Ultrastructure of ascosporogenesis in Nannizzia gypsea. Ascosporogenesis in Nannizzia gypsea was studied by electron microscopy. Development of ascospores began with the formation of an ascus vesicle composed of two paired unit membranes. Myelin figures consisting of coiled or concentric membranes were regularly connected with the growing ascus vesicle. Both the ascus vesicle and the myelin figures possessed an electron-dense line between paired membranes, and both were stained by the periodic acid-silver methenamine technique. Invagination of the ascus vesicle about the haploid nuclei resulted in eight uninucleate prospores bounded by two concentric membranes. Spore wall material was deposited between the two membranes of the prospores, and deposition was greatest in areas of the wall overlying stacked elements of endoplasmic reticulum. A single myelin figure surrounded by a polysaccharide halo was observed in the spore.


Intradermal catabolism of rabbit IgG and its fragments. Disappearance of rabbit EgG and its Fc fragment from the injected sites in skin and also the relationship to reverse passive cutaneous anaphylaxis (RPCA) were studied using guinea pigs.  Rabbit IgG and its Fc fragment, trace labelled with -125-I, disappeared from the injected sites with half-lives of 12--14 and 6--7 hr, respectively.  The shorter half-life of the Fc fragment explained why its activity to provoke RPCA disappeared more rapidly than that of intact IgG as the sensitization period was prolonged.  The ability to provoke RPCA did not seem to influence the persistence of Fc fragment in the sites, since F(ab')2 as well as Fab fragments had the same half-life as Fc fragment.


Assembly of a rod-shaped virus.  Disk aggregate of cucumber green mottle mosaic virus protein and its function. Although it is established that in the assembly of tobacco mosaic virus (TMV) in vitro, a 20 S disk aggregate of protein is essential for the initiation of the reaction, there is no agreement as to whether subsequent rod elongation proceeds by the addition of protein subunits or disks.  Cucumber green mottle mosaic virus (CGMMV) is a rod-shaped virus closely related to TMV.  It was observed directly by electron microscopy that CGMMV-protein also formed a single- or double-layer disk aggregate under conditions where reconstitution with the protein and TMV-RNA proceeds efficiently.  Whichever forms, a single or double layer, a disk aggregate is required for the initiation of reconstitution, but cannot participate in rod elongation.  These results obtained with CGMMV support our model proposed for the mechanism of assembly of TMV and rod-shaped viruses; i.e., the assembly occurs in two steps; disk aggregates of protein are essential only for initiation, and rod eleongation proceeds by subsequent addition of subunits.


Low molecular weight components (g-chains) of myosin from rabbit skeletal muscle.  Separation, amino acid compositions and contents in myosin. Low molecular weight components (g1, g2, and g3) were isolated from rabbit skeletal muscle myosin and their amino acid compositions were analyzed. One mole tryptophan was found in g1 and in g2, but none in g3. One mole of acetic acid was found per mole of each g-chain and it was concluded that the N-terminal groups of all three g-chains are acetylated. The minimum molecular weight of the g-chains were estimated from their amino acid compositions. It was estimated by SDS-disc electrophoresis that 1 mole of myosin contained 0.90, 1.7, and 0.63 moles of g1, g2, and g3, respectively. Similar values were obtained with psoas muscle myosin, but in heavy meromyosin prepared from skeletal muscle myosin the content of g2 was much lower, and that of g3 was much higher.


Demonstration by affinity chromatography of the cell-free synthesis of ribonuclease-specific immunoglobulin. An immunoglobulin specific for RNase [EC 3.1.4.22] has been synthesized in a cell-free system containing either lymph node microsomes or polysomes or spleen polysomes from rats previously immunized with RNase.  The synthesis of anti-RNase immunoglobulin was demonstrated by affinity chromatography using an RNase-Sepharose column.  Supporting evidence for the cell-free synthesis of immunoglobulin was obtained by separating the synthesized immunoglobulin from other proteins by DEAE-Sephadex A-25 column chromatography and by neutralization of RNase activity with the separated immunoglobulin.  Lymph nodes and spleen had an almost equal capacity to synthesize the immunoglobulin to RNase.  Under our experimental conditions, 5 to 15 per cent of the total protein synthesis was directed toward immunoglobulin synthesis.


A simple chemical method for the determination of dermatan sulfate in the presence of chondroitin 4-sulfate and chondroitin 6-sulfate. A simple chemical method for the determination of individual mucopolysaccharides in mixtures of dermatan sulfate and chondroitin sulfates by means of a single reagent was established, utilizing the difference in reaction rates of these polysaccharides with orcinol.  To each 1 ml of a sample mixture of standard dermatan sulfate and standard chondroitin sulfate (either 4- or 6-sulfate) was added 3 ml of orcinol reagent and the resulting solution was heated in a boiling-water bath.  After 20 and 60 min reaction, absorbances at 660 nm were measured and the concentrations of individual mucopolysaccharides were calculated.  High reproducibility was observed for the determination of dermatan sulfate in the presence of chondroitin sulfates.  In addition, orcinol reaction for 90 min employing D-glucuronolactone as a standard appeared to be of practical value in the estimation of the uronic acid content of these mucopolysaccharides.


Gel electrophoresis of amylose in the presence of sodium dodecyl sulfate. It has been shown that sodium dodecyl sulfate (SDS) is capable of forming stable complexes with amylose and that fractionation of short-chain amyloses can be effected by SDS-gel electrophoresis.  Using a well-defined smylose fraction (molecular weight 4,000), the thermodynamic parameters pertaining to SDS-amylose interaction have been evaluated by means of frontal gel chromatography.  The results are as follows: association constant (K)=5.0 times 10-3-M-minus 1 at 25 degrees (pH 9.4); standard free energy change (delta G degrees)=-5.1 kcal/mole; standard enthalpy change (delta H degrees)=-5.8 kcal/mole; standard entropy change (delta Sdegrees)=-2.3(e.u.) and the maximum number of binding sites for SDS (n)=1.  In the presence of 0.5--1 percent SDS, amylose migrates toward the anode upon gel electrophoresis, giving a compact band.  High resolution of amylose fractions (released by treatment of amylopectin with debranching enzyme) has been attained using pore-size gradient gel electrophoresis.


Novel monofunctional substrates of polynucleotide phosphorylase.  The "single-addition" of 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide. A method was developed for stepwise wynthesis of oligonucleotides of difined wequence using 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphates as substrates for polynucleotide phosphorylase [ED 2.7.7.8].  Polynucleotide phosphorylase from Thermus thermophilus catalyzed the transfer of one 2'(3')-blocked ADP to the 3'-terminus of the primer trinucleoside diphosphate, ApApA.  The product was 2'(3')-substituted triadenylyladenosine.  The blocking group, dihydrocinnamoyl, could be removed completely from the product without destruction of the phosphodiester bond using alpha-chymotrypsin [ED 3.4.21.1] at neutral pH.


Demonstration of thyroxine-stimulated incorporation of amino acid into peptide linkage in mitochondria-free system. The observation that thyroxine stimulated in vitro protein synthesis in the absence of mitochondria (Carter, W.J., Faas, F.H., and Wynn, J (1971) J. Biol. Chem. 246, 4973-4977) has been disputed on the basis that radioactivity incorporated into protein did not represent peptide synthesis but incorporation of labeled contaminants present in the L-(U-14C) valine precursor (Sokoloff, L., and Roberts, P.A. (1972 Fed. Proc. 31, 1525). The question of mitochondrial requirement is important in determining whether thyroxine has a direct action on the polysome or causes the release of stimulatory factors from mitochondria. In this paper, thyroxine stimulation of peptide synthesis in mitochondria-free systems has been confirmed. Peptide synthesis is required for the thyroxine effect since it is dependent on the presence of polysomes and an energy source in the reaction mixture and is abolished by puromycin. The thyroxine effect is not due to incorporation of labeled contaminants since hydrolysis of labeled protein recovered from control and thyroxine-treated reaction mixtures yields the labeled amino acid precursor as the only radioactive product. Thyroxine stimulates polyuridylic acid-directed polyphenylalanine synthesis, providing further evidence that thyroxine is stimulating peptide synthesis rather than incorporation of radioactive contaminants by mechanisms other than peptide synthesis. Although thyroxine stimulates polyphenylalanine synthesis, it does not influence polyuridylic acid hydrolysis measured in the same reaction. Therefore, thyroxine stimulation of peptide synthesis is not due to prevention of hydrolysis of nucleic acid components of the reaction mixture. Thyroxine does not influence the size or specific activity of the free valine pool in the reaction mixture, indicating that observed increases in valine incorporation reflect increased peptide synthesis rather than increased specific activity of the valine precursor.  The fact that thyroxine stimulates peptide synthesis using (14C)aminoacyl-tRNA precursors strengthens this conclusion. Therefore, thyroxine stimulation of protein labeling is dependent on the presence of peptide synthesis and cannot be explained by incorporation of labeled contaminants, prevention of RNA hydrolysis, or change in the specific activity of the amino acid precursor. Thyroxine causes a genuine increase in peptide synthesis by a direct action at the polysomal level.


N-Methylglutamate synthetase. Substrate-flavin hydrogen transfer reactions probed with deazaflavin mononucleotide. N-Methylglutamate synthetase, reconstituted from apo-protein with 5-deazaFMN, catalyzes the reversible formation of N-methylglutamate via the same two-step mechanism previously elucidated for native enzyme (Reactions 1 and 2).(See article). This conclusion is based on the observation that: 1. Enzyme-bound deazaFMN (gamma-max equals 410, 338, epsilon410 equals 10,400 m-minus 1 cm-minus 1) is reduced by L-glutamate, N-methyl-L-glutamate but not D-glutamate. At saturating concentrations of L-glutamate Reaction 1 proceeds at 1% of the rate observed with FMN-reconstituted enzyme. 2. Substrate-reduced deazaFMN enzyme is reoxidized by methylamine or ammonia. 3. A glutaryl enzyme intermediate, isolated by Sephadex G-25 chromatography, contains radioactivity when prepared from [U-14C]glutamate, [alpha-3H]glutamate, or N-[glutaryl U-14C]methylglutamate; however, this intermediate is not labeled from N[methyl 14C]methylglutamate. 4. The amount of radioactivity incorporated into the intermediate is stoichiometric with the amount of deazaFMN reduced during its formation. 5. Intermediate prepared with [U-14C]glutamate yields alpha-[14C]ketoglutarate when denatured with acid and N-[glutaryl-U-14C]methylglutamate when incubated with methylamine. In the absence of methylamine deazaFMN enzyme intermediate slowly decays to yield alpha-hydroxyglutarate. 6. The rate of deazaFMN glutaryl enzyme intermediate formation at a fixed glutamate concentration is equal to the rate of the over-all reaction while the rate of intermediate reaction with methylamine is approximately 50 times greater than the over-all reaction. DeazaFMN enzyme intermediate prepared with [alpha-3H]-glutamate yields [3H]deazaFMNH2 when denatured with acid or phenol and N-[3H]methylglutamate when incubated with methylamine. These results show that the alpha-hydrogen of glutamate is transferred to deazaFMNH2, presumably at the 5 position, during Reaction 1 and that the same hydrogen is utilized for the reformation of the alpha C-H bond during Reaction 2. These results provide the first direct evidence for enzymic hydrogen transfer from substrate to flavin.


High sensitivity automated sequence determination of polypeptides. We report the development of a high sensitivity Edman method for use in the automated protein sequenator. With the use of a radioactive coupling reagent, [35S]phenylisothiocyanate, and minor modifications in the sequenator program, sequence analyses have been performed on nanomole quantities of protein. The radioactive phenylthiohydantoin derivatives produced in the degradation are identified at the 10 to 100 pmol level by two-dimensional thin layer chromatographic procedures with autoradiography and are quantitated by scintillation counting. This high sensitivity approach, which is about 100 times more sensitive than conventional automated Edman procedures, has allowed continuous amino acid assignments for 15 or more cycles on quantities of protein less than 5 nmol. It has been employed in the NH2-terminal sequence analysis of several proteins whose sequences were previously undetermined.


The effect of adenosine triphosphate on the tricarboxylate transporting system of rat liver mitochondria. ATP has two significant effects on the mitochondrial tricarboxylate transporting system. First, it alters the concentration gradients at equilibrium for the substrates of this transporter. ATP (2MM) caused the uptake of 10 nmol of citrate into the mitochondria coincident with the output of a similar amount of L-malate. This redistribution was dependent on ATP transport, the effect being inhibited by atractyloside and mimicked by the nonmetabolizable derivative adenylyl imidodiphosphate. A mechanism to account for these observations is proposed. Secondly, preincubation of mitochondria with ATP resulted in a 2- to 3-fold increase in the K-m of the mitochondrial citrate transporter. This effect of ATP was not produced by ADP and P-i, nor by N, N, N1, N1-tetramethyl-p-phenylenediamine and ascorbate. It was prevented by the addition of rotenone and antimycin A. This effect of ATP was observed in the presence of oligomycin and could not be attributed to a change in the content of the known tricarboxylate carrier inhibitor, palmitoyl-CoA, nor to the ATP concentration. The origin of possible regulatory factor (or factors) is discussed.


Identification of a chloroplast ribosomal protein altered by a chloroplast mutation in Chlamydomonas. Direct evidence is presented that a chloroplast gene mutation in Chlamydomonas reinhardi alters one of the chloroplast ribosomal proteins. Proteins of 30 S subunits of chloroplast ribosomes from mutant strains, carrying maternally inherited antibiotic resistances, were compared with those from the wild type strain by CM-cellulose column chromatography and gel electrophoresis. When 30 S ribosomal proteins from a [3H]arginine-labeled streptomycin-resistant strain and a [14C]arginine-labeled wild type strain, or vice versa, were cochromatographed on a CM-cellulose column, one peak (Peak 17) was absent from the mutant profile. Instead, a pronounced peak was observed to elute at a slightly lower ionic strength than Peak 17 in the region of Peak 16. The molecular weights in both Peak 16 and Peak 17 regions determined by discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis were indistinguishable, approximately 18,000. Thus, a chloroplast gene mutation to streptomycin resistance has altered the chromatographic behavior of a chloroplast ribosomal protein of the 30 S subunit. We interpret the additional protein in the mutant eluting at Peak 16 as most likely the mutationally altered form of the Peak 17 protein.


Size of the polyadenylic acid region of newly synthesized globin messenger ribonucleic acid. The size of the polyadenylic acid region of newly synthesized globin mRNA was determined on mRNA isolated from nucleated erythroid spleen cells of anemic mice. The globin mRNA was purified by a combination of affinity chromatography on oligodeoxythymidylate-cellulose (oligo-(dT)-cellulose) and sucrose density  gradient centrifugation. The purified RNA was shown to be globin mRNA by virtue of its ability to direct the synthesis of mouse alpha- and beta-globin chains in a cell-free system and by the presence of two bands migrating identically with authentic mouse alpha- and beta-globin mRNA when subjected to electrophoresis in polyacrylamide gels in the presence of formamide. When labeled for 1 hour with [3H]adenosine, the newly synthesized radioactive mRNA also migrated as two bands in these gels but they moved slower than the main bands suggesting that they have higher molecular weights. The polyadenylic acid region of the mRNA was isolated from the T1 and pancreatic RNase digestion mixture by acrylamide sodium dodecyl sulfate gel electrophoresis. The polyadenylic acid region was found to contain approximately 150 adenylate residues. As it is known that globin mRNA isolated from reticulocytes contains only 40 to 60 residues, it follows that at least 150 adenylic acid residues are added to the globin mRNA soon after its synthesis and that some of these are removed during the subsequent maturation of the erythroid cell.


Activity of wheat seedling nuclease toward single-stranded nucleic acids. Procedures have been developed for the facile preparation of wheat seedling nuclease in highly purified form. The preparation appears to be homogeneous by many physical criteria; however, analytical gel electrophoresis reveals 12 protein bands, only one of which is catalytically active. It is suggested that most of the inactive species are artifacts formed from active enzyme by processes which increase the negative charge of the protein. The enzyme rapidly catalyzes the hydrolysis of denatured DNA and RNA to acid-soluble products and also of the 3'-phosphomonoester linkage of a variety of 3'-mononucleotides. The enzyme has very little, if any, activity toward native DNA with respect to the production of acid-soluble substances; however, athe succeeding paper demonstrates that native DNA is cleaved at a few specific loci to yield large duplex DNA fragments. The nuclease has been characterized as having endonucleolytic activity towards denatured DNA and primarily exonucleolytic activity towards RNA. The mononucleotides produced under the influence of the enzyme bear 5'-phosphomonoester groups. Various lines of evidence indicate a relatively high preference of the enzyme for the hydrolysis primarily of 3'-phosphoester linkages of adenylic acid units and secondarily of thymidylic or uridylic acid units in DNA and in ribohomopolymers, respectively. Corresponding linkages involving cytidylic acid and especially guanylic acid are relatively resistant. The 3'-nucleotidase activity of the enzyme at pH 5.0 towards the various mononucleotides fits the same pattern; i.e. nucleotides containing adenine are hydrolyzed most rapidly, followed in decreasing order by those containing thymine or uracil, cytosine, and guanine.


The structure of arthropod and mollusc hemocyanins. The hemocyanins from molluscs and from arthropods differ in the size of their polypeptide chains. A variety of physical techniques including sodium dodecyl sulfate polyacrylamide gel electrophoresis and column chromatography in sodium dodecyl sulfate and guanidine HCl indicate that the polypeptide chain of mollusc hemocyanin has a molecular weight of 290,000. These results were corroborated by quantitative end group analyses. Several experiments designed to count the number of tryptophan and methionine-containing peptides in the hemocyanin from the whelk Busycon canaliculatum indicate that sequence homology within the polypeptide chain of the mollusc hemocyanins accounts for their large size. Digestion of the native protein with subtilisin produces a 50,000-dalton fragment in high yield which corresponds to one binding site for oxygen. On the other hand, the polypeptide chain molecular weight of lobster hemocyanin is 76,000 to 78,000 and this seems to be a general property of all arthropod hemocyanins. The pigment from lobster consists of two very similar polypeptide chains which are not present in equal amount. Analysis of the cysteine-containing and of the tryptophan-containing tryptic peptides confirms the value of the molecular weight. However, separation of fragments which contain methionine indicates that there is sequence homology withing the polypeptide chain of this protein. It is concluded that the mollusc and arthropod hemocyanins have little structural similarity.


Metabolism of resorcinylic compounds by bacteria. Purification and properties of orcinol hydroxylase from Pseudomonas putida 01. Orcinol hydroxylase (EC 1.14.13.6), which catalyzes the first reaction of orcinol catabolism in Pseudomonas putida 01, has been purified to homogeneity, and crystallized. Orcinol hydroxylase catalyzes the hydroxylation of orcinol with equimolar consumption of O2 and NADH (or NADPH) to 2, 3, 5-trihydroxytoluene, which is nonenzymically oxidized to a quinone. The visible absorption spectrum of the enzyme shows maxima at 373 and 454 nm and a shoulder at 480 nm. FAD can be dissociated from the protein. Reconstitution of enzymic activity was achieved with FAD, and to a limited extent by FMN. The enzyme has a molecular weight of 63,000 to 68,000 and contains 1 mol of FAD per mol of protein. K-m values for the three substrates orcinol, NADH, and O2 are 0.03, 0.13, and 0.07mM, RESPECTIVELY. The molecular activity of the crystalline enzyme is 1560 min minus 1. In the absence of orcinol, NADH is only slowly oxidized with formation of H2O2. Several analogs of orcinol also serve as substrates for hydroxylation, namely resorcinol, 4-methylresorcinol, and 4-bromoresorcinol. Other analogs, m-cresol, m-ethylphenol, 4-ethylresorcinol, and phloroglucinol, mimic orcinol as effectors, in that they (a) accelerate electron flow from NADH to the flavin and (b) decrease the apparent K-m for NADH but not to the same extent as the substrates that are hydroxylated. The latter compounds are not hydroxylated. Instead H2O2 accumulates as the only product of O2 reduction. The enzyme therefore behaves either as a hydroxylase or an oxidase. The ratio of hydroxylase to oxidase activities of the enzyme is decreased by an increase in the temperature of incubation; at 60 degrees the reaction with orcinol is almost 50% uncoupled from hydroxylation. The apparent K-m values for the effectors are in good agreement with the D-D values obtained for orcinol, resorcinol, and m-cresol. K-D values were obtained by measurement of the effector-induced perturbations of the visible absorption spectrum of the flavoprotein by difference absorption spectroscopy. The circular dichroism spectrum of orcinol hydroxylase is also altered in the presence of orcinol. The participation of the flavin in the over-all reaction is demonstrated by its rapid reduction under anaerobic conditions by NADH in the presence or orcinol, resorcinol, or m-cresol. Subsequent introduction of oxygen restores the oxidized form and yields H2O2 when m-cresol is the effector, but not when orcinol is the effector. Transfer of reducing equivalents from the reduced flavoprotein to free FAD may also occur. Reduction of orcinol hydroxylase by NADH in the absence of an effector is 10-4-fold slower than in the presence of an effector. The minimal structural requirements for effectors appear to be a 1,3-dihydroxy or 1-alkyl-3-hydorxybenzene, but only the former are substrates for hydroxylation.


Mechanism of immunoglobulin A polymerization. Employing mercaptoethylamine as a reducing agent, it was demonstrated by analytical ultracentrifugation and polyacrylamide gel electrophoresis that polymeric immunoglobulin A (IgA) was reduced to a 10 S dimer and 7 S monomer, and that dimer IgA was more resistant to reductive cleavage than the higher polymers. When dimer and monomer IgA were subjected to electrophoresis in polyacrylamide gels in 8 M urea or chromatographed on Bio-Gel P-200 equilibrated in 4 M guanidine HCl, there was no dissociations into H, L, or J chains, suggesting that the interchain disulfide bridges between H--H, L--H, and H--J were intact and that mercaptoethylamine produced selective cleavage of intersubunit bonds. Only the dimer, with a sedimentation coefficient of 10.2 S, released J chain upon reduction with dithiothreitol. Polymers of IgA were reduced with mercaptoethylamine and subsequently alkylated with [14C]-iodoacetamide and the dimer and monomer isolated. The results demonstrated that the isolated dimer contained 2 mol of [14C]labeled S carboxyamidomethylcysteine per mol of dimer, while the monomer contained 1 mol of --SH per mol of monomer. The labeled dimer was then completely reduced with dithiothreitol and alkylated with [14C]iodoacetamide and J chain isolated. It was shown that the J chain contained no 14C-labeled sulfhydryl groups, while the monomer contained 1 mol of --SH per mol of monomer. These results suggest that J chain is disulfide-bonded to only two of the subunits of polymeric IgA and that the remaining subunits in the higher polymers are disulfide-bonded one to the other. This is similar to the model previously suggested for 19 S immunoglobulin M (IgM). The sulfhydryl data also suggests that polymeric IgA may not be a covalently bonded circular structure as has been shown for IgM. However, no conclusions can be made from this study regarding the structure of pentameric IgA, since this species was present in very small amounts in our polymer preparation.


Purification and properties of homoserine transacetylase from Bacillus polymyxa. Homoserine transacetylase (EC 2.3.1.31), the first enzyme of methionine biosynthesis, has been purified to near homogeneity from extracts of a methionine auxotroph of Bacillus polymyxa. The enzyme is subject to rapid irreversible inactivation. Its half-life at 0 degrees is 15 min and much less at higher temperatures, but ethylene glycol affords some protection. In addition, Zn2+ reversibly inhibits the enzyme with a K-I of 3 muM. The enzyme has a molecular weight of about 40,000 and consists of a single polypeptide chain. Besides catalyzing the acetyl transfer from acetyl-CoA to L-homoserine, homoserine transacetylase promotes a homoserine-O-acetylhomoserine exchange reaction in the absence of CoA, suggesting the formation of an acetyl-enzyme intermediate. The results of kinetic studies are consistent with a ping-pong mechanism. Homoserine transacetylase is subject to multivalent feedback inhibition by L-methionine and S-adenosylmethionine. Analysis of the inhibition data and specificity studies suggest that the inhibitors bind to separate sites on the enzyme which are distinct from the active site. Inhibition is competitive with respect to both substrates, and the saturation curves for the inhibitors, as well as substrate saturation curves in the absence or presence of the inhibitors, are hyperbolic. The absence of cooperativity is, in fact, a property which would be expected in a monomeric allosteric enzyme such as homoserine transacetylase.


Regulation of homoserine transacetylase in whole cells of Bacillus polymyxa. The levels of homoserine transacetylase (EC 2.3.1.31) in Bacillus polymyxa grown in minimal medium can vary over a 40-fold range, depending on whether methionine limits growth or is present in excess. This suggests that the synthesis of the enzyme is under control by methionine or one of its metabolites. The stability of homoserine transacetylase in growing cells was measured after repression of further synthesis by the addition of methionine. At 30 degrees, the enzyme was stable for 2 hours, whereas at 37 degrees it decayed with a half-life of 40 min. This contrasts with the striking instability in cell-free extracts described in the preceding paper (Wyman, A., and Paulus, H. (1975) J. Biol. Chem. 250, 3897-3903). The properties of homoserine transacetylase were also studied in cells of B. polymyxa that had been made permeable to small molecules by treatment with toluene. They differed in two important respects from those of the enzyme in cell-free extracts described in the preceding paper: the enzyme was relatively stable, with a half-life of 15 min at 37 degrees, and responded in a sigmoid manner to increasing concentrations of the inhibitors L-methionine and S-adenosylmethionine. These observations suggest that homoserine transacetylase is an oligomeric protein within the bacterial cell but dissociates into monomers in cell-free extracts. When B. polymyxa was transferred at 39 degrees from a rich medium to one without amino acids, growth resumed only very slowly. The growth lag after shift-down was not observed at 37 degrees or in the presence of methionine or cystathionine. This phenomenon appears to be due to a need for derepression of homoserine transacetylase upon shift-down which is thwarted at 39 degrees by the rapid thermal inactivation of the enzyme. A possible physiological function of the striking thermolability of the first enzyme in methionine biosynthesis is discussed.


Poly(8-bromoadenylic acid): synthesis and characterization of an all-syn polynucleotide. Poly(8-bromoadenylic acid) (poly(8-BrA)) has been synthesized by polymerization of 8-BrADP with polynucleotide phosphorylase in the presence of oligonucleotide primers. In the absence of oligonucleotides, significant (i.e. more than 1%) polymerization does not occur. Oligo(I) primer was removed selectively from the polymer with ribonuclease T1 to yield the homopolymer, poly(8-BrA). End group analysis, based on quantitative infrared measurement of the (Ip)3I-primed polymer, indicates an average degree of polymerization of about 70 residues. The primed polymers and the homopolymer appear to have similar helical structures, probably double-stranded with mutual hydrogen bonding interaction of BrA residues. Preliminary NMR observations of poly(8-BrA) with a tetrainosinic acid primer at the 5' ends of the polymer chain ((Ip)3I-(8-BrA)n) are consistent with the existence of a rigid helical structure below the melting range of the primed polymer. Above the melting range (81 degrees) the H1' coupling constants of (Ip)3I-(8-BrA)n and of polyadenylic acid (poly(A)) suggest a significantly higher population of C3' endo conformation of ribose residues in the primed polymer than in poly(A) at 81 degrees.


Metabolism of glucosyl [13H]ceramide by human skin fibroblasts from normal and glucosylceramidotic subjects. Metabolic utilization of glucosyl [3H]ceramide (glucocerebroside) by human skin fibroblasts from normal and glucosylceramidotic subjects was examined in cell culture. Exogenous glucosyl [3H]ceramide in the culture medium did not influence activity of "acid" beta glucosidase in either cell type. Expectedly in the lipidotic cells, the enzymatic activity was markedly (similar to 20-fold) lower. Normal cells were found preferentially to utilize exogenous tritium-labeled glucosylceramide for a source of precursors for phopholipid biosynthesis. The fatty acid and the sphingosine components of sphingomyelin, and the fatty acid components of phosphatidylcholine and phosphatidylethanolamine, but not phosphatidylserine and phosphatidylinositol, were tritium-labeled. In contrast, glucosylceramidotic cells utilized labeled glucosylceramide far more for synthesis of lactosylceramide (lactocerebroside) and also higher neutral glycosphingolipid homologues. These experimental findings suggest that glucosyl [3H]ceramide is hydrolyzed in normal skin fibroblasts to [3H]ceramide and further to [3H]dihydrosphingosine and 3H-labeled fatty acids. These compounds are subsequently incorporated into cellular phospholipids. Flux of glucosyl [3H]ceramide through this catabolic sequence and reincorporation of its breakdown products into phospholipids predominates in normal skin fibroblasts. In contrast, it is greatly reduced in glucosylceramidotic skin fibroblasts. Consequently, a greater amount of glucosyl [3H]ceramide remains intact for the synthesis of more highly glycosylated glycosphingolipids in the latter cells.


Cholesterol ester formation in cultured human fibroblasts. Stimulation by oxygenated sterols. Incubation of monolayers of cultured human fibroblasts with oxygenated sterols (25-hydroxycholesterol, 7-ketocholesterol, or 6-ketocholestanol) markedly enhanced the rate at which the cells esterified their endogenous cholesterol and produced an increase in the cellular content of cholesterol esters. The enhanced esterification capacity was associated with an increase in the activity of a membrane-bound fatty acyl-CoA:cholesteryl acyltransferase. Incubation of cells for 5 hours with 5 mug/ml of 25-hydroxycholesterol produced an 8-fold increase in the specific activity of this enzyme when assayed in cell-free extracts. Since the oxygenated sterols that elevated the activity of fatty acyl-CoA:cholesteryl acyl-transferase also suppressed the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the data suggest that the processes of cholesterol ester formation and cholesterol synthesis in human fibroblasts are regulated in a reciprocal manner by coordinate changes in the activities of these two membrane-bound enzymes.


The functional significance of vitamin K action. Difference in phospholipid binding between normal and abnormal prothrombin. In comparison to normal prothrombin, the abnormal prothrombin produced in response to vitamin K antagonists has been found to bind much less tightly, if at all, to phospholipid surfaces. As a consequence, the activation of abnormal prothrombin by Factor Xa and Ca2+ is not accelerated by the addition of phospholipid to the mixture while the activation of normal prothrombin under these conditions is greatly accelerated by phospholipid addition. In the absence of phospholipid, however, the rate of activation of abnormal prothrombin by Factor Xa and Ca2+ in both the presence and absence of Factor Va is indistinguishable from that of normal prothrombin. The distribution of the partial proteolysis products during activation by Factors Xa, Va, and Ca2+ also appears to be the same for both prothrombins. These observations provide an explanation for the function in prothrombin activation of the gamma-carboxyglutamate residues formed in the vitamin K-dependent carboxylation of prothrombin.


The kinetic characteristics of inhibition of hepatic cholesterogenesis by lipoproteins of intestinal origin. In these studies intestinal lipoproteins were injected intravenously into recipient rats in order to study the kinetic characteristics of cholesterol uptake by the liver cell and inhibition of the cholesterol synthetic pathway. Net cholesterol uptake from circulating intestinal lipoproteins took place only in the liver, and only this tissue manifested inhibition of cholesterol syntheses. Cholesterol uptake by the liver, quantified by a rise in the cholesterol ester content, was a linear function of time and of the amount of lipoprotein cholesterol administered to the animals. Using groups of rats that were either fed cholesterol or injected intravenously with intestinal lipoproteins as a bolus or as a continuous infusion, there was generally a correlation between inhibition of the rate of cholesterol synthesis and the cholesterol ester content of the liver. However, there was no consistent quantitative relationship between these two variables suggesting either that cholesterol ester was not the immediate effector of the inhibition or, alternatively, that there was intracellular localization of the effector at the site of control of the rate-limiting enzyme in the cholesterogenic pathway.


Tryptophan hydroxylase. Purification and some properties of the enzyme from rabbit hindbrain. Tryptophan hydroxylase from rabbit hindbrain has been extensively purified. It is estimated that the enzyme is between 85 and 90% pure and has a molecular weight of 230, 000. Sodium dodecyl sulfate gel electrophoresis shows that the enzyme is composed of two subunits very close in molecular weight (57, 500 and 60, 900). The substrate specificity and the reaction stoichiometry catalyzed by the enzyme in the presence of 6, 7-dimethyltetrahydropterin, 6-methyltetrahydropterin, and tetrahydrobiopterin have been determined. The effect of some natural occurring phospholipids on the purified enzyme was investigated.


Biologic and physical properties of succinylated and glycosylated Acinetobacter glutaminase-asparaginase. Acinetobacter glutaminase-asparaginase was chemically modified by succinylation and glycosylation with glycopeptides from human fibrin and gamma-globulin. These modifications markedly prolonged the half-lives of the enzyme in mice, rats, and rabbits. The plasma half-life in mice increased with decreasing isoelectric point. Glycosylation caused greater prolongation in rodents than succinylation. The kinetic properties of the modified enzymes were unchanged. Succinylation protected the enzyme from trypsin digestion. Glycosylated preparations had less heat inactivation than native and succinylated enzyme. Sedimentation equilibrium studies on a succinylated preparation showed reversible dissociation to a dimer (71, 400 g/mol) with an association constant of 1.3 times 10-6 liters/mol. This dissociation was identical with native enzyme, except for a 3% increase in molecular weight due to succinate groups. Sedimentation equilibrium studies on glycosylated preparations showed mixtures of molecular weight from 60, 000 to over 180, 000. Gel filtration and active enzyme sedimentation showed active polymers, but no active species smaller than tetramer.


Cobalt stimulation of heme degradation in the liver. Dissociation of microsomal oxidation of heme from cytochrome P-450. The administration of cobalt to rats caused a marked increase in the oxidative degradation of heme (hematin, iron protoporphyrin-IX) BY HEPATIC MICROSOMAL ENZYMES. The onset of this enzyme stimulation was very rapid, beginning within 2 hours after injection of the metal and reaching its maximum in 16 to 24 hours. During the rapid phase of stimulation, i.e. the first 2 to 4 hours, when heme oxidation was 450% above control values, there was a significant decrease in microsomal oxidative N-demethylation activity and in microsomal oxidative Ndemethylation activity and in microsomal content of heme with an insignificant decrease in cytochrome P-450 content. Within 24 hours the oxidative activity of the microsomal electron transport chain for drugs was decreased to about 30% of the control. However, during the same period the oxidation of heme approached levels 800% above control. During this period there was a further decrease in the microsomal content of heme with a significant decrease in cytochrome P-450 content and an increase in the activity of delta-aminolevulinate synthetase. The activity of delta-aminolevulinate synthetase reached its maximum within 8 hours after cobalt treatment. Repeated injections (at 24-hour intervals) of cobalt were necessary to maintain these changes in microsomal enzyme activities since, after single injections of the metal, these parameters returned to normal within 72 hours. The inducing effect of cobalt on the oxidation of heme could be inhibited by the administration of actinomycin D and puromycin. Furthermore, this stimulatory effect could not be elicited by in vitro treatment of microsomes with cobalt nor could the effect be attributed to any soluble components of the cytoplasm. Cobalt protoporphyrin-IX was less effective than cobalt chloride in stimulating heme oxidation. 3-Amino-1, 2, 4-triazole did not enhance hepatic heme oxidation activity, while allylisopropylacetamide decreased this activity. The oxidative degradation of heme was found not to be cytochrome P-450 dependent since the highly increased levels of heme oxidation in microsomes from cobalt-treated animals could be retained despite the fact that the cytochrome P-450 content of such microsomes was decreased to spectrally undetectable amounts and drug oxidation was eliminated by treatment of the microsomes with 4 M urea. These findings exclude an obligatory role for cytochrome P-450 in the oxidation of heme compounds, although the possibility that this process is a heme-dependent oxidation is not ruled out.


The hepatic adenylate cyclase system. I. Evidence for transition states and structural requirements for guanine nucloetide activiation. Previous studies have shown that guanine nucleotides, acting at a site termed nucleotide regulatory site, are required for activation of hepatic adenylate cyclase and that glucagon facilitates this process. This study shows that only guanine nucleotides containing triphosphate groups at the 5' position of ribose (or 3'-deoxyribose) are capable of activating the enzyme. The terminal phosphate is not utilized in the activation process since 5'-guanylylimidodiphosphate (Gpp(NH)p and 5'-guanylyl methylenediphosphonate, analogues of GTP that are not utilized in transferase or hydrolase reactions, stimulate enzyme activity. The nucleotides bind in their free form at the regulatory site; chelation by magnesium ion shifts the apparent concentration dependence for activation by Gpp(nh)p. GDP inhibits competitively Gpp(NH)p-stimulated activity and inhibits basal activity and activities stimulated by glucagon. Activation of the enzyme by Gpp(NH)p is a slow process; the length of the lag time increases as an inverse function of nucleotide concentration and is as long as 4 min before onset of increased enzyme activity. Following pretreatment with Gpp(NH)p and extensive washing of hepatic membranes, the enzyme displays immediate increases in activity with rates that are a function of the nucleotide concentration during pretreatment; the rates remain constant for at least 6 min despite the absence of Gpp(NH)p in the medium. Studies with labeled Gpp(NH)p show that the intact nucleotide remains firmly bound to the membranes after extensive washing, suggesting that the persistence of adenylate cyclase activity may be related to slow dissociation of the nucleotide from the regulatory site. Addition of 1 nM glucagon, a submaximal concentration, does not abolish the lag phase of Gpp(NH)p activation even at saturating concentration of the nucleotide (1 muM or higher). The maximal steady state rate is achieved under these conditions. Addition of 2 muM glucagon, a saturating hormone concentration, does not alter the steady state rate but abolishes the lag phase of Gpp(NH)p activation. The transient kinetics of Gpp(NH)p activation and the effects of glucagon thereon are discussed in terms of a three state model in which the guanine nucleotide induces the formation of an intermediate transition state that displays no increase in enzyme activity over the basal state and which slowly isomerizes to a high activity state of the adenylate cyclase system; glucagon acts by accelerating the rate of isomerization.


The hepatic adenylate cyclase system. III. A mathematical model for the steady state kinetics of catalysis and nucleotide regulation. This paper presents a steady state kinetic model for hepatic adenylate cyclase. The activity of the enzyme has been assayed in the presence of a range of concentrations of magnesium, adenylylimidodiphosphate (App(NH)p), 5'-guanylylimidodiphosphate (Gpp(NH)p), and in the presence and absence of saturating concentrations of glucagon. The data were tested against proposed models using an iterative least squares curve fitting program (SAAM25) and confidence estimates for the model parameters were obtained. Hepatic adenylate cyclase is viewed as an enzyme having three characteristic states of catalytic function (E, E', E''). Each state has its own intrinsic activity in carrying out the catalysis of MgApp(NH)p-3 minus to form cyclic adenosine 3':5'-monophosphate. It is shown, in agreement with a proposal by de Haën, that unchelated substrate can inhibit adenylate cyclase activity. It is further concluded that this inhibition is principally due to App(NH)pH-3 minus. The three catalytic states differ markedly in their susceptibility to inhibition as well as in their Vmax, but the Km for MgApp(NH)p-2 minus is essentially the same for all states. The state transitions induced by Gpp(NH)p and by hormone are considered. Gpp(NH)p binding to state E causes transformation to state E'. State E' undergoes spontaneous transformation to state E''. Glucagon augments the transition from E' to E''. We conclude that the activating species of Gpp(NH)p is an unchelated form, most probably Gpp(NH)p-4 minus. Our results indicate that state E' is significantly more susceptible to inhibition by App(NH)pH-3 minus than the other two states. Certain phenomena occurring in fat cell adenylate cyclase are discussed in light of our findings in hepatic adenylate cyclase.


The partial amino acid sequence of trypsin inhibitor II from garden bean, Phaseolus vulgaris, with location of the trypsin and elastase-reactive sites. The amino acid sequences of garden bean (Phaseolus vulgaris) trypsin inhibitor II and a related molecular species II' have been examined. The entire sequence of II' has been determined with the exception of five internal residues. The garden bean inhibitors are highly homologous to the Bowman-Birk soybean inhibitor and lima bean trypsin inhibitor IV. The trypsin-reactive site has been located in the second half of the molecule, while the first reactive site has been found to be directed against elastase. Garden bean inhibitor II (and II') is thus a double-headed inhibitor, simultaneously inhibiting 1 molecule of trypsin and 1 of elastase.


Characterization of the binding of 125-I-labeled epidermal growth factor to human fibroblasts. Epidermal growth factor (EGF) was labeled with 125-I by a lactoperoxidase technique. The unlabeled, monoiodinated and diiodinated species were separated by DEAE-cellulose chromatography and found to possess equivalent biological activities. The binding of monoiodinated epidermal growth factor to human fibroblasts was specific in that unrelated polypeptides did not affect the binding reaction. The binding reaction was a saturable process and was time- and temperature-dependent. A Scatchard analysis of the binding data indicated that each cell was capable of binding approximately 100, 000 molecules of 125-I-EGF. The apparent dissociation constant for the binding reaction was calculated to be 2.7 to 4.3 times 10-minus 10 M. Subsequent to the binding of 125-I-EGF to the fibroblasts, the growth factor was degraded by a cell-mediated proteolysis and [125-I]monoiodotyrosine appeared in the medium. The extent of degradation was reduced by the protease inhibitors, tosyl-L-lysine chloromethyl ketone and the benzyl ester of guanidobenzoic acid. Active binding sites of 125-I-egf appeared to be present in some but not all cell types. These results demonstrated that cells derived from a number of species (human, mouse, rat, and chick) possessed receptors that interacted with this mouse-derived growth factor.


Purification of yeast alpha-isopropylmalate isomerase. High ionic strength hydrophobic chromatography. alpha-Isopropylmalate isomerase, the second enzyme specific for leucine biosynthesis, can be purified from extracts of yeast utilizing a chromatographic procedure that allows separation of proteins in the presence of high concentrations of (NH4)2SO4. The purification procedure utilizes the stabilizing effect of glycerol and (NH4)2SO4 on the isomerase and their opposing effects on protein retention on valine-Sepharose and leucine-Sepharose. The method effectively separates the isomerase from fumarase, a stable internal marker protein that was co-purified in early steps. High ionic strength hydrophobic chromatography, based on differential retention as a function of the length of the hydrophobic sidearm and ionic strength, yields approximately 200-fold purified alpha-isopropylmalate isomerase and may be of general utility in purifying unstable enzymes requiring high ionic strength.


Proteolytic digestion of the micellar complex of f1 coat protein and deoxycholate. The major coat protein of bacteriophage f1 radioactively labeled with specific amino acids was solubilized with deoxycholate and digested with trypsin or alpha-chymotrypsin. The degree of proteolysis of the coat protein was assayed by gel filtration chromatography of the digest in the presence of deoxycholate. Hydrolysis occurred at residues in the hydrophilic termini of the coat, releasing peptides containing proline, lysine, and phenylalanine. No cleavage occurred at the tyrosine or methionine residues in the hydrophobic core. However, chymotrypsin could cleave somewhat at these residues in the absence of deoxycholate. A model for the topography of the micellar complex of coat protein and deoxycholate is presented in which the hydrophobic sequence of the coat is bound to deoxycholate within a micelle, while the hydrophilic termini of the coat project from the micelle.


UDPGlucose 4-epimerase from Saccharomyces fragilis. Allosteric kinetics with UDP-glucose as substrate. UDPglucose 4-epimerase from Saccharomyces fragilis catalyzes a freely reversible reaction between UDP-galactose and UDP-glucose. With UDP-galactose as the substrate the enzyme shows a classical hyperbolic kinetics but when UDP-glucose is used as the substrate a distinct allostericity is observed. As a consequence, at low concentrations of UDP-glucose, the enzyme fails to establish the equilibrium at a significant rate. Glucose 6-phosphate acts as a strong activator for the enzyme with low concentrations of UDP-glucose as the substrate. In view of these rather unusual kinetic data for an enzyme catalyzing a freely reversible reaction, UDPglucose 4-epimerase may play a regulatory role in controlling the flux of galactose metabolism.


Early systemic hypertension after surgical closure of atrial septal defect. The systemic haemodynamic changes occurring in the immediate post-operative period were examined in a series of patients undergoing closure of atrial septal defect (A.S.D.) and in a consecutive series of all patients undergoing open-heart surgery in this Unit. Significant systemic hypertension was observed in 47.5% of patients in the A.S.D. series. In an attempt to explain this finding, several other haemodynamic variables were examined in relation to the systemic blood pressure. There was no correlation between central venous pressure or blood loss and systemic blood pressure. However, there was an inverse correlation between blood balance and systolic and diastolic blood pressure at 4 hours after operation. The explantation of this finding is unclear but it may be due to the effects on atrial receptors of lower right atrial and pulmonary artery pressures following closure of the A.S.D. Although the post-operative systemic hypertension was transient in most cases, it may produce adverse effects in the early post-operative period and therefore treatment with hypotensive agents should be considered in severe cases.


Considerations on occlusive diseases of the leg arteries and determination of muscle blood flow by Xe-133 clearance method. Muscle blood flow in the anterior tibial and gastrocnemius muscles of 40 limbs with thromboangitis obliterans with occlusions only in the leg arteries was measured by Xe-133 non-ischemic work method. All cases with complete occlusion at the origin of the anterior tibial artery had remarkably abnormal curves in the anterior tibial muscle. All cases, except one, with occlusion of the posterior tibial artery at its origin or at the origin of the peroneal artery had moderate abnormal clearance curves in the gastrocnemius muscle. Even if the peroneal artery was completely occluded the clearance curve in the gastrocnemius muscle was normal. In producing intermittent claudication in the calf without occlusion proximally to the popliteal artery, the sural arteries, the posterior tibial artery and the muscle nutritive arteries are very important.


A clinico-radiological survey of deep venous thrombosis of the leg. In a clinico-radiological survey of 150 cases of varicose veins of the leg, 44 had primary varicosities with no dermal complications; their venograms showed patency of the deep veins of the leg. In 106 cases presenting dermal complications, venographic evidence of deep venous occlusion was present. The dermal complications of varicosities are, therefore, always secondary to deep venous occlusion. Deep venous occlusion of the leg was confined to the lower 1/3 of the leg in 58% of the cases occurring as a complication of silent varices. In 15% it involved the upper 1/3. This lesion complicated trauma and surgical operations. In 5% thrombosis occurred in the muscular sinusoids as a complication of septicaemia. In 22%, deep venous occlusion in lower 1/3 of the leg extended to the middle and upper thirds with a corresponding ascending aggravation of the clinical picture. The underlying factors in the pathogenesis of dermal complications are discussed as well as the role of the lymphatics. The reported classification of deep venous occlusion of the leg may prove of value if a venographic study is not feasible. A review of the various types of treatment is presented in the light of these informations.


Mitral insufficiency in an incomplete form of Marfan syndrome corrected with valve replacement. A 14-year-old boy with an incomplete form of the Marfan syndrome developed an acute episode of severe congestive heart failure following exertion. The preoperative studies indicated a mitral insufficiency, and a floppy, thickened mitral valve was removed and replaced with a Starr-Edwards prosthesis. Microscopically, the valve showed increase in collagen with central myxomatous transformation. Seven months postoperatively the patient is doing well and has no symptoms of heart failure.


Effect of athrombogenic therapy, especially high dose therapy of dipyridamole, after prosthetic valve replacement. In order to prevent thrombo-embolism after prosthetic valve replacement, a high dose therapy with 450 mg/day of Dipyridamole and 3,000 mg/day of Aspirin was carried out for 1 year and 2 months in 91 cases (26 cases with aortic valve replacement, 40 cases with mitral valve replacement and 25 cases with multiple valve replacement). In the treated group, the incidence was 1.9% in cases more than 5 years after valve replacement and 2.9% in cases less than 5 years after valve replacement. In contrast, in the control group of 89 cases (47 cases of aortic valve replacement, 30 cases of mitral valve replacement and 12 cases of multiple valve replacement), the incidence was 9.1% and 14.7%, respectively. Thus, in both time intervals following prosthetic valve replacement the incidence of thrombo-embolism in the treated group was significantly lower than in the control group.


Protein-losing enteropathy in lymphoedema. Surgical investigation and treatment. Patients with primary lymphoedema have been tested for protein loss from the gut by the chromium chloride  test. In a proportion of patients there is significant loss which may drop the plasma proteins thereby increasing oedema. Three patients were treated by abdominal exploration, mesenteric lymphography and resection of the worst affected length of gut. There was clinical and biochemical improvement and no complication.


Improved techniques for lumbar aortography. The use of a large volume technique (60-100 ml of contrast) for lumbar aortography is recommended for routine use in the investigation of peripheral arterial disease of the legs. With this method lesions are less likely to be missed and more information is obtained. Using the modern contrast media such as iothalamate, the complications due to contrast medium are not significantly greater than with the conventional volume technique. In Leriche syndrome, although the proximal end of the arterial occlusion can be demonstrated using conventional volume anortography, the distal end, and the arteries below the occlusion can usually only be shown by using a relatively large volume of contrast. Movement of a patient with a lumbar aortography needle in position is safe. Additional projections such as obliques to show the origins of the profunda femoris arteries or the arteries of the feet, can be taken if required. Complications due to stripping of the aortic wall are diminished if a blocked ended single side opening lumbar aortography needle is used.


The subclavian artery in 16 patients with complete cervical ribs. In a series of 16 patients presenting with symptoms due to the presence of a complete cervical rib, 8 complained of neurological symptoms only and 8 presented with major vascular symptoms. There were 21 complete cervical ribs in this series, 5 patients having bilateral cervical ribs, and these were excised. At operation particular attention was paid to the anatomy and pathology of the subclavian artery in relation to the cervical rib. Two anatomical variants were present. In type A cervical rib (16 cases) the subclavian artery crossed the first rib medial to the exostosis and all patients with major vascular symptoms were in this category. In type B (5 cases) the subclavian artery crossed the first rib lateral to the exostosis and symptoms, when present, were neurological rather than vascular. The two groups can be distingushed clinically and this may be of prognostic value. Post-stenotic dilatation of the subclavian artery acompanied 15 of the 21 complete cervical ribs and was attributed to compression of the artery between the cervical rib and the anterior scalene muscle. In 8 instances the post-stenotic dilatation was complicated by aneurysm and peripheral thrombo-embolism and this was regarded as a secondary phenomenon due to intermittent trauma at cost-clavicular level. A follow-up of up to 9 years would indicate that post-stenotic dilatation of mild or moderate degree is adequately treated by resection of the cervical rib and exostosis on first rib. When an aneurysm is present with localised disruption of the arterial wall with mural thrombus, it is necessary also to excise the aneurysm for, otherwise, there is a risk of further thrombo-embolic episodes.


The long term behaviour of autogeneou vein by-pass grafts. The results of the follow-up of 50 consecutive atuogeneous vein bypass grafts during 1958-65 are described. Arteriography was performed at regular intervals throughout the follow-up lasting from 8-15 years. 11 grafts failed in less than 2 years and were considered technical failures. Thereafter, of the 37 cases on whom arteriographic information is available, a further 5 grafts failed. Deterioration of the graft itself occurred in 19 percent, proximal anastomosis dilatation it 16 percent, and distal anastomosis narrowing in 25 percent. Improvement of flow to the lower leg vessels did not accelerate progression of the long saphenous vein at the time of operation is the limiting factor in longterm graft deterioration. The majority of grafts remained patient without degeneration for up to 15 years.


Mitochondrial and cytoplasmic ribosomes and their activity in blood and culture form Trypanosoma brucei. Ribosomes of Trypanosoma brucei, a parasitic, flagellated protozoan (order Kinetoplastida), were identified on sucrose density gradients by their radioactively labeled nascent peptides. Ultraviolet absorption revealed only cytoplasmic ribosomes which served as internal sedimentation markers. Synthesis on cytoplasmic ribosomes was completely inhibited by cycloheximide. In the presence of this antibiotic, nascent peptides were associated with ribosomes of lower sedimentation coefficient than the cytoplasmic ribosomes. Chloramphenicol blocked synthesis on these ribosomes which are probably the mitochondrial ribosomes. These ribosomes differed from the cytoplasmic ribosomes in several ways. Their sedimentation coefficient was about 72S rather than 84S. The stability of the 72S ribosomes was less sensitive to pancreatic ribonuclease and low Mg-++ concentrations, dissociating below 0.1 mM Mg++. The 72S ribosomes were more sensitive to elevated KCl concentrations, dissociation above 0.25 M. Protein synthetic activity associated with the 72S class of ribosomes was found in trypanosomes grown in rats. Under these conditions no cytochromes or fully active Krebs cycle is present in these cells and respiration is insensitive to cyanide.


Methods for the freeze-fracturing of nerve tissue cultures and cell monolayers. Two methods for freeze-cleaving of thin tissue layers are presented. Whereas a simple technique can be employed to fracture continuous, relatively firm tissue layers, a more sophisticated technique employing special carriers is needed to fracture very thin and incomplete layers, e.g., the fiber outgrowth of cultured nerve tissue or sparsely seeded isolated cells. Both methods basically consist of freezing the specimens sandwiched between two small metal carriers which are then fractured apart so that the cleavage plane runs through the tissue. In the resulting replicas of such thin specimens, large membrane areas are exposed, and new information is provided on the topography of membrane properties in entire cells or cell processes. The technique should also be useful for studies on the interactions of cells grown in culture.


Effects of actinomycin D on the association of newly formed ribonucleoproteins with the cistrons of ribosomal RNA in Triturus oocytes. The effect of actinomycin D(AMD) on the association of the nascent ribonucleo-protein (RNP) fibrils containing the precursors of ribosomal RNA (pre-rRNA) with their template deoxyribonucleoprotein (rDNP) strands has been studied in lampbrush stage oocytes from Triturus alpestris. Ovary pieces were incubated in vitro either in media containing radioactive ribonucleosides and then, for various times, in solutions containing 25 mug/ml AMD, or were directly exposed to the drug. The ultrastructure of the nucleoli and the nuclear periphery was studied by electron microscopy of thin sections and positively stained spread preparations of isolated nuclear contents, and by light and electron microscope autoradiography. The fate of the labeled pre-rRNA was followed by gel electrophoresis of RNA extracted from manually isolated nuclei. Our results show that the growing fibrils which contain the nascent pre-rRNA progressively detach from the DNP strands, the majority being released between 45 and 180 min after application of the drug. The release pattern seems to be random and does not show preference for regions close to the initiator or terminator sites of the transcribed rDNP units. There is a pronounced tendency to removal of groups of adjacent mascent fibrils. The effect of the drug is very heterogeneous. Even after 3 h of treatment with AMD the nucleoli exhibit several individual transcriptional units which appear almost completely covered with lateral fibrils. Autoradiography revealed that most of this released RNP remains within the confinements of the nucleoli which show some foci of aggregation and condensation of fibrillar components but no clear "segregation" phenomenon. In the gel-electrophoretic analysis, a significant but moderate decrease of labeled pre-rRNA was noted only in the first stable pre-rRNA component, whereas pre-rRNA classes of lower molecular weight are very stable under these conditions. The results are discussed in relation to the stability of rDNA transcription complexes and as a basis for an explanation of the ultrastructural changes which are generally observed in nucleoli of AMD-treated cells. It is postulated that inhibition of transcription results in a slow but progressive release of the arrested incomplete RNP fibrils from the template.


Acute arterial occlusion and the contraceptive pill. Two female patients in their thirties suffered peripheral arterial occlusion, in close association with the ingestion of the oral contraceptive pill. While current investigative technique cannot directly implicate the pill, it is felt that in the two patients presented it was an important aetiological factor, no co-existing arterial pathology being detected.


Carotid endarterectomy. A review of 104 operations. A follow-up of 104 carotid endarterectomies in 90 patients is presented. The operations were performed under general anaesthesia with controlled positive pressure ventilation and with peroperative EEG-registration. The patients have been divided into four groups: Group 1: Patients with no neurological symptoms. Group 2: Patients with transitory ischemic attacks (TIA). Group 3: Patients with neurological deficits. Group 4: Patients with cerebro-lesional symptoms. The specific neurological symptoms as well as the early surgical results and the results of the follow-up are presented. An evaluation of different kinds of X-ray examinations and of EEG-registration has been performed.


The permeability barrier in mammalian epidermis. The structural basis of the permeability barrier in mammalian epidermis was examined by tracer and freeze-fracture techniques. Water-soluble tracers (horesradish peroxidase, lanthanum, ferritin) were injected into neonatal mice or into isolated upper epidermal sheets obtained with staphylococcal exfoliatin. Tracers percolated through the intercellular spaces to the upper stratum granulosum, where further egress was impeded by extruded contents of lamellar bodies. The lamellar contents initially remain segregated in pockets, then fuse to form broad sheets which fill intercellular regions of the stratum corneum, obscuring the outer leaflet of the plasma membrane. These striated intercellular regions are interrupted by periodic bulbous dilatations. When adequately preserved, the interstices of the stratum corneum are wider, by a factor of 5-10 times that previously appreciated. Freeze-fracture replicas of granular cell membranes revealed desmosomes, sparse plasma membrane particles, and accumulating intercellular lamellae, but no tight junctions. Fractured stratum corneum displayed large, smooth, multilaminated fracture faces. By freeze-substitution, proof was obtained that the fracture plane had diverted from the usual intramembranous route in the stratum granulosum to the intercellular space in the stratum corneum. We conclude that: (a) the primary barrier to water loss is formed in the stratum granulosum and is subserved by intercellular deposition of lamellar bodies, rather than occluding zonules; (b) a novel, intercellular freeze-fracture plane occurs within the stratum corneum; (c) intercellular regions of the stratum corneum comprise an expanded, structurally complex, presumably lipid-rich region which may play an important role in percutaneous transport.


Intramembrane organization of specialized contacts in the outer plexiform layer of the retina. A freeze-fracture study in monkeys and rabbits. Freeze-fracture analysis of the neural connections in the outer plexiform layer of the retina of primates (Macaca mulatta and Macaca arctoides) demonstrates a remarkable diversity in the internal structure of the synaptic membranes. In the invaginating synapses of cone pedicles, the plasma membrane of the photoreceptor ending contains an aggregate of A-face particles, a hexagonal array of synaptic vesicle sites, and rows of coated vesicle sites, which are deployed in sequence from apex to base of the synaptic ridge. The horizontal cell dendrites lack vesicle sites and have two aggregates of intramembrane A-face particles, one at the interface with the apex of the synaptic ridge, the other opposite the tip of the invaginating midget bipolar dendrite. Furthermore, the horizontal cell dendrites are interconnected by a novel type of specialized junction, characterized by: (a) enlarged intercellular cleft, bisected by a dense plate and traversed by uniformly spaced crossbars; (b) symmetrical arrays of B-face particles arranged in parallel rows within the junctional membranes; and (c) a layer of dense material on the cytoplasmic surface of the membranes. The plasmalemma of the invaginating midget bipolar dendrite is unspecialized. At the contact region between the basal surface of cone pedicles and the dendrites of the flat midget and diffuse cone bipolar cells, the pedicle membrane has moderately clustered A-face particles, but no vesicle sites, whereas the adjoining membrane of the bipolar dendrites contains an aggregate of B-face particles. The invaginating synapse of rod spherules differs from that of cone pedicles, because the membrane of the axonal endings of the horizontal cells only has an A-face particle aggregate opposite the apex of the synaptic ridge. Specialized junctions between horizontal cell processes, characterized by symmetrical arrays of intramembrane B-face particles, are also present in the neuropil underlying the photoreceptor endings. Small gap junctions connect the processes of the horizontal cells; other gap junctions probably connect the bipolar cell dendrites which make contact with each cone pedicle. Most of the junctional specializations typical of the primate outer plexiform layer are also found in the rabbit retina. The fact that specialized contacts between different types of neurons interacting in the outer plexiform layer have specific arrangements of intramembrane particles strongly suggests that the internal structure of the synaptic membranes is intimately correlated with synaptic function.


Nonspecific aorto-arteries.

Nonspecific aorto-arteries. In the USSR, like in other countries, aorto-arteritis is no rarity. Onehundred and twentysix patients with aorto-arteritis have been studied at the A.N. Bakulev Institute for Cardio-Vascular surgery during the past 13 years (81 women and 45 men from 8 to 49 years of age). Morphologic changes consisted in inflammatory infiltration of the adventitia and, to a lesser degree, of the media as well as in reactive hyperplasia of the intima. The predominant features in the chronic stage were those of sclerosis and fibrosis of the adventitia and the media which in combination with the intimal hyperplasia resulted in stenosis or occlusion of the aorta and its main branches. The clinical picture and symptpmatology depend largely on the localization, form, and severity of the lesions. In aorto-arteritis ischemia of various organs and hypertension can be eliminated only by radical reconstructive surgery of the aorta and its branches. The most adequate restoration of the blood flow in the major vessels is provided by resection combined with replacement. It is not quite clear yet whether endarterectomy is possible and justified. Radical surgery was performed in 80 patients. In addition, different palliative and explorative operations were performed in 10 patients. The majority of patients had resection with replacement of the brachiocephalic arteries, descending thoracic aorta, abdominal aorta, renal and visceral arteries. Blood flow in the major vessels was restored in 69 patients. Early postoperative thrombosis of the prosthesis occurred in 7 patients. Immediate postoperative mortality was 13.3 percent. Sixtytwo patients were followed up from 1 to 9 years after radical surgery. Persistent normalization or significant improvement of the systemic and regional hemodynamics was found in 53 patients. Late thrombosis of the aorto-renal branch or aorto-femoral prosthesis occurred in 4 patients. There were three late deaths.


Release of growth hormone from ox pituitary slices after pronase treatment. Proteolytic enzymes have been used both to modify properties of the cell membrane and to dissociate cells from many tissues including pituitary (4, 5, 12). Exposure of secretory tissues to pronase can alter their secretory response. Thus incubation of pancreatic islets of Langerhans in the presence of low concentrations of pronase increased the subsequent release of insulin in the presence of stimulatory and nonstimulatory glucose concentrations (7). The purpose of the present investigation was to determine whether low concentrations of pronase have the same stimulatory effect on the release of a pituitary hormone, growth hormone. Such an effect on hormone release could be of some importance in view of the development of dissociated cell systems as models for the study of the control of hormone release (4, 5).


Substructure of the glomerular slit diaphragm in freeze-fractured normal rat kidney. In the renal glomerulus, the narrow slits between adjacent epithelial podocytes are bridged by a diaphragm (2, 8, 11). In rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde (TAG), it has recently been discovered that this diaphragm has a highly ordered, isoporous substructure (9). It consists of a regular array of alternating cross bridges extending from the podocyte plasma membranes to a centrally running filament. This zipperlike pattern results in two rows of rectangular pores, approximately 40 X 140 A in cross section, dimensions consistent with the proposed role of the diaphragm as an important filtration barrier to plasma proteins (6). In the present study, we found in freeze-cleaved and in freeze-etched normal rat glomeruli that the surface of the slit diaphragm has an appearance conforming to the pattern found in sectioned material.


Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules. Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.


Identification of actin in situ at the ectoplasm-endoplasm interface of Nitella. Microfilament-chloroplast association. Using a glycerination procedure designed to avoid excessive plasmolysis or disruption of the ectoplasm, microfilaments in bundles at the ectoplasm-endoplasm interface of Nitella internode cell segments were found to bind rabbit heavy meromyosin (HMM) in situ. All HMM arrowheads in a bundle seem to have the same polarity and many lie in register as judged from the electron micrographs; the arrowhead periodicity is approximately  380 . The decorated microfilaments are thus similar to those seen in negatively stained cytoplasmic suspensions of internode cells. In glycerinated material, as well as in suspensions, the microfilaments are closely associated with chloroplasts. The microfilaments lie adjacent to or are attached to the chloroplast envelope. The results provide further evidence that the microfilaments thought to play a role in cytoplasmic streaming in vivo in Nitella consist of actin and suggest that they may be anchored to the chloroplasts.


Membrane proteins synthesized by rabbit reticulocytes. Intact rabbit reticulocyte cells synthesize two predominant species of polypeptides which are components of the cell plasma membrane. Previous work (Lodish, H. F. 1973. Proc. Natl. Acad. Sci. U. S. A. 70:1526-1530.) showed that these proteins were synthesized by polyribosomes not attached to membranes. We show here that both polypeptides are confined to the cytoplasmic surface of the cell membrane. These studies utilized iodination of whole cells and of membranes with lactoperoxidase, and digestion of whole cells and membranes with chymotrypsin, One of these proteins is synthesized as a precursor, and about 20-40 amino acids are removed after it is incorporated into the membrane, We discuss the probable sites of synthesis of these and other classes of membrane proteins.


Structural and transcriptional features of the mouse spermatid genome. A whole-mount electron microscope technique has allowed direct visualization of the transcription process in mouse spermatids. Thes observations have been supported by light and electron microscope autoradiographic techniques that employ [3H]uridine and [3H]arginine in attempts to clarify mechanisms of RNA synthesis and their relationship to nuclear histone changes throughout spermiogenesis. Early spermatid genomes are dispersed almost completely, whereas in later spermiogenic steps the posterior or flagellar nuclear region is readily dispersed and the anterior or subacrosomal nuclear region remains compact. Display of genome segments permits identification of regions where transcription complexes, presumably heterogeneous nuclear RNA species, are seen related to chromatin. These complexes appear as ribonucleoprotein chains, some of them of considerable length, decreasing progressively in number in late spermiogenic steps. This decrease coincides with diminishing rates of [3H]uridine incorporation. Two distinct patterns of chromatin have been identified: a beaded chromatin type associated with transcription complexes encounterd in early spermatids; and a smooth chromatin type not involved in transcriptive activity observed in advanced spermiogenic genomes. Protein particles staining densely with phosphotungstic acid become apparent in nuclei of spermatids after [3H]arginine incorporation becomes significant. There is no structural or autoradiographic evidence for the presence of nucleoli during spermiogenesis. From these data and from previous experimental findings, we conclude that: (a) spermatogonia, spermatocytes and Sertoli cells are transcriptionally expressed into heterogeneous nuclear RNA and preribosomal RNA species whereas transcription in spermatids is predominantly heterogeneous nuclear RNA; and (b) the modification of the chromatin patterns in late spermiogenic steps indicates a stabilized genome that restricts transcriptive functions.


Electron microscope autoradiographic study of intestinal absorption of decanoic and octanoic acids in the rat. Intestinal absorption of [3H]octanoic acid and [3H]decanoic acid was investigated in the rat by electron microscope autoradiography. The common duct (bile and pancreatic common duct) of the rats was diverted and a loop of the duodenum was cannulated 24 h later. The lipid mixture to be investigated was introduced into each experimental loop, and after 15 min or less the loop was removed. One part of each loop was used to determine the distribution of radioactivity in different lipid fractions, and an autoradiographic study was performed on the other part of the loop. Radioactivity distribution studies confirmed that medium chain fatty acids are absorbed in their nonesterified form and established that these fatty acids are absorbed much more rapidly than oleic acid. Autoradiographic studies indicated that the medium chain fatty acids are taken up in a molecular or aggregate molecular form, leave the epithelial cells by way of the lateral plasma membrane, and are next found in the blood capillaries. Our results suggest that the Golgi complex does not play an important role in the absorption of unesterified fatty acids.


Isolation and separation of highly enriched fractions of viable mouse gastric parietal cells by velocity sedimentation. Methods of tissue dissociation and cell separation have been modified to obtain highly enriched fractions of mouse gastric parietal cells. Suspension of gastric mucosal cells are prepared by pronase digestion of the glandular portion of the stomach from adult mice. By utilizing the velocity sedimentation technique to separate cells of different sizes it is possible to recovery parietal cells, which are larger than the other cell types, in fractions with purity of 75-95%. The homogeneity of cell fractions has been assessed by light and electron microscopy. The ability of the isolated cells to exclude the dye trypan blue, to incorporate labeled substrate, to consume oxygen, and to retain their structural integrity indicates that they are viable and still capable of functional activity.


Changes in distribution pattern of cytoplasmic filaments in human melanocytes during ultraviolet-mediated melanin pigmentation. The role of the 100-Angstrom filaments in the elongation of melanocytic dendrites and in the movement and transfer of melanosomes. Human melanocytes characteristically contain 100-A filaments. These 100-A filaments shift from the perinuclear area to the center of the dendritic processes and are in close association with melanosomes during the different stages of UV-mediated melanin pigmentation. We suggest that these 100-A filaments in human melanocytes participate in the elongation of the dendrites and in the transfer of melanosomes.


Formation of temporary flagellar structures during insect organogenesis. Cilia and flagella are rare in nongerminal tissues of anthropods, and are generally thought to be restricted to sperm and sensory cells in insects (2). Whitten (5) has reported the presence of kinetosomes at the base of mitotrichia in the dipteran fly Sarcophaga bullata, but reports no evidence of the organization of fibrous elements characteristic of cilia and or flagella. During an ultrastructural analysis of morphogenesis of the colleterial gland of the silk moth Hyalophora cecropia, we found the first example of paired flagella associated with an insect secretory cell. These structures are also unusual in that they serve a temporary role in morphogenesis and subsequently disappear at the terminal stages of differentiation.


Quantitative measurement of the course of bean callus differentiation. Two strains of callus have been isolated from bean hypocotyl and grown on a defined maintenance medium supplemented with 2 mg/l. 2:4-dichlorophenoxyacetic acid (2:4D) and 2% sucrose. Root initiation was observed in one strain and formation of nodules containing xylem and phloem in both strains after transfer to an induction medium supplemented with 1 mg/l. naphthyleneacetic acid, 0-2 mg/l. kinetin and 3% sucrose, after 3 transfers to maintenance medium. The number of nodules per gramme increased 10-fold between 6 and 12 days after transfer, and thereafter remained constant. Phenylalanine ammonia lyase (PAL) activity rose to a maximum value when the rate of nodule formation was greatest, and decreased after the maximum nodule concentration was reached. The final constant value for PAL activity was above that of callus grown on maintenance medium. Beta I leads to 3 glucan synthetase activity rose to a maximum 15 days after transfer, and then fell gradually to a level above that measured in callus on maintenance medium. Callus was transferred from maintenance medium after 3, 4, 5 and 6 transfers. The concentration of nodules after 21 days on induction medium decreased as the callus was kept in culture. No further differentiation could be induced after 6 transfers. The fall in nodule formation was paralleled by a decrease in PAL and betaI leads to 3 glucan synthetase activities measured 21 days after transfer.


An electron-microscope study of the lampbrush chromosomes of the newt Triturus cristatus. The ultrastructure of lampbrush chromosomes has been examined in sections of end-embedded spread preparations, where the nuclear sap was dispersed prior to fixation, and in oocyte nuclei fixed entire, in 60-kV and i-MV electron microscopes. In spread preparations the axial chromomeres are seen to be organized as regularly spaced, unravelled skeins of DNP, each with a skein width of some 30 nm, though in some chromomeres there are regions where the DNP is much more densely packed. In both unravelled and dense regions, the 'ultimate' DNP fibres, wherever they can be identified, appear to be some 5 nm wide and thrown into loose coils. The unravelled state, although it clearly reflects an orderly packaging of the non-transcribing DNP, is an artifact of preparation; in sections of entire nuclei all chromomeres are seen to consist of DNP fibrils in the more densely packed state. The interchromeric fibril is single, and some 10 nm or less wide; it shows no sign of transcriptional activity. In sections of end-embedded preparations the RNPmatrix of most lateral loops, where transcription occurs, is seen to be made up of particles, each uniformly some 30 nm in diameter and strung together in linear array. These RNP particles are equally evident in sections of whole nuclei. In many loops the strings of particles are wound back on themselves to form regularly spaced, dense aggregates, each some 200-300 nm wide or wider; the larger aggregates can be resolved in the light micrpscope. The RNP particles are of the same dimensions throughout the lengths of individual lateral loops, and of substantially the same dimensions in loops of different gross morphologies. It is suggested that as each successive short length of RNA is transcribed from loop axis DNA, a protein associates with this RNA and winds it up to form a 'manageable' package, allowing transcription to proceed.


Light- and electron-microscope observations on certain leukocytes in a teleost fish and a comparison of the envelope-limited monolayers of chromatin structural units in different species. Previously it was shown that the nuclear envelope-limited sheets of chromatin, monolayers of nucleoprotein structural units, are present in blood cells from 4 classes of vertebrates. Now we show that sheets of similar width are present in certain leukocytes of a fifth class, a teleost fish. We describe the fine structure of leukocytes in peripheral blood and in the main haematopoietic organ, kidney. We also examined the granulocytes of connective tissue in intestine. By May-Grunwald-Giemsa staining and electron microscopy heterophilic granulocytes, cosinophils but no basophils could be recognized in peripheral blood and kidney. Problems in classification of the cells are discussed. In one group (A) of 5 fish, sheets occurred at a frequency of roughly 1% in heterophilic (type 1) granulocytes and lymphocytes from peripheral blood. No sheets were found in a second group (B) of 5 fish. Kidney and intestine were examined in some fish from both groups and no sheets were present. In an atypical group (C) sheets were found in the eosinophilic (type 2) granulocytes from peripheral blood of one fish and in lymphocytes from connective tissue of intestine in another. Sheets were usually associated with nuclei of irregular shape and their width averaged 36 nm. We tabulate data from other workers on occurence and width of sheets. They are found in all the main classes of tissue in mammals, namely blood and other connective tissues, in epithelial, nervous, germinal tissue and muscle, as well as in invertebrate and certain plants. Their nearly constant width, average value 35 nm, provides very convincing evidence for the hypothesis that the molecules of DNA and protein are organized into the same fundamental structural units, irrespective of species. We discuss the variable incidence of sheets among different cell types and the factors which might determine this.


Rifampicin inhibition of the plastid rRNA synthesis of Marchantia polymorpha. The effect of rifampicin on the synthesis of plastid rRNA in Marchantia polymorpha was studied in vivo. As bacterial rRNA and plastid rRNA have the same electrophoretic mobilities, this study was possible only after a method for inhibiting bacterial contamination was developed. It was established that 91-100% of the rRNA synthesized by cultures of bacteria from Marchantia, after a labelling period of 3 and 9 h by 32-P, is inhibited by 10 mug/ml of rifampicin. The same inhibition was observed when Marchantia was labelled for 3 h in the presence of 10 mug/ml of rifampicin, showing that no plastid rRNA was synthesized under out conditions, but only bacterial RNA. However, when labelling was continued for 9 h two important peaks of rRNA (23 and 19 s) were labelled in the presence of 10 or 20 mug/ml of rifampicin. These peaks are of chlorophastic origin as confirmed by the following facts: the labelling is light-activated; plastids isolated from  thalli labelled for 12 h also show these two radioactive peaks. Cytoplasmic rRNA is synthesized under certain conditions. The synthesis of plastid rRNA is inhibited by higher concentrations of rifampicin, a concentration of 250 mug/ml producing at least 75% inhibition. Marchantia, a primitive multicellular plant, differs in this respect from higher plants, which seem to be, in most cases, insensitive to rifampicin


Kinetic aspects of synchrony in suspension cultures of Acer pseudoplatanus L. Synchronous divisions have been induced in cell suspension cultures of sycamore (Acer pseudoplatanus L.) initiated at low density from stationary phase, nitrate-starved cells. The pattern of division and DNA replication synchrony is unusual in that it does not show progressive decay but rather a sharp transition to asynchrony. Feulgen densitometry data indicate that in terms of an S-phase synchrony index, DNA replication becomes more highly synchronized as cell density rises and interphase duration is reduced. In comparison with asynchronous cultures the duration of mitosis appears to be reduced during synchronous growth. The persistence of division and S-phase synchrony and the apparent shortening of interphase and mitosis in these sycamore cultures suggests the operation of intercellular entrainment. The departure from synchrony during the interphase which precedes initiation of asynchronous division remains unexplained.


The fine structure and cell kinetics of mouse epidermis after wounding. A variable amount of cornified tissue removed from mouse dorsal epidermis results in stimulation of the entire basal layer. Stimulation does not appear to be dependent on damage to an indiviaual epidermal proliferative unit (EPU). The immediate reaction to wounding is a rapid movement of cells from the basal layer to the differentiating compartment resulting in depopulation of the basal layer, which is followed by a burst in DNA-synthetic activity. The result of the increased transit of cells through the epidermis is that various aspects of keratinization can appear abnormal. The Langerhans cells show several changes, often appearing suprabasal and becoming smaller, rounded cells with a less-clear cytoplasm and fewer granules. The initial migratory reaction results in a largely normal epidermis on the third day. This reaction is followed by a transient hyperplasia which reaches its peak on the sixth to seventh day and gradually returns to normal by the fourteenth to fifteenth day. The hyperplasia is characterized by a loss of the ordered stacking of cornified cells which become shorter and thicker than normal. There is a return to the stacked state beginning on the tenth day. The Langerhans frequency is apparently at its lowest on days 6-7 when the proliferation levels are at their maximum. An inverse relationship appears to exist between relative Langerhans cell frequency and cell proliferation rate. The data suggest that the frequency of Langerhans granules also changes during the course of the hyperplasia, peak levels being observed just before the decline in proliferative activity.


Data security.

Data security. Data security is a rich and complex subject dealing with the protection of the computing capability from all threats to its continuity. Some fundamental elements of the process of achieving a reasonable, prudent measure of that protection are considered.


The large data base file structure dilemma. This paper first presents a brief tutorial on the principal random file organization methods for handling two major applications--Transaction oriented systems and information storage and retrieval systems. It then addresses a particular large data base dilemma, not satisfactorily resolved by any of these methods, and which is currently under active investigation. Two approaches to a solution are described. One is called the hybrid inverted list; the other is based upon an old technique called super-imposed coding. The former has been implemented and has recently been installed in an operational system. Some statistics related to file characteristics in this application are provided, but operational cost and performance statistics are not yet available.


Large data base at the Lawrence Livermore Laboratory. The Lawrence Livermore Laboratory Octopus network includes a central storage facility of over 10-12 bits. This facility is accessed through a directory mechanism which permits flexible organization and general sharing of files by many time-sharing users. The chief problems of the system are not unusual: the maintenance of high transfer rates, of reliability and availability, and of sufficient storage capacity.


The management of a large data base in IRIS. IRIS (Illinois Resource Information System) is a retrieval and analysis system for land-use planners. The system stores the attributes of land parcels. Currently two major data bases are in use: a 78,000 parcel data base covering eight counties around Chicago and a 16,000 parcel data base covering six counties around Chicago.


Input/output considerations for large data bases. Input/output devices, format designs, and programming for rejecting defective input, updating and correcting input, and for producing a variety of outputs from the input are discussed, especially from the viewpoint of a large data base and that of the information needs of the users.


Data compression of large document data bases. Consideration is given to a document data base that is structured for information retrieval purposes by means of an inverted index and term dictionary. Vocabulary characteristics of various fields are described, and it is shown how the data base may be stored in a compressed form by use of restricted variable length codes that produce a compression not greatly in excess of the optimum that could be achieved through use of Huffman codes. The coding is word oriented. An alternative scheme of word fragment coding is described. It has the advantage that it allows the use of a small dictionary, but is less efficient with respect to compression of the data base.


Lockheed experience in processing large data bases for its commercial information retrieval service. The last few years have seen the emergence of the information retrieval services industry. This industry consists of three tiers: data base producers, retrieval service vendors, and information centers who service the end user. The experience of Lockheed Information Systems as a retrieval service vendor is recounted as it pertains to its system and to its relationship with the data base suppliers. Government competition is seen as a potential threat to the industry.


Chemical Abstracts Service approach to management of large data bases. When information handling is "the business," as it is at Chemical Abstract Service (CAS), the total organization must be involved in information management. Since 1967, when, as a result of long-range planning efforts, CAS adopted a "data-base approach" to management of both the processing system and the distribution of information files, CAS has been grappling with the problems of managing large collections of information in computer-based systems. This paper describes what has been done at CAS in the management of large files and what we see as necessary, as a result of our experience, to improve and complete the information management system that is the foundation of our production processes.


TOSAR--a system for the structural formula-like representation of concept connections in chemical publications. A system is described for representing graphically the conceptual contents of a document, such as preparations and processes delineated in a patent or journal article.


A comparison of the performance of some similarity and dissimilarity measures in the automatic classification of chemical structures. A group of 39 structures with local anesthetic activity has been classified automatically by calculating similarity or dissimilarity coefficients between pairs of structure diagrams and applying cluster analysis to the results. The performance of a number of similarity and dissimilarity coefficients has been compared using the relationship between structure and property. Simple coefficients and a distance function give more satisfactory results than functions using probabilistic weighting or standardized distance.


Search strategies and user interface.

Search strategies and user interface. This paper discusses what in The Mead Corporation have found to be certain aspects of search strategy with respect to searching large data bases and certain problem areas that remain. The paper defines some problem areas, especially problem areas in the freeing-up of the interface language between the user and the search process. Four specific areas in the interface language are defined, and a recommendation is made concerning basic research into some linguistic meanings for the four areas.


[Angiography of the cervico-cephalic vessels of the dog.  The carotid system]. The authors report an experimental study of the pathology and cervico-cerebral hemodynamics in the dog and discuss the normal anatomy of the carotid arteries in the dog.  These results include the interpretation of 30 selective angiographies and numerous anatomical dissections.  Many points distinguish the carotid arteries in the dog from those in man.  Firstly, in their distribution, where the internal carotid artery appears particularly narrow the internal maxillary artery particularly large.  Then and above all, their function, it seems that external carotid artery is more important than the internal carotid artery in the dog, so that the blood supply of the central nervous system seems to be obtained almost entirely through the vertebro-basilar system.  Finally, there exists between each network of internal carotid arteries, the external carotid artery and the vertebral arteries numerous anastomoses.  Contrary to the distrubution of arteries in man, these anastomoses usually function permanently.