Characterization of 5'-terminal methylation of human alpha- and beta-globin mRNAs. After incubating peripheral blood of various anemic individuals with [methyl-3H]methionine, 5'-terminal cap structures and the sequential methylation of human globin mRNAs were determined. Human alpha- and beta-globin mRNAs have the same methylated nucleotides at their 5' ends as the alpha- and beta-globin mRNAs of mouse and rabbit: m7G(5')ppp(5')[m6Am/Am]pCmp. The order of methyl group addition to human globin mRNA is also similar to that of mouse globin mRNA in that methylation of the 5'-terminal guanosine and the ribose of adenosine occurs earlier than that of the base of adenosine and the ribose of cytidine. Thus, both the cap structures and the order of methylation are conserved in the globin mRNAs of these species, suggesting that variation in the structure of the 5' terminus of globin mRNAs may alter the function of these molecules.


A soluble lipid.protein complex from bovine adrenal medulla chromaffin granules. A unique soluble lipoprotein has been isolated from aqueous lysates of bovine adrenal medulla chromaffin granules by DEAE-cellulose chromatography and gel filtration. Chloroform/methanol extracts of this complex contain sphingomyelin, lecithin, and cholesterol. Gel filtration in aqueous media indicate an approximate molecular weight of 900,000 for the complex. Incubation with sodium dodecyl sulfate causes dissociation to a low molecular weight polypeptide; prolonged treatment with guanidine HCl does not promote dissociation at all. Amino acid analysis revealed a high content of hydrophobic amino acids. Analysis of the tryptic fingerprint indicates that a single type of polypeptide chain is present. The complex appears to contain approximately five copies of polypeptide per aggregate.


Bovine tendons. Aging and collagen cross-linking. The level of cross-linking between the polypeptide chains of the collagen molecules in bovine tendons of different ages has been assessed by measuring quantitatively through densitometry the changes in the ratios of individual cyanogen bromide peptides separated on polyacrylamide gels. An increase in the number of cross-links in mature, as compared to young, tendons correlates with a depletion in the proportion of the free, COOH-terminal peptides alpha1-CB6 and alpha2-CB3,5 and with an increase in a broad distribution of peptides moving slowly in the gels: these peptides are not seen in digests of acid-soluble collagen. Some of these peptides which are presumably cross-linked migrate more slowly than beta components and collagen alpha chains and are apparently of a higher molecular weight. No increase in cross-linked peptides is detectable beyond the age of maturation; this analysis refutes, at least in this tissue, the common presumption that progressive cross-linking occurs in collagen through an animal's lifetime.


Glutathione transferases. Catalysis of nucleophilic reactions of glutathione. Homogeneous preparations of the glutathione transferases from rat liver have been tested for their ability to catalyze a number of diverse nucleophilic reactions of GSH. Although disulfide interchange with GSSG or L-cystine, and cis-trans isomerization of maleic acid, are clearly promoted by thiols in solution, the reactions were not catalyzed by the glutathione transferases. In contrast, certain more hydrophobic analogs of these compounds were found to serve as substrates. The transferases also catalyze the glutathione-dependent release of p-nitrophenol from p-nitrophenyl acetate and p-nitrophenyl trimethylacetate. These observations are consistent with the formulation that catalysis may result from close juxtaposition of sufficiently electrophilic, nonpolar compounds with GSH on the enzyme surface.


Homocysteine biosynthesis in green plants. Physiological importance of the transsulfuration pathway in Chlorella sorokiniana growing under steady state conditions with limiting sulfate. The physiological roles of the transsulfuration and direct sulfhydration pathways in Chlorella sorokiniana growing under steady state photoautotrophic conditions with limiting sulfate were studied by following the patterns of assimilation of 35SO4(2-) into sulfur amino acids. The labeling patterns expected of each pathway were defined by means of models based on the rates of net synthesis of the terminal pools of GSH, protein cysteine, and protein methionine. The labeling patterns observed are entirely consistent with the transsulfuration pathway and inconsistent with the direct sulfhydration pathway. By analysis of the amounts of radioactivity present in key intermediates at labeling times as short as 1 s, it was demonstrated that direct sulfhydration makes no detectable contribution to homocysteine biosynthesis, and if operative contributes no more than approximately 3% of the total homocysteine biosynthesized. From the combined determinations of the initial rates of labeling and net rates of synthesis of the various sulfur amino acids, a tentative working model is presented that summarizes our best current estimates of the major fluxes of sulfur in the experimental system. The labeling data further showed that soluble cysteine consists of at least two pools. One pool, termed "rapidly turning over" cysteine comprises less than 1% of the total soluble cysteine, and is the precursor of GSH, protein cysteine, and, almost certainly, cystathionine. The other pool, "slowly turning over" cysteine, appears to be in equilibrium with "rapidly turning over" cysteine, but not to be further metabolized.


The reaction of ferrous leghemoglobin with hydrogen peroxide to form leghemoglobin(IV). Ferrous leghemoglobin reacts with hydrogen peroxide to form the stable product, leghemoglobin(IV). The reaction follows second order kinetics (k = 2.24 X 10(4) M-1 S-1 at 20 degrees C) and may be regarded as a single-step, two-electron oxidation. Ferric leghemoglobin is not an intermediate. The oxidation state of leghemoglobin(IV) is established by reductive titration with dithionite; 2 eq of dithionite are required to convert 1 mol of leghemoglobin(IV) to ferrous leghemoglobin. An outstanding property of leghemoglobin(IV) is its stability, little change is noted after 12 h at 25 degrees C. Leghemoglobin(IV) differs from the higher oxidation states of other hemoglobins and myoglobins in that it does not react with hydrogen peroxide to form the oxygenated protein.


Identification of essential lysyl and cysteinyl residues in spinach ribulosebisphosphate carboxylase/oxygenase modified by the affinity label N-bromoacetylethanolamine phosphate. We reported earlier (Schloss, J. V., and Hartman, F. C. (1977) Biochem. Biophys. Res. Commun. 77, 230-236) that N-bromoacetylethanolamine phosphate is an affinity label for spinach ribulosebisphosphate carboxylase/oxygenase. We now show inactivation to be correlated directly with the alkylation either of a single lysyl residue (in the presence of Mg2+) or of 2 different cysteinyl residues (in the absence of Mg2+), consistent with the likelihood that these residues are located in the active site region. This proposition is further supported by the demonstration that the residues are protected from alkylation by substrate, a competitive inhibitor, or the transition state analog 2-carboxyribitol bisphosphate. Tryptic peptides that contain the modified residues have been isolated and sequenced. One of the 2 cysteinyl residues that are subject to alkylation is only 3 residues distant in sequence from the lysyl residue modified by bromoacetylethanolamine phosphate. This lysyl residue is identical with 1 of the 2 lysyl residues alkylated by the previously described affinity label, 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate (Stringer, C. D., and Hartman, F. C. (1978) Biochem. Biophys, Res. Commun. 80, 1043-1048).


Properties of crystalline leucine dehydrogenase from Bacillus sphaericus. The distribution of bacterial leucine dehydrogenase (L-leucine:NAD+ oxidoreductase, deaminating, EC 1.4.1.9) was investigated, and Bacillus sphaericus (IFO 3525) was found to have the highest activity of the enzyme. Leucine dehydrogenase, which was purified to homogeneity and crystallized from B. sphaericus, has a molecular weight of about 245,000 and consists of six identical subunits (Mr = 41,000). The enzyme catalyzes the oxidative deamination of L-leucine, L-valine, L-isoleucine, L-norvaline, L-alpha-aminobutyrate, and L-norleucine, and the reductive amination of their keto analogues. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. D-Enantiomers of the substrate amino acids inhibit competitively the oxidation of L-leucine. The enzyme activity is significantly reduced by both sulfhydryl reagents and pyridoxal 5'-phosphate. Purine and pyrimidine bases, nucleosides and nucleotides have no effect on the enzyme activity. Initial velocity and product inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by alpha-ketoisocaproate and ammonia, and the products are released in the order of L-leucine and NAD+. The Michaelis constants are as follows: L-leucine (1 mM), NAD+ (0.39 mM), NADH (35 micrometer), alpha-ketoisocaproate (0.31 mM), and ammonia (0.2 M). The pro-S hydrogen at C-4 of the dihydronicotinamide ring of NADH is exclusively transferred to the substrate; the enzyme is B-stereospecific.


Amino acid sequence of hemerythrin from Themiste dyscritum. The amino acid sequence of hemerythrin from the sipunculid worm, Themiste dyscritum, was determined by sequenator analyses of the S-pyridylethylated protein and fragments derived by further chemical and enzymatic cleavages. The fragments were obtained by cleavage of the intact protein with hydroxylamine, trypsin digestion of citraconylated intact protein, and subdigestion with Staphylococcal protease V8. The COOH-terminal sequence was determined using carboxypeptidases A and B and amino acid analyses. The polypeptide chain was found to contain 113 amino acids. Since heterogeneity was observed at no more than two positions in the amino acid sequence, the native octameric protein appears to be composed of identical subunits. By combining information derived from sequence analyses and x-ray crystallographic studies, it has been possible to identify amino acids responsible for the tertiary and quaternary structure of the protein as well as amino acids serving as iron ligands at the oxygen-binding site.


Purification and characterization of the cytotoxic Cerebratulus A toxins. Mucus secreted from the skin of a marine worm, Cerebratulus lacteus, contains a family of polypeptide cytotoxins (A toxins) in addition to the previously reported polypeptide neurotoxins (B toxins). The A toxins were purified by Sephadex G-50 chromatography and then CM-cellulose gradient chromatography at pH 7.5 and pH 3.5. The three most abundant A toxins (designated according to their order of CM-cellulose elution) were homogeneous by gel electrophoreses, amino acid composition, and by NH2-terminal and COOH-terminal partial sequence analyses. Each of the three A toxins consists of a single basic polypeptide chain of 93 to 99 residues, cross-linked by three or four disulfide bonds, lacking reducing sugar and cysteinyl residues. The three A toxins rapidly lysed human red cells and Ehrlich ascites tumor cells at 1 to 10 microgram/ml concentrations. On a molar basis toxin A-III is about 4 times more active than melittin (bee venom lysin) and over 10 times more active than cardiotoxin (elapid snake lysin) upon human red cells. Purified A toxins lacked phospholipase A activity. The cytoxins as well as the neurotoxins were concentrated within the body wall integument.


RNA synthesis in rat liver cells with different DNA contents. The DNA content of mature rat liver parenchymal cells falls into three distinct classes with 1:2:4 amounts of DNA. These classes represent 2 N, 4 N, and 8 N cells. We have measured the changing proportions of these cells from 0.75 to 24 months of age. We have obtained a useful approximation for the numbers of each ploidy class, for cellular preparations from liver, as a function of the amount of DNA and age. Multiple linear regression analysis revealed that the average DNA content per cell is given by the relationship DNA/cell = 41.28(1/2)--324.14 Age1/4 + 499.37 Age1/8--207.24, with a multiple correlation coefficient of 0.990. Measurements of the amount of RNA synthesized in a 15-min period revealed that the relative proportions were 1.2:1.0 for 4 N:2 N cells of young rats, 1.3:1.1.:1.0 for 8 N:4 N:2 N cells of intermediate ages, and 1.1:1.0 for 8 N:4 N cells of old rats. The absolute amounts of RNA synthesized by each ploidy class declined with age.


Enzymatic synthesis of a glucose-containing oligosaccharide-lipid involved in glycosylation of proteins. Earlier studies on the biosynthesis of an oligosaccharide-lipid with the structure (alpha-Man)4-6-beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc-P-P-dolichol have been extended to show that enzymes in membrane preparations of hen oviduct catalyze synthesis of a Glc-containing oligosaccharide-lipid that is similar in properties to the Glc-free oligosaccharide-lipid. Conditions that enable enzymatic preparation of Man-, GlcNAc-, or Glc-labeled oligosaccharide-lipid have been established. Experiments with isolated [Glc-14C]oligosaccharide-lipid as substrate demonstrated that the oligosaccharide chain was transferred en bloc from the lipid to an endogenous membrane protein of apparent Mr = 25,000. In addition, an exogenous soluble protein, S-carboxymethylated alpha-lactalbumin, was shown to serve as acceptor of the oligosaccharide chain from the Glc-containing oligosaccharide-lipid. Studies on the metabolic fate of the Glc residues in these proteins are reported in the accompanying paper (Chen, W. W., and Lennarz, W. J. (1978) J. Biol. Chem. 253, 5780-5785).


Enzymatic excision of glucosyl units linked to the oligosaccharide chains of glycoproteins. Studies in the accompanying paper (Chen, W. W., and Lennarz, W. J. (1978) J. Biol. Chem. 253, 5774-5779) showed that hen oviduct membranes catalyze synthesis of a Glc-containing oligosaccharide-lipid and that the oligosaccharide moiety of this compound is transferred en bloc to an endogenous protein as well as to an exogenous, soluble protein. In this study we have established that the endogeneous proteins in oviduct membranes are devoid of Glc. In view of these findings, the possibility that Glc was excised from the oligosaccharide chain after it had been transferred to protein was examined. Kinetic studies involving the use of endogenous membrane proteins as acceptors of the oligosaccharide chain suggested that the Glc residues were released without significant degradation of the core oligosaccharide. Direct evidence for the presence of a membrane-bound glucosidase was obtained using either free, Glc-containing oligosaccharide or Glc-containing oligosaccharide linked to S-carboxymethylated alpha-lactalbumin as substrates. Experiments utilizing [3H]Glc- and [14C]GlcNAc-labeled, glycosylated S-carboxymethylated alpha-lactalbumin established that, under conditions leading to extensive removal of [3H]Glc, there was essentially no degradation of the [14C]GlcNAc-labeled oligosaccharide core.


Physical and functional properties of adenylate cyclase from mature rat testis. The mature rat testis contains two forms of adenylate cyclase. One is membrane-bound and hormone-responsive; the other is water-soluble and not activated by hormones (Braun, T., and Dods, R. F. (1976) Pnoc. Natl. Acad. Sci. U.S.A. 72, 1097-1101). The water-soluble adenylate cyclase is a globular protein which is much smaller than the enzyme which is solubilized from membranes of mature rat testis by Triton X-100. Its physical properties are: sedimentation coefficient, 3.8 S; Stokes radius, 34 A; molecular weight, 56,000; frictional ratio, 1.2. Inclusion of protease inhibitors during enzyme extraction did not affect the fraction of total enzyme activity which was water-soluble, nor did autolysis for 1 h at 25 degrees C. The physical properties of the membrane-bound adenylate cyclase were determined after solubilization with Triton X-100. In detergent, the values are: sedimentation coefficient, 6.7 S; Stokes radius, 68 A; partial specific volume, 0.73 ml/g; molecular weight, 191,000; frictional ratio, 1.6. The fact that the partial specific volume of the enzyme in detergent is the same as that of a typical water-soluble protein shows that the Triton X-100-solubilized enzyme does not bind a large quantity of detergent. This indicates that it does not have extensive hydrophobic regions on its surface. The detergent-solubilized adenylate cyclase of the mature rat testis is similar in this respect to adenylate cyclase solubilized from the rat renal medulla (Neer, E. J. (1974) J. Biol. Chem. 249, 6527-6531).


Immunological properties of fractionated avian erythroid nuclei. Chicken reticulocyte (polychromatic primitive erythrocyte) and erythrocyte chromatin was fractionated by ultrasound shearing and salt precipitation into three fractions differing in their activities to support the in vitro RNA synthesis. The transcriptionally active fraction of chicken reticulocyte chromatin which represented only about 0.5% of the total nuclear DNA contained essentially all the chromatin-associated endogenous RNA. Approximately 2% of this endogenous reticulocyte RNA hybridized to globin cDNA probe and could be translated in vitro into polypeptides which coelectrophoresed with the in vitro translation product of isolated chicken globin mRNA or chicken globin marker. Each of the three fractions had a characteristic distribution of chromosomal proteins and endogenous RNA. Polyacrylamide gel electrophoresis of the chromosomal proteins showed differences in their distribution among individual fractions of the same cell type and among corresponding fractions of reticulocyte or erythrocyte chromatin. Antisera produced against dehistonized reticulocyte chromatin were specific for reticulocyte but not erythrocyte chromatin. When reacted with each of the differentially templating chromatin fractions, it was found that reticulocyte-specific antibodies were highly reactive with the template-active fraction of reticulocytes, but essentially nonreactive with any other reticulocyte fraction. This same antiserum was not significantly reactive toward any erythrocyte fraction. The antigenicity of the template-active fraction of reticulocytes was abolished after pronase or DNase II digestion, but only partially diminished after DNase I digestion.


Purification of the "corrinoid" enzyme involved in the synthesis of acetate by Clostridium thermoaceticum. A corrinoid enzyme has been purified to approximately 80% homogeneity from Clostridium thermoaceticum. It catalyzes the formation of acetate from N5-methyltetrahydrofolate and pyruvate in combination with the required supplementary enzymes which are supplied by an extract that has been treated with propyl iodide. The enzyme was purified by chromatography on a folate affinity column and a DEAE-Bio-Gel column and by ultrafiltration. The molecular weight as determined by sedimentation equilibrium is 158,000 and the sedimentation coefficient is 10.5 S. By gel electrophoresis in sodium dodecyl sulfate, the subunit molecular weight was found to be 40,000, thus, the enzyme may be a tetramer of four similar subunits. The results of electron microscopy confirmed the tetrameric structure. In the absence of sodium dodecyl sulfate, two bands of similar intensity were observed by electrophoresis, but both yielded the 40,000 molecular weight subunit in the presence of sodium dodecyl sulfate. These results indicate the two bands represent either two different molecular weight forms of the enzyme or two differently charged isoenzymes. The enzyme is quite labile being sensitive to dilution, aerobic conditions, and light. Dithiothreitol and glycerol were found to stabilize the enzyme. The cofactor requirements for acetate synthesis have been determined. ATP, thiamin pyrophosphate, S-adenosylmethionine, and Fe2+ were found to be required for maximum activity and the Km values were determined. High concentrations of methyltetrahydrofolate, pyruvate, and S-adenosylmethionine were found to inhibit the synthesis of acetate.


Effect of comonomer ratio on hydrocortisone diffusion from sustained-release composite capsules. The daily in vitro release of hydrocortisone from composite polymer capsules is reported here for over 120 days. Increase in vinyl acetate comonomer content of the ethylene-vinyl acetate copolymer matrix brought about an increase in the diffusion rate. Variation in the initial drug content of the capsules from 40 mg to 20 mg affects the daily drug release less significantly than the variation in copolymer ratio. The correlation between vinyl acetate comonomer content and the percent crystallinity of the copolymer matrix is suggested as one of the possible major factors in controlling diffusion rate from this drug-polymer system. The diffusion constant (D) calculated was 0.212 X 10(10) cm2/sec when the copolymer carrier has 30% vinyl acetate content and 0.430 X 10(11) cm2/sec when the copolymer carrier has 20% vinyl acetate content for capsules with 20 mg initial drug content, and 0.118 X 10(-11) cm2/sec and 0.226 X 10(-11) cm2/sec, respectively, for capsules with 40 mg initial drug content.


Thrombus formation in a staged capillary membrane oxygenator associated with a microaggregate blood filter: a scanning electron microscope study. Blood contact at interfaces in extracorporeal devices is a source of traumas. Proteins are very rapidly adsorbed; then, depending on which proteins are left various degrees of platelet aggregation follow and thrombi develop. The scanning electron microscope reveals very instructive information on the morphology of the blood deposits which adhere to foreign surfaces. Oxygenators such as the Awad D, which is a staged one made of silicone rubber, become "thrombus invaded" after prolonged extracorporeal circulation in spite of adequate heparinization of the blood. In most cases, evaluation of membranes and devices is assessed with respect to transfer of blood gases. Careful examination of the morphology of deposits should be developed. This study shows the importance of blood flow rate, design, and materials.


Compatibility and long-term stability of glass-ceramic implants. The biocompatible and bioactive glass-ceramic material Ceravital, which has been developed, can be adapted to the physiological conditions of the organs by apropriately monitoring its chemical composition. The material exhibits a remarkable long-term stability in in vitro solubility tests and when used as implants in animal experiments.


The appearances of wear on polyethylene--a comparison of in vivo and in vitro wear surfaces. An examination has been made of the wear surfaces of 21 ultra-high-molecular-weight polyethylene components of hip and knee joint prostheses removed from patients. Both light optics and scanning electron microscope systems of examination have been used. The appearances are compared with specimens subjected to two different in vitro wear tests. Surface appearances related to the wear experience have been classified into seven modes. At least four of these are evidences of what may be four different wear mechanisms. Entrapment of bone-cement particles between articulating surfaces is fundamentally extraneous but is seen to generate the most severe wear. In the absence of bone-cement particles, in vivo, wear seems to be mild. Wear in vitro produces similar surface modes but with different relative emphasis.


Compliance as a factor effecting the patency of a copolyurethane vascular graft. A new solid-wall vascular graft which has a compliance approximating that of the natural artery has been prepared from a copolyether-urethane material. Six of nine of these compliant grafts implanted in dogs were patent on removal, the longest implant time being 77 days for a 4-mm I.D. femoral artery graft. This is in contrast to noncompliant grafts of the same copolyurethane in which failure usually occurred within 48 hr.


Evaluation of sunlight stability of polyurethane elastomers for maxillofacial use. I. The evaluation of the efficiency of polymer additives with special emphasis on uv absorbers and antioxidants in polyurethane elastomers has been completed. Aliphatic polyurethanes were chosen for this study because their properties closely relate to the requirements of maxillofacial prosthesis. The polyurethane elastomers were either synthesized by ourselves or formulated according to the manufacturer's recommendation. Eleven different types of uv absorbes, coupled with one antioxidant, were incorporated into the polyurethane systems. The Atlas twin-lamp carbon arc Weatherometer was used as the source of uv. The samples were periodically withdrawn for examination of yellowing and tackiness. It was found that, although the incorporation of uv stabilizers enhanced the uv resistance of polyurethanes, the problem of tackiness resulting from uv aging was not solved satisfactorily. The phenomenon of yellowing, however, was significantly improved, mainly due to the aliphatic structure of polyurethanes. The most promising uv absorbers are Tinuvin 770 and the combination of Tinuvin 328, ZnO, and an antioxidant. Their effectiveness in other polyurethane systems is not known and further research is underway to explore this field. Hopefully, these findings will greatly assist the successful application polyurethane elastomers in maxillofacial prosthesis.


Force production decay rate in Alastik modules. The production and decay of forces produced by Alastik modules was studied under conditions simulating clinical usage over time period of 2 hr to 4 weeks. Force decay was moderate and all Kx modules tested produced approximately 1 lb of force at the end of the 4-week test period. Therefore, from a clinical standpoint, modules need not be changed more frequently than once every 4 weeks.


The effect of the cooling rate of dental alloys on their amalgamation properties. Silver-tin alloys of various compositions (60-80 atomic % Ag) have been prepared from the melt using cooling rates from about 50 degrees C/hr to about 10(6) degrees C/sec. Structural differences due to the different cooling rates were shown by metallographic and x-ray diffraction techniques. From each composition and cooling rate, powders were then prepared and amalgamated. The amalgams were tested for Hg content, dimensional change during setting, and tensile strength. Changes in cooling rates had some effect on the relative amounts of phases present but primarily affected the grain sizes. In general, the faster the cooling rate, the smaller the grain size; the smaller the grain size, the faster and more uniform the amalgamation; the more uniform the amalgamation, the stronger the amalgam. Storage of the alloy powders at room temperatures slowed their amalgamation rates, destroyed their uniformity,and resulted in weaker amalgams. However, no phase changes were observed by x-ray diffraction in the freshly comminuted alloys after aging at room temperature for two months.


The effect of shear, specific surface, and air interface on the development of blood emboli and hemolysis. Heparinized dog blood was exposed to shear and foreign surfaces in conicylindrical test cells. The cells were injection molded from polycarbonate and were filled using a technique that avoided contact of the blood with air. Particulate-matter formation was measured and was found to be dominated by the surface-to-blood-volume ratio and to be independent of shear rate. Hemolysis was also measured and was found to vary linearly with shear rate and to increase with increasing surface-to-blood volume ratio. Thus, at low shear rates and high specific surface conditions, the degree of hemolysis was found to be minimal while particulate-matter formation was high. The results suggest that the safety of extracoporeal perfusion procedures cannot be inferred from hemolysis measurements alone. In one series of tests, a gas-blood interface was generated at a rate equivalent to the rate of surface renewal in conventional disc oxygenators. The gas-blood interface failed to contribute significantly to the damage indices, which suggests that the apparent superiority of membrane oxygenators may be a result of factors other than the absence of a blood-gas interface.


Hydraulic permeability of hollow-fiber membranes. Two experimental methods have been developed to characterize the pressure-driven flow of a liquid through the walls of single hollow-fiber membranes. In one method, the permeation rate is measured directly by following the air-water interface in a pressurized pipet connected to a hollow fiber which is sealed at one end. With the second method, the permeation rate is determined by tracking the decent of an air bubble inside a pressurized hollow fiber sealed at one end. Results obtained by the two experimental methods are in good agreement. Darcy's law is used to analyze the experimental data because the Darcy permeability constant, k is an intrinsic property of the material. The membrane dimensions and the Darcy permeability constant for reconstituted collagen hollow fibers are shown to be quite sensitive to hydrogen ion concentration and ionic strength. A conceptual link between the Darcy permeability constant and the membrane microstructure is obtained by combining the the aligned-rod structural model for reconstituted collagen proposed by Kramer with a hydrodynamic calculation due to Happel and electrical double-layer theory. On the average, the predictions of this model for volume fraction solids and microstructure dimensions are in reasonable agreement with experimental observations.


Maxillofacial prostheses of chlorinated polyethylene. There is clearly a need for maxillofacial prosthetic materials with improved properties. The chlorinated polyethylenes are thermoplastic elastomers which have particularly promising properties, and were used by us to prepare improved maxillofacial prostheses. Suitable CPE resins were compounded with other polymers and with pigments on a heated rubber mill to form thin sheets in a variety of shades. These were heated at 190 degrees C for 10 min and placed between heated linotype mold halves. The prosthesis was formed in a hand press. Sometimes heating and pressing were repeated. After cooling in water, the prosthesis was removed and hand-shaded with oil-soluble dyes. Physical properties were evaluated using standard techniques; skin irritation studies were conducted by 14-day insult patch tests on rabbits. Clinical evaluations were conducted on human volunteers. Parallel evaluations were conducted on commerically available materials for comparison. The CPE was superior to all of the three commerical materials in most properties, and comparable to the better of the three in the remaining properties. On balance, CPE was significantly superior. Early results indicate that the materials and techniques required are easily handled in the dental lab and that the final prosthesis has excellent aesthetic and patient acceptability.


Kinematics of the wrist. I. An experimental study of radial-ulnar deviation and flexion-extension. The kinematics of the wrist during radial-ulnar deviation and flexion-extension were studied in several ways. In six fresh cadaver wrists, the forearm was fixed in full pronation, each motion was constrained to one plane, and two metal markers were placed in each of the finger metacarpals, as well as in the radius and all of the carpal bones except the pisiform and greater multangular. Radial ulnar and flexion-extension movements in these wrists were studied roentgenographically. In the wrists of six normal volunteers, a similar roentgenographic analysis was carried out and the trajectories of wrist motions also were studied using light-emitting diodes. Finally, roentgenographic measurements were made on 100 wrists of normal subjects. From these studies, it was concluded that: (1) during each of these motions, rotation occurs about a fixed axis located within the head of the capitate, and the location of each axis is not changed by the position of the hand in either plane; (2) the distance from the base of the third metacarpal to the distal articular surface of the radius (the carpal height), measured along the proximally projected axis of the third metacarpal on posteroanterior roentgenograms, is constant throughout radial-ulnar deviation of the normal wrist and can be used as a measure of carpal collapse; and (3) the perpendicular distance of the fixed axis of rotation for radial-ulnar deviation from the distally projected longitudinal axis of the ulna can be used as a quantitative measurement of the amount of translation of the carpus in pathological conditions.


Myelodysplasia. The influence of the quadriceps and hip abductor muscles on ambulatory function and stability of the hip. In an analysis of motor function, ambulatory function, and hip stability in sixty-five patients with myelodysplasia, four motor-function groups based on the strength of the quadriceps and hip abductor muscles were identified. Retrospectively, it was evident that based on these groups, it would have been possible to predict which hips would remain stable, what level of ambulatory function the patients could achieve, and whether treatment to reduce and stabilize the hips was indicated. Fifty-seven of fifty-eight hips in the twenty-nine patients with functioning quadriceps muscles but non-functioning hip-abductor muscles were either subluxated or dislocated. Thirty-nine of the remaining forty-six patients with functioning quadriceps muscles could walk. In this series, three operative procedures were used to treat hip subluxation: varus osteotomy, varus osteotomy combined with iliopsoas transfer, and iliopsoas transfer alone. One shelf procedure was also done. Varus osteotomy was the best procedure for hip subluxation while posterior iliopsoas transfer, either alone or in combination with a varus osteotomy, was of questionable value. Treatment of hip instability (subluxation or dislocation) in patients without quadriceps function was not necessary.


Arthroscopic examination of the knee. Polypuncture technique with percutaneous intra-articular manipulation. Many of the limitations of conventional diagnostic arthroscopy of the knee have been largely overcome through the development of techniques that permit manipulation of intra-articular structures through paired, coordinated entry sites. Ten accessory entry sites are described. This technique was tested in sixty-three fresh knees from cadavera and was employed in fifty-eight knees of ninety patients who required arthroscopy between September 1976 and July 1977. The technique provided information of diagnostic significance that was not obtainable on preliminary conventional arthroscopic examination in 74 per cent of the clinical trials, and it resulted in no complications. It increased the accuracy and realiability of diagnostic arthroscopy of the knee and at times helped to avoid an exploratory arthrotomy while facilitating the planning of therapeutic arthrotomy.


Arthrographic findings in Legg-Calvé-Perthes disease and transient synovitis of the hip. Bilateral arthrography of the hip was performed in thirty-seven patients with unilateral Legg-Calvé-Perthes disease. Measurements made on the plain roentgenograms and on the arthrograms of the diameter of the femoral head, the width of the medial joint space, and the thickness of the acetabular cartilage were compared. The acetabulum-head quotients also were calculated. When present, evidence of flattening of the femoral head cartilage and of pooling of the contrast material also was noted. These data showed that in the early stages of Legg-Calvé-Perthes disease the articular cartilage is thickened, and that the cartilaginous femoral head is deformed and protrudes from the acetabulum. Arthrograms made with the hip in different positions also indicated the extent and location of collapse of the (osteochondroal) femoral head and therefore proved useful as a means to determine the best therapeutic position of the hip to provide coverage of the head. In three patients with severe transient synovitis, thickening of the articular cartilage resembling that seen in Legg-Calvé-Perthes disease was demonstrated.


Role of the posterior calf muscles in normal gait. The role of the calf muscles during the single-limb stance phase of gait was assessed in fifteen normal subjects and in seven with either an amputation or a neuromuscular deficit. Normal activity of the muscles, paralysis by nerve blocks, and stability versus instability of the ankle were studied. Pelvic displacement and velocity, step length, and step time were evaluated for each limb during the gait cycle, and the contribution of activity of calf muscles to each was determined. Limb-segment position, lower-limb joint angles, over-all cadence, velocity, and stride length were measured to reveal compensatory mechanisms in the absence of muscle strength in the calf. The results indicate that the force produced by activity of calf muscles is used to restrain the body's own forward momentum and not used to propel it further. Absence of the calf muscle causes an immediate compensatory reaction directed toward maintaining over-all stability rather than speed.


Function of the triceps surae during gait. Compensatory mechanisms for unilateral loss. We studied the gait of a woman who was normal except for surgical excision of the gastrocnemius and soleus muscles. She was able to compensate for nearly all of her abnormalites of gait by excessive lateral pelvic tilt and prolonged quadriceps activity. Her mild disability, as regards gait, consisted of inability to increase walking speeds beyond the normal pacing. However, despite uneven step lengths, she had uniform forward progression. She had excessive dorsiflexion of the ankle and diminished plantar flexion on the involved side even though the retained plantar flexors could provide 38 per cent of her normal plantar flexor strength.


Scapulocostal stabilization for scapular winging in facioscapulohumeral muscular dystrophy. A surgical procedure has been devised to prevent scapular winging and to increase shoulder abduction in facioscapulohumeral muscular dystrophy. The operation improves the limited shoulder abduction and scapular winging caused by the loss of scapular fixation  to the thoracic cage, while passive abduction at the glenohumeral joint remains normal. Preventing scapular malrotation by eliminating scapulothoracic motion increases shoulder abduction. This is achieved by fastening the scapula to several underlying ribs with fascia. The operation has resulted in a 37 per cent increase in abduction, increased shoulder strength and endurance, and the prevention of scapular winging.


A new type of fracture-dislocation of the thoracolumbar spine. Ten instances of fracture-dislocation of the thoracolumbar spine are described in which the lower vertebra was displaced forward under the upper one by forward slipping of the posterior articular processes. The mechanism involved is a posterior lumbosacral shearing force. All patients had neurological deficits of different degrees of severity and all but three patients recovered. Surgical treatment was indicated because of instability in four patients and was done in three of the four. Surgical reduction was always difficult. A stable and painless spine was achieved in all patients surveyed, regardless of the kind of treatment given. One patient died of other injuries.


Fractures of the femoral shaft treated surgically. Comparative results of early and delayed operative stabilization. In a study of the results of operative reduction and internal stabilization of eighty-nine femoral fractures, operative reduction was performed within six days in fifty-four of the fractures and between seven and thirty days in thirty-five. Dual Eggers plates were used in sixty-three fractures; intramedullary nails, in eighteen; a nail-plate, in six; and a single plate, in two fractures. The criteria for ultimate healing were roentgenographic demonstration of bridging callus and the ability of the patient to bear full, unsupported weight on the extremity. Healing that occurred later than  nine months after fracture was considered to be delayed. In the group treated by early operative management, the incidence of delayed union was 18 per cent and of non-union, 18 per cent. In the group treated by delayed operative management, the incidence of delayed union was 3 per cent and there were no instances of non-union. All ten non-unions, ten of the eleven delayed unions, and five of the six infections occurred in the early operative group.


Fluid balance within the canine anterolateral compartment and its relationship to compartment syndromes. Fluid homeostasis within muscle compartments is maintained by four pressures: capillary blood pressure, capillary blood oncotic pressure, tissue-fluid pressure, and tissue fluid oncotic pressure. As determined in the canine anterolateral compartment, capillary blood pressure is 25 +/- 3 millimeters of mercury; capillary blood oncotic pressure, 26 +/- 3 millimeters of mercury, tissue-pbessure, -2 +/- 2 millimeters of mercury; and tissue-fluid oncotic pressure, 11 +/- 1 millimeters of mercury. The wick technique allows direct measurement of tissue-fluid pressure in skeletal muscle and, with minor modifications, is adapted to collect microsamples of interstitial fluid for determinations of tissue-fluid oncotic pressure. The wick technique detects very slight fluctuations in intracompartmental pressure such as light finger compression, injection of small volumes of fluid, and even pulsation due to adjacent arterial pressure. Adjacent muscle compartments may contain different tissue-fluid pressure due to impermeable osseofascial barriers. Our results obtained in canine muscle compartments pressurized by infusion of autologous plasma suggest that risks of muscle damage are significant at intracompartmental pressures greater than thirty millimeters of mercury.


A fluted femoral intramedullary rod. Biomechanical analysis and preliminary clinical results. An intramedullary rod for the femur was designed and compared with rods commonly used. Its bending strength was increased 40 to 80 per cent and its torsional rigidity was increased 230 to 3,000 per cent compared with the other devices tested. In forty patients with traumatic and pathological fractures and osteotomies of the femur, excellent fixation was achieved and very early weight-bearing was possible. In all fresh fractures and non-infected non-unions, union occurred very promptly and bone-grafting was not required in most instances. The stregth and rigidity of the device made it exceptionally useful for non-unions, pathological fractures of the femur, and resection-fusions of the knee.


Structural changes in intact tubular bone after application of rigid plates with and without compression. The influence of a rigid plate on the structure of intact tubular bone was studied in forty adult rabbits using a four-hole commercial-steel DCP-ASIF plate attached to each tibiofibular bone, with and without compression. The morphological changes in the bone underlying the plate were examined and were assessed quantitatively at one day to thirty-six weeks after operation. The rigid plate induced porotic changes in the cortical bone, which were evident three weeks after the operation and increased rapidly between twelve and eighteen weeks. At thirty-six weeks, resorption cavities occupied 40.6 per cent of the cortical wall of the bones plated with compression and 41.2 per cent of the wall of those plated without compression. The changes were visible slightly earlier in the presence of compression. A rigid plate brought about a statistically significant increase both in the outer diameter of the bone and in the diameter of the medullary space as a result of subperiosteal new-bone formation and concomitant subendosteal bone resorption.


Geometric total knee replacement for treatment of the rheumatoid knee. The results of fifty-four geometric total knee arthroplasties in forty-four patients with definite or classic rheumatoid arthritis were analyzed twenty-four to sixty-four months after the surgical procedure. There were no operative deaths, no postoperative infections, and no known pulmonary emboli. Three patients required additional operative procedures on the knee: one, a patellectomy for pain one year after arthroplasty; the second, resection of tibial bone and reinsertion of the tibial component to correct a flexion contracture; and the third, replacement of the tibial component because of loosening. The relief of pain and increase in ability to carry out the activities of daily living were dramatic in these patients, who, because of the limitations imposed by their rheumatoid arthritis, applied minimum stress on the prosthetic knees. Postoperatively, the average arc of knee flexion was 104 degrees.


Somatosensory evoked potentials during Harrington instrumentation for scoliosis. The somatosensory evoked potential can be obtained in the anesthetized patient during corrective surgery on the spine. The techniques of anesthesia and somatosensory evoked potential recordings described herein were utilized in fifty-five patients during surgical correction of scoliosis with Harrington instrumentation and spine fusion. No detectable complications were encountered and no neurological morbidity ensued in our series. This method may prove to be of significant value when potential injury to the spinal cord may be encountered during correction of spinal deformities.


Intraoperative awakening to monitor spinal cord function during Harrington instrumentation and spine fusion. Description of procedure and report of three cases. Since Vauzelle and associates first reported in 1973 on the functional monitoring of spinal cord activity during surgery on the spine, we have used intraoperative awakening routinely in patients undergoing surgery involving distraction of the spinal column. Of the 166 consecutive patients studied, three were discovered to have a neural deficiency when they were awakened during surgery. These deficits disappeared immediately on release of the distracting force. Although it is unlikely that all three patients would have proceeded to paraplegia, we believe that this is a safe and sensitive test which will identify patients in danger of neural complications.


Experimental induction of gene activity in the salivary gland chromosomes of Trichosia pubescens (Diptera: Sciaridae). During the course of experiments with larvae of Trichosia pubescens, we have unexpectedly found that diethyl ether or chloroform anesthesia induces a large puff in a specific band in the polytene chromosomes of the salivary glands. This puff develops a few minutes after the treatment, attaining its maximum size after 60-100 min, and regresses completely 200 min after its activation. Through autoradiography, an intense incorporation of RNA precursors into that puff was observed. A few other smaller puffs are also induced by the treatment. The treatment with diethyl ether or chloroform does not induce puffing in the polytene cells of malpighian tubules and of midgut.


Cell fractionation studies on the guinea pig pancreas. Redistribution of exocrine proteins during tissue homogenization. A double-label protocol was used to estimate the extent of leakage and relocation artifacts that affect exocrine pancreatic proteins in cell fractionation experiments. Guinea pig pancreatic lobules were pulsed in vitro with a mixture of 14C-amino acids to enable the lobules to produce and process endogenously labeled exocrine proteins. At the end of the pulse (10 min) or after an appropriate chase interval, the lobules were homogenized in 0.3 M sucrose to which a complete mixture of 3H-labeled exocrine pancreatic proteins was added as an exogenous tracer. The distribution of both labels was studied in each cell fraction of interest at the level of TCA-insoluble proteins and individual exocrine proteins resolved by using a two-dimensional gel system. Based on the premises that the exogenous and endogenous label behave identically during homogenization-fractionation and that all endogenously labeled exocrine proteins found in the postmicrosomal supernate come from intracellular compartments ruptured during tissue homogenization, a series of equations was derived to quantitate leakage and adsorption and to define the ratio of endogenous label still in its primary location to total label (primary location index or PLI) for each cell fraction. Leakage was found to be uniform for all exocrine proteins, but unequal in extent from different cell compartments (condensing vacuoles is greater than zymogen granules is greater than rough endoplasmic reticulum) ; it increased with exposure to shearing forces especially in the case of zymogen granules and condensing vacuoles, and was substantially reduced from rough microsomes by adding 10 mM KCl to the homogenization media. Relocation of exogenous label by adsorption to other subcellular components was extensive (approximately 55%), uneven (free polysomes is greater than rough microsomes is greater than smooth microsomes and zymogen granules), preferential (cationic proteins are massively adsorbed to ribosomes and membranes, resulting in a complementary enrichment of the post-microsomal supernate with anionic exocrine proteins), and reversible (with successive 50-100 mM KCl washes). After correction for adsorption and leakage, the kinetics of intracellular transport derived from cell fractionation data were found to be nearly identical to those obtained from quantitative autoradiographic studies.


Inhibition by insulin of the formation of autophagic vacuoles in rat liver. A morphometric approach to the kinetics of intracellular degradation by autophagy. Electron microscopic morphometry has demonstrated a rapid decrease in the fractional volume of autophagic vacuoles (AV) in hepatocytes of adult male rats after the intraperitoneal administration of insulin (5 U/kg of body weight). Except for a significant decrease in glycogen to about one-half its initial value, no major changes in the composition of the remaining cytoplasm, or in the average volume of the single hepatocyte, were seen. The decrease found in the AVs is attributed to an inhibition of the formation of new AVs-probably the morphologic counterpart of the well-known anticatabolic effects of insulin. The decay of the fractional volume of the AVs appeared to follow first-order kinetics. Thus, the termination of the "life" of an AV by destruction of its contents may not depend directly on the "age" of the AV. The average half-life of the AVs amounted to approximately 9 min. Similar values were found for the different types of AVs, except for those containing glycogen. The half-life of these AVs was approximately 18 min. From the half-life values and from the "segregated fractions" at time zero, which were different for the different cytoplasmic components, rates of removal from the cytoplasm by autophagy were calculated. Expressed as "percent per day", the following rates were found: whole cytoplasm, 2.3; mitochondria, 3.9; microbodies, 8.9; and glycogen, 1.1. The results indicate that autophagy, to some extent, is selective and plays an important, but not an exclusive, role in intracellular turnover.


Membrane potentials and resistances of giant mitochondria. Metabolic dependence and the effects of valinomycin. The membrane potentials and resistances of giant mitochondria from mice fed cuprizone have been studied. They were found to correspond approx. 10-20 mV, positive inside, and 2 M omega, respectively. These properties were found to be independent of the metabolic state. The microelectrodes were in the inner mitochondrial space since (a) the potentials in the presence of valinomycin depended on the K+ concentration of the medium and magnitude of the K+ diffusion potentials was consistent with the presence of a high internal concentration of K+, (b) almost identical results were obtained with mitochondria from which the external membrane had been removed and the cristae were evaginated, and (c) punch-through experiments, in which the microelectrodes were advanced until they emerged through the other side of the mitochondria, showed an identical membrane potential both in the presence and in the absence of valinomycin. The potentials were stable under a variety of conditions and showed no sign of decay of membrane leakiness. Detailed evidence that the impaled mitochondria are metabolically viable will be presented in a separate publication.


Assays of the metabolic viability of single giant mitochondria. Experiments with intact and impaled mitochondria. Single giant mitochondria isolated from mice fed cuprizone were assayed for their metabolic viability. Two tests were devised. One test optically detected the accumulation of calcium phosphate within the mitochondria under massive loading conditions (including the presence of succinate and ATP). The accumulation corresponds to a test of energy coupling from either electron transport or the hydrolysis of ATP since it is blocked by either antimycin A or oligomycin. The other assay tested for the production of ATP from ADP and Pi, using myofibrils. Myofibrils prepared from glycerinated rabbit psoas muscle contract only in the presence of ATP and not in the presence of ADP. Myofibrillar contraction is unaffected by the presence of antimycin A or oligomycin. However, myofibrils in the presence of mitochondria that are phosphorylating ADP to ATP do contract. This contraction is blocked by antimycin A and/or oligomycin. Hence, the ATP which causes myofibrillar contraction is produced by oxidative phosphorylation. At low mitochondrial concentration, only the myofibrils in close proximity with mitochondria contract in the presence of ADP. Therefore the assay can be used to test the viability of individual mitochondria. Individual giant mitochondria were found to be viable, using both of these assays. Comparable results were obtained in mitochondria impaled with microelectrodes. The potentials and resistances were unaffected by concomitant calcium phosphate accumulation or oxidative phosphorylation.


Microtubules in cone myoid elongation in the teleost retina. The myoids of retinal cone cells of the blue-striped grunt (Haemulon sciurus) undergo significant elongation during dark adaptation of the retina. Longitudinally oriented microtubules are present in myoids both before and after elongation. Injection of colchicine into the vitreous of the eye in vivo disrupts the microtubules in the myoids and prevents dark-adaptive myoid elongation. Counts of microtubules in transverse sections along the lengths of elongating myoids show that there is a uniform decrease in the number of microtubules at any one point along the myoid as the myoid elongates. The magnitude of the decrease is proportional to the extent of the elogation. The product of the mean myoid microtubule number (determined from counts at progressive intervals along the myoid) and the myoid length remains essentially constant during myoid elongation, indicating that the total quantity of microtubules in the myoid does not increase with elogation. Serial section tracings of the microtubules along the myoids suggest that individual microtubules do not extend the length of the myoid and that the myoid microtubular apparatus consists of bundles of overlapping shorter microtubules. We propose that elongation of the myoid is accompanied by sliding redistribution of microtubules along the length of the myoid, and that the sliding may be generated by interaction between microtubules in regions where they closely overlap in bundles. We find no evidence for the involvement of discrete, electron-dense microtubular organizing centers in myoid elogation.


Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography. The distribution of acetylcholinesterase (ACHe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which ACHe concentration is an invariant feature of endplate morphology and what, if any aspects may be related to their fast speed of response. We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is n  average of 6.4 +/- 2.1 X 10(7) DFP-binding sites per endplate, of which 29% (1.8 X 10(7)) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe site are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites/micron 2 of surface area of pre-and postjunctional membrane. This stacking density of DFP-binding sites per surface area of membrane ( probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.


Form of the postsynaptic density. A serial section study. Through the use of serial sectioning of dog cerebral cortex tissue, holes or perforations could be revealed in the larger postsynaptic densities (PSDs), in confirmation of the earlier work of Peters and Kaisermann-Abramof (1969. Z. Zellforsch. Mikrosk. Anat. 100:487-506). These holes appeared in serial sections which happened to be cut both parallel and normal to the plane of the synaptic junction. Cleft material was absent in that part of the synaptic cleft opposite this hole. Somestimes the presynaptic membrane opposite the hole was indented into the presynaptic cell. In addition, most of the synaptic vesicles in the presynaptic cell close to the membrane were clustered at that part of the membrane opposite the edge of the density disk. The meaning of the hole and of the other features mentioned above for the function of the density is not known at present.


In vitro polymerization of microtubules from HeLa cells. Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro has become routine, the application of this method to non-neural cultured cells has been less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. We have developed a method for preparing microtubules from HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through two complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C in vitro. The microtubules obtained are morphologically similar to brain microtubules in electron micrographs, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the second polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after two cycles of polymerization and depolymerization has an apparent mol wt of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.


Cell-to-cell communication and ovulation. A study of the cumulus-oocyte complex. Cell-to-cell communication was characterized in cumulus-oocyte complexes from rat ovarian follicles before and after ovulation. Numerous, small gap junctional contacts were present between cumulus cells and oocytes before ovulation. The gap junction are formed on the oocyte surface by cumulus cell processes that transverse the zona pellucida and contact the oolemma. The entire cumulus mass was also connected by gap junctions via cumulus-cumulus interactions. In the hours preceding ovulation, the frequency of gap junctional contacts between cumulus cells and the oocyte was reduced, and the cumulus was disorganized. Electrophysiological measurements indicated that bidirectional ionic coupling was present between the cumulus and oocyte before ovulation. In addition, iontophoretically injected fluorescein dye was tranferred between the oocyte and cumulus cells. Examination of the extent of ionic coupling in cumulus-oocyte specimens before and after ovulation revealed that ionic coupling between the cumulus and oocyte progressively decreased as the time of ovulation approached. In postovulatory specimens, no coupling was detected. Although some proteolytic mechanism may be involved in the disintegration of the cumulus-oocyte complex, neither the cumulus cells nor the oocyte produced detectable levels of plasminogen activator, a protease which is synthesized by membrana granulosa cells. In summary, cell communication is a characterisitc feature of the cumulus-oocyte complex, and this communication is terminated near the time of ovulation. This temporal pattern of the termination of communication between the cumulus and the oocyte may indicate that communication provides a mechanism for regulating the maturation of the oocyte during follicular development before ovulation.


The effect of protein degradation on cellular growth characteristics. The role of protein degradation in cellular proliferation was investigated by measurements of the rates of degradation of labile and stable proteins for a number of cell types under various growth conditions. The rate of protein degradation was found to be a relatively invariant parameter in that it did not change after strong inhibition of protein synthesis with cychloheximide or histidinol, it was the same in both exponential and stationary phase, and it did not correlate with the presence or absence of malignant transformation. Using three different cell types with widely differing division rates, the rate of cell division and DNA synthesis (in %/hr) was found to be precisely equal to the rate of protein accumulation (in %/hr) , i.e., to the rate of protein synthesis minus the rate of protein degradation. Division rates between the different cell types appeared to be determined chiefly by the rate of protein synthesis though, especially at low division rates, the rate of protein degradation could represent a large component of the protein accumulation rate.


The isolation of a low molecular weight serum factor which enhances the viability of SV3T3 cells. A serum factor which enhances the growth/viability of SV40 transformed 3T3 mouse fibroblasts, but not untransformed 3T3 cells in tissue culture has been partially purified from calf serum. The purification, which involves acidification to pH 2, chromatography on Sephadex G-100 at pH 2 followed by either Sephadex G-25 or BioGel P-2, results in material exhibiting two ninhydrin positive spots on thin layer chromatography and five to six dansylatable bands on SDS gels. This substance, termed Peak III or Serum Factor III, has been found in every serum examined thus far (including calf, rat, mouse, goat, lamb, rabbit, pig, horse, and chicken) and appears to be a low molecular weight, heat-insensitive molecule. Attempts to characterize it by chemical analyses and specific enzymatic inactivation have been either negative or inconclusive. Its biological mode of action is presently obscure. While it does not effect DNA synthesis, it does greatly increase the viability of SV3T3 cultures growing in low serum (0.15-0.30% calf serum) medium and, when added to "stationary phase" cells, restores healthy proliferation. This and evidence reported by others suggest that Peak III may affect SV3T3 cells in either overcoming a potentially lethal block in G1 or in passage through G2/M.


Binding and internalization of 125I thrombin in chick embryo fibroblasts: possible role in mitogenesis. Human alpha thrombin acts as a mitogen for cultures of resting chick embryo fibroblasts (CEF) in serum free medium. The use of 125I-labeled thrombin shows that thrombin specifically binds to CEF and that after a lag of approximately 30 to 60 minutes it can not be removed by subsequent exposure to trypsin. The entry of 125I thrombin into the trypsin-insensitive domain is not inhibited to any great extent by excess unlabelled thrombin. The cell-associated thrombin retains its native molecular weight and its catalytic activity toward synthetic amide substrates. It appears to be located in the crude nuclear fraction of homogenized CEF cells. The association of thrombin with CEF is specific, since the non-mitogenic serine protease chymotrypsin is internalized to a much lesser extent than thrombin. The data are discussed in terms of a possible intracellular site for thrombin's mitogenic action.


Transport and accumulation of purine bases by Crithidia fasciculata. The capacity for the rapid transport of purine bases by Crithidia fasciulata is found only in cells starved for purines. Cells grown in complete medium transport poorly. Rapid transport capability appears and then disappears during growth of purine-depleted cultures. This rapid transport appears to occur by a process of mediated diffusion. Two mechanisms are involved, one of low velocity and high affinity, the other of high velocity and low affinity. Accumulation of the bases within the cell occurs by their rapid conversion to ribonucleotides by phosphoribosyltransferases.


Deoxyglucose and 3-O-methylglucose transport in untreated and ATP-depleted Novikoff rat hepatoma cells. Analysis by a rapid kinetic technique, relationship to phosphorylation and effects of inhibitors. Detailed time courses of uptake of labeled 3-O-methyl-D-glucose and 2-deoxy-D-glycose by untreated and ATP-depleted Novikoff rat hepatoma cells were determined as function of concentration (0.2-10 mM) by a rapid mixing/sampling technique which allows uptake measurements in time intervals as short as 1.5 seconds. Intracellular accumulation of 3-O-methylglucose in untreated and ATP-depleted cells and of deoxyglucose in ATP-depleted cells to equilibrium followed pseudo-first order kinetics and initial velocities were computed from overall time courses of substrate accumulation. Initial velocity was a Michaelis-Menten function of exogenous substrate concentration. The estimated kinetic constants for zero-trans transport of 3-O-methylglucose were about the same for untreated and ATP-depleted cells (Kztm = 1.73 +/- 0.24 mM; Vztmax = 28.8 +/- 3.6 pmoles/microliter cell H2O. sec) and were similar to those for deoxyglucose transport in ATP-depleted cells (Kztm = 0.65 +/- 0.1 mM; Vztmax = 19.6 +/- 1.6 pmoles/microliter cell H2O. sec). Similar kinetic parameters were obtained for the transport of D-glucose and D-galactose in ATP-depleted cells. The transport of 3-O-methylglucose and deoxyglucose  were inhibited by each other in a simple competitive manner with apparent Ki's similar to their transport Km's. In untreated cells, in which deoxyglucose was phosphorylated, intracellular steady-state levels of free deoxyglucose accumulated within 10 to 20 seconds of incubation regardless of its concentration in the medium. Thereafter, the rate of deoxyglucose incorporation into total cell material reflected the rate of phosphorylation rather than the transport rate. The rate of deoxyglucose transport exceeded the initial rate of its phosphorylation by 20-40 %. The intracellular steady-state-levels observed during the first 2 minutes of incubation decreased from about 40% of equilibrium level at 0.2 mM deoxyglucose to about 8% at 10 mM. Computer fits of a kinetic equation describing transport and phosphorylation as independent processes operating in tandem to these data are consistent with the observed kinetic constants for hexose transport and hexokinase activity with deoxyglucose as substrate. Upon longer incubation (2-10 minutes) the rate of deoxyglucose uptake by the phosphorylating cells decreased progressively, concomitant with a decrease in intracellular ATP and an increase in intracellular deoxyglucose to equilibrium levels. It is demonstrated that the rate of deoxyglucose uptake, measured at two or more minutes, seriously underestimates the hexose transport rate and yields misleading conclusions regarding the extent and type of inhibition by transport inhibitors, such as persantin or cytochalasin B. Persantin inhibited hexose transport in a simple non-competitive manner (Ki = 20 muM) indicating that the drug affects the function of the hexose carrier.


Localization of pyruvate kinase isozymes in bovine kidney and comparison of these patterns with those of lactate dehydrogenases and aldolases. Electrophoretic and immunofluorescence analysis were used to study the distribution of pyruvate kinase isozymes in the bovine kidney. Electrophoretic analysis demonstrated the presence of large amounts of K4 plus small amounts of K-M hybrids in cortical, medullary, and papillary sections cut from the kidney. Nearly all of the K-L hybrids seen in whole kidney extracts were found in cortical sections. Immunofluorescence of frozen sections revealed the presence of type L subunits in the tubules but the complete absence of this subunit type in flomeruli. Glomeruli do contain large quantities of pyruvate kinase isozymes, probably K4 and K-M hybrids, that cross-react with antibodies produced against type M pyruvate kinase. Type L-containing forms of pyruvate kinase and aldolase type B both appear to be found in cell types thought to be capable of catalyzing of gluconeogenesis, while type K pyruvate kinase and type A aldolase are found in predominantly glycolytic cell types of the kidney. Lactate dehydrogenase isozymic patterns appear to be less closely correlated with glycolytic versus gluconeogenic functions of the kidney but may be determined more directly by other metabolic functions.


Factors influencing endothelial cell proliferation in vitro. The relative roles of blood cell products and plasma factors on endothelial cell proliferation were evaluated by studying the proliferative response of human umbilical vein endothelial cells to cell free plasma derived serum (CFPDS), whole blood serum (WBS), platelet released factors, fibroblast growth factor and macrophage conditioned medium in vitro. Human adult arterial smooth muscle cells were treated in a similar manner for comparison. The rate of endothelial cell proliferation was directly related to the concentrations of both WBS and CFPDS. Grwoth rate in WBS was marginally greater than that observed in CFPDS during early culture, however, similar confluent densities were achieved. The addition of platelet released factors to CFPDS did not further stimulate endothelial cell proliferation. In contrast smooth muscle cells were quiescent in CFPDS despite increasing serum concentrations, but proliferated actively in response to platelet released factors. Both human macrophage conditioned medium and fibroblast growth factor increased endothelial cell proliferation significantly when compared with CFPDS alone. It is concluded that endothelial cell proliferation in preconfluent cultures is dependent on plasma factors while human vascular smooth muscle cells also require cell derived mitogens such as platelet growth factor to proliferate. The release of a substance by human macrophages mitogenic for endothelial cells may be involved in endothelial cell proliferation in vivo.


DNA replication in human lymphocytes during aging. An analysis of the accuracy of protein and DNA synthesis in human lymphocytes with respect to aging has been carried out. The response of human peripheral lymphocytes, from young and old adults, to phytohemagglutinin was measured at varying temperatures. This should provide a sensitive test for the accumulation of altered thermolabile proteins that are rate limiting in the response to phytohemagglutinin. At 37 degrees C the rate of thymidine incorporation as well as the induction of DNA polymerase in phytohemagglutinin-stimulated lymphocytes from old and young adults were similar. Also at elevated temperatures, the thermosensitivity of DNA replication in lymphocytes from young and old adults was the same. DNA polymerase was purified from PHA-stimulated lymphocytes from young and old adults. The fidelity of DNA synthesis using poly (dC) as a template was similar with both enzymes. However, DNA polymerase-alpha purified from old adults was thermolabile compared to the enzyme from young adults. Thus, while the lymphocytes from old individuals may have heat labile proteins, they do not limit their proliferative capacity.


The role of myonuclei in muscle regeneration: an in vitro study. It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell division to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or "freed" as a result of the trauma, play any role in muscle regeneration remains unresolved. In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub-cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H3-thymidine and their ultimate fate, either as "free" myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub-cultured. In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re-enter the mitotic cycle.


Lack of a correlation between polyamine synthesis and DNA synthesis by cultured rat liver cells and fibroblasts. Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely alpha-hydrazino ornithine and methylglyoxal bis (guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth-stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G1) to enable them to enter from G1 into S phase and traverse S phase at normal rates in the absense of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content.


Membrane bound and cellular cationic changes associated with insulin stimulation of cultured cells. Insulin was employed as a stimulant in our continuing investigations of the molecular mechanisms involved in the coordinate control of cellular metabolism and growth. Incubation of chicken embryo fibroblasts for 16 hours in media containing 0-0.1 U insulin/ml resulted in a 17-fold increase in the rate of 3H-thymidine incorporation into DNA. Concomitantly, there were graded increases in intracellular K+ (14%) AND Mg2+ (22%) and no significant change in Ca2+. These changes in cation content occurred within 10 to 30 minutes and preceded the changes in 3H-thymidine incorporation. Insulin produced a consistent graded decrease in externally bound Mg2+ and Ca2+ and a concomitant increase in bound Na+ and K+ with no significant change in the rates of K+ and Mg2+ efflux. The results are consistent with the concept of Mg2+ as a second messenger for insulin action, as well as with the more general hypothesis that Mg2+ is the centtral agent in the coordinate control of metabolism and growth in animal cells.


A human serum protein system affecting muscle contractility: characterization of the five components and their reaction sequence. A human serum protein system that affects muscle contractility, previously reported to be a calcium transport system (CTS), was fractionated into five protein components, A, B1, Bfi, B3 and C. The system was assayed on frog heart muscle in vitro by its capacity to increase contractile force and at high concentrations to cause contracture. With separated fractions, the reaction sequence was divided into three separate steps: B1 and B3 interacted with the heart in the first step, B2 and B3 in the second, and A and C in the third. Increased contractility occurred only after the third step. Partial purification, with preservation of biological activity, was achieved for B1, B2, A and C. B1 has a M.W. of about 170,000, as determined by its elution from Sephadex G-200. B2, a labile protein of M.W. 220,000, was one of the few serum proteins precipitated by M.W. 5000,000 dextran sulfate at pH 8.0. This was the basis for the preparation of highly purified B2, which was used to produce rabbit anti-B2 antibody. Experiments with this antibody proved that B2 became bound to the heart surface. CTS-A, the smallest protein of the system, had a M.W. of approximately 130,000. The M.W. of C was over 300,000. Purified C antigen induced rabbit antibody that inhibited the action of C without affecting the other components.


Diffusible factor(s) controlling density inhibition of 3T3 cell growth: a new approach. In 3T3 Swiss mouse fibroblasts, incorporation of phosphate into cells and phosphorylation of small organic compounds were increased by shaking dense cultures. This response was not obtained with SV40 transformed Swiss 3T3 cells (SV-3T3). It appeared likely that these results could be accounted for by an inhibitor released from 3T3 cells but not from SV-3T3 cells. Our new method of co-incubation of sparse and dense cultures allowed us to demonstrate inhibition of growth and phosphate metabolism in sparse 3T3 cultures which were shaken in the presence of dense cultures. The inhibition was much less when the cultures were co-cultivated but not shaken. The inhibition of phosphate incorporation in acid-soluble and acid-insoluble fractions of sparse cultures was observed as early as 20 minutes of co-incubation in the presence of dense cultures, so this inhibition is not the result of depletion of growth factors in the medium. Our experiments suggest that an inhibitor(s) was released from dense cultures of 3T3 cells.


Analysis of the penetrable space within the nucleus. Radioactive glycogen molecules have been used as passive probes to compare cavity systems within nuclei and isolated chromatin. Isolated chromatin was found to possess a narrow range of microspaces with mean effective diameters between 4.0 and 4.5 nm (40 and 45 A) depending on shape assumptions. Intact nuclei contained a far larger class of free spaces with average diameters in the order of 11.0-15.0 nm. This clearly shows that DNase-I (diam. 4.1 nm) can penetrate and occupy a large proportion of nuclear space even though this enzyme does not readily attack the undisturbed nuclear structure. A structure which simulated the pattern of penetrability and incorporated other known properties of chromatin was used to explain this DNase-I resistance of intact nuclei in terms of an ordered, compact, local structure interspersed by much larger spaces. A system for this local packing is suggested and the functional implications of this type of organization considered.


Follicle cell bridges in the mosquito ovary: syncytia formation and bridge morphology. In the mosquito, Culex pipiens quinqefasciatus, the follicle cells enveloping the oocyte and the nurse cells are connected by intercellular bridges. The bridges are formed by incomplete cytokinesis, and they persist for more than 30 h after their formation. Reconstructions from serial sections showed that one syncytial group contained at least 32 cells; several cells continued outside the series. The cells in a syncytium divide asynchronously; this results in an irregular, branched orgainzation. The bridges may be either embedded in the cytoplasm of the cells, or they may form an extracellular connexion.


The blood/germ cell barrier in male Schistocerca gregaria: the time of its establishment and factors affecting its formation. Tracer studies carried out on the testes of the insect Schistocerca gregaria demonstrate a blood/germ cell barrier which is established prior to the onset of meiosis in the 3rd instar. Ecdysterone treatment in vitro prematurely stimulates the formation of the barrier in the testicular follicles of 2nd instar hoppers; this effect is antagonized by juvenile hormone. The follicles may be a store for the barrier-stimulating factor because testis extract also induces premature production of the barrier.


Studies of the endoplasmic reticulum and plasma membrane-bound ribosomes in erythropoietic cells. Erythropoietic cells of 5 species, including man, contain endoplasmic reticulum present as individual cisternae or tubules scattered throughout the cytoplasm of all stages except mature RBCs. The endoplasmic reticulum is mainly agranular but occurs frequently as a variant of granular ER which is characterized by an asymmetrical and irregular distribution of ribosomes along one cytoplasmic face. In most cells, the endoplasmic reticulum occurs in close proximity to mitochondria or the plasma membrane , suggesting that the organelle may be involved in functions related to these structures, e.g. haem biosynthesis. Endoplasmic reticulum is more abundant in early than in late erythroid cells. Its exact role in RBC development is unclear. Since endoplasmic reticulum could account for 'plasma membrane-bound ribosomes' reported in lysed reticulocytes, studies were performed which ruled out this possibility and which suggested that such ribosomes were an artifact of the lysing conditions. Hypotonic lysis in less than 20 vol. of magnesium-containing buffers yielded ghosts variably contaminated by ribosomes and other structures. Lysis of reticulocytes in 20-30 vol. of magnesium-free buffer or homogenization of whole cells or crude membrane fractions in hypotonic buffer removed virtually all contaminating ribosomes from the purified membrane fraction.


Sulphate metabolism in the exocrine pancreas. II. The production of sulphated macromolecules by the mouse exocrine pancreas. The types of sulphated macromolecules produced by the exocrine pancrease were investigated. To determine whether this tissue utilized inorganic sulphate for protein production, the in-vitro behaviour of material labelled with 35S-sulphate was compared with material labelled with [3H]leucine (secretory proteins). While incubating tissue slices in the presence of cycloheximide resulted in an immediate and nearly complete inhibition of protein synthesis, a similar decrease in production of sulphated material was not observed until after 2 h of incubation in the presence of the drug. Likewise, the kinetics of pilocarpine-induced discharge of radioactive material from pancreatic slices pulse-labelled with either 3H-Leu. or 35S-sulphate was compared. During the first 90 min of stimulation sulphated macromolecules were detected in chase medium 10-15 min prior to the appearance of 3H-labelled secretory proteins. That in-vitro behaviour of sulphated material differed from radioleucine-labelled material is indicative of the fact that the pancreas utilizes inorganic sulphate for the production of macromolecules other than secretory proteins. Lipid and proteoglycan fractions were prepared from pancreatic tissue 4 h after intraperitoneal injection of radiosulphate. The recovery of a significant amount of radioactivity in both fractions deomonstrated the ability of the pancreas to use inorganic sulphate for the production of both sulphated lipids and sulphated proteoglycans. The possible function of sulphated macromolecules in pancreatic secretion is discussed.


Osmoregulation in the alga Vacuolaria virescens. Structure of the contractile vacuole and the nature of its association with the Golgi apparatus. The contractile vacuole of the chloromonadophycean alga Vacuolaria virescens is a permanent structure that possesses a specialized membrane: subunits of this membrane have a diameter of 21-24 nm and in places are arranged in a regular hexagonal pattern. The lateral walls of these subunits form regularly spaced bristles or pegs which extend inwards from the trilaminar membrane for a distance of 13-15 nm. The contractile vacuole is situated immediately above an extensive Golgi apparatus that covers most of the anterior surface of the nucleus. Vesicles of Golgi origin give rise to subsidiary vacuoles which in turn empty into the contractile vacuole. Golgi vesicles, subsidiary vacuoles and the contractile vacuole contain similar electron-dense material. It is suggested that this material might be a highly hydrophilic substance which will attract water from the cytoplasm into the Golgi vesicles, subsidiary vacuoles and contractile vacuole from whence it is discharged from the cell. This method of osmoregulation is compared to that occurring in other algae and protozoa.


Effect of ultraviolet or laser light irradiation on the growth of Micrasterias thomasiana. The effect of ultraviolet on laser light on growth of lobes in a green alga, Micrasterias thomasiana was investigated. During plasmolysis, the cell wall and the outermost region of the cytoplasm were separately irradiated with an ultraviolet or laser microbeam. Growth of the lobes was more effectively inhibited by irradiation of the cytoplasm than of the cell wall when the dose of irradiation was weak. Growth inhibition was observed in the irradiated cytoplasm where no apparent decrease in velocity of plasma streaming was seen, which suggested that irradiation caused more damage to the mechanism of exocytosis than to the migration of secretion vesicles. Strong irradiation resulted in gelatinization of the cytoplasm. With strong doses of irradiation on both the cytoplasm and cell wall, growth was completely inhibited.


Translational movements of macrophages through media of different viscosities. The extent of the migration of guinea-pig peritoneal macrophages from capillary tubes over the surfaces of plastic Petri dishes, was quantitated by planimetry. It was observed that the amount of migration decreased as the viscosity of the medium was increased by the addition of agarose. An essential component of translational movement is the formation of adhesions between cells and their substrata. It is suggested on the basis of the present migration and adhesion experiments, that the increased viscosity reduces migration by retarding the formation of adhesions between the cells and their substrata. The experiments do not support the suggestion that agarose adsorbed to the dish surface affects the adhesion-dependent arm of migration or that viscous 'drag' on whole cells (over the viscosity range of 0.71 to 3.9 x 10(-3) N s m-2) reduces their movements.


Genetic aspects of intracellular motility: cortical localization and insertion of trichocysts in Paramecium tetraurelia. Wild-type and selected mutants of Paramecium tetraurelia were subjected to detailed analysis of the trichocyst phenotype. The characteristics of morphology, saltatory motility, and cortical insertion of trichocysts were found to be genetically distinguishable. In mutants with defective trichocyst saltatory motility, other intracellular motility systems (i.e. mitochondrial saltatory motility, and cyclosis) appeared to be unaffected. Saltatory motility appears to be an essential intermediate step in the functional development of the trichocyst, since it serves to transport the trichocyst from its site of assembly, deep in the cytoplasm, to the cortex, where it is inserted and becomes discharge-competent. If trichocyst saltatory motility is lost, as in the mutants ndA and tam8, then trichocysts are not transported to the cortex and are therefore not inserted. However, the successful transport of a trichocyst to the cortex does not guarantee the proper insertion of that trichocyst, as in the case of the mutant am. Apparently there are independent steps involved specifically with trichocyst insertion. The gross morphology of the trichocyst appears to be independent of its saltatory motility, and to a degree, independent of cortical insertion.


Cell interactions in the developing somite; in vivo comparisons between amputated (AM/AM) and normal mouse embryos. Light and electron microscopy coupled to a quantitative analysis form a powerful technique for the analysis of cell behaviour in mutant and normal tissues. Using this approach the morphology of the cells and their contacts are examined in the somites of a recessive mouse mutant, amputated, and of its normal littermates. The results of the analysis show that cell density is the same in both mutant and normal sclertome but that mutant cells tend to form small clumps whereas normal cells disperse individually. There is a correspondingly greater area of cell contact per cell in the mutant. Filopodia are equally numerous in the mutant and normal but where in the normal they stretch across wide intercellular spaces to make contact at their tips with other cells, in the mutant they form a tangled web sticking back on to the surface of the cell of origin and adjacent cells. The appearance of mutant and normal sclerotome is compared with presomitic cells and with the cells of other mutants whose abnormal development has been shown to depend on cell contact morphology and behaviour.


Cell movement and the mechanism of invasiveness: a survey of the behaviour of some normal and malignant cells implanted into the developing chick wing bud. A survey of the behaviour of a variety of normal and malignant tumours and cells has been carried out to gain insights into the mechanisms of tumour invasiveness. The tumours and cells were implanted into the developing chick wing bud, which is a loose mesenchyme bounded by ectoderm. The distribution of the grafted cells was examined histologically after one or two days. The special feature of this assay is that the behaviour of cells is tested in a 3-dimensional tissue. Cells from 3 different carcinomas, mouse lung tumour, rat bladder tumour and human breast tumour did not invade the mesenchyme, whereas trophoblast, sarcoma 180, cultured hamster fibroblasts (BHK, PyBHK, Nil 8, HSV Nil 8) and neuroblastoma cells did. Cells from embryonic pigmented retina and heart ventricle were non-invasive. These results suggest that cell movement may not be a common feature of all invasive tumours. The cells that did move into the mesenchyme appeared to do so by various mechanisms. Lack of contact inhibition of movement, although probably involved in the invasiveness of sarcoma 180 cells, does not appear to be necessary for invasion: cells that have been shown to exhibit contact inhibition of movement (BHK and PyBHK) also invade. Both normal and transformed cells (BHK and PyBHK; Nil 8 and HSV Nil 8) moved into the mesenchyme. Other invading cells, such as trophoblast, neuroblastoma and to a small extent, HSV Nil 8 cells, destroy the adjacent host tissue and this may be important in the invasiveness of these cells. The patterns of invasion and interactions with the host tissue were varied. Trophoblast and the fibroblasts were often elongated along the basement membrane at the ectoderm/mesenchyme border and also closely apposed to the endothelial linings of blood vessels. Sarcoma 180 and neuroblastoma cells clustered around nerves. The embryonic tissues and neuroblastoma cells were often associated with blood vessels. These results are discussed in relation to tumour invasion. A striking finding was that the carcinoma cells were frequently found positioned within the wing ectoderm on the basement membrane. This affinity of carcinoma cells for the epithelium rather than the mesenchyme leads to a reappraisal of the mechanisms involved in the invasiveness of carcinomas.


Ruthenium red-positive surface layer, extracellular filamentous material and intercellular junctions in hybrids between tumour and normal cells: abundant gap junctions correlate with density-dependent inhibition of growth. The cell surface and intercellular junctions of transformed and non-transformed hybrid cells, obtained from fusion of murine mammary adenocarcinoma cells (TA3B) and normal rat embryofibroblasts (REF), were compared with those of the parental cells, using ruthenium red (RR) staining of cells fixed in situ. An RR-positive layer of variable thickness was found on the surface of all cell types. Measurements of the thickness of this layer on the free surface of the cell cultures deomonstrated a significant difference between transformed and non-transformed cells. The thickness distribution of the RR layer was similar on the surface of REF and non-transformed hybrid cells, but there was significant variation among all the transformed cell lines. Extracellular filamentous RR-positive material, usually in direct contact with the cell surface, was present in confluent cultures of REF and non-transformed hybrid cells, but was absent in the transformed cell lines. Gap junctions were few in both parental cell lines and rare or absent in transformed hybrids; a large increase in number and size of gap junctions was found in non-transformed hybrids. Abundance of long gap junctions was correlated with density-dependent inhibition of growth, since non-transformed hybrids grew in single layers whereas both normal and tumour parental cell types, and transformed hybrids grew in multilayers. Tight junctions were frequently encountered in TA3B and transformed hybrid cells but were not seen in REF cells and only occasionally seen in non-transformed hybrids. Intermediate-type junctions occurred in all cells, but desmosomes were found only rarely in TA3B cells and never in the other cell types.


Cellular actin and junction formation during reaggregation of adult rat hepatocytes into epithelial cell sheets. Aggregation of adult rat hepatocytes, isolated by the collagenase perfusion technique, was studied by ultrastructural methods and the indirect immunofluorescence technique using anti-actin antibodies. In a primary culture the cells rapidly made contact with each other by filopodia-like structures, as seen by scanning electron microscopy. In a few hours this led to stable adhesion of the cells. No identifiable junction formation occurred during the first 17 h in culture. Within 48 h the cells had formed epithelial cell sheets with junctional complexes consisting of tight junctions, bile canaliculus-like structures and zonula adhaerens-type junctions. The distribution of cytoplasmic actin fluorescence remained homogenous in the contacting cells during the first 24 h in culture, as seen with anti-actin antibodies by the indirect immunofluorescence technique. The first short, fluorescent actin filaments appeared in the periphery of the developing lamellipodia of the spreading cell islands. In organized epithelial cell sheets these filaments were seen as long fibres ending at the perinuclear region of the marginal cells. In the submarginal cells fluorescent actin fibres were distinct at the junctional regions of the cells. This filamentous fluorescence seemed to extend throughout the entire cell sheet in an organized manner and correspond to the apical layer of parallel microfilaments seen in transmission electron microscopy. Our results suggest that filamentous actin plays a role in the contact-induced spreading of the cells and in the maintenance of the internal organization of the epithelial cell sheets.


Modulation of the effect of colchicine and cytochalasin B on fibroblast aggregates by the substratum. In previous investigations, we have shown that aggregates of normal human fibroblasts displayed a remarkable number of morphogenetic properties in suspension and on a substratum. The present investigation has attempted to illustrate more clearly the role of colchicine- and cytochalasin-sensitive structures in the observed ultrastructural properties of the fibroblasts in this system. Cytochalasin B and colchicine alter the surface ultrastructure of the cells as well as the behaviour of the cells during aggregate outgrowth. In addition, the effects of both drugs on the cells in the aggregates are modulated by the attachment of the aggregates to a substratum.