm cultivated in vitro. These changes were similar to those of embryonic heart differentiation. RNAs from other sources isolated under identical conditions and synthetic polynucleotides were not effective. In the absence of inducer CEH-RNA, the embryonic cells remained undifferentiated. The dependence of heart-like differentiation on a specific RNA species thus offers an opportunity to test the gene-regulating role of the RNA in an experimentally analyzable biological system.


DNA polymerase-alpha. Purification and structural characterization of the near homogeneous enzyme from human KB cells. In this report we describe the purification and structural characterization of a near homogeneous preparation of DNA polymerase-alpha that we have obtained from cultured human KB cells. When analyzed by nondenaturing gel electrophoresis, velocity gradient centrifugation, and isoelectric focusing, the enzyme activity demonstrates a constancy of gel mobility, sedimentation coefficient, and isoelectric point during the final three chromatographic steps of the purification. Native gel electrophoresis of the penultimate polymerase fraction at seven concentrations of acrylamide reveals co-migration of the enzyme activity with a single discernible protein band, at constant specific activity, and indicates that the polymerase protein is electrophoretically homogeneous at this stage. The purified enzyme has a sedimentation coefficient of 7.1 to 7.2 S at high or low ionic strength, a molecular weight gel filtration of 149,000, and an isoelectric point of pH 5.0 to 5.2. Sodium dodecyl sulfate gel analyses demonstrate that the polymerase-alpha molecule is a dimer comprised of two dissimilar subunits of 76,000 and 66,000 daltons that are present in equimolar ratio.


Isolation and characterization of an avian hepatic binding protein specific for N-acetylglucosamine-terminated glycoproteins. An hepatic receptor which recognizes and binds specifically to serum glycoproteins bearing terminal, nonreducing N-acetylglucosamine residues has been purified to homogeneity by affinity chromatography from chicken liver. The isolated binding protein has been characterized as a water-soluble glycoprotein in which sialic acid, galactose, mannose, and glucosamine comprise 8% of the total molecule. The binding reaction is a saturable process and is proportional to receptor concentration. Evidence has been adduced to indicate the presence of a single high affinity binding site with a dissociation constant of 1.4 x 10(-9) M. A single subunit has been identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate with an estimated molecular weight of 26,000. The chemical and physical properties of the avian protein have been evaluated with respect to the analogous hepatic protein, of mammalian origin, which exhibits a binding specificity for galactose-terminal serum glycoproteins.


Nuclease B. A possible precursor of nuclease A, an extracellular nuclease of Staphylococcus aureus. During purification of nuclease (redisignated as nuclease A in the present studies) from the culture media of Staphylococcus aureus strain Foggi, three enzymacally active second species (nucleases B1, B2, and B3) were isolated as a mixture by ion exchange chromatography. Examination of the amino acid sequence of these second species indicates that nucleases B1, B2, and B3 apparently contain the same sequence as that of nuclease A with an extra sequence Ser-Gln-Thr-Asp-Asx-Gly-Val-Asx-Arg-Ser-Gly-Ser-Glu-Asp-Pro-Thr-Val-Tyr-Ser linked through a peptide bond to the NH2 terminus of the nuclease A portion. In nuclease B1 the 2 residues indicated by Asx are aspartic acid, in nuclease B2 the first and the second Asx from the NH2 terminus are aspartic acid and asparagine, respectively, and in nuclease B3 both Asx are asparagine. These second species do not contain a significant amount of carbohydrate. The extra amino acid sequence appears to be flexible and does not interfere with the ordered structure and function of the nuclease A portion. The nuclease A portion was recovered, in part, from a mixture of these nuclease B species after digestion with staphylococcal protease in the presence of ligands, deoxythymidine 3',5'-diphosphate, and calcium ion. Thus, these nuclease B species may be closely related to, if not identical with, a precursor of nuclease A. Similar second species of nuclease have been found in strain V8.


Detection of a variant of protein 3, the major transmembrane protein of the human erythrocyte. A variant of the major transmembrane protein of the human erythrocyte has been detected following proteolytic digestion of intact erythrocytes. Pronase digestion of normal erythrocytes gives rise to a 60,000 molecular weight fragment of Protein 3, while digestion of erythrocytes with the variant protein produces two fragments of 60,000 and 63,000 molecular weight when peptides are separated by sodium dodecyl sulfate-acrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (Laemmli, U. K. (1970) Nature 227, 680-685). The two fragments cannot be resolved if electrophoresis is conducted using the continuous phosphate or Tris/acetate buffer systems. This increased molecular weight of the variant fragment does not appear to be due to increased glycosylation, since neither sialic acid residues nor terminal galactose units can be detected. Furthermore, the transmembrane segment of Protein 3 can be detected after proteolytic digestion at both the external and cytoplasmic membrane surfaces. These transmembrane segments of both the normal and the variant peptide have identical molecular weights of 20,000 to 21,000. These results suggest that the increased molecular weight of the variant peptide is due to the incorporation of an additional segment into that region of the molecule which is exposed at the cytoplasmic side of the membrane.


Human lymphoblastoid interferon. Large scale production and partial purification. Human lymphoblastoid interferon was produced on an 800-liter scale (2.6 X 10(9) units) by induction of Namalva cells with Newcastle disease virus, strain B1. The interferon was partially purified by anti-leukocyte interferon affinity chromatography, sulfopropyl Sephadex ion exchange chromatography, isoelectric focusing, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recovery of interferon after gel electrophoresis varied from 11 to 33% based on the original crude material, with about 35,000-fold purification. The gel electrophoresis resolved the antiviral activity into two components with apparent molecular weights of 18,000 and 22,000; treatment with glycosidases resulted in all the activity being associated with the lower molecular weight species. Interferon activity could be completely (85 to 113%) recovered from the gels by elution into a buffer containing sodium dodecyl sulfate. The presence of sodium dodecyl sulfate did not appear to affect the assay of interferon. The protein could also be completely (75 to 106%) eluted from gels stained with coomassie blue, again with no loss in activity.


Purification of rat liver nuclear protein kinase NII. Rat liver nuclear protein kinase activity (NII), which is eluted from DEAE-Sephadex columns, has been purified approximately 1500-fold from solubilized nuclear protein. The method of purification involved chromatography of protein eluted from DEAE-Sephadex successively on phosvitin-Sepharose, mixed histone-Sepharose, and histone H2b-Sepharose followed by gel filtration on Sephadex G-200. Resulting preparations are homogeneous by polyacrylamide gel electrophoresis. The enzyme consists of three polypeptides with molecular weights of 42,000 (alpha), 39,000 (alpha'), and 26,000 (beta) which are present in the ratio 1:1:2 indicating that the enzyme has a minimum tetrameric subunit composition of alphaalpha'beta2. The molecular weight and s20,w of the purified enzyme were 123,000 and 7.0, respectively, as determined by sucrose density gradient centrifugation in 0.4 M NaCl. The enzyme has maximal activity with phosvitin as substrate and is not stimulated by 10(-5) to 10(-4) M cAMP or cGMP using H2b as substrate.


Genetic differences inthe te of histidase synthesis in inbred mice. Inbred strains of mice fall into two groups with respect to their liver histidase activity levels, high strains having approximately twice as much activity as low strains. Analysis of the F1, F2, and backcross progeny of the mating of a high activity strain (C57BL/6J) and a low activity strain (C3H/HeJ) indicates that the difference between the strains is determined by a single genetic locus with two alleles exhibiting additive inheritance. No differences with respect to various physical and kinetic parameters were found in studies of partially purified histidase from both strains. Quantitation of the amount of enzyme present by immunotitration showed that the amount of enzyme antigen is proportional to the level of enzyme activity in the two strains. Measurements of the relative rates of histidase synthesis by combined radiochemical and immunological techniques showed that the relative rate of synthesis was closely correlated with the amount of enzyme present. Rates of enzyme degradation in the two strains, measured by recovery of activity after irreversible inhibition with nitromethane, were the same.


Reversible inhibition of adenine nucleotide translocation by long chain acyl-CoA esters in bovine heart mitochondria and inverted submitochondrial particles. Comparison with atractylate and bongkrekic acid. Isolated beef heart mitochondria incubated with atractylate and oleoyl coenzyme A at concentrations below 5 micrometer produced an immediate and significant inhibition of adenine nucleotide translocation, whereas inhibition by bongkrekic acid, which required preincubation with the mitochondria, was less rapid and a concentration of 50 micrometer was required for maximum effect. In sonicated submitochondrial particles, which are inverted with the inner face of the membrane exposed, the adenine nucleotide translocase was much more sensitive to inhibition by bongkrekic acid but was now insensitive to atractylate. The characteristics of the inhibition of the adenine nucleotide translocase by oleoyl-CoA were similar qualitatively and quantitatively in isolated mitochondria and "inside out" submitochondrial particles. Thus, in contrast to both atractylate and bongkrekic acid which bind to the membrane asymmetrically, long chain acyl-CoA esters have the capacity to bind and inhibit the adenine nucleotide translocase from both sides of the inner mitochondrial membrane.


Interaction of phosphoglycerate kinase with human erythrocyte membranes. The purpose of this study was to investigate the interaction of phosphoglycerate kinase with the human erythrocyte ghost membrane. Ghosts prepared in 0.1 mM EDTA and 17 mM Tris buffer (pH 7.5) have about 230 molecules of phosphoglycerate kinase/ghost. No additional binding is observed after incubating soluble enzyme with these leaky ghosts. This binding is tight but reversible with Kd = 7.1 X 10(-10) M. The enzyme can be eluted significantly from the membrane by incubation with 0.15 M NaCl and it rebinds to the membrane when the depleted ghosts are incubated with rabbit muscle phosphoglycerate kinase. Ligand binding studies show that NADH and NAD have opposite effects on the binding of the enzyme to the membrane; NAD (1.0 MM) favors binding while NADH (0.25 MM) does not. Similarly, ADP (0.2 mM) favors binding while ATP does not. ATP elutes the membrane-bound enzyme with Kd = 0.058 mM. MgSO4 also stimulates dissociation of the membrane-bound phosphoglycerate kinase (Kd = 0.36 mM), an effect which appears to be due to the magnesium ion. ADP (0.2 mM) can counteract the negative effect of MgSO4 (1.0 mM) on binding of phosphoglycerate kinase to the membrane. We have been unable so far to find tight coupling of the (Na+-K+)-ATPase with the membrane-bound phosphoglycerate kinase.


Selective extraction of membrane-bound proteins by phospholipid vesicles. Extraction of membrane proteins from erythrocytes into sonicated phosphatidylcholine vesicles is described. In a process involving phospholipid and neutral lipid exchange, cell membrane proteins associate with the vesicles and can be separated from the cells by centrifugation. The protein transfer appears to be reversible; phospholipid vesicles mediate the delivery of small amounts of previously extracted protein into cell membranes. Prior to extraction, all but one of the proteins are accessible to lactoperoxidase iodination, and lipid analysis indicates that primarily the outer monolayer of the cell is involved in phospholipid exchange. Among the extracted proteins is acetylcholinesterase which is removed much more efficiently by this procedure than by concentrated salt solutions. The most abundant proteins of the erythrocyte membrane are not represented in the vesicle extract.


Mechanism for oxygen exchange in the chloroplast photophosphorylation system. The oxygen exchange that occurs between water and the gamma-PO3 of ATP in light-activated chloroplast lamellae was found to proceed with close to full equilibration of the oxygens before ATP returned to the medium. This is in contrast to the entry of approximately one water oxygen when ATP is synthesized from ADP and P1 in the same system. In the latter case, the limitation is kinetic, however, not steric, as shown by the presence of some molecules containing more than one water-derived oxygen in the gamma-PO3. The different extents of exchange can be explained by a relatively faster rate of dissociation of ATP from the chloroplast coupling factor during synthesis from ADP and P1 relative to its dissociation in the absence of net phosphorylation. To determine the mechanism of gamma-PO3:H2O exchange, its rate was compared with the rate of reversible cleavage of ATP as detected by betagamma bridge to beta nonbridge 18O scrambling in [Pbeta-18O-Pgamma]ATP (Midelfort, C. F., and Rose, I. A. (1976) J. Biol. Chem. 251, 5881-5887). The scrambling reaction, which depends on cleavage of the PbetaO--Pgamma bond, was found to occur in nearly the same fraction of ATP molecules that experienced gamma-PO3:H2O exchange in the same incubation, suggesting that the latter is due to multiple cycles of reversible ATP hydrolysis on the chloroplast coupling factor, i.e. [ATP-H2O in equilibrium ADP-Pi].


Adhesion among neural cells of the chick embryo. I. An immunological assay for molecules involved in cell-cell binding. An immunologically based method for the quantitative assay of molecules involved in cell adhesion is described. Three  observations served as a basis for this assay: (a) cells obtained by trypsinization of retinal tissue aggregated rapidly, provided they had been allowed to recover in culture from the dissociation process; (b) treatment of chick retinal cells with Fab' fragments from rabbit antibodies against these cells prevented their aggregation; and (c) incubation of these antibody fragments with antigens released by retinal cells in culture neutralized their ability to inhibit aggregation. The amount of neutralizing antigen was determined by measuring the rates of cell aggregation in the presence and absence of antibody and antigen using a particle counter. Although adhesion was inhibited by anti-retinal cell antibodies, it was not affected by lectins or anti-carbohydrate antibodies that also were bound to the cell surface. Together, the results suggest that the inhibition involved blockade or inactivation of particular cell surface molecules and that the retinal cell antigens capable of neutralizing the antibodies represented these molecules or their fragments. In the accompanying paper, we describe the use of this assay for the purification from culture supernatants of a cell surface molecule involved in cell to cell adhesion.


Adhesion among neural cells of the chick embryo. II. Purification and characterization of a cell adhesion molecule from neural retina. The aggregation of cells from dissociated neural retinas of chick embryos can be inhibited by antibodies prepared against whole retinal cells. In order to identify the antigens involved, substances released by retinal tissues in culture were tested for their ability to neutralize specifically the inhibition by antibody of cell adhesion. Using this assay, three active polypeptides from the culture supernatant were purified 500-fold by gel filtration and polyacrylamide gel electrophoresis. Rabbit antibodies prepared against these purified supernatant activities inhibited cell adhesion and reacted only with the three polypeptides. Immunoprecipitation by the specific antibodies of 3H-labeled proteins from a detergent extract of embryonic retinal cell membranes yielded a polypeptide having a Mr of 140,000 in sodium dodecyl sulfate. This precipitation was inhibited in the presence of the three culture supernatant polypeptides that had activity, suggesting that they contained antigenic determinants in common with the 140,000 Mr surface component. They therefore represent all or parts of this cell surface molecule that were released into solution during tissue culture. The data are consistent with the hypothesis that the 140,000 Mr polypeptide is intimately involved in initial adhesion among neural cells.


Zinc ion-induced assembly of tubulin.

Zinc ion-induced assembly of tubulin. Zinc ion-induced assembly of tubulin was followed using electron microscopy and turbidimetric measurements. A scheme utilizing repeated cycles of assembly and disassembly was used to prepare tubulin and microtubule-associated proteins (MAPs) (Shelanski, M. L., Gaskin, F., and Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 765-768). Tubulin was further purified by phosphocellulose chromatography to remove the MAPs (Weingarten, M., Lockwood, A. H., Hwo, S-Y, and Kirschner, M. W. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1858-1862). In tubulin preparations containing MAPs and added GTP, Zn2+-induced sheets of 15 to 60 protofilaments oriented in parallel. In the absence of MAPs and/or added GTP, Zn2+ induced the formation of sheets which wrapped quite specifically and serial sections were often consistent with a tubular structure of approximately 220 nm. The assembly of recycled tubulin + GTP and 0 to 1 mM Zn2+ was analyzed by A350 as a function of time at 30 degrees. The greater the concentration of Zn2+, the shorter the lag time, the faster the rate after the lag, and the greater the plateau value of A350. Although turbidimetric measurements can be used to quantitate microtubules, they are not quantitative for Zn2+-induced sheets.


An in vitro and in vivo analysis of anodized tantalum capacitive electrodes: corrosion response, physiology, and histology. Oxidation-reduction reactions which can destroy high current-density metal-stimulating electrodes are avoided when using capacitive electrodes. The results of in vitro and in vivo testing of anodized, high surface area, sintered tantalum electrodes are presented. The corrosion response of the electrodes is excellent; there is no evidence of dissolution of the electrode. A deposit forms on the surface of the electrodes, but has little effect on the voltage response to constant current stimulation. The physiological and histopathological results indicate the capacitive tantalum electrode to be the safest yet tested.


Evaluation of hip arthroprostheses by means of body environment simulators. Laboratory tests carried out by means of body environment simulators have proved to be very important for the evaluation of endoprostheses potentially useful for implantation. The present paper examines the problems connected with the design of such a simulator. The results obtained with an initial, very primitive simulator concerning the outlines of the phenomena of wear, corrosion, and friction and some wear properties of commercially available hip endoprostheses are presented. From these results, the motivation that led to the design of a more advanced simulator is presented. This latter simulator is also described.


Temperature control of a bone cement by addition of a crystalline monomer. The peak temperature reached during the polymerization of a bone cement was reduced by addition of solid N-vinylcarboazole to the powder component. It was also necessary to saturate the liquid component with N-vinylcarbazole to prevent dissolution from the modified powder component on mixing. It is believed that this technique is generally applicable and opens up the possibility of achieving temperature control of bone cements with choice from a range of crystalline monomers.


Degradation rates of oral resorbable implants (polylactates and polyglycolates): rate modification with changes in PLA/PGA copolymer ratios. This study determined the difference in rate of degradation between pure polymers of lactic acid (pla), glycolic acid (PGA), and various ratios of copolymers of these two substances. Fast-cured and slow-cured polyglycolide was compared with copolymers of glycolide/lactide intermixed in ratios of 75:25, 50:50, and 25:75, as well as pure polylactide. A total of 420 rats were implanted with carbon-14 and tritium-labeled polymers in bone and soft tissue. At intervals of 1, 2, 3, 5, 7, 9, and 11 months, groups of five animals with the implants in bone and five with the implants in the abdominal wall were sacrificed. The implant area as well as tissue from the liver, spleen, kidney, lung and some muscle tissue was analyzed for radioactivity along with the urine and feces collected throughout the experiment. Half-lives of the different polymers and copolymers were calculated from the radioactivity present in the implant area for each time interval. Half-life of the polymers and copolymers decreased from 5 months for 100% PGA to 1 week with 50:50 PGA:PLA copolymer and rapidly increased to 6.1 months for 100% PLA. Fast-cured PGA had a half-life in tissue of 0.85 months. No difference in rate of degradation was seen in soft tissue or bone. No significant radioactivity was detected in urine, feces, or tissue samples. From this study, it is concluded that control of degradation rate of the implant could best be attained by varying the composition of PLA and PGA between 75% and 100% PLA along with a corresponding 25% to 0% PGA. This would provide a half-life range of the implant of from 2 weeks to 6 months.


Collagen sponge: theory and practice of medical applications. Theoretical as well as practical-clinical applications of one form of collagen (collagen sponge) as a biodegradable material is reviewed. The role of porosity of the sponge and surface characteristics of the meshwork in relation to cell ingrowth are considered essential features of collagen sponge. Rate of resorption and antigenicity could be controlled by graded crosslinking of collagenous framework. Four basic examples of clinical use of collagen sponge are presented: as wound (burn) dressing material, as a matrix, for bone and cartilage repair, as an intravaginal contraceptive diaphragm, and as surgical tampons.


Immobilization of urea cycle enzymes. II. Characterization of immobilized argininosuccinate synthetase. Argininosuccinate synthetase (EC 6.3.4.5) was immobilized on CNBr-activated Sepharose 4B. Properties of the immobilized enzyme are described and compared with those of the native enzyme. The immobilized enzyme was much more stable than the native enzyme at 37 degrees C. It was further stabilized in the presence of the assay reagents. The optimum pH of the immobilized enzyme shifted towards alkalinity (approximately 0.5 unit). The apparent Michaelis constants measured for the immobilized enzyme were not greatly different from those measured for the native enzyme. Urea formation from citrulline was confirmed in a continuous column reactor by the coimmobilized argininosuccinate synthetase, argininosuccinate lyase (EC 4.3.2.1), and arginase (EC 3.5.3.1).


Blood-compatible materials by fluid perfusion. The feasibility of preventing platelet adhesion to porous foreign surfaces in contact with blood by passing (perfusing) a physiologic fluid through the pores into the blood was tested. Porous-walled tubes of Teflon (Gore-tex) and aluminosilicate ceramic were perfused with lactated Ringer's solution at rates between 3.13 and 0.3 cc/min per cm2 of perfused surfaced and exposed to heparinized and unheparinized flowing human whole blood for periods of 3-6 min. Under these conditions, with 0.159 cm I.D. tubes and a mean blood flow rate of 8.3 cm/sec, the adhesion of all blood cells was prevented, compared with the adhesion of 1 to 8 X 10(6) platelets/cm2 on the nonperfused controls. The adhesion of plasma proteins was also markedly reduced as determined by bromophenol blue staining. The critical perfusion rate to prevent platelet adhesion to Gore-tex (0.5 micron pore size, 60% pore volume) was found to be between 0.04 and 0.3 cc/(min-cm2). The boundary layer produced by a perfused segment of porous tubing prevented platelet adhesion for several centimeters downstream from the perfusing segment under experimental conditions used.


Thromboresistance of graft-type copolymers with hydrophilic-hydrophobic microphase-separated structure. Graft-type copolymers composed of hydrophilic poly(hydroxyethyl methacrylate) (PHEMA) as a backbone and a small amount of hydrophobic poly(methylmethacrylate) (PMMA) as a long branch exhibit excellent thromboresistance. The interrelationship between the thromboresistance and microstructure of the graft-type copolymers as well as the water structure in the hydrated polymers has been investigated. It was concluded that both the structure of hydrophobic domain dispersed in the hydrophilic matrix and the water bound to the polymer may be responsible for the excellent thromboresistance of the graft-type copolymers.


The natural history and management of congenital short tibia with dysplasia or absence of the fibula. Forty-three patients with unilateral congenital short tibia with partial or complete absence of the fibula are reviewed. The factors influencing the degree of leg shortening at maturity are considered. Serial radiographic measurements of leg length in fourteen patients covering an average observation period of 9-3 years support the hypothesis that the relative difference in growth between the two limbs remains remarkably constant. By estimating the percentage difference between the normal and abnormal leg lengths on the first measurable radiograph it is therefore possible to predict the likely shortening at maturity. This method of prediction allows the surgeon to make the decision to proceed to Syme's amputation or to the use of an extension prosthesis at about one year of age when the child starts to walk.


The vascularity and remodelling of subchondrial bone and calcified cartilage in adult human femoral and humeral heads. An age- and stress-related phenomenon. A quantitative study of the vascularity and a qualitative study of the remodelling of the calcified cartilage and subchondral bone end-plate of adult human femoral and humeral heads were performed with respect to age. In the femoral head the number of vessels per unit area was found to fall 20% from adolescence until the seventh decade and in the humeral head 15% until the sixth decade. Thereafter an increase was noted in the femur but none in the humerus. More vessels were present at all ages in the more loaded areas of the articular surfaces: 25% more for the femur and 15% more for the humerus. The degree of active remodelling by endochondral ossification declined 50% from adolescence until the seventh decade in the femoral head, and 30% until the sixth decade in the humeral head, rising thereafter to levels comparable to those found at young ages. More remodeling was noted in the more loaded areas at all ages.


Benign osteoblastoma as a cause of osteomalacia. A report of two cases. Two patients, a Japanese man and woman both aged eighteen, developed symptoms and signs of osteomalacia over a period of five years. Each of them had a benign osteoblastoma, one in the right fourth metacarpal and the other in the uppermost third of the right humerus. Resection of the tumours without any treatment by vitamin D resulted in rapid cure of the osteomalacia. Attempts to prove a phosphaturic humoral substance or vitamin D antagonist in the osteoblastoma of the humerus were unsuccessful, probably due to prompt excretion from the tumour cells.


Fibrous interposition causing valgus deformity after fracture of the upper tibial metaphysis in children. The development of genu valgum in a child after a fracture of the upper metaphysis of the tibia with a medial gap is due to the interposition of a flap of fibrous tissue consisting of pes anserinus and periosteum avulsed from the lower fragment. The ensuing biomechanical disturbance induces bowing of the shaft and asymmetrical growth at both ends of the bone. Four cases of established deformity are reported, together with two cases of fresh fracture successfully treated by surgical clearance of the fibrous tissue from the gap.


The natural history of the patient with an infected total hip replacement. Deep infection, the most serious local complication of total hip replacement, prompted a study of the records of 135 patients (137 hips) thus afflicted in a nationwide survey of Canada. Particular attention has been paid to the natural history of the infection, and the problems of diagnosis are described. Twenty-one patients died after the insertion, or removal, of the prosthesis, and of the survivors of the original 135 patients only eighteen have been able to retain the prosthesis without further problems with the wound. The remaining patients had the prosthesis removed, and most dry wounds. Certain suggestions are made on management. The advice that a second total hip prosthesis should be inserted after a deep infection of the first implant is not supported.


The results of the pseudarthrosis after removal of an infected total hip prosthesis. Twenty-nine patients with thirty pseudarthroses after removal of infected total hip prostheses have been reviewed one to six years later. Six hips still had a discharging sinus. Complete removal of all cement was found to be essential for healing, and various points in the technique of its clearance have been made. In this respect radio-opaque cement was a great advantage, and lateral guttering of the femur was most effective. Compared with the situation before replacement considerable relief of pain was obtained in most patients but there was much less improvement in function.


An assessment of the value of examination of the hip in the newborn. The Norwich Health District (population 400,000) has been studied during a five-year period in an attempt to assess the value of examination of the hip in the newborn. It is confirmed that complicated deliveries produce higher rates of neonatal instability. But, in sixteen out of the seventeen cases with hip dislocations diagnosed late, delivery had been normal. It is estimated that after normal deliveries, hospital doctors detect only 50%, and family doctors only 28%, of cases of instability of the hip. It is concluded: 1) that the problem of congenital dislocation of the hip will not be eliminated by neonatal examination alone, even if skill in clinical examination could be improved and maintained; 2) that repeated examinations should be considered mandatory until the child is walking. In this regard, the value of the sign of limitation of abduction requires study; 3) that the public should be made aware by an appropriate health education programme that the problem of congenital dislocation of the hip has not yet been solved.


Rotational displacement of the lower tibial epiphysis due to trauma. A rare form of injury to the distal tibial growth plate is described. Only two patients with such an injury have been reported previously. The injury, rotational displacement of the distal tibial epiphysis with posterior displacement of the fibula but without fracture, can be reduced easily and appears to have no lasting ill-effects.


The results of early operation in talipes quino-varus. A preliminary report. The results of operation performed within the first six months of life upon seventy-seven resistant club feet are presented. The indications for and the rationale of early operation are discussed. Particular attention has been paid to the relationship between the age at operation and the outcome more than four years later; the results were greatly superior when operation was undertaken early. Two surgical techniques are compared, the postero-medial release proving better than a simple posterior release. The relationship between clinical and radiological assessment is discussed, and also the influence of the results reported upon future practice.


Evaluation of a computerised image analyser for studying alterations in radiographic bone density in the rat. A computerised image analyser has been used experimentally to determine its possible usefulness in quantifying bone density. Rats were prepared for this purpose and ash weight/volume readings were obtained on femora which had been radiographed and studied to estimate radiographic density by means of the image analyser. The findings indicated that rats can be prepared by administering oestradiol or by allowing an additional week's growth, both of which give a statistically significant increase of bone density. The computerised image analyser was able to detect these differences but the correlations, although real and linear, between ash weight/volume and radiographic density were not large. Further refinements in techniques will be needed before the system is applied clinically.


Fracture of the patella treated by total excision. A long-term follow-up. Thirty-one patients have been reviewed four and a half to thirteen years after total excision of the patella for fracture. This operation did not give the uniformly excellent results previously reported by some authors. The type of incision used was unimportant in the long term. Immobilisation in plaster-of-Paris for any period between one and eight weeks after operation had no adverse effect on the long-term results. There was no correlation between the amount of calcification or ectopic bone formation found in the patellar tendon and the degree of function or discomfort in the joint. There was no evidence that osteoarthritis is an inevitable sequel to patellectomy in man. Maximal recovery of knee function may take up to three years after patellectomy. In this series 22% of patients had excellent results, 39% good results and 39% poor results, according to defined criteria. The implications of these findings are discussed.


Macrodactyly in the foot.

Macrodactyly in the foot. Seven patients with macrodactyly in the foot are reported. None showed any stigmata of neurofibromatosis and all were found to have excessive accumulation of fibro-fatty tissue as the most striking pathological feature. It is suggested that this may represent the basic lesion in this condition. The literature is reviewed and attention is drawn to the differences between macrodactyly in the hand and in the foot.


Kienböck's disease in cerebral palsy.

Kienböck's disease in cerebral palsy. Five cases of Kienböck's disease occurring in a group of fifty-three adults with cerebral palsy are described. The increased incidence of the disease is attributed to the flexed posture habitual in the affected wrist and to an effect on the pattern of blood supply to the lunate.


Aetiology of osteochondritis dissecans. Failure to establish a familial background. In a family study in which thirty-four patients with osteochondritis dissecans and eighty-six of their first-degree relatives were examined clinically and radiologically, only one relative was found to have osteochondritis dissecans. No association with other forms of osteochondritis, endocrinological abnormalities or dwarfism was found.


Lead shot appendicitis in northern native people. Acute perforated appendicitis due to large numbers of inspissated lead shot occurred in two inuit who hunted and ate game regularly. Both lived in remote villages distant from a hospital.


Radiologic features of miliary tuberculosis in children and adults. We reviewed the radiologic features of 19 children and 26 adults with miliary tuberculosis. Children had a very high prevalence of associated lymph node enlargement compared to the adults (95% vs. 12%). Pulmonary consolidation was more common in children (42% vs. 12%). Pleural effusions were uncommon in both groups. The miliary pulmonary lesions cleared faster in children. Evidence of previous tuberculosis was seen in 31% of the adults but none of the children. Most of these differences in radiologic features between children and adults probably represent differences in pathogenesis for hematogenous spread in the two groups.


Radiologic studies of larynx after radiotherapy for carcinoma. Antero-posterior linear tomography was used in conjunction with a xeroradiographic lateral view of the neck to study 35 cases of carcinoma of larynx previously treated by irradiation. Although false negatives and false positives did occur, this method is reasonably reliable in the diagnosis or exclusion of recurrent malignancy.


Bony proliferation of terminal toe phalanges in psoriasis: the "ivory" phalanx. Twenty-eight per cent of our patients with psoriatic arthritis involving the feet demonstrated osseous proliferation and increased radiodensity of the terminal phalanges. This sign, the "ivory" phalanx, is a poorly recognized radiographic manifestation of the disease, but is particularly important in the absence of articular abnormality or of resorption of the tufts. It is almost invariably associated with nail diseases of the same digit.


Osteopathia striata revisited.

Osteopathia striata revisited. Two cases of osteopathia striata are presented, including an unusual case of unilateral involvement with a normal bone scan. The association with focal dermal hypoplasia is discussed. Prominent normal bony trabeculations should not be mistaken for osteopathia striata.


[Angiographic findings in chondrosarcomas]. In chondrosarcomas, angiography may be helpful in confirming malignancy, delineating the anatomic extent and detecting recurrence. The angiographic findings in three chondrosarcomas are demonstrated including a vertebral one, abrupt narrowing of tumor vessels, neovascularlity and accumulation of contrast medium during the capillary phase. Changing calibre of arteries and neovascularity are better seen on selective angiograms and after subtraction technique.


Knee joint bursal abnormalities in rheumatoid arthritis. Twenty-five patients with rheumatoid arthritis presented with knee joint bursal abnormalities. Nineteen patients presented with symptoms simulating thrombophlebitis. Sixteen had a rupture of the posterior joint capsule without a communicating semimembranosus-gastrocnemius bursa (S-G), with extension ventrally between the soleus and gastrocnemius muscles or between the skin and muscle. Nine had an enlarged S-G bursa with or without rupture. Two patients had additional cephalad ruptures: one from the posterior compartment and the other one from the suprapatellar bursa.


[Pelvic lipomatosis].

[Pelvic lipomatosis]. A case of pelvic lipomatosis is presented and the literature is reviewed. The radiologic signs enable the radiologist to make an accurate diagnosis. This should avoid unnecessary laparatomy.


Polypoid carcinoid tumor of the stomach. A case report and a review of the literature. A gastric carcinoid presented as a polyp with a stalk.


Pancreaticoduodenal artery aneurysms in gas abscess pancreatitis. Aneurysms of the small pancreatic and peripancreatic arteries have been reported in chronic pancreatitis, pancreatic pseudocysts, atherosclerosis, trauma, and on a congenital basis. This paper presents for the first time an example of aneurysm formation in acute gas-abscess pancreatitis.


A simplified technique for selective and superselective abdominal angiography. A simplified technique for selective abdominal angiography utilizing the Simmons Femoral-Cerebral catheter is described. The technique affords a rapid simple approach to selective and superselective catheterization of the celiac, superior mesenteric and inferior mesenteric arteries.


Serum stimulation of human fibroblasts: effect on non-histones of nuclear ribonucleoprotein and chromatin. In quiescent human fibroblasts stimulated to proliferate by fresh medium plus 15% serum, no changes were seen in the incorporation of 3H tryptophan into the protein of nuclear ribonucleoprotein during the first three hours following re-feeding. This was in contrast to non-histone chromosomal proteins where the incorporation increased by 90% within ten minutes. The density of the formaldehyde fixed nuclear ribonucleoprotein in CsCl was 1.43-1.44 g/ml and this also did not change following stimulation. The electrophoretic profile of the proteins of nuclear ribonucleoprotein on SDS gels exhibited a predominant band corresponding to a molecular weight of 44,000 closely trailed by a band at 47,000 and other bands at higher molecular weight. This pattern was not altered by serum stimulation and the same was true for the more complex electrophoretic profile of the chromatin proteins. Following a 10-minute pulse of 3H-tryptophan at ten minutes after stimulation, there was a selective increase in the labeling of non-histone chromosomal protein of molecular weight 59,000; no change was seen in the labeling of any protein of nuclear ribonucleoprotein.


Adhesive selectivity is exhibited in vitro by cells from adjacent tissues of the embryonic chick retina. Cells from anatomically remote organs of developing embryos do not normally encounter one another. If intercellular adhesive selectivity has a role in determining or maintaining the position of cells in organs, it should be exhibited between cells which are found in close proximity within a single organ. To test this hypothesis, a collecting cell-sheet assay for intercellular adhesive selectivity has been applied to cells from 3 layers of the retina of the embryonic chick. The finding of a modest degree of selectivity of adhesion between cells from the neural retina and choroid supports the hypothesis. The observed selective low adhesiveness for self of the upper surface of pigmented retina epithelium emphasizes the likely morphogenetic significance of the topographical distribution of intercellular adhesiveness.


An ultrastructural study of the relationship between the plasma membrane and the cell wall of the coenocytic alga hydrodictyon africanum. A wall/plasma membrane unit was prepared from Hydrodictyon africanum by microdissection. A replica of the inner surface of the membrane was made and by freeze-fracture of the whole cell, 2 corresponding internal fracture faces were obtained. The large coenocytes of the alga were plasmolysed and the wall separated by cutting it away. Its inner surface was directly viewed in the electron microscope after shadowing with Pt/C. Particles were found on the outer half of the internal fracture face of the membrane which were oriented in the same 2 directions as the microfibrils laid down at the inner surface of the wall. No structures were found at the inner surface of the membrane. Some evidence was obtained for a structural connexion between the innermost layers of the wall and the plasma membrane.


Junctional structures in hydra.

Junctional structures in hydra. The sealing and communicating junctions present in hydra have been examined using conventional staining, lanthanum tracer and freeze-fracturing techniques. The presence of distinct types of gap and septate junctions has been confirmed. Combined lanthanum tracer and freeze-fracture results have provided a more detailed understanding of these junctional structures. A model has been constructed which demonstrates the various aspects of the junction seen at different sectioning angles. The probable lengths of septa within septate junctions and the junctional 'maze' formed by them is discussed because of its bearing on the 'sealing' nature of the junction and also, to some extent, on its permeability to tracers such as lanthanum.


Observations on the number of nuclei within the fibres of some red and white muscles. Nuclei have been enumerated in muscle fibres of different physiological properties within adult rats and rabbits. Almost invariably, and regardless of muscle type, there is a direct relationship between the cross-sectional area (or fibre breadth) of muscle fibres and the number of nuclei within them. The one exception occurred in muscles of older rats where increased nuclear numbers do not always appear to result in broader muscle fibres. The greater complement of nuclei in broader fibres is accompanied by larger amounts of cell substance per nucleus. Confirming early observations in the literature, red fibres of the slow-phasic type have more nuclei than have white, fast-phasic fibres of similar breadth. These conclusions are not vitiated by differences in the number of nuclei within capillaries or in satellite cells, by differences in nuclear length or by variation in the degree to which fibres are contracted. In respect of their complement of nuclei, and the average amount of cell substance formed per nucleus the small red fibres that occur within muscles of predominantly fast-phasic character appear to be fast-rather than slow-phasic in type. When the number of nuclei observed per fibre is plotted against fibre cross-sectional area, the shapes of the resulting distributions suggest that estimates of muscle nuclei may be valuable not only as an index of growth potential, but of the extent to which that potential is expressed. In one muscle, the above distribution was of a form which indicated that some fibres may have formed abnormally large amounts of protein per nucleus. However, this was not adequately confirmed. Various factors have been investigated that are relevant to the accuracy of enumerating nuclei and measuring fibre breadths.


Lanthanum penetration in crayfish nervous system: observations on intact and 'desheathed' preparations. Central neural connectives and peripheral nerves from the crayfish Procambarus clarkii were incubated in 5 mM lanthanum solutions in physiological saline, for periods from 15 min to 2 h. The tracer only rarely reaches the axon surfaces in the perineurium-ensheathed connectives, penetrating the elaborate perineurial layer slowly. In peripheral nerves, on the other hand, where the perineurium is extermely attenuated and interrupted by open extracellular clefts, inward movement of lanthanum to the axon surfaces occurs readily. When the perineurial layer of the neural connectives is removed by 'desheathing', penetration of the tracer to the level of the axolemma occurs rapidly, implicating the perineurium as the major site of restriction of entry of large ions and exogenous molecules. This conclusion is discussed in relation to recent electrophysiological studies on K+ movements. In both peripheral nerves and desheathed connectives, the transcellular tubular lattice system present in crustacean glial cells appears to serve as a route for the entry of tracer to the axon surfaces, and is more direct than the long and complex extracellular pathway formed by the interdigitations of the extensive glial cell processes.


Freeze-fracture replication and surface sublimation of frozen collagen fibrils. Collagen fibrils from various rapidly frozen mammalian tissues were examined by the technique of straightforward freeze-fracture, freeze-fracture followed by sublimation and surface sublimation technique. Fixed and non-fixed specimens cryoprotected either with glycerol or dimethylsulphoxide (DMSO), as well as non-cryoprotected ones, were examined. Fibrils were observed either with a banded pattern or as helically twisted strands, and in some cases as a combination of both arrangements. Minimally processed fibrils presented a clearcut banded appearance, while the helical pattern seemed to be associated with the presence of the cryoprotectant, glycerol and DMSO. It is concluded that the banded appearance is in all probability a normal feature of the collagen fibrils in vivo, and that the helical pattern is an abnormal feature resulting from various pretreatments of tissue.


Quantitative studies of rDNA in amphibians. The proportion of the genome that is complementary to 18 and 28 s has been determined for 12 species of amphibians. The group of species chosen for the experiment includes 5 related species belonging to the same genus (Plethodon) as well as species belonging to distant taxonomic groups whose C-values range from 3 to 62 pg. Hybridization of rRNA (18 S + 28 S) to whole chromosomal DNA to saturation level indicates that the proportion of rDNA decreases with increasing DNA content. The results presented in this paper do not support the hypothesis of Nardelli, Amaldi & Lava-Sanchez that the number of ribosomal cistrons in various amphibians can always be expressed as a power of 2.


Studies on the template activity of 'isolated' Xenopus erythrocyte nuclei. II. The effects of cytoplasmic extracts. The effects of exposing nuclei isolated from Xenopus erythrocytes to cytoplasmic extracts prepared from various sources on their template activity are described. The cytoplasm of rat liver cells and the immature blood cells of Xenopus contain factors which stimulate RNA synthesis in these essentially inactive nuclei, whereas the cytoplasm of many other cells does not yield such factors. The active factors in rat liver cytoplasmic extracts appear to be proteins of molecular weight about 40000 Daltons. The implications of these results for genetic control in eukaryotes are discussed.


Inversion heterozygosity in females of the newt Notophthalmus viridescens and its influence on chiasma distribution. In a wild population of the American newt Notophthalmus viridescens 15 females out of a total of 94 were found to be heterozygous for a paracentric inversion which includes almost the whole of the longer arm of the smallest chromosome (XI). The inversion was recognized in preparations of lampbrush chromosomes because it transfers the sequential loops, which normally lie close to the telomere, to a position neighbouring the centromere. Because of inversion the transcriptional polarity of the sequential loops is reversed vis-à--vis the chromosome as a whole. In normal bivalents XI (both in male and female meiosis) each arm pair generally forms a single chiasma close to the telomeres (proterminal localization). In bivalents XI heterozygous for the inversion no chiasmata are formed between the mutually inverted longer arm pairs, presumably because they fail to synapse, but chiasma frequency in the non-inverted shorter arm pairs is increased, and the normal restraint on chiasma distribution in this arm pair is lifted. An explanation is offered in terms of the availability of recombination nodules, and the time of their association with the synaptonemal complex.