Glycosphingolipids of human KB cells grown in monolayer, suspension, and synchronized cultures.

Studies have been carried out on the glycosphingolipids of human KB cells grown in monolayer and suspension culture, and by synchronization of the latter with a double thymidine (2mM) block. Glycosphingolipids were identified tentatively by thin layer chromatography, gas-liquid chromatography, and combined gas-liquid chromatography and mass spectrometry. The predominant gangliosides in the these cells were AcNeu-Gal-Glc-Cer and AcNeu-Gal-GalNAc-Gal-(AcNeu). Glc-Cer. Theprincipal neutral glycosphingolipids were Glc-Cer, Gal-Glc-Cer, Gal-Gal-Glc-Cer, and GalNAc-Gal-Gal-Glc-Cer. Incubation of KB cells (grown in monolayer and subsequently in suspension culture) for 48 hours with D-[1-14Clgalactose resulted in appreciable incorporation of radioactivity into all of the principal glycosphingolipids of these cells. These experiments confirmed that KB cells are capable of synthesizing their constituent glycosphingolipids. KB cells grown in suspension culture showed A 2- to 3-fold increase inthe concentration of Glc-Cer, Gal-Glc-Cer, GalNAc-Gal-Gal-Glc-Cer, and AcNeu-Gal1NAc-Gal-Gal-Glc-Cer, and AcNeu-Gal-Ga1NAcGal-(AcNeu)-Glc-Cer. Thus, the occurrence of tissue culture-dependent changes in the level of glycosphingolipids is demonstrated. Perhaps messages governing the synthesis of glycosphingolipids are translated earlier in thecell cycle under certain conditions of growth and are affected by cell-cell contact and cell adhesion.


Regulation of long chain fatty acid activation in heart muscle.

Regulation of fatty acid activation was studied in whole tissue homogenates of rat heart. The palmityl-CoA synthestase activity was proportional to the fatty acid to albumin ratio in the incubation medium with maximal activity occurring at a molar ratio of about 5. Fatty acyl-CoA synthetase activity was inhibited by products of the reaction (AMP, pyrophosphate, and palmityl-CoA). The apparent Ki for palmityl-CoA inhibition was 5 muM and this inhibition could be relieved by CoA-SH or albumin. The Km for CoA-SH in the absence of palmityl-CoA was 7 muM and was increased to 24 muM by addition of 8 muM palmityl-CoA. Cytosolic and mitochondrial levels of CoA-SH and carnitine were estimated in whole tissue homogenates of heart and liver. From 90 to 100% of whole tissue CoA was recovered in the mitochondrial fraction of heart muscle and it was estimated that the cytosolic concentration of free CoA-SH probably never exceeds its Km value for fatty acid activation in this tissue. Therefore, the rate of fatty acid activation would be expected to depend on the availability of CoA-SH in the cytosolic space. By adjusting the concentration of CoA-SH in the cytosol to the rate of acetyl-CoA oxidation, carnitineacetyl-CoA transferase may function in cardiac muscle to couple the rate of fatty acid activation in the cytosolic compartment to acetyl-CoA oxidation in the mitochondria. Approximately 30% of whole tissue CoA-SH was located in the cytosolic space in liver. Heart muscle has about twice as much carnitine as liver but in both tissues 100% of whole tissue carintine was located in the cytosolic space. The ratio of carnitine to CoA-SH in the cytosolic space was estimated to be about 100 in heart and 17 in liver. This high ratio in cardiac muscle may function to channel fatty acids toward oxidation rather than toward synthesis of complex lipids.


Isolation and partial characterization of a 40 S ribosomal subunit-transfer ribonucleic acid binding factor from rabbit reticulocytes.

A factor that catalytically promotes the codon-directed, GTP-independent binding of tRNA to 40 S ribosomal subunits has been isolated from the postribosomal supernatant and the ribosomal wash of rabbit reticulocytes. The factor is a heat labile, sulfhydryl reagent-sensitive protein of a molecular weight of approximately 50,000. It consists of two non-identical subunits of Mr equals approximately 30,000 and 20,000. Its basic character has been confirmed by the high ratio of basic amino acids to nonamidic aspartic and glutamic acid present in the purified protein. Formation of a factor promoted 40 S-poly(U)-phenylalanyl-tRNA initiation complex causes a shift in the Mg-2+ concentration optimum for polyphenylalanine synthesis from 8 mM to 4mM.


Role of the surface excess of palmitoyl coenzyme A in the 1-acylglycerol-3-phosphate acyltransferase reaction catalyzed by microsomes.

Surface excess values for palmitoyl-coenzyme A have been determined at the air-water interface. In the bulk concentration range of 0.23 to 3.7 muM, the surface concentration of palmitoyl-CoA ranges from 0.7 to 1.4 times 10- minus 10 mol/cm-2. The molecules of palmitoyl-CoA in the surface layer behave as if they were in a monolayer with each molecule occupying a limiting molecular area of 79 A-2. The distribution of palmitoyl-CoA between bulk and surface phases can be described by a Langmuir adsorption isotherm with an equilibrium constant of 0.33 muM. This constant is identical to the apparent Km for palmitoyl-CoA in the 1-acylglycerol-3-phosphate acyltransferase reaction catalyzed by microsomes. The results of this study, together with those from earlier work, suggest that the observed saturation behavior of the enzymatic reaction reflects the formation of a positive surface excess of palmitoyl-CoA in the vicinity of the catalytic site.


Partial purification and characterization of aspartate aminotransferases from seedling oat leaves.

As relatively little information is available on the properties of aspartate aminotransferase from photosynthetic tissue, isolation and characterization of the two major electrophoretically distinct forms of this enzyme from seedling oat leaf homogenates were undertaken. These two forms are designated I for the more anionic form and II for the less anionic form. Form I, 80 to 90% of the total activity, has been purified to a specific activity of 120 mumol/min/mg of protein (1100-fold) and is estimated to be 90 to 95% homogeneous, as judged by analytical polyacrylamide gel electrophoresis. Form II, 10 to 20% of the total activity, has been purified to a specific activity of approximately 6 mumol/min/mg of protein (300-fold). Both forms exhibit optimal activity at pH 7.5. Michaelis constants do not differ greatly between forms I and II and are similar to those reported for the pig heart cytosolic enzyme as well as aspartate aminotransferase from other plant sources. A molecular weight of 130,000 for the purified aspartate aminotransferase I was estimated by sedimentation equilibrium centrifugation; molecular weights of the two forms are similar as estimated by sucrose density gradient centrifugation. No activation by pyridoxal phosphate has been observed during purification.


Hydrocarbon chain packing and molecular motion in phospholipid bilayers formed from unsaturated lecithins. Synthesis and properties of sixteen positional isomers of 1,2-dioctadecenoyl-sn-glycero-3-phosphorylcholine.

Isomers of cis-octadecenoic acid, with the double bond in each position in the hydrocarbon chain, were used to synthesize the corresponding 1,2-diacyl-sn-glycero-3phosphorylcholines (lecithins). Differential thermal analysis of the lecithins, as a function of water content, permitted evaluation of the limiting transition temperature (Tc) of each isomer. Values of Tc plotted against double bond position fell on a smooth curve with a minimum at minus 22 degrees for the dioctadec-9'-enoyl compound. The presence of a "pretransition" endotherm in differential thermal analysis of 1,2-dioctadec-15'-and 1,2-dioctadec-16'-enoyl-sn-glycero-3-phosphorylcholine implies the existence of two beta crystalline forms. This was not observed with any of the other lecithins. Enthalpy and entropy data were then obtained from differential scanning calorimetry measurements. Values of delta H were lower (7.6 plus or minus 0.1 kcal mol- minus 1) when the center of unsaturation was near the middle of the hydrocarbon chain than they were (9.6 kcal mol- minus 1) when the center of unsaturation was close to either end of the chain. However, values of delta S showed no consistent variation with double bond position. Four positional isomers of 1-octadec-cis-enoyl-2-octadecanoyl-sn-glycero-3-phosphorylcholine were synthesized. With the double bond near the middle of the chain or close to the terminal group, the Tc values of the mixed acid lecithins were higher than those of the corresponding dioctadecenoyl lecithins. 13-C nuclear magnetic resonance relaxation measurements were used to obtain information about chain motion of selected 1,2-dioctadec-cis-enoyl-sn-glycero-3-phosphorylcholines at a temperature (52 degrees) above the Tc values. Spin-lattice relaxation times of the resolved resonances indicated that location of double bonds near the middle, as compared to either end, of the hydrocarbon chain favors enhanced molecular motion along the length of the chins and especially at the terminal methyl end. In the gel state, the minimum interaction potential energy of hydrocarbon chains in bilayers formed from dioctadecenoyl lipids appears to be minimized by localization of the double bond near the middle of the chains. It is suggested that in the case of homogeneous chains the double bond primarily affects the cooperativity of interactions and has very little steric effect on van der Waals' contacts. By contrast, in bilayers of mixed lecithins, with heterogeneous chains, the steric effect may become dominant, depending on double bond position. These differences in chain packing in the gel state are promulgated beyond the phase transition to the liquid crystalline state as an enhancement of chain motion as the temperature rises above Tc.


The activation of coagulation factor X. Identity of cleavage sites in the alternative activation pathways and characterization of the COOH-terminal peptide.

Bovine Factor X can be activated by two alternative pathways. The first, favored at high concentrations of the complex of tissue factor and Factor VII, is initiated by the action of Factor VII on Factor X to cleave an activation peptide from the NH2 terminus of the heavy chain, to produce alpha-Xa. This is then converted autocatalytically to another form of Factor Xa, beta-Xa, by the loss of a 17-residue glycopeptide from the COOH terminus of the heavy chain, in a lipid-dependent reaction. The alternative pathway, favored at lower activator concentrations, is initiated by the action of Factor Xa on Factor X, in the presence of lipid, to release the same COOH-terminal peptide as is produced in the conversion of alpha-Xa to beta-Xa. The intermediate produced by the loss of this peptide from Factor X,I1, can be activated directly to beta-Xa by the tissue factor-Factor VII complex, with the loss of the same NH2-terminal peptide as is produced in the conversion of Factor X to alpha-Xa. The autocatalytic activation of Factor X by Factor Xa described previously occurs to a marked extent only at very low activator concentrations, and has been shown to proceed largely by the loss of the normal NH2-terminal peptide from the heavy chain of I1-Initial experiments show that neither peptide affects the rate of coagulation by either the extrinsic or intrinsic pathways. The amino acid sequences have been determined on both sides of the peptide cleavages, and it has been shown that the cleavage sites are the same, regardless of the pathway of activation. The amino acid sequence and carbohydrate composition of the COOH-terminal peptide have been determined. The carbohydrate moiety is attached via an O-glycosidic linkage at a threonine residue, and contains galactosamine but no glucosamine.


Purification of papain-solubilized histocompatibility antigens from a cultured human lymphoblastoid line, RPMI 4265.

HL-A antigens having specificities HL-A2, HL-A7, HL-A12 have been solubilized by papain treatment of membrane preparations from the cultured human lymphoblastoid cell line RPMI 4265 and purified about 80-fold by chromatography on carboxymethylcellulose, Sephadex G-150, and diethylaminoethylcellulose columns. Separation of HL-A2 from a mixture of HL-A7 and HL-A12 was achieved on the final DEAE-cellulose column. The yield was about 1 mg of protein of each antigen preparation per 100 g of packed, frozen cells. On sodium dodecyl sulfate gel electrophoresis both preparations showed two polypeptide bands. The smaller subunit of 12,000 daltons is common to all HL-A preparations and has been shown to be identical with beta2-microglobulin. The larger subunit is a glycopeptide and in the HL-A7, 12 preparation was resolved into a duplex of 34,000 and 37,000 daltons. The HL-A2 preparation showed a single band at 34,000 daltons. On isoelectric focusing under nondenaturing conditions, the preparation showed multiple bands, all of which contained both subunits and retained antigenic activity. On isoelectric focusing in the presence of 6 M urea a single band for beta2-microglobulin and multiple bands for the larger subunit were seen. This charge heterogeneity of the larger subunit has been shown to be due to variable amounts of sialic acid. When HL-A antigen preparations were subjected to Sephadex G-100 chromatography in the presence of 3 M KCl, no separation of the two subunits was observed.


Effects of hypophysectomy, growth hormone, and thyroxine on protein turnover in heart.

Cardiac atrophy following hypophysectomy was accompanied by decreased heart content of RNA and polysomes and increased levels of ribosomal subunits, suggesting that protein synthesis was restricted by a reduced supply of ribosomes and an imbalance between rates of peptide-chain initiation and elongation. During perfusion in vitro, provision of palmitate restored the normal balance between rates of initiation and elongation but protein synthesis was lower in hearts of hypophysectomized than normal rats, reflecting the lower RNA content of hearts from hormone-deficient animals. After the period of atrophy had passed, or after treatment with growth hormone and thyroxine, heart RNA content and rates of protein synthesis were equal to or greater than those found in normal hearts. When plasma levels of amino acids, glucose, fatty acids, and insulin, and rates of beating and ventricular pressure development observed in normal and hypophysectomized rats were simulated during in vitro perfusion, hearts from hormone-deficient rats had reduced rates of protein synthesis but unaltered rates of degradation. Cathepsin D activity in heart homogenates (+ Triton X-100) was elevated during cardiac atrophy when expressed per g of tissue but not when expressed per heart.


The epinephrine-sensitive adenylate cyclase of rat liver plasma membranes. Role of guanyl nucleotides.

The epinephrine sensitivity in vitro of the adenylate cyclase system in liver plasma membranes from adrenalectomized rats was increased by the addition of 1 to 100 muM GTP or GDP in the incubation medium. Basal and glucagon-stimulated cyclase activities were also enhanced by GTP and GDP. These effects occurred even in the absence of an ATP-regenerating system. They were mimicked by 5'-guanyl diphosphonate and a series of guanyl derivatives, indicating that the structural requirement for the GTP action is not very stringent. Guanyl nucleotides did not increase the affinity of the adenylate cyclase system for the activating hormones, nor did they protect the enzyme activity against denaturation. Their synergic effect was due to an enhancement of the affinity of the enzyme for the substrate MgATP and also to an increase of the maximal velocity of the reaction. It is proposed that the guanyl nucleotides act directly and primarily upon the catalytic component of the cyclase system, independently of their effects on the binding of the activating hormones to liver plasma membrane. Since the activating effects of epinephrine and glucagon are similar in the presence of GTP, but not in its absence, it is suggested that the lower efficiency of epinephrine under normal conditions is not due to intrinsic membrane characteristics, but rather, to superimposed extraneous modulations.


Specific hydrolysis of the cohesive ends of bacteriophage lambda DNA by three single strand-specific nucleases.

Procedures have been worked out for Aspergillus nuclease S1 and mung been nuclease to quantitatively cleave off both of the 12-nucleotide long, single-stranded cohesive ends of lambdaDNA. This cleavage is indicated by the almost complete elimination of the repair incorporation of radioactive nucleotides by DNA polymerase into the digested DNA. With S1 nuclease, cleavage was complete at 10 degrees as well as at 30 degrees. Under the conditions for quantitative cleavage of the single-stranded regions there was no digestion of the double-stranded lambdaDNA. The mung bean nuclease cleaved off the cohesive ends completely at 30 degrees but at 5 degrees, the cleavage was not complete even at high enzyme concentration. The nearest neighbor analysis of the repaired DNA indicates that at 5 degrees about four nucleotides remained undigested. The mung bean nuclease also introduced, under the conditions used, some nicks into double-stranded DNA as determined by the repair incorporation. The Escherichia coli exonuclease VII cleaved off part of the cohesive ends of lambdaDNA, leaving two nucleotides on each end as single-stranded tails.


Properties of chromatin subunits from developing trout testis.

When a sample of trout testis nuclei is digested with micrococcal nuclease, the DNA is cleaved almost entirely to discrete fragments approximately 200 base pairs long and multiples thereof. The same DNA fragments can be obtained when isolated chromatin, as opposed to intact nuclei, is nuclease digested. These DNA fragments can also be found in discrete chromatin "subunits" isolated from nuclease-digested nuclei. Sedimentation through sucrose gradients or velocity sedimentation in an analytical ultracentrifuge separates these chromatin subunits into 11 S (monomer), 16 S (dimer), and 22 S (trimer) etc. species. Subunits can also be fractionated on a Sepharose 2B column equilibrated and run in low salt. High salt (greater than 40 mM NaCl) or divalent cations (congruent to 5 mM) cause subunit precipitation. Chromatin subunits have a protein to DNA ratio of approximately 1.2 and contain all the histones, including the trout-specific histone T. There are, however, no detectable nonhistone chromosomal proteins. Mg-2+ precipitates of the 11 S chromatin monomers, when pelleted, are thin and clear, while oligomer Mg-2+ pellets are thick and white. This could reflect a more symmetrical or ordered packing of 11 S monomers, which are deficient in histone I. This histone may cross-link the larger oligomers, resulting in a disordered Mg-2+ complex. These results are consistent with the subunit model of chromatin structure, based on 200 base pair long regions of DNA associated with histones. These subunits would be separated by nuclease-sensitive DNA spacer regions and cross-linked by histone I.


Replication of phage G4. A novel and simple system for the initiation of deoxyribonucleic acid synthesis.

Conversion in vitro of single-stranded circular DNA of phage G4 (related to phage phiX174) to the double-stranded replicative form (RF-II) depends on a novel and relatively simple group of three proteins: a priming protein of approximately 65,000 daltons, the DNA unwinding protein, and the DNA polymerase III holoenzyme. Stimulation by ATP and GTP suggests an RNA synthetic step in the priming of DNA synthesis. The synthetic strand in the RF-II contains a small gap at a unique position relative to the template strand; the 5' end of the gap is about 250 nucleotide residues (5% of the genome length) away from the single site of cleavage by a restriction endonuclease (Eco RI).


Integrated steady state rate equations and the determination of individual rate constants.

Integrated steady state rate equations have been used to determine the kinetic constants (Vs, Ks, Vp, and Kp) and rate constants (k1, k2, k3, and k4) of the reversible enzyme mechanism: (see article). The fumarase reaction has been used as a model to illustrate the procedures for determining these constants. In contrast to initial velocity studies, the values of the constants have been obtained by examining the enzyme reaction in only one direction rather than in both forward and reverse directions. To accomplish this, a new procedure is described for fitting data to integrated rate equations which eliminates problems encountered when data are analyzed graphically. The advantages of examining on enzyme reaction in one direction with these new procedures allow this method to be extended to the examination of enzymes with simple mechanisms where initial velocities are difficult to measure because either the substrate or product is not readily available, or because the reaction is not readily reversible.


A new carboxylation reaction. The vitamin K-dependent incorporation of H-14-CO3- into prothrombin.

The bovine plasma zymogen prothrombin contains a number of gamma-carboxyglutamic acid residues which are not found in an abnormal prothrombin produced when cattle are given the vitamin K antagonist dicoumarol. These modified glutamic acid residues appear to be formed post-translationally by a reaction which requires vitamin K. It has been shown that postmitochondrial supernates from vitamin K-deficient rats incorporate added H-14-CO3- minus into microsomal proteins upon the addition of vitamin K. This incorporation is dependent upon the presence of the prothrombin precursor in the microsomal preparations, and upon factors which are present in the postmicrosomal supernatant. Most of the radioactive protein which can be obtained from the microsomal pellet by extraction with 0.25% Triton X-100 has been identified as prothrombin and it can be shown that all of the radioactivity is in the amino-terminal activation fragment of prothrombin. This portion of the protein has previously been shown to contain the gamma-carboxyglutamic acid residues. Hydrolysis of the purified radioactive prothrombin resulted in a loss of 50% of the radioactivity and subsequent chromatography of the amino acid hydrolyzate demonstrated that the remaining radioactivity was entirely in glutamic acid. These results are consistent with the hypothesis that all of the H-14-CO3- minus was incorporated into the carboxyl groups of gamma-carboxyglutamic acid residues.


Unique cathepsin D-type proteases in rat thoracic duct lymphocytes and in rat lymphoid tissues.

Two unique cathepsin D-type proteases apparently present only in rat thoracic duct lymphocytes and in rat lymphoid tissues are described. One, termed H enzyme, has an apparent molecular weight of similar to95,000; the other, termed L enzyme, has an apparent molecular weight of similar to45,000, in common with that of most cathepsins D from other tissues and species. Both enzymes differ from cathepsin D, however, by a considerably greater sensitivity to inhibition by pepstatin and by a smaller degree of inhibition by an antiserum which inhibits rat liver cathepsin D. H enzyme is converted to L enzyme by treatment with beta-mercaptoethanol; the relationship between the two enzymes remains unknown. H and L enzyme have been detected in rat lymphoid tissues and in mouse spleen, but they are not present in other rat tissues (liver, kidney, adrenals), rabbit tissues, calf thymus, bovine spleen, or human tonsils. As measured on acid-denatured bovine hemoglobin as substrate, both enzymes have pH activity curves identical with that of rat liver cathepsin D, with optimal activity at pH 3.6. Activity on human serum albumin is much less and also shows an optimum at pH 3.6; hence, neither enzyme has the properties of cathepsin E. Thiol-reactive inhibitiors have no effect on the activity of H and L enzyme; thus they do not belong to the B group of cathepsins. Additional information, discussed in this paper, leads us to conclude that partially purified H and L enzymes are cathepsin D-type proteases.


Physical and chemical properties of beta-glucuronidase from the preputial gland of the female rat.

In order to obtain sufficient quantities of beta-glucuronidase for use in structural studies, the enzyme was purified from its richest known source, the female rat preputial gland, by a method similar to that of Ohtsuka and Wakabayashi (1969) (Enzymologia 12, 109). The purified enzyme has an S-o20, w of 12.5 S and a D-o20, w of 4.3 times 10- minus 7 cm-2 S-minus 1. Sedimentation diffusion and sedimentation equilibrium yielded molecular weights of 267,000 and 283,000, respectively. The limiting viscosity (3.6 ml/g) and the f/fo (1.08 at sigma equals to 0.2 g of H2O/g of protein) indicate that the enzyme is a typical globular protein possessing little asymmetry. The circular dichroism spectrum indicates approximately 14% alpha-helix and a far greater amount of random coli than beta structure. The enzyme is acidic, having an isoelectric point of 6.15. In electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate the enzyme exhibits a single band at molecular weight 72,000, a result indicating that the enzyme consists of four subunits of similar molecular weight. Tryptic peptide mapping suggests that the subunits are identical.


The preparation and chemical composition of the multiple forms of beta-glucuronidase from the female rat preputial gland.

Beta-Glucuronidase isolated from the preputial gland of the female rat has previously been shown to be a tetrameric glycoprotein. We have now separated the enzyme into several molecular forms by chromatography on hydroxylapatite columns. The three major forms (A, B, and C) have a very similar or identical amino acid composition, and kinetic and stability studies on forms B and C disclosed no differences between these two forms. However, from C contained much more carbohydrate than forms A and B, which were very similar in carbohydrate composition. The sugars in forms A and B are mannose (2.8%), glucosamine (1.9%), fucose (0.2%), galactose (0.16%), and glucose (0.17%). Form C is a little higher in mannose content, but, more distinctively, is much richer in fucose (0.6%), galactose (1.1%), and glucose (1.5%). The presence of glucose was established by paper chromatography as well as by gas-liquid chromatography, and several special experiments were performed to rule out the possibility that this hexose was present in a persistent contaminant. Direct chemical analysis for sialic acid consistently showed the absence of this sugar in the enzyme. The fact that the carbohydrate-protein linkage is alkali-stable suggests that the linkage involves an asparaginyl-N-acetylglucosamine residue. The NH2-terminal amino acid in the polypeptide chain is leucine.


The alkylation of hemoglobin S by nitrogen mustard. High resolution proton nuclear magnetic resonance studies.

Sickle cell hemoglobin (Hb S) treated with nitrogen mustard (bis(beta-chloroethyl)methylamine hydrochloride) gives two reaction products, one labile and one stable. After dialysis against buffer solution, the remaining stable product is found to inhibit the polymerization of deoxyhemoglobin S. High resolution proton nuclear magnetic resonance has been used to study the structure and function of this stable product and to investigate the nature of the binding sites of nitrogen mustard to the hemoglobin molecule. The NMR results suggest that the nitrogen mustard treatment of Hb S does not alter the heme environment or the subunit interfaces of the hemoglobin molecule. Moreover, the NMR spectra have also shown that the nitrogen mustard reacts with the beta2 histidines of the hemoglobin molecule and have suggested that several other surface amino acid residues of the hemoglobin molecule are also affected by the nitrogen mustard alkylation. These NMR findings are in good agreement with the data obtained from biochemical studies of nitrogen mustard-treated Hb S. The NMR spectra also indicate that nornitrogen mustard (which is also effective in inhibiting sickling) binds with the hemoglobin molecule in a manner identical with nitrogen mustard. Sulfur mustard, on the other hand, produces no observable changes in the aromatic proton resonances, which is consistent with the fact that it does not inhibit the polymerization of deoxy-Hb S.


Enzymes that hydrolyze fungal cell wall polysaccharides. The carbonhydrate constitution of mycodextranse, an endo-alpha (1 yields 4)-D-glucanase from Pencillium melinii.

The chemical constitution of the carbohydrate portion of mycodextranase, an exocellular endo-alpha(1 yields 4) D-glucanase of Penicillium melinii, has been investigated. At least 80% of the carbohydrate, consisting exclusively of mannose and glucose, is released from protein by treatment of the enzyme with 0.05 M potassium hydroxide plus 1 M sodium borohydride or 0.5 M sodium hydroxide at 50 degrees. There is concomitant destruction of 60% of the threonine and 15% of the serine of the treated enzyme and an increase in absorption, at 241 nm, of the treated protein's spectrum, indicative of an O-glycosidic beta-hydroxyamino acyl linkage between untreated protein and its associated carbohydrate. Mannose is the monosaccharide involved in this linkage. Smith degradation, methylation, and glycosidase digestions of the carbohydrate indicate that it is present in mycodextranase as side chains of mannose, glucosyl alpha(1 yields 2)-mannose, and mannosyl alpha(1 yields 2)-glucosyl alpha(1 yields 2)-mannose units with each enzyme molecule bearing a calculated average of 25 side chains. Separation of pronase glycopeptides by gel filtration on Sephadex G-25 revealed that 96% of the carbohydrate is present in the highest molecular weight fraction which contains 60% of the threonine of mycodextranase but only 3.5% of the aromatic acids judged by its absorbance at 280 nm. Further fractionation of this glycopeptide component on Sephadex G-75 indicates carbohydrate is restricted to two fractions, one containing 71% by weight of the threonine and serine of mycodextranase and 56% of its carbohydrate. These results suggest carbohydrate chains of mycodextranase are clustered in a few threonin-rich regions along the polypeptide chain rather than being separated from each other by nonglycosylated areas.


A new type of carbohydrate-protein linkage in a glycopeptide from normal human urine.

A glycopeptide, 3-O-beta-D-glucopyranosyl-alpha-L-fucopyranosyl-L-threonine, has been isolated from normal human urine. The glycopeptide was isolated by gel chromatography, preparative zone electrophoresis, paper chromatography, and high voltage electrophoresis. The average yield of the glycopeptide was in the range of 0.2 to 0.3 mg/liter of urine. Sugar analysis and amino acid analysis gave equimolar amounts of glucose, fucose, and threonine. Linkages and sequential order were established by methylation analysis of the glycopeptide after degradation of the amino acid residue with ninhydrin. The permethylated product was analyzed on gas liquid chromatography and mass spectrometry. Anomeric configuration was deduced from optical rotation.


Elongation of the interchain disulfide bridges of insulin.  A synthetic analog.

The sythesis and isolation in purified form of an analog of insulin with the interchain disulfide bridges elongated by a methylene group is described.  This analog differs from the parent molecule in that the cystein residues occupying positions A-7 and A-20 and involved in the formation of the two interchain disulfide bridges of insulin have been replaced by homocysteine residues.  For the synthesis of this compound the Hcy-7, 20-A chain of sheep insulin was chemically synthesized and isolated in the S-sulfonated form.  Conversion of the latter product to the sulfhydryl derivative and combination with the S-sulfonated form of the B chain of sheep insulin yielded the [Hcy-7, 20-A] insulin.  Isolation of the analog from the combination mixture was effected by chromatography on a carboxymethylcellulose column with acetate buffer (pH 3.3) and an exponential sodium chloride gradient.  This analog, by the mouse convulsion assay methods and in doses at least 40-fold higher than those normally used for insulin assay, was inactive.  By the radioimmunoassay method this synthetic analog was found to possess a potency of 2 i.u./mg.  It is concluded that the biological activity of insulin depends critically on a particular geometry conferred on the molecule by the proper placement of the A and B chains.


Separation and evaluation of the covalent and noncovalent interactions which contribute to the binding of pyridoxal 5'-phosphate to D-serine apodehydratase.

The equilibrium constant (KX) for the reaction D-serine dehydratase + pyridoxamine-P in equilibrium KX D-serine apodehydratase: pyridoxamine-P + pyridoxal-P was determined.  At 25 degreees, pH 7.80, KX increases from 5.4 times 10-minus 5 to 21 times 10-minus 5 as T/2 is increased from 0.33 to 0.66.  A value of 1.3 times 10-minus 4 M at 25 degrees, pH 7.80, T/2 0.33 for the equilibrium constant (KPMP) for dissociation of pyridoxamine-P from D-serine apodehydratase was determined from the ratio of the equilibrium constant for dissociation of pyridoxal-P from holoenzyme to KX.  Pyridoxamine-P and the thiazolidine, formed from pyridoxal-P and cysteine, were found to have similar affinities for D-serine apodehydratase.  Using the affinities of these derivatives as a measure of the noncovalent interactions between cofactor and protein, it was possible to estimate the contribution of the Schiff base linkage to the stability of the complex formed between pyridoxal-P and protein.  The covalent Schiff base linkage in the holoenzyme was found to be no more stable than the Schiff base linkage formed between 6-aminocaproic acid and pyridoxal-P.  The contribution of noncovalent interactions to the stability of the cofactor-protein complex was shown to be at least 20 to 40 times greater than the contribution of the covalent Schiff base linkage.


Two-dimensional gel analysis of soluble proteins.  Charaterization of guinea pig exocrine pancreatic proteins.

A two-dimensional gel technique using slab gel isoelectric focusing in the first dimension and sodium dodecyl sulfate gradient gel electrophoresis in the second dimension has been developed for the separation of soluble proteins larger than 10,000 daltons.  The technique is sensitive to 0.6 mug of protein and recovery of radiolabeled proteins averages 90%.  Analysis of secretory protein from the guinea pig exocrine pancreas shows the presence of 19 distinct high molecular weight proteins.  Each of these proteins has been characterized by isoelectric point, molecular weight, and proportionate mass.  Thirteen of the 19 proteins have been identified by actual or potential enzymatic activity,accounting for 96% of the protein mass resolved by the two-dimensional gels.


Modification of isoleucine-16 acetylated delta-chymotrypsin.

Activation of acetylated chymotrypsinogen with trypsin leads to catalytically active acetylated delta-chymotrypsin containing NH2-terminal isoleucine. The importance of the cationic terminus to the control of the active conformation of acetylated delta-chymotrypsin has been demonstrated (Oppenheimer, H. L., Labouesse, B., and Hess, G. P. (1966) J. Biol. Chem. 241, 2720). Later studies appeared to suggest that the modification of isoleucine-16 of delta-chymotrypsin is not accompanied by the loss of catalytic activity as measured by the hydrolysis of N-acetyl-L-tyrosine ethyl ester (Agarwal, S. P., Martin, C. J., Blair, T. T., and Marini, M.A. (1971)Biochem. Biophys. Res. Commun. 43, 510; Blair, T. T., Marini, M. A., Agarwal, S. P., and Martin, C. J. (1971)  FEBS Lett. 1486) or by the loss of active site content (Ghelis, C., Garel, J. R., and Labouesse, J. (1970) Biochemistry 9, 3902). In the present studies, controlled acetylation of the terminal alpha-aminogroup of acetylated delta-chymotrypsin with acetic anhydride led to a progressive loss of active sites of the enzyme. Determination of the catalytic and kinetic properties of the modified enzyme with the specific ester substrate N-acetyl-L-tyrosine ethyl ester or the nonspecific substrates p-nitrophenyl acetate and cinnamyol imidazole gave nearly identical results. With N-acetyl-L-tyrosine ethyl ester as substrate, the Km (app) values for acetylated delta-chymotrypsin (1.0 plus or minus 0.1 mM) and the modified enzyme (0.67 plus or minus 0.05 mM) are nearly identical and the kcat value is reduced to about 25% in the latter enzyme species. This value correlates well with about 20% of the active sites in this enzyme as measured by the rapid initial liberation of p-nitrophenol. With p-nitrophenyl acetate as substrate, the acylation rate constants (0.13 plus or minus 0.04 s(-1) at pH 6.0, 25 degrees, in 3.3% acetonitrile) and the deacylation rate constants (0.01 s(-1) at pH 8.5, 25 degrees, in 3.3% acetonitrile) are identical for the acetyl isoleucine-16 and the isoleucine-16 enzymes. Furthermore, the residual enzyme activity could be correlated well with the residual NH2-terminal isoleucine content and with the moles of [1--14C]acetyl groups incorporated per mol of the enzyme. The activity associated with the modified enzyme can be attributed to the enzyme species in which isoleucine-16 of acetylated delta-chymotrypsin is not acetylated. These data are in general agreement with the studies of Ghelis et al. (1970) but are in disagreement with the results of Blair et al. (1971) and of Agarwal et al. (1971) and confirm the hypothesis that the final conformation of acetylated delta-chymotrypsin containing an acetylated NH2 terminus is catalytically inactive and resembles acetylated zymogen in many of its physical properties.


The specificity of lambda exonuclease. Interactions with single-stranded DNA.

The lambda exonuclease, an enzyme that has been implicated in genetic recombination, rapidly and processively degrades native DNA, starting at the 5' terminus. The enzyme will also degrade the 5'-terminated strand at a single-stranded branch. The experiments reported here reveal various interactions of the enzyme with single-stranded DNA. The rate of digestion is related inversely to the length of single strands. Chains of 100 nucleotides are digested at about 10% the rate of digestion of native DNA. Digestion of the single-stranded ends of lambda DNA does not appear to occur processively. The enzyme binds to circular as well as linear single strands and the affinity for single strands is also related inversely to the chain length. In an equimolar mixture of single- and double-stranded DNA the action of lambda exonuclease on the latteris about half-inhibited. At 20 degrees the initiation of digestion at the 5' terminus of duplex DNA is blocked sterically when such DNA has 3'-terminal single strands that are longer than 100 nucleotides. Information about these properties is important for the practical use of lambda exonuclease as well as for reflections on the role of the enzyme in genetic recombination.


Expression of differentiated activities in reaggregated brain cell cultures.

Dissociated fetal mouse brain cells are allowed to reassociate in rotation culture to form aggregates.  After several weeks these reaggregated brain cell cultures show markedly increased specific activities of monoamine oxidase, lactate dehydrogenase, and the brain-specific protein S-100, while catechol-O-methyltransferase activity increases slightly.  Similar changes in these activities are found during mouse brain maturation. The amounts of monoamine oxidase, catechol-O-methyltransferase, and S-100 were also determined in surface cultures of fetal mouse brain cells, as well as glioma and neuroblastoma cell lines.  The fetal brain and glial cell cultures possess much higher activities than the cultured neuroblastoma cells.  However, lactate dehydrogenase activity was highest in the glioma and lowest in the surface cultures of fetal brain cells.


The size and detergent binding of membrane proteins.

Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins.  In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles.  A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII).  These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein.  A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent.  Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell.


Binding of colchicine to purified microtubule protein.

The binding of colchicine to tubulin, purified by two cycles of assembly-disassembly, has been studied. Equilibrium studies indicated a dissociation constant which declined during incubation approaching a minimum value of approximately 0.30 times 10- minus 6 M after 13 hours of incubation. Because tubulin is unstable during prolonged incubation (t1/2 of 5.2 hours for free tubulin, t1/2 of 12.5 hours for tubulin bound to colchicine), the equilibrium Kd was felt to be an overestimation of the true Kd. The rate constant of dissociation (k-1 equal to 0.009 hour- minus 1 hour- minus 1) and the rate constant of association (k1 equal to 0.37 times 10-6 M-minus 1) were measured under conditions designed to circumvent or correct for tubulin instability. The dissociation constant determined by the ratio k-1/k1 was 0.024 times -minus 6 M. To determine whether the discrepancy between the "equilibrium" and "kinetic" determined dissociation constants could be accounted for on the basis of tubulin instability, the binding reaction was computer-simulated using the measured association and dissociation rate constants and the rate constants for decay of bound and free tubulin. Computer simulation was in close agreement with the experimentally determined behavior of the reaction during a 13-hour incubation. It is concluded that the Kd determined by equilibrium methodology results in a considerable overestimation due to the instability of tubulin, and that the best estimate for the Kd of the colchicine-tubulin equilibrium is the value determined by the ratio of the rate constants.


The kinetic behavior of an artificial bienzyme membrane.

Artificial membranes bearing immobilized enzymes can be used to study some effects of membrane structure on enzyme kinetic behavior.  The bienzyme system described is a mixture of beta-glucosidase and glucose oxidase.  Gluconolactone, the product of thesecond enzyme, is an inhibitor of the first one.  The resulting feedback effect has been compared using a mixed two-enzyme membrane, two separated one-enzyme membranes, and astirred bienzyme solution.  The feedback effect is quicker and more efficient in the two-enzyme membrane than in solution; it is slower and less efficient in the case of the separated one-enzyme membranes.  Effects of enzyme proximity in the structure are discussed.  Conclusions are drawn concerning the efficiency of feedback mechanisms when enzymes are embedded within a single structure.


The synthesis of a DNA duplex corresponding to the icosanucleotide sequence at the 5' end of messenger RNA from the gene N of bacteriophage lambda.

In connection with work on the nucleotide sequence of the promoter for the gene N of bacteriophage lambda as well as a study of the mechanism of transcription, a 20-unit long DNA duplex corresponding to the known sequence at the 5' end of the above gene transcript has been synthesized. For synthesis, the required duplex was divided into the following deoxyribooligonucleotides: a) the dodecanucleotide, d-A-T-C-A-G-C-A-G-G-A-C-G (II); b) the octanucleotide, d-C-A-C-T-G-A-C-C- (IV); c) the hexanucleotide, d-G-C-T-G-A-rU (I); and d) dodecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T (III). All of the four olignucleotides were chemically synthesized and characterized by extensive chromatographic and fingerprinting methods (after labeling the 5' ends with[32P]phosphate group). Longer polynucleotides (an icosa- and an octadecanucleotide) were prepared by polynucleotide ligase-catalyzed joining of segments I and III and by joining segments II and IV. The use of the octadecanucleotide, d-T-C-A-G-T-G-C-G-T-C-C-T-G-C-T-G-A-rU, in work on the sequence analysis of the promoter is described in the accompanying paper. The octadecanucleotide and icosanucleotide were hybridized together to give the double-stranded duplex.


Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites.

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently.


Embryonal cell surface recognition.  Extraction of an active plasma membrane component.

Plasma membranes obtained from different neural regions of the chicken embryo have previously been shown to specifically bind to homotypic cells and prevent cell aggregation (Merrell, R., and Glaser, L. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 2794-2798). Proteins responsible for the specific inhibition of cell aggregation have been solubilized from the plasma membrane of neural retina and optic tectum by delipidation with acetone followed by extraction with lithium diiodosalicylate. The extracts show the same regional and temporal specificity as previously shown for plasma membrane recognition by the same cells (Gottlieb, D. I., Merrell, R., and Glaser, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1800-1802). Two micrograms of the most purified protein fraction inhibits the aggregation of 2.5 times 10(-4) cells under standard assay conditions. This represents a 20-fold increase in specific activity compared to whole membranes.


Active alanine transport in isolated brush border membranes.

Uptake of L-alanine against a concentration gradient has been shown to occur with isolated brush border membranes from rat small intestine. An alanine transport system, displaying the following characteristics, was shown: (a) L-alanine was taken up and released faster than D-alanine; (b) Na+ as well as Li+ stimulated the uptake of both stereoisomers; (c) the uptake of L- and D-alanine showed saturation kinetics; (d) countertransport of L-alanine was shown; (e) other neutral amino acids inhibited L-alanine but not D-alanine entry when an electrochemical Na+ gradient across the membrane was present initially during incubation. No inhibition occurred in the absence of a Na+ gradient. The electrogenicity of L-alanine transport was established by three types of experiments: (a) Gradients of Na+ salts across the vesicle membrane (medium concentration greater than intravesicular concentration) supported a transient uptake of L-alanine above equilibrium level, and the lipophilic anion SCN- was the most effective counterion. (b) A gradient of K= across the membrane (vesicle greater than medium) likewise supported active transport of L-alanine into the vesicles provided the K= conductance of the membrane was increased with valinomycin. (c) Similarly, a proton gradient (vesicle greater than medium) in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an agent known to increase the proton conductance of membranes, produced an overshooting L-alanine uptake. A consideration of the possible forces, existing under the experimental conditions, suggests that the gradients of SCN-, K+ in the presence of valinomycin, and H+ in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone contribute to the driving force for L-alanine transport by creating a diffusion potential. Since the presence of Na+ was required in all experiments with active L-alanine transport these results support the existence of a transport system in the brush border membrane which catalyzes the co-transport of Na+ and L-alanine across this membrane.


Procollagen assembly and secretion in embryonic chick bone.

Chick embryo skull  bones incorporated radioactive proline and cystein into procollagen in short term organ culture.  Pulse-chase experiments showed that individual precursor chains (pro-alpha1 and pro-alpha2) were formed first and that these were subsequently linked by disulfide bounds into trimers.  Radioautography showed that labeled material was secreted 30 min after adding label to the cells, and electrophoretic analyses showed that after this time completed labeled collagen molecules appeared.  Conversion from disulfide-linked procollagen to collagen proceeded in more than one step.  An intermediate form consisting of shorter chains, which were still trimerically linked, was found.


Scheuermann's kyphosis. Results of surgical treatment by posterior spine arthrodesis in twenty-two patients.

Twenty-two patients with Scheuermann's kyphosis were treated with correction, Harrington instrumentation (in twenty-one), and posterior spine fusion. This procedure relieved pain and corrected deformity in all patients but loss of correction of 5 degrees or more occurred in sixteen of the twenty-two patients, including all those whose initial kyphosis was greater than 70 degrees. The incidence of complications was so high that this procedure should ordinarily be performed only for disabling pain or spinal cord compression.


The association of scoliosis and congenital heart defects.

The effects of cardiac surgery on patients with congenital heart defects and the subsequent development of scoliosis were studied. A group of 998 patients with congenital heart defects who were less than sixteen years old were operated on at the Mayo Clinic during the ten-year period 1950 through 1959. Standing roentgenograms of the spine were made of 377 of the patients ten years or more after surgery. The ages of the patients at follow-up ranged from ten years and seven months to thirty-five years and three months, with a mean age of twenty-one years and four months. The average length of follow-up was fourteen years and eleven months. Of the 377 patients, thirty-two (8.5 per cent) had curves greater than 20 degrees. The female:male ratio of patients with congenital heart defects was 1:1, whereas of those who developed scoliosis it was 5:3. There was no correlation between scoliosis and the following: patient's sex, cardiac abnormality, size or side of the heart, side of the aortic arch, presence of cyanosis, age at surgery, number and type of surgical incisions, number and side of ribs removed, or number and type of surgical procedures.


Progression of congenital scoliosis due to hemivertebrae and hemivertebrae with bars.

From 315 cases of congenital scoliosis seen from 1925 to 1970, sixty cases of scoliosis or kyphoscoliosis due only to a hemivertebra, hemivertebrae, or a unilateral bar associated with the hemivertebrae were selected, classified into six types, and analyzed. Progression occurred in all but the balanced hemivertebrae group in two patients with cervical-high thoracic curves due to multiple hemivertebrae. Location of the hemivertebra or hemivertebrae and the presence of unilateral bars are the major determinants of deformity. The rate of progression of the scoliosis was variable, ranging from 1 to 33 degrees per year (average, 4 degrees per year).


Degenerative spondylolisthesis. Predisposing factors.

Study of twenty skeletons and two hundred patients with degenerative spondylolisthesis established that it occurred four times more frequently in females, six to nine times more frequently at the interspace between the fourth and fifth lumbar vertebrae than at adjoining levels, three times more frequently in blacks than in whites, and four times more frequently the fifth lumbar vertebra was sacralized. It did not occur before the fifth decade or in conjunction with spina bifida or isthmic spondylolisthesis. The slipping occurs as a result of degenerative disease of the articular processes, but it never exceeds 30 per cent. When symptoms are severe and unrelieved by conservative treatment (10 per cent of our patients), decompression laminectomy and excision of the medial portion of the articular processes affords relief of pain. The predisposing factor is a straight, stable lumbosacral joint which puts abnormal stress on the intervertebral joint between the fourth and fifth lumbar vertebrae, leading to decompensation of disc and ligaments, hypermobility, and degeneration of the articular processes allowing forward slipping.


Silastic arthroplasty of the metacarpophalangeal joints in rheumatoid arthritis.

Fifty rheumatodi patients (fifty hands) had Silastic joint implants in 144 metacarpophalangeal joints. Average follow-up time was 2.5 years (maximum 3.5 and minimum 1.5). At clinical examination, gross power, range of motion, lessening of ulnar deviation, relief of pain, and appearance were determined. The results were correlated with preoperative indications. Postoperatively, there was no significant gain in gross power. The range of motion was somewhat increased and the lessening of ulnar deviation was marked. Relief of pain was achieved. Complications included fracture of four of the 144 implants, one infection, and six cases of tendon adhesions.


Opponensplasty in intrinsic-muscle paralysis of the thumb in leprosy.

For eighty-six cases of leprous paralysis of the intrinsic muscles of the hand, a modified Bunnell opponensplasty (transfer of a finger flexor) was done. Good results were seen in eighteen and fair results in sixty-three. The five poor results occurred because of infection, weakness, or deformity. Adjuvant procedures, as needed, were interphalangeal arthrodeis of the thumb, capsulorrhaphy of the wrist, and additional tendon transfers.


Femoral fractures in conjunction with total hip replacement.

We reviewed thirty-eight cases, in thirty-six patients, of fracture of the femur distal to the base of the neck incurred in conjunction with total hip replacement. There were thirteen preoperative, eighteen intraoperative, and seven postoperative fractures. The cases were contributed by thirteen surgeons. We found that the preoperative fractures with pre-existing disease in the hip joint were effectively treated by primary total hip replacement using custom-made femoral components with long necks or long stems, or both. The intraoperative femoral fractures usually occurred while the surgeon was reaming the canal, seating the femoral component, or manipulating the femur in patients who were predisposed to fracture. Theoretically these lesions can be treated like preoperative fractures, but this demands immediate access to custom-made femoral components with long necks or long stems, or both, along with an appreciation of the extent and significance of the fracture. Inadequate fixation was found to lead to painful non-union or late loosening of the femoral component in four of eighteen patients. Postoperative fractures occurred too rarely for us to draw any definite conclusions about management, except to say that surgical treatment can be hazardous and traction has been successful in this series and in other reports. Prophylactic measures, however, may help to prevent postoperative femoral fractures. Most of these fractures occur through a cortical defect near the tip of the femoral component. A long-stem femoral component may help to prevent postoperative fractures whenever a proximal cortical defect of the femur is present preoperatively or is created at surgery.


Paralytic dislocation of the hip in myelodysplasia. The role of the adductor transfer.

In thirty-nine patients with myelodysplasia at the third, fourth, or fifth lumbar level and subluxation or dislocation of the hip (fifty-nine hips involved), the following surgical approaches were utilized to balance the muscles about the hip: In one group (twenty hips in eleven patients) the origins of the adductors longus, brevis, and gracilis were transferred to the ischial tuberosity, and in fifteen hips in that group the iliopsoas also was transferred to the greater trochanter when the abductors were severely weakened. In the second group (thirty-nine hips in twenty-eight patients), only the iliopsoas transfer was done. Group I patients demonstrated improved muscle balance with less severe flexion-adduction contractures; increased power in abduction and extension; and a higher incidence of stable hips at follow-up.


Osteochondritis dissecans in Legg-Calvé-Perthes disease.

Seven cases of osteochondritis dissecans following Legg-Calvé-Perthes disease are presented and the literature is reviewed. This complication could not be predicted during the early stages of the Legg-Calvé-Perthes process. A high index of suspicion is necessary to recognize the lesion. Conservative treatment is suggested.


Normal postural stability and steadiness: quantitative assessment.

A force platform was utilized to provide a new and sensitive means to measure postural steadiness and stability. Normal standards are presented for the center of pressure of the vertical supportive force during standing and sustained weight-shifting of normal men in three age groups. Two distinguishing characteristics were seen for normal upright posture: (1) a large area of stability over which weight can be safely shifted and maintained, and (2) steadiness such that the center of pressure fluctuates incessantly, traversing large total excursions, while remaining remarkably close to the mean point. The youngest age group showed the greatest steadiness and stability and the oldest group showed the least.


Bone composition and phosphatase activity in magnesium deficiency in rats.

The effect of magnesium deficiency on phosphatase activity and bone composition was determined in the femora of young rats. In the right distal metaphysis the acid and alkaline phosphatase activities were decreased in magnesium-deficient rats, and the activity of bone alkaline phosphatase in the incubation mixture after adding magnesium was significantly greater in the magnesium-deficient than in the control rats. In the left distal metaphysis the water content was significantly lower in the magnesium-deficient rats at the fifth week but not at the third week. Conversely, the ash content of metaphyseal bone was significantly increased in magnesium-deficient rats at the fifth week, but not at the third week. The magnesium and phosphorus contents were abnormally low in the deficient bone at both periods. The calcium content was increased in the deficient bone at the third week, but not at the fifth week.


The effects of magnesium deficiency on the host response to intramuscular bone matric implanted in the rat.

HCl-demineralized mid-diaphyseal allogeneic segments of tibia were implanted in the lumbar muscles of rats fed either a magnesium-deficient or a magnesium-supplemented diet for one week prior to implantation and for three weeks thereafter. Histological studies, histochemical mucopolysaccharide determinations, tests for protease content of undemineralized bone tissue, and microradiography showed that in the normal hosts mostly trabecular bone grew into the heterotopic site, whereas in the deficient rats a large fibrous covering formed about the implant and in many instances cartilage was present in multiple locations under the fibrous coat. In some instances cartilage wedges, not preceded by multinucleated giant cells (matrixclasts), invaded the implant. This fibrocartilaginous response to an exogenous inductor resembled the modified growth pattern of bone recently reported in magnesium-deficient rats.


In vitro wear of articular cartilage.

Seeking a reliable chemical index of the wear of articular cartilage during in vitro experiments, the contents of hydroxyproline, hexosamine, and the amino acid composition of adult bovine articular cartilage were determined as functions of depth from the surface. The hydroxyproline content, expressed as per cent of dry weight of tissue, was constant throughout the thickness of the tissue except in a surface region approximately twenty-five micrometers thick; the hexosamine content in this region was less than in the interior of the tissue; the collagen content was higher and the amino acid composition was less like that of pure collagen here than in the interior, indicating that the content of noncollagenous protein in the superficial layer of cartilage was greater than that in the interior. It was also evident that adult bovine articular cartilage contains significant amounts of collagen with a low hydroxylysine content, presumably Type I as well as Type II. Since the content of hydroxyproline is constant throughout the cartilage and the collagen and proteoglycan constituents of the intact tissue are relatively insoluble, the hydroxyproline content of the lubricant and the wear debris can be used to measure the extent of wear of articular cartilage during in vitro experiments. However, approximately 10 per cent of the hydroxyproline and 50 to 60 per cent of the glycosaminoglycans of the wear debris are dissolved in the lubricating fluid. Therefore, both the lubricant (solvent) and the solid wear debris must be analyzed to determine the amount of cartilage wear.


A simplified compression arthrodesis of the shoulder.

An initial series of eleven consecutive patients who had arthrodesis of the shoulder by means of a simplified compression method was reviewed. The operation was successful in all patients and subjectively satisfactory in all but one.


A fixed-ankle, below-the-knee orthosis for the management of genu recurvation in spastic cerebral palsy.

A fixed-ankle, below-the-knee molded plastic splint designed to maintain the ankle in 5 degrees of dorsiflexion and correct any foot deformity was used on eighteen extremities of twelve children with spastic cerebral palsy and genu recurvatum. After an average follow-up of twenty-six months, the genu recurvatum was well controlled and gait was improved.


Complications of halo-pelvic distraction for scoliosis.

A survey has been undertaken of the various complications of halo-pelvic distraction in 118 patients with scoliosis prior to spinal fusion. In the first sixty-two patients the standard solid distraction rods were employed. The neurological complications included ten cases of cranial nerve lesions and two cases of paraplegia, one of them permanent. Springs were then incorporated in the distraction rods so as to allow direct readings of the distraction forces, and a total force of 18 kilograms was not exceeded in the last fifty-six patients. No further serious neurological complications occurred, but the amount of correction achieved in the adolescent and juvenile idiopathic types of scoliosis was reduced.


Infantile idiopathic scoliosis. Causative factors, particularly in the first six months of life.

An attempt has been made to determine the aetiological factors in infantile idiopathic scoliosis from a clinical, genetic and epidemiological survey of 134 infants, ninety-seven of whom developed a curve in the first six months of life. Plagiocephaly was present in all cases; mental retardation occurred in 13 per cent of males with progressive scoliosis; congenital dislocation of the hip occurred in 3-5 per cent of cases and congenital heart disease in 2-5 per cent; and inguinal hernia was found in 7-4 per cent of males. Approximately 3 per cent of parents and 3 per cent of sibs had the same deformity, thirty times the general population frequency for the Edinburgh area. Other positive findings included an excess of breech presentations and of premature, low birthweight males, and a preponderance of curves developing in the winter months. Infants with progressive scoliosis tended to have older mothers and to come from poorer families. Only three children all with resolving scoliosis, habitually lay prone in early infancy, in marked contrast to North American infants where this posture is usual. The almost complete absence of infantile idiopathic scoliosis in North America is noted and it is thought that the two facts may be related. The aetiology is likely to be multifactorial, with a genetic tendency to the deformity which is either "triggered off" or prevented by external factors.


Familial scoliosis. A clinical report.

Scoliosis appeared in fifteen members of a family in three generations. The eight members examined showed an idiopathic type of scoliosis. Father-to-son transmission occurred more than once. Although at first appearance transmission of the scoliosis is as an autosomal dominant trait, the concept of polygenic inheritance, as has been shown in previous population studies, could also explain the inheritance in this family.


Excision of tumours of humerus and femur, with restoration by internal prostheses.

A personal experience is recounted of operations in cases of tumour involving the humerus or femur with restoration by endoprostheses. Twenty-four patients were treated in this way from 1950 to 1969 inclusive and have been followed up for between four and twenty-four years. The patients selected for treatment have presented chondrosarcoma (ten), so-called benign giant-cell tumour of bone, usually recurrent (nine), angiomatous osteolysis (two), seemingly single thyroid or renal metastasis (two), and ununited pathological fracture after irradiation of a tumour (one). Development of the prostheses from early beginnings is outlined. Some points in surgical management are referred to. The complications and results are recorded.


Surgical prophylaxis of subluxation and dislocation of the hip in cerebral palsy.

The clinical and radiological state of the hips of a group of children with cerebral palsy treated without operation is compared with that in a group treated by operation to correct adduction and flexion deformity and to obtain balanced action in the hip muscles. In the first group, 11 per cent of hips were dislocated, 28 per cent subluxated, 46 per cent dysplastic and 15 per cent normal. In the second group no hip was dislocated, 13 per cent were subluxated, 35 per cent dysplastic and 52 per cent normal. Surgical intervention is indicated clinically for a range of abduction diminishing to less than 45 degrees and--on radiological criteria--for early dysplastic changes, especially a break in Shenton's line, irrespective of the patient's age, severity of involvement or neurological maturity. Prevention of subluxation or dislocation improves function and diminishes the liability to develop a painful hip in adolescence or early adult life.


A study of the "capsular" shadow in disorders of the hip in children.

Displacement and blurring of the soft-tissue shadows about the hip has been described in several conditions, particularly in transient synovitis. A study was made between such displacements and the posture commonly adopted by an "irritable hip". Examination of radiographs of normal hips, and of those in cases of transient synovitis and Perthes' disease showed that the appearance of "capsular swelling" is related to the position of lateral rotation and abduction. This was confirmed by anatomical dissections of the lateral plane, which appears to be an intermuscular plane lying anterior to the hip, and an explantation is given for the blurring which may accompany its lateral displacement. "Capsular swelling" appears to be a radiological artefact.


Metatarsal osteotomy for metatarsalgia.

An oblique osteotomy in the distal half of the metatarsal shaft is described for the treatment of metatarsalgia due to prolapse of one or more of the middle three metatarsal heads. Thirty-eight patients who have had this operation have been followed up for a period of from two to five years. The operation is simple, recovery is rapid and symptoms have been well relieved.


Surgical treatment of chronic dislocation of the sterno-clavicular joint.

The costo-clavicular ligament is always ruptured in dislocation at the sterno-clavicular joint. Anterior, superior or posterior displacement of the medial end of the clavicle may occur. Acute dislocation usually responds to conservative treatment and operation is seldom required. Chronic, or recurrent, dislocation may cause pain and disability on strenuous activity and necessitate surgical treatment. The operation of tenodesis of the subclavius tendon with capsulorrhaphy described by Burrows (1951) has been adopted. The intraarticular meniscus is often damaged and displaced, and may block reduction; its removal is then necessary. In addition, a threaded Stinmann pin transfixing the joint has been found useful to maintain the stability of reduction. The operation has been performed on five patients, four of whom had excellent results. The fifth patient disrupted the repair in a drinking bout shortly after the operation.


Hypoxaemia after fractures.

Serial arterial blood-gas analyses showed a phase of primary hypoxaemia in thirty-two out of fifty fracture patients (64 per cent) without head, chest or abdominal injury. The incidence was greater in those with shaft fractures of the femur or tibia or both, than in those with fractured hips, and was related to the severity of injury and the nature of the accident. Most affected subjects were already hypoxaemic on admission to hospital; the arterial PO2 commonly fell to between 60 and 70 millimetres of mercury, and the episode generally lasted a few days. The hypoxaemia was generally subclinical but four patients developed mild fat embolism. Early hypoxaemia was not found in six patients admitted with only soft-tissue injuries. One or more subsequent attacks of subclinical hypoxaemica, each lasting a few days, occurred in half of those previously affected. Most episodes followed fracture operation or manipulation. Pulmonary thromboembolism seemed responsible in two patients, but it could be excluded in others given oral anticoagulant prophylaxis from soon after admission. Pulmonary fat embolism is the most likely explanation of the primary episodes and could account for most of the subsequent periods of hypoxaemia.


Late results of transfer of the tibial tubercle for recurrent dislocation of the patella.

The authors wished to determine the late results of the Hauser operation, with special reference to the development of osteoarthritis. Predisposing factors associated with recurrent dislocation of the patella were also investigated. Thirty-five patients with forty-four surgically treated knees attended for review, ten to twenty-five (average sixteen) years after operation. Two patients had subsequently undergone excision of the patella. Ten patients gave a family history of recurrent dislocation of the patella and seven patients showed generalised joint laxity. Pain was present in eight knees before operation and was present in thirty-three knees (75 per cent) at the time of review. Patella crepitus was present in thirty-seven out of forty-two knees (88 per cent) at review. Osteoarthritis was present in thirty out of forty-two knees (70 per cent). The incidence increases with time since operation and the present age of the patient. It is concluded that the Hauser operation prevents further dislocation but does not prevent the development of osteoarthritis. It is possible that a simple soft-tissue operation which effectively prevents dislocation might achieve the same results.


Phosphate compounds in bone scanning.

Bone scanning with radioactive isotopes has been used to study a wide variety of disorders. Recently certain phosphate compounds, labelled with technetium, have been used as bone scanning agents. The comparative merits of three technetium-labelled phosphate compounds currently available for bone scanning--pyrophosphate, tripolyphosphate and ethylhydroxydiphosphonate (EHDP) have been compared in rabbits. Each substance was injected into ten rabbits and blood was withdrawn at regular intervals. The animals were killed at four hours and the blood and tissue samples were assayed for radioactivity. The results show that EHDP has a more rapid blood clearance than the other two agents, with a resultant improvement in the bone to soft-tissue ratio. Of the three substances investigated technetium-labelled EHDP was the best and might allow the technique of scanning to be used on a wide scale for the general study of bone and its pathology.


Massive osteolysis of the humerus with spontaneous recovery.

An elderly woman presented with a pathological fracture of the right humerus. Progressive dissolution of the shaft of this bone took place over six months. No cause could be established and the patient refused biopsy. With only simple splintage for treatment the humeral shaft gradually reformed and re-ossified over a period of two years. The patient has been under review for four and a half years and no further pathology has come to light. The cause of the osteolysis remains obscure.


Animal experiments in radiotherapy I -- small animals.

This is a brief review of the contribution that the small laboratory animal, in particular the mouse, has made to experimental radiotherapy. Despite the great differences between mouse and man (e.g. size and life span), there are similarities of tissue cell kinetics and radiation response which greatly aid in the mouse-to-man extrapolation. The crucial advantage of being able to perform experiments with spontaneous tumors transplanted into syngeneic hosts is discussed. Examples are given of work using the tumor control dose (TCD50) endpoint, the endpoint dilution assay, two different clone-counting assays, and the tumor regrowth system. The sophistication that can be achieved with an animal as small as a mouse is illustrated by two studies: one of prolonged intra-arterial catheterization, the other of blood flow measurements in an irradiated limb.


Motility in Echinosphaerium nucleofilum. I. An analysis of particle motions in the axopodia and a direct test of the involvement of the axoneme.

The motion of particles in the axopodia of Echinosphaerium nucleofilum is saltatory. In the present study, photokymograph records of 123 motions from six axopodia have been analyzed. Particles followed rectilinear paths of from 1 to 15 mum while in continuous motion at an average velocity of 0.66 plus or minus 0.32 mum/s. The velocity of the particles was variable in 36% of the cases measured. Some motions were punctuated by pauses either before continuing in the same direction or reversing. Frequently, several particles were moving at the same velocity, but neighboring particles showed no motion or moved in the opposite direction. Two particles occasionally contacted one another and travelled as a unit for varying lengths of time but subsequently moved independently. These motions reflect the underlying mechanism of motive force production. Furthermore, a glass microneedle can be substituted for the microtubular axoneme in the axopodia. In these artificial axopodia, bidirectional particle motions occurred which were similar to those in normal axopodia. Colchicine, at the threshold dose for axonemal dissolution, had no affect on these particel motions. It is concluded that the microtubular axoneme is not responsible for particle motions and also that individual microtubules are unlikely candidates for motive force production in this system.


Motility in Echinosphaerium nucleofilum. II. Cytoplasmic contractility and its molecular basis.

Echinosphaerium nucleofilum exhibits at least three kinds of movement: locomotion by the bending and shortening of its many axopodia, feeding by means of food-cup pseudopodia formed from its cortical cytoplasm, and saltatory motion of cytoplasmic particles, especially in the cortex and axopodia. Since previously presented evidence indicated that the microtubular axoneme is not essential for particle motion, the cytoplasm was investigated for the possible existence of contractile behavior and for the possible presence of linear elements other than microtubules. Cytoplasm can be isolated in physiological media in which rigor, relaxation, and contraction can be induced, as in muscle, by manipulating the concentrations of calcium ions and magnesium-adenosine triphosphate. Contraction is initiated by calcium ions at concentrations above 2.4 times 10-minus 7 M. The rigor-to-relaxation transition occurs at subthreshold calcium concentrations on the addition of 10-minus 3 M ATP. Negatively stained preparations of isolated cytoplasm show two types of filaments: thin filaments identified as cytoplasmic actin by virtue of their binding heavy meromyosin from striated muscle in characteristic arrowhead arrays, and thicker filaments which do not strictly resemble myosin aggregates from muscle or amoeba but could conceivably by myosin aggregated in an unfamiliar form.


Microfilamentous system and secretion of enzyme in the exocrine pancreas. Effect of cytochalasin B.

The microfilaments in the acinar cell of the exocrine pancreas are essentially located in the apical part of the cell: thin microfilaments (50 A), cytochalasin B (CB)-sensitive, form the axis of the microvilli and a network lying beneath the apical membrane; thicker filaments (100 A), at least partly CB-insensitive, form bundles parallel to the plasma cell membrane and the desmosomal links. CB interaction with the acinar cell of the exocrine pancreas involves at least two sites: a membrane site involved in the inhibitory effect of CB on the monosaccharide transport and a less sensitive site at the filamentous level at least partly responsible for the inhibitory effect of CB in the secretion of the exportable enzyme from the pancreatic cell. CB did not alter the energy balance of the acinar cell nor the exchanges of 15-Ca between the extracellular medium and the pancreatic tissue. CB (2 times 10-minus 7 and 2 times 10-minus 6 M) has secretagogue properties whereas CB (2 times 10-minus 5 M) has inhibitory effect on stimulated secretion and secretagogue properties. The mechanism of these secretory effects is not yet explained. The analysis presented in this investigation affords strong evidence for the involvement of the microfilamentous network in the last steps of the secretory cycle in the acinar cell of the exocrine pancreas.


Demonstration of ricin-binding sites on the outer face of azurophil and specific granules of rabbit polymorphonuclear leukocytes.

The presence of carbohydrate residues on the outer surface of PMN granules has been demonstrated by the use of ricin-conjugated ferritin. The binding of the lectin was inhibited by alpha-lactose. No difference in the binding densities of azurophil or specific granules was observed.


Oriented thick and thin filaments in Amoeba proteus.

Actin and myosin filaments as a foundation of contractile systems are well established from ameba to man (3). Wolpert et al. (19) isolated by differential centrifugation from Amoeba proteus a motile fraction composed of filaments which moved upon the addition of ATP. Actin filaments are found in amebas (1, 12, 13) which react with vertebrate heavy meromyosin (HMM), forming arrowhead complexes as vertebrate actin (3, 9), and are prominent within the ectoplasmic tube where some of them are attached to the plasmalemma (1, 12). Thick and thin filaments possessing the morphological characteristics of myosin and actin have been obtained from isolated ameba cytoplasm (18, 19). In addition, there are filaments exhibiting ATPase activity in amebas which react with actin (12, 16, 17). However, giant ameba (Chaos-proteus) shapes are difficult to preserve, and the excellent contributions referred to above are limited by visible distortions occurring in the amebas (rounding up, pseudopods disappearing, and cellular organelles swelling) upon fixation. Achievement of normal ameboid shape in recent glycerination work (15) led us to attempt other electron microscope fixation techniques, resulting in a surprising preservation of A. proteus with a unique orientation of thick and thin filaments in the ectoplasmic region.


Endothelial cell junctions.

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.


In vivo recovery of muscle contraction after alpha-bungarotoxin binding.

Acetylcholine receptors were inactivated in vivo at the mouse neuromuscular junction using alpha-bungarotoxin (alpha-BTX). It was found that neurally produced muscle contraction recovered within 4-8 days (halftime similar to 3 days). Actinomycin D interfered with this recovery, but did not affect normal nerve-stimulated muscle contraction. If the response was initially eliminated by [125-I]alpha-BTX and the end plates examined by EM autoradiography, no evidence of mass internalization of bound radioactivity during recovery was seen. The fine structure of the end plates and muscle was unaltered during the post-alpha-BTX recovery period.


Colchicine inhibition of plasma protein release from rat hepatocytes.

Colchicine, both in vitro and in vivo, inhibits secretion of albumin and other plasma proteins. In vitro, secretion by rat liver slices is inhibited at 10-minus 6 M with maximal effect at 10-minus 5 M. Inhibition of secretion is accompanied by a concomitant retention of nonsecreted proteins within the slices. Colchicine does not inhibit protein synthesis at these concentrations. Vinblastine also inhibits plasma protein secretion but lumicolchicine, griseofulvin, and cytochalasin B do not. Colchicine also acts in vivo at 10-25 mumol/100 g body weight. Inhibition of secretion is not due to changes in the intracellular nucleotide phosphate levels. Colchicine, administered intravenously, acts within 2 min and its inhibitory effect lasts for at least 3 h. Colchicine has no effect on transport of secretory proteins in the rough or smooth endoplasmic reticulum but it causes these proteins to accumulate in Golgi-derived secretory vesicles.


Morphologic evidence of photoreceptor differentiation of pinealocytes in the neonatal rat.

The pineal body and the retina of the neonatal Sprague-Dawley rat were studied by light and electron microscopy, and the morphologic differentiation of the parenchymal cells of the pineal body was compared with that of the developing photoreceptor cells of the retina. Between the ages of 4 and 12 days after birth, some of the developing pinealocytes were observed to become elongated and polarized, with their nuclei located at one pole. "Synaptic" ribbons were observed within the cell body. At the opposite pole the cells developed elongated cell processes that initially contained microtubules and ribosomes. These cell processes projected into luminal spaces and were attached by structures resembling zonulae adherentes to the adjacent cells. Extending from the tips of the cell processes, cilia with a 9 + 0 arrangement were observed. Lamellated and vesicular membranes were noted at the tips of the cilia. Such morphologic differentiation, however, could be observed only in rats younger than 17 days. Comparison of the morphologic features of the neonatal pinealocytes with those of the developing retinal photoreceptor cells showed much similarity. It is suggested that the pinealocytes of the neonatal rat undergo "photoreceptor-like" differentiation during a transient neonatal period. Such morphologic differentiation may provide an explanation for light-induced biochemical changes described in neonatal rats whose eyes had been enucleated.


Lack of dynein arms in immotile human spermatozoa.

Sermatozoa from two brothers who are not twins were found to be straight and immotile. Examinations of the sperm showed that oxygen consumption and lactic acid production were normal; viability tests showed that the percentage of dead sperm was not increased. The ultrastructural appearance of the sperm tail was normal except for a complete lack of dynein arms and some irregularities in the arrangement of the accessory fibers and the longitudinal columns of the fibrous sheath. The mitochondrial apparatus and the sperm head conform to the conventional model. According to the sliding-filament hypothesis first proposed by Afzelius (1959. J. Biophys. Biochem. Cytol. 5:269.), the arms are responsible for the bending movements of the tail. The simplest explanation for the simultaneous lack of arms and sperm motility appears to be that the two brothers have a genetic disorder involving production, assembly, or attachment of the dynein arms.


Mouse sperm basic nuclear protein. Electrophoretic characterization and fate after fertilization.

Mouse sperm were labeled in vivo with [3H]arginine. The sperm were then followed autoradiographically from the time of label incorporation until after fertilization. The label was completely lost from the sperm head after fertilization, during the oocyte's second meiotic division. That the [3H]arginine was incorporated into a sperm-specific basic protein was demonstrated by fractionating acid extracts of epididymal and ejaculated sperm with polyacrylamide gel electrophoresis. All the histone fractions were resolved in the epididymal extracts, but in addition a band was present that migrated faster than histone F2al and slower than the salmon protamine used as a marker. This new fraction (proposed name: musculine) was also present in ejaculated sperm; it was shown to be the only fraction that was labeled. Musculine therefore represents the end product of a histone transition in mice. It is, however, according to our electrophoretic characterization, not identical to the classical fish protamines. Rather, musculine resembles bovine sperm nuclear protein. Since the loss of this fraction from the sperm head was coincident with the rearrangement of the male genome, before its resumption of transcription, it is suggested that musculine is involved in the control of chromatin that accompanies spermiogenesis and fertilization.


Laser-induced spreading arrest of Mytilus gill cilia.

Using a "slit camera" recording technique, we have examined the effects of local laser irradiation of cilia of the gill epithelium of Mytilus edulis. The laser produces a lesion which interrupts epithelial integrity. In artificial sea water that contains high K+ or is effectively Ca++ free, metachronism of the lateral cilia continues to either side of the lesion with only minor perturbations in frequency synchronization and wave velocity, such as would be expected if metachronal wave coordination is mechanical. However, in normal sea water and other appropriate ionic conditions (i.e., where Ca++ concentration is elevated), in addition to local damage, the laser induces distinct arrest responses of the lateral cilia. Arrest is not mechanically coordinated, since cilia stop in sequence depending on stroke position as well as distance from the lesion. The velocity of arrest under standard conditions is about 3 mm/s, several orders of magnitude faster than spreading velocities associated with diffusion of materials from the injured region. Two responses can be distinguished on the basis of the kinetics of recovery of the arrested regions. These are (a) a nondecremental response that resembles spontaneous ciliary stoppage in the gills, and (b) a decremental response, where arrest nearer the stimulus point is much longer lasting. The slower recovery is often periodic, with a step size approximating lateral cell length. Arrest responses with altered kinetics also occur in laterofrontal cilia. The responses of Mytilus lateral cilia resemble the spreading ciliary arrest seen in Elliptio and arrest induced by electrical and other stimuli, and the decremental response may depend upon electrotonic spread of potential change produced at the stimulus site. If this were coupled to transient changes in Ca++ permeability of the cell membrane, a local rise in Ca++ concentration might inhibit ciliary beat at a sensitive point in the stroke cycle to produce the observed arrest.


Morphological alterations and ganglioside sialyltransferase activity induced by small fatty acids in HeLa cells.

Incubation of HeLa cells in the presence of millimolar concentrations of propionate, butyrate, or pentanoate increases the specific activity of CMP-sialic acid:lactosylceramide sialyltransferase 7-20-fold within 24 h. Longer-chain saturated fatty acids or acetate are much less effective, decanoate showing no induction. Unsaturated fatty acid analogs of butyrate and other compounds are ineffective. Only the three most effective compounds also produce characteristic smooth extended cell processes in HeLa cells. Butyrate (5 mM) induces the sialyltransferase after a 4-h lag, producing maximum specific activity by 24 h. The amount of sialyl-lactosylceramide, the glycolipid product of the enzyme, increases during that time 3.5 times more than in control cultures. No other glycosphingolipid enzyme is significantly altered by butyrate exposure. The cellular shape changes occur 2-3 h later than the increase of sialyltransferase activity, and both processes require the continuous presence of inducer and the synthesis of RNA and protein but not the synthesis of DNA or the presence of serum.


Role of sulfhydryl groups in cellular adhesiveness.

Aggregation of chick embryonic liver and kidney cells was completely abolished by a treatment with carboxypyridine disulfide which binds -SH groups. The effect could be reversed by a subsequent treatment with some thiols. Inhibition of RNA synthesis or respiratory metabolism did not prevent cell aggregation. Cell adhesion is discussed in the light of these observations.


Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.


Sodium and calcium transport in cat red cells.

The transport of radioactive sodium and calcium in high sodium low potassium cat red blood  cells has been studied under various experimental conditions. In these cells, calcium uptake was found to increase by 25-fold when cell volume was decreased from 1.0 normal to 0.55. Increasing cell volume from 1.0 to 1.1 normal was found to decrease calcium-uptake by 30%. N-ethylmaleimide, NEM, phloretin, colchicine, and vinblastine were found to inhibit Na-uptake by these cells. Among these four agents, phloretin was the most potent inhibitor of Na-influx. It appears that phloretin produces its effect through its interaction with the cell membrane and not with cell interior.


A light- and electron-microscope study of the oocyte nucleus during development of the antral follicle in the prepubertal mouse.

The ordered changes which occur in the structural organization of the mouse oocyte nucelus during the preparatory, the maturative and the preovulatory stages of antral follicle development, have been studied under both light and electron microscopy. All observations have been made on those antral follicles whose development is initiated on postnatal day 14 and completed by postnatal day 28 in prepubertal animals of the ICR albino mouse strain. The formed entities that can be recognized within the oocyte nucleus during that period are the condensing bivalents, the heterochromatic knobs, the nucleolus and the extranucleolar bodies. At the onset of antral follicle development, the highly unravelled dictyate bivalents are seen to take on a lampbrush-type configuration. Subsequent condensation of these lampbursh bivalents appears to be a very gradual and lengthy process that extends over almost the entire period of antral follicle development. The shortening and thickening of the lampbrush bivalents are best interpreted as resulting from the withdrawal of their lateral loop-like projections into the chromosome axes and from the focal aggregation of these axes into compact chromatin masses. Electron-opaque granules, which appear within the oocyte nucleus during the preparatory and maturative follicle stages, are seen to be intimately associated with these condensing bivalents. A number of Feulgen-positive heterochromatic knobs make their appearance in contact with certain bivalents during the preparatory follicle stage. These knobs are not reincorporated as such into the condensing chromatin masses and undergo disintegration and dissolution during the preovulatory follicle stage. The size, shape and ultrastructural features of the nucleolus remain unchanged thoughout the period of antral follicle development. Breakdown and dissolution of the nucleolar mass is a swift process that takes place only in the fully mature preovulatory follicle and more or less concomitantly with the dismantling of the nuclear envelope. The extranucleolar bodies increase noticeably in size during the preparatory and the maturative follicle stages; they shrink in size and undergo dissolution during the preovulatory stage of antral follicle development. An attempt is made to interpret these morphological changes in the light of current knowledge concerning the architectural and functional organization of the oocyte nucleus in general during meiotic prophase. The relevant observational evidence would be consistent with the view that, during antral follicle deveopment, the mouse oocyte nucleus is not, as too often assumed, in a period of arrested evolution; its formed components undergo structural, maturational and functional changes which are of significance not only for the resumption of the first meiotic prophase but also for the early development of the embryo.


Drosophila cell fusion induced by wheat germ agglutinin.

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.


Adhesion of Phytophthora palmivora zoospores: electron microscopy of cell attachment and cyst wall fibril formation.

Zoospores of Phytophthora palmivora adhered to a plastic film surface were examined by electron microscopy. Three stages of adhesion were compared: (1) non-adhesive, unencysted zoospores, (2) adhered incipient cysts, and (3) adhered mature cysts. Thin sections of incipient cysts revealed cells attached to the film surface through the partially discharged contents of the so-called peripheral vesicles; this seems to be the first step in cell adhesion. In mature cysts, the adhesive appeared to have been compacted into an electron-dense deposit binding the cyst wall to the plastic surface. The adhesion zone was also examined in face view after lysing attached incipient cysts with sodium dodecyl sulphate. Cyst wall microfibrils were seen together with an amorphous substance (presumably the adhesive material). The microfibrils were in various stages of formation. Seemingly, adhesion and microfibril formation take place concurrently. The possibility was considered that the material contained in the peripheral vesicles serves in both cell adhesion and microfibril elaboration.